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CA 02564791 2006-10-27
WO 2005/123131 PCT/US2005/020792
HIGH PRESSURE SPRAY-DRY OF BIOACTIVE MATERIALS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and benefit of a prior U.S.
Provisional
Application number 60/579,850, High Pressure Spray-Dry of Antibodies, by Vu
Truong-Le,
et al., filed June 14, 2004. This application is a continuation in part of
prior U.S. Utility
Patent Application 10/738,971, "High Pressure Spray-Dry of Bioactive
Materials", by Vu
Truong-Le, et al., filed December 16, 2003, and a prior U.S. Provisional
Application
number 60/434,377, "High Pressure Spray-Dry of Bioactive Materials", by Vu
Truong-Le,
et al., filed December 17, 2002. The full disclosure of the prior applications
is incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The invention is in the field of spray-dry particle formation and
preservation
of bioactive materials. The present invention provides, e.g., formulations for
high pressure
spray drying of bioactive materials, such as peptides, polypeptides, proteins,
viruses,
bacteria, antibodies, cells, liposomes, vaccines and/or the like. High
pressure spraying
allows fine spray droplets to be dried, e.g., in a shorter time, at a lower
temperature, with
less concomitant degradation of sensitive molecules. Formulations of bioactive
material for
high pressure spraying include, for example, the bioactive material, amino
acids and sugars.
High pressure spraying produces powder particles wherein the incorporated
bioactive
material can be more readily reconstituted at higher concentrations. The
present invention
provides methods and systems to precisely control spray droplet size and
powder particle
size by adjustment of process variables.
BACKGROUND OF THE INVENTION
[0003] Methods to preserve biologic materials in storage have a long history,
from
the preservation of food to the preservation of modern pharmaceutical
compositions.
Biological materials have been dried, salted, frozen, cryoprotected, spray
dried, and freeze-
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dried. Optimal methods of preservation can depend on the acceptable degree of
degradation, the desired storage time, and the nature of the biological
material.
[0004] For centuries, food has been preserved for later consumption by drying.
Food harvested in times of plenty was laid out in the sun to remove excess
water. Drying
can malce the food unsuitable for growth of spoilage bacteria and fungi.
Autolytic
processes, in which plant and animal tissues self destruct, can also be
prevented by drying.
Salting food can provide a similar preservative effect. Dried and salted food
usually
experiences a loss of fresh appearance and nutritional value. Drying and
salting bioactive
materials, such as enzymes and pharmaceuticals, can destroy activity by heat,
oxidation,
water removal, production of radicals and peroxides, photobleaching, and the
like, that
denature the material.
[0005] Spray drying has been used in food processing and pharmaceutical
production with some advantages over salting or slow drying. Water can be
quickly
removed by spraying a fine mist of the dissolved biological molecules into a
stream of hot
gasses. The dried particles can have a large surface to volume ratio for
speedy
reconstitution with aqueous buffers. In Platz et al., U.S. Patent number
6,165,463,
"Dispersible Antibody Compositions and Methods for Their Preparation and Use",
for
example, dry powder particles are prepared by spray drying for inhaled
administration of
pharmaceuticals to patients. The biological molecules, in a dilute solution,
are sprayed at
moderate pressures (e.g., 80 psi) into a stream of hot gasses (e.g., 98-
105°C) for primary
drying, then the particles are further dehydrated by prolonged exposure to
high temperatures
(e.g., 67°C). Although such processes are suitable for food and rugged
biomolecules,
sensitive molecules can be denatured, or otherwise inactivated, by the stress,
long drying
periods, and high temperatures of these methods.
[0006] Freezing can be an effective way to preserve biological molecules. Cold
temperatures can slow degradation reaction kinetics. Freezing can reduce the
availability of
water to degradation reactions and contaminant microbes. Ice can reduce
oxidation of the
molecules by blocl~ing contact with air. However, freezing can have negative
effects such
as concentration of salts that can denature proteins in the unfrozen zones of
solution, or the
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formation of sharp ice crystals that can pierce cell structures. Some of the
damage caused
by freezing can be mitigated by the addition of cryoprotectants which prevent
denaturation
by lowering the freezing temperature and inhibiting formation of ice crystals.
Even in cases
where freezing and thawing degradation can be avoided, continuous operation of
refrigeration equipment can make preservation by storage in a freezer
inconvenient and
expensive.
[0007] Freeze-drying processes have many of the benefits of freezing and
drying.
Degradation is suspended by freezing then water removal makes the product more
stable for
storage. Drying by sublimation of the frozen water into a vacuum can avoid the
high heat
of some spray drying processes. The lyophilized product can be quite stable in
storage even
at room temperatures. However, the molecules can still experience denaturing
salt
concentrations during the freezing step. In addition, many freeze-drying
protocols call for
prolonged secondary drying steps at high temperatures to reduce moisture
content. Bulky
calves of lyophilized material can be slow to reconstitute and must be finely
ground for
delivery by inhalation.
[0008] A need remains for compositions and methods to prepare stable particles
containing bioactive materials without loss of purity due to excessive heat,
chemical, or
shear stress. It would be desirable to have formulations for high pressure
spray drying of
bioactive materials that would enhance the stability and reduce reconstitution
time for
resultant powder particles. The present invention provides these and other
features that will
become apparent upon review of the following.
SUMMARY OF THE INVENTION
[0009] The present invention provides, e.g., methods to prepare stable
compositions
of bioactive materials including, but not limited to, peptides, polypeptides,
proteins, viruses,
bacteria, antibodies, cells, liposomes, vaccines and/or the like with low
process
denaturation. Methods of preparing powder particles, e.g., by spray drying
viscous
solutions at high pressures reduce shear stress and heat stress degradation.
The invention
provides adjustments in process parameters to precisely tune the size of
sprayed droplets
and dried powder particles. Stability and shelf life are increased for the
powder particles
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high pressure spray dried from formulations having suitable concentrations of
sugars and
amino acids. Powder particles sprayed from such formulations can be
reconstituted without
undue aggregation and quickly reconstituted to high concentrations.
[0010] Preferred formulations for spray drying of bioactive materials by
methods of
the invention include, for example, amino acids and sugars. The amino acids
can act, e.g.,
as zwitterions, antioxidants, buffers, stabilizers, bulking agents,
solubilizers, and/or the like,
to improve qualities of the powder particle product. The sugars can act, e.g.,
as viscosity
enhancing agents, stabilizers, bulking agents, solubilizing agents, and/or the
like. In one
aspect of the invention, the formulation for spray drying therapeutic
bioactive material
includes from about 4% to about 10% by weight of the therapeutic bioactive
material, from
about 0.1 mM to about 50 mM total of one or more amino acids, from about 0.5%
to about
4% by weight of a sugar, and water. Optionally, the bioactive material is a
virus present in
the liquid formulation at from about 103 TCID5o/mL to about 1012 TCIDSO/mL.
The
formulation can optionally include, e.g., surfactants and polymers. A
preferred formulation
comprises about 8% by weight of the bioactive material, about 10 mM histidine
(pH 6.0),
about 0.5% arginine, and about 2% sucrose.
[0011] Antibody bioactive materials in certain formulations are typically
monoclonal antibodies that can act as therapeutic agents on administration to
a patient. The
antibody is often an IgG. Specifically, antibodies to be used in the invention
include, but
are not limited to, synthetic antibodies, polyclonal antibodies, monoclonal
antibodies,
recombinantly produced antibodies, multispecific antibodies (including bi-
specific
antibodies), human antibodies, humanized antibodies, chimeric antibodies,
intrabodies,
single-chain Fvs (scFv) (e.g., including monospecific and bi-specific, etc.),
Fab fragments,
F(ab') fragments, disulfide-linlced Fvs (sdFv), anti-idiotypic (anti-Id)
antibodies, and
epitope-binding fragments, and antibodies conjugated to toxins. In many
embodiments, the
formulation comprises about 8% of the antibody by weight.
[0012] Preferred amino acids for bioactive material formulations of the
invention
include, e.g., glycine, histidine and arginine. In a preferred embodiment, the
amino acids
include from about 1 mM to about 20 mM histidine or from about 0.1% to about
2% of
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arginine by weight. In a more preferred embodiment, the one or more amino
acids comprise
about 10 mM histidine and about 30 mM arginine.
[0013] Preferred sugars for bioactive material formulations include, e.g.,
sucrose,
trehalose, and mannitol. Many of the formulations include about 2% of the
sugar by
weight.
[0014] Other excipients in bioactive material formulations include polymers
and
surfactants. For example the formulation can include from about 0.01% to about
0.2%
polyoxyethylenesorbitan monooleates or polyethylene glycol sorbitan
monolaurates. The
formulations can include, e.g., from about 0.5% to about 0.05% polyvinyl
pyrrolidone
(PVP).
[0015] In methods of spraying bioactive materials, the formulations are spray
dried
using high pressures to form fine dry powder particles. Such powder particles
can typically
be quicl~ly reconstituted to a concentration of about 200 mg bioactive
material per ml or
more while retaining a purity and activity substantially unchanged from the
pre-dried
formulation.
[0016] Methods of the invention for high pressure spray drying bioactive
materials
include preparing an aqueous formulation containing the bioactive material, a
sugar and an
amino acid; spraying the formulation at high pressure; and drying the spray
droplets. For
example, the methods can include preparing powder particles of an bioactive
material by
preparing an aqueous suspension or solution comprising: from about 4% to about
10% by
weight of the bioactive material, and preferably having about 8% of the
bioactive material
by weight, from about 0.1 mM to about 50 mM total of one or more amino acids,
and from
about 0.5% to about 4% by weight of a sugar; spraying the suspension or
solution through a
nozzle at a high pressure to form a mist of fine droplets; drying the droplets
to form powder
particles; and, recovering the particles. Optionally, the bioactive material
is a virus present
in the liquid formulation at from about 103 TCIDSO/mL to about 1012 TCIDSO/mL.
In
preferred embodiments, the bioactive material comprises a peptide sequence of
any of SEQ
ID NOs. 1 to 20, or a conservative variation thereof, the sugar is sucrose,
and the drying
CA 02564791 2006-10-27
WO 2005/123131 PCT/US2005/020792
chamber drying gas outlet temperature ranges from about 40°C to about
60°C during drying
of the particles.
[0017] In certain embodiments of the methods, antibody formulations are
sprayed.
The antibody can be any type of antibody, such as defined above, and the like.
Preferred
antibodies for high pressure spray drying methods of the invention include,
but are not
limited to, e.g.,: anti-RSV, anti-hMPV, anti-avb3 integrin, anti-avb5
integrin, anti-alpha
IIb/beta 3 integrin, anti-alpha 4 integrin, anti-EphA2, and anti-EphA4, anti-
EphB4, anti-IL9,
anti-IL4, anti-IL5, anti-IL13, anti-IL15, anti-CTLA4, anti-PSA, anti-PSMA,
anti-CEA, anti-
cMET, anti-CSa, anti-TGF-beta, anti-HMGB-1, anti-interferons alpha and anti-
interferon
alpha receptor, anti-IFN beta and gamma, anti-chitinase, anti-TIRC7, anti-T-
cell, MT-103
BiTE~, anti-EpCam, anti-Her2/neu, anti-IgE, anti-TNF-alpha, anti-VEGF, anti-
EGF and
anti-EGF receptor, anti-CD22, anti-CD19, anti-Fc, anti-LTA, anti-Flk-1, and
anti-Tie-1. In
particular, antibodies for production of powder particles by the methods
include antibodies
having the any of the peptide sequences of SEQ ID NOs. 1 to 20.
[0018] Process parameters of the methods can be adjusted to obtain powder
particles
with desired characteristics. Favored spray pressures for the formulation
and/or pressurized
atomization gas ranging from about 800 psi to about 1800 psi, or about 1300
psi. Favored
formulation spray droplets range in diameter from about 3 ~.m to about 30 Vim.
It is
preferred to spray the formulation into a particle formation vessel that acts
as, or is in fluid
connection with, a drying chamber. The drying chamber can have a drying gas
inlet and an
outlet. Preferred drying conditions include a drying gas outlet temperature
during particle
formation comprises a temperature ranging from about 40°C to about
60°C. Preferred
powder particle average diameters range from about 2 ~m to about 10 ~.m, e.g.,
for rapid
reconstitution or pulmonary administration.
[0019] The dried powder particles containing bioactive materials can be
reconstituted from the powder particles, e.g., by addition of water to provide
a solution or
suspension containing about 200 mg of the antibodies per milliliter. The
reconstituted
solution or suspension can be administered, e.g., to a human patient by
subcutaneous
injection to treat a disease state.
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[0020] The methods of preparing stable particles can also include, e.g.,
preparing an
aqueous suspension or solution (formulation) with a bioactive material and a
viscosity
enhancing agent, spraying the formulation through a nozzle at high pressure to
form a mist
of fine droplets, drying the droplets to form powder particles, and recovering
the particles.
The viscosity enhancing agent can be present in a concentration, e.g.,
sufficient to provide a
5% or more viscosity increase, or a 0.05 centipoise or more viscosity
increase, over the
formulation without viscosity enhancing agent.
[0021] The bioactive materials of the method can include peptides,
polypeptides,
proteins, viruses, bacteria, antibodies, cells, liposomes, and/or the lilce.
For example, the
bioactive material can be present in the process formulation at a
concentration ranging from
about 1 mg/ml to about 200 mg/ml, from about 5 mg/ml to about 80 mg/ml, or
about 50
mg/ml. Optionally, the bioactive material can be, e.g., a virus present in the
formulation in
a titer ranging from about 2 log FFU (focus forming units)/ml to 12 log
FFU/ml, or about 8
log FFU/ml.
[0022] The viscosity enhancing agents can be, e.g., a polyol and/or a polymer.
For
example, the polyol can be trehalose, sucrose, sorbose, melezitose, glycerol,
fructose,
mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, palactose,
glucose,
mannitol, xylitol, erythritol, threitol, sorbitol, raffinose, and/or the like.
Exemplary polymer
viscosity enhancing agents can include starch, starch derivatives,
carboxymethyl starch,
hydroxyethyl starch (HES), dextran, dextrin, polyvinyl pyrrolidone (PVP),
human serum
albumin (HSA), inulin, gelatin, and/or the like. The viscosity enhancing
agents of the
invention can be present in the formulation, e.g., an amount ranging from
about 0.1 weight
percent to about 20 weight percent, 2 weight percent to 8 weight percent, or 6
weight
percent. Optionally, the viscosity enhancing agent can be present in a
concentration, e.g.,
sufficient to provide a 50%, a 0.05 centipoise, or a 100 centipoise increase
in viscosity, or
more.
[0023] The solution or suspension of the method can include a surfactant
and/or a
zwitterion. Surfactants in the method can include, e.g., polyethylene glycol
sorbitan
monolaurates (e.g., Tween 80), polyoxyethylenesorbitan monooleates (e.g.,
Tween 20), or
bloclc polymers of polyethylene and polypropylene glycol (e.g., Pluronic F68),
and/or the
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like. Zwitterions of the method can include, e.g., arginine, histidine,
glycine, and/or the
like. The average size of sprayed droplets can be adjusted by varying the
concentration of
surface active agents in the formulation, e.g., preferably in the presence of
sucrose.
[0024] High pressure spraying through nozzles in the method can include, e.g.,
high
pressure spraying of liquid, atomization with a high pressure gas, and/or
spraying into a
cold fluid. Spraying can be by high pressure nitrogen gas atomization. The
nozzle can have
an internal diameter ranging, e.g., from about 50 ~m to about 500 Vim, from
about 75 ~m to
about 150 ~,m, or the nozzle orifice can have an internal diameter of about
100 [um. The
high pressure spraying nozzle can be an atomizing nozzle with channels for a
high pressure
atomizing gas, e.g., to enhance dispersal of the sprayed droplets. The high
pressure
atomizing gas, such as nitrogen, can have a pressure and/or temperature at
least 10% or 15%
away from a critical point for the gas.
[0025] The method of the invention can include, e.g., spray freeze-drying the
suspension and/or solution droplets. The fine droplets can be, e.g., immersed
in a cold fluid
to freeze the droplets. The cold fluid can be, e.g., gaseous or liquid argon,
helium, carbon
dioxide, and/or nitrogen. The cold fluid can range in temperature, e.g., from
about
-80°C to about -200°C. The droplets can be dried, e.g., by
applying a vacuum and raising
the temperature of the environment around the droplets to form powder
particles (e.g.,
freeze dried). The vacuum can be a gas pressure less than about 200 Torr or
less than about
Torn.
[0026] Solutions or suspensions can be sprayed at high pressure to create a
fine mist
of droplets. The high pressure can be, e.g., between about 200 psi and about
2500 psi,
between about 1000 psi and 1500 psi, or about 1300 psi. The fine mist can
include droplets
with an average diameter between about 2 ~,m and about 200 Vim, between about
3 hum and
about 70 ~,m, between about 5 hum and about 30 hum, or about 10 Vim.
[0027] Droplets can be dried to form powder particles, e.g., by displacement
of the
gas from the fine mist with a drying gas to remove water vapor and spray
gasses. The
drying gas can be, e.g., a substantially inert gas, such as nitrogen at a
temperature between
about 25°C and about 99°C, about 35°C and about
65°C, or about 55°C. The powder
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WO 2005/123131 PCT/US2005/020792
particles of the invention can have an average size ranging from about 0.1 ~.m
to about 100
~,m, or from about 2 ~,m to about 10 ~.m.
[0028] The method of the invention can provide a high process yield without
significant reduction in product purity. For example, the method can have a
process yield
(e.g., specific activity retention) ranging from about 40 percent to about 98
percent, or about
90 percent. The product purity of a protein bioactive material can remain high
through
spraying, e.g., with less than about 5 percent, 4 percent 3 percent, 2
percent, or less total
aggregates and fragments on reconstitution of the powder particles. The
product purity or
specific activity of a protein bioactive material or viability of a virus
bioactive material can
be substantially the same before and after the drying of droplets.
[0029] Powder particles can be used, e.g., to administer the bioactive
material
according to the methods of the invention. The powder particles can be
delivered to a
mammal by inhalation through the nasal and/or pulmonary route. Alternately,
the powder
particles can be reconstituted with an aqueous buffer for delivery of the
bioactive material
by injection. Powder particles of the method can be reconstituted into a
formulation of
bioactive material at a concentration ranging, e.g., from about 1 mg/ml to
about 400 mg/ml,
or 5 mg/ml to about 200 mg/ml. Substantially isotonic (an osmolality within
about 10% of
physiological values) reconstituted material can comprise antibodies at a
concentration of
about 200 mg/rnl.
[0030] Compositions of the invention can be, e.g., stable powder particles
readily
reconstituted to solutions of highly pure bioactive materials at high
concentrations.
Compositions of the invention can be, e.g., particles containing a bioactive
material made
by the process of preparing an aqueous formulation with the bioactive material
and a
viscosity enhancing agent, spraying the formulation through a nozzle at high
pressure to
form a mist of fine droplets, drying the droplets to form powder particles,
and recovering
the particles. The viscosity enhancing agent can be present, e.g., at a
concentration
adequate to provide a 5% or more increase in viscosity, or a 0.5 centipoise
increase in
viscosity, over the suspension of solution without the viscosity enhancing
agents.
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[0031] The bioactive materials can be peptides, polypeptides, proteins,
viruses,
bacteria, antibodies, cells, liposomes and/or the like. Bioactive materials
can be present in
the process formulation at a concentration ranging, e.g., from about 1 mg/ml
to about 200
mglml, about 5 mg/ml to about 80 mg/ml, or about 50 mg/ml. Viral bioactive
materials,
such as influenza virus, can be present in formulations at a titer ranging
from about 2 log
FFU/ml to about 12 log FFIJ/ml, or about 8 log FFU/ml. In the powder particle
product, the
bioactive material can be present, e.g., in the powder particles in an amount
ranging from
about 0.1 weight percent or less to about 80 weight percent.
[0032] In one exemplary embodiment, the bioactive material of the composition
can
be present in the process formulation in an amount ranging from about 0.5
weight percent
to about 20 weight percent, or about 8 weight percent. The viscosity enhancing
agent of the
composition can include, e.g., a polyol, such as sucrose or trehalose, or a
polymer, such as
hydroxyethyl starch (HES), dextran, dextrin, inulin, or polyvinyl pyrrolidone
(PVP). The
sucrose can be present in the formulation in an amount ranging from about 1
weight percent
to about 10 weight percent, or about 6 weight percent. The aqueous formulation
can contain
antibodies in combination with arginine and sucrose. Optionally, the viscosity
enhancing
agents can include PVP.
[0033] A composition containing a bioactive material can be, e.g., powder
particles
with a ratio of excipients (other total solids on drying) to the bioactive
material ranging
from about 1/100 to about 100/1, about 2/3 to about 3/2, or about 1/1. The
bioactive
material composition of powder particles can incorporate, e.g., sucrose in an
amount
ranging from about 30 weight percent to about 60 weight percent. The powder
particles can
contain less than about 5 percent moisture.
[0034] The bioactive material in powder particles can be quite stable, e.g.,
with less
than about 3% aggregates on reconstitution of the powder particles after
storage at about
4°C for 1 year, 5 years, or about 7 years. Bioactive materials dried to
powder particles
using formulations and methods of the invention can have, e.g., less than
about 3%
aggregates on reconstitution of the powder particles after storage at about
25°C for 0.1
years, 0.5 years, 1 year, or about 1.5 years, or more.
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[0035] The bioactive material compositions of the invention can be
reconstituted
powder particles. For example, an aqueous buffer can be added to the powder
particles to
form a reconstituted formulation of bioactive material. Such a solution can
be, e.g.,
substantially similar to the formulation sprayed in the process. Optionally,
the powder
particles can be reconstituted with appropriate buffers to provide desired
characteristics
such as isotonicity and/or high bioactive material concentrations. The
reconstituted solution
or suspension of the bioactive material can have, e.g., a concentration
ranging from less
than about 0.1 mg/ml to about 500 mg/ml. In a preferred embodiment, the powder
particles
can be reconstituted in 10 minutes or less, e.g., to a concentration of
bioactive material of
about 200 mg/ml. In another prefeiTed embodiment, the powder particles can be
reconstituted to a substantially isotonic formulation containing a bioactive
material
concentration of up to about 200 mg/ml.
[0036] A composition of reconstituted bioactive material can comprise a 50
mg/ml
to 500 mg/ml solution, or more, with less than about 3 percent aggregates or
fragments. In a
preferred embodiment, the bioactive materials are reconstituted at a
concentration of 200
mg/ml or more. Such compositions can be manufactured by the process of
preparing an
aqueous formulation of the bioactive material with a viscosity enhancing
agent, spraying the
formulation through a nozzle at high pressure to form a mist of fine droplets,
drying the
droplets to form powder particles, recovering the particles, and
reconstituting the particles
in an aqueous solution. The composition can be prepared from a formulation
increased in
viscosity with the viscosity enhancing agent by 50%, 0.05 centipoise, or more.
The
formulations for spraying drying particles of bioactive material will
typically include, e.g.,
significant amounts of amino acids and sugars.
[0037] The compositions of the invention can include, e.g., a polyol and/or
polymer
viscosity enhancing agents. The polyols of the compositions can be, e.g.,
trehalose, sucrose,
sorbose, melezitose, glycerol, fructose, mannose, maltose, lactose, arabinose,
xylose, ribose,
rhamnose, palactose, glucose, mannitol, xylitol, erythritol, threitol,
sorbitol, raffinose,
and/or the like. The polymers of the compositions can be, e.g., starch, starch
derivatives,
carboxymethyl starch, inulin, hydroxyethyl starch (HES), dextran, dextrin,
polyvinyl .
pyrrolidone (PVP), human serum albumin (HSA), gelatin, and/or the like. The
formulation
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in the process of malting the compositions can have viscosity enhancing
agents, e.g., in an
amount between about 0.1 weight percent and about 20 weight percent, or about
5 weight
percent.
[0038] The aqueous solution or suspension sprayed in the process of the
composition can include, e.g., zwitterions, such as arginine, histidine,
glycine, and/or the
like. Arginine can be present in the process formulation in an amount, e.g.,
between about
0.1 weight percent to about 5 weight percent, or about 0.5 weight percent. In
a preferred
embodiment, the compositions of the invention are prepared from formulations
containing
sucrose at concentrations ranging from about 0.4% to about 4% and arginine at
concentrations ranging from about 0.1% to about 0.5%.
[0039] The aqueous solution or suspension sprayed in the process of the
composition can include, e.g., a surfactant. The surfactant can be, e.g.,
polyethylene glycol
sorbitan monolaurates, polyoxyethylenesorbitan monooleates, block polymers of
polyethylene and polypropylene glycol, e.g., Tween 80, Tween 20, Pluronic F68,
and/or the
like.
[0040] The present invention provides processes of making compositions by high
pressure spraying, e.g., with atomizing high pressure nitrogen gas, andlor
into a cold fluid.
The process for preparing the composition can provide, e.g., immersion of the
fine droplets
in a cold fluid, thereby freezing the droplets, followed by drying the frozen
droplets by
applying a vacuum and raising the temperature of the droplets.
[0041] Powder particles of the composition can vary, e.g., in average particle
diameter (size), formula, and component proportions. For example, the average
size of the
powder particles can range from about 0.1 ~,m to about 100 ~,m, or from about
2 ~,m to
about 10 Vim. The powder particles can contain sucrose in an amount ranging,
e.g., from
about 20 weight percent to about 60 weight percent, or about 40 weight
percent. The
powder particle composition can contain arginine ranging in concentration from
about 1%
to about 20% by weight, or about 5% by weight. The composition of powder
particles can
contain PVP ranging in concentration from about 0% to about 5%, or about 0.05%
to about
0.5% by weight.
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[0042] The size of spray droplets can be controlled in systems and methods of
the
invention by adjusting one or more parameters. For example, the size of
droplets or
particles can be controlled by adjusting the percent surface active agent in
the formulation,
adjusting a spraying pressure, adjusting an atomizing gas pressure, adjusting
a viscosity,
adjusting the total solids in the formulation, adjusting a flow rate of the
formulation,
adjusting a mass flow ratio (formulation flow to atomizing gas flow),
adjusting a
temperature of the formulation, and/or the like.
[0043] Compositions of the invention include, e.g., dry powder particles with
an
average particle size ranging from about 2 ~m to about 200 [gym, a particle
density of about
1, and 40 weight percent to about 60 weight percent bioactive materials with
more than
about 90 percent purity (non-aggregated and non-fragmented). In preferred
embodiments
the particle size is less than 10 hum and the bioactive material purity is 97%
or more. The
composition of dry particles can be stable with, e.g., bioactive materials
less than about 3%
aggregated on reconstitution of the powder particles after storage at about
4°C for about 1
year to about 7 years. The composition of powder particles on reconstitution
after storage at
about 25°C for about 0.1 years to about 1.5 years can have, e.g., less
than about 3%
aggregates. Such powder particle compositions can include, e.g., about 40
weight percent
to about 60 weight percent sucrose or trehalose, and/or arginine.
[0044] In a preferred composition of the invention, particles containing a
virus are
prepared by: preparing an aqueous formulation suspension or solution
containing the virus
and sucrose, spraying the suspension or solution through a nozzle at high
pressure to form a
mist of fine droplets, drying the droplets to form powder particles, and
recovering the
particles. The presence of the viscosity enhancing agent in the suspension can
increase
viscosity by 50%, 0.05 centipoise, or more. High pressure spraying can be by
atomization
with a gas at temperatures and pressures at least 10% away from a critical
point for the gas.
The virus can include influenza virus. Using the methods and formulations of
the invention,
viability of the virus is not reduced significantly in the recovered
particles.
13
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DEFINITIONS
[0045] Before describing the present invention in detail, it is to be
understood that
this invention is not limited to particular described methods or biological
materials, which
can, of course, vary. It is also to be understood that the terminology used
herein is for the
purpose of describing particular embodiments only, and is not intended to be
limiting. As
used in this specification and the appended claims, the singular forms "a",
"an" and "the"
can include plural referents unless the context indicates otherwise. Thus, for
example,
reference to "a polyol" can include a combination of two or more polyols;
reference to
"sugars" can include mixtures of one or more sugars, and the like.
[0046] Unless defined otherwise, all technical and scientific terms used
herein have
the same meaning as commonly understood by one of ordinary skill in the art to
which the
invention pertains. Although any methods and materials similar or equivalent
to those
described herein can be used in the practice for testing of the present
invention, the
preferred materials and methods are described herein. In describing and
claiming the
present invention, the following terminology will be used in accordance with
the definitions
set out below.
[0047] The term "particle size", as used herein, generally refers to the
average
physical diameter of particles.
[0048] The term "specific activity", in the context of bioactive materials of
the
invention refers to the bioactivity (determinable, e.g., by an appropriate
bioassay) relative to
the amount of agent. A highly pure, undenatured bioactive material can have,
e.g., a high
specific activity. A denatured bioactive material can have a low specific
activity. A highly
pure bioactive material can be low in fragments, dimers, trimers, and
aggregates, as
measured, e.g., by a size exclusion chromatography.
[0049] The term "high pressure spraying", as used herein, refers to spraying a
formulation fed through an orifice at a pressure greater than used for
standard spray dryers.
High pressures can be, e.g., greater than about 200 psi. Preferred high
pressure spraying
pressures range from about 1000 psi to about 2000 psi. High pressure spraying
can include,
e.g., pressurization and/or atomization of the formulation with a gas at a
pressure more than
14
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WO 2005/123131 PCT/US2005/020792
10% away, or more than 15% away, from a critical pressure (at a given
temperature) and/or
from a critical temperature (at a given pressure) for the gas.
[0050] The term "viscosity enhancing agent", as used herein, refers to
molecular
species in the formulations of the invention that significantly increase the
viscosity of the
formulation. For example, a molecular species can be a viscosity enhancing
agent in a
formulation in an amount that substantially increases the viscosity of the
formulation and
significantly reduces shear stress denaturation of proteins sprayed in the
formulation.
Preferred viscosity enhancing agents include, e.g., polyols, polymers, sugars,
and
polysaccharides.
[0051] The term "bioactive materials", as used herein, refers to peptides,
polypeptides, proteins, viruses, bacteria, antibodies, cells, liposomes,
vaccines and/or the
like, or as commonly referred to by those of shill in the art.
[0052] The term "therapeutic bioactive material" is a bioactive material, as
defined
above, which is suitably formulated to be administered to a human or animal
subject in need
of a therapy provided by the bioactivity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] Figure 1 shows a chart comparing droplet size versus mass flow ratio
(MFR)
for solutions sprayed at high pressure versus solutions sprayed at lower
pressures.
[0054] Figure 2 shows a chart presenting critical temperature and pressure
points of
phase transition for a gas.
[0055] Figure 3 shows chart of droplet size versus atomization pressure for a
solution containing viscosity enhancing agents and/or surface active agents.
[0056] Figures 4A and 4B show charts of dry powder particle size versus mass
flow
ratio and atomization pressure, respectively.
[0057] Figure 5 is a schematic diagram of an exemplary high pressure spray
nozzles.
[0058] Figure 6 shows a chart of droplet size versus liquid feed rate for
combinations of pressures and atomizing nozzle orifice internal diameters.
CA 02564791 2006-10-27
WO 2005/123131 PCT/US2005/020792
[0059] Figure 7 shows chromatographic charts indicating the viscosity
enhancing
agent prevention of denaturation in the high pressure spray-drying process.
[0060] Figure 8 shows chromatographic charts indicating the high purity, high
concentration, and high stability of reconstituted compositions of the
invention.
[0061] Figure 9 is a schematic diagram of an exemplary high pressure spray dry
system.
[0062] Figures 10A-D are schematic diagrams of exemplary triple-inlet high
pressure spray nozzles.
DETAILED DESCRIPTION
[0063] The present invention provides compositions and methods for preparing
stable particles containing bioactive materials, such as, e.g., peptides,
polypeptides,
proteins, viruses, bacteria, antibodies, cells, liposomes, vaccines and/or the
like. The
method includes, e.g., quick drying of spray droplets into particles without
high drying gas
heat by using high spray pressures to inject a fine mist into a warm stream of
drying gas.
Favored formulations for spray drying of therapeutic bioactive materials
include amino
acids and sugars to form stable easily reconstituted powder particles.
[0064] The methods of the invention provide preferred formulations for high
pressure spray drying of therapeutic bioactive material. The formulations can
provide stable
powder particles that are readily reconstituted to high concentrations. The
formulations can
include, e.g., from less than about 4% to about 10% by weight of the
therapeutic bioactive
material, from about 0.1 mM to about 50 mM total of one or more amino acids,
and from
about 0.5% to about 4% by weight of a sugar in an aqueous solution or
suspension. Where
the bioactive material is a virus, e.g., in an attenuated live virus vaccine,
the virus can be
present in the liquid formulation in an amount ranging from about 103 TCmSo/mL
to about
1012 TCmSO/mL, or from about 105 TCmso/mL to about 10~ TCmso/mL. Other
constituents can be added to the formulation, e.g., to provide desirable
benefits in stability,
reconstitution time, and physical characteristics.
16
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WO 2005/123131 PCT/US2005/020792
[0065] The method of the invention generally provides, e.g., spray drying of
bioactive materials in a composition with viscosity enhancing agents at a high
pressure to
produce fine droplets that dry quickly to powder particles with little initial
loss of in purity
or viability. The high initial purity and protective effects of excipients
provide, e.g., a long
shelf life and excellent stability for powder particles storage. The fine
powder particles and
highly soluble excipients allow ready reconstitution of bioactive materials to
a high
concentration with high specific activity.
[0066] The methods of the invention to prepare powder particles include high
pressure spraying of formulations with, e.g., about 8% bioactive material,
about 0.5%
arginine, and about 2% of sucrose, into a dry powder. Alternately, where the
bioactive
material is a live virus, the bioactive material can be present at much lower
mass ratio, e.g.,
with attenuated virus present at from about 103 TCIDso (50% Tissue Culture
Infecting
Dose)/mL to about 1012 TCIDSO/mL. In one aspect or the invention, dried powder
particles
can be administered to human patients by reconstitution to a concentration of
about 200
mg/ml bioactive material for subcutaneous injection.
METHODS OF HIGH PRESSURE SPRAY DRYING
[0067] Methods of the invention combine high pressure spraying with protective
formulations for fast drying of pure and stable bioactive materials. The
methods of the
invention include production of powder particles containing bioactive
materials, such as
peptides, polypeptides, proteins, viruses, bacteria, antibodies, cells,
liposomes, vaccines
and/or the like, e.g., by preparing an aqueous formulation of the bioactive
material with a
sugar and amino acid, spraying the suspension or solution through a nozzle at
high pressure
to form a mist of fine droplets, drying the droplets to form powder particles,
and recovering
the particles for storage or immediate use.
[0068] The methods can be modified to provide suitable products depending on,
e.g., the sensitivity of the bioactive material, the expected storage
conditions, and the
proposed route of administration. A variety of viscosity enhancing agents,
such as, e.g.,
polyols and polymers, are available which can provide desirable
characteristics, in addition
to shear stress protection, including antioxidation, hydrogen bonding with the
bioactive
17
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WO 2005/123131 PCT/US2005/020792
material to replace water of molecular hydration, high solubility to aid in
reconstitution, and
safety for injection in humans. High pressure to spray formulations of the
bioactive
material can be provided, e.g., by hydraulic pressure, pressurized gases, or
high pressure
pumps, such as HPLC pumps. Drying of droplets can be achieved, e.g., by
freezing and
sublimation, warm streams of humidity and/or temperature controlled drying
gasses, and/or
suspension in a fluidized bed. Recovering the particles can include separation
of particles
by size, filtering, settling, filling into sealed containers, and the like.
Particles of the
invention can be used, e.g., to administer the bioactive material by
inhalation, to reconstitute
for administration by injection, to store analytical reference samples for
long term
references, and/or the like.
Preparing_a Formulation of a Bioactive Material for Sprayin~
[0069] A bioactive material of interest can be added to a solution comprising
a sugar
and amino acid to prepare the spray drying formulation of the invention.
Additional
excipients can be added to enhance solubility of components, reduce oxidation,
increase
viscosity, add bulk, reduce surface tension, reduce the porosity of the
particles, control pH,
and/or the like.
[0070] Individual constituents can play multiple roles as components in a
formulation. For example, an amino acid can be a stabilizer, buffer,
antioxidant, bulking
agent, etc. A sugar can be a stabilizer, reconstitution accelerator,
cryoprotectant, bulking
agent, viscosity enhancing agent, etc. A formulation component, such as
viscosity
enhancing agent, excipient, buffer, sugar, amino acid, surfactant, stabilizer,
and/or the like,
can be represented by the cumulative different individual constituents that
contribute to the
role of the component in the formulation.
[0071] Although the preferred bioactive materials of the invention are
antibodies
and vaccines, methods and formulations can be applied to , e.g., industrial
reagents,
analytical reagents, pharmaceuticals, therapeutics, and the like. Bioactive
materials of the
invention include, e.g., peptides, polypeptides, proteins, viruses, bacteria,
antibodies,
monoclonal antibodies, cells, liposomes, and/or the like. Preparation steps
for liquid
18
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WO 2005/123131 PCT/US2005/020792
formulations of these materials can vary depending on the unique sensitivities
of each
material.
[0072] Liquid formulations for spraying can be prepared by mixing the
bioactive
material, sugar, amino acids, and additional excipients, in an aqueous
solution. Many
bioactive materials, such as antibodies, can dissolve readily into an aqueous
solution. Other
bioactive materials, such as, e.g., some peptides, viruses, bacteria, and
liposomes can be
particles that exist as a suspension in the formulation. Whether the bioactive
material can
exist in a solution or suspension, it is often necessary, e.g., to avoid
severe conditions of
shear stress or high temperatures when mixing them into a formulation. Where
other
formulation constituents require heat or strong stirring to bring into
solution, they can, e.g.,
be dissolved separately then gently blended with the bioactive material after
cooling.
[0073] The total solids in the final formulation are generally, e.g., high, to
help
provide the high viscosity and/or quick low temperature drying aspects of the
invention.
For example, process formulations for spraying in the invention can include
from about 5
percent to about 50 percent total solids (residual on drying), from about 10
percent to 20
percent total solids, or about 15 percent total solids. The formulations for
high pressure
spraying can have a viscosity significantly greater than that of water at room
temperature
(0.01 poise), and greater than the viscosity of the bioactive material
formulation without
addition of supplementary viscosity enhancing agents. For example, addition of
the
viscosity enhancing agent can increase the viscosity of the formulation for
spraying by 0.02
centipoise, 0.05 centipoise, 0.1 centipoise, 0.5 centipoise, 1 centipoise, 5
centipoise, 10
centipoise, 0.5 poise, 1 poise, 5 poise, 10 poise, or more. In another aspect,
addition of the
viscosity enhancing agent can increase the viscosity of the formulation for
spraying by 1%,
5%, 25%, 50%, 100%, 500%, or more. In a preferred embodiment, viscosity
enhancing
agents are present at a concentration sufficient to increase the viscosity by
0.05 centipoise or
more, or sufficient to increase the viscosity of the formulation by 5% or
more. In a
preferred embodiment, the addition of a viscosity enhancing agent provides a
significant
(e.g., measurable) reduction in bioactive material deactivation, fragmentation
or aggregation
compared to the same formulation without the additional viscosity enhancing
agent.
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WO 2005/123131 PCT/US2005/020792
[0074] The concentration of bioactive material in the formulation can vary
widely,
depending on, e.g., the specific activity, concentration of excipients, route
of administration,
and/or intended use of the material. Where the bioactive material is, e.g., an
antibody for
therapeutic administration by inhalation or injection, or a liposome for
topical
administration, the required concentration can be higher. Where the bioactive
material is a
peptide vaccine, live attenuated virus, killed virus for vaccination, or
bacteria, for example,
the required concentration of material can be quite low. In general, bioactive
materials can
be present in the solutions or suspensions of the invention at a
concentration, e.g., between
less than about 1 mg/ml to about 200 mg/ml, from about 5 mg/ml to about 80
mg/ml, or
about 50 mg/ml. Viral particles can be present in the formulations in amounts,
e.g., ranging
from about 10 pg/ml to about 50 mg/ml or about 10 ug/ml; or, e.g., present in
the liquid
formulation in an amount ranging from about 103 TCIDso/mL to about 101
TC~SO/mL.
[0075] Viscosity enhancing agents of the invention are generally, e.g., sugars
or
water soluble polymers which can be dissolved or effectively suspended into
the solution or
suspension at concentrations high enough to provide significant protection
against shear
disruption or denaturation of the bioactive material. In general, effective
amounts of
viscosity enhancing polymers are lower than effective amounts required for
sugars due to
the higher viscosity produced by longer molecules in solution. Viscosity
enhancing agents
can be present in the formulations of the invention in amounts, e.g., between
about 0.05
weight percent to about 30 weight percent, from about 0.1 weight percent to
about 20
weight percent, or about 2 weight percent to about 6 weight percent. Many
viscosity
enhancing agents are carbohydrates that can provide, e.g., protective effects
to bioactive
materials under other process stresses, such as, e.g., freezing and drying.
[0076] The formulation of the invention can include, e.g., a surfactant
compatible
with the particular bioactive material involved. A surfactant can enhance
solubility of other
formulation components to avoid aggregation or precipitation at higher
concentrations.
Surface active agents can, e.g., lower the surface tension of the formulation
so that bioactive
materials are not denatured at gas-liquid interfaces, and/or so that spraying
forms finer
droplets. Surfactants can be present in the solutions or suspensions of the
invention in an
CA 02564791 2006-10-27
WO 2005/123131 PCT/US2005/020792
amount ranging from about 0.005 percent to about 1 percent, from about 0.01
percent to
about 0.5 percent, or about 0.02 percent.
Formulations for S~ra~Dryin~ Bioactive Materials
[0077] Formulations of the invention can be particularly useful for spraying
stable
powder particles with good reconstitution characteristics. Formulations
particularly useful
for spray drying of bioactive material can include, e.g., 4% to 10% of the
bioactive material
by weight, 0.5% to 4% of a sugar and from about 0.1 mM to about 50 mM of amino
acids.
The formulations can beneficially also include, e.g., surfactants, polymers,
and/or buffers
providing a pH at or below about pH 6. In a preferred embodiment, a
formulation for high
pressure spray drying of bioactive materials includes, e.g., about 8% of the
bioactive
material by weight, about 10 mM histidine, about 0.5% arginine and about 2%
sucrose at
about pH 6. In other preferred embodiments, formulations for high pressure
spray drying of
vaccines includes, e.g., from about 103 TC~so/mL to about 1012 TCIDso/mL
attenuated
virus, about 10 mM histidine, about 0.5% arginine and about 2% sucrose at
about pH 6.
[0078] Therapeutic bioactive materials benefiting from the particular
formulations
can include, e.g., peptides, polypeptides, proteins, viruses, bacteria,
antibodies, cells,
liposomes, vaccines and/or the like.
[0079] Antibodies of the formulations include, but are not limited to, an
antibody
having the sequence, or containing a sequences of a complementarity
determining region
(CDR), or containing sequences that are merely conservative variations of
novel sequences
in: 1) an anti-RSV antibody disclosed in U.S. Patent 5,824,307; Johnson S, et
al.,
"Development of a Humanized Monoclonal Antibody (MEDI-493) with Potent In
Vitro and
In Vivo Activity Against Respiratory Syncytial Virus." J. Infect Dis., 176(5):
1215-24,
(1997, Nov); U.S. Patent 6,656,467, or U.S. Published Application 20030091584;
or 2) an
a~(33 disclosed in U.S. Patent 6,531,580, U.S. Application Number 20030166872,
or Wu, H.
et al., "Stepwise In Vitro Affinity Maturation of Vitaxin, an a"(33-Specific
Humanized
mAb", Proc Natl Acad Sci U S A, 26; 95(11): 6037-42, (1998 May); or 3) an anti-
EphA2
antibody disclosed in U.S. Patent Application Publication No. 20040091486. In
a preferred
embodiment, a bioactive material is an antibody as defined above. Specifically
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contemplated antibodies include, but are not limited to: anti-RSV, anti-hMPV,
anti-avb3
integrin, anti-avb5 integrin, anti-alpha IIb/beta 3 integrin, anti-alpha 4
integrin, anti-EphA2,
and anti-EphA4, anti-EphB4, anti-IL9, anti-III, anti-ILS, anti-IL13, anti-
IL15, anti-
CTLA4, anti-PSA, anti-PSMA, anti-CEA, anti-cMET, anti-CSa, anti-TGF-beta, anti-
HMGB-1, anti-interferons alpha and anti-interferon alpha receptor, anti-IFN
beta and
gamma, anti-chitinase, anti-TIRC7, anti-T-cell, MT-103 BITE~, anti-EpCam, anti-
Her2/neu, anti-IgE, anti-TNF-alpha, anti-VEGF, anti-EGF and anti-EGF receptor,
anti-
CD22, anti-CD19, anti-Fc, anti-LTA, anti-Flk-1, and anti-Tie-1.
[0080] Vaccine antigens to be used in the invention include, but are not
limited to,
viral vaccines which can be live whole virus vaccines, killed whole virus
vaccines, subunit
vaccines, purified or recombinant viral antigens, recombinant virus vaccines,
anti-idiotype
antibodies, cancer vaccines, and DNA vaccines. In certain preferred
embodiments, a
bioactive material is a vaccine antigen. Specifically contemplated vaccines
comprise one or
more antigens from the following: Epstein Barr virus, Streptoccocus,
pneumococcal, RSV
(respiratory syncytial virus), PIV (para-influenza virus), hMPV (human
metapneumovirus),
EphA2 cancer vaccine, HPV (human papilloma virus) HPV-16, HPV-18, CMV
(cytomegalo virus), Pneumocystis carinii, Influenza virus, rubella, measles,
mumps,
anthrax, botulism, ebola, chicken pox, shingles, small pox, polio, yellow
fever, hepatitis B,
Rift Valley fever, tuberculosis, viral meningitis, pandemic flu, avian flu,
and adenovirus.
[0081] Sugars can provide many useful characteristics to formulations of
bioactive
materials for spray drying. Sugars in the formulations can enhance stability,
accelerate
reconstitution, reduce shear denaturation during spraying and administration,
etc. In the
present invention, it is preferred that sugars be present in the formulation
for high pressure
spray drying in an amount ranging from about 0.1% to about 8% by weight or
more, from
about 0.5% to about 4%, from about 1% to about 3%, or about 2%. Preferred
sugars are
generally not reducing sugars. Exemplary sugars for spray drying biomaterial
formulations
include sucrose, mannitol and/or trehalose.
[0082] Amino acids can be included in formulations for high pressure spray
drying
of bioactive materials, e.g., to enhance stability, buffer the pH, provide
readily soluble bulk,
and/or the like. Formulations for spraying bioactive materials, in the
invention can include,
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WO 2005/123131 PCT/US2005/020792
e.g., amino acids in amounts ranging from about 0.05 mM to about 100 mM, from
about 0.1
mM to about 50 mM, from about 1 mM to about 30 mM, or about 20 mM total amino
acids.
Preferred amino acids include, e.g., glycine, leucine, histidine and arginine.
Preferred
histidine concentrations in the formulations range from about 2 mM to about 20
mM, or
about 10 mM. Preferred arginine concentrations in the formulations range from
about 5
mM to about 50 mM, from about 10 mM to about 40 mM or about 30 mM (about 0.5%
by
weight). Of course, formulations free of histidine or arginine are envisioned,
but generally
less preferred.
[0083] In a preferred embodiment, at least one amino acid in the formulation
is a
small hydrophobic amino acid, as such Leucine. Use of small hydrophobic amino
acids in
the formulations can particularly benefit the properties of powders sprayed in
the presence
of organic solvents. For example, leucine in combination with an ethanol-
mediated spray
drying process can improve the dispersibility and flowability of resultant
powders. Inter-
particle cohesion and reconstitution time can also be reduced for powders
sprayed using a
combination of solvent and small hydrophobic amino acid. Preferred solvent
spraying
techniques can include spraying with formulations having 0-2% w/v leucine
and/or
mannitol.
[0084] Surface active agents can be included in formulations for high pressure
spray
drying of bioactive materials, e.g., to enhance stability and reduce
reconstitution times for
the bioactive material. Surfactants can be present in the formulations in
amounts ranging
from 0% to about 2%, from about 0.001% to about 1%, from about 0.01% to about
0.5%,
from about 0.05% to about 0.2%, or about 0.1%. Preferred surfactants for the
high pressure
spraying formulations include 0.01 % to about 0.2% of nonionic detergents,
such as
polyoxyethylenesorbitan monooleates (Tween-20) or polyethylene glycol sorbitan
monolaurates (Tween-80).
[0085] In addition to amino acids and sugars, polymers can be added to the
formulations for high pressure spray drying of bioactive materials. A
preferred polymer is
polyvinyl pyrrolidone (PVP). Polymers, when present in the formulation, are
preferred in
amounts ranging from about 0.01% to about 2%, from about 0.05% to about 0.5%,
or about
0.1% to about 0.2%.
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WO 2005/123131 PCT/US2005/020792
[0086] In particular embodiments of high pressure spray drying, the bioactive
materials are antibodies described as having a sequence of any of SEQ ID NOs.
1 to 20, or a
conservative variation thereof. Conservative amino acid substitutions, in one
or a few
amino acids in an amino acid sequence are substituted with different amino
acids with
highly similar properties, are also readily identified as being highly similar
to a disclosed
construct. Such conservative variations of each disclosed sequence are a
feature of the
present invention. One of skill will recognize that individual substitutions,
deletions or
additions which alter, add or delete a single amino acid or a small percentage
of amino acids
(typically less than 5%, more typically less than 4%, 2% or 1%) in an encoded
sequence are
"conservatively modified variations" where the alterations result in the
deletion of an amino
acid, addition of an amino acid, or substitution of an amino acid with a
chemically similar
amino acid. Thus, "conservative variations" of a listed polypeptide sequence
of the present
invention include substitutions of a small percentage, typically less than 5%,
more typically
less than 2% or 1 %, of the amino acids of the polypeptide sequence, with a
conservatively
selected amino acid in the same conservative substitution group. The
conservative
variations particularly include those which do not substantially change the
specificity or
affinity of the identified antibodies.
[0087] Table 1 -- Conservative Substitution Groups
1 Alanine (A) Serine (S) Threonine
(T)
2 Aspartic acid Glutamic acid (E)
(I7)
3 Asparagine Glutamine (Q)
(N)
4 Arginine (R) Lysine (K)
Isoleucine Leucine (L) Methionine Valine (V)
(I) (M)
6 Phenylalanine Tyrosine (Y) Trytophan
(F) (W)
In Table 1, substitution of an amino acid with another amino acid of the same
group can be
considered a conservative variation substitution.
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WO 2005/123131 PCT/US2005/020792
S~rayin~ the Formulation
[0088] Formulations of the invention are sprayed, e.g., from a spray nozzle at
high
pressure to produce a fine mist of droplets. Spray parameters can vary, e.g.,
according to
the viscosity of the solution, the desired particle size, the intended method
of drying, the
design of atomization nozzles, and/or sensitivities of the bioactive material.
[0089] High pressure spraying has significant advantages over lower pressure
spraying methods, e.g., because of the fine droplets, and ultimately, fine dry
powder
particles thus obtained. As shown in Figure 1, high pressure spraying (plot
10) can provide
droplet sizes less than 10 ~m with mass flow ratios (MFR - the ratio of
atomizing gas mass
flow per liquid mass flow) less than 1, whereas standard (lower pressure
atomizing nozzles,
plot 11) can require MFRS in the range of about 15 to obtain droplet sizes
less than 10 hum.
High pressure spraying can provide a significant reduction in the use of
atomizing gasses
while spraying finer average droplet sizes than obtainable with lower pressure
spray
methods. Optionally, high pressure spraying can be practiced without
simultaneous
discharge of atomizing gas, i.e., spraying of high pressure liquid from a
nozzle without a jet
of gas.
[0090] The formulation can be sprayed from a nozzle at a pressure effective in
providing the desired droplet size. Higher pressures generally provide, e.g.,
smaller droplet
sizes. When the solution is more viscous, e.g., a higher pressure can be
required to provide
the desired droplet size. The presence of a surfactant, e.g., often lowers the
pressure
required to provide the desired droplet size in high pressure spraying
processes. Where
formulations are atomized by spraying in the presence of a pressurized gas
flow, the mass
flow ratio can affect droplet sizes. The spray pressures of the invention can
be, e.g.,
between about 200 psi (pounds per square inch) and about 5000 psi, between
about 500 psi
and 2500 psi, 1000 psi and 1500 psi, or can be about 1300 psi. The size of
spray droplets
andlor dried particles can be controlled by, e.g., adjusting the percent
surface active agent in
the formulation, adjusting a spraying pressure, adjusting an atomizing gas
pressure,
adjusting a viscosity, adjusting the total solids in the formulation,
adjusting a flow rate of
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the formulation, adjusting a mass flow ratio, adjusting a temperature of the
formulation,
and/or the like.
[0091] Where the spray of droplets is atomized with a high pressure atomizing
gas,
the atomizing gas can have, e.g., a pressure or temperature at least 10%, or
at least 15%, or
at least 20%, away from a critical point for the gas. As shown in Figure 2,
pressurization
and/or cooling of many gasses can lead to a phase transition from the gas
state to a liquid or
solid state. These transitions from the gas state can take place at critical
pressures andJor
critical temperatures. It is an aspect of the invention that in some
embodiments, atomizing
gasses are more than 10%, more than 15%, or more than 20% below the critical
pressure for
the gas at a given temperature. It is an aspect of the invention that in some
embodiments,
atomizing gasses are more than 10%, more than 15%, or more than 20% above the
critical
temperature (as measured in degrees Kelvin) for the gas at a given pressure.
[0092] In one embodiment, the formulation includes both a viscosity enhancing
agent and a surface active agent, e.g., to provide improved control of sprayed
droplet size at
a given spray pressure. In the presence of viscosity enhancing agents, sprayed
droplet sizes
are generally greater than for solutions without viscosity enhancing agents.
In the presence
of surface active agents, sprayed droplet sizes are generally smaller than for
solutions
without surface active agents. However, when formulations include both a
viscosity
enhancing agent and a surface active agent, some useful and unexpected results
can be
obtained. A chart of droplet size versus atomization pressure can be prepared
to show
relationships between pressures, surface active agents, viscosity enhancing
agents and
droplet sizes, as shown for example in Figure 3. At some pressures, e.g., 900
to 1100 psi,
pure water 30 can spray into smaller droplet sizes than for water with surface
active agent
(Tween 80) and/or viscosity enhancing agent (Sucrose). At other pressures,
e.g., from about
1300 psi to about 2200 psi, solutions or suspensions containing surface active
agent can
spray into droplet sizes smaller than for pure water. At a certain enhanced
surfactant
control ranges of spray pressures, surface active agents can exert a
particularly significant
influence on the droplet size of solutions or suspensions containing viscosity
enhancing
agents. For example, at 1500 psi the average droplet size of 20% sucrose
solution 31 can be
more than for water at about 14 Vim, but the average droplet size can be less
than for water
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WO 2005/123131 PCT/US2005/020792
at about 8 ~m for 20% sucrose solution with 0.1% Tween 80 32. In one
embodiment of the
invention, the droplet size of sprayed formulations is controlled at a
particular atomization
pressure by adjustment of the surface active agent concentration. For example,
incremental
adjustments of surface active agent concentrations can provide tuned droplet
sizes even if
other parameters, such as orifice internal diameter, viscosity enhancing agent
concentration,
pressure, and MFR are held constant. Enhanced surfactant control ranges can be
determined empirically for bioactive agent, surface active agent, viscosity
enhancing agent
combinations of interest.
[0093] Methods of the invention particularly suitable for high pressure spray
drying
of bioactive materials can include, e.g., spraying preferred formulations of
the bioactive
material with sugars and amino acids to form a mist of droplets, and drying
the droplets to
form powder particles. The formulation can be constituted as described above
in the
Formulations for Spray Drying Bioactive Materials section, e.g., with from
about 4% to
about 10% bioactive material by weight (or optional described virus
concentrations), from
about 0.1 mM to about 50 mM amino acid, and from about 0.5% to about 4% of a
sugar.
Spraying can be at a high pressure, e.g., from about 200 psi to about 5000
psi, from about
800 psi to about 1800 psi, from about 1000 psi to about 1500 psi, or about
1300 psi. High
pressure spraying of the bioactive material can produce any suitable size of
droplets, but
preferred droplets will produce dry powder particles ranging in size from
about 0.5 ~m to
about 100 Vim, from about 1 hum to about 50 hum, from about 2 [um to about 20
Vim, from
about 7 ~,m to about 18 ~,m or from about 10 ~.m to about 15 Vim. For example,
the
droplets, before drying, can range in size from about 1 ~m to about 200 ~,m,
from about 2
~m to about 100 Vim, from about 3 ~m to about 30 ~,m, from about 10 ~,m to
about 20 hum
or about 15 Vim.
[0094] Droplet sizes can be affected by the mass flow ratio (MFR) of atomizing
gas
and the formulation. Under conditions of low MFR for a given atomizing
pressure, as
shown on the left side of the chart in Figure 4A, larger particles are formed.
Under
conditions of higher MFR for a given atomizing pressure, as shown on the right
side of the
chart, smaller powder particles are formed on drying of the sprayed droplets.
One
27
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explanation for this observation can be that higher relative flows of
atomizing gas are able
to disrupt a given fluid flow into smaller droplets. In many cases, average
droplet size (and
final dried particle sizes) can be tuned by adjusting the flow rate of a
formulation to be high
pressure sprayed while any atomizing gas pressure remains constant.
Optionally, the MFR
can be varied to adjust droplet size by varying the pressurized atomizing gas
flow while the
flow of formulation is held constant, as shown in Figure 4B.
[0095] In a preferred embodiment, formulations are high pressure spray-dried
with
an atomizing stream of pressurized nitrogen gas. Atomization with the nitrogen
gas stream
can contribute to reduced droplet sizes as a given pressure as compared to
direct high
pressure spraying without a atomizing gas. Nitrogen has an advantage over
atomization
with pressurized air in that it is relatively inert and can protect bioactive
materials, e.g.,
from oxidation. Nitrogen has advantages over carbon dioxide in that it does
not form acids
in aqueous solutions and has a greater capacity to hold water vapor. Nitrogen
is less
expensive than other substantially inert gasses, such as helium and argon,
which can also be
used in high pressure spray dry processes. Appropriate nozzles for high
pressure spraying
with atomizing nitrogen include, e.g., dual channel atomizing nozzles and
nozzles with T
intersections of liquid with the atomizing gas. As shown in Figure 4B,
particle sizes of
dried droplets generally decrease with higher atomization pressures at a given
MFR.
[0096] In another preferred embodiment, the bioactive material, can be spray
ch-ied
as a formulation in the presence of an organic solvent. Typically the
formulation and
solvent are sprayed from a nozzle along with a high pressure atomization gas,
such as, e.g.,
nitrogen and/or carbon dioxide. For example, the formulation can be introduced
into a
triple-inlet effervescent atomization nozzle along with separate inputs of
solvent and gas.
One nozzle inlet can be dedicated to a high pressure atomization gas (e.g.,
nitrogen or C02),
one inlet dedicated to the (liquid) formulation and active ingredients, and
one inlet
dedicated to organic solvents that modify evaporation behavior of the
droplets. Methanol
and/or ethanol (1-50 °Io v/v concentration range relative to the total
formulation plus solvent
sprayed) have been found to improve evaporative efficiency and, e.g.,
affecting particle
surface morphology, powder particle size, and/or particle density. These
changes can help
improve powder dispersibility and flowability aimed at enhancing deep lung
delivery.
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[0097] High pressure spray drying processes can be scaled up, e.g., by
spraying
larger volumes of formulations. Larger volumes can be sprayed, e.g., by using
multiple
spray nozzles, by spraying at higher pressures, by spraying at a higher
formulation flow
rate, and/or by spraying through a larger internal diameter spray orifice.
Figure 5 shows
some examples of high pressure spray nozzle configurations. Figure 5B shows a
high
pressure liquid spray nozzle with a constrictor at the orifice. When spraying
from an
atomizing nozzle, e.g., as shown in Figures 5A and 5C, the MFR can change with
the flow
rate of the formulation resulting with changed droplet sizes at a given
atomizing gas
pressure. This is because as the flow rate of the liquid increases, the flow
of atomizing gas
can become restricted. For example, as shown in Figure 6, as the liquid feed
rate increases
for a formulation being atomized with a 2500 psi gas through a 250 ~,m
orifice, the droplet
size begins to increase in a nonlinear fashion at a liquid flow rate of about
30 ml/min (plot
60). This is due to restriction of the atomizing gas flow by the flow of
liquid and resultant
drop in the MFR. Such a rapid increase in droplet size can be delayed by
employing an
atomizing nozzle with a larger orifice internal diameter, as shown in plot 61
for a
formulation being atomized with a 1170 psi gas through a 500 ~.m orifice.
[0098] Triple-inlet spray nozzles can have any functional configuration. For
example, the nozzles can have inlets to "T" intersections, radially arrayed
inlets, or staged
combination of input fluids. Figure 10A shows how gas, formulation and solvent
can be
combined and sprayed from a nozzle having a T intersection of fluid inlets.
Figure 10B
shows radial introduction and combination of fluids (Figure 10C is a cross-
section through
10B, as indicated). Figure 10D shows preliminary combination of formulation
and solvent
before aspiration with a gas flow at the nozzle outlet.
[0099] Molecular, particulate, and cellular bioactive materials sensitive to
shear
stress can experience denaturation or deactivation when sprayed at high
pressure. This
problem can be reduced, e.g., by spraying with a viscosity enhancing agent.
Figure 7, for
example, shows size exclusion analyses of a solution of antibodies before and
after spray
drying. Figure 7A shows a size exclusion chromatograph of the antibody before
spraying.
Figure 7B shows a size exclusion chromatograph of the antibody after spraying
without
effective amounts of a viscosity enhancing agent, wherein the amount of
aggregate 70 has
29
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WO 2005/123131 PCT/US2005/020792
increased about 6-fold and fragments 71 have increased slightly. Aggregates of
the
antibody can have a lowered specific activity due to shear stress denaturation
of the
antibody protein and associated abnormal hydrophobic interactions between the
antibody
molecules. Figure 7C shows a size exclusion chromatograph of the same antibody
which
has been protected from aggregation and fragmentation by including a viscosity
enhancing
agent in the solution before spraying.
[0100] The spray nozzle of the invention can be adapted to provide the desired
fine
mist of droplets. The nozzle can have, e.g., a conduit feeding the formulation
at high
pressure to a spray orifice that has an internal diameter of between about 50
~.m and about
500 Vim, between about 75 hum and about 250 Vim, or about 100 Vim. Wider
diameter
orifices can provide, e.g., higher production rates but can result in larger
droplet sizes. The
nozzle can be configured as an atomizer, i.e., with a second channel routing a
pressurized
gas into the stream of formulation, to aid in the dispersal of the droplets.
The nozzles can
include additional channels, e.g., for blending of additional fluids (e.g.,
solvents) into the
stream.
[0101] The process formulation can be sprayed from the nozzle at high pressure
to
form fine droplets that are readily dried into desired powder particles of the
invention. The
droplets can be sprayed, e.g., into a stream of inert warm drying gas, into a
vacuum of 200
Torr or less, or into a freezing stream or pool of a cold fluid. The clioplets
can have an
average diameter of about 2 ~,m to about 200 ~,m, about 3 ~,m to about 70 ~.m,
about 5 ~m
to about 30 ~,m, or about 10 ~,m. If the droplets are frozen, e.g., in a cold
stream of gaseous
or liquid, argon, helium, carbon dioxide, or nitrogen, at between about -
80°C to about -
200°C, they can be dried by sublimation to form particles about the
same size as the
droplets but having a low density (and a lower aerodynamic diameter). If the
formulation is
high in total solids, the dried particles can be, e.g., larger and/or more
dense.
Drying, the Droplets
[0102] Sprayed droplets can be dried to form powder particles. Droplets
sprayed
using methods of the invention can be dried, e.g., without excessively hot
temperatures to
provide high recovery of particles with high purity, high specific activity,
and high stability.
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WO 2005/123131 PCT/US2005/020792
Drying can be, e.g., by exposure to a temperature, humidity, and/or pressure
controlled
environment. Drying can be by sublimation of ice, vacuum drying, contact with
drying
gasses, suspension in a fluidized bed, retention in a drying chamber, and/or
the like.
Primary drying generally includes, e.g., removal of liquid or ice water bulk
from the
droplets of the formulation. Secondary drying generally includes, e.g.,
removal of trapped
moisture and/or water of hydration from particles to a level of 15 percent
residual moisture,
percent residual moisture, 5 percent residual moisture, 3 percent residual
moisture, 1
percent residual moisture, or less.
[0103] Drying can be by, e.g., spraying the droplets into a stream of drying
gas
controlled for humidity and/or temperature. Drying parameters can be
controlled, e.g., to
provide conditions necessary to obtain particles with the desired activity,
density, residual
moisture, and/or stability. Drying parameters can be controlled to provide the
desired
particle characteristics within a time frame compatible with process
requirements, such as
drying time, drying chamber retention time, agglomeration prevention, etc. The
gas can be,
e.g., an inert gas, such as nitrogen, that displaces the water vapor, and
other gases emanating
from the sprayed mist of formulation. The drying gas can be the same gas as
the high
pressure spray gas, e.g., to facilitate drying gas recycling. The gas can be
dry, e.g;, with a
low relative humidity, to absorb moisture and speed evaporation of the
droplets. The gas
can be, e.g., controlled to a temperature between about 10°C to about
90°C, about 15°C and
about 70°C, between 25°C and about 60°C, or about
35°C to about 55°C. The temperature
of drying gas at a drying chamber inlet can be controlled to provide a drying
gas
temperature at the drying chamber outlet ranging from about 30°C to
about 80°C, from
about 40°C to about 60°C, or about 50°C. Drying
temperatures can remain, e.g., below the
glass transition temperature (Tg) of the particle constituents to avoid
changing the porosity,
density, stability, andlor reconstitution time of the particles. The small
particle sizes, spray
plume size, spray plume turbulence, and high total solids of the invention
can, e.g., allow
for short drying times and cooler drying temperatures that will not
substantially degrade
many sensitive bioactive materials.
[0104] The droplets can be dried, e.g., by application of a vacuum (gas
pressures
less than atmospheric pressure, such as 200 Torr, about 100 Torr, about 50
Torr, about 10
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WO 2005/123131 PCT/US2005/020792
Torr, or less) to the sprayed mist or partially dried particles. Vacuum drying
has the benefit,
e.g., of quickly "boiling" or sublimating away water from the droplets while
reducing the
temperature of the droplets. The temperature of the droplets falls as latent
heat is lost
during the phase transition of liquid water to gas. Thus, vacuum drying can
significantly
reduce heat stress on the bioactive material. In the case of droplets frozen
in a stream of
cold fluid, or frozen by the loss of latent heat during drying processes,
vacuum pressures
can sublimate water directly from the solid ice phase to the gas phase
providing freeze-dried
(lyophilized) particles.
[0105] Secondary drying conditions can be used, e.g., to further lower the
moisture
content of particles. Particles can be collected in a chamber and held at a
temperature
between about 20°C and about 99°C, about 25°C and about
65°C, or about 35°C and 55°C,
e.g., in a vacuum (pressure below atmospheric), for from about 2 hours to
about 5 days, or
about 4 hours to about 48 hours, to reduce residual moisture. Secondary drying
can be
accelerated by providing an updraft of drying gasses in the chamber to create
a fluidized bed
suspension of powder particles. Particles with lower residual moisture
generally show
better stability in storage with time. Secondary drying can continue until the
residual
moisture of the powder particles is between about 0.5 percent and about 10
percent, or less
than about 5 percent. At very low residual moisture values, some bioactive
material
molecules can be denatured by loss of water molecules of hydration. This
denaturation can
often be mitigated by providing hydrogen binding molecules, such as sugars,
polyols,
and/or polymers, in the process formulation.
[0106] Powder particles of the invention can have a size, e.g., suitable to
the
handling, reconstitution, and/or administration requirements of the product.
For example,
powder particles of bioactive materials for administration by intranasal
delivery by
inhalation can be larger, at between about 20 ~m to about 150 ~m or more, than
for deep
pulmonary delivery by inhalation, at between about 2 pm to about 10 ~m
(average physical
diameter). The average particle size for products that reconstitute slowly can
be smaller to
speed dissolution of the particles. Spray freeze-dried particles can have,
e.g., a lower
density, because the ice can be removed from droplets without collapse of a
cake structure
of the remaining solids. Such particles can have, e.g., a physically larger
acceptable size for
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inhaled administration due to their lower aerodynamic radius. Freeze-dried
particles can,
e.g., be larger than particles dried from liquid droplets and still retain
quick reconstitution
properties due to the porous nature of freeze-dried particles. Freeze dried
powder particles
of the invention can have average physical diameters, e.g., between about 0.1
~m and about
200 Vim, or between about 2 ~m and about 100 Vim, or about 10 ~.m.
[0107] Drying spray mist droplets of formulations that include bioactive
materials
other than viruses or antibodies can generally proceed as described above. In
preferred
embodiments, high pressure sprayed droplets of bioactive material formulations
come into
contact with a drying gas having a temperature, e.g., from about 30°C
to about 80°C. It can
be preferred to dry the droplets in a drying chamber having a drying gas inlet
and a drying
gas outlet. Preferred outlet gas temperatures for drying of high pressure
sprayed droplets of
the invention containing antibodies range from about 30°C to bout
80°C, from about 40°C to
about 60°C, or about 50°C. Preferred drying gasses include air
or inert gasses, such as, e.g.,
nitrogen. It is further preferred to recycle gas from the drying chamber
outlet, e.g., by
removing water and by adjusting the temperature before returning the drying
gas to dry
additional droplets in the drying chamber. Dried powder particles of bioactive
material
formulations can be recovered from the drying chamber or other collection
vessel. The
powder particles can be administered as powder particles, e.g., by inhalation,
dry injection,
or by injection on reconstitution.
[0108] The average size and size uniformity of particles can be controlled,
e.g., by
adjusting spraying parameters andlor by adjusting drying parameters. For
example, average
droplet size can be affected by nozzle size, solution pressures, solution
viscosity, and
solution constituents, etc., as described above in the Spraying the
Formulation section
above. Average particle size, and size distribution, can be affected by drying
conditions that
affect shrinkage or agglomeration of particles, such as, e.g., the use of
freeze-drying, the
completeness of drying, the neutralization of static charges, particle density
during drying,
the rate of drying, the temperature of drying, andlor the lilce. The average
size and size
uniformity of particles can be selected as described in the Recovery of
Particles section,
below.
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Recovery of Particles
[0109] Powder particles of the invention can be physically recovered from the
process stream, e.g., by settling or filtration. The recovery of bioactive
material activity
(e.g., antibody titer or plaque forming units) in the spray drying process is
the product of the
physical recovery times the specific activity (measured activity per material
mass) of
recovered agent.
[0110] Physical recovery of powder particles can depend, e.g., on the amount
of
material retained or expelled by the spray-drying equipment and losses
incurred due to
particle size selection methods. For example, material containing the
bioactive material can
be lost in the plumbing, and on surfaces of the spray-drying equipment.
Solutions or
particles can be lost in the process, e.g., when an undesired agglomeration of
spray droplets
grows and falls out of the process stream or when under sized droplets dry to
minute
particles that are carried past a collection chamber in a process waste gas
stream. Process
yields (the percent recovery of input bioactive material through the process)
of the invention
can range, e.g., from about 40 percent to about 98 percent, about 90 percent,
or more.
[0111] Particles of a desired average size and size range, can be selected,
e.g., by
filtration, settling, impact adsorption, and/or other means known in the art.
Particles can be
sized by screening them through one or more filters with uniform pore sizes.
Large
particles can by separated by allowing them to fall from a suspension of
particles in a
moving stream of liquid or gas. Smaller particles can be separated by allowing
them to be
swept away in a stream of liquid or gas moving at a rate at which larger
particles settle.
Large particles can be separated by surface impact from a turning gas flow
that carries away
particles with less momentum.
[0112] Recovery of active bioactive material can be affected, e.g., by
physical
losses, agent disruption, denaturation, aggregation, fragmentation, oxidation,
and/or the lilce,
experienced during the spray-dry process. The methods of the invention offer
improved
recovery of bioactivity over the prior art, e.g., by providing spray dry
techniques that reduce
shear stress, reduce drying time, reduce drying temperatures, and/or enhance
stability. For
example, monoclonal antibodies spray dried by the methods of the invention can
experience
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less than 4 percent aggregation and fragmentation on initial production and
after in storage
for up to about 7 years at 4°C. Methods of the invention can provide
dried powder having
bioactive material substantially unchanged activity or viability compared to
the same
bioactive material in the formulation before high pressure spraying.
Administration of the Bioactive material
[0113] Where it is appropriate, the bioactive material of the invention can be
administered, e.g., to a mammal. Bioactive materials of the invention can
include, e.g.,
peptides, polypeptides, proteins, viruses, bacteria, antibodies, cells,
liposomes, and/or the
like, and as defined herein. Such agents can act as therapeutics, nutrients,
vaccines,
pharmaceuticals, prophylactics, and/or the like, that can provide benefits on
administration
to a patient, e.g., by gastrointestinal absorption, topical application,
inhalation, and/or
injection. Optionally, cells or tissues can come in contact with the bioactive
materials of the
invention to provide a biological effect or response.
[0114] The bioactive material can be administered to a patient by topical
application. For example, the powder particles can be mixed directly with a
salve, carrier
ointment, and/or penetrant, for application to the skin of a patient.
Alternately, the powder
particles can, e.g., be reconstituted in an aqueous solvent before admixture
with other
ingredients before application.
[0115] Bioactive materials of the invention can be administered by inhalation.
Dry
powder particles about 10 ~,m in aerodynamic diameter, or less, can be inhaled
into the
lungs for pulmonary administration. Optionally, powder particles about 20 ~.m,
and greater,
in aerodynamic diameter can be administered intranasally, or to the upper
respiratory tract,
where they are removed from the air stream by impact to the mucus membranes of
the
patient. The powder particles can alternately be reconstituted to a suspension
or solution for
inhalation administration as an aqueous mist.
[0116] Bioactive materials of the invention can be administered by injection.
The
powder particles can be administered directly under the skin of a patient
using, e.g., a jet of
high pressure air. More commonly, the powder particles can be, e.g.,
reconstituted with a
sterile aqueous buffer for injection through a hollow syringe needle. Such
injections can be,
CA 02564791 2006-10-27
WO 2005/123131 PCT/US2005/020792
e.g., intramuscular, intra venous, subcutaneous, intrathecal, intraperitoneal,
and the like, as
appropriate. Powder particles of the invention can be reconstituted to a
solution or
suspension with a bioactive material concentration of from less than about 1
mg/ml to about
500 mg/ml, or from about 5 mg/ml to about 400 mg/ml, or about 200 mg/ml, as
appropriate
to the dosage and handling considerations. Reconstituted powder particles can
be further
diluted, e.g., for multiple vaccinations, administration through IV infusion,
and the like.
COMPOSITIONS OF THE INVENTION
[0117] Compositions of the invention are generally bioactive materials, such
as
antibodies, in dry powders prepared using the methods of the invention.
Numerous
combinations of bioactive materials, processing steps, process parameters, and
composition
constituents, as described herein, are available to suit the intended use of
the composition.
[0118] The compositions of the invention provide, e.g., powder particles
containing
a bioactive material which are made by preparing an aqueous formulation of the
bioactive
material (e.g., a therapeutic antibody or vaccine) and a viscosity enhancing
agent, spraying
the formulation through a nozzle at high pressure to form a mist of fine
droplets, drying the
droplets to form powder particles, and recovering the particles, as is
described in the
Methods sections, above. In a particular embodiment of the composition, the
powder
particles contain antibodies as the bioactive material that can be
reconstituted into a 200
mg/ml solution, 400 mg/ml solution, or more concentrated solution, with the
antibodies
having less than about 3 percent aggregates or fragments. The compositions of
the
invention include, e.g., stable powder particles and highly concentrated
solutions of
bioactive materials with high purity and high specific activity. Powder
particles containing
viral bioactive materials can be prepared by high pressure spraying a
suspension of the
virus, sucrose, and a surface active agent. Particle compositions of viruses
are often
processed from liquid formulations with the virus present in an amount ranging
from about
101 TCIDso/mL to about 1012 TCll~so/mL, or from about 106 TCIDSo/mL to about
10~
TCIDSO/mL. Dried powder particle compositions of the invention can provide
virus present
in an amount, e.g., from about 101 TCIDSO/g to not more than 1012 TCIDso/g.
Dried powder
particle compositions can provide virus present in an amount, e.g., of about
101 TCIDSO/g,
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WO 2005/123131 PCT/US2005/020792
about 102 TCIDso/g, about 103 TC>Dso/g, about 104 TC>Dso/g, about 10s
TC>Dso/g, about
10~ TCIDso/g, about 107 TC)Dso/g, about 108 TC>Dso/g, about 10~ TC>Dso/g,
about lOlo
TC>l~so/g, or about 1011 TCIDso/g.
Powder Particles
[0119] Powder particles of the invention are dried droplets of the process
formulations of the invention. The particles include, e.g., stable bioactive
materials in a
dried matrix of excipients, such as the sugar, amino acid, surfactants, polyol
and/or polymer
viscosity enhancing agents. The particles range in average physical diameter
(size), e.g.,
from about 0.1 ~m to about 200 ~.m, about 1 ~,m to about 100 hum, about 2 ~m
to about 30
Vim, about 4 ~.m to about 20 ~m or 15 hum, or about 7 ~,m to about 10 Vim. The
bioactive
material can be present in the powder particles in a ratio ranging, e.g., from
less than about
1/100 to about 100/1, about 1/5 to about 5/1, or about 2/3 to about 3/2, or
about 1/l, with
respect to excipients, by weight. In one embodiment, powder particles of the
invention
average about 5 ~.m in diameter with about 55 weight percent of an antibody,
about 15
weight percent arginine, about 2 weight percent polyvinyl pylTOlidone, about
33 weight
percent sucrose, and about 5% moisture. In another embodiment, a composition
of the
invention comprises dry powder particles with about 55 weight percent of an
antibody,
about 21 weight percent arginine, about 1 weight percent polyvinyl
pyrrolidone, about 14
weight percent sucrose, and about 5% moisture. In another embodiment, the
composition of
dry powder particles includes, e.g., a live attenuated virus at about 0.01% by
weight, about
15 percent arginine, 70 percent polyol, and less than 5 percent moisture.
Bioactive materials
[0120] Bioactive materials of the composition (powder particles) include, for
example, antibodies, peptides, polypeptides, proteins, viruses, bacteria,
cells, liposomes,
and/or the like and as defined herein. Bioactive materials in the powder
particles of the
invention can be, e.g., highly pure and active at the time of drying the
powder particles, due
to the reduced shear stress, the low drying temperatures, protective
excipients, and the short
drying times used in their preparation. Bioactive materials are, e.g., stable
in the powder
particles due to the low initial process degradation and protective aspects of
the composition
37
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WO 2005/123131 PCT/US2005/020792
excipients. Bioactive materials of the composition can be, e.g., reconstituted
at high
concentrations without degradation due to the high surface to volume ratio of
the particles
and the solubility enhancements provided by the excipients of the composition.
[0121] Formulations, for high pressure spray-drying according the invention
contain, e.g., the bioactive materials of the invention in amounts ranging
from less than
about 1 mg/ml to about 400 mg/ml, from about 5 mg/ml to about 200 mg/ml, or
about 50
mg/ml. Bioactive materials in the dry powder particles of the invention can be
present in
amounts ranging, e.g., from less than about 0.1 weight percent to about 80
weight percent,
from about 40 weight percent to about 60 weight percent, or about 50 weight
percent.
Bioactive materials of the reconstituted composition can be present in
concentrations
ranging, e.g., from less than about 0.1 mg/ml to about 500 mg/ml, from about 5
mg/ml to
about 400 mg/ml, about 100 mg/ml to about 300 mg/ml, or about 200 mg/ml. In
one aspect
of the invention, the bioactive material is a virus present in the suspension
to be sprayed at a
titer ranging from about 2 log FFU/ml to about 12 log FFU/ml, or about 3 log
FFU (focus
forming units) to 13 log FFU per gram of dry powder particles.
Viscosit~Enhancin~ Agents
[0122] Viscosity enhancing agents of the composition include, e.g., polyols
and/or
polymers that can provide protection to bioactive materials against shear
stress when the
solutions or suspensions of the invention are sprayed at high pressure. The
viscosity
enhancing agents can ultimately become a significant part of the powder
particle bulk and
provide additional benefits. For example, the viscosity enhancing agents in
the particles
can, e.g., help stabilize the bioactive material by providing hydrogen bonding
replacement
for water molecules of hydration lost in drying, increase the solubility of
the particles for
quicker reconstitution at high concentrations, provide a glassy matrix to
retard reaction
kinetics, and physically block destabilizing molecules (such as oxygen) from
gaining access
to the bioactive material.
[0123] Polyols useful as viscosity enhancing agents should be, e.g.,
compatible with
the intended use of the composition. For example, viscosity enhancing agents
in particles
intended for injection into humans should be generally recognized as safe.
Viscosity
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WO 2005/123131 PCT/US2005/020792
enhancing polyols can include, e.g., trehalose, sucrose, sorbose, melezitose,
glycerol,
fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose,
palactose, glucose,
mannitol, xylitol, erythritol, threitol, sorbitol, raffinose, and/or the like.
Non-reducing
sugars are generally recommended, e.g., where the bioactive material is a
peptide, in order
to avoid chemical modification of the side chains.
[0124] Polymers useful as viscosity enhancing agents can include, e.g.,
starch,
starch derivatives, carboxymethyl starch, inulin, hydroxyethyl starch (HES),
dextran,
dextrin, polyvinyl pyrrolidone (PVP), human serum albumin (HSA), gelatin,
and/or the like.
Many polymers are, e.g., more viscous in solution by weight than polyols so
can often
provide adequate shear stress protection at lower concentrations.
[0125] Viscosity enhancing agents can be present in the formulations of the
invention before spray-drying in amounts between about 0.1 weight percent to
about 20
weight percent, between about 2 weight percent and ~ weight percent, or about
6 weight
percent. In many embodiments, polyol viscosity enhancing agents are present at
about 2 to
6 weight percent in the formulation, while polymer viscosity enhancing agents
are present at
about 0.5 to 2 weight percent. Viscosity enhancing agents are preferably
present in the
formulations of the inventions at concentrations sufficient to increase the
viscosity of the
formulation by about 5°70 or more, or by 0.05 centipoise or more.
Other Excipients
[0126] The compositions of the invention can include additional excipients
(e.g., not
solvent or the bioactive material) to provide appropriate characteristics and
benefits. For
example, the compositions can include surfactants, zwitterions, buffers, and
the like.
[0127] Surfactants can be included in the formulations of the invention, e.g.,
to
increase the solubility of composition constituents, and/or to reduce surface
tension.
Surfactants can, e.g., increase the suspension or solubility of certain
bioactive materials by
surrounding them with charged or hydrogen bonding groups. Surfactants can help
in
reconstitution of powder particles by, e.g., accelerating the dissolution of
the excipient
matrix on exposure to water. By reducing surface tension, surfactants can
reduce
aggregation and conformational changes that can occur with some bioactive
materials at the
39
CA 02564791 2006-10-27
WO 2005/123131 PCT/US2005/020792
air/liquid interface of droplets during spraying. Surfactants of the
formulations can include,
e.g., any appropriate surfactant, such as polyethylene glycol sorbitan
monolaurates,
polyoxyethylenesorbitan monooleates, or block polymers of polyethylene and
polypropylene glycol, e.g., Tween 80, Tween 20, or Pluronic F68. Surfactants
can be
present in the formulations in amounts between about 0.01 weight percent to
about 2 weight
percent, between about 0.02 weight percent and 0.5 weight percent, between
about 0.1
weight percent and 0.3 weight percent, or about 0.2 weight percent. Surface
active agents
can provide benefits in the control of droplet and particle sizes, as
described above.
[0128] Zwitterions, such as amino acids, can be included in the compositions,
e.g.,
as counter ions to charged groups of the bioactive materials or surfactants.
The presence of
these counter ions can, e.g., help the bioactive materials retain non-
denatured
conformations, prevent aggregation, and inhibit adsorption of charged
bioactive materials
onto surfaces of processing equipment. The zwitterions can, e.g., help protect
the bioactive
materials against deamidation reactions, act as antioxidants, and provide pH
buffering
capacity. Zwitterions of the invention can include, e.g., arginine, leucine,
histidine, glycine,
and/or the like. Zwitterions can be present in the powder particles of the
invention in
amounts ranging between about 0.1 percent and about 20 percent, between about
0.5
percent and about 15 percent, between about 1 percent and about 10 percent, or
about 7
percent of the total solids.
[0129] Buffers can be included in the compositions of the invention, e.g., to
control
pH, increase product stability, and/or to increase the comfort of
administration. Buffers of
the composition can include, e.g., phosphate, carbonate, citrate, glycine,
amino acids,
acetate, and the like.
EXAMPLES
[0130] The following examples are offered to illustrate, but not to limit the
claimed
invention.
CA 02564791 2006-10-27
WO 2005/123131 PCT/US2005/020792
EXAMPLE 1 - HIGH PRESSURE SPRAY DRYING OF ANTIBODIES
[0131] Antibodies are generally high pressure spray dried under the following
conditions to provide desired powder particles. Formulations of about 8%
monoclonal
antibody flowing at about 1-2 mL/min through a 150 hum nozzle at 1300 psi with
15- 25
mL/min nitrogen flow are sprayed into a drying chamber. The drying chamber
(Buchi 191
model) has a 30 m3/hr flow of nitrogen drying gas (3- 7% RH at 24°C)
from a chamber inlet
at 60°C to 80°C to an outlet at 40°C to 60°C.
[0132] An aqueous solution formulation is prepared to contain 8 weight percent
of
monoclonal antibody against RSV, 2 weight percent sucrose, 0.2 weight percent
PVP, 10
mM histidine, 0.5 weight percent arginine, and 0.2 weight percent Tween-20, pH
6Ø The
formulation is sprayed from a nozzle at about 1300 psi to provide droplets
with an average
diameter of about 10 Vim. The droplets are dried in a stream of dry nitrogen
gas ranging in
temperature from about 60°C inlet to about 45°C outlet to
produce powder particles with an
average diameter of about 4 ~,m and a moisture less than 5 percent. The powder
particles
are initially reconstituted into solutions with antibody concentrations of up
to 200 mg/ml
and with less than 3 percent total aggregates and fragments.
[0133] An aqueous solution formulation is prepared to contain 8 weight percent
of
monoclonal antibody against a~(33 integrin, 2 weight percent sucrose, 10 mM
histidine pH
6.0, 0.5 weight percent arginine, and 0.2 weight percent Tween-80. The
formulation is
sprayed from a nozzle at about 1300 psi to provide droplets with an average
diameter of
about 10 Vim. The droplets are dried in a stream of dry nitrogen gas ranging
in temperature
from about 60°C inlet to about 45°C outlet to produce powder
particles with an average
diameter of about 4 hum and a moisture less than 5 percent.
[0134] An aqueous solution was prepared to contain 8 weight percent of a
monoclonal antibody, 6 weight percent sucrose, 0.2 weight percent PVP, and 2
weight
percent arginine. The solution was sprayed from a nozzle at about 1150 psi to
provide
droplets with an average diameter of about 10 Vim. The droplets were dried in
a stream of
dry nitrogen gas ranging in temperature from about 60°C to about
45°C to produce powder
particles with an average diameter of about 4 ~m and a residual moisture less
than 5
41
CA 02564791 2006-10-27
WO 2005/123131 PCT/US2005/020792
percent. The powder particles were initially reconstituted into solutions with
antibody
concentrations of up to 500 mg/ml and with less than 3 percent total
aggregates and
fragments. Figure 8 shows the antibody after reconstitution at high
concentrations and
storage for nine days, or more, at 50°C. The powder particles remained
stable with trend
analysis predicting stability, with less than 3 percent aggregates, over about
7 years in
storage at 4°C, or for about 1.5 years in storage at 25°C.
[0135] In another example of stability for high pressure spray dried
formulations, an
aqueous solution was prepared to contain 8 weight percent of a monoclonal
antibody, 6
weight percent sucrose, 0.002% Tween 20, and 2 weight percent arginine. The
solution was
sprayed from a nozzle at about 1300 psi into an inlet nitrogen drying gas
temperature of
about 60°C, with a drying chamber outlet temperature of about
45°C. Stability data indicate
the dried powder particles should form only about 1.5% additional aggregates
after more
than 6 years in storage at 4°C or after about 2 years in storage at
25°C.
[0136] In another example, a low tonicity, fast dissolving formulation was
high
pressure spray-dried to prepare stable powder particles. An aqueous solution
was prepared
to contain 8 weight percent of a monoclonal antibody, 2 weight percent
sucrose, 0.008%
Tween 20, and 0.5 weight percent arginine for high pressure spraying with
atomizing
nitrogen at 1300 psi into an inlet nitrogen drying gas temperature of about
60°C, with a
drying chamber outlet temperature of about 45°C. The dried powder was
reconstituted to
an antibody concentration of 180 mg/ml with a dissolution time of only 10
minutes using
orbital shaking at room temperature. Such a formulation can have practical
benefits of
quick preparation for injection and reduced pain and irritation at the site of
injection.
Stability data indicate more than 2 years in storage at 4°C before the
fomnation of 2%
additional aggregates in the dried powder.
EXAMPLE 2 - HIGH PRESSURE SPRAY DRYING OF LIVE VIRUS
[0137] An aqueous solution was prepared of live influenza virus at about 7.5
log
FFU/ml in formula AV047r (5% sucrose, 2% trehalose, 10 mM methionine, 1%
arginine,
0.2% Pluronic F68, 50 mM KP04, pH 7.2) was high pressure sprayed at 1300 psi
into a
42
CA 02564791 2006-10-27
WO 2005/123131 PCT/US2005/020792
drying chamber with a 55°C inlet temperature. Reconstitution of the dry
powder showed no
significant viability loss with a titer of about 7.5 log FFU/ml. The
formulation required 23
days at a 37°C accelerated storage temperature to experience a 1 log
loss of viability.
EXAMPLE 3 - A HIGH PRESSURE SPRAY DRY SYSTEM
[0138] A high pressure spray drying system can include, e.g., a high pressure
pumping system to deliver formulation to a high pressure spray nozzle, and a
spray drying
system to carry droplets and particles in a stream of conditioned gasses. As
shown in Figure
9, formulation 90, with a bioactive material, is transferred from a holding
container to high
pressure spray nozzle 91 using high pressure pump 92. High pressure gas from
gas source
93 is pumped through high pressure gas pump 94 to atomize the formulation into
a fine mist
spray of droplets 95 into particle formation vessel 96. Temperature controlled
gas 97 is
drawn by fan 98 in a stream that displaced water vapor from the spray to dry
droplets 95
into powder particles 99. Powder particles 99 were transferred to secondary
drying
chamber 100 where residual moisture is removed to an acceptable level. The
powder
particle product settled into collection vessel 101 at the bottom of drying
chamber 100 for
recovery.
[0139] High pressure spraying can be accomplished in a variety of ways known
in
the art, such as by high pressure spraying directly from a high pressure
nozzle, atomizing
the spray with a jet of gasses, and/or high pressure spraying into a cold
fluid. For high
pressure spraying, the formulation can be fed to the nozzle by a high pressure
pump, such as
a HPLC pump, or by application of a high pressure gas on the holding
container. For
atomized spraying, a pressurized gas can be released from outlets near the
spray outlet
orifice to further disrupt and disperse the sprayed droplets. For spray freeze
drying, the
droplets can be sprayed in to a cold (e.g., about -~0°C, or less) gas
or liquid in the particle
formation vessel.
[0140] Drying the droplets with a temperature controlled gas can include
displacement of spray gasses and evaporation of water into a temperature,
humidity, and/or
pressure controlled gas. Fan 98 can draw a stream of gas 97 into the spray of
droplets 95 to
displace spray gasses, such as water vapor, and/or volatile solution
components.
43
CA 02564791 2006-10-27
WO 2005/123131 PCT/US2005/020792
Temperature controller 102 can be a heater or refrigeration system to adjust
the gas
temperature before it enters particle formation vessel 96. The gas can flow
through
humidity controller 103 (a condenser coil or desiccant) to remove moisture. A
vacuum
pump in fluid contact with the collection vessel can remove gasses from the
drying chamber
to speed evaporation from liquid droplets or to lyophilize frozen droplets.
Drying gasses
can be routed through filters, dryers, heat exchangers, activated charcoal
beds, or other
devices to recondition the gas for recycling through the particle formation
and drying
chambers. The process gasses can recirculate in a closed system of conduit or
the system
can be enclosed in an environmental control chamber. For example, the
recycling loop can
include an environmental control chamber, e.g., into which the entire spray
dry system has
been placed. Temperature and humidity sensors in the recirculating gasses can
be adapted
to regulate heating, cooling, andlor humidity control devices.
EXAMPLE 4 - ANTIBODY AMINO ACID SEQUENCES
[0141] The present invention includes spray drying of antibodies disclosed in:
U.S.
Patent number 5,824,307, "Human-Murine Chimeric Antibodies Against Respiratory
Syncytial Virus, to Johnson, et al., flied August 15, 1994; Johnson S, et al.
"Development
of a Humanized Monoclonal Antibody (MEDI-493) with Potent In Vitro and In Vivo
Activity Against Respiratory Syncytial Virus." J. Infect Dis. 1997
Nov;176(5):1215-24;
U.S. Patent number 6,656,467, "Ultra High Affinity Neutralizing Antibodies",
to Young et
al., filed January 26, 2001; U.S. Published Application 20030091584, Methods
of
Administering/Dosing Anti-RSV Antibodies for Prophylaxis and Treatment", by
Young,
filed November 28, 2001; U.S. Patent number 6,531,580 "Anti-av(33 Recombinant
Human
Antibodies and Nucleic Acids Encoding Same", to Huse et al., filed June 24,
1999; U.S.
Application number 20030166872, " Anti-av(33 Recombinant Human Antibodies,
Nucleic
Acids Encoding Same and Methods of Use", by Huse et al., filed November 25,
2002; Wu,
H. et al. "Stepwise In Vitro Affinity Maturation of Vitaxin, an av(33-Specific
Humanized
mAb", Proc Natl Acad Sci U S A. 1998 May 26;95(11):6037-42; and, U.S. Patent
Application Publication number 20040091486, "EphA2 Agonistic Monoclonal
Antibodies
44
CA 02564791 2006-10-27
WO 2005/123131 PCT/US2005/020792
and Methods of Use Thereof", by Kinch et al., filed May 12, 2003. Each of
these references
is hereby incorporated by reference in their entirety.
[0142] The following table includes preferred amino acid sequences for
antibodies
useful in formulations and methods of the invention.
Table 2 - Amino Acid Seguences of Antibodies
[0143] A) Sequences of Anti-RSV antibodies comprise one or more of the
following sequences, as published in application number 20030091584, and B)
Sequences
of Anti-av(33 antibodies comprise one or more of the following sequences, as
published in
U.S. patent number 6,531,580.
Table 2
Cross Reference
of Sequence
Identification
Numbers in This
Specification
to Those Found
in the Published
in Application
Number 20030091584
and U.S. Patent
Number 6,531,580
A. SEQ ID Sequence Source SEQ ID Numbers From Application
Numbers Number 20030091584
This Specification
SEQ ID NO 1 Heavy chain CDR1 SEQ ~ NO 1: TSGMSVG
SEQ ll~ NO 2 Heavy chain CDR SEQ ID NO 2: IWWDDKKDYNPSLKS
2
SEQ ID NO 3 Heavy chain CDR SEQ ID NO 3: SMITNWYFDV
3
SEQ ~ NO 4 Light chain CDR1 SEQ ID NO 4: KCQLSVGYNgI
SEQ ID NO 5 Light chain CDR2 SEQ ID NO 5: DTSKLAS
SEQ ID NO 6 Light chain CDR3 SEQ ID NO 6: FQGSGYPFT
SEQ ID NO 7 Heavy Chain VariableSEQ ID NO 7
Region
SEQ ID NO 8 Light Chain VariableSEQ >D NO 8
Region
SEQ ID NO 9 Heavy chain CDR1 SEQ ID NO 10: TAGMSVG
SEQ ID NO 10 Light Chain VariableSEQ 117 NO 11
Region
CA 02564791 2006-10-27
WO 2005/123131 PCT/US2005/020792
SEQ B7 NO 11 Heavy chain CDR2 SEQ m NO 19:
IWWDDKKHYNPSLI~D
SEQ m NO 12 Heavy chain CDR3 SEQ m NO 20: DNBFNFYFDV
SEQ m NO 13 Light chain CDR1 SEQ m NO 39: SASSRVGYMFi
SEQ m NO 14 Heavy Chain VariableSEQ ~ NO 48
Region
B. SEQ ms Sequence Source SEQ m From Patent
This Specification Number 6,531,580
SEQ m NO 15 Heavy chain CDR1 SEQ m NO 34: Gly-Phe-Thr-Phe-Ser-
Ser-Tyr-Asp-Met-Ser.
SEQ m NO 16 Light chain CDR3 SEQ m NO 90: Gln-Gln-Ser-Gly-Ser-
Trp-Pro-Leu-Thr.
SEQ ~ NO 17 Heavy chain CDR2 SEQ m NO 102: Lys-Val-Ser-Ser-Gly-
Gly-Gly-Ser-Thr-Tyr-Tyr-Leu-Asp-Thr-
Val-Gln-Gly.
SEQ m NO 18 Heavy chain CDR3 SEQ m NO 106: His-Leu-His-Gly-Ser-
Phe-Ala-Ser
SEQ m NO 19 Light chain CDR1 SEQ m NO 110: Gln-Ala-Ser-Gln-Ser-
lle-Ser-Asn-Phe-Leu-His
SEQ ~ NO 20 Light chain CDR2 SEQ m NO 112: Tyr-Arg-Ser-Gln-Ser-
Ile-Ser.
[0144] Preferred antibodies against RSV for use in the formulations and
methods of
the invention include those with heavy chain peptide sequences including CDR1
SEQ m
NOs 1 or 9, CDR2 SEQ m NOs 2 or 11, and/or CDR3 SEQ m NOs 3 or 12; or
conservative variations thereof. More preferred antibodies against RSV include
heavy
chain variable regions with peptide sequence SEQ m NOs 7 or 14, or
conservative
variations thereof.
46
CA 02564791 2006-10-27
WO 2005/123131 PCT/US2005/020792
[0145] Preferred antibodies against RSV for use in the formulations and
methods of
the invention include those with light chain peptide sequences including CDR1
SEQ ID
NOs 4 or 13, CDR2 SEQ ID NO 5, and/or CDR3 SEQ ID NO 6; or conservative
variations
thereof. More preferred antibodies against RSV include light chain variable
regions with a
peptide sequence of SEQ ID NOs 8 or 10, or conservative variations thereof.
[0146] Most preferred antibodies against RSV for use in the formulations and
methods of the invention include those with heavy chain peptide sequences
including CDR1
SEQ ID NOs 1 or 9, CDR2 SEQ ID NOs 2 or 11, and/or CDR3 SEQ ID NOs 3 or 12;
and,
with light chain peptide sequences including CDR1 SEQ IDs 4 or 13, CDR2 SEQ ID
5,
and/or CDR3 SEQ ID 6; or conservative variations thereof.
[0147] With regard to antibodies against integrin av(33, preferred antibodies
for use
in the formulations and methods of the invention include those with heavy
chain peptide
sequences including CDR1 SEQ ID NO 15, CDR2 SEQ ID NO 17, and/or CDR3 SEQ ID
NO 18; or conservative variations thereof. Preferred antibodies against av(33
include those
with light chain peptide sequences including CDR1 SEQ ID NO 19, CDR2 SEQ ID NO
20,
and/or CDR3 SEQ ID NO 16; or conservative variations thereof. Most preferred
antibodies
include heavy chain peptide sequences including CDR1 SEQ ID NO 15, CDR2 SEQ ID
NO
17, and CDR3 SEQ ID NO 18; and light chain peptide sequences including CDR1
SEQ ID
NO 19, CDR2 SEQ ID NO 20, and CDR3 SEQ ~ NO 16; or conservative variations
thereof.
EXAMPLE 5 - FORMULATIONS FOR SPRAYING WITH SOLVENTS
[0148] The data table below describes the formulation combination that was
used
with the EtOH spray drying process.
47
CA 02564791 2006-10-27
WO 2005/123131 PCT/US2005/020792
FormulationMab: F~ccips ProcessPowderMC PowderParticleParticleOther
F~cptl.
# Initial Soln Loss Yield DensitySiae Siae Parameters
Concs. % % % /mL Dv50 Dv90
M493SD-% wtv A Theor um um
1 8*: 2 Sucn 0.5 0.31 60.72.13 5.07 11.30 60C Inlet
a Ar
2 8: 1 Sucr: 2 0.22 74.62.180.32 3.51 6.19 60C Inlet
Mann
3 8: 2 Mann : 0.5 -0.3473.92.360.30 3.51 5.91 60C Inlet
Ar
4 8: 1 Sucr : 2 0.06 65.71.420.36 3.11 5.66 60C Inlet
Mann : 0.5 Leu
8: 2 Sucr : 0.5 0.09 62.52.660.31 3.68 6.05 60C Inlet
Leu
6 8: 2 Sucr : 0.5 0.78 54.72.320.14 4.04 7.31 60C Inlet
Ar , 20 % EtOH
7 8: 1 Sucr : 2 0.07 40.01.750.13 4.30 7. 60C Inlet
Mann : 0.5 Leu i8
+ 20 % EtOH
8 8; 2 Sucr: 0.5 0.45 49.2 0.38 60C Inlet
Ar +30% Water
9 8:1 Sucr:2Mann:10.14 26.9 0.07 60Clnlet
Leu+20% EtOH
9b 8: 1 Sucr : 2 68.6 0.32 60C Inlet
Mann : 1 Leu
8: 1 Sucr : 2 0.14 23.8 0.04 60C Inlet
Mann : 1.5 Leu
+ 20% EIOH
11 8: 1 Sucr : 2 0.13 19.2 0.06 60C Inlet
Mann : 2 Leu
+ 20% EtOH
12a 8: 1 Sucr: 2 57.7 0.29 4o Solids
Mann : 1 Leu 60C Inlet
12b 8: 1 Sucr : 2 46.4 0.28 4 % Solids
Mann : 1 Leu 90C Inlet
13 8: 1 Sucr: 2 5.2 0.02 2o Solids
Mann : 1 Leu, 50.o EtCH
50% EtON
*'8'=8% mAbconcentrationinformulation
[0149] It is understood that the examples and embodiments described herein are
for
illustrative purposes only and that various modifications or changes in light
thereof will be
suggested to persons skilled in the art and are to be included within the
spirit and purview of
this application and scope of the appended claims.
[0150] While the foregoing invention has been described in some detail for
purposes
of clarity and understanding, it will be clear to one skilled in the art from
a reading of this
disclosure that various changes in form and detail can be made without
departing from the
true scope of the invention. For example, all the techniques and apparatus
described above
can be used in various combinations without undue experimentation.
[0151] All publications, patents, patent applications, and/or other documents
cited in this
application are incorporated by reference in their entirety for all purposes
to the same extent
as if each individual publication, patent, patent application, and/or other
document were
individually indicated to be incorporated by reference for all purposes.
48
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