Note: Descriptions are shown in the official language in which they were submitted.
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EXCIPIENT COMPOUNDS FOR BIOPOLYMER FORMULATIONS
RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application No.
62/940,683
filed November 26, 2019! The entire contents of the above application are
incorporated by
reference herein.
FIELD OF APPLICATION
100011 This application relates generally to formulations for delivering
biopolymers.
to
BACKGROUND
100021 Biopolymers may be used for therapeutic or non-therapeutic purposes.
Biopolymer-
based therapeutics, such as antibody or enzyme formulations, are widely used
in treating
disease. Non-therapeutic biopolymers, such as enzymes, peptides, and
structural proteins,
have utility in non-therapeutic applications such as household, nutrition,
commercial, and
industrial uses.
100031 Biopolymers used in therapeutic applications must be formulated to
permit their
introduction into the body for treatment of disease. For example, it is
advantageous to deliver
antibody and protein/peptide biopolymer formulations by subcutaneous (SC) or
intramuscular
(IM) routes under certain circumstances, instead of administering these
formulations by
intravenous (IV) injections. In order to achieve better patient compliance and
comfort with
SC or IM injection though, the liquid volume in the syringe is typically
limited to 2 to 3 ccs
and the viscosity of the formulation is typically lower than about 20
centipoise (cP) so that
the formulation can be delivered using conventional medical devices and small-
bore needles.
These delivery parameters do not always fit well with the dosage requirements
for the
formulations being delivered.
100041 Antibodies, for example, may need to be delivered at high dose levels
to exert their
intended therapeutic effect. Using a restricted liquid volume to deliver a
high dose level of an
antibody formulation can require a high concentration of the antibody in the
delivery vehicle,
sometimes exceeding a level of 150 mg/mL. At this dosage level, the viscosity-
versus-
concentration plots of the formulations lie beyond their linear-nonlinear
transition, such that
the viscosity of the formulation rises dramatically with increasing
concentration. Increased
viscosity, however, is not compatible with standard SC or IM delivery systems.
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[0005] As an additional concern, solutions of biopolymer-based therapeutics
are also prone
to stability problems, such as precipitation, fragmentation, oxidation,
deamidation, hazing,
opalescence, denaturing, liquid-liquid phase separation, and gel formation,
reversible or
irreversible aggregation. The stability problems limit the shelf life of the
solutions or require
special handling.
[0006] As an example, one approach to producing protein formulations for
injection is to
transform the therapeutic protein solution into a powder that can be
reconstituted to form a
suspension suitable for SC or IM delivery. Lyophilization is a standard
technique to produce
protein powders. Freeze-drying, spray drying, and even precipitation followed
by super-
to critical-fluid extraction have been used to generate protein powders for
subsequent
reconstitution. Powdered suspensions are low in viscosity before re-
dissolution (compared to
solutions at the same overall dose) and thus may be suitable for SC or TM
injection, provided
the particles are sufficiently small to fit through the needle. However,
protein crystals that are
present in the powder have the inherent risk of triggering immune response.
The uncertain
protein stability/activity following re-dissolution poses further concerns.
There remains a
need in the art for techniques to produce low viscosity protein formulations
for therapeutic
purposes while avoiding the limitations introduced by protein powder
suspensions.
100071 In addition to the therapeutic applications of proteins described
above, biopolymers
such as enzymes, peptides, and structural proteins can be used in non-
therapeutic
applications. These non-therapeutic biopolymers can be produced from a number
of different
pathways, for example, derived from plant sources, animal sources, or produced
from cell
cultures.
100081 The non-therapeutic proteins can be produced, transported, stored, and
handled as a
granular or powdered material or as a solution or dispersion, usually in
water. The
biopolymers for non-therapeutic applications can be globular or fibrous
proteins, and certain
forms of these materials can have limited solubility in water or exhibit high
viscosity upon
dissolution. These solution properties can present challenges to the
formulation, handling,
storage, pumping, and performance of the non-therapeutic materials, so there
is a need for
methods to reduce viscosity and improve solubility and stability of non-
therapeutic protein
solutions.
100091 Proteins are complex biopolymers, each with a uniquely folded 3-D
structure and
surface energy map (hydrophobic/hydrophilic regions and charges). In
concentrated protein
solutions, these macromolecules may strongly interact and even inter-lock in
complicated
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ways, depending on their exact shape and surface energy distribution. "Hot-
spots" for strong
specific interactions lead to protein clustering, increasing solution
viscosity. To address these
concerns, a number of excipient compounds are used in biotherapeutic
formulations, aiming
to reduce solution viscosity by impeding localized interactions and
clustering. These efforts
are individually tailored, often empirically, sometimes guided by in silico
simulations.
Combinations of excipient compounds may be provided, but optimizing such
combinations
again must progress empirically and on a case-by-case basis.
WOW] There remains a need in the art for a truly universal approach to
reducing viscosity
in protein formulations at a given concentration under nonlinear conditions.
There is an
to additional need in the art to achieve this viscosity reduction while
preserving the activity of
the protein and avoiding stability problems. It would be further desirable to
adapt the
viscosity-reduction system to use with formulations having tunable and
sustained release
profiles, and to use with formulations adapted for depot injection. In
addition, it is desirable
to improve processes for producing proteins and other biopolymers.
SUMMARY OF THE INVENTION
100111 Disclosed herein, in embodiments, are liquid formulations comprising a
protein and
an excipient compound selected from the group consisting of hindered amines,
aromatic or
anionic aromatics, functionalized amino acids, oligopeptides, short-chain
organic acids, low
molecular weight aliphatic polyacids, diones and sulfones, zwitterionic
excipients, and
crowding agents with hydrogen bonding elements, wherein the excipient compound
is added
in a viscosity-reducing amount. In embodiments, the protein is a PEGylated
protein and the
excipient is a low molecular weight aliphatic polyacid. In embodiments, the
excipient
compound is a hindered amine compound.
100121 In embodiments, the hindered amine can be selected from the group
consisting of
anunonium chloride, ammonium bromide, ammonium fluoride, ammonium acetate,
ammonium citrate, monoethanolamine hydrochloride, monoethanolamine
hydrobromide,
monoethanolamine hydrofluoride, monoethanolamine acetate, monoethanolamine
citrate,
diethanolamine hydrochloride, diethanolamine hydrobromide, diethanolamine
hydrofluoride,
diethanolamine acetate, diethanolamine citrate, triethanolamine hydrochloride,
triethanolamine hydrobromide, triethanolamine hydrofluoride, triethanolamine
acetate,
triethanolamine citrate, urea hydrochloride, urea hydrobromide, urea
hydrofluoride, urea
acetate, and urea citrate. In embodiments, the modified amine is a conjugate
acid of
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ammonia or ammonium hydroxide. In embodiments, the hindered amine compound is
a
conjugate acid of ammonia or ammonium hydroxide.
100131 In embodiments, the formulation is a pharmaceutical composition, and
the
pharmaceutical composition comprises a therapeutic protein, wherein the
excipient
compound is a pharmaceutically acceptable excipient compound. In embodiments,
the
formulation is a non-therapeutic formulation, and the non-therapeutic
formulation comprises
a non-therapeutic protein. In embodiments, the therapeutic protein is selected
from the group
consisting of bevaciztunab, trastuzumab, adalimumab, infliximab, etanercept,
darbepoetin
alfa, epoetin alfa, cetwcimab, pegfilgrastim, filgrastim, and rituximab. In
embodiments, the
to excipient compound is formulated as a concentrated excipient solution.
In embodiments, the
viscosity-reducing amount reduces viscosity of the formulation to a viscosity
less than the
viscosity of a control formulation. In embodiments, the viscosity of the
formulation is at
least about 10% less than the viscosity of the control formulation, or is at
least about 30% less
than the viscosity of the control formulation, or is at least about 50% less
than the viscosity of
the control formulation, or is at least about 70% less than the viscosity of
the control
formulation, or is at least about 90% less than the viscosity of the control
formulation. In
embodiments, the viscosity is less than about 100 cP, or is less than about 50
cP, or is less
than about 20 cP, or is less than about 10 cP. In embodiments, the excipient
compound has a
molecular weight of <5000 Da, or <1500 Da, or <500 Da In embodiments, the
formulation
contains at least about 1 mg/ml of the protein, or at least about 25 mg/mL of
the protein, or at
least about 50 mg/mL of the protein, or at least about 100 mg/mL of the
protein, or at least
about 200 mg/mL of the protein In embodiments, the formulation comprises
between about
0.001 mg/mL to about 60 mg/mL of the excipient compound, or comprises between
about 0.1
mg/mL to about 50 mg/mL of the excipient compound, or comprises between about
t mg/mL
to about 40 mg/mL, or comprises between about 5 mg/mL to about 30 mg/mL of the
excipient compound. In embodiments, the formulation has an improved stability
when
compared to the control formulation. The improved stability can be manifested
as a decrease
in the formation of visible particles, subvisible particles, aggregates,
turbidity, opalescence,
or gel. In embodiments, the formulation has an improved stability, wherein the
improved
stability is determined by comparison with a control formulation, and wherein
the control
formulation does not contain the excipient compound. In embodiments, the
improved
stability prevents an increase in particle size as measured by light
scattering. In embodiments,
the improved stability is manifested by a percent monomer that is higher than
the percent
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monomer in the control formulation, wherein the percent monomer is measured by
size
exclusion chromatography. In embodiments, the excipient compound is a hindered
amine,
which can be caffeine. In embodiments, the excipient compound is a hindered
amine. In
embodiments, the hindered amine is selected from the group consisting of
caffeine,
theophylline, tyramine, imidazole, aspartame, saccharin, acesulfame potassium,
pyrimidinone, 1,3-dimethyluracil, triaminopyrimidine, pyritnidine, and
theacrine. In
embodiments, the hindered amine is caffeine, procaine, lidocaine, imidazole,
aspartame,
saccharin, and acesulfame potassium. In embodiments, the hindered amine is
caffeine. In
embodiments, the hindered amine is a local injectable anesthetic compound. The
hindered
to amine can possess an independent pharmacological property, and the
hindered amine can be
present in the formulation in an amount that has an independent
pharmacological effect. In
embodiments, the hindered amine can be present in the formulation in an amount
that is less
than a therapeutically effective amount. The independent pharmacological
activity can be a
local anesthetic activity. In embodiments, the hindered amine possessing the
independent
pharmacological activity is combined with a second excipient compound that
further
decreases the viscosity of the formulation. The second excipient compound can
be selected
from the group consisting of caffeine, theophylline, tyramine, procaine,
lidocaine, imidazole,
aspartame, saccharin, and acesulfame potassium. In embodiments, the
formulation can
comprise an additional agent selected from the group consisting of
preservatives, surfactants,
sugars, polysaccharides, arginthe, proline_ hyaluronidase, stabilizers,
solubilizers, co-
solvents, hydrotropes, and buffers.
100141 Further disclosed herein are methods of treating a disease or disorder
in a mammal
in need thereof, comprising administering to said mammal a liquid therapeutic
formulation,
wherein the therapeutic formulation comprises a therapeutically effective
amount of a
therapeutic protein, and wherein the formulation further comprises an
pharmaceutically
acceptable excipient compound selected from the group consisting of hindered
amines,
aromatics and anionic aromatics, functionalized amino acids, oligopeptides,
short-chain
organic acids, low molecular weight aliphatic polyacids, diones and sulfones,
zwitterionic
excipients, and crowding agents with hydrogen bonding elements; and wherein
the
therapeutic formulation is effective for the treatment of the disease or
disorder. In
embodiments, the therapeutic protein is a PEGylated protein, and the excipient
is a modified
amine. In embodiments, the modified amine can be selected from the group
consisting of
ammonium chloride, ammonium bromide, ammonium fluoride, ammonium acetate,
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ammonium citrate, monoethanolamine hydrochloride, monoethanolamine
hydrobromide,
monoethanolamine hydrofluoride, monoethanolamine acetate, monoethanolamine
citrate,
diethanolamine hydrochloride, diethanolamine hydrobromide, diethanolamine
hydrofluoride,
diethanolamine acetate, diethanolarnine citrate, triethanolamine
hydrochloride,
triethanolamine hydrobromide, triethanolamine hydrofluoride, triethanolamine
acetate,
triethanolamine citrate, urea hydrochloride, urea hydrobromide, urea
hydrofluoride, urea
acetate, and urea citrate. In embodiments, the modified amine is a conjugate
acid of
ammonia or ammonium hydroxide. In embodiments, the protein is a PEGylated
protein and
the excipient compound is a low molecular weight aliphatic polyacid. In
embodiments, the
to therapeutic protein is a PEGylated protein, and the excipient compound
is an aromatic
compound, which can be a phenol or a polyphenol, and the polyphenol can be
tannic acid. In
embodiments, the excipient is a hindered amine. In embodiments, the
formulation is
administered by subcutaneous injection, or an intramuscular injection, or an
intravenous
injection. In embodiments, the excipient compound is present in the
therapeutic formulation
in a viscosity-reducing amount, and the viscosity-reducing amount reduces
viscosity of the
therapeutic formulation to a viscosity less than the viscosity of a control
formulation. In
embodiments, the excipient compound is prepared as a concentrated excipient
solution. In
embodiments, the therapeutic formulation has an improved stability when
compared to the
control formulation, In embodiments, the excipient compound is essentially
pure.
100151 Disclosed herein, in embodiments, are methods of improving stability of
a liquid
protein formulation, comprising: preparing a liquid protein formulation
comprising a
therapeutic protein and an excipient compound selected from the group selected
from the
group consisting of hindered amines, aromatics and anionic aromatics,
functionalized amino
acids, oligopeptides, short-chain organic acids, low molecular weight
aliphatic polyacids,
diones and sulfones, zwitterionic excipients, and crowding agents with
hydrogen bonding
elements, wherein the liquid protein formulation demonstrates improved
stability compared
to a control liquid protein formulation, wherein the control liquid protein
formulation does
not contain the excipient compound and is otherwise substantially similar to
the liquid protein
formulation. In embodiments, the excipient compound is formulated as a
concentrated
excipient solution. The stability of the liquid formulation can be a chemical
stability
manifested by resistance to a chemical reaction selected from the group
consisting of
hydrolysis, photolysis, oxidation, reduction, deamidation, disulfide
scrambling,
fragmentation, and dissociation. The stability of the liquid formulation can
be a cold storage
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conditions stability, a room temperature stability or an elevated temperature
stability. The
improved stability of the liquid protein formulation can be is manifested by a
percent
monomer that is higher than the percent monomer in the control formulation,
wherein the
percent monomer is measured by size exclusion chromatography. The stability of
the liquid
formulation can be a mechanical stability, which can be manifested by an
improved tolerance
for a stress condition selected from the group consisting of agitation,
pumping, filtering,
filling, and gas bubble contact.
100161 Also disclosed herein, in embodiments, are liquid formulations
comprising a protein
and an excipient compound selected from the group consisting of hindered
amines, aromatics
to and anionic aromatics, functionalized amino acids, oligopeptides, short-
chain organic acids,
low molecular weight aliphatic polyacids, diones and sulfones, zwitterionic
excipients, and
crowding agents with hydrogen bonding elements, wherein the presence of the
excipient
compound in the formulation results in a more stable protein-protein
interaction or improved
protein-protein interaction characteristics, which can be measured by the
protein diffusion
interaction parameter kD, or the second virial coefficient B22. In
embodiments, the
formulation is a therapeutic formulation, and comprises a therapeutic protein.
In
embodiments, the formulation is a non-therapeutic formulation, and comprises a
non-
therapeutic protein.
100171 The invention also encompasses a liquid formulation comprising a
protein and an
excipient compound, wherein the excipient compound is a pyrimidine and wherein
the
excipient compound is added in a viscosity-reducing amount. In certain
aspects, the
pyrimidine is a methyl-substituted pyrimidine. Non-limiting examples of
pyrimidines are
pyrimidine, pyrimidinone, triaminopyrimidine, 1,3-dimethyluracil, 1-
methyluracil,
methyluracil, 1,3-diethyluracil, 5-methyluracil, 6-methyluracil, uracil, 1,3-
dimethyl-
tetrahydro primidinone, thymine, 1-methylthyrnine, 0-4-Tnethylthymine,
dimethylthymine, dimethylthymine dimer, theacrine, cytosine, 5-methylcytosine,
and 3-
methylcytosine. In certain specific aspects, the pyrimidine is 1,3-
dimethyluracil. In additional
aspects, the invention is directed to a liquid formulation comprising a
protein and an
excipient compound selected from the group consisting of 3-atninopyridine,
dicyclomine, 1-
methyl-2-pyrrolidone, phenylserine, DL-3-phenylserine, and cycloserine,
wherein the
excipient compound is added in a viscosity-reducing amount. The liquid
formulation can be
a pharmaceutical composition, wherein the protein is a therapeutic protein and
the excipient
is a pharmaceutically acceptable excipient. Examples of therapeutic proteins
include, but are
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not limited to, therapeutic antibodies (e.g., bevacizumab, trastuzumab,
adalimumab,
inflixirnab, etanercept, cetuxirnab, rituxirnab, ipilimurnab, and omalizumab)
and PEGylated
proteins. Alternatively, the liquid formulation can be a non-therapeutic
formulation, wherein
the protein is a non-therapeutic protein. The viscosity-reducing amount of the
excipient in
the liquid formulation disclosed herein can be about 250 mg/ml or less;
between about 10
mg/mL and about 200 mg/mL; between about 10 mg/tnL and about 120 mg/mL;
between
about 20 mg/mL and about 120 mg/mL; between about 1 mg/mL and about 100 mg/mL;
between about 2 mg/mL and about 80 mg/mL; between about 5 mg/mL and about 50
mg/rnL;
between about 10 mg/mL and about 40 mg/mL; between about 1 InM and about 400
mM;
to between about 2 tn.M and about 150 inM; between about 5 mM and about 100
InM; or
between about 15 rnM and about 50 rnM. In additional aspects, the viscosity-
reducing
amount of the excipient compound reduces the viscosity of the formulation to a
viscosity less
than the viscosity of a control formulation, wherein the control formulation
does not contain
the excipient but is otherwise identical on a dry weight basis to the
therapeutic formulation;
for example, the viscosity of the liquid formulation can be at least about
10%, at least about
30%, at least about 50%, at least about 70%, or at least about 90% less than
the viscosity of
the control formulation. In additional aspects, the liquid formulation has a
viscosity less than
about 100 cP, less than about 50 cP, less than about 20 cP, or less than about
10 cP. In yet
further aspects, the liquid formulation comprises an additional agent selected
from the group
consisting of preservatives, surfactants, sugars, polysaccharides, arginine,
prolate,
hyaluronidase, stabilizers, solubilizersõ co-solvents, hydrotropes, and
buffers.
100181 The invention further encompasses methods of reducing the viscosity of
a liquid
formulation comprising a protein, for exa.mple, a therapeutic protein or a non-
therapeutic
protein, comprising adding to the liquid formulation a viscosity-reducing
amount of a
pyrimidine as described herein.
100191 in yet additional aspects, the invention is directed a method of
reducing the
viscosity of a liquid formulation comprising a protein, for example, a
therapeutic protein or a
non-therapeutic protein comprising adding to the liquid formulation a
viscosity-reducing
amount of an excipient selected from the group consisting of 3-aminopyridine,
dicyclotnine,
1-methyl-2-pyrrolidone, phenylserine, DL-3-phenylserine, and cycloserine.
100201 The invention additionally encompasses a method of treating a disease
or disorder
in a mammal in need thereof, comprising administering to said mammal a liquid
therapeutic
formulation, wherein the liquid therapeutic formulation comprises a
therapeutically effective
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amount of a therapeutic protein, wherein the liquid therapeutic formulation
further comprises
a viscosity-reducing amount of a pharmaceutically acceptable excipient
compound, wherein
the excipient compound is a pyrimidine; and wherein the therapeutic
formulation is effective
for the treatment of the disease or disorder.
[0021] In additional aspects, the invention includes a method of treating a
disease or
disorder in a mammal in need thereof, comprising administering to said mammal
a liquid
therapeutic formulation, wherein the liquid therapeutic formulation comprises
a
therapeutically effective amount of a therapeutic protein, wherein the liquid
therapeutic
formulation further comprises a viscosity-reducing amount of a
pharmaceutically acceptable
to excipient compound selected from the group consisting of 3-
atninopyridine, dicyclomine, 1-
methy1-2-pyrrolidone, phenylserine, DL-3-phenylserine, and cycloserine, and
wherein the
therapeutic formulation is effective for the treatment of the disease or
disorder.
[0022] Further disclosed herein, in embodiments, are methods of improving a
protein-
related process comprising providing the liquid formulation as described
herein, and
employing it in a processing method. hi embodiments, the processing method
includes
filtration, pumping, mixing, centrifugation, purification, membrane
separation,
lyophilization, or chromatography.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIG. 1 shows a graph of particle size distributions for solutions of a
monoclonal
antibody under stressed and non-stressed conditions, as evaluated by Dynamic
Light
Scattering. The data curves in FIG. 1 have a baseline offset to allow
comparison: the curve
for Sample 1-A is offset by 100 intensity units and the curve for Sample 1-FT
is offset by
200 intensity units in the Y-axis.
[0024] FIG. 2 shows a graph measuring sample diameter vs. multimodal size
distribution
for several molecular populations, as evaluated by Dynamic Light Scattering.
The data
curves in FIG. 2 have a baseline offset to allow comparison: the curve for
Sample 2-A is
offset by 100 intensity units and the curve for Sample 2-FT is offset by 200
intensity units in
the Y-axis.
[0025] FIG. 3 shows a size exclusion chromatogram of monoclonal antibody
solutions with
a main monomer peak at 8-10 minutes retention time. The data curves in FIG. 3
have a
baseline offset to allow comparison: the curves for Samples 2-C, 2-A, and 2-FT
are offset in
the Y-axis direction.
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DETAILED DESCRIPTION
100261 Disclosed herein are formulations and methods for their production and
use that
permit the delivery of concentrated protein solutions. In embodiments, the
approaches
disclosed herein can yield a lower viscosity liquid formulation or a higher
concentration of
therapeutic or nontherapeutic proteins in the liquid formulation, as compared
to traditional
protein solutions. In embodiments, the approaches disclosed herein can yield a
liquid
formulation having improved stability when compared to a traditional protein
solution. A
stable formulation is one in which the protein contained therein substantially
retains its
physical and chemical stability and its therapeutic or nontherapeutic efficacy
upon storage
to under storage conditions, whether cold storage conditions, room
temperature conditions, or
elevated temperature storage conditions. Advantageously, a stable formulation
can also offer
protection against aggregation or precipitation of the proteins dissolved
therein. For example,
the cold storage conditions can entail storage in a refrigerator or freezer.
In some examples,
cold storage conditions can entail storage at a temperature of 10 C or less.
In additional
examples, the cold storage conditions entail storage at a temperature from
about 2' to about
10 C. In other examples, the cold storage conditions entail storage at a
temperature of about
4 C. In additional examples, the cold storage conditions entail storage at
freezing
temperature such as about -20 C or lower. In another example, cold storage
conditions entail
storage at a temperature of about -20 C to about 0 C. The room temperature
storage
conditions can entail storage at ambient temperatures, for example, from about
10 C to about
C. Elevated storage conditions can entail storage at a temperature greater
than about 30 C.
Elevated temperature stability, for example at temperatures from about 30 C to
about 50 C,
can be used as part of an accelerated aging study to predict the long-term
storage at typical
ambient (10-30 C) conditions.
25 100271 It is well known to those skilled in the art of polymer science
and engineering that
proteins in solution tend to form entanglements, which can limit the
translational mobility of
the entangled chains and interfere with the protein's therapeutic or
nontherapeutic efficacy. In
embodiments, excipient compounds as disclosed herein can suppress protein
clustering due to
specific interactions between the excipient compound and the target protein in
solution.
30 Excipient compounds as disclosed herein can be natural or synthetic, and
desirably are
substances that the FDA generally recognizes as safe ("GRAS").
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1. Definitions
1041281 For the purpose of this disclosure, the tem "protein" refers to a
sequence of amino
acids having a chain length long enough to produce a discrete tertiary
structure, typically
having a molecular weight between 1-3000 kDa. In some embodiments, the
molecular weight
of the protein is between about 50-200 kDa; in other embodiments, the
molecular weight of
the protein is between about 20-1000 kDa or between about 20-2000 kDa. In
contrast to the
term "protein," the term "peptide" refers to a sequence of amino acids that
does not have a
discrete tertiary structure. A wide variety of biopolymers are included within
the scope of the
term "protein." For example, the term "protein" can refer to therapeutic or
non-therapeutic
to proteins, including antibodies, aptamers, fusion proteins, PEGylated
proteins, synthetic
polypeptides, protein fragments, lipoproteins, enzymes, structural peptides,
and the like.
a Therapeutic Biopolymers Definitions
100291 Those biopolymers having therapeutic effects may be termed "therapeutic
biopolymers." Those proteins having therapeutic effects may be termed
"therapeutic
proteins." The therapeutic protein contained in a therapeutic formulation may
also be termed
its "protein active ingredient."
100301 As non-limiting examples, therapeutic proteins can include mammalian
proteins
such as hormones and prohonnones (e.g., insulin and proinsulin, glucagon,
calcitonin, thyroid
hormones (T3 or T4 or thyroid-stimulating hormone), parathyroid hormone,
follicle-
stimulating hormone, luteinizing hormone, growth hormone, growth hormone
releasing
factor, and the like); clotting and anti-clotting factors (e.g., tissue
factor, von Willebrand's
factor, Factor VIIIC, Factor IX, protein C, plasminogen activators (urokinase,
tissue-type
plasminogen activators), thrombin); cytokines, chemokines, and inflammatory
mediators;
interferons; colony-stimulating factors; interleulcins (e.g., IL-1 through IL-
10); growth factors
(e.g., vascular endothelial growth factors, fibroblast growth factor, platelet-
derived growth
factor, transforming growth factor, neurotrophic growth factors, insulin-like
growth factor,
and the like); albumins; collagens and elastins; fibrin sealants;
hematopoietic factors (e.g.,
erythropoietin, thrombopoietin, and the like); osteoinductive factors (e.g.,
bone
morphogenetic protein); receptors (e.g., integrins, cadherins, and the like);
surface membrane
proteins; transport proteins; regulatory proteins; antigenic proteins (e.g., a
viral component
that acts as an antigen); and antibodies. The term "antibody" is used herein
in its broadest
sense, to include as non-limiting examples monoclonal antibodies (including,
for example,
full-length antibodies with an immunoglobulin Fc region), single-chain
molecules, bi-specific
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and multi-specific antibodies, diabodies, antibody-drug conjugates, antibody
compositions
having polyepitopic specificity, polyclonal antibodies (such as polyclonal
immunoglobulins
used as therapies for immune-compromised patients), and fragments of
antibodies (including,
for example, Fe, Fab, Fv, and F(ab')2). Antibodies can also be termed
"immunoglobulins."
An antibody is understood to be directed against a specific protein or non-
protein "antigen,"
which is a biologically important material; the administration of a
therapeutically effective
amount of an antibody to a patient can complex with the antigen, thereby
altering its
biological properties so that the patient experiences a therapeutic effect.
100311 In embodiments, the proteins are PEGylated, meaning that they comprise
to poly(ethylene glycol) ("PEG") and/or poly(propylene glycol) ("PPG")
units. PEGylated
proteins, or PEG-protein conjugates, have found utility in therapeutic
applications due to their
beneficial properties such as solubility, pharmacokinetics, pharinacodynamics,
immunogenicity, renal clearance, and stability. Non-limiting examples of
PEGylated
proteins are PEGylated versions of cytokines, hormones, hormone receptors,
cell signaling
factors, clotting factors, antibodies, antibody fragments, peptides, aptamers,
and enzymes. In
embodiments, the PEGylated proteins can be interferons (PEG-IFN), PEGylated
anti-vascular
endothelial growth factor (VEGF), PEGylated human growth hormones (HUH),
PEGylated
mutein antagonists, PEG protein conjugate drugs, Adagen, PEG-adenosine
deaminase, PEG-
thicase, Pegaspargase, PEGylated granulocyte colony-stimulating factors
(GCSF),
Pegfilgrastim, Pegloticase, Pegvisomant, Pegaptanib, Peginesatide, PEGylated
erythropoiesis-stimulating agents, PEGylated epoetin-a, PEGylated epoetin-I3,
methoxy
polyethylene glycol-epoetin beta, PEGylated antihemophilic factor VIII,
PEGylated
antihemophilic factor IX, and Certolizumab pegol.
100321 PEGylated proteins can be synthesized by a variety of methods such as a
reaction of
protein with a PEG reagent having one or more reactive functional groups. The
reactive
functional groups on the PEG reagent can form a linkage with the protein at
targeted protein
sites such as lysine, histidine, cysteine, and the N-terminus. Typical
PEGylation reagents
have reactive functional groups such as aldehyde, maleimide, or succinimide
groups that have
specific reactivity with targeted amino acid residues on proteins. The
PEGylation reagents
can have a PEG chain length from about 1 to about 1000 PEG and/or PPG
repeating units.
Other methods of PEGylation include glyco-PEGylation, where the protein is
first
glycosylated and then the glycosylated residues are PEGylated in a second
step. Certain
PEGylation processes are assisted by enzymes like sialyltransferase and
transglutaminase.
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[0033] While the PEGylated proteins can offer therapeutic advantages over
native, non-
PEGylated proteins, these materials can have physical or chemical properties
that make them
difficult to purify, dissolve, filter, concentrate, and administer. The
PEGylation of a protein
can lead to a higher solution viscosity compared to the native protein, and
this generally
requires the formulation of PEGylated protein solutions at lower
concentrations.
[0034] It is desirable to formulate protein therapeutics in stable, low
viscosity solutions so
they can be administered to patients in a minimal injection volume. For
example, the
subcutaneous (SC) or intramuscular (IM) injection of drugs generally requires
a small
injection volume, preferably less than 2 mL. The SC and IM injection routes
are well suited
to to self-administered care, and this is a less costly and more accessible
form of treatment
compared with intravenous (IV) injection which is only conducted under direct
medical
supervision. Formulations for SC or TM injection require a low solution
viscosity, generally
below 30 cP, and preferably below 20 cP, to allow easy flow of the therapeutic
solution
through a narrow-gauge needle with minimal injection force. This combination
of small
injection volume and low viscosity requirements present a challenge to the use
of PEGylated
protein therapeutics in SC or IM injection routes.
[0035] A "treatment" includes any measure intended to cure, heal, alleviate,
improve,
remedy, or otherwise beneficially affect the disorder, including preventing or
delaying the
onset of symptoms and/or alleviating or ameliorating symptoms of the disorder.
Those
patients in need of a treatment include both those who already have a specific
disorder, and
those for whom the prevention of a disorder is desirable. A disorder is any
condition that
alters the homeostatic wellbeing of a mammal, including acute or chronic
diseases, or
pathological conditions that predispose the mammal to an acute or chronic
disease. Non-
limiting examples of disorders include cancers, metabolic disorders (e.g.,
diabetes), allergic
disorders (e.g., asthma), dermatological disorders, cardiovascular disorders,
respiratory
disorders, hematological disorders, musculoskeletal disorders, inflammatory or
rheumatological disorders, autoimrnune disorders, gastrointestinal disorders,
urological
disorders, sexual and reproductive disorders, neurological disorders, and the
like. The term
"mammal" for the purposes of treatment can refer to any animal classified as a
mammal,
including humans, domestic animals, pet animals, farm animals, sporting
animals, working
animals, and the like. A "treatment" can therefore include both veterinary and
human
treatments. For convenience, the mammal undergoing such "treatment" can be
referred to as
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a "patient." In certain embodiments, the patient can be of any age, including
fetal animals in
neer .
100361 Formulations containing therapeutic proteins in therapeutically
effective amounts
may be termed "therapeutic formulations," In embodiments, a treatment involves
providing a
therapeutically effective amount of a therapeutic formulation to a mammal in
need thereof A
"therapeutically effective amount" is at least the minimum concentration of
the therapeutic
protein administered to the mammal in need thereof, to effect a treatment of
an existing
disorder or a prevention of an anticipated disorder (either such treatment or
such prevention
being a 'therapeutic intervention"). Therapeutically effective amounts of
various therapeutic
to proteins that may be included as active ingredients in the therapeutic
formulation may be
familiar in the art; or, for therapeutic proteins discovered or applied to
therapeutic
interventions hereinafter, the therapeutically effective amount can be
determined by standard
techniques carried out by those having ordinary skill in the art, using no
more than routine
experimentation. In embodiments, a therapeutic formulation comprises a
therapeutically
effective amount of a protein active ingredient and an excipient, with or
without other
optional components.
b. Non-Therapeutic Blopolymers Definitions
100371 Those biopolymers used for non-therapeutic purposes (i.e., purposes not
involving
treatments), such as household, nutrition, commercial, and industrial
applications, may be
termed "non-therapeutic biopolymers." Those proteins used for non-therapeutic
purposes
may be termed "non-therapeutic proteins." Formulations containing non-
therapeutic proteins
may be termed "non-therapeutic formulations." The non-therapeutic proteins can
be derived
from plant sources, animal sources, or produced from cell cultures; they also
can be enzymes
or structural proteins. The non-therapeutic proteins can be used in in
household, nutrition,
commercial, and industrial applications such as catalysts, human and animal
nutrition,
processing aids, cleaners, and waste treatment.
100381 An important category of non-therapeutic proteins is the category of
enzymes_
Enzymes have a number of non-therapeutic applications, for example, as
catalysts, human
and animal nutritional ingredients, processing aids, cleaners, and waste
treatment agents.
Enzyme catalysts are used to accelerate a variety of chemical reactions.
Examples of enzyme
catalysts for non-therapeutic uses include catalases, oxidoreductases,
transferases, hydrolases,
lyases, isomerases, and ligases. Human and animal nutritional uses of enzymes
include
nutraceuticals, nutritive sources of protein, chelation or controlled delivery
of micronutrients,
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digestion aids, and supplements; these can be derived from amylase, protease,
tiypsin,
lactase, and the like. Enzymatic processing aids are used to improve the
production of food
and beverage products in operations like baking, brewing, fermenting, juice
processing, and
winemaking. Examples of these food and beverage processing aids include
amylases,
cellulases, pectinases, glucanases, lipases, and lactases. Enzymes can also be
used in the
production of biofuels. Ethanol for biofuels, for example, can be aided by the
enzymatic
degradation of biomass feedstocks such as cellulosic and lignocellulosic
materials. The
treatment of such feedstock materials with cellulases and ligninases
transforms the biomass
into a substrate that can be fermented into fuels. In other commercial
applications, enzymes
to are used as detergents, cleaners, and stain lifting aids for laundry,
dish washing, surface
cleaning, and equipment cleaning applications. Typical enzymes for this
purpose include
proteases, cellulases, amylases, and lipase& In addition, non-therapeutic
enzymes are used in
a variety of commercial and industrial processes such as textile softening
with cellulases,
leather processing, waste treatment, contaminated sediment treatment, water
treatment, pulp
bleaching, and pulp softening and debonding. Typical enzymes for these
purposes are
amylases, xylanases, cellulases, and ligninases.
100391 Other examples of non-therapeutic biopolymers include fibrous or
structural
proteins such as keratins, collagen, gelatin, elastin, fibroin, actin,
tubulin, or the hydrolyzed,
degraded, or derivatized forms thereof These materials are used in the
preparation and
formulation of food ingredients such as gelatin, ice cream, yogurt, and
confections; they area
also added to foods as thickeners, theology modifiers, mouthfeel improvers,
and as a source
of nutritional protein. In the cosmetics and personal care industry, collagen,
elastin, keratin,
and hydrolyzed keratin are widely used as ingredients in skin care and hair
care formulations.
Still other examples of non-therapeutic biopolymers are whey proteins such as
beta-
lactoglobulin, alpha-lactalbumin, and serum albumin. These whey proteins are
produced in
mass scale as a byproduct from dairy operations and have been used for a
variety of non-
therapeutic applications.
2. Measurements
100401 In embodiments, the protein-containing formulations described herein
are resistant
to monomer loss as measured by size exclusion chromatography (SEC) analysis.
In SEC
analysis as used herein, the main analyte peak is generally associated with
the target protein
contained in the formulation, and this main peak of the protein is referred to
as the monomer
peak. The monomer peak represents the amount of target protein, e.g., a
protein active
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ingredient, in the monomeric state, as opposed to aggregated (dimeric,
trimeric, oligomeric,
etc.) or fragmented states. The monomer peak area can be compared with the
total area of the
monomer, aggregate, and fragment peaks associated with the target protein.
Thus, the
stability of a protein-containing formulation can be observed by the relative
amount of
monomer after an elapsed time; an improvement in stability of a protein-
containing
formulation of the invention can therefore be measured as a higher percent
monomer after a
certain elapsed time, as compared to the percent monomer in a control
formulation that does
not contain the excipient.
1004111 In embodiments, an ideal stability result is to have from 98 to 100%
monomer peak
to as determined by SEC analysis. In embodiments, an improvement in
stability of a protein-
containing formulation of the invention can be measured as a higher percent
monomer after
exposure to a stress condition, as compared to the percent monomer in a
control formulation
that does not contain the excipient when such control formulation is exposed
to the same
stress condition. In embodiments, the stress conditions can be a low
temperature storage,
high temperature storage, exposure to air, exposure to gas bubbles, exposure
to shear
conditions, or exposure to freeze/thaw cycles.
100421 In embodiments, the protein-containing formulations as described herein
are
resistant to an increase in protein particle size as measured by dynamic light
scattering (DLS)
analysis. In DLS analysis, as used herein, the particle size of the protein in
the protein-
containing formulation can be observed as a median particle diameter. Ideally,
the median
particle diameter of the target protein should be relatively unchanged when
subjected to DLS
analysis, since the particle diameter represents the active component in the
monomeric state,
as opposed to aggregated (dimeric, trimeric, oligomeric, etc.) or fragmented
states. An
increase of the median particle diameter could represent an aggregated
protein. Thus, the
stability of a protein-containing formulation can be observed by the relative
change in median
particle diameter after an elapsed time.
100431 In embodiments, the protein-containing formulations as described herein
are
resistant to forming a polydisperse particle size distribution as measured by
dynamic light
scattering (DLS) analysis. In embodiments, a protein-containing formulation
can contain a
monodisperse particle size distribution of colloidal protein particles. In
embodiments, an
ideal stability result is to have less than a 10% change in the median
particle diameter
compared to the initial median particle diameter of the formulation. In
embodiments, an
improvement in stability of a protein-containing formulation of the invention
can be
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measured as a lower percent change of the median particle diameter after a
certain elapsed
time, as compared to the median particle diameter in a control formulation
that does not
contain the excipient. In embodiments, an improvement in stability of a
protein-containing
formulation of the invention can be measured as a lower percent change of the
median
particle diameter after exposure to a stress condition, as compared to the
percent change of
the median particle diameter in a control formulation that does not contain
the excipient when
such control formulation is exposed to the same stress condition. In
embodiments, the stress
conditions can be a low temperature storage, high temperature storage,
exposure to air,
exposure to gas bubbles, exposure to shear conditions, or exposure to
freeze/thaw cycles. In
to embodiments, an improvement in stability of a protein-containing
formulation therapeutic
formulation of the invention can be measured as a less polydisperse particle
size distribution
as measured by DLS, as compared to the polydispersity of the particle size
distribution in a
control formulation that does not contain the excipient when such control
formulation is
exposed to the same stress condition.
100441 In embodiments, the protein-containing formulations of the invention
are resistant to
precipitation as measured by turbidity, light scattering, and/or particle
counting analysis. In
turbidity, light scattering, or particle counting analysis, a lower value
generally represents a
lower number of suspended particles in a formulation. An increase of
turbidity, light
scattering, or particle counting can indicate that the solution of the target
protein is not stable.
Thus, the stability of a protein-containing formulation can be observed by the
relative amount
of turbidity, light scattering, or particle counting after an elapsed time. In
embodiments, an
ideal stability result is to have a low and relatively constant turbidity,
light scattering, or
particle counting value. In embodiments, an improvement in stability of a
protein-containing
formulation of the invention can be measured as a lower turbidity, lower light
scattering, or
lower particle count after a certain elapsed time, as compared to the
turbidity, light scattering,
or particle count values in a control formulation that does not contain the
excipient. In
embodiments, an improvement in stability of a protein-containing formulation
as described
herein can be measured as a lower turbidity, lower light scattering, or lower
particle count
after exposure to a stress condition, as compared to the turbidity, light
scattering, or particle
count in a control formulation that does not contain the excipient when such
control
formulation is exposed to the same stress condition. In embodiments, the
stress conditions
can be a low temperature storage, high temperature storage, exposure to air,
exposure to gas
bubbles, exposure to shear conditions, or exposure to freeze/thaw cycles.
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3. Therapeutic Formulations
100451 In one aspect, the formulations and methods disclosed herein provide
stable liquid
formulations of improved or reduced viscosity, comprising a therapeutic
protein in a
therapeutically effective amount and an excipient compound. In embodiments,
the
formulation can improve the stability while providing an acceptable
concentration of active
ingredients and an acceptable viscosity. In embodiments, the formulation
provides an
improvement in stability when compared to a control formulation; for the
purposes of this
disclosure, a control formulation is a formulation containing the protein
active ingredient that
is substantially similar or identical on a dry weight basis in every way to
the therapeutic
to formulation except that it lacks the excipient compound. In embodiments,
the formulation
provides an improvement in stability under the stress conditions of long-term
storage,
elevated temperatures such as 25-45 C, freeze/thaw conditions, shear or
mixing, syringing,
dilution, gas bubble exposure, oxygen exposure, light exposure, and
lyophilization. In
embodiments, improved stability of the protein- containing formulation is in
the form of
lower percentage of soluble aggregates, particulates, subvisible particles, or
gel formation,
compared to a control formulation. In embodiments, improved stability of the
protein-
containing formulation is in the form of higher biological activity compared
to a control
formulation. In embodiments, improved stability of the protein-containing
formulation is in
the form of improved chemical stability, such as resistance to a chemical
reaction such as
hydrolysis, photolysis, oxidation, reduction, dearnidation, disulfide
scrambling,
fragmentation, or dissociation. In embodiments, improved stability of the
protein-containing
formulation is manifested by a decrease in the formation of visible particles,
subvisible
particles, aggregates, turbidity, opalescence, or gel.
100461 It is understood that the viscosity of a liquid protein formulation can
be affected by
a variety of factors, including but not limited to: the nature of the protein
itself (e.g., enzyme,
antibody, receptor, fusion protein, etc.); its size, three-dimensional
structure, chemical
composition, and molecular weight; its concentration in the formulation; the
components of
the formulation besides the protein; the desired pH range; the storage
conditions for the
formulation; and the method of administering the formulation to the patient.
Therapeutic
proteins most suitable for use with the excipient compounds described herein
are preferably
essentially pure, i.e., free from contaminating proteins. In embodiments, an
"essentially
pure" therapeutic protein is a protein composition comprising at least 90% by
weight of the
therapeutic protein, or preferably at least 95% by weight of therapeutic
protein, or more
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preferably, at least 99% by weight of the therapeutic protein, all based on
the total weight of
therapeutic proteins and contaminating proteins in the composition. For the
purposes of
clarity, a protein added as an excipient is not intended to be included in
this definition. The
therapeutic formulations described herein are intended for use as
pharmaceutical-grade
formulations, i.e., formulations intended for use in treating a mammal, in
such a form that the
desired therapeutic efficacy of the protein active ingredient can be achieved,
and without
containing components that are toxic to the mammal to whom the formulation is
to be
administered.
[0047] In embodiments, the therapeutic formulation contains at least 1 mg/mL
of protein
to active ingredient. In other embodiments, the therapeutic formulation
contains at least 10
mg/mL of protein active ingredient. In other embodiments, the therapeutic
formulation
contains at least 25 mg/mL of protein active ingredient. In other embodiments,
the
therapeutic formulation contains at least 25 mg/mL of protein active
ingredient. In other
embodiments, the therapeutic fomndation contains at least 100 mg/mL of protein
active
ingredient. In other embodiments, the therapeutic formulation contains at
least 200 mg/mL
of protein active ingredient. In yet other embodiments, the therapeutic
formulation solution
contains at least 300 mg/mL of protein active ingredient. Generally, the
excipient
compounds disclosed herein are added to the therapeutic formulation in an
amount between
about 0.001 to about 60 mg/mL. In embodiments, the excipient compound can be
added in
an amount of about 0.1 to about 50 mg/mL. In embodiments, the excipient
compound can be
added in an amount of about 1 to about 40 mg/mL. In embodiments, the excipient
can be
added in an amount of about 5 to about 30 mg/mL.
[0048] In certain embodiments, the therapeutic formulation solution contains
at least 300
mg/mL of protein active ingredient. In certain aspects, the excipient
compounds disclosed
herein are added to the therapeutic formulation in an amount between about 1
to about 300
mg/mL, or in amounts between about 5 to about 300 mg/mL. In embodiments, the
excipient
compound can be added in an amount of about 10 to about 200 mg/mL. In
embodiments, the
excipient compound can be added in an amount of about 5 to about 100 mg/mL, or
about 20
to about 100 mg/mL. In embodiments, the excipient compound can be added in an
amount of
about 10 to about 75 mg/mL, or about 20 to about 75 mg/mL. In embodiments, the
excipient
can be added in an amount of about 15 to about 50 mg/mL.
[0049] Excipient compounds of various molecular weights are selected for
specific
advantageous properties when combined with the protein active ingredient in a
formulation.
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Examples of therapeutic formulations comprising excipient compounds are
provided below.
In embodiments, the excipient compound has a molecular weight of <5000 Da In
embodiments, the excipient compound has a molecular weight of <1000 Da In
embodiments, the excipient compound has a molecular weight of <500 Da.
[0050] In embodiments, the excipient compounds disclosed herein is added to
the
therapeutic formulation in a viscosity-reducing amount. In embodiments, a
viscosity-
reducing amount is the amount of an excipient compound that reduces the
viscosity of the
formulation at least 10% when compared to a control formulation; for the
purposes of this
disclosure, a control formulation is a formulation containing the protein
active ingredient that
to is substantially similar or identical on a dry weight basis in every way
to the therapeutic
formulation except that it lacks the excipient compound. In embodiments, the
viscosity-
reducing amount is the amount of an excipient compound that reduces the
viscosity of the
formulation at least 30% when compared to the control formulation. In
embodiments, the
viscosity-reducing amount is the amount of an excipient compound that reduces
the viscosity
of the formulation at least 50% when compared to the control formulation. In
embodiments,
the viscosity-reducing amount is the amount of an excipient compound that
reduces the
viscosity of the formulation at least 70% when compared to the control
formulation. In
embodiments, the viscosity-reducing amount is the amount of an excipient
compound that
reduces the viscosity of the formulation at least 90% when compared to the
control
formulation.
[0051] In embodiments, the viscosity-reducing amount yields a therapeutic
formulation
having a viscosity of less than 100 cP. In other embodiments, the therapeutic
formulation has
a viscosity of less than 50 cP. In other embodiments, the therapeutic
formulation has a
viscosity of less than 20 cP. In yet other embodiments, the therapeutic
formulation has a
viscosity of less than 10 cP. The term "viscosity" as used herein refers to a
dynamic viscosity
value when measured by the methods disclosed herein.
[0052] In embodiments, the therapeutic formulations are administered to a
patient at high
concentration of therapeutic protein. High concentration solutions of
therapeutic proteins
formulated with the excipient compounds described herein can be administered
to patients
using syringes or pre-filled syringes. In embodiments, the therapeutic
formulations are
administered to patients in a smaller injection volume and/or with less
discomfort than would
be experienced with a similar formulation lacking the therapeutic excipient.
In embodiments,
the therapeutic formulations are administered to patients using a narrower
gauge needle, or
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less syringe force that would be required with a similar formulation lacking
the therapeutic
excipient. In embodiments, the therapeutic formulations are administered as a
depot
injection. In embodiments, the therapeutic formulations extend the half-life
of the therapeutic
protein in the body. These features of therapeutic formulations as disclosed
herein would
permit the administration of such formulations by intramuscular or
subcutaneous injection in
a clinical situation, i.e., a situation where patient acceptance of an
intramuscular injection
would include the use of small-bore needles typical for IM/SC purposes and the
use of a
tolerable (for example, 2-3 cc) injected volume, and where these conditions
result in the
administration of an effective amount of the formulation in a single injection
at a single
to injection site. By contrast, injection of a comparable dosage amount of
the therapeutic protein
using conventional formulation techniques would be limited by the higher
viscosity of the
conventional formulation, so that a SC/IM injection of the conventional
formulation would
not be suitable for a clinical situation.
[0053] Therapeutic formulations in accordance with this disclosure can have
certain
advantageous properties consistent with improved stability. In embodiments,
the therapeutic
formulations are resistant to shear degradation, phase separation, clouding
out, precipitation,
oxidation, deamidation, aggregation, and/or denaturing. In embodiments, the
therapeutic
formulations are processed, purified, stored, syringed, dosed, filtered,
and/or centrifuged
more effectively, compared with a control formulation.
[0054] In embodiments, the therapeutic formulations disclosed herein are
resistant to
monomer loss as measured by size exclusion chromatography (SEC) analysis. In
SEC
analysis, the main analyte peak is generally associated with the active
component of the
formulation, such as a therapeutic protein, and this main peak of the active
component is
referred to as the monomer peak. The monomer peak represents the amount of
active
component in the monomeric state, as opposed to aggregated (dimeric, trimeric,
oligorneric,
etc.) protein. Thus, the stability of a therapeutic formulation can be
observed by the relative
amount of monomer after an elapsed time. In embodiments, an improvement in
stability of a
therapeutic formulation as disclosed herein can be measured as a higher
percent monomer
after a certain elapsed time, as compared to the percent monomer in a control
formulation that
does not contain the excipient. In embodiments, an improvement in stability of
a therapeutic
formulation as disclosed herein can be measured as a higher percent monomer
after exposure
to a stress condition, as compared to the percent monomer in a control
formulation that does
not contain the excipient after exposure to the stress condition. In
embodiments, the
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therapeutic formulations of the invention are resistant to an increase in
protein particle size as
measured by dynamic light scattering (DLS) analysis. In DLS analysis, the
particle size of
the therapeutic protein can be observed as a median particle diameter.
Ideally, the median
particle diameter of the therapeutic protein should be relatively unchanged.
An increase of
the median particle diameter, therefore, can represent an aggregated protein.
Thus, the
stability of a therapeutic formulation can be observed by the relative change
in median
particle diameter after an elapsed time. In embodiments, the therapeutic
formulations as
disclosed herein are resistant to forming a polydisperse particle size
distribution as measured
by dynamic light scattering (DLS) analysis. In embodiments, an improvement in
stability of
to a therapeutic formulation of the invention can be measured as a lower
percent change of the
median particle diameter after a certain elapsed time, as compared to the
median particle
diameter in a control formulation that does not contain the excipient. In
embodiments, an
improvement in stability of a therapeutic formulation as disclosed herein can
be measured as
a lower percent change of the median particle diameter after exposure to a
stress condition, as
compared to the percent change of the median particle diameter in a control
formulation that
does not contain the excipient. In other words, in embodiments, improved
stability prevents
an increase in particle size as measured by light scattering. In embodiments,
the stress
conditions can be a low temperature storage, high temperature storage,
exposure to air,
exposure to gas bubbles, exposure to shear conditions, or exposure to
freeze/thaw cycles. In
embodiments, an improvement in stability of a therapeutic formulation as
disclosed herein
can be measured as a less polydisperse particle size distribution as measured
by DLS, as
compared to the polydispersity of the particle size distribution in a control
formulation that
does not contain the excipient.
100551 In embodiments, the therapeutic formulations as disclosed herein are
resistant to
precipitation as measured by turbidity, light scattering, or particle counting
analysis. In
embodiments, an improvement in stability of a therapeutic formulation as
disclosed herein
can be measured as a lower turbidity, lower light scattering, or lower
particle count after a
certain elapsed time, as compared to the turbidity, light scattering, or
particle count values in
a control formulation that does not contain the excipient. In embodiments, an
improvement
in stability of a therapeutic formulation as disclosed herein can be measured
as a lower
turbidity, lower light scattering, or lower particle count after exposure to a
stress condition, as
compared to the turbidity, light scattering, or particle count in a control
formulation that does
not contain the excipient. In embodiments, the stress conditions can be a low
temperature
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storage, high temperature storage, exposure to air, exposure to gas bubbles,
exposure to shear
conditions, or exposure to freeze/thaw cycles.
100561 In embodiments, the therapeutic excipient has antioxidant properties
that stabilize
the therapeutic protein against oxidative damage. In embodiments, the
therapeutic
formulation is stored at ambient temperatures, or for extended time at
refrigerator conditions
without appreciable loss of potency for the therapeutic protein. In
embodiments, the
therapeutic formulation is dried down for storage until it is needed; then it
is reconstituted
with an appropriate solvent, e.g., water. Advantageously, the formulations
prepared as
described herein can be stable over a prolonged period of time, from months to
years. When
to exceptionally long periods of storage are desired, the formulations can
be preserved in a
freezer (and later reactivated) without fear of protein denaturation. In
embodiments,
formulations can be prepared for long-term storage that do not require
refrigeration.
100571 In embodiments, the excipient compounds disclosed herein are added to
the
therapeutic formulation in a stability-improving amount. In embodiments, a
stability-
improving amount is the amount of an excipient compound that reduces the
degradation of
the formulation at least 10% when compared to a control formulation; for the
purposes of this
disclosure, a control formulation is a formulation containing the protein
active ingredient that
is substantially similar on a weight basis to the therapeutic formulation
except that it lacks the
excipient compound. In embodiments, the stability-improving amount is the
amount of an
excipient compound that reduces the degradation of the formulation at least
30% when
compared to the control formulation. In embodiments, the stability-improving
amount is the
amount of an excipient compound that reduces the degradation of the
formulation at least
50% when compared to the control formulation. In embodiments, the stability-
improving
amount is the amount of an excipient compound that reduces the degradation of
the
fommlation at least 70% when compared to the control formulation. In
embodiments, the
stability-improving amount is the amount of an excipient compound that reduces
the
degradation of the formulation at least 90% when compared to the control
formulation.
100581 Methods for preparing therapeutic formulations may be familiar to
skilled artisans.
The therapeutic formulations of the present invention can be prepared, for
example, by
adding the excipient compound to the formulation before or after the
therapeutic protein is
added to the solution. The therapeutic formulation can, for example, be
produced by
combining the therapeutic protein and the excipient at a first (lower)
concentration and then
processed by filtration or centrifugation to produce a second (higher)
concentration of the
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therapeutic protein. Therapeutic formulations can be made with one or more of
the excipient
compounds with chaotropes, kosmotropes, hydrotropes, and salts. Therapeutic
formulations
can be made with one or more of the excipient compounds using techniques such
as
encapsulation, dispersion, liposome, vesicle formation, and the like. Methods
for preparing
therapeutic formulations comprising the excipient compounds disclosed herein
can include
combinations of the excipient compounds. In embodiments, combinations of
excipients can
produce benefits in lower viscosity, improved stability, or reduced injection
site pain. Other
additives may be introduced into the therapeutic formulations during their
manufacture,
including preservatives, surfactants, sugars, sucrose, trehalose,
polysaccharides, arginine,
to proline, hyaluronidase, stabilizers, buffers, and the like. As used
herein, a pharmaceutically
acceptable excipient compound is one that is non-toxic and suitable for animal
and/or human
administration.
3. Non-Therapeutic Formulations
100591 In one aspect, the formulations and methods disclosed herein provide
stable liquid
formulations of improved or reduced viscosity, comprising anon-therapeutic
protein in an
effective amount and an excipient compound. In embodiments, the formulation
improves the
stability while providing an acceptable concentration of active ingredients
and an acceptable
viscosity. In embodiments, the formulation provides an improvement in
stability when
compared to a control formulation; for the purposes of this disclosure, a
control formulation
is a formulation containing the protein active ingredient that is
substantially similar or
identical on a dry weight basis in every way to the non-therapeutic
formulation except that it
lacks the excipient compound.
100601 It is understood that the viscosity of a liquid protein formulation can
be affected by
a variety of factors, including but not limited to: the nature of the protein
itself (e.g., enzyme,
structural protein, degree of hydrolysis, etc.); its size, three-dimensional
structure, chemical
composition, and molecular weight; its concentration in the formulation; the
components of
the formulation besides the protein; the desired pH range; and the storage
conditions for the
formulation.
100611 In embodiments, the non-therapeutic formulation contains at least 25
mg/mL of
protein active ingredient. In other embodiments, the non-therapeutic
formulation contains at
least 100 mg/mL of protein active ingredient. In other embodiments, the non-
therapeutic
formulation contains at least 200 mg/mL of protein active ingredient. In yet
other
embodiments, the non-therapeutic formulation solution contains at least 300
mg/mL of
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protein active ingredient. Generally, the excipient compounds disclosed herein
are added to
the non-therapeutic formulation in an amount between about 0.001 to about 60
mg/mL. In
embodiments, the excipient compound can be added in an amount of about 0.1 to
about 50
mg/mL. In embodiments, the excipient compound can be added in an amount of
about 1 to
about 40 mg/mL. In embodiments, the excipient can be added in an amount of
about 5 to
about 30 ing/mL,
[0062] In certain embodiments, the non-therapeutic formulation solution
contains at least
300 mg/mL of protein active ingredient In certain aspects, the excipient
compounds
disclosed herein are added to the therapeutic formulation in an amount between
about t to
to about 300 mg/mL, or in amounts between about 5 to about 300 mg/mL. In
embodiments, the
excipient compound can be added in an amount of about 10 to about 200 mg/mL.
In
embodiments, the excipient compound can be added in an amount of about 5 to
about 100
mg/mL, or about 20 to about 100 mg/mL, In embodiments, the excipient compound
can be
added in an amount of about 10 to about 75 mg/mL, or about 20 to about 75
mg/mL. In
embodiments, the excipient can be added in an amount of about 15 to about 50
mg/mL.
[0063] Excipient compounds of various molecular weights are selected for
specific
advantageous properties when combined with the protein active ingredient in a
formulation.
Examples of non-therapeutic formulations comprising excipient compounds are
provided
below. In embodiments, the excipient compound has a molecular weight of <5000
Da. In
embodiments, the excipient compound has a molecular weight of <1000 Da In
embodiments, the excipient compound has a molecular weight of <500 Da
[0064] In embodiments, the excipient compounds disclosed herein is added to
the non-
therapeutic formulation in a viscosity-reducing amount. In embodiments, a
viscosity-
reducing amount is the amount of an excipient compound that reduces the
viscosity of the
formulation at least 10% when compared to a control formulation; for the
purposes of this
disclosure, a control formulation is a formulation containing the protein
active ingredient that
is substantially similar or identical on a dry weight basis in every way to
the therapeutic
formulation except that it lacks the excipient compound. In embodiments, the
viscosity-
reducing amount is the amount of an excipient compound that reduces the
viscosity of the
formulation at least 30% when compared to the control formulation. In
embodiments, the
viscosity-reducing amount is the amount of an excipient compound that reduces
the viscosity
of the formulation at least 50% when compared to the control formulation. In
embodiments,
the viscosity-reducing amount is the amount of an excipient compound that
reduces the
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viscosity of the formulation at least 70% when compared to the control
formulation. In
embodiments, the viscosity-reducing amount is the amount of an excipient
compound that
reduces the viscosity of the formulation at least 90% when compared to the
control
formulation.
100651 In embodiments, the viscosity-reducing amount yields a non-therapeutic
formulation having a viscosity of less than 100 cP. In other embodiments, the
non-
therapeutic formulation has a viscosity of less than 50 cP. In other
embodiments, the non-
therapeutic formulation has a viscosity of less than 20 cP. In yet other
embodiments, the non-
therapeutic formulation has a viscosity of less than 10 cP, The term
"viscosity" as used
to herein refers to a dynamic viscosity value.
100661 Non-therapeutic formulations in accordance with this disclosure can
have certain
advantageous properties. In embodiments, the non-therapeutic formulations are
resistant to
shear degradation, phase separation, clouding out, oxidation, deamidation,
aggregation,
precipitation, and denaturing. In embodiments, the therapeutic formulations
can be
processed, purified, stored, pumped, filtered, and centrifuged more
effectively, compared
with a control foimulation.
100671 In embodiments, the non-therapeutic excipient has antioxidant
properties that
stabilize the non-therapeutic protein against oxidative damage. In
embodiments, the non-
therapeutic formulation is stored at ambient temperatures, or for extended
time at refrigerator
conditions without appreciable loss of potency for the non-therapeutic
protein. In
embodiments, the non-therapeutic formulation is dried down for storage until
it is needed;
then it can be reconstituted with an appropriate solvent, e.g., water.
Advantageously, the
formulations prepared as described herein is stable over a prolonged period of
time, from
months to years. When exceptionally long periods of storage are desired, the
formulations are
preserved in a freezer (and later reactivated) without fear of protein
denaturation. In
embodiments, formulations are prepared for long-term storage that do not
require
refrigeration.
100681 In embodiments, the excipient compounds disclosed herein are added to
the non-
therapeutic formulation in a stability-improving amount. In embodiments, a
stability-
improving amount is the amount of an excipient compound that reduces the
degradation of
the formulation at least 10% when compared to a control formulation; for the
purposes of this
disclosure, a control formulation is a formulation containing the protein
active ingredient that
is substantially similar or identical on a dry weight basis to the therapeutic
formulation except
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that it lacks the excipient compound. In embodiments, the stability-improving
amount is the
amount of an excipient compound that reduces the degradation of the
formulation at least
30% when compared to the control fomnilation. In embodiments, the stability-
improving
amount is the amount of an excipient compound that reduces the degradation of
the
formulation at least 50% when compared to the control formulation. In
embodiments, the
stability-improving amount is the amount of an excipient compound that reduces
the
degradation of the formulation at least 70% when compared to the control
formulation. In
embodiments, the stability-improving amount is the amount of an excipient
compound that
reduces the degradation of the formulation at least 90% when compared to the
control
to formulation.
[0069] Methods for preparing non-therapeutic formulations comprising the
excipient
compounds disclosed herein may be familiar to skilled artisans. For example,
the excipient
compound can be added to the formulation before or after the non-therapeutic
protein is
added to the solution. The non-therapeutic formulation can be produced at a
first (lower)
concentration and then processed by filtration or centrifugation to produce a
second (higher)
concentration. Non-therapeutic formulations can be made with one or more of
the excipient
compounds with chaotropes, kosmotropes, hydrotropes, and salts. Non-
therapeutic
formulations can be made with one or more of the excipient compounds using
techniques
such as encapsulation, dispersion, liposome, vesicle formation, and the like.
Other additives
can be introduced into the non-therapeutic formulations during their
manufacture, including
preservatives, surfactants, stabilizers, and the like.
4. Excipient Compounds
[0070] Several excipient compounds are described herein, each suitable for use
with one or
more therapeutic or non-therapeutic proteins, and each allowing the
formulation to be
composed so that it contains the protein(s) at a high concentration. Some of
the categories of
excipient compounds described below are: (1) hindered or modified amines; (2)
aromatics
and anionic aromatics; (3) functionalized amino acids; (4) oligopeptides; (5)
short-chain
organic acids; (6) low-molecular-weight aliphatic polyacids; and (7) diones
and sulfones.
1007111 In embodiments, one or more viscosity-reducing excipient compounds can
be added
to the formulation simultaneously or sequentially. In embodiments, at least
one of the
viscosity-reducing compounds is a hindered amine. In embodiments, the at least
one
excipient compound is a pyrimidine, a methyl-substituted pyrimidine, or a
phenethylamine.
A pyrimidine is a compound comprising at least one pyrimidine ring; it is to
be understood
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that the pyrimidine ring can be optionally substituted and/or can be part of a
fused ring
system, for example, part of a fused bicyclic or tricyclic ring system.
Exemplary pyrimidines
include, for example, pyrimidine, pyrimidinone, triaminopyrimidine, purine,
adenine, and
guanine. A methyl-substituted pyrimidine is a compound comprising at least one
methyl-
substituted pyrimidine ring. Exemplary methyl-substituted pyrimidines are 1,3-
dimethyluracil, 1-methyluracil, 3-methyluracil, 5-methyluracil, and 6-
methyluracil.
Additional exemplary pyrimidines are described in more detail below. In
certain aspects, the
pyrimidine or the methyl-substituted pyrimidine is not caffeine or a xanthine.
In yet
additional aspects, the pyrimidine is a compound comprising at least one
pyrimidine ring,
to wherein the pyrimidine ring is not part of a fused ring system. In
embodiments, the at least
one excipient compound is selected from the group consisting of caffeine,
saccharin,
acesulfame potassium, aspartame, theophylline, taurine, 1-methyl-2-
pyrrolidone, 2-
pynrolidinone, niacinamide, and imidazole. In embodiments, the at least one
excipient
compound is selected from the group consisting of caffeine, taurine,
niacinamide, and
imidazole. In embodiments, the at least one excipient compound is selected
from the group
consisting of uracil, 1-methyluracil, 6-methyluracil, 5-rnethyluracil, 1,3-
dimethyluracil,
cytosine, 5-methylcytosine, 3-methylcytosine, thymine, 1-methylthymine, 0-4-
methylthymine, 1,3-dimethylthymine, and dimethylthymine dimer. In embodiments,
the at
least one excipient compound is selected from the group consisting of
diphenhydramine,
phenetlrylamine, N-methylphenethylamine, N,N-dimethylphenethylamine, beta-3-
dihydroxyphenethylamine, beta-3-dihydroxy-N-methylphenethylamine, 3-
hydroxyphenethylamine, 4-hydroxyphenethylamine, tyrosinol, tyramine, N-
methyltyramine,
and hordenine. In embodiments, the at least one excipient compound is an
anionic aromatic
excipient, and, in some embodiments, the anionic aromatic excipient can be 4-
hydroxybenzenesulfonic acid. In embodiments, the viscosity-reducing amount is
between
about 1 mg/mL to about 100 mg/mL of the at least one excipient compound, or
the viscosity-
reducing amount is between about 1 mM to about 400 mM of the at least one
excipient
compound, or the viscosity-reducing amount is an amount from about 2 mM to
about 150
tnIVI., or the viscosity-reducing amount is an amount from about 2 mg/mL to
about 80
mg/mL, or the viscosity-reducing amount is an amount between about 5 mg/mL and
about 50
mg/mL, or the viscosity-reducing amount is an amount between about 10 mg/mL
and about
mg/mL, In embodiments, the viscosity-reducing amount is an amount between
about 2
mM and about 150 mM,, or is between about 5 mM and about 100 mM, or is between
about
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mM to about 75 mM, or is between about 15 mM and about 50mM. In embodiments,
the
carrier solution comprises an additional agent selected from the group
consisting of
preservatives, sugars, polyols, polysaccharides, arginine, proline,
surfactants, stabilizers, and
buffers.
5 100721 Without being bound by theory, the excipient compounds described
herein are
thought to associate with certain fragments, sequences, structures, or
sections of a therapeutic
protein that otherwise would be involved in inter-particle (i.e., protein-
protein) interactions.
The association of these excipient compounds with the therapeutic or non-
therapeutic protein
can mask the inter-protein interactions such that the proteins can be
formulated in high
to concentration without causing excessive solution viscosity. In
embodiments, the excipient
compound can result in more stable protein-protein interaction; protein-
protein interaction
can be measured by the protein diffusion parameter kD, or the osmotic second
virial
coefficient 822, or by other techniques familiar to skilled artisans.
100731 Excipient compounds advantageously can be water-soluble, therefore
suitable for
use with aqueous vehicles. In embodiments, the excipient compounds have a
water solubility
of >1 mg/mL. In embodiments, the excipient compounds have a water solubility
of >10
mg/mL. In embodiments, the excipient compounds have a water solubility of >100
mg/mL.
In embodiments, the excipient compounds have a water solubility of >500 mg/mL.
100741 Advantageously for therapeutic protein formulations, the excipient
compounds can
be derived from materials that are biologically acceptable and are non-
immunogenic, and are
thus suitable for pharmaceutical use. In therapeutic embodiments, the
excipient compounds
can be metabolized in the body to yield biologically compatible and non-
immunogenic
byproducts.
a. Excipient Compound Category 1: Hindered or
Modified Amines
100751 High concentration solutions of therapeutic or non-therapeutic proteins
can be
formulated with hindered or modified amine small molecules as excipient
compounds. As
used herein, the term "hindered amine" refers to a small molecule containing
at least one
bulky or sterically hindered group, consistent with the examples below. As
used herein, the
term "modified amine" refers to a small molecule containing an amine
functional group but
without a bulky or sterically hindered group, as described below in more
detail. Hindered or
modified amines can be used in -the free base form, in the protonated form, or
a combination
of the two. In protonated forms, the hindered or modified amines can be
associated with an
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anionic counterion such as chloride, hydroxide, bromide, iodide, fluoride,
acetate, formate,
phosphate, sulfate, or carboxylate.
100761 Hindered amine compounds useful as excipient compounds can contain
secondary
amine, tertiary amine, quatemary ammonium, pyridinium, pyrrolidone,
pyrrolidine,
piperidine, morpholine, or guanidinium groups, such that the excipient
compound has a
cationic charge in aqueous solution at neutral pH. The hindered amine
compounds also
contain at least one bulky or sterically hindered group, such as cyclic
aromatic,
cycloaliphatic, cyclohexyl, or alkyl groups. In embodiments, the sterically
hindered group
can itself be an amine group such as a dialkylamine, trialkylamine,
guanidinium,
to or quaternary ammonium group. Without being bound by theory, the
hindered amine
compounds are thought to associate with aromatic sections of the proteins such
as
phenylalanine, tryptophan, and tyrosine, by a cation pi interaction. In
embodiments, the
cationic group of the hindered amine can have an affinity for the electron
rich pi structure of
the aromatic amino acid residues in the protein, so that they can shield these
sections of the
protein, thereby decreasing the tendency of such shielded proteins to
associate and
agglomerate.
100771 In embodiments, the hindered amine excipient compounds has a chemical
structure
comprising imidazole, imidazoline, or imidazolidine groups, or salts thereof,
such as
imidazole, 1-methylimidazole, 4-methylimidazole, 1-hexy1-3-methylimidazolium
chloride, 1-
ethylimidazole, 4-ethylimidazole, 1-hexy1-3-ethylimidazolium chloride,
imidazoline, 2-
imidazoline, imidazolidone, 2-imidazolidone, histamine, 4-methylhistamine,
alpha-
methylhistamine, betahistine, beta-alanine, 2-methyl-2-imidazoline, 1-buty1-3-
methylimidazolium chloride, butyl imidazole, uric acid, potassium urate,
betazole, carnosine,
spermine, spermidine, aspartame, saccharin, acesulfame potassium, xanthine,
theophylline,
theobromine, caffeine, and anserine. In embodiments, the hindered amine
excipient
compound is a pyrimidine selected from the group consisting of pyrimidine,
pyrimidinone,
triaminopyrimidine, 1,3-dimethyluracil, 1-methyluracil, 3-methyluracil, 1,3-
diethyluracil, 6-
methyluracil, uracil, 1,3-dimethyl-tetrahydro pyrimidinone, thymine, 1-
methylthymine, 0-4-
methylthytnine, 1,3-dimethylthymine, dimethylthymine dimer, theacrine,
cytosine, 5-
methylcytosine, 3-methylcytosine, purine, guanine, and adenine. In other
aspects, the
hindered amine excipient compound is selected from the group consisting of 1-
methy1-2-
pyffolidone, phenylserine, DL-3-phenylsenne, cycloserine, dicyclomine, and
cysteamine. In
embodiments, the hindered amine excipient compound is selected from the group
consisting
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of guanidinoacetate, dimethylethanolamine, ethanolatnine,
dimethylarninoethanol,
dimethylaminopropylamine, triethanolamine, 1,3-diaminopropane, 1,2-
diaminopropane,
polyetheramines, Jeffamine brand polyetheramines, polyether-monoamines,
polyether-
diamines, polyether-triamines, 1-(1-adamantypethylamine, hordenine,
benzylamine,
dimethylbenzylamine, dimethylcyclohexylamine, diethylcyclohexylamine,
dicyclohexylmethylatnine, hexamethylene bigttanide, poly(hexamethylene
bigttanide),
imidazole, dimethylglycine, meglumine, agmatine, agrnatine sulfate,
cliazabicyclo[2.2.2]octane, tetramethylethylenediatnine, N,N-
dimethylethanolamine,
ethanolamine phosphate, glucosamine, choline chloride, phosphocholine,
niacinamide,
to isonicotinatnide, N,N-diethyl nicotinatnide, nicotinic acid, nicotinic
acid sodium salt,
isonicotinic acid, tyramine, N-methyltyrarnine, 3-aminopyridine, 4-
aminopyridine, 2,4,6-
trimethylpyridine, 3-pyridine methanol, dipyridamole, nicotinarnide adenosine
dinucleotide,
biotin, folic acid, folinic acid, folinic acid calcium salt, moipholine, N-
methylpyrrolidone, 2-
pyrrolidinone, procaine, lidocaine, dicyandiamide-taurine adduct, 2-
pyridylethylamine,
dicyandiamide-benzyl amine adduct, dicyandiamide-alkylamine adduct,
dicyandiamide-
cycloalkylamine adduct, and dicyandiamide-aminomethanephosphonic acid adducts.
In
embodiments, a hindered amine compound consistent with this disclosure is
formulated as a
protonated ammonium salt. In embodiments, a hindered amine compound consistent
with
this disclosure is formulated as a salt with an inorganic anion or organic
anion as the
counterion. In embodiments, high concentration solutions of therapeutic or non-
therapeutic
proteins are formulated with a combination of caffeine with a benzoic acid, a
hydroxybenzoic
acid, Of a benzenesulfonic acid as excipient compounds. In embodiments, the
hindered amine
excipient compounds are metabolized in the body to yield biologically
compatible
byproducts. In some embodiments, the hindered amine excipient compound is
present in the
formulation at a concentration of about 250 mg/ml or less. In additional
embodiments, the
hindered amine excipient compound is present in the formulation at a
concentration of about
10 mg/ml to about 200 mg/ml, or at a concentration of about 10mg/m1 to about
120 mg/ml. In
yet additional aspects, the hindered amine excipient compound is present in
the formulation
at a concentration of about 20 mg/nil to about 120 mg/ml.
100781 In embodiments, viscosity-reducing excipients in this hindered amine
category may
include methylxamhines such as caffeine and theophylline, although their use
has typically
been limited due to their low water solubility. In some applications it may be
advantageous
to have higher concentrated solutions of these viscosity-reducing excipients
despite their low
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water solubility. For example, in processing it may be advantageous to have a
concentrated
excipient solution that can be added to a concentrated protein solution so
that adding the
excipient does not dilute the protein below the desired final concentration.
In other cases,
despite its low water solubility, additional viscosity-reducing excipient may
be necessary to
achieve the desired viscosity reduction, stability, tonicity etc. of a final
protein formulation.
In embodiments, a highly concentrated excipient solution may be formulated (i)
as a
viscosity-reducing excipient at a concentration 1.5 to 50 times higher than
the effective
viscosity-reducing amount, or (ii) as a viscosity-reducing excipient at a
concentration 1.5 to
50 times higher than its literature reported solubility in pure water at 298 K
(e.g., as reported
to in The Merck Index; Royal Society of Chemistry; Fifteenth Edition,
(April 30, 2013)), or
both.
[0079] Certain co-solutes have been found to substantially increase the
solubility limit of
these low solubility viscosity-reducing excipients, allowing for excipient
solutions at
concentrations multiple times higher than literature reported solubility
values. These co-
solutes can be classified under the general category of hydrotropes. Co-
solutes found to
provide the greatest improvement in solubility for this application were
generally highly
soluble in water (> 0.25 M) at ambient temperature and physiological pH, and
contained
either a pyridine or benzene ring. Examples of compounds that may be useful as
co-solutes
include aniline Ha, isoniacinamide, niacinamide, n-methyltyramine HC1, phenol,
procaine
HC1, resorcinol, saccharin calcium salt, saccharin sodium salt, sodium
aminobenzoic acid,
sodium benzoate, sodium parahydroxybenzoate, sodium metahydroxybenzoate,
sodium 2,5-
dihydroxybenzoate, sodium salicylate, sodium sulfanilate, sodium
parahydroxybenzene
sulfonate, synephrine, and tyramine HC1.
[0080] In embodiments, certain hindered amine excipient compounds can possess
other
pharmacological properties. As examples, xanthines are a category of hindered
amines
having independent pharmacological properties, including stimulant properties
and
bronchodilator properties when systemically absorbed. Representative xanthines
include
caffeine, aminophylline, 3-isobuty1-1-methylxanthine, paraxanthine,
pentoxifylline,
theobromine, theophylline, and the like. Methylated xanthines are understood
to affect force
of cardiac contraction, heart rate, and bronchodilation. In some embodiments,
the xanthine
excipient compound is present in the formulation at a concentration of about
30 mg/ml or
less.
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[0081] Another category of hindered amines having independent pharmacological
properties are the local injectable anesthetic compounds. Local injectable
anesthetic
compounds are hindered amines that have a three-component molecular structure
of (a) a
lipophilic aromatic ring, (b) an intermediate ester or amide linkage, and (c)
a secondary or
tertiary amine. This category of hindered amines is understood to interrupt
neural conduction
by inhibiting the influx of sodium ions, thereby inducing local anesthesia The
lipophilic
aromatic ring for a local anesthetic compound may be formed of carbon atoms
(e.g., a
benzene ring) or it may comprise heteroatoms (e.g., a thiophene ring).
Representative local
injectable anesthetic compounds include, but are not limited to, amylocaine,
articaine,
to bupivicaine, butacaine, butanilicaine, chlorprocaine, cocaine,
cyclomethycaine,
dimethocaine, editocaine, hexylcaine, isobucaine, levobupivacaine, lidocaine,
metabutethamine, metabutoxycaine, mepivacaine, meprylcaine, propoxycaine,
prilocaine,
procaine, piperocaine, tetracaine, trimecaine, and the like. The local
injectable anesthetic
compounds can have multiple benefits in protein therapeutic formulations, such
as reduced
viscosity, improved stability, and reduced pain upon injection. In some
embodiments, the
local anesthetic compound is present in the formulation in a concentration of
about 50 mg/m1
or less.
[0082] In embodiments, a hindered amine having independent pharmacological
properties
is used as an excipient compound in accordance with the formulations and
methods described
herein. In some embodiments, the excipient compounds possessing independent
pharmacological properties are present in an amount that does not have a
pharmacological
effect and/or that is not therapeutically effective. In other embodiments, the
excipient
compounds possessing independent pharmacological properties are present in an
amount that
does have a pharmacological effect and/or that is therapeutically effective.
In certain
embodiments, a hindered amine having independent pharmacological properties is
used in
combination with another excipient compound that has been selected to decrease
formulation
viscosity, where the hindered amine having independent pharmacological
properties is used
to impart the benefits of its pharmacological activity. For example, a local
injectable
anesthetic compound can be used to decrease formulation viscosity and also to
reduce pain
upon injection of the formulation. The reduction of injection pain can be
caused by
anesthetic properties; also, a lower injection force can be required when the
viscosity is
reduced by the excipients. Alternatively, a local injectable anesthetic
compound can be used
to impart the desirable pharmacological benefit of decreased local sensation
during
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formulation injection, while being combined with another excipient compound
that reduces
the viscosity of the formulation.
100831 In other embodiments, a viscosity-reducing excipient is selected based
on its
physiological impact or lack thereof on a potential patient For example, while
certain
substituted phenethylamines are understood to modulate various
neurotransmitters, such as
the monoamine neurotransmitter systems, and these may have various
psychotropic effects
(e.g., stimulant, hallucinogenic, or entactogenic effects) because of their
impact on the central
nervous system, it can be desirable to employ a viscosity-reducing
phenethylamine excipient
in a viscosity-reducing amount that does not produce psychotropic effects, or
that does not
to produce clinically problematic psychotropic effects, or that may produce
psychotropic effects
in a dose-related manner, but does not produce psychotropic effects at the
dosage to be found
in a specific formulation. Similarly, it can be desirable to employ other
viscosity-reducing
excipients that do not produce other physiological effects (e.g.,
cardiovascular, respiratory,
gastrointestinal, genitourinary, and the like), or that do not produce
clinically problematic
physiological effects, or that may produce physiological effects in a dose-
related manner, but
do not produce physiological effects at the dosage to be found in a specific
formulation.
100841 In embodiments, the modified amine excipient can be a conjugate acid of
a weak
base. As use herein, the term "conjugate acid" refers to a protonated form of
a base that has
some acidity; such a molecule is therefore known as the conjugate acid of the
base. Weak
bases whose conjugate acids are included as modified amine excipients include,
for example,
ammonia (NI-13), ammonium hydroxide (NFLOH), alkartolamines, and urea.
Modified amine
excipients can be produced by reacting such weak bases with acids to produce
the conjugate
acids thereof Exemplary modified amine excipients include conjugate acids of
such weak
bases, such as ammonium chloride, ammonium bromide, ammonium fluoride,
ammonium
acetate, ammonium citrate, monoethanolamine hydrochloride, monoethanolamine
hydrobromide, monoethanolamine hydrofluoride, monoethanolamine acetate,
monoethanolamine citrate, diethanolamine hydrochloride, diethanolamine
hydrobromide,
diethanolamine hydrofluoride, diethanolamine acetate, diethanolamine citrate,
triethanolamine hydrochloride, triethanolamine hydrobromide, triethanolamine
hydrofluoride,
triethanolamine acetate, triethanolamine citrate, urea hydrochloride, urea
hydrobromide, urea
hydrofluoride, urea acetate, urea citrate. In embodiments, the weak base is
ammonia or
ammonium hydroxide, and the modified amine is a conjugate acid thereof In
certain
embodiments, the weak base is an ethoxylated amine such as an alkanolamine,
for example
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monoethanolamine, diethanolamine, and triethanolamine, and the modified amine
is the
conjugate acid thereof.
100851 In embodiments, the monoethanolamine weak base can be produced by the
synthetic route of reacting ammonia with ethylene oxide, or it can be produced
by
decarboxylation of serine or phosphatidylserine, optionally with the aid of a
decarboxylase
enzyme. In other embodiments, the weak base is urea The weak base can be
modified with
an acid (such as HF, HC1, HBr, H3PO4., H2SO4, acetic acid, citric acid, and
the like) to
produce the conjugate acid excipient before it is introduced to the solution
containing the
protein. In other embodiments, the weak base can be modified with the acid to
form the
to modified amine excipient in the presence of the protein in solution.
100861 Modified amines, such as those disclosed above, are particularly useful
in reducing
the viscosity of formulations comprising PEGylated proteins. Without being
bound by
theory, it is believed that the protonated amine groups can alter the hydrogen
bonding and
salvation of the PEG segments of the PEGylated protein, leading to a
conformational change
in the backbone of the PEG chain and fundamentally altering the PEG solution
structure such
that the solution viscosity is reduced.
b. Excipient Compound Category 2: Aromatics
and Anionic Aromatics
100871 High concentration solutions of therapeutic or non-therapeutic proteins
can be
formulated with aromatic small molecule compounds as excipient compounds. The
aromatic
excipient compounds can contain an aromatic functional group such as phenyl,
benzyl, aryl,
alkylbenzyl, hydroxybenzyl, phenolic, hydroxyaryl, heteroaromatic group, or a
fused
aromatic group. The aromatic excipient compounds also can contain a functional
group such
as carboxylate, oxide, phenoxide, sulfonate, sulfate, phosphonate, phosphate,
or sulfide.
Aromatic excipients can be anionic, cationic, or uncharged.
100881 A charged aromatic excipient can be described as an acid, a base, or a
salt (as
applicable), and it can exist in a variety of salt forms. Without being bound
by theory, a
charged aromatic excipient compound is thought to be a bulky, sterically
hindered molecule
that can associate with oppositely charged segments of a protein, so that they
can shield these
sections of the protein, thereby decreasing the interactions between protein
molecules that
render the protein-containing formulation viscous. For example, an anionic
aromatic
excipient can associate with cationic segments of a protein, so that they can
shield these
sections of the protein, thereby decreasing the interactions between protein
molecules that
render the protein-containing formulation viscous.
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[0089] In embodiments, examples of aromatic excipient compounds include
anionic
aromatic compounds such as salicylic acid, aminosalicylic acid, hydroxybenzoic
acid,
aminobenzoic acid, para-aminobenzoic acid, benzenesulfonic acid,
hydroxybenzenesulfonic
acid, naphdialenesulfonic acid, naphthalenedisulfonic acid, hydroquinone
sulfonic acid,
sulfanilic acid, vanillic acid, vanillin, vanillin-taurine adduct,
aminophenol, anthranilic acid,
cinnamic acid, coumaric acid, caffeic acid, isonicotinic acid, folic acid,
folinic acid, folinic
acid calcium salt, phenylserine, DL-3-phenylserine, adenosine monophosphate,
deoxyadenosine, guanosine, deoxyguanosine, indole acetic acid, potassium
urate, furan
dicarboxylic acid, furan-2-acrylic acid, 2-furanpropionic acid, sodium
phenylpyruvate,
to sodium hydroxyphenylpyruvate, dihydroxy benzoic acid, trihydroxybenzoic
acid, pyrogallol,
benzoic acid, and the salts of the foregoing acids. In embodiments, the
anionic aromatic
excipient compounds are formulated in the ionized salt form. In embodiments,
an anionic
aromatic compound is formulated as the salt of a hindered amine, such as
dimethylcyclohexylammonitun hydroxybenzoate. In embodiments, the anionic
aromatic
excipient compounds are formulated with various counterions such as organic
cations. In
embodiments, high concentration solutions of therapeutic or non-therapeutic
proteins are
formulated with anionic aromatic excipient compounds and caffeine. In
embodiments, the
anionic aromatic excipient compound is metabolized in the body to yield
biologically
compatible byproducts.
[0090] In embodiments, examples of aromatic excipient compounds include
phenols and
polyphenols. As used herein, the term "phenol" refers an organic molecule that
consists of at
least one aromatic group or fused aromatic group bonded to at least one
hydroxyl group and
the term "polyphenol" refers to an organic molecule that consists of more than
one phenol
group. Such excipients can be advantageous under certain circumstances, for
example when
used in formulations with high concentration solutions of therapeutic or
nontherapeutic
PEGylated proteins to lower solution viscosity. Non-limiting examples of
phenols include
the benzenediols resorcinol (1,3-benzenediol), catechol (1,2-benzenediol) and
hydroquinone
(1,4-benzenediol), the benzenetriols hydroxyquinol (1,2,4-benzenetriol),
pyrogallol (1,2,3-
benzenetriol), and phloroglucinol (1,3,5-benzenetriol), the benzenetetrols
1,2,3,4-
Benzenetetrol and 1,2,3,5-Benzenetetrol, and benzenepentol and benzenehexol.
Non-limiting
examples of polyphenols include tannic acid, ellagic acid, epigallocatechin
gallate, catechin,
tannins, ellagitannins, and gallotannins. More generally, phenolic and
polyphenolic
compounds include, but are not limited to, flavonoids, lignarts, phenolic
acids, and stilbenes.
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Flavonoid compounds include, but are not limited to, anthocyanins, chalcones,
dihydrochalcones, dihydroflavanols, flavanols, flavanones, flavones,
flavonols, and
isoflavonoids. Phenolic acids include, but are not limited to, hydroxybenzoic
acids,
hydroxycinnamic acids, hydroxyphenylacetic acids, hydroxyphenylpropanoic
acids, and
hydroxyphenylpentanoic acids. Other polyphenolic compounds include, but are
not limited
to, alkylmethoxyphenols, allcylphenols, curcuminoids, hydroxybenzaldehy des,
hydroxybenzoketones, hydroxycinnamaldehydes, hydroxycotunarins,
hydroxyphenylpropenes, methoxy phenols, naphtoquinones, hydroquinones,
phenolic
terpenes, resveratrol, and tyrosols. In embodiments, the polyphenol is tannic
acid. In
to embodiments, the phenol is gallic acid. In embodiments, the phenol is
pyrogallol. In
embodiments, the phenol is resorcinol. Without being bound by theory, the
hydroxyl groups
of phenolic compounds, e.g., gallic acid, pyrogallol, and resorcinol, form
hydrogen bonds
with ether oxygen atoms in the backbone of the PEG chain and thus form a
phenol/PEG
complex that fundamentally alters the PEG solution structure such that the
solution viscosity
is reduced. Polyphenolic compounds, such as tannic acid, derive their
viscosity-reducing
properties from their respective phenol group building blocks, such as gallic
acid, pyrogallol,
and resorcinol. The specific organization of the phenol groups within a
polyphenolic
compound can give rise to complex behavior in which a viscosity reduction
attained by the
addition of a phenol is enhanced by the addition of a lower quantity of the
respective
polyphenol.
c. Excipient Compound Category 3:
Functionalized Amino Acids
100911 High concentration solutions of therapeutic or non-therapeutic proteins
can be
formulated with one or more functionalized amino acids, where a single
functionalized amino
acid or an oligopeptide comprising one or more functionalized amino acids may
be used as
the excipient compound. In embodiments, the functionalized amino acid
compounds
comprise molecules ("amino acid precursors") that can be hydrolyzed or
metabolized to yield
amino acids. In embodiments, the functionalized amino acids can contain an
aromatic
functional group such as phenyl, benzyl, aryl, alkylbenzyl, hydroxybenzyl,
hydroxymyl,
heteroaromatic group, or a fused aromatic group. In embodiments, the
functionalized amino
acid compounds can contain esterified amino acids, such as methyl, ethyl,
propyl, butyl,
benzyl, cycloalkyl, glyceryl, hydroxyethyl, hydroxypropyl, PEG, and PPG
esters. In
embodiments, the functionalized amino acid compounds are selected from the
group
consisting of arginine ethyl ester, arginine methyl ester, arginine
hydroxyethyl ester, and
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arginine hydroxypropyl ester. In embodiments, the functionalized amino acid
compound is a
charged ionic compound in aqueous solution at neutral pH. For example, a
single amino acid
can be derivatized by forming an ester, like an acetate or a benzoate, and the
hydrolysis
products would be acetic acid or benzoic acid, both natural materials, plus
the amino acid. In
embodiments, the functionalized amino acid excipient compounds are metabolized
in the
body to yield biologically compatible byproducts.
d. Excipient Compound Category 4: Oligopeptides
100921 High concentration solutions of therapeutic or non-therapeutic proteins
can be
formulated with oligopeptides as excipient compounds. In embodiments, the
oligopeptide is
to designed such that the structure has a charged section and a bulky
section. In embodiments,
the oligopeptides consist of between 2 and 10 peptide subunits. The
oligopeptide can be bi-
functional, for example a cationic amino acid coupled to a non-polar one, or
an anionic one
coupled to a non-polar one. In embodiments, the oligopeptides consist of
between 2 and 5
peptide subunits. In embodiments, the oligopeptides are homopeptides such as
polyglutamic
acid, polyaspartic acid, poly-lysine, poly-arginine, and poly-histidine. In
embodiments, the
oligopeptides have a net cationic charge. In other embodiments, the
oligopeptides are
heteropeptides, such as Trp2Lys3. In embodiments, the oligopeptide can have an
alternating
structure such as an ABA repeating pattern. In embodiments, the oligopeptide
can contain
both anionic and cationic amino acids, for example, Arg-Glu. Without being
bound by
theory, the oligopeptides comprise structures that can associate with proteins
in such a way
that it reduces the intermolecular interactions that lead to high viscosity
solutions; for
example, the oligopeptide-protein association can be a charge-charge
interaction, leaving a
somewhat non-polar amino acid to disrupt hydrogen bonding of the hydration
layer around
the protein, thus lowering viscosity. In some embodiments, the oligopeptide
excipient is
present in the composition in a concentration of about 50 mg/nil or less.
e. Excipient Compound Category 5: Short-chain organic acids
100931 As used herein, the term "short-chain organic acids" refers to C2-C6
organic acid
compounds and the salts, esters, or lactones thereof This category includes
saturated and
unsaturated carboxylic acids, hydroxy functionalized carboxylic acids, and
linear, branched,
or cyclic carboxylic acids. In embodiments, the acid group in the short-chain
organic acid is
a carboxylic acid, sulfonic acid, phosphonic acid, or a salt thereof
100941 In addition to the four excipient categories above, high concentration
solutions of
therapeutic or non-therapeutic proteins can be formulated with short-chain
organic acids, for
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example, the acid or salt forms of sorbic acid, valeric acid, propionic acid,
glucuronic acid,
caproic acid, and ascorbic acid as excipient compounds. Examples of excipient
compounds
in this category include potassium sorbate, taurine, sodium propionate,
calcium propionate,
magnesium propionate, and sodium ascorbate.
f Excipient Compound Category 6: Low molecular weight aliphatic polyacids
100951 High concentration solutions of therapeutic or non-therapeutic
PEGylated proteins
can be formulated with certain excipient compounds that enable lower solution
viscosity,
where such excipient compounds are low molecular weight aliphatic polyacids.
As used
herein, the term "low molecular weight aliphatic polyacids" refers to organic
aliphatic
to polyacids having a molecular weight < about 1500, and having at least
two acidic groups,
where an acidic group is understood to be a proton-donating moiety. The acidic
groups can
be in the protonated acid form, the salt form, or a combination thereof. Non-
limiting
examples of acidic groups include carboxylate, phosphonate, phosphate,
sulfonate, sulfate,
nitrate, and nitrite groups. Acidic groups on the low molecular weight
aliphatic polyacid can
be in the anionic salt form such as carboxylate, phosphonate, phosphate,
sulfonate, sulfate,
nitrate, and nitrite; their counterions can be sodium, potassium, lithium, and
ammonium_
Specific examples of low molecular weight aliphatic polyacids useful for
interacting with
PEGylated proteins as described herein include maleic acid, tartaric acid,
glutaric acid,
malonic acid, itaconic acid, citric acid, ethylenediaminetetraacetic acid
(EDTA), aspartic
acid, glutamic acid, alendronic acid, etidronic acid and salts thereof Further
examples of low
molecular weight aliphatic polyacids in their anionic salt form include
phosphate (PO4.3),
hydrogen phosphate (HP043), dihydrogen phosphate (1121304), sulfate (S042),
bisulfate
(HS0c), pyrophosphate (P20741, hexametaphosphate, carbonate (C032), and
bicarbonate
(HCO3). The counterion for the anionic salts can be Na, Li, K, or ammonium
ion. These
excipients can also be used in combination with excipients. As used herein,
the low molecular
weight aliphatic polyacid can also be an alpha hydroxy acid, where there is a
hydroxyl group
adjacent to a first acidic group, for example glycolic acid, lactic acid, and
gluconic acid and
salts thereof In embodiments, the low molecular weight aliphatic polyacid is
an oligomeric
form that bears more than two acidic groups, for example polyacrylic acid,
polyphosphates,
polypeptides and salts thereof. In some embodiments, the low molecular weight
aliphatic
polyacid excipient is present in the composition in a concentration of about
50 mg/ml or less.
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g. Excipient Compound Category 7: Diones and sulfones
100961 An effective viscosity-reducing excipient can be a molecule containing
a sulfone,
sulfonamide, or dione functional group that is soluble in pure water to at
least 1 g/L at 298K
and having a net neutral charge at pH 7. Preferably, the molecule has a
molecular weight of
less than 1000 g/mol and more preferably less than 500 g/mol. The diones and
sulfones
effective in reducing viscosity have multiple double bonds, are water soluble,
have no net
charge at pH 7, and are not strong hydrogen bonding donors. Not to be bound by
theory, the
double bond character can allow for weak pi-stacking interactions with
protein. In
embodiments, at high protein concentrations and in proteins that only develop
high viscosity
to at high concentration, charged excipients are not effective because
electrostatic interaction is
a longer-range interaction. Solvated protein surfaces are predominantly
hydrophilic, making
them water soluble. The hydrophobic regions of proteins are generally shielded
within the 3-
dimensional structure, but the structure is constantly evolving, unfolding,
and re-folding
(sometimes called "breathing") and the hydrophobic regions of adjacent
proteins can come
into contact with each other, leading to aggregation by hydrophobic
interactions. The pi-
stacking feature of dione and sulfone excipients can mask hydrophobic patches
that may be
exposed during such "breathing." Another other important role of the excipient
can be to
disrupt hydrophobic interactions and hydrogen bonding between proteins in
close proximity,
which will effectively reduce solution viscosity. Dione and sulfone compounds
that fit this
description include dimethylsulfone, ethyl methyl sulfone, ethyl methyl
sulfonyl acetate,
ethyl isopropyl sulfone, bis(methylsulfonyOmethane, methane sulfonamide,
methionine
sulfone, sodium bisulfite, menadione sodium bisulfite, 1,2-cyclopentanedione,
1,3-
cyclopentanedione, 1,4-cyclopentanedione, and butane-2,3-dione.
h. Excipient Compound Category 8: Zwitterionic excipients
[0097] Solutions of therapeutic or non-therapeutic proteins can be formulated
with certain
zwitterionic compounds as excipients to improve stability or reduce viscosity.
As used herein,
the term "zwitterionic" refers to a compound that has a cationic charged
section and an
anionic charged section. In embodiments, the zwitterionic excipient compounds
are amine
oxides. In embodiments, the opposing charges are separated from each other by
2-8 chemical
bonds. In embodiments, the zwitterionic excipient compounds can be small
molecules, such
as those with a molecular weight of about 50 to about 500 g/mol, or can be
medium
molecular weight molecules, such as those with a molecular weight of about 500
to about
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2000 g/mol, or can be high molecular weight molecules, such as polymers having
a molecular
weight of about 200010 about 100,000 g/mol.
100981 Examples of the zwitterionic excipient compounds include (3-
carboxypropyl)
trimethylammonium chloride, 1-aminocyclohexane carboxylic acid,
homocycloleucine, 1-
methy1-4-imida7oleacetic acid, 3-(1-pyridinio)-1-propanesulfonate, 4-
arninobenzoic acid,
alendronate, aminoethyl sulfonic acid, aminohipptuic acid, aspartame,
aminotris
(methylenephosphonic acid) (ATMP), calcobutrol, calteridol, cocatnidopropyl
betaine,
cocamidopropyl hydroxysultaine, creatine, cytidine, cytidine monophosphate,
diaminopimelic acid, diethylenetriaminepentaacetic acid, dimethyl
phenylalanine,
to methylglycine, sarcosine, dimethylglycine, zwitterionic dipeptides
(e.g., Arg-Glu, Lys-Glu,
His-Glu, Mg-Asp, Lys-Asp, His-Asp, Glu-Arg, Glu-Lys, Glu-His, Asp-Mg, Asp-Lys,
Asp-
His), diethylenetriamine penta(methylene phosphonic acid) (DTPMP), dipahnitoyl
phosphatidylcholine, ectoine, ethylenediamine tetra(methylenephosphonic acid)
(EDTMP),
folate benzoate mixture, folate niacinamide mixture, gelatin, hydroxyproline,
iminodiacetic
acid, isoguvacine, lecithin, myristatnine oxide, nicotinamide adenine
dinucleotide (NAD),
aspartic acid, N-methyl aspartic acid, N-methylproline, lysine, N-trimethyl
lysine, omithine,
oxolinic acid, risendronate, allyl cysteine, S-allyl-L-cysteine, somapacitan,
taurine, theanine,
trigonelline, vigabatrin, ectoine, 4-(2-hydroxyethyl)-1-
piperazineethanesulfonate, o-
octylphosphoryl choline, nicotinamide mononucleotide, trig,lycine,
tetraglycine, 13-
guanidinopropionic acid, 5-aminolevulinic acid hydrochloride, picolinic acid,
lidofenin,
phosphocholine, 1-(5-Carboxypenty1)-4-methylpyridin-1-ium bromide, L-anserine
nitrate, L-
glutathione reduced, N-ethyl-L-glutamine, N-methyl proline, (Z)-14N-(2-
arninoethyl)-N-(2-
ammonioethyl) amino]diazen-l-ium-1,2-diolate (DETA-NONOate), (Z)-14N-(3-
aminopropy1)-N-(3-arnmoniopropyflamino]diazen-1-ium-1,2-diolate (DPTA-NONate),
and
zOledronic acid.
100991 Not to be bound by theory, the zwitterionic excipient compounds can
exert viscosity
reducing or stabilizing effects by interacting with the protein, for example
by charge
interactions, hydrophobic interactions, and steric interactions, causing the
proteins to be more
resistant to aggregation, or by affecting the bulk properties of the water in
the protein
formulation, such as an electrolyte contribution, a surface tension reduction,
a change in the
amount of unbound water available, or a change in dielectric constant.
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i. Excipient Compound Categoiy 9: Crowding agents with hydrogen bonding
elements
1001001 Solutions of therapeutic or non-therapeutic proteins can be formulated
with
crowding agents with hydrogen bonding elements as excipients to improve
stability or reduce
viscosity. As used herein, the term "crowding agent" refers to a formulation
additive that
reduces the amount of water available for dissolving a protein in solution,
increasing the
effective protein concentration. In embodiments, crowding agents can decrease
protein
particle size or reduce the amount of protein unfolding in solution. In
embodiments, the
crowding agents can act as solvent modifiers that cause structuring of the
water by hydrogen
to bonding and hydration effects. In embodiments, the crowding agents can
reduce the amount
of intermolecular interactions between proteins in solution. In embodiments,
the crowding
agents have a structure containing at least one hydrogen bond donor element
such as
hydrogen attached to an oxygen, sulfur, or nitrogen atom. In embodiments, the
crowding
agents have a structure containing at least one weakly acidic hydrogen bond
donor element
having a pKa of about 6 to about 11. In embodiments, the crowding agents have
a structure
containing between about 2 and about 50 hydrogen bond donor elements. In
embodiments,
the crowding agents have a structure containing at least one hydrogen bond
acceptor element
such as a Lewis base. In embodiments, the crowding agents have a structure
containing
between about 2 and about 50 hydrogen bond acceptor elements. In embodiments,
the
crowding agents have a molecular weight between about 50 and 500 g/mol. In
embodiments,
the crowding agents have a molecular weight between about 100 and 350 g/mol.
In other
embodiments, the crowding agents can have a molecular weight above 500 g/mol,
such as
raffinose, inulin, pullulan, or sinistrins.
1001011 Examples of the crowding agent excipients with hydrogen bonding
elements include
1,3-Dimethy1-3,4,5,6-tetrahydro-2(1H)-pyrimidione, 15-crown-5, 18-crown-6, 2-
butanol, 2-
butanone, 2-phenoxyethanol, acetaminophen, allantoin, arabinose, meglumine,
arabitol,
benzyl acetonacetate, benzyl alcohol, chlorobutanol, cholestanoltetraacetyl-b-
glucoside,
cinnamaldehyde, cyclohexanone, deoxyribose, diethyl carbonate, dimethyl
carbonate,
dimethyl isosorbide, dimethylacetamide, dimethylformamide, dimethylol ethylene
urea,
dimethyluracil, epilactose, erythritol, erythrose, ethyl lactate, ethyl
maltol, ethylene
carbonate, fonnamide, fucose, galactose, genistein, gentisic acid
ethanolamide,
gluconolactone, glyceraldehyde, glycerol, glycerol carbonate, glycerol formal,
glycerol
urethane, glycyrrhizic acid, gossypin, harpagoside, hederacoside C,
icodextrin, iditol,
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imidazolidone, inositol, inulins, isomaltitol, kojic acid, lactitol,
lactobionic acid, lactose,
lactulose, lyxose, madecassoside, maltotriose, mangiferin, mannose, melzitose,
methyl
lactate, methylpyrrolidone, mogroside V. N-acetylgalactosamine, N-
acetylglucosamine, N-
acetylneuraminic acid, N-methyl acetamide, N-methyl formamide, N-methyl
propionamide,
pentaerythritol, pinoresinol diglucoside, glucuronic acid, piracetam, propyl
gallate, propylene
carbonate, psicose, pullulan, pyrogallol, quinic acid, raffinose, rebaudioside
A, rhamnose,
ribitol, ribose, ribulose, saccharin, sedoheptulose, sinistrins, solketal,
stachyose, sucralose,
tagatose, t-butanol, tetraglycol, triacetin, N-acetyl-d-maimosamine, nystose,
kestose,
turanose, acarbose, D-saccharic acid 1,4-lactone, thiodigalactoside, fucoidan,
hydroxysafflor
to yellow A, shikimic acid, diosmin, pravastatin sodium salt, D-altrose, L-
gulonic gamma-
lactone, neomycin, rubusoside dihydroartemisinin, phloroglucinol, naringin,
baicalein,
hesperidin, apigenin, pyrogallol, morin, salsalate, kaempferol, myricetin,
31,4%7-
trihydroxyisoflavone, (+)-taxifolin, silybin, perseitol difonnal, 4-
hydroxyphenylpyruvic acid,
sulfacetamide, isopropyl 3-D-1-thiogalactopyranoside, ethyl 2,5-
dihydroxybenzoate,
spectinomycin, resveratrol, quercetin, kanamycin sulfate, 1-(2-
Pyrimidyl)piperazine, 2-(2-
pyridyflethylamine, 2-imidazolidone, DL-1,2-isopropylideneglycerol, metformin,
xylylenediamine, x-xylylenediamine, demeclocycline, tripropylene glycol,
tubeimoside 1,
verbenaloside, xylitol, and xylose.
6. Protein/Excipient Solutions: Properties and
Processes
1001021 In certain embodiments, solutions of therapeutic or non-therapeutic
proteins are
formulated with the above-identified excipient compounds, or combinations
thereof
("excipient additives"), such as hindered or modified amines, aromatics,
functionahzed
amino acids, oligopeptides, short-chain organic acids, low molecular weight
aliphatic
polyacids, diones and sulfones, zwitterionic excipients, and crowding agents,
to result in
improved protein-protein interaction characteristics or protein self-
interactions as measured
by the protein diffusion interaction parameter, kD, by biolayer
interferometiy, by surface
plasmon resonance, or by determining the second virial coefficient, 822, or
similar method.
As used herein, an "improvement" in one or more protein-protein interaction
parameters
achieved by test formulations using the above-identified excipient compounds
or
combinations thereof can refer to a decrease in attractive protein-protein
interactions when a
test formulation is compared under comparable conditions with a comparable
formulation
that does not contain the excipient compounds or excipient additives.
Measurements of kD
and 822 can be made using standard techniques in the industry and can be an
indicator of
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improved solution properties or stability of the protein in solution. For
example, a highly
negative kD value can indicate that the protein has strong attractive
interactions, and this can
lead to aggregation, instability, and theology problems. When formulated in
the presence of
certain of the above-identified excipient compounds or combinations thereof,
the same
protein can have an improved proxy parameter of a less negative kD value, or a
kD value
near or above zero, with this improved proxy parameter being associated with
an
improvement in a formulation-related parameter.
1001031 In embodiments, certain of the above-described excipient compounds or
combinations thereof, such as hindered or modified amines, aromatics,
functionalized amino
to acids, oligopeptides, short-chain organic acids, low molecular weight
aliphatic polyacids,
diones and sulfones, zwitterionic excipients, and/or crowding agents are used
to improve a
protein-related process, such as the manufacture, processing, sterile filling,
purification, and
analysis of protein-containing solutions, using processing methods such as
filtration,
syringing, transferring, pumping, mixing, heating or cooling by heat transfer,
gas transfer,
centrifugation, chromatography, membrane separation, centrifugal
concentration, tangential
flow filtration, radial flow filtration, axial flow filtration,
lyophilization, and gel
electrophoresis. These processes and processing methods can have improved
efficiency due
to the lower viscosity, improved solubility, or improved stability of the
proteins in the
solution during manufacture, processing, purification, and analysis steps.
Additionally,
equipment-related processes such as the cleanup, sterilization, and
maintenance of protein
processing equipment can be facilitated by the use of the above-identified
excipients due to
decreased fouling, decreased denaturing, lower viscosity, and improved
solubility of the
protein, and parameters associated with the improvement of these processes are
similarly
improved.
EXAMPLES
1001041 Materials:
= Bovine gamma globulin (BGG), >99% purity, Sigma Aldrich
= Histidine, Sigma Aldrich
= Other materials described in the examples below were from Sigma
Aldrich unless
otherwise specified.
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Example 1: Preparation of formulations containing excipient compounds and test
protein
1001051 Formulations were prepared using an excipient compound and a test
protein, where
the test protein was intended to simulate either a therapeutic protein that
would be used in a
therapeutic formulation, or anon-therapeutic protein that would be used in a
non-therapeutic
formulation. Such formulations were prepared in 50 mM histidine hydrochloride
with
different excipient compounds for viscosity measurement in the following way.
Histidine
hydrochloride was first prepared by dissolving 1.94 g histidine (Sigma-
Aldrich, St. Louis,
MO) in distilled water and adjusting the pH to about 6.0 with 1 M hydrochloric
acid (Sigma-
Aldrich, St Louis, MO) and then diluting to a final volume of 250 nit with
distilled water in
to a volumetric flask. Excipient compounds were then dissolved in 50 mM
histidine HC1. Lists
of excipients are provided below in Examples 4, 5, 6, and 7. In some cases
excipient
compounds were adjusted to pH 6 prior to dissolving in 50 mM histidine HCI. hi
this case
the excipient compounds were first dissolved in deionized water at about 5 wt%
and the pH
was adjusted to about 6.0 with either hydrochloric acid or sodium hydroxide.
The prepared
salt solution was then placed in a convection laboratory oven at about 150
degrees Fahrenheit
(about 65 degrees C) to evaporate the water and isolate the solid excipient.
Once excipient
solutions in 50 mM histidine HCl had been prepared, the test protein (bovine
gamma globulin
("BCC") (Sigma-Aldrich, St. Louis, MO)) was dissolved at a ratio of about
0.336 g BOG per
1 inL excipient solution. This resulted in a final protein concentration of
about 280 mg/tnL.
Solutions of BOG in 50 mM histidine HC1 with excipient were formulated in 20
inL vials and
allowed to shake at 100 rpm on an orbital shaker table overnight. The
solutions were then
transferred to 2 inL microcentrifuge tubes and centrifuged for ten minutes at
2300 rpm in an
IEC MicroMax microcentrifuge to remove entrained air prior to viscosity
measurement.
Example 2: Viscosity measurement
11301061 Viscosity measurements of formulations prepared as described in
Example 1 were
made with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,
Middleboro,
MA). The viscometer was equipped with a CP-40 cone and was operated at 3 rpm
and 25
degrees C. The formulation was loaded into the viscometer at a volume of 0.5
mL and
allowed to incubate at the given shear rate and temperature for 3 minutes,
followed by a
measurement collection period of twenty seconds. This was then followed by 2
additional
steps consisting of 1 minute of shear incubation and subsequent twenty-second
measurement
collection period. The three data points collected were then averaged and
recorded as the
viscosity for the sample.
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Example 3: Protein concentration measurement
1001071 The concentration of the protein in the experimental solutions was
determined by
measuring the absorbance of the protein solution at a wavelength of 280 nm in
a lUVNIS
Spectrometer (Perkin Elmer Lambda 35). First the instrument was calibrated to
zero
absorbance with a 50 mM histidine buffer at pH 6. Next the protein solutions
were diluted by
a factor of 300 with the same histidine buffer and the absorbance at 280 nm
recorded. The
final concentration of the protein in the solution was calculated by using the
extinction
coefficient value of 1.264 rnL/(mg x cm).
Example 4: Formulations with hindered amine excipient compounds
to 1001081 Formulations containing 280 mg/triL BGG were prepared as
described in Example
1, with some samples containing added excipient compounds. In these tests, the
hydrochloride salts of dimethylcyclohexylamine (DMCHA),
dicyclohexylmethylamine
(DCHMA), dimethylaminopropylamine (DMAPA), triethanolamine (TEA),
dimethylethanolamine (DMEA), and niacinamide were tested as examples of the
hindered
amine excipient compounds. Also, a hydroxybenzoic acid salt of DMCHA and a
taurine-
dicyandiamide adduct were tested as examples of the hindered amine excipient
compounds.
The viscosity of each protein solution was measured as described in Example 2,
and the
results are presented in Table 1 below, showing the benefit of the added
excipient compounds
in reducing viscosity.
TABLE 1
Excipient
Test
Viscosity Viscosity
Excipient Added
Concentration
Number
(GP) Reduction
(mg/mL)
4.1 None
0 79 0%
4+2 DMCHA-HCI
28 50 37%
4.3 DMCHA-HCI
41 43 46%
4.4 DMCHA-HCI
50 45 43%
4.5 DMCHA-HCI
82 36 54%
4.6 DMCHA-HCI
123 35 56%
4.7 DMCHA-HCI
164 40 49%
4.8 DMAPA-HCI
87 57 28%
4.9 DMAPA-HCI
40 54 32%
4.10 DCHMA-HCI
29 51 35%
4.11 DCHMA-HCI
50 51 35%
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Excipient
Test
Viscosity Viscosity
Excipient Added
Concentration
Number
(cP) Reduction
(mg/mL)
4.14 TEA-HC1
97 51 35%
4.15 TEA-HC1
38 57 28%
4.16 DMEA-HC1
51 51 35%
4.17 DMEA-HC1
98 47 41%
4.20 DMCHA-hydroxybenzoate
67 46 42%
4.21 DMCHA-hydroxybenzoate 92
42 47%
4.22 Product of Example 8
26 58 27%
4.23 Product of Example 8
58 50 37%
4.24 Product of Example 8
76 49 38%
4.25 Product of Example 8
103 46 42%
4.26 Product of Example 8
129 47 41%
4.27 Product of Example 8
159 42 47%
4.28 Product of Example 8
163 42 47%
4.29 Niacinamide
48 39 51%
4.30 N-Methyl-2-pyrrolidone
30 45 43%
4.31 N-Methyl-2-pyrrolidone
52 52 34%
Example 5: Formulations with anionic aromatic excipient compounds
1001091 Formulations of 280 mg/mL BGG were prepared as described in Example 1,
with
some samples containing added excipient compounds. The viscosity of each
solution was
measured as described in Example 2, and the results are presented in Table 2
below, showing
the benefit of the added excipient compounds in reducing viscosity.
TABLE 2
Test
Excipien.t Viscosity Viscosity
Excipient Added
Concentration
Number
(cP) Reduction
(mg/mL)
5.1 None 0 79
0%
5.2 Sodium aminobenzoate 43 48
39%
5.3 Sodium hydroxybenzoate 26 50
37%
5.4 Sodium sulfanilate 44 49
38%
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Excipient
Test
.. Viscosity Viscosity
Excipient Added
Concentration
Number
(cP) Reduction
(mg,/mL)
5.5 Sodium sulfanilate 96 42
47%
5.6 Sodium indole acetate 52 58
27%
5.7 Sodium indole acetate 27 78
1%
5.8 Vanillic acid, sodium salt 25 56
29%
5.9 Vanillic acid, sodium salt 50 50
37%
5.10 Sodium salicylate
25 57 28%
5.11 Sodium salicylate
50 52 34%
5.12 Adenosine monophosphate
26 47 41%
5.13 Adenosine monophosphate
50 66 16%
5.14 Sodium benzoate
31 61 23%
5.15 Sodium benzoate
56 62 22%
Example 6: Formulations with oligopeptide excipient compounds
1001101 Oligopeptides (n=5) were synthesized by NeoBioLab Inc. (Woburn, MA) in
>95%
purity with the N terminus as a free amine and the C terminus as a free acid.
Dipeptides
(11=2) were synthesized by LifeTein LLC in 95% purity. Formulations of 280
mg/mL BOG
were prepared as described in Example 1, with some samples containing the
synthetic
oligopeptides as added excipient compounds. The viscosity of each solution was
measured as
described in Example 2, and the results are presented in Table 3 below,
showing the benefit
of the added excipient compounds in reducing viscosity.
to
TABLE 3
Excipient
Test
. Viscosity Viscosity
Excipient Added
Concentration
Number
(cP) Reduction
(mg/mL)
6.1 None 0 79
0%
6.2 ArgX5 100 55
30%
6.3 ArgX5 50 54
32%
6.4 HisX5 100 62
22%
6.5 HisX5 50 51
35%
6.6 HisX5 25 60
24%
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Excipient
Test
. Viscosity Viscosity
Excipient Added
Concentration
Number
(mg/mL) ('k) Reduction
6.7 Trp2Lys3 100 59
25%
6.8 Trp2Lys3 50 60
24%
6.9 AspX5 100 102
-29%
6.10 AspX5
50 82 -4%
6.11 Dipeptide LE (Leu-Glu)
50 72 9%
6.12 Dipeptide YE (Tyr-Glu)
50 55 30%
6.13 Dipeptide RP (Mg-Pro)
50 51 35%
6.14 Dipeptide FtIC (Arg-Lys)
50 53 33%
6.15 Dipeptide RH (Mg-His)
50 52 34%
6.16 Dipeptide RR (Mg-Mg)
50 57 28%
6.17 Dipeptide RE (Arg-Glu)
50 50 37%
6.18 Dipeptide LE (Leu-Glu)
100 87 -10%
6.19 Dipeptide YE (Tyr-Glu)
100 68 14%
6.20 Dipeptide RP (Mg-Pro)
100 53 33%
6.21 Dipeptide RIC (Arg-Lys)
100 64 19%
6.22 Dipeptide RH (Arg-His)
100 72 9%
6.23 Dipeptide RR (Mg-Mg)
100 62 22%
6.24 Dipeptide RE (Mg-Gin)
100 66 16%
Example 8: Synthesis of guanyl taurine excipient
100111111 Guanyl taurine was prepared following method described in U.S. Pat.
No.
2,230,965. Taurine (Sigma-Aldrich, St Louis, MO) 3.53 parts were mixed with
1.42 parts of
dicyandiamide (Sigma-Aldrich, St. Louis, MO) and grinded in a mortar and
pestle until a
homogeneous mixture was obtained. Next the mixture was placed in a flask and
heated at
200 C for 4 hours. The product was used without further purification.
Example 9: Protein formulations containing excipient compounds
[00112] Formulations were prepared using an excipient compound and a test
protein, where
to the test protein was intended to simulate either a therapeutic protein
that would be used in a
therapeutic formulation, or a non-therapeutic protein that would be used in a
non-therapeutic
formulation. Such formulations were prepared in 50 rnivi aqueous histidine
hydrochloride
buffer solution with different excipient compounds for viscosity measurement
in the
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following way. Histidine hydrochloride buffer solution was first prepared by
dissolving 1.94
g histidine (Sigma-Aldrich, St Louis, MO) in distilled water and adjusting the
pH to about
6.0 with 1 M hydrochloric acid (Sigma-Aldrich, St. Louis, MO) and then
diluting to a final
volume of 250 mL with distilled water in a volumetric flask. Excipient
compounds were then
dissolved in the 50 mM histidine HC1 buffer solution. A list of the excipient
compounds is
provided in Table 4. In some cases, excipient compounds were dissolved in 50
mM histidine
BCE and the resulting solution pH was adjusted with small amounts of
concentrated sodium
hydroxide or hydrochloric acid to achieve pH 6 prior to dissolution of the
model protein. In
some cases, excipient compounds were adjusted to pH 6 prior to dissolving in
50 mM
to histidine HC1. In this case the excipient compounds were first dissolved
in deionized water at
about 5 wt% and the pH was adjusted to about 6.0 with either hydrochloric acid
or sodium
hydroxide. The prepared salt solution was then placed in a convection
laboratory oven at
about 150 degrees Fahrenheit (65 degrees C) to evaporate the water and isolate
the solid
excipient. Once excipient solutions in 50 mM histidine HC1 had been prepared,
the test
protein, bovine gamma globulin (Sigma-Aldrich, St. Louis, MO) was dissolved at
a ratio to
achieve a final protein concentration of about 280 mg/mL. Solutions of BGG in
50 mM
histidine HCI with excipient were formulated in 20 mL vials and allowed to
shake at 100 rpm
on an orbital shaker table overnight. The solutions were then transferred to 2
mL
microcentrifuge tubes and centrifuged for ten minutes at 2300 rpm in an IEC
MicroMax
microcentrifuge to remove entrained air prior to viscosity measurement.
[00113] Viscosity measurements of formulations prepared as described above
were made
with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,
Middleboro, MA).
The viscometer was equipped with a CP40 cone and was operated at 3 rpm and 25
degrees
C. The formulation was loaded into the viscometer at a volume of 0.5 nth and
allowed to
incubate at the given shear rate and temperature for 3 minutes, followed by a
measurement
collection period of twenty seconds. This was then followed by 2 additional
steps consisting
of 1 minute of shear incubation and subsequent twenty-second measurement
collection
period. The three data points collected were then averaged and recorded as the
viscosity for
the sample. Viscosities of solutions with excipient were normalized to the
viscosity of the
model protein solution without excipient. The normalized viscosity is the
ratio of the
viscosity of the model protein solution with excipient to the viscosity of the
model protein
solution with no excipient.
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TABLE 4
Excipient
Normalized
Test
Viscosity
Excipient Added Concentration Viscosity
Ndumber
(mg/mL)
(cP) Reuction
9.1 DMCHA-HC1 120 0.44
56%
9.2 Niacinatnide 50 0.51
49%
9.3 Isonicotinamide 50 0.48
52%
9.4 Tyramine HCI 70 0.41
59%
9.5 Histamine HCI 50 0.41
59%
9.6 Imidazole HO 100 0.43
57%
9.7 2-methyl-2-imidazoline HO 60 0.43
57%
1-butyl-3-methylimidazoliurn
9.8 100 0.48
52%
chloride
9.9 Procaine HC1 50 0.53
47%
9.10 3-aminopyridine
50 0.51 49%
9.11 2,4,6-trimethylpyridine
50 0.49 51%
9.12 3-pyridine methanol
50 0.53 47%
Nicotinamide adenine
9.13 20 0.56 44%
dinucleotide
9.15 Sodium phenylpyruvate
55 0.57 43%
9.16 2-Pyrrolidinone
60 0.68 32%
9.17 Morpholine HC1
50 0.60 40%
9.18 Agmatine
sulfate 55 0.77 23%
1-butyl-3-methylimidazolium
9.19 60 0.66 34%
iodide
9.21 L-Anserine nitrate
50 0.79 21%
1-hexy1-3-methylimidazolitun
9.22 65 0.89 11%
chloride
9.23 N,N-diethyl nicotinamide
50 0.67 33%
9.24 Nicotinic acid, sodium salt
100 0.54 46%
9.25 Biotin
20 0.69 31%
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Example 10: Preparation of formulations containing excipient combinations and
test protein
1001141 Formulations were prepared using a primary excipient compound, a
secondary
excipient compound and a test protein, where the test protein was intended to
simulate either
a therapeutic protein that would be used in a therapeutic formulation, or a
non-therapeutic
protein that would be used in a non-therapeutic formulation. The primary
excipient
compounds were selected from compounds having both anionic and aromatic
functionality,
as listed below in Table 5. The secondary excipient compounds were selected
from
compounds having either nonionic or cationic charge at pH 6 and either
irnidazoline or
benzene rings, as listed below in Table 5. Formulations of these excipients
were prepared in
to 50 inM histidine hydrochloride buffer solution for viscosity measurement
in the following
way. Histidine hydrochloride was first prepared by dissolving 1.94 g histidine
(Sigma-
Aldrich, St Louis, MO) in distilled water and adjusting the pH to about 6.0
with 1 M
hydrochloric acid (Sigma-Aldrich, St. Louis, MO) and then diluting to a final
volume of 250
nit with distilled water in a volumetric flask. The individual primary or
secondary excipient
compounds were then dissolved in 50 mM histidine HO. Combinations of primary
and
secondary excipients were dissolved in 50 niM histidine HCl and the resulting
solution pH
adjusted with small amounts of concentrated sodium hydroxide or hydrochloric
acid to
achieve pH 6 prior to dissolution of the model protein. Once excipient
solutions had been
prepared as described above, the test protein (bovine gamma globulin (BGG)
(Sigma-
Aldrich, St. Louis, MO)) was dissolved into each test solution at a ratio to
achieve a final
protein concentration of about 280 mg/mL. Solutions of BGG in 50 mNI histidine
HC1 with
excipient were formulated in 20 inL vials and allowed to shake at 100 rpm on
an orbital
shaker table overnight. The solutions were then transferred to 2 nth
microcentrifuge tubes
and centrifuged for ten minutes at 2300 rpm in an 1EC MicroMax microcentrifuge
to remove
entrained air prior to viscosity measurement.
1001151 Viscosity measurements of formulations prepared as described above
were made
with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,
Middleboro, MA).
The viscometer was equipped with a CP40 cone and was operated at 3 rpm and 25
degrees
C. The formulation was loaded into the viscometer at a volume of 0.5 rriL and
allowed to
incubate at the given shear rate and temperature for 3 minutes, followed by a
measurement
collection period of twenty seconds. This was then followed by 2 additional
steps consisting
of 1 minute of shear incubation and a subsequent twenty-second measurement
collection
period. The three data points collected were then averaged and recorded as the
viscosity for
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the sample. Viscosities of solutions with excipient were normalized to the
viscosity of the
model protein solution without excipient and summarized in Table 5 below. The
normalized
viscosity is the ratio of the viscosity of the model protein solution with
excipient to the
viscosity of the model protein solution with no excipient. The example shows
that a
combination of primary and secondary excipients can give a better result than
a single
excipient.
TABLE 5
Primary Excipient
Secondary Excipient
Test Concentration
Concentration Normalized
Name
Name
Number (mg/mL)
(mg/mL) Viscosity
10.1 Salicylic Acid 30
None 0 0.79
10.2 Salicylic Acid 25
Imidazole 4 0.59
4-hydroxybenzoic
10.3 30
None 0 0.61
acid
4-hydroxybenzoic
10.4 25
Imidazole 5 0.57
acid
10.5
4-hydroxybenzene 31
None 0 0.59
sulfonic acid
Primary Excipient
Secondary Excipient
4-hydroxybenzene
10.6 26
Itnidazole 5 0.70
sulfonic acid
4-hydroxybenzene
10.7 25 Caffeine 5 0.69
sulfonic acid
10.8 None 0
Caffeine 10 0.73
10.9 None 0
Imidazole 5 0.75
Example 11: Preparation of formulations containing excipient combinations and
test protein
to 1001161 Formulations were prepared using a primary excipient compound, a
secondary
excipient compound and a test protein, where the test protein was intended to
simulate a
therapeutic protein that would be used in a therapeutic formulation, or a non-
therapeutic
protein that would be used in a non-therapeutic formulation. The primary
excipient
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compounds were selected from compounds having both anionic and aromatic
functionality,
as listed below in Table 6. The secondary excipient compounds were selected
from
compounds having either nonionic or cationic charge at pH 6 and either
imidazoline or
benzene rings, as listed below in Table 6. Formulations of these excipients
were prepared in
distilled water for viscosity measurement in the following way. Combinations
of primary and
secondary excipients were dissolved in distilled water and the resulting
solution pH adjusted
with small amounts of concentrated sodium hydroxide or hydrochloric acid to
achieve pH 6
prior to dissolution of the model protein. Once excipient solutions in
distilled water had been
prepared, the test protein (bovine gamma globulin (BOG) (Sigma-Aldrich, St.
Louis, MO))
to was dissolved at a ratio to achieve a final protein concentration of
about 280 mg/mL.
Solutions of BGG in distilled water with excipient were formulated in 20 na
vials and
allowed to shake at 100 rpm on an orbital shaker table overnight. The
solutions were then
transferred to 2 mL microcentrifuge tubes and centrifuged for ten minutes at
2300 rpm in an
IEC MicroMax microcentrifuge to remove entrained air prior to viscosity
measurement.
1001471 Viscosity measurements of formulations prepared as described above
were made
with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,
Middleboro, MA).
The viscometer was equipped with a CP-40 cone and was operated at 3 rpm and 25
degrees
C. The formulation was loaded into the viscometer at a volume of 0.5 mL and
allowed to
incubate at the given shear rate and temperature for 3 minutes, followed by a
measurement
collection period of twenty seconds. This was then followed by 2 additional
steps consisting
of 1 minute of shear incubation and a subsequent twenty-second measurement
collection
period. The three data points collected were then averaged and recorded as the
viscosity for
the sample. Viscosities of solutions with excipient were normalized to the
viscosity of the
model protein solution without excipient and summarized in Table 6 below. The
normalized
viscosity is the ratio of the viscosity of the model protein solution with
excipient to the
viscosity of the model protein solution with no excipient. The example shows
that a
combination of primary and secondary excipients can give a better result than
a single
excipient
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TABLE 6
Primary Excipient
Secondary Excipient
Test Concentration
Concentration Normalized
Name
Name
Number (mg/mL)
(mg/mL) Viscosity
11.1 Salicylic Acid 20
None 0 0.96
11.2 Salicylic Acid 20
Caffeine 5 0.71
11.3 Salicylic Acid 20
Niacinamide 5 0.76
11.4 Salicylic Acid 20
Imidazole 5 0.73
Example 12: Preparation of formulations containing excipient compounds and PEG
[00118] Materials: All materials were purchased from Sigma-Aldrich, St, Louis,
MO.
Formulations were prepared using an excipient compound and PEG, where the PEG
was
intended to simulate a therapeutic PEGylated protein that would be used in a
therapeutic
formulation. Such formulations were prepared by mixing equal volumes of a
solution of PEG
with a solution of the excipient Both solutions were prepared in a Tris buffer
consisting of 10
mM Tris, 135 mM NaCl, and 1 mM trans-cinnamic acid at pH of 73.
to [00119] The PEG solution was prepared by mixing 3 g of Poly(ethylene
oxide) average Mw
¨1,000,000 (Aldrich Catalog # 372781) with 97 g of the Tris buffer solution.
The mixture
was stirred overnight for complete dissolution.
[00120] An example of the excipient solution preparation is as follows: An
approximately
80 mg/mL solution of citric acid in the Tris buffer was prepared by dissolving
0.4 g of citric
acid (Aldrich cat 251275) in 5 mL of the Tris buffer solution and adjusted the
pH to 7.3
with minimum amount of 10 M NaOH solution.
[00121] The PEG excipient solution was prepared by mixing 0.5 mL of the PEG
solution
with 0.5 ml. of the excipient solution and mixed by using a vortex for a few
seconds. A
control sample was prepared by mixing 0.5 mL of the PEG solution with 0.5 mL
of the Tris
buffer solution.
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Example 13: Viscosity measurements of formulations containing excipient
compounds and
PEG
[00122] Viscosity measurements of the formulations prepared were made with a
DV-ITT LV
cone and plate viscometer (Brookfield Engineering, Middleboro, MA). The
viscometer was
equipped with a CP-40 cone and was operated at 3 rpm and 25 degrees C. The
formulation
was loaded into the viscometer at a volume of 0.5 inL and allowed to incubate
at the given
shear rate and temperature for 3 minutes, followed by a measurement collection
period of
twenty seconds. This was then followed by 2 additional steps consisting of 1
minute of shear
incubation and subsequent twenty second measurement collection period. The
three data
to points collected were then averaged and recorded as the viscosity for
the sample.
[00123] The results presented in Table 7 show the effect of the added
excipient compounds
in reducing viscosity.
TABLE 7
Excipient
Test
. Viscosity Viscosity
Excipient
Concentration
Number
(mg/mL) (cP) Reduction
131 None
0 1048 0%
13.2 Citric acid Na salt
40 56.8 44%
13.3 Citric acid Na salt
20 73,3 28%
13.4 glycerol phosphate
40 71.7 30%
13.5 glycerol phosphate
20 83.9 18%
13.6 Ethylene diamine
40 84.7 17%
13.7 Ethylene diamine
20 83.9 15%
13.8 EDTA/K salt
40 67.1 36%
13.9 EDTA/K salt
20 76.9 27%
13.10 EDTA/Na salt
40 68.1 35%
13.11 EDTA/Na salt
20 77.4 26%
13.12 D-Gluconic acid/K salt
40 80.32 23%
13.13 D-Gluconic acid/K salt
20 88.4 16%
13.14 D-Gluconic acid/Na salt
40 81.24 23%
13.15 D-Gluconic acid/Na salt
20 86.6 17%
13.16 lactic acicUK salt
40 80.42 23%
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Excipient
Test
. Viscosity Viscosity
Excipient
Concentration
Number
(cP) Reduction
(mg/mL)
13.17 lactic acid/K salt
85,1 19%
13.18 lactic acid/Na salt
40 86.55 17%
13.19 lactic acid/Na salt
20 87,2 17%
13.20 etidronic acid/K salt
24 71.91 31%
13.21 etidronic acid/K salt
12 80.5 23%
13.22 etidronic acid/Na salt
24 71.6 32%
13.23 etidronic acid/Na salt
12 79.4 24%
Example 14: Preparation of PEGylated BSA with 1 PEG chain per BSA molecule
[00124] To a beaker was added 200 mL of a phosphate buffered saline (Aldrich
Cat. #
P4417) and 4 g of BSA (Aldrich Cat. # A7906) and mixed with a magnetic bar.
Next 400 mg
of methoxy polyethylene glycol maleimide, MW=5,000, (Aldrich Cat. #63187) was
added.
The reaction mixture was allowed to react overnight at room temperature. The
following day,
20 drops of HCI 0.1 M were added to stop the reaction. The reaction product
was
characterized by SDS-Page and SEC which clearly showed the PEGylated BSA. The
reaction
mixture was placed in an Amicon centrifuge tube with a molecular weight cutoff
(MWCO) of
to 30,000 and concentrated to a few milliliters. Next the sample was
diluted 20 times with a
histidine buffer, 50 mM at a pH of approximately 6, followed by concentrating
until a high
viscosity fluid was obtained. The final concentration of the protein solution
was obtained by
measuring the absorbance at 280 nm and using a coefficient of extinction for
the BSA of
0.6678. The results indicated that the final concentration of BSA in the
solution was 342
mg/mL.
Example 15: Preparation of PEGylated BSA with multiple PEG chains per BSA
molecule
[00125] A 5 mg/mL solution of BSA (Aldrich A7906) in phosphate buffer, 25 mM
at pH of
7.2, was prepared by mixing 0.5 g of the BSA with 100 mL of the buffer. Next 1
g of a
methoxy PEG propionaldehyde Mw=20,000 (JenKem Technology, Plano, TX 75024) was
added followed by 0.12 g of sodium cyanoborohydride (Aldrich 156159), The
reaction was
allowed to proceed overnight at room temperature. The following day the
reaction mixture
was diluted 13 times with a Tris buffer (10 mM Tris, 135 mM NaCl at pH=7.3)
and
concentrated using Amicon centrifuge tubes MWCO of 30,000 until a
concentration of
approximately 150 mg/mL was reached.
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Example 16: Preparation of PEGylated lysozyme with multiple PEG chains per
lysozyme
molecule
1001261 A 5 mg/mL solution of lysozyme (Aldrich L6876) in phosphate buffer, 25
mI1/4/1 at
pH of 7.2, was prepared by mixing 0.5 g of the lysozyme with 100 mL of the
buffer. Next 1 g
of a methoxy PEG propionaildehyde Mw=5,000 (JenKem Technology, Plano, TX
75024) was
added followed by 0.12 g of Sodium cyanoborohydride (Aldrich 156159). The
reaction was
allowed to proceed overnight at room temperature. The following day the
reaction mixture
was diluted 49 times with the phosphate buffer, 25 mM at pH of 7.2, and
concentrated using
Amicon centrifuge tubes MWCO of 30,000. The final concentration of the protein
solution
to was obtained by measuring the absorbance at 280 nm and using a
coefficient of extinction for
the lysozyme of 2.61 The final concentration of lysozyme in the solution was
140 mg/mL.
Example 17: Effect of excipients on viscosity of PEGylated BSA with 1 PEG
chain per BSA
molecule
1001271 Formulations of PEGylated BSA (from Example 14 above) with excipients
were
prepared by adding 6 or 12 milligrams of the excipient salt to 0.3 mL of the
PEGylated BSA
solution. The solution was mixed by gently shaking and the viscosity was
measured by a
RheoSense microVisc equipped with an A10 channel (100 micron depth) at a shear
rate of
500 sec'. The viscometer measurements were completed at ambient temperature.
1001281 The results presented in Table 8 shows the effect of the added
excipient compounds
in reducing viscosity.
TABLE 8
Test Excipient
Concentration Viscosity Viscosity
Excipient
Number (mg/mL)
(cP) Reduction
17.1 None 0
228.6 0%
Alpha-Cyclodextrin
17.2 20 151.5 34%
sulfated Na salt
17.3 K acetate 40
89.5 60%
Example 18: Effect of excipients on viscosity of PEGylated BSA with multiple
PEG chains
per BSA molecule
1001291 A formulation of PEGylated BSA (from Example 15 above) with citric
acid Na salt
as excipient was prepared by adding 8 milligrams of the excipient salt to 0.2
mL of the
PEGylated BSA solution. The solution was mixed by gently shaking and the
viscosity was
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measured by a RheoSense microVisc equipped with an A10 channel (100 micron
depth) at a
shear rate of 500 sec* The viscometer measurements were completed at ambient
temperature. The results presented in Table 9 shows the effect of the added
excipient
compounds in reducing viscosity.
TABLE 9
Excipient
Test
Viscosity Viscosity
Excipient Added Concentration
Number
(mg/mL) (cP) Reduction
18.1 None 0
56.8 0%
18.2 Citric acid Na salt 40
43.5 23%
Example 19: Effect of excipients on viscosity of PEGylated lysozyme with
multiple PEG
chains per lysozyme molecule
to 1001301 A formulation of PEGylated lysozyme (from Example 16
above) with potassium
acetate as excipient was prepared by adding 6 milligrams of the excipient salt
to 0.3 mL of
the PEGylated lysozyme solution. The solution was mixed by gently shaking and
the
viscosity was measured by a RheoSense microVisc equipped with an A10 channel
(100
micron depth) at a shear rate of 500 sec* The viscometer measurements were
completed at
is ambient temperature. The results presented in the next table
shows the benefit of the added
excipient compounds in reducing viscosity.
TABLE 10
Excipient Viscosity Viscosity
Test Number Excipient
Concentration (mg/mL)
(cP) Reduction
19.1 None 0 24.6
0%
19.2 K acetate 20 22.6
8%
Example 20: Protein formulations containing excipient combinations
20 1001311 Formulations were prepared using an excipient
compound or a combination of two
excipient compounds and a test protein, where the test protein was intended to
simulate a
therapeutic protein that would be used in a therapeutic formulation. These
formulations were
prepared in 20 mNI histidine buffer with different excipient compounds for
viscosity
measurement in the following way. Excipient combinations were dissolved in 20
inNI
25 histidine (Sigma-Aldrich, St. Louis, MO) and the resulting
solution pH adjusted with small
amounts of concentrated sodium hydroxide or hydrochloric acid to achieve pH 6
prior to
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dissolution of the model protein. Excipient compounds for this Example are
listed below in
Table 11. Once excipient solutions had been prepared, the test protein (bovine
gamma
globulin (BGG) (Sigma-Aldrich, St. Louis, MO)) was dissolved at a ratio to
achieve a final
protein concentration of about 280 mg/mL. Solutions of BGG in the excipient
solutions were
formulated in 5 mL sterile polypropylene tubes and allowed to shake at 80-100
rpm on an
orbital shaker table overnight. The solutions were then transferred to 2 inL
microcentrifuge
tubes and centrifuged for about ten minutes at 2300 rpm in an IEC MicroMax
microcentrifuge to remove entrained air prior to viscosity measurement.
1001321 Viscosity measurements of formulations prepared as described above
were made
to with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,
Middleboro, MA).
The viscometer was equipped with a CP-40 cone and was operated at 3 rpm and 25
degrees
Centigrade. The formulation was loaded into the viscometer at a volume of 0.5
inL and
allowed to incubate at the given shear rate and temperature for 3 minutes,
followed by a
measurement collection period of twenty seconds. This was then followed by 2
additional
steps consisting of 1 minute of shear incubation and subsequent twenty second
measurement
collection period. The three data points collected were then averaged and
recorded as the
viscosity for the sample. Viscosities of solutions with excipient were
normalized to the
viscosity of the model protein solution without excipient, and the results are
shown in Table
11 below. The normalized viscosity is the ratio of the viscosity of the model
protein solution
with excipient to the viscosity of the model protein solution with no
excipient.
TABLE 11
Excipient A
Excipient B
Normalized
Test #
Name Conc. (mg/mL) Name Conc.
(mg/mL) Viscosity
20.1 None 0 None
0 1.00
20.2 Aspartame 10 None
0 0.83
20.3 Saccharin 60 None
0 0.51
20.4 Acesulfame K 80 None
0 0.44
20.5 Theophylline 10 None
0 0.84
20.6 Saccharin 30 None
0 0.58
20.7 Acesulfame K 40 None
0 0.61
20.8 Caffeine 15
Taurine 15 0.82
20.9 Caffeine 15
Tyramine 15 0.67
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Example 21: Protein formulations containing excipients to reduce viscosity and
injection
vain
1001331 Formulations were prepared using an excipient compound, a second
excipient
compound, and a test protein, where the test protein was intended to simulate
a therapeutic
protein that would be used in a therapeutic formulation. The first excipient
compound,
Excipient A, was selected from a group of compounds having local anesthetic
properties.
The first excipient, Excipient A and the second excipient, Excipient B are
listed in Table 12.
These formulations were prepared in 20 irtM histidine buffer using Excipient A
and Excipient
B in the following way, so that their viscosities could be measured.
Excipients in the amounts
to disclosed in Table 12 were dissolved in 20 mM histidine (Sigma-Aldrich,
St Louis, MO) and
the resulting solutions were adjusted with small
amounts of concentrated sodium
hydroxide or hydrochloric acid to achieve pH 6 prior to dissolution of the
model protein.
Once excipient solutions had been prepared, the test protein (bovine gamma
globulin (1360)
(Sigma-Aldrich, St. Louis, MO)) was dissolved in the excipient solution at a
ratio to achieve
a final protein concentration of about 280 mg/mL. Solutions of BOG in the
excipient
solutions were formulated in 5 inL sterile polypropylene tubes and allowed to
shake at 80-
100 rpm on an orbital shaker table overnight. BOG-excipient solutions were
then transferred
to 2 mL microcentrifuge Tubes and centrifuged for about ten minutes at 2300
rpm in an IEC
MicroMax microcentrifuge to remove entrained air prior to viscosity
measurement.
1001341 Viscosity measurements of the formulations prepared as described above
were made
with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,
Middleboro, MA).
The viscometer was equipped with a CP-40 cone and was operated at 3 rpm and 25
degrees
Centigrade. The formulation was loaded into the viscometer at a volume of 0.5
mt. and
allowed to incubate at the given shear rate and temperature for 3 minutes,
followed by a
measurement collection period of twenty seconds. This was then followed by 2
additional
steps consisting of 1 minute of shear incubation and subsequent twenty second
measurement
collection period. The three data points collected were then averaged and
recorded as the
viscosity for the sample. Viscosities of solutions with excipient were
normalized to the
viscosity of the model protein solution without excipient, and the results are
shown in Table
12 below. The normalized viscosity is the ratio of the viscosity of the model
protein solution
with excipient to the viscosity of the model protein solution with no
excipient.
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TABLE 12
Excipient A
Excipient B
Normalized
Test # Conc.
Conc.
Name Name
Viscosity
(mg/mL)
(mg/mL)
21.1 None 0 None
0 1.00
21.2 Lidocaine 45 None
0 0.73
21.3 Lidocaine 23 None
0 0.74
21.4 Lidocaine
10 Caffeine 15 0.71
21.5 Procaine HCI 40 None
0 0.64
21.6 Procaine MCI 20
Caffeine 15 0.69
Example 22: Formulations containing excipient compounds and PEG
1001351 Formulations were prepared using an excipient compound and PEG, where
the PEG
was intended to simulate a therapeutic PEGylated protein that would be used in
a therapeutic
formulation, and where the excipient compounds were provided in the amounts as
listed in
Table 13. These formulations were prepared by mixing equal volumes of a
solution of PEG
with a solution of the excipient. Both solutions were prepared in DI-Water,
1001361 The PEG solution was prepared by mixing 16.5 g of poly(ethylene oxide)
average
to Mw ¨100,000 (Aldrich Catalog # 181986) with 83.5 g of DI water. The
mixture was stiffed
overnight for complete dissolution.
1001371 The excipient solutions were prepared by this general method and as
detailed in
Table 13 below: An approximately 20 mg/mL solution of potassium phosphate
tribasic
(Aldrich Catalog # P5629) in DI-water was prepared by dissolving 0.05 g of
potassium
phosphate in 5 mL of DI-water. The PEG excipient solution was prepared by
nixing 0.5 mL
of the PEG solution with 0.5 mL of the excipient solution and mixed by using a
vortex for a
few seconds. A control sample was prepared by mixing 0.5 mL of the PEG
solution with 0.5
mL of DI-water. Viscosity was measured and results are recorded in Table 13
below.
TABLE 13
T Excipient
est
Viscosi
Excipient Concentration
Viscosity (cP) ty
Number
Reduction (%)
(mg/mL)
22.1 None 0
79.7 0
22.2 Citric acid Na salt 10
74.9 6.0
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Excipient
Test
Viscosity
Excipient Concentration
Viscosity (cP)
Number
Reduction (%)
(mg/mL)
Potassium
22.3 10 72.3 9.3
phosphate
Citric acid Na
22.4 salt/Potassium 10/10
69.1 13.3
phosphate
22.5 Sodium sulfate 10
75.1 5.8
Citric acid Na
22.6 10/10 70.4 11.7
salt/Sodium sulfate
Example 23: Improved processing of protein solutions with excipients
1001381 Two BGG solutions were prepared by mixing 0.25 g of solid BGG (Aldrich
catalogue number 65009) with 4 ml of a buffer solution. For Sample A: Buffer
solution was
20 mM histidine buffer (pH=6.0). For sample B: Buffer solution was 20 m1VI
histidine buffer
containing 15 ing/m1 of caffeine (pH=6). The dissolution of the solid BGG was
carried out by
placing the samples in an orbital shaker set at 100 rpm. The buffer sample
containing caffeine
excipient was observed to dissolve the protein faster. For the sample with the
caffeine
excipient (Sample B) complete dissolution of the BGG was achieved in 15
minutes. For the
to sample without the caffeine (Sample A) the dissolution needed 35
minutes.
11101391 Next the samples were placed in 2 separate Amicon Ultra 4 Centrifugal
Filter Unit
with a 30,000 molecular weight cut off and the samples were centrifuged at
2,500 rpm at 10
minute intervals. The filtrate volume recovered after each 10 minute
centrifuge run was
recorded. The results in Table 14 show the faster recovery of the filtrate for
Sample B. In
addition, Sample B kept concentrating with every additional run, but Sample A
reached a
maximum concentration point and further centrifugation did not result in
further sample
concentration.
TABLE 14
Centrifiige time
Sample A filtrate collected (mL) Sample B filtrate collected (ml.)
(min)
10 0.28
0.28
0.56 0.61
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Centrifuge time
Sample A filtrate collected (mL) Sample B filtrate collected (mL)
(min)
30 0.78
0.88
40 0.99
1.09
50 1.27
1.42
60 1.51
1.71
70 1.64
1.99
80 1.79
2.29
90 1.79
2.39
100 1.79
2.49
Example 24: Protein formulations containing multiple excipients
[00140] This example shows how the combination of caffeine and arginine as
excipients has
a beneficial effect on decreasing viscosity of a BGG solution. Four BGG
solutions were
prepared by mixing 0.18g of solid BGG (Aldrich catalogue number G5009) with
0.5 mL of a
20 inM Histidine buffer at pH 6. Each buffer solution contained different
excipient or
combination of excipients as described in the table below. The viscosity of
the solutions was
measured as described in previous examples. The results show that the hindered
amine
excipient, caffeine, can be combined with known excipients such as arginine,
and the
to combination has better viscosity reduction properties than the
individual excipients by
themselves.
TABLE 15
Sample Excipient(s) added
Viscosity Viscosity Reduction (%)
(cP)
A None
130.6 0
Caffeine (10 mg/m1)
87.9 33
Caffeine (10 mg/m1) / Arginine
(25 mg/m1)
66.1 49
Arginine (25 mg/m1)
76.7 41
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1001411 Arginine was added to 280 mg/mL solutions of BOG in histidine buffer
at pH 6. At
levels above 50 mg/mL, adding more arginine did not decrease viscosity
further, as shown in
Table 16.
TABLE 16
Arginine added (mg/mL) Viscosity
(cP) Viscosity reduction (%)
0 79.0
0%
53 40.9
48%
79 46.1
42%
105 47.8
40%
132 49.0
38%
158 48.0
39%
174 50.3
36%
211 51.4
35%
1001421 Caffeine was added to 280 mg/mL solutions of BGG in histidine buffer
at pH 6. At
levels above 10 mg/ml, adding more caffeine did not decrease viscosity
further, as shown in
Table 17.
TABLE 17
Caffeine added (mg/mL) Viscosity (cP) Viscosity reduction (%)
0 79
0%
60 31%
62 23%
22 50
45%
to
Example 25: Preparation of solutions of co-solutes in deionized water
1001431 Compounds used as co-solutes to increase caffeine solubility in water
were obtained
from Sigma-Aldrich (St. Louis, MO) and included niacinamide, proline, procaine
HC1,
ascorbic acid, 2,5-dihydroxybenzoic acid, lidocaine, saccharin, acesulfame K,
tyramine, and
15 aminobenzoic acid. Solutions of each co-solute were prepared
by dissolving dry solid in
deionized water and in some cases adjusting the pH to a value between pH of
about 6 and pH
of about 8 with 5 M hydrochloric acid or 5 M sodium hydroxide as necessary.
Solutions
were then diluted to a final volume of either 25 mL or 50 mL using a Class A
volumetric
flask and concentration recorded based on the mass of compound dissolved and
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volume of the solution. Prepared solutions were used either neat or diluted
with deionized
water.
Example 26: Caffeine solubility testing
1001441 The impact of different co-solutes on the solubility of caffeine at
ambient
temperature (about 23 C) was assessed in the following way. Dry caffeine
powder (Sigma-
Aldrich, St. Louis, MO) was added to 20 mL glass scintillation vials and the
mass of caffeine
recorded. 10 mL of a co-solute solution prepared in accordance with Example 25
was added
to the caffeine powder in certain cases (as recorded in Table 18); in other
cases (as recorded
in Table 18), a blend of a co-solute solution and deionized water was added to
the caffeine
to powder, maintaining a final addition volume of 10 mL. The volume
contribution of the dry
caffeine powder was assumed to be negligible in any of these mixtures. A small
magnetic
stir bar was added to the vial, and the solution was allowed to mix vigorously
on a stir plate
for about 10 minutes. After about 10 minutes the vial was observed for
dissolution of the dry
caffeine powder, and the results are given in Table 18 below. These
observations indicated
that niacinamide, procaine HC1, 2,5-dihydroxybenzoic acid sodium salt,
saccharin sodium
salt, and tyramine chloride salt all enabled dissolution of caffeine to at
least about four times
the reported caffeine solubility limit (-16 mg/rnL at room temperature
according to Sigma-
Aldrich).
TABLE 18
Co-solute
Caffeine
Test No.
Observation
Name
Conc. (mg/mL) (mg/mL)
26.1 Proline 100 50
DND
26.2 Niacinamide 100 50
CD
26.3 Niacinamide 100 60
CD
26.4 Niacinamide 100 75
CD
26.5 Niacinamide 100 85
CD
26.6 Niacinamide 100 100
CD
26.7 Niacinamide 80 85
CD
26.8 Niacinamide 50 80
CD
26.9 Procaine HC1 100 85
CD
26.10 Procaine HC1 50 80
CD
26.11 Niacinamide 30 80
DND
26.12 Procaine HC1 30 80
DND
26.13 Niacinamide 40 80
MD
26.14 Procaine HC1 40 80
DND
26.15 Ascorbic acid, Na 50 80
DND
26.16 Ascorbic acid, Na 100 80
DND
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Co-solute
Caffeine
Test No.
Observation
Name
Conc. (mg/mL) (mg/mL)
26.17 2,5 DHBA, Na 40 80
CD
26.18 2,5 DHBA, Na 20 80
MD
26.19 Lidocaine HC1 40 80
DND
26.20 Saccharin, Na 90 80
CD
26.21 Acesulfame K 80 80
DND
26.22 Tyrairiine HC1 60 80
CD
26.23 Na Aminobenzoate 46 80
DND
26.24 Saccharin, Na 45 80
DND
26.25 Tyramine HC1 30 80
DND
CD=completely dissolved; NED=mostly dissolved; DND=did not dissolve
Example 27: Impact of higher caffeine concentrations on protein formulations
1001451 Formulations were prepared using a primary excipient compound, a
secondary
excipient compound and a test protein, where the test protein was intended to
simulate a
therapeutic protein that would be used in a therapeutic formulation. The
primary excipient
compounds were selected from compounds having both low solubility and
demonstrated
viscosity reduction. The secondary excipient compounds were selected from
compounds
having higher solubility and either pyridine or benzene rings. Such
formulations were
to prepared in 20 mM histidine buffer with different excipient compounds
for viscosity
measurement in the following way: excipient combinations were dissolved in 20
triM
histidine buffer solution and the resulting solution pH adjusted with small
amounts of
concentrated sodium hydroxide or hydrochloric acid to achieve pH 6 prior to
dissolution of
the model protein. In certain experiments, the primary excipient was added in
amounts
greatly exceeding its room temperature solubility limit as reported in
literature. Once the
excipient solutions had been prepared, the test protein (bovine gamma globulin
(BGG,
Sigma-Aldrich, St. Louis, MO)) was dissolved at a ratio to achieve a final
protein
concentration of about 280 mg/inL. Solutions of BGG in the excipient solutions
were
formulated in 20 niL vials and allowed to shake at 100 rpm on an orbital
shaker table
overnight. The solutions were then transferred to 2 mL microcentrifuge tubes
and centrifuged
for ten minutes at 2300 rpm in an IEC MicroMax inicrocentrifuge to remove
entrained air
prior to viscosity measurement.
1001461 Viscosity measurements of formulations prepared as described above
were made
with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,
Middleboro, MA).
The viscometer was equipped with a CP40 cone and was operated at 3 rpm and 25
degrees
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Centigrade. The formulation was loaded into the viscometer at a volume of 0.5
inL and
allowed to incubate at the given shear rate and temperature for 3 minutes,
followed by a
measurement collection period of twenty seconds. This was then followed by 2
additional
steps consisting of 1 minute of shear incubation and subsequent twenty second
measurement
collection period. The three data points collected were then averaged and
recorded as the
viscosity for the sample, as shown in Table 19 below. Viscosities of solutions
with excipient
were normalized to the viscosity of the model protein solution without
excipient. The
normalized viscosity is the ratio of the viscosity of the model protein
solution with excipient
to the viscosity of the model protein solution with no excipient.
TABLE 19
T No. Primary Excipient
Secondary Excipient Normalized
est
Name Conc. (mg/mL)
Name (mg/mL) Viscosity
27.1 Caffeine 15 None 0
0.75
Primary Excipient
Secondary Excipient Normalized
Test No.
Name Conc. (mg/mL)
Name (mg/mL) Viscosity
27.2 Caffeine 15 Saccharin 14
0.62
27.3 Procaine
Caffeine 15 HC1
20 0.61
27.4 Caffeine 60 Niacinamide 50
0.61
27.5 Procaine
Caffeine 60
50 0.60
HC1
27.6 Caffeine 60 Saccharin 50
0.66
Example 28: Improved stability of adalimumab solutions with caffeine as
excipient
1001471 The stability of adalimumab solutions with and without caffeine
excipient was
evaluated after exposing samples to 2 different stress conditions: agitation
and freeze-thaw.
The adalimumab drug formulation HUMIRA was used, having properties described
in
more detail in Example 32. The HUMIRA sample was concentrated to 200 mg/ml
adalimumab concentration in the original buffer solution as described in
Example 32; this
concentrated sample is designated "Sample 1". A second sample was prepared
with ¨200
mg/mL of adalimumab and 15 mg/mL of added caffeine as described in Example 33;
this
concentrated sample with added caffeine is designated "Sample 2". Both samples
were
diluted to a final concentration of 1 mg/m1 adalimumab with the diluents as
follows: Sample
1 diluent is the original buffer solution, and Sample 2 diluent is a 20 mM
histidine, 15 mg/ml
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caffeine, pH=5. Both HUNIIRA dilutions were filtered through a 0.22 it
syringe filter. For
every diluted sample, 3 batches of 300 pl each were prepared in a 2 ml
Eppendorf tube in a
laminar flow hood. The samples were submitted to the following stress
conditions: for
agitation, samples were placed in an orbital shaker at 300 rpm for 91 hours;
for freeze-thaw,
samples were cycled 7 times from -17 to 30 C for an average of 6 hours per
condition.
Table 20 describes the samples prepared.
TABLE 20
Sample # Excipient added
Stress condition
1-C None
None
1-A None
Agitation
1-FT None
Freeze-Thaw
2-C 15 mg/mL caffeine
None
2-A 15 mg/mL caffeine
Agitation
2-FT 15 mg/mL caffeine
Freeze-Thaw
to 1001481 Evaluation of stability by Dynamic Light Scattering (DLS)
1001491 A Brookhaven Zeta Plus dynamic light scattering instrument was used to
measure
the hydrodynamic radius of the adalimumab molecules in the samples and to look
for
evidence of the formation of aggregate populations. Table 21 shows the DLS
results for the 6
samples described in Table 20; some of them (1-A, 1-FT, 2-A, and 2-FT) had
been exposed
to stress conditions (stressed Samples), and others (1-C and 2-C) had not been
stressed. In the
absence of caffeine as an excipient, the stressed Samples 1-A and 1-FT showed
higher
effective diameter than non-stressed Sample 1-C and in addition they show a
second
population of particles of significantly higher diameter; this new grouping of
particles with a
larger diameter is evidence of aggregation into subvisible particles. The
stressed samples
containing the caffeine (Samples 2-A and 2-FT) only display one population at
a particle
diameter similar to the unstressed Sample 2-C. These results demonstrate the
effect of adding
caffeine to reduce or minimize the formation of aggregates or subvisible
particles. Table 21
and Figures 1, 2, and 3 show the DLS data, where a multimodal particle size
distribution of
the monoclonal antibody is evident in stressed samples that do not contain
caffeine.
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TABLE 21
Effective Diameter of %
by Diameter of % by
Sample # Diameter
Population Intensity of Population Intensity of
(am) #1 (nm)
Population #1 #2 (nm) Population #2
1-C 10.9 10.8
100
1-A 11.5 10.8 87
28.9 13
1-FT 20.4 11.5 66
112.2 44
2-C 10.5 10.5
100 - -
2-A 10.8 10.8
100 - -
2-FT 11.4 11.4
100 - -
1001501 Tables 22A and Table 22B display the DLS raw data of adalimumab
samples
showing the particle size distributions. G(d) is the intensity- weighted
differential size
distribution. C(d) is the cumulative intensity-weighted differential size
distribution.
TABLE 22A
Sample 1-C
Sample 1-A Sample 1-FT
Diameter (3(d) C(d) Diameter C(d) Diameter
G (d) 6(d) C(d)
(nm) (nm)
(nm)
10.6 14 4 9.3 13
3 8.2 12 2
10.6 53 20 9.8 47
15 9.2 55 13
10.7 92 46 10.3 87
37 10.3 98 32
10.8 100 76 10.8
100 63 11.5 100 52
10.9 61 93 11.4 67
80 12.9 57 63
10.9 22 100 12
27 87 14.5 14 66
26.1 4 88 89.3 5 67
27.5 10 91 100.1 27 72
28.9 13 94 112.2 52 83
30.5 13 97 125.7 52 93
32.1 7 99 140.8 30 99
33.8 4 100 157.8 7 100
TABLE 22B
Sample 2-C Sample 2-A
Sample 2-FT
Diameter Diameter
Diameter
G (d) C(d) G (d) C(d) 6(d) C(d)
(nm) (nm)
(nm)
10.3 14 4 10.6 7
2 11.3 28 9
10.4 52 19 10.6 43
16 11.3 64 29
10.5 91 46 10.7 79
40 11.4 100 60
10.5 100 75 10.8 100
71 11.5 79 85
10.6 62 93 10.8 64
91 11.5 43 98
10.7 23 100 10.9 29
100 11.6 7 100
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Example 29: Evaluation of stability by size-exclusion chromatography (SEC)
1001511 SEC was used to detect any subvisible particulates of less than about
0.1 microns in
size from the stressed and unstressed adalimumab samples described above. A
TSKgel
SuperSW3000 column (Tosoh Biosciences, Montgomeryville, PA) with a guard
column was
used, and the elution was monitored at 280 nm. A total of 10 gl of each
stressed and
unstressed sample was eluted isocratically with a pH 6.2 buffer (100 m114
phosphate, 325 m114
NaC1), at a flow rate of 0.35 mUmin. The retention time of the adalimumab
monomer was
approximately 9 minutes. The data showed that samples containing caffeine as
an excipient
did not show any detectable aggregates. Also, the amount of monomer in all 3
samples
to remained constant.
Example 30: Viscosity reduction of HERCEPTIN
1001521 The monoclonal antibody trastuzumab (HERCEPTIN*)from Genentech) was
received as a lyophilized powder and reconstituted to 21 mg/mL in DI water.
The resulting
solution was concentrated as-is in an Amicon Ultra 4 centrifugal concentrator
tube (molecular
weight cut-off, 30 KDa) by centrifuging at 3500 rpm for 1.5 hrs. The
concentration was
measured by diluting the sample 200 times in an appropriate buffer and
measuring
absorbance at 280 nm using the extinction coefficient of 1.48 mL/mg. Viscosity
was
measured using a RheoSense microVisc viscometer.
1001531 Excipient buffers were prepared containing salicylic acid and caffeine
either alone
or in combination by dissolving histidine and excipients in distilled water,
then adjusting pH
to the appropriate level. The conditions of Buffer Systems 1 and 2 are
summarized in Table
23.
TABLE 23
Salicylic Acid
Caffeine Osmolality
Buffer System #
pH
concentration
concentration (mOsin/kg)
1 10 mg/mL 10
mg/mL 145 6
2 15
mg/mL 86 6
1001541 HERCEPTIN solutions were diluted in the excipient buffers at a ratio
of ¨1:10 and
concentrated in Amicon Ultra 15 (MWCO 30 KDa) concentrator tubes.
Concentration was
determined using a Bradford assay and compared with a standard calibration
curve made
from the stock HERCEPTIN sample. Viscosity was measured using the RheoSense
microVisc. The concentration and viscosity measurements of the various
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solutions are shown in Table 24 below, where Buffer System 1 and 2 refer to
those buffers
identified in Table 23.
TABLE 24
Solution with 10 mg/mL Caffeine
Solution with 15 mg/mL
Control solution with no added
+ 10 ing/rn1 Salicylic Acid added
Caffeine added (Buffer
excipients
(Buffer System 1)
System 2)
Antibody
Antibody Antibody
Viscosity Viscosity
Viscosity
Concentration Concentration
Concentration
(cP) (cP)
(cP)
(mg/mL)
(mg/mL) (mg/mL)
37.2 215 9.7
244 23.4 236
9.3 161 7.7 167
12.2 200
3.1 108 2.9 122
5.1 134
1.6 54 2.4 77
2.1 101
1001551 Buffer system 1, containing both salicylic acid and caffeine, had a
maximum
viscosity reduction of 76% at 215 mg/mL compared to the control sample. Buffer
system 2,
containing just caffeine, had viscosity reduction up to 59% at 200 mg/mL.
Example 31: Viscosity reduction of AVASTIN
1001561 AVASTIN (bevacizumab formulation marketed by Genentech) was received
as a
to 25 mg/ml solution in a histidine buffer. The sample was concentrated in
Amicon Ultra 4
centrifugal concentrator tubes (MWCO 30 1CDa) at 3500 rpm. Viscosity was
measured by
RheoSense microVisc and concentration was determined by absorbance at 280 nm
(extinction coefficient, 1.605 inL/mg). The excipient buffer was prepared by
adding 10
mg/mL caffeine along with 25 rnM histidine HCl. AVASTIN stock solution was
diluted
with the excipient buffer then concentrated in Amicon Ultra 15 centrifugal
concentrator tubes
(MWCO 30 I(Da). The concentration of the excipient samples was determined by
Bradford
assay and the viscosity was measured using the RheoSense microVisc. Results
are shown in
Table 25 below.
TABLE 25
Concentration Viscosity without Viscosity with
10 mg/mL % Viscosity Reduction
(mg/mL) added excipient (cP) added caffeine
excipient (cP) from Excipient
266 297
113 62%
213 80 22
73%
190 21
13 36%
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1001571 AVASTIN showed a maximum viscosity reduction of 73% when concentrated
with 10 mg/mL of caffeine to 213 mg/ml when compared to the control AVASTIN
sample.
Example 32: Profile of HUNEIRA
1001581 HUMIRA (AbbVie Inc_, Chicago, IL) is a commercially available
formulation of
the therapeutic monoclonal antibody adalimumab, a TNF-alpha blocker typically
prescribed
to reduce inflammatory responses of autoimrriune diseases such as rheumatoid
arthritis,
psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative
colitis, moderate to
severe chronic psoriasis and juvenile idiopathic arthritis. HUMIRA is sold in
0.8 mL single
use doses containing 40 mg of adalimumab, 4.93 mg sodium chloride, 0,69 mg
sodium
to phosphate monobasic dihydrate, 1.22 mg sodium phosphate dibasic
dihydrate, 0.24 mg
sodium citrate, 1.04 mg citric acid monohydrate, 9.6 mg mannitol and 0.8 mg
polysorbate-80.
A viscosity vs. concentration profile of this formulation was generated in the
following way.
An Amicon Ultra 15 centrifugal concentrator with a 30 kDa molecular weight cut-
off (EMD-
Millipore, Billerica, MA) was filled with about 15 mL of deionized water and
centrifuged in
a Sorvall Legend RT (Kendro Laboratory Products, Newtown, CT) at 4000 rpm for
10
minutes to rinse the membrane. Afterwards the residual water was removed and
2.4 mL of
HUMIRA liquid formulation was added to the concentrator tube and was
centrifuged at
4000 rpm for 60 minutes at 25 'C. Concentration of the retentate was
determined by diluting
10 microliters of retentate with 1990 microliters of deionized water,
measuring absorbance of
the diluted sample at 280 tun, and calculating the concentration using the
dilution factor and
extinction coefficient of 1.39 mL/mg-cm. Viscosity of the concentrated sample
was
measured with a microVisc viscometer equipped with an A05 chip (RheoSense, San
Ramon,
CA) at a shear rate of 250 sec-1 at 23 C. After viscosity measurement, the
sample was
diluted with a small amount of filtrate and concentration and viscosity
measurements were
repeated. This process was used to generate viscosity values at varying
adalimumab
concentrations, as set forth in Table 26 below.
TABLE 26
Adalimumab concentration (mg/mL)
Viscosity (cP)
277
125
253
63
223
34
202
20
182
13
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Example 33: Reformulation of HUMIRA with viscosity-reducing excipient
1001591 The following example describes a general process by which HUMIRA was
reformulated in buffer with viscosity-reducing excipient. A solution of the
viscosity-reducing
excipient was prepared in 20 inIVI histidine by dissolving about 0.15 g
histidine (Sigma-
Aldrich, St. Louis, MO) and 0.75 g caffeine (Sigma-Aldrich, St. Louis, MO) in
deionized
water. The pH of the resulting solution was adjusted to about 5 with 5 M
hydrochloric acid.
The solution was then diluted to a final volume of 50 mL in a volumetric flask
with deionized
water. The resulting buffered viscosity-reducing excipient solution was then
used to
reformulate HUMIRA at high InAb concentrations. Next, about 0.8 mL of HUMIRA
was
to added to a rinsed Atnicon Ultra 15 centrifugal concentrator tube with a
30 kDa molecular
weight cutoff and centrifuged in a Sorvall Legend RT at 4000 rpm and 25 C for
8-10
minutes. Afterwards about 14 mL of the buffered viscosity-reducing excipient
solution
prepared as described above was added to the concentrated HUMIRA in the
centrifugal
concentrator. After gentle mixing, the sample was centrifuged at 4000 rpm and
25 C for
about 40-60 minutes. The retentate was a concentrated sample of HUMIRA
reformulated in
a buffer with viscosity-reducing excipient. Viscosity and concentration of the
sample were
measured, and in some cases then diluted with a small amount of filtrate to
measure viscosity
at a lower concentration. Viscosity measurements were completed with a
microVisc
viscometer in the same way as with the concentrated HUMIRA formulation in the
previous
example. Concentrations were determined with a Bradford assay using a standard
curve
generated from HUMIRA stock solution diluted in deionized water.
Reformulation of
HUMIRA with the viscosity-reducing excipient gave viscosity reductions of 30%
to 60%
compared to the viscosity values of HUMIRA* concentrated in the commercial
buffer
without reformulation, as set forth in Table 27 below.
TABLE 27
Adalimumab concentration (mg/mL)
Viscosity (cP)
290
61
273
48
244
20
205
14
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Example 34: Preparation of formulations containing caffeine, a secondary
excipient and test
protein
[00160] Formulations were prepared using caffeine as the excipient compound or
a
combination of caffeine and a second excipient compound, and a test protein,
where the test
protein was intended to simulate a therapeutic protein that would be used in a
therapeutic
formulation. Such formulations were prepared in 20 mN1 histidine buffer with
different
excipient compounds for viscosity measurement in the following way. Excipient
combinations (Excipients A and B, as described in Table 28 below) were
dissolved in 20 mM
histidine (Sigma-Aldrich, St. Louis, MO) and the resulting solution pH
adjusted with small
to amounts of concentrated sodium hydroxide or hydrochloric acid to achieve
pH 6 prior to
dissolution of the model protein. Once excipient solutions had been prepared,
the test protein
(bovine gamma globulin (BGG) (Sigma-Aldrich, St. Louis, MO)) was dissolved at
a ratio to
achieve a final protein concentration of about 280 mg/mL. Solutions of BGG in
the excipient
solutions were formulated in 20 nal, glass scintillation vials and allowed to
shake at 80-100
rpm on an orbital shaker table overnight. The solutions were then transferred
to 2 mL
rnicrocentrifuge tubes and centrifuged for about ten minutes at 2300 rpm in an
IEC
MicroMax microcentrifuge to remove entrained air prior to viscosity
measurement.
[00161] Viscosity measurements of formulations prepared as described above
were made
with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,
Middleboro, MA).
The viscometer was equipped with a CP40 cone and was operated at 3 rpm and 25
degrees
Centigrade. The formulation was loaded into the viscometer at a volume of 0.5
inL and
allowed to incubate at the given shear rate and temperature for 3 minutes,
followed by a
measurement collection period of twenty seconds. This was then followed by 2
additional
steps consisting of 1 minute of shear incubation and subsequent twenty second
measurement
collection period. The three data points collected were then averaged and
recorded as the
viscosity for the sample. Viscosities of solutions with excipient were
normalized to the
viscosity of the model protein solution without excipient. The normalized
viscosity is the
ratio of the viscosity of the model protein solution with excipient to the
viscosity of the model
protein solution with no excipient.
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TABLE 28
Excipient A Excipient
B
Normalized Viscosity
Name Conc. (mg/mL) Name
Conc. (mg/mL)
0
0 1.00
Caffeine 15
0 0.77
Sodium
Caffeine 15
12 0.77
acetate
Sodium
Caffeine 15
14 0.78
sulfate
Caffeine 15 Aspartic acid
20 0.73
CaCl2
Caffeine 15
15 0.65
dihydrate
Dimethyl
Caffeine 15
25 0.65
Sulfone
Caffeine 15 Arginine
20 0.63
Caffeine 15 Leucine
20 0.69
Caffeine 15 Phenylalanine
20 0.60
Caffeine 15 Niacinamide
15 0.63
Caffeine 15 Ethanol
22 0.65
Example 35: Preparation of formulations containing dimethyl sulfone and test
protein
1001621 Formulations were prepared using dimethyl sulfone (Jarrow Formulas,
Los Angeles,
CA) as the excipient compound and a test protein, where the test protein was
intended to
simulate a therapeutic protein that would be used in a therapeutic
formulation. Such
formulations were prepared in 20 inM histidine buffer for viscosity
measurement in the
following way. Dimethyl sulfone was dissolved in 20 mM histidine (Sigma-
Aldrich, St.
to Louis, MO) and the resulting solution pH adjusted with small amounts of
concentrated
sodium hydroxide or hydrochloric acid to achieve pH 6 and then filtered
through a 0.22
micron filter prior to dissolution of the model protein. Once excipient
solutions had been
prepared, the test protein (bovine gamma globulin (BGG) from Sigma-Aldrich,
St. Louis,
MO) was dissolved at a concentration of about 280 mg/mL. Solutions of BGG in
the
excipient solutions were formulated in 20 mL glass scintillation vials and
allowed to shake at
80-100 rpm on an orbital shaker table overnight. The solutions were then
transferred to 2 niL
microcentrifuge tubes and centrifuged for about ten minutes at 2300 rpm in an
IEC
MicroMax microcentrifuge to remove entrained air prior to viscosity
measurement.
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1001631 Viscosity measurements of formulations prepared as described above
were made
with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,
Middleboro, MA).
The viscometer was equipped with a CP-40 cone and was operated at 3 rpm and 25
degrees
Centigrade. The formulation was loaded into the viscometer at a volume of 03
inL and
allowed to incubate at the given shear rate and temperature for 3 minutes,
followed by a
measurement collection period of twenty seconds. This was then followed by 2
additional
steps consisting of 1 minute of shear incubation and subsequent twenty second
measurement
collection period. The three data points collected were then averaged and
recorded as the
viscosity for the sample. Viscosities of solutions with excipient were
normalized to the
to viscosity of the model protein solution without excipient. The
normalized viscosity is the
ratio of the viscosity of the model protein solution with excipient to the
viscosity of the model
protein solution with no excipient.
TABLE 29
Dimethyl sulfone concentration (mg/mL)
Normalized viscosity
0
1.00
15 0.92
30 0.71
50 031
30 0.72
Example 36: Preparation of formulations containing tannic acid
1001641 In this Example, a high molecular weight poly(ethylene oxide) (PEG)
molecule is
used as a model compound to mimic the viscosity behavior of a PEGylated
protein. A
control sample (#36.1 in Table 30) was prepared by mixing PEG (Sigma-Aldrich,
Saint
Louis, MO) with viscosity averaged molecular weight (MV) of 1,000,000 with
deionized
(DI) water to make approximately 1.8 % by weight PEG solution in sterile 5 tnL
polypropylene centrifuge tubes. Two samples, #36.2 and 36.3, with tannic acid
(TA,
Spectrum Chemicals, New Brunswick, NJ) were prepared with TA:PEG mass ratios
of
approximately 1:100 and 1:1000 respectively by mixing the appropriate amounts
PEG, TA,
and 1 mM KC1 prepared in DI water. All samples were placed on a Daigger
Scientific
(Vernon Hills, IL) Labgenius orbital shaker at 200 rpm overnight. Viscosity
measurements
were made on a microVISC rheometer (RheoSense, San Ramon, CA) at 20 C and a
wall
shear rate of 250 s-t. After measurement, the viscosities were normalized to
the respective
PEG concentrations to account for the concentration dependence of the
viscosity. The PEG
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concentrations, TA concentrations, viscosities, normalized viscosities, and
viscosity
reductions for the samples 36.1, 36.2, and 36.3 are listed in Table 30 below.
The addition of
TA to concentrated PEG solutions substantially decreases the solution
viscosity by up to
approximately 40 %,
TABLE 30
PEG TA
% Reduction in
Viscosity Normalized viscosity
Sample concentration concentrationnormalized viscosity
(cP)
(Viscosity/ wt. % PEG)
(wt. %) (wt %)
from control
36.1 1.79 0 128
71.4 N/A
36.2 1.79 1.78 x 10-2 75.0
41.9 41.3
363 1.80 1.71 x 103 84.5
46.9 34.3
Example 37: Formulations with excipient dose profile
[00165] Formulations were prepared with different molar concentrations of
excipient and a
to test protein, where the test protein was intended to simulate a
therapeutic protein that would
be used in a therapeutic formulation. Such formulations were prepared in 20 mM
histidine
buffer for viscosity measurement in the following way. Stock solutions of 0
and 80 mM
caffeine excipient were prepared in 20 mM histidine (Sigma-Aldrich, St. Louis,
MO) and the
resulting solution pH adjusted with small amounts of concentrated sodium
hydroxide or
is hydrochloric acid to achieve pH 6 prior to dissolution of the model
protein. Additional
solutions at various caffeine concentrations were prepared by blending the two
stock
solutions at various volume ratios. Once excipient solutions had been
prepared, the test
protein (bovine gamma globulin (BOG, Sigma-Aldrich, St. Louis, MO)) was
dissolved at a
ratio to achieve a final protein concentration of about 280 mg/mL by adding
0.7 mL solution
20 to 0.25 g lyophilized protein powder. Solutions of BGG in the excipient
solutions were
formulated in 5 mL sterile polypropylene tubes and allowed to shake at 100 rpm
on an orbital
shaker table overnight to dissolve. The solutions were then transferred to 2
mL
microcentrifuge tubes and centrifuged for five minutes at 2400 rpm in an TEC
MicroMax
microcentrifuge to remove entrained air prior to viscosity measurement.
25 [00166] Viscosity measurements of formulations prepared as described
above were made
with a microVisc viscometer (RheoSense, San Ramon, CA). The viscometer was
equipped
with an A-10 chip having a channel depth of 100 microns and was operated at a
shear rate of
250 s-1 and 25 degrees Centigrade_ To measure viscosity, the formulation was
loaded into the
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viscometer, taking care to remove all air bubbles from the pipette. The
pipette with the loaded
sample formulation was placed in the instrument and allowed to incubate at the
measurement
temperature for five minutes. The instrument was then run until the channel
was fully
equilibrated with the test fluid, indicated by a stable viscosity reading, and
then the viscosity
recorded in centipoise. The results of these measurements are listed in Table
31.
TABLE 31
Normalized
Test # Excipient conc. (mM) Excipient conc. (mg/mL) Viscosity (cP)
.
Viscosity
37.1 20 39
77 0.92
37.2 50 9.7
65 0.78
37.3 10 1.9
70 0.84
37.4 5 1.0
67 0.81
37.5 30 5.8
63 0.76
37.6 40 7.8
65 0.78
37.7 0 0.0
83 1.00
37.8 70 13.6
50 0.60
37.9 80 15.5
50 0.60
37.10 60 11.7
57 0.69
Example 38: Preparation of formulations containing phenolic compounds
to 1001671 In this Example, a high molecular weight poly(ethylene oxide)
(PEG) molecule is
used as a model compound to mimic the viscosity behavior of a PEGylated
protein. A
control sample was prepared by mixing PEG (Sigma-Aldrich, Saint Louis, MO)
with a
viscosity averaged molecular weight (MV) of 1,000,000 with deionized (DI)
water to
approximately 1.8 % by weight PEG in sterile 5 mL polypropylene centrifuge
tubes. The
phenolic compounds gallic acid (GA, Sigma-Aldrich, Saint Louis, MO),
pyrogallol (PG,
Sigma-Aldrich, Saint Louis, MO) and resorcinol (R, Sigma-Aldrich, Saint Louis,
MO) were
tested as excipients as follows. Three PEG containing samples were prepared
with added
GA, PG and R at excipient:PEG mass ratios of approximately 3:50, 1:2, and 1:2
respectively
by mixing the appropriate amounts PEG, excipient, and DI water. Samples were
placed on a
Daigger Scientific (Vernon Hills, IL) Labgenius orbital shaker at 200 rpm
overnight.
Viscosity measurements were made on a microVISC rheometer (RheoSense, San
Ramon,
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CA) at 20 C and a wall shear rate of 250 s-1. After measurement, the
viscosities were
normalized to the respective PEG concentrations to account for the
concentration dependence
of the viscosity. The PEG concentrations, excipient concentrations,
viscosities, normalized
viscosities, and viscosity reductions for the control and excipient-containing
samples are
listed in Table 32 below.
TABLE 32
Normalized % Reduction
PEG Excipient
Sample Excipient
Viscosity viscosity in normalized
conc. conc. (wt.
fi added
(cP) (Viscosity/ viscosity from
(wt. %) %)
wt. % PEG)
control
38.1 1.80 None 0.000 134 74.5
N/A
38.2 1.80 GA 0.206 120 67.0
10.1
38.3 1.80 PG 0.870 106 59.2
19.6
38.4 1.80 R 1.00 113 63.1
15.3
Example 39: Preparation of formulations containing excipients having a
pyrimidine ring and
test protein
to 1001681 Formulations were prepared using an excipient compound having a
structure that
included one pyrimidine ring and a test protein, where the test protein was
intended to
simulate a therapeutic protein that would be used in a therapeutic
formulation. Such
formulations were prepared in 20 inM histidine buffer with different excipient
compounds in
the following way. Excipient compounds as listed in Table 33 were dissolved in
an aqueous
solution of 20 mNI histidine (Sigma-Aldrich, St Louis, MO) and the resulting
solution pH
was adjusted with small amounts of concentrated sodium hydroxide or
hydrochloric acid to
achieve pH 6 prior to dissolution of the model protein. Once the excipient
solutions had been
prepared, the test protein (bovine gamma globulin (BGG, Sigma-Aldrich, St.
Louis, MO))
was dissolved in each at a ratio to achieve a final protein concentration of
280 mg/mL.
Solutions of BOG in the excipient solutions were formulated in 20 rut glass
scintillation vials
and agitated at 100 rpm on an orbital shaker table overnight. The solutions
were then
transferred to 2 inL microcentrifuge tubes and centrifuged for ten minutes at
2300 rpm in an
IEC MicroMax microcentrifuge to remove entrained air prior to viscosity
measurement.
1001691 Viscosity measurements of formulations prepared as described above
were made
with a DV-IIT LV cone and plate viscometer (Brookfield Engineering,
Middleboro, MA).
The viscometer was equipped with a CP-40 cone and was operated at 3 rpm and 25
degrees
Centigrade. The formulation was loaded into the viscometer at a volume of 0.5
inL and
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allowed to incubate at the given shear rate and temperature for 3 minutes,
followed by a
measurement collection period of twenty seconds. This was then followed by 2
additional
steps consisting of 1 minute of shear incubation and subsequent twenty second
measurement
collection period. The three data points collected were then averaged and
recorded as the
viscosity for the sample. Viscosities of solutions with excipient were
normalized to the
viscosity of the model protein solution without excipient. The normalized
viscosity is the
ratio of the viscosity of the model protein solution with excipient to the
viscosity of the model
protein solution with no excipient. Results of these tests are shown in Table
33.
TABLE 33
Excipient
Excipient Normalized
Sample ft Excipient Added
MW (g/mol) Cone (mg/mL)
Viscosity
39.1 Pyrimidinone 96.1 14
0.73
39.2 1,3-Dimethyluracil 140.1 22
0.67
39.3 Tiiaminopyrimidine 125.1 20
0.87
39.4 Caffeine 194 15
0.77
39.5 Pyrimidinone 96.1 7
0.82
39.6 1,3-Dimethyluracil 140.1 11
0.76
39.7 Pyrimidine 80.1 13
0.78
39.8 Pyrimidine 80.1 6.5
0.87
39.9 None n/a 0
0.97
39.10 None n/a 0
1.00
39.11 None 0
0.95
39.12 Theacrine 224 15
0.73
39.13 Theacrine 224 15
0.77
to
Example 40: Preparation of formulations containing excipients and a PEG model
compound
1001701 Materials for this example included a linear PEG molecule with 20,000
molecular
weight, abbreviated as PEG-20K_ Formulations were prepared using excipient
compounds
having a structure including a conjugate acid of a weak base, and a PEG-20K
model
compound, where the PEG-20K model compound was intended to simulate a
PEGylated
protein.
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1001711 First, the viscosity profile of PEG-20K at different concentrations in
water and in 20
mM citrate buffer was determined as a baseline condition, as shown in Table 34
below with
n=3 replicate measurements per condition.
TABLE 34
PEG-20K (mg/mL) Water solution
20 mM citrate buffer solution
concentration viscosity (cP) SD
viscosity (cP) SD
240 80.64
0.67 83.99 1.08
180 35.20
0.49 35.64 0.66
210 54.81
0.61 58.25 1.08
150 2178
0.43 24.37 0.54
120 14.98
0.12 15.23 0.29
90 7.86
0.08 7.96 0.20
1001721 Next, excipient stock solutions were prepared at 1M concentration in
20 mM citrate
buffer. The pH of the stock solutions was adjusted to about 5. PEG-20K
solutions were
prepared by mixing 140 mL of 300 mg/mL PEG-20K stock with 20-60 pit of
excipient stock.
Depending on amount of excipient spiked in, the final sample volume was
adjusted to 200 pt
to by adding 40-0 L of citrate buffer. The blended solutions
containing the PEG-20K and the
excipients were all prepared with citrate buffer adjusted to pH 4.9-5.0, so
any weak base
excipients were converted to their conjugate acids in the buffer solution.
Viscosity of each
PEG-20K sample containing 210 mg/mL of the PEG-20K with different excipients
was
measured on MicroVISC for at least three times, and the results are summarized
in Table 35
is below.
TABLE 35
Excipient added to the 210 mg/mL PEG 20K solution
Viscosity (cP) SD
none (control)
59,3
NH4OH (0.3 M)
52.6 0.8
NH4OH (0.1 M)
54.4 0.5
Proline
55.6 1.7
trigonelline (0.1M)
56.2 3.5
imidazole (0.1 M)
56.5 0.9
NaF (0.1M)
56.9 1.2
NH4F (0,6M)
57.5 2,9
a-cyclodextrin sulfate K (2%)
57.6 0.2
glycerol phosphate
57.9 1.2
dicyclomine (0.05 M) 58.3 0.3
ammonium sulfate (0.1 M)
58.4 0.5
L-isoleucine
58.6 0.5
Boric acid (0,1 M)
58.8 0.5
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Excipient added to the 210 mg/mL PEG 20K solution
Viscosity (GP) SD
spennine
58.8 3.6
Alanine (0.3 M)
59.0 2_8
potassium acetate
59.1 0.2
urea (0.1 M)
59.4 2.0
ammonium acetate
59.5 0.5
Tris (0.1M)
59.8 1.3
NH4F (0.1M)
60.3 1.8
ZnC12 (0.1M)
60.6 0_3
Glycine (0.3 M)
60.9 1_3
Sorbitol (5%)
66.0 0.1
Example 41: Improving Tangential Flow Filtration using caffeine
1001731 400 inL of human gamma globulin (Octagam, Octapharma, USA) at a
concentration
of 35 mg/mL was prepared by diluting the stock at 100 mg/mL into phosphate
buffered saline
(PBS). The buffer was prepared by dissolving 1.8 mM KH2PO4, 10 mM Na2HPO4, 137
mM
NaC1, 2.7 mM KO in 1 L of Milli-Q water. A caffeine PBS solution was prepared
by
dissolving 50 inN1 caffeine, 1.8 mM IC1El2Pa4, 10 mM Na2HPO4, 137 mM NaCl, 2.7
mM KC1
in 1 L of Milli-Q water. Tap water was purified with a Direct-Q 3 UV
purification system
from EMD Millipore (Billerica., MA) to produce the DI water. The human gamma
globulin
to (HGG) solution was transferred to a reservoir of a Labscale tangential
flow filtration (TFF)
system (Millipore, Billerica, MA) equipped with 30 KDa MWCO Pellicon XL TFF
cassette
(Millipore, Billerica, MA). Prior to use, the cassette was flushed with Milli-
Q water followed
by PBS and a water permeability test was carried out to ensure membrane
integrity and
efficiency_ The HGG solution was pumped using a Quattroflow pump (Cole-
Partner, IL)
through the cassette with the retentate line going back to the sample
reservoir and the
permeate collected in a graduated measuring cylinder. A stirrer bar ensured
proper mixing of
the feed with the retentate. The feed pump was set to deliver 120 ma/min feed
to the cassette.
The retentate restrictor was used to get the transmembrane pressure (TMP)
roughly in the 20
to 30 psi range, and it was ensured that the TMP remained constant throughout
the run by
adjusting the feed pump and retentate restrictor. Data logging of the pressure
and flow rates
was carried out and samples taken every 30 minutes. To calculate the feed
concentration,
samples were analyzed by SE-FIPLC where 50 mg was loaded onto an Agilent 1100
HPLC
system fitted with TSKgel SuperSW3000 column (30 cm x 4.6 mm ID, Tosoh
Bioscience,
King of Prussia, PA) and Agilent G135 1B Diode array detector. PBS was used as
mobile
phase at a flow rate of 0.35 mL/min. The protein concentration was calculated
by integrating
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the area under the peaks. The feed concentration plotted as a function of time
was used to
compare TFF efficiency in presence of caffeine with the TFF using the control
system.
Higher percent concentration change from the initial feed concentration was
observed in a
shorter time with the caffeine as compared to the control, as shown in Table
36 below,
demonstrating increased TFF efficiency.
TABLE 36
Control system, Control
Caffeine system, Caffeine
Time (min) protein cone system,
protein cone system,
(mWmL) A, change
(mg/mL) % change
0 38_62 0
37.07 0
30 67.42 74.6
82.11 121.5
60 78.14
1023 120.63 225.4
90 101.94
163.9 141.88 282.7
120 140.81
264.6 198.96 436.7
150 162.39
320.4 222.50 500.2
180 226.06
485.3 286.68 673.3
210 254.76
559.6 305.39 723.8
240 281.58
629.0
270 291.99
656:0
Example 42: Using caffeine to improve purification yield from Protein A resin
to 1001741 Research-grade omalizumab, purchased from Bioceros (Utrecht, The
Netherlands)
at 15 mg/mL in 20 triM sodium phosphate, pH 7 buffer was used as test sample.
This protein
solution was filtered through a 0.2 pm polyethersulfone (PES) filter. The
filtered material
was mixed in a 1:1 ratio with a binding buffer that consisted of 20 niVI
sodium phosphate at
pH 7 in DI water. Tap water was purified with a Direct-Q 3 UV purification
system from
EMD Millipore (Billerica, MA) to produce the DI water. Protein-A purification
was
performed using a HiTrap Protein-A HP 1 mL column from GE Healthcare (Chicago,
IL). 10
mL of binding buffer was used for column equilibration, followed by loading 30
mg of
protein. The column was then washed with 5 mL of binding buffer to remove
unbound
protein. Bound omalizumab was eluted from the column in 1 mL fractions using
either 0_1 M
glycine buffer at pH 3.5 as the control buffer or by using 0.1 M glycine, 50
mM caffeine
buffer at pH 3.5. The control buffer was prepared by dissolving 7.5 g of
glycine into DI
water, adjusting the pH to 3.5 using 6M HC1 and adjusting the volume to 1 L.
Caffeine buffer
was prepared by dissolving 7.5 g of glycine and 10 g of caffeine into DI
water, adjusting the
pH to 3.5 using 6M MCI and adjusting the volume to 1 L. Five 1 mL fractions
were
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collected; these eluted fractions were labeled El, E2, E3, E4, and E5.
Finally, Protein-A was
regenerated by washing the column with 5 niL of 0.1 M glycine pH 3.0 buffer.
The flowrate
for each step was 1 mL/min, which was maintained by a Fusion 100 infusion pump
(Chemyx,
Stafford, TX). 10 mL NonnJect Luer Lok syringes were used (Henke Sass Wolf,
Tuttlingen,
Germany, reference number 4100-000V0).
[00175] The 5 eluted fractions, El, E2, E3, E4, and E5, were assayed for total
protein
content by high performance size-exclusion chromatography (SEC) analysis. SEC
analysis
was performed using a TSKgel SuperSW3000 column (30 cm x 4.6 mm ID, Tosoh
Bioscience, King of Prussia, PA) connected to an Agilent HP 1100 HPLC system.
PBS was
to used as mobile phase at a flow rate of 0.35 mL/min at room temperature.
The protein
concentration was monitored by absorbance at 280 nm using a G1315B diode array
detector.
The total amount of protein eluted from the Protein-A resin was determined by
integrating the
chromatograms, and about 8% increase in yield was observed in the presence of
caffeine, as
shown in Table 37 below.
TABLE 37
Protein concentration in fractions
Protein concentration in
Sampk
eluted without caffeine
fractions eluted with caffeine
Fraction
containing buffer (mg/mL)
containing buffer (mg/mL)
El 0.277
1.13
E2 2.14
6.72
E3 6.58
4.67
E4 2.24
1.40
E5 1.06
0.708
Total yield 12.29
14.63
% recovery 44.38
52.83
Example 43: Excipients for stabilization during low pH hold
1001761 Research-grade ipilimumab, purchased from Bioceros (Utrecht, The
Netherlands) at
15 ing/mL in 20 niM sodium phosphate, pH 7 buffer was used as test sample. The
protein
solution was filtered through a 0.2 pm polyethersulfone (PES) filter.
Raffinose pentahydrate
was obtained from Sigma (Si Louis, MO). The excipient stock was prepared by
dissolving the
raffinose pentahydrate at a concentration of 1 M in 0A5 M glycine buffer, pH
175. The
buffer was prepared by dissolving 7.5 g of glycine in 0.9 L Milli-Q water,
adjusting the pH to
2.75 using 1 M HO, and making the volume to 0.1 L. One control formulation and
three
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excipient-containing formulations were prepared by adding the excipient to the
ipilimumab
solution, with a final excipient concentration of 0 rnNI, 100 mM, 200 m.M and
400 inNI and a
final ipilimumab concentration of 2 mg/mL. The samples were incubated
overnight at the
acidic pH (2.75) for 24 h and the samples were analyzed by SE-HPLC where 50 mg
was
loaded onto an Agilent 1100 HPLC system fitted with TSKgel SuperSW3000 column
(30 cm
x4,6 mm ID, Tosoh Bioscience, King of Prussia, PA) and Agilent G135 1B diode
array
detector. PBS was used as mobile phase at a flow rate of 0.35 mL/min. The
monomer protein
(ipilimumab) concentration was calculated by integrating the areas under the
monomer peak.
The monomer fraction from an untreated sample not exposed to the low pH was
normalized
to to 100 % and the monomeric fractions of the treated samples expressed as
percentage change
of this untreated sample. The results in Table 38 below, show that the
presence of raffinose in
the samples resulted in a higher percentage monomeric form of the ipilimumab
after the low
pH hold.
TABLE 38
Sample % of ipilimumab
in the monomeric form
0 triM raffinose
38.69
100 mM raffinose
52.68
200 mM raffinose
63.07
400 mM raffinose
78.88
Untreated
100
Example 44: Buffer and excipient preparation
1001771 A stock 20 inM histidine hydrochloride (His HC1) buffer was prepared
for use in
formulating excipients and protein buffer exchange. Two liters of His HCI was
prepared by
dissolving 6.206 grams of histidine (Sigma-Aldrich, St Louis, MO) in Type 1
ultrapure
water. The solution of dissolved histidine was titrated to pH 6.0 using
concentrated
hydrochloric acid. The His HO solution was then brought up to 2 liters using a
volumetric
flask and filtered through a 0.2 gm membrane bottle-top filter device (Sigma
Aldrich, St.
Louis, MO). Excipients to be tested in Example 46 (listed in Table 39) were
prepared as
excipient solutions for subsequent testing as follows. Each excipient was were
prepared at
10X (1 M) by dissolving it in this His HC1 buffer described above and
adjusting the pH with
concentrated sodium hydroxide or concentrated hydrochloric acid. Each
excipient solution
was then filtered using 0.2 pm membrane filter.
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Example 45: Protein solution preparation
10411781 Two test proteins, purified omalizumab purchased from Bioceros (The
Netherlands)
and human serum derived polyclonal IgG (Octagam 10%), were buffer exchanged
into His
HCl (as prepared in Example 44) using 20 kDa molecular weight cut-off dialysis
cassettes
(Fisher Scientific). Each protein solution was transferred into the dialysis
cassette attached to
a buoy and placed in a flask for buffer exchanges. A total of 3 buffer
exchanges were
performed into > 50x the starting protein volume. Upon the final buffer
exchange step, the
protein solution was removed from the dialysis cassette and filtered through
0.2 p.m
membrane filter and protein concentration was measured via A280 by diluting
100-fold into
to His HCl buffer. 100 pL was then transferred to a UV clear 96 half-well
microplate (Greiner
Bio-One, Austria), and absorbance measured at a wavelength of 280 nm with a
Synergy HT
plate reader (BioTek, Winooski, VT). The blanked, pathlength corrected A280
measurement
was then divided by the respective extinction coefficient and multiplied by
the dilution factor
to determine the protein concentration. A subsequent concentration step was
needed to
concentrate the protein in preparation for dynamic light scattering (DLS)
viscosity
measurements in Example 46. Concentration was performed using Amicon-15
centrifugal
devices with a 30 kDa molecular weight cut-off (EMD Millipore, Billerica, MA)
and
concentrated to 175 mg/mL based on tetentate mass in the centrifugal device by
centrifuging
at 4000 x g on a benchtop centrifuge (Sorvall Legend RT).
Example 46: DLS measurement of the diffusion interaction parameter
[00179] In this Example, the diffusion interaction parameter (kD) of a dilute
protein solution
was measured by DLS in the presence of 0.1 M excipient solution. The
excipients being
tested are listed in Table 39 For each excipient, a 0.2M solution of the
excipient was prepared
separately from the previously prepared 1 M excipient stock. The kD was
measured by DLS
using 5 different concentrations of omalizumab (prepared as described in
Example 45)
ranging from 10 mg/mL to 0.6 mg/mL in the presence of 0.1M excipient. An
identical set of
control samples was prepared, containing the same concentrations of omalizumab
in the
absence of any excipient. For each test sample, 20 pL of protein solution was
combined with
20 pL of 0.2 M excipient solution (1:1 mixture) onto a 384-well plate (Aurora
Microplates,
Whitefish, MT). After loading the samples, the well plate was shaken on a
plate shaker to
mix the contents for 5 minutes. Upon mixing, the well plate was centrifuged at
400 x g in a
Sorvall Legend RT for 1 minute to force out any air pockets. The well plate
was then loaded
into a DynaPro II DLS plate reader (Wyatt Technologies Corp., Goleta, CA) and
the
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diffusion coefficient of each sample was measured at 25 C. For each excipient,
the measured
diffusion coefficient was plotted as a function of protein concentration, and
the slope of the
linear fit of the data was recorded as the Ica In this example, each
measurement was
normalized to a control average and reported as a percent to the control.
These results are
shown in Table 39 below.
TABLE 39
% change in kD value from
Excipients Protein
control
3-(1-Pyridinio)-1-propanesulfonate Omalizumab 22%
Aspartic Acid
Omalizumab 72%
Ornithine
Omalizumab 49%
beta-alanine
Omalizumab 25%
Lysine Omalizumab 47%
Trigonelline
Omalizumab 28%
(3-carboxypropyl)
Omalizumab 59%
trimethylammoniurn chloride
Aminohippuric acid
Omalizumab 20%
Arginine
Omalizumab 81%
1-Hexy1-3-methylimidazolium
Omalizumab 25%
chloride
Naa. (200 inM)
Omalizumab 70%
ethanolamine HCL
Omalizumab 25%
Spennidine
Omalizumab 54%
4-arninopyridine
Omalizumab 25%
Lysine Omalizumab 66%
cysteamine HCl
Omalizumab 38%
x-xylylenediamine
Omalizumab 56%
nicotinic acid
Omalizumab 18%
quinic acid
Omalizumab 19%
1,3-diaminopropane
Omalizumab 62%
lactobionic acid
Omalizumab 47%
Glutamic acid
Omalizumab 35%
Sodium Ascorbate
Omalizumab 35%
sodium propionate
Omalizumab 33%
Quinic acid
Omalizumab 27%
sodium benzoate
Omalizumab 33%
Glucuronic acid
Omalizumab 32%
Hydroxybenzoic acid
Omalizumab 50%
sodium bisulfite
Omalizumab 57%
Salicylic acid
Omalizumab 43%
Etidronate
Omalizumab 56%
Acesulfame K+ salt
Omalizumab 49%
Calcium propionate
Omalizumab 83%
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% change in Id) value from
Excipients Protein
control
Citric acid Omalizumab
47%
hydroquinone sulfonic acid Omalizumab
36%
Menadione sodium bisulfite Omalizumab
25%
2-dimethylaminoethanol Omalizumab
31%
2-methyl-2-imidazoline Omalizumab
17%
cycloserine Omalizumab
9%
3-aminopyridine Omalizumab
6%
4-aminopyridine Omalizumab
23%
agmatine sulfate Omalizumab
69%
cytidine Omalizumab
29%
ethanolamine Omalizumab
29%
meglumine Omalizumab
171%
morpholine Omalizumab
17%
triethanolamine Omalizumab
40%
Example 47: Viscosity measurement by DLS
1001801 Purified omalizumab purchased from Bioceros (The Netherlands) and
human serum
derived polyclonal IgG (Octagam 10%, Pfizer) were used as model protein
systems to
explore viscosity effects of excipients. Concentrated stock solutions of
excipients (listed in
Tables 40 and 41) were prepared at 10X (1M) in His HC1 buffer, following the
protocol
described in Example 44. Omalizumab was buffer exchanged using Amicon-15
centrifugal
(30 kDa MWCO) devices into His HCI buffer and concentrated to 175 mg/mL based
on
retentate mass in the centrifugal device. Excipient and concentrated protein
were combined in
to a 200 tiL PCR tube, adding 1-part 10X excipient and 9 parts protein. An
additional 2 pL of a
5-fold diluted solution of polyethylene glycol surface modified gold
nanoparticles
(nanoComposix, San Diego, CA) was added to each PCR tube and mixed thoroughly
by
inversion. A control sample was prepared identically, except without adding
any excipient.
Each sample (test samples and the control) was transferred to a 384-well
microplate (Aurora
Microplates, Whitefish, MT) in duplicate (25 pia per well) and centrifuged at
400 x g for 1
minute before analysis. A DynaPro II DLS plate reader (Wyatt Technology Corp.,
Goleta,
CA) was used to measure apparent particle size of gold nanoparticles at 25 C.
The ratio of
the apparent particle size of the gold nanoparticle to the known panicle size
of the gold
nanoparticle in water was used to determine the viscosity of the protein
formulation
according to the Stokes-Einstein equation. In this Example, each measurement
was
normalized to a control average and reported as a percent reduction compared
with the
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control, and standard deviation is shown, and the results are shown in Tables
40 and 41
below.
TABLE 40
Excipient (100 mM) Protein % Reduction Std. dev.
Dimethyluracil hIgG
36.5% 1.5%
Hordenine hIgG 33.7% 0.7%
acesulfame K hIgG
32.6% 2.5%
Nicotinamide hIgG
26.8% 2.1%
Arginine higG
21.8% 3.0%
Aspartame hIgG 20.0% 2A%
Saccharin hIgG 17.5% 3.7%
3-(1-Pyridinio)-1-propanesulionate hIgG
15.0% 1.9%
Caffeine hIgG
19.8% 7.8%
Imidazole hIgG 8.7% 3.3%
Tyramine higG 3.6% 4.4%
Control hIgG
0.0% 2.8%
Dimethylglycine higG
2.5% 10.0%
4-aminopyridine hIgG
29.7% 2.9%
nicotinamide/caffeine hIgG 30.0% 1.0%
nicotinamide/caffeine hIgG 33.4% 3.5%
hordenine HCI hIgG
32.9% 5.5%
Dimethyluracil/arginine hIgG
22.2% 0.6%
Jeffamine M600 higG
16.5% 2.1%
Dimethyluracil hIgG
18.1% 9.4%
diethylnicotinamide hIgG
11.1% 2.8%
arginine HC1 hIgG
14.6% 6.9%
arginine/glutamic acid hIgG
17.5% 11.2%
nicotinamide hIgG
12.1% 5.8%
serine/theonine hIgG
5.8% 5.1%
isonicotinamide hIgG
8.8% 8.5%
(3-carboxylpropyl) trimethyl
hIgG
5.2% 12.1%
ammonium chloride
TABLE 41
Excipient (100 mM)
Protein A Reduction Std. dev.
4-(2-hydroxyethyl)-1-
omalizumab
71% 1.3%
piperazineethanesulfonic aci d
0-(octylphosphoryl)choline
omalizumab 38% 0.1%
Nicotinamide mononucleotide
ornalizurnab 61% 8%
Itaconic acid omalizumab 54% 1%
3-(1-Pyridinio)-1-propanesulfonate
omalizumab 4% 0.2%
N-methyl aspartic acid omalizumab 73% 0.6%
L-Ornithine omalizumab 82% 0.0%
CA 03159377 2022-5-25
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WO 2021/108427
PCT/US2020/062051
Excipient (100 mM)
Protein A. Reduction Std. dev.
2-dimethylaminoethanol
omalizumab 65% 10.6%
2-methyl-2-imidazoline
omalizumab 49% 12,6%
cycloserine
omalizumab 33% 1.2%
3-aminopyridine
omalizumab 59% 4.0%
4-aminopyridine
omalizumab 73% 2.3%
agmatine sulfate
omalizumab 85% 1.8%
cytidine
omalizumab 68% 1.6%
diphenhydramine
omalizumab 81% 0.1%
ethanolamine
omalizumab 93% 1.6%
meglumine
omalizumab 91% 0.2%
rnoipholine
omalizumab 78% 2.7%
Example 48: Viscosity measurements by viscometer
1001811 Excipient solutions for those excipients listed in Tables 42 and 43
were prepared at
0.1 M or 0.075 M in His HO buffer and pH adjusted using concentrated sodium
hydroxide or
concentrated hydrochloric acid. Ornalizumab and human IgG were buffer
exchanged into
each excipient formulation using Amicon-15 centrifugal devices (30 kDa MWCO).
After
buffer exchange, the protein solution was concentrated up to 150 mg/mL for
omalizumab and
250 mg/tnL for human IgG based on retentate mass in the centrifugal device.
Control
formulations were prepared in identical manner except in the absence of the
excipient.
to
Viscosity measurements were performed on a
RheoSense micro-viscometer using an A05
chip enclosed in a temperature-controlled enclosure set to 25 C. The shear
rate was set to 250
The viscosity of each formulation was measured 3 times and then diluted by
adding 20 tit
of the respective buffer and viscosity was measured again. This was repeated 5-
6 times each
to generate viscosity data for 5-6 different protein concentrations. Protein
concentration was
measured by absorbance at 280 tun using an Agilent 1100 series high pressure
liquid
chromatography instrument paired with a size exclusion column (TOSOH TSKgel
SuperSW3000). A scatter plot was generated by plotting viscosity as a function
of
concentration for each excipient formulation. An exponential trendline was
fitted to each
formulation and the viscosity at a concentration was calculated based on the
exponential fit
with the equation y = a*etbsio, where y is viscosity in cP units, x is
concentration of protein in
mg/mL, a and b are fitting parameters for the equation, and R2 is the
statistical coefficient of
determination. For this example, the viscosity is reported as a function of a
fixed
concentration and results are given in Tables 42 and 43 below.
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TABLE 42
Exponential Equation Calculations
Excipient Protein Buffer
Cale. Viscosity
a
R2
@ 250 mg/mL
Caffeine human IgG His HC1 pH 5.5 0.469 0.0187
0.9194 503
nicotinamide human IgG His HC1 pH 5.5 0.6136 0.0198
0.9329 86.6
hordenine HC1 human IgG His HC1 pH 5.5 0.4116 0.0197
09535 56.7
1,3-dimethyluracil human IgG His HC1 pH 5.5 0.0586 0.0282 0.9929
67.6
control human IgG His HC1 pH 5.5 0.2059 0.0248
0.9995 101.5
TABLE 43
Exponential Equation
Excipient Protein Buffer
a Calc. Viscosity
R2
@120 mg/mL
Sulfanilic Acid omalizumab His HC1 pH 6.0 1.2194 0.0185
0.8218 11.2
Nicotinic acid omalizumab His HC1 pH 6.0 1.0171 0.0244
0.8041 19.0
Omithine omalizumab His HCI pH 6.0 0.1068 0.0419
0.6874 16.3
control omalizumab His HC1 pH 6.0 0.8156 0.0319
0.9763 37.5
1,3 diaminopropane ornalizumab His HC1 pH 6.0 0.1498 0.0274 0.9835
4.0
Example 49: BLI measurement of self-interaction
1001821 In this example, biolayer interferometry (BLI) tests were done with a
ForteBio Octet
Red-96 instrument. Amine reactive second-generation (AR2G) biosensors
(Molecular
Devices, CA) were conjugated with omalizumab to detect protein self-
interaction in the
presence of excipients. Excipient solutions for the excipients listed in Table
44 were prepared
to at 0.1 M in His HC1 buffer. 20 mIVI sodium phosphate, pH 6.4 buffer was
prepared by
dissolving 1.679 g of dibasic sodium phosphate, heptahydrate (Sigma, St.
Louis) and 1.895 g
of monobasic sodium phosphate, monohydrate (Sigma, St. Louis) in DI water and
adjusting
the volume to 1 L. Research-grade omalizumab, purchased from Bioceros
(Utrecht, The
Netherlands) was buffer exchanged using Amicon-15 centrifugal (30 kDa MWCO)
devices
into phosphate buffer at pH 6.4. This omalizumab stock solution at 15 mg/mL in
20 inM
sodium phosphate, pH 6.4 buffer was further buffer exchanged using Sephadex G-
25 PD-10
desalting columns (GE Healthcare Life Sciences) and eluted with the 20 InM
sodium
phosphate, pH 6.4 buffers containing the prepared excipient at 0.1 M. The
control was
similarly prepared by using Sephadex G-25 PD-10 desalting columns (GE
Healthcare Life
Sciences) and eluted with the 20 inM sodium phosphate, pH 6.4 buffer. Protein
concentration
was measured using UV clear 96 half-well microplate (Greiner Bio-One,
Austria), and
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absorbance measured at a wavelength of 280 nm with a Synergy FIT plate reader
(BioTek,
Winooski, VT). Protein concentration was adjusted to 5 mg/mL by diluting in
the prepared
excipient buffers. In a black bottom 96-well microplate (Greiner Rio-One,
Austria), 250 pt
of each excipient solution at 0.1 M in 20 mM sodium phosphate, pH 6.4 buffer
was
transferred to column B and 250 pa, of the 5 mg/ml omalizumab solution
containing 0.1 M
excipients was transferred to column C. The 96-well plate was set up so the
columns
represented individual formulations and rows distinguished protein-containing
formulations.
The tray was then transferred into a ForteBio Octet Red-96 for analysis. The
omalizumab
conjugated biosensors were dipped into the formulations containing no protein
for 120
to seconds to generate a baseline. Biosensors were then removed and dipped
into formulations
containing protein for 300 seconds. In this example, we reported the delta in
a percent of
binding signal at 300 seconds compared to the binding signal of the control,
and the results
are summarized in Table 44 below.
TABLE 44
Excipient Binding (nm) at
300 s % change
Control 7
0%
Omithine 2.5
64%
iminodiacetic acid 1.25
82%
nicotinic acid 0.5
93%
sulfanilic acid 0.3
96%
EQUIVALENTS
1001831 While specific embodiments of the subject invention have been
disclosed herein, the
above specification is illustrative and not restrictive. While this invention
has been
particularly shown and described with references to preferred embodiments
thereof, it will be
understood by those skilled in the art that various changes in form and
details may be made
therein without departing from the scope of the invention encompassed by the
appended
claims. Many variations of the invention will become apparent to those of
skilled art upon
review of this specification. Unless otherwise indicated, all numbers
expressing reaction
conditions, quantities of ingredients, and so forth, as used in this
specification and the claims
are to be understood as being modified in all instances by the term "about."
Accordingly,
unless indicated to the contrary, the numerical parameters set forth herein
are approximations
that can vary depending upon the desired properties sought to be obtained by
the present
invention.
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