Note: Descriptions are shown in the official language in which they were submitted.
CA 02154164 2004-03-04
EXCIPIENT STABILIZATION OF POLYPEPTIDES
TREATED WITH ORGANIC SOLVENTS
Field of the Invention
This invention relates to the use of excipients to
stabilize both dry and aqueous formulations of polypeptides
treated with organic solvents.
Background of the Invention
Pharmaceutical preparations of polypeptides are
sensitive to denaturation and degradation upon formulation
and storage. Polyols have been used to stabilize proteins
and other macromolecules in aqueous formulations and in air
I5 drying or lyophilization from aqueous solutions.
U.S. 4,297,344 discloses stabilization of coagulation
_ factors II and VIII, antithrombin III, and plasminogen
against heat by adding selected amino acids such as
glycine, alanine, hydroxyproline, glutamine, and
aminobutyric acid, and a carbohydrate such as a
monosaccharide, an oligosaccharide, or a sugar alcohol.
European Patent Application Publication No. 0 303 746
discloses stabilization of growth promoting hormones with
polyols consisting of non-reducing sugars, sugar alcohols,
sugar acids, pentaerythritol, lactose, water-soluble
dextrans, and Ficoll; amino acids, polymers of amino acids
having a charged side group at physiological pH, and
choline salts.
European Patent Application Publication No. 0 193 917
discloses a biologically active composition for slow
release characterized by a water solution of a complex
between a protein and a carbohydrate.
Australian Patent Application No. AU-A-30771J89
discloses stabilization of growth hormone using glycine and
mannitol.
U.S. 5,096,885 discloses a formulation of hGH for
lyophilization containing glycine, mannitol, a non-ionic
surfactant, and a buffer.
The use of polyethylene glycols to stabilize proteins
is reviewed in Pharm. Res. 8:285-291, 1991.
*-trademark
WO 94/19020 rF _ ~ PCT/LJS94/01666
2
Examples of the use of trehalose and other polyols for
the stabilization of proteins during drying in aqueous
systems include the following.
U.S. 4,891,319 discloses the preservation of sensitive
proteins and other macromolecules in aquecus systems by '
drying at ambient temperatures and at atmospheric pressure
in the presence of trehalose.
U.S. 5,149,653 discloses a method of preserving live
viruses in an aqueous system by drying in a frozen state or
at ambient temperature, in the presence of trehalose.
Polyols have also been used to stabilize dry drug
formulations as, for example, in WO 8903671, filed May 5,
1989, which discloses the addition of a stabilizer such a
gelatin, albumin, dextran, or trehalose to a mixture of a
finely powdered drug suspended in a oily medium.
Treatment of a polypeptide with an organic solvent
such as methylene chloride poses the problem of
denaturation of the polypeptide of interest. Thus, it is
an object of this invention to provide a method for
stabilizing polypeptides in aqueous formulations treated
with organic solvents.
It is another object of the invention to stabilize dry
polypeptides treated with organic solvents.
It is another object of the invention to provide a
method for stabilization of encapsulated polypeptides.
It is another object of the invention to provide a
polypeptide stabilized by an excipient for use in a
controlled release formulation, wherein the polypeptide is
treated with an organic solvent.
~L~ARY OF THE INVENTION
One aspect of the invention is a method of stabilizing
a polypeptide against denaturation when treated with an
organic solvent, wherein the method comprises admixing the
polypeptide with a polyol, wherein the molecular weight of
the polyol is,less than about 70,000 kD.
Another aspect of the invention is a method of
formulating a polypeptide comprising
11
~O 94/19020 . - ' .' PCT/US94/01666
3
a) admixing the polypeptide in an aqueous solution
with a polyol having a molecular weight less than about
70,000 kD; and
b) treating the polypeptide in the aqueous solution
with an organic solvent.
Another aspect of the invention is a method of
formulating a dry polypeptide for controlled release
comprising
a) admixing the polypeptide with an excipient, wherein
said excipient is a polyol having a molecular weight less
than about 70,000 kD; and
b) treating the product of step a) with an organic
solvent.
Another aspect of the invention is a composition for
controlled release of a polypeptide comprising a
polypeptide admixed with an excipient, the excipient being
a polyol having a molecular weight less than about 70,000
kD, wherein the polypeptide admixed with the excipient is
treated with an organic solvent and is encapsulated in a
polymer matrix.
DETAILED DESCRIPTION OF THE INVENTION
A. DEFID1ITIOI'TS
The term "polyol" as used herein denotes a hydrocarbon
including at least two hydroxyls bonded to carbon atoms.
Polyols may include other functional groups. Examples of
polyols useful for practicing the instant invention include
sugar alcohols such as mannitol and trehalose, and
polyethers.
The term "polyether" as used herein denotes a
hydrocarbon containing at least three ether bonds.
Polyethers may include other functional groups. Polyethers
useful for practicing the invention include polyethylene
glycol (PEG).
The term "dry polypeptide" as used herein denotes a
polypeptide which has been subjected to a drying procedure
such as lyophilization such that at least 50~ of moisture
has been removed.
WO 94/19020 4 PCT/iJS94/01666~
X15 ~~.~ 4
The term "encapsulation" as used herein denotes a
method for formulating a therapeutic agent such as a
polypeptide into a composition useful for controlled
release of the therapeutic agent. Examples of
encapsulating materials useful in the instant inven~ion "
include polymers or copolymers of lactic and glycolic
V
acids, or mixtures of such polymers and/or copolymers,
commonly referred to as "polylactides."
The term "admixing" as used herein denotes the
addition of an excipient to a polypeptide of interest, such
as by mixing of dry reagents or mixing of a dry reagent
with a reagent in solution or suspension, or mixing of
aqueous formulations of reagents.
The term "excipient" as used herein denotes a non-
therapeutic agent added to a pharmaceutical composition to
provide a desired consistency or stabilizing effect.
The term "organic solvent" as used herein is intended
to mean any solvent containing carbon compounds. Exemplary
organic solvents include methylene chloride, ethyl acetate,
dimethyl sulfoxide, tetrahydrofuran, dimethylformamide, and
ethanol.
"Treating" a polypeptide with an organic solvent as
used herein refers to mixing a dry polypeptide with an
organic solvent, or making an emulsion of a polypeptide in
an aqueous formulation with an organic solvent, creating an
interface between a polypeptide in an aqueous formulation
with an organic solvent, or extracting a polypeptide from
an aqueous formulation with an organic solvent.
"Polypeptide" as used herein refers generally to
peptides and proteins having more than about 10 amino
acids.
B. GENERAL METHODS
In general, both aqueous formulations and dry
polypeptides may be admixed with an excipient to provide a
stabilizing effect before treatment with an organic
solvent. An aqueous formulation of a polypeptide may be a
polypeptide in suspension or in solution. Typically an
aqueous formulation of the excipient will be added to an
aqueous formulation of the polypeptide, although a dry
~O 94119020 , $ ~ ,. , : pCT/US94/01666
excipient may be added, and vice-versa. An aqueous
formulation of a polypeptide and an excipient may be also
dried by lyophilization or other means. Such dried
formulations may be reconstituted into aqueous formulations
5 before treatment with an organic solvent.
The excipient used to stabilize the polypeptide of
interest will typically be a polyol of a molecular weight
less than about 70,000 kD. Examples of polyols that maybe
used include trehalose, mannitol, and polyethylene glycol.
Typically, the mass ratio of trehalose to polypeptide will
be 100:1 to 1:100, preferably 1:1 to 1:10, more preferably
1:3 to 1:4. Typical mass ratios of mannitol to polypeptide
will be 100:1 to 1:100, preferably 1:1 to 1:10, more
preferably 1:1 to 1:2. Typically, the mass ratio of PEG to
polypeptide will be 100:1 to 1:100, preferably 1:1 to 1:10.
Optimal ratios are chosen on the basis of an excipient
concentration which allows maximum solubility of
polypeptide with minimum denaturation of the polypeptide.
The formulations of the instant invention may contain
a preservative, a buffer or buffers, multiple excipients,
such as polyethylene glycol (PEG) in addition to trehalose
or mannitol, or a nonionic surfactant such as Tween~
surfactant. Non-ionic surfactants include a polysorbate,
such as polysorbate 20 or 80, etc., and the poloxamers,
such as poloxamer 184 or 188, Pluronic~ polyols, and other
ethylene/polypropylene block polymers, etc. Amounts
effective to provide a stable, aqueous formulation will be
used, usually in the range of from about 0.1~(w/v) to about
30~(w/v).
Buffers include phosphate, Tris, citrate, succinate,
acetate, or histidine buffers. Most advantageously, the
buffer is in the range of about 2 mM to about 100 mM.
Preferred buffers include sodium succinate and potassium
phosphate buffers.
Preservatives include phenol, benzyl alcohol, meta-
cresol, methyl paraben, propyl paraben, benzalconium
chloride, and benzethonium chloride. The preferred
preservatives are 0.2-0.4~(w/v) phenol and 0.7-1~(w/v)
benzyl alcohol, although the type of preservative and the
concentration range are not critical.
WO 94/19020 ~ ~, ~ . PCT/US94/01666
6
In general, the formulations of the subject invention
may contain other components in amounts not detracting from
the preparation of stable forms and in amounts suitable for
effective, safe pharmaceutical administration. For
example, ether pharmaceutically acceptable excipients well
known to those skilled in the art may form a part of the
subject compositions. These include, for example, various
bulking agents, additional buffering agents, chelating
agents, antioxidants, cosolvents and the like; specific
examples of these could include trihydroxymethylamine salts
("Tris buffer"), and disodium edetate.
Polypeptides of interest include glycosylated and
unglycosylated polypeptides, such as growth hormone, the
interferons, and viral proteins such as HIV protease and
gp120.
The stabilized polypeptide of the instant invention
may be formulated for sustained release, especially as
exposure to organic solvents is a common step in many of
such preparations. Suitable examples of sustained-release
preparations include semipermeable matrices of solid
hydrophobic polymers containing the polypeptide, which
matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices
include polyesters, hydrogels [e. g., poly(2-hydroxyethyl-
methacrylate) as described by Langer, et al., J. Biomed.
Mater. Res., 15:167-277 (1981) and Langer, Chem. Tech.,
12:98-105 (1982) or poly(vinylalcohol)], polylactides (U. S.
Pat No. 3,773,919, EP 58,481), copolymers of L-glutamic
acid and gamma ethyl-L-glutamate (Sidman, et al.,
Biopolymers, 22:547-556 [1983]), non-degradable ethylene-
vinyl acetate (Langer, et al., supra), degradable lactic
acid-glycolic acid copolymers such as the Lupron DepotTM
(injectable microspheres composed of lactic acid-glycolic .
acid copolymer and leuprolide acetate), and poly-D-(-)-3-
hydroxybutyric acid (EP 133,988). While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable
release of molecules for over 100 days, certain hydrogels
release polypeptides for shorter time periods. When
encapsulated polypeptides remain in the body for a long
time, they may denature or aggregate as a result of
~O 94/19020 PCT/US94/01666
7
exposure to moisture at 37°C, resulting in a loss of
biological activity and possible changes in immunogenicity.
Rational strategies can be devised for polypeptide
stabilization depending on the mechanism involved. For
example, if the aggregation mechanism is discovered to be
intermolecular S-S bond formation through thio-disulfide
interchange, stabilization may be achieved by modifying
sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives,
and developing specific polymer matrix compositions.
Sustained-release ligand analogs or antibody
compositions also include liposomally entrapped
polypeptides. Liposomes containing polypeptides are
prepared by methods known per se: f7E 3,218,121; Epstein,
et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985);
Hwang, et a3., Proc. Natl. Acad. Sci. USA, 77:4030-4034
(1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP
142,641; Japanese patent application 83-118008; U.S. Pat.
No. 4,485,045 and U.S. Pat. No. 4,544,545; and EP 102,324.
Ordinarily the liposomes are of the small (about 200-800
Angstroms) unilamelar type in which the lipid content is
greater than about 30 mol. ~ cholesterol, the selected
proportion being adjusted for the optimal ligand analogs
therapy. Liposomes with enhanced circulation time are
disclosed in U.S. Pat. No. 5,013,556.
EXpERTMFNTAL EX_AMpLES
EXAMPLE I.
Stabilization of Ac~ruP~"~ Formulations
Recombinant human growth hormone (hGH) and recombinant
human gamma-interferon (hIFN-y) were formulated with
various excipients for analysis of the excipient effects on
stabilization in the organic solvent, methylene chloride.
Optimal formulations were generally those that yielded the
maximum soluble polypeptide concentration and the greatest
recovery of native polypeptide after treatment with
methylene chloride. The maximum solubility of hGH in each
solution was determined through the continuous addition of
hGH lyophilized in ammonium bicarbonate buffer to the
WO 94/19020 PCT/US94/01666
8
solution and the solubility limit was defined as the
concentration at which addition of polypeptide resulted in
precipitation. The maximum solubility of hIFN-Y was
measured by adding a concentrated stock solution (264 mg/ml
hIFN-'y, 10 mM Na succinate, pH 5; to~concentrated excipient '
solutions. The apparent solubility limit of hIFN-y was not
observed for any of the formulations at these conditions, '
but long term storage of the stock solution did result in
precipitation as the result of a pH increase (final
solution, pH 6). Both polypeptide formulations were tested
for stability in methylene chloride by addition of 100 ~1
of the polypeptide solution to 1 ml of methylene chloride.
The mixture was then sonicated for 30 sec. After
sonication, the polypeptide was extracted from the organic
phase by dilution into 50 ml of excipient-free buffer (50
mM phosphate buffer, pH 8 for hGH; 10 mM succinate buffer,
pH 5 for hIFN-y). The amount of soluble polypeptide
recovered was determined by ultraviolet absorbance
measurements and the amount of monomeric polypeptide was
assessed by size exclusion chromatography.
Both polypeptides were tested for stability with
trehalose, mannitol, carboxymethylcellulose (CMC), Tween~
20 surfactant, dextran, gelatin, and polyethylene glycol.
Previous studies with hGH indicated that formulations
containing an equal mass ratio of polypeptide and mannitol
stabilized the polypeptide from denaturation and provided a
maximum soluble polypeptide concentration of 200 mg/ml (100
mM phosphate, 200 mg/ml mannitol, pH 8). Trehalose ' '
formulations containing mass ratios of excipient to
polypeptide of 1:4 and 1:3 yielded the highest
concentration of soluble polypeptide, 400 mg/ml and 300
mg/ml, respectively. In addition, when the lyophilized
polypeptide in these formulations was treated with
methylene chloride, complete recovery of soluble monomeric
hGH was achieved. hGH formulations containing mannitol or _
mannitol with PEG resulted in similar recovery of monomeric
hGH, but the mass ratio (excipient/polypeptide) required to
prevent denaturation was greater than that of the trehalose
formulations (mannitol: 1:1; mannitollPEG 1:1 or 1:2;
trehalose: 1:3 or 1:4) (Table I). Therefore, trehalose
~154~64
~O 94/19020 . PCT/US94/01666
9
provided high hGH solubility and protection from
denaturation in methylene chloride at a lower mass
concentration. In the absence of excipients, the
solubility of hGH was much lower (about 106 mg/ml) and the
polypeptide °Nas more susceptible to denaturation.
Table I
Methylene chloride testing
of nh"o..."~.. s.nv r_~~__~ _
as v
Formulations Soluble Monomer yV Maximum Solubilityc
Recoveredb (mg/ml)
(mg/ml)
100 mg/ml PEG 12.2 96.4
(3350 MW)
50 m /ml PEG 34,7 89.6
m /ml PEG 37.2 128.3
100 m /mI Mannitol 66.0 gg.p
50 m /ml Mannitol 46.2 106
10 m /ml Mannitol 56.0 106
100 m /ml Dextran 34.6 112.6
70
50 m /ml Dextran 70 64.6 146.1
10 m /ml Dextran 70 38.4 167.1
2~ CMC 44.7 91.9
100 m /m1 Trehalose 113.9 267.3
50 m /ml Trehalose 82.8 275
10 m /ml Trehalose 92.0 267.3
4 mg/ml PEG (3350 102.6 243.7
MW),
96 m /ml Mannitol
10 mg/ml PEG (3350 104.0 184
MW), 90 m /ml Mannitol
mg/ml PEG (3350 139.9 240
MW)
80 m /ml Mannitol
2~ Gelatin 21.9 70.5
100 mg/ml PEG 69.2 131.5
(1000 MW)
50 m /ml PEG 84.3 246.5
10 m /ml PEG 126.5 226.3
4 mg/ml PEG (1000 122.3 230.3
MW)
96 m /ml Trehalose
10 mg/ml PEG (1000 58.4 218.7
MW)
90 m /ml Trehalose
20 mg/ml PEG (1000 75.3 207.5
MW)
80 m lml Trehalose
~ No excipient 65 0 ~n~ ~
10
a All solutions contained 10 mM NaP04, pH 8.
. b Polypeptide extracted out of methylene chloride tfraction of
total) as determined by absorbance at 278 nm multiplied by the
fraction of monomer recovered from SEC-HPLC. Polypeptide treated at
15 maximum solubility.
c Maximum solubility was defined as the maximum amount of
unformulated hGH that would dissolve in each buffer.
For studies with hIFN-y, both mannitol and trehalose
20 were the best excipients tested. When mannitol was used at
WO 94/19020 ~ ~ ~ '~ PCT/US94/0166
a mass ratio (excipient/polypeptide) of 1:3, the amount of
soluble dimer in solution (as determined by size exclusion
chromatography) after methylene chloride treatment was
equivalent to the amount in the starting material.
5 However, the mannitol formulations yielded less than 6u'~ '
recovery of total soluble polypeptide. In contrast, the
trehalose formulation with a mass ratio of 1:2.5 gave an '
80~ recovery of total soluble polypeptide and the same
fraction of soluble dimer (as determined by size exclusion
10 chromatography, denoted native SEC-HPLC) as the starting
material (Table II). Excipient-free polypeptide
formulations treated with methylene chloride retained 10~
of the initial soluble dimer (as determined by native SEC-
HPLC) after methylene chloride treatment and the total
soluble polypeptide recovery was less than 60~. When
assayed by size exclusion chromatography in 0.2M NaP04/0.1~
SDS at pH 6.8 (denoted SDS SEC-HPLC), all formulations were
greater than 99~ monomer before and after methylene
chloride treatment.
For both polypeptides, a dramatically lower recovery
of monomeric polypeptide was observed after methylene
chloride treatment for all formulations containing Tween~
20 surfactant. Although other surfactants have not been
studied, it is likely that hydrophobic molecules such as
Tween~ 20 surfactant stabilize the denatured polypeptides
while sugars such as mannitol and trehalose stabilize the
native polypeptide.
Table II
Methylene chloride testing
of aqueous hIFN-v formv.lationR
Formulations % Soluble % Intact Soluble
Polypeptide Dimerc Dimerd
Recoveredb
0.01 Tween~ 20 * 36.1 49.0 11.1
0.01 Tween~ 20 * 59.0 69.0 25.6
62 m /ml Mannitol
5 m /ml Mannitol 58.3 72.7 56.8
50 m /ml Mannitol 62.9 83.4 70.3
5 m /ml Trehalose 117 34.2 53.6
50 m /ml Trehalose 75.6 61.3 62.1
1~ CMC 78.2 62.5 65.5
[No excipient ~ 51.6 ~ 6.0 7
~.~~4I64
~O 94/19020 ~ PCT/US94/01666
11
a All solutions with excipient contained 134 mg/ml hIFN-y,10 mM
sodium succinate, pH 5; "no excipient" formulation contained 256.3
mg/ml protein, 10 mM sodium succinate, pH 5Ø
b Polypeptide extracted out of methylene chloride (fraction of
total) as determined by absorbance at i80 nm.
~ Amount of intact dimer measured by SEC-HPLC native method. All
formulations yielded >99$ monomer when assayed by the SEC-HPLC SDS
method.
d Soluble dimer concentration (mg/ml) based on the amount of soluble
polypeptide recovered and the fraction of dimer (native SEC-HPLC
method).
* Polypeptide concentration in these formulations was 62.8 mg/ml.
Example II
Stabs ~ ization of Drv amd
Aaueous Formulations for Encap~~ilation
In the development of a long acting formulation for
recombinant human growth hormone the use of a biodegradable
polymeric matrix for sustained release of hGH was
investigated. The polymer used for this application was a
copolymer of lactic and glycolic acids which is often
referred to as poly(lactic/glycolic acid) or PLGA. To
incorporate hGH into this polymer, the PLGA must be
dissolved in a water immiscible solvent. The most commonly
used solvent for dissolution of PLGA has been methylene
chloride which provides both water immiscibility and PLGA
solubility.
In general, for production of,hGH-PLGA microspheres,
the polypeptide was added to a solution of methylene
chloride containing PLGA. In initial studies, the
polypeptide was added in the form of a milled lyophilized
powder. After polypeptide addition, the methylene chloride
solution was then briefly homogenized and the solution was
added to an emulsification bath. This process resulted in
the extraction of methylene chloride with the concomitant
formation of PLGA microspheres containing hGH. The
polypeptide released from these microspheres was then
studied to determine the integrity of hGH after
incorporation into the microspheres. Assessment of
released hGH was performed by analytical size exclusion
chromatography (SEC-HPLC) as well as other techniques.
Size exclusion chromatography indicated that hGH was
released from the PLGA microspheres in the form of the
WO 94/19020 f PCT/US94/01666r
12
native monomer, aggregates, and an unknown structure which
eluted between the monomer and dimer. The unknown
polypeptide structure has been extensively studied and has
been shown to be a conformational variant of hGH. In
addition, the ::ame aggregates and conformational variant
can be obtained by treatment of hGH with methylene
chloride. Thus, the use of methylene chloride in the '
process may cause denaturation and aggregation of hGH.
The release of monomeric native hGH from the PLGA
microspheres is required for a successful long acting
formulation. Previous studies investigated several organic
solvents as alternatives to methylene chloride. This
research indicated that hGH was susceptible to damage by
several organic solvents. Since methylene chloride
provided the desired solvent properties (i.e. water
immiscibility, PLGA dissolution, etc.) for PLGA microsphere
production and other solvents did not significantly improve
hGH stability, methylene chloride was chosen for the
production of the PLGA microspheres. The polypeptide used
for the solvent study and in the PLGA production process
was formulated and lyophilized in ammonium bicarbonate
buffer at pH 7. Therefore, this study was performed to
develop formulations which would stabilize hGH during the
production of the PLGA microspheres.
A. Methods
1. Pretaaration of hGH Formulations
For development of a methylene chloride stable
formulation, hGH lyophilized in ammonium bicarbonate was
reconstituted in the desired buffer and allowed to
dissolve. Undissolved polypeptide was removed by
centrifugation at 13,000 rpm for 1 min.
For each lyophilization, indicted below, the hGH
concentration was 10 mg/ml. The residual moisture of these
formulations was not determined, but the same
lyophilization cycle was used in each case.
Milling of lyophilized protein was performed with a
pressure driven impaction mill and resulted in a fine
particulate of hGH.
2. M~thylene Chloride Testing of hGH Formulations
0 94/19020 ~
PCT/LTS94/01666
13
The effect of methylene chloride on hGH stability was
determined by adding hGH to a solution of methylene
chloride. For solid hGH conditions, the ratio of
polypeptide mass (mg) to volume of organic solvent (ml) was
40 mg/ml. For the aqueous hGH conditions, 100 ~.1 of hGFi in
a buffered solution was added to 1.0 ml of methylene
chloride to assess the effects of each buffer system on
stabilization of hGH in methylene chloride. After
polypeptide addition, the samples were sonicated for 30
seconds in a 47 kHz bath sonicator (Cole Parmer, Model
08849-00) to simulate the homogenization step in the
microsphere production process. If the formulation
stabilized hGH against denaturation in this test, it was
further assessed by homogenization in methylene chloride.
After sonication or homogenization, the polypeptide was
extracted from the methylene chloride by dilution into a 50
fold excess of 5 mM NaHP04, pH 8. The amount and quality
of the polypeptide extracted in this step was determined by
polypeptide concentration measurements (absorbance at 278
nm) and size exclusion HPLC (SEC-HPLC). The preferred
stable formulation was one that yielded the maximum
recovery of monomeric polypeptide without the formation of
conformational variants or aggregates larger than dimers.
B. Results
1. Fxc;p;ent Stud; Ps of hGH Sta~h;lization
Initial studies of hGH lyophilized in ammonium
bicarbonate investigated the solubility of the polypeptide
in different buffers at various pH conditions. From these ~ -
studies, it was determined that hGH had the maximum
stability and solubility in phosphate buffer (5 - 10 mM) at
pH 8, and thus additional studies were performed with hGH
in this buffer.
Initial attempts to prevent aggregation of hGH
utilized Tween~ 80 surfactant in the formulation buffer.
As shown in Table III, methylene chloride testing of these
aqueous formulations indicated that low Tween~ surfactant
concentrations (0.1~ Tween~ 80 surfactant) with 10 mg/ml
mannitol provided good recovery of soluble monomeric
polypeptide. However, the best results in this experiment
were obtained for hGH which was formulated in 10 mg/ml
WO 94/19020 ~ ~ ~ ~ 14 PCT/LJS94/01666
mannitol without Tween~ 80 surfactant (5 mM NaHP04, pH 8).
Higher concentrations of Tween~ surfactant in the
formulation buffer resulted in increased aggregation and
decreased recovery of soluble polypeptide. For each case
shown in Table III, the formulations provided greater '
stabilization of hGH than the milled polypeptide which was
lyophilized in ammonium bicarbonate.
Table III
Methylene chloride testing
of aqueous hGH formulations
Soluble
Polypeptide
(Mass
Fraction
of
Total)
% % % % % %
FormulationsPolypeptidebAreac TrimerDimer Inter- Monomer
Recovered Recovery mediate
1~ Tween~ 85.7 90.0 0.5 3.4 1.1 94.9
80
0.1~ Tween~ 70.9 98.3 2.0 3.6 1.8 92.6
80
1~ Tween~ 65.0 97.8 3.3 3.4 3.4 90.0
80
10 mg/ml
Mannitol
0.1$ Tween~ 70.9 98.3 0.0 2.2 0.0 97.8
80
10 mg/ml
Mannitol
10 mg/ml 97.6 101.1 0.0 2.6 0.0 97.4
PEG
(3350 MW)
10 mg/ml 76.4 97.7 1.7 2.8 1.6 93.9
PEG
10 mg/ml
Mannitol
5 mM NaP04, 55.3 99.4 0:0 3.2 0.0 96.8
H 8
5 mM NaP04, 91.7 99.8 0.0 1.8 0.0 98.2
pH 8
10 mg/ml
Mannitol
All solutions contain 5 mM NaP04, pH 8
Polypeptide extracted out of methylene chloride (fraction of total)
as determined by absorbance at 278 nm.
SEC-HPLC results for polypeptide extracted into buffer after
methylene chloride treatment
Methylene chloride testing of solid hGH formulations '
are shown in Table IV. These results indicated that the
formulation which best stabilized the protein was 5 mM
KP04, 2.5 mg/ml trehalose.
~~~4~.~4
~O 94/19020 ~ PCT/US94I01666
Table Iv
Methylene chloride testing
of solid rhGH formulations
Soluble
Protein
(Mass
Fraction
of Total)
% % Areac % % % Inter-%
FormulationsProi.flinbRecovery Trimer Dimer mediate Monomer
Recovered
Milled Solids
NH4C03 44.5 85.4 7.5 5.9 7.3 79.2
5 mM NaP04, 85.7 100. 0.0 2.1 0.0 97.8
H 8
5 mM NaP04, 87.6 100. 0.0 3.0 0.0 97.0
pH 8
10 mg/ml
Mannitol
Homogenized
Solidsd
5 mM KP04, 97.3 100. 0.0 2.2 0.0 97.8
pH 8,
2.5 mg/ml
Trehalose
5 mM NaP04, 96.8 100. 0.0 2.0 0.0 98.0
pH 8
10 mg/ml
Mannitol
0.3 M Na 94.3 100. 0.0 4.2 0.0 95.8
Succinate,
10 mg/ml
Mannitol,
H 7
5 a All samples lyophilized at 10 mg/ml rhGH with buffer and
excipients as shown.
Protein extracted out of methylene chloride (fraction of total) as
determined by absorbance at 278 nm.
SEC-HPLC results for protein extracted into buffer after methylene
10 chloride treatment.
Solid lyophilized formulations were homogenized in methylene
chloride at 25,000 rpm for 1 min.
Further studies were performed to determine whether a
15 surfactant could stabilize the methylene chloride-
polypeptide interface. Thus, Tween~ surfactant was added
to the methylene chloride phase and mixed with solid hGH
(KP04, pH 8). The addition of Tween~ surfactant to the
methylene chloride phase did not improve the stability of
the solid~hGH (KP04, pH 8) as shown in Table V. In
addition, the use of the surfactant, Span~ 80 surfactant,
in the methylene chloride phase did not improve the
stability of the solid hGH (KP04, pH 8). Further attempts
with Tween~ surfactant in the methylene chloride phase were
unsuccessful for the more stable solid hGH formulation
(Mannitol, KP04, pH 8). These results along with the
WO 94/19020 ' ~ ~ ~' : 16 PCT/US94/01666~
aqueous studies indicated that Tween~ surfactant is
preferably not used with these formulations since it
promotes aggregation and decreases the solubility of
methylene chloride treated hGH.
Table V
Effect of Tween~ surfactant in the
methylene chloride phase on solid hGH stab3.litv
Soluble
polypeptide
(Mass
Fraction
of Total)
% % % % % %
Taeen~ polypeptideaAreab Trimer Dimer IntermediateMonomer
in MeCl2 Recovered Recovery
0.01 40.8 98.7 5.2 13.0 0.0 81.8
Twe en~
80
0.1$ 40.8 102.9 8.0 14.0 0.0 77.9
Tween~
80
1 $ 53.8 97.3 7.0 11.6 0.0 81.4
Tween~
80
a Polypeptide extracted out of methylene chloride (fraction of total)
as determined by absorbance at 278 nm.
SEC-HPLC results for polypeptide extracted into buffer after
methylene chloride treatment
To increase the amount of polypeptide loaded into the
microspheres, the amount of excipient should be minimized.
Therefore, lower concentrations of mannitol (2 and 5 mglml)
with 10 mg/ml hGH were used in the formulation buffer (10
mM NaHP04, pH 8) and the aqueous solutions were tested for
methylene chloride stability. These mannitol
concentrations yielded 20~ less soluble monomer than the 10
mg/ml mannitol formulation. Significant reductions in the
mannitol concentration would sacrifice the quality of the
released polypeptide. Alternative excipients at lower
concentrations were also attempted. Carboxymethylcellulose
(CMC) at 0.5, 2, and 5 mg/ml was used in the aqueous
formulation (10 mg/ml hGH, 10 mM NaHP04, pH 8). CMC at 0.5
mg/ml provided the same fraction of soluble monomer as the
10 mg/ml mannitol formulation, but the amount of
polypeptide recovered in the aqueous phase was 15~ lower.
Equal mass mixtures of CMC and mannitol (1 mg/ml and 2.5
mg/ml of each) were also attempted to provide stability at
lower excipient concentrations. The use of 2.5 mg/ml of
each excipient provided comparable results to the 10 mg/ml
~O 94119020 ~~ ~ , PCT/US94/01666
17 4 r :;
mannitol formulation. The 0.5 mg/ml CMC and 2.5 mg/ml each
of CMC and mannitol formulations were therefore lyophilized
to assess their use for microencapsulation.
To assess formulations for use in the aqueous form,
each lyophilized material. was reconstituted to the maximum
solubility which was defined as the polypeptide
concentration where additional polypeptide would not
dissolve in the solution. The maximum concentration of hGH
in this experiment was achieved with the formulation
lyophilized in 10 mg/ml mannitol. This formulation was
successfully reconstituted with 5 mM NaHP04 buffer, pH 8 to
200 mg/ml of hGH (200 mg/ml mannitol, 100 mM KP04) without
precipitation of the polypeptide. The formulation without
excipients (KP04, pH 8) provided the second best solubility
at 165 mg/ml of hGH. However, attempts to reconstitute the
CMC and CMC/mannitol formulations at high polypeptide
concentrations were not successful. In both cases, the
formulation formed a paste at concentrations greater than
100 mg/ml. Methylene chloride testing of the pastes formed
from the CMC and CMC/mannitol formulations revealed that
the amount of polypeptide recovered was significantly
reduced (less than 75~ recovery) compared to the mannitol
formulation, but the soluble fraction was greater than 95~
monomer. Since a gel-like formulation may have utility for
stabilizing the inner aqueous phase in the process, another
thickening agent, gelatin was also attempted. To maintain
a low excipient concentration while still obtaining a gel
for the final formulation (200 mg/ml hGH), the gelatin
formulation was tested at 0.5 mg/ml gelatin, 10 mg/ml hGH,
10 mM KP04, pH 8. Methylene chloride testing of this
formulation yielded recovery of soluble monomer which was
comparable to the 10 mg/ml mannitol formulation.
Therefore, this formulation was also lyophilized for
further analysis. Reconstitution of the lyophilized
polypeptide at 200 mg/ml hGH (10 mg/ml gelatin, 100 mM
KP04, pH 8) resulted in the formation of a paste which had
properties similar to those of the CMC/mannitol and CMC
formulations at the same hGH concentration.
VVO 94/19020 PCT/LJS94/01666
~~~ ~ 18
Example III
Stability of rhGH Formulations in Ethyl Acetate
Microencapsulation of proteins in biodegradable
polymers often requires the use of organic solvents to
solubilize the polymer. The polymer, typically PLGA, "
polylactide (PLA), or polyglycolide (PGA), is first
dissolved in an organic solvent that is not completely
miscible with water. The common organic solvents used in
this process are methylene chloride and ethyl acetate.
These two solvents have very different physical and
chemical properties. Therefore, it was necessary to assess
the stability of rhGH formulations in both solvents.
The testing of rhGH formulations for stability in
ethyl acetate was performed by a method similar to the one
used for the methylene chloride studies in the examples
above. Solutions of rhGH at 10 mg/ml were prepared by
adding lyophilized solid rhGH (ammonium bicarbonate
formulation) to each formulation. As shown in Table VI,
the formulations were prepared with 5 mM KP04, pH 8 and
contained different excipients, PEG (3350 MW), mannitol,
trehalose, and Tween~ 20, or combinations of excipients.
Each rhGH formulation (100 uL) was added to 1 mL of ethyl
acetate and sonicated for 30 sec to form an emulsion. This
emulsion was then mixed with 10 mL of 5 mM KP04, pH 8
resulting in an overall dilution of rhGH by 100 fold. The
rhGH extracted into the buffer was analyzed by size
exclusion HPLC. Several formulations yielded greater than
100 recovery of soluble protein indicating that the amount
of protein added to the emulsion was greater than the
estimated amount (0.1 mL x 10 mg/mL = 1 mg) as the result
of the accuracy in volume measurements. In addition, the
recovery of soluble protein and the amount of monomer
recovered were generally greater than the rhGH in the same
formulation treated with methylene chloride. Overall, the
recovery of soluble protein was greatest for trehalose (1 &
2 mg/mL), trehalose with PEG (10 mg/mL each), mannitol
with PEG (10 mg/mL each) and mannitol with Tween~ 20 (10
mg/mL each). However, only the trehalose (1 & 2 mglml) and
the mannitol with Tween~ 20 (10 mg/mL each) also had a high
monomer content (greater then 97~). The mannitol/Tween~ 20
~~~~~6~
~O 94/19020 PCT/US94/01666
19
formulation does not allow adequate solubility for a double
emulsion microencapsulation procesys. and it requires a 4:1
excipient to protein ratio (by mass)~k~'fh~~~,-the optimum
formulation in these experiments was the 1 mg/mL trehalose
formulation (1:10 excipient to protein ratio and high rhGH
solubility).
Table v=
Ethyl acetate testing of agueous rhGFi
formulations as degcr;har~ ;" +.~,e ~-e~..
%
Formulations Recoveryb Soluble
Soluble Protein
(Mass
Fraction
of Total)
% Large % %
Aggreg. Dimer Monomer
No exci Tent 98.9 2.3 3.2 94.5
10 mg/mL PEG (3350 99.8 2.7 2.3 94.9
MW )
5 m /mL PEG 108.5 1.7 3.0 95.2
2 m /mL PEG 107.2 1.8 3.8 94.3
10 m /mL Mannitol 96.6 1.7 3.6 94.7
2 m /mL Mannitol 86.3 4.1 3.8 92.2
10 m /mL Trehalose 100.1 1.8 4.5 93.7
2 m /mL Trehalose 119.8 0.4 2.0 97.7
1 m /mL Trehalose 111.1 0.6 2.3 97.1
10 mg/mL PEG (3350 115.6 3.8 2.9 93.3
MW)10 m /mL Trehalose
2 mg/mL PEG (3350 93.0 0.8 3.1 96.1
MW)2 m /mL Trehalose
1 mg/mL PEG (3350 MW) 95.8 4.5 3.3 92.2
1 m /mL Trehalose
10 mg/mL PEG(3350 MW) 116.3 1.2 2.5 96.3
10 m /mL Mannitol
2 mg/mL PEG (3350 MW) 106.5 1.7 2.7 95.6
2 m /mL Mannitol
0.1~ Tween~ 20 122.8 0.8 1.6 97.6
10 m /mL Mannitol
a All initial test solutions contained 10 mg/mL rhGH and 5 mM KP04,
pH 8 except three of the formulations which were at rhGH
concentrations less than 10 mg/mL (no excipient: 9.39 mg/mL; 10 mg/mL
mannitol/10 mg/mL PEG: 7.84 mg/mL; 10 mg/mL mannitol: 9.71 mg/mL).
b SEC-HPLC results for protein extracted into buffer after ethyl
acetate treatment. The percent recovery of soluble protein was
defined as the ratio of the concentrations from the total peak area
2 0 of the sample and the appropriate controls (same formulation) times
100. The control rhGH concentration was determined by absorbance at
278 nm and the sample rhGH concentration was calculated as a 100 fold
dilution of the stock material based on the dilutions used in the
overall method (0.1 mL in 1 mL EtAc added to 10 mL buffer).
WO 94/19020 PCT/US94/01666~
Example IV
Stability of Spray Dried
~f,~H Formulations in Oraanic Solvents
Trie double emulsion technique (water-in-oil-in-water)
5 for microencapsulation can only provide moderate loading of
drug in the final product. The drug loading is limited by
the solubility of the drug in water and the volume of
aqueous drug that can be added to the polymer in organic
solvent . Volumes of greater than 0.5 mL of drug per gram
10 of polymer typically result in a large initial burst of
drug from the microspheres. To avoid these difficulties, a
solid drug formulation can be used in place of the aqueous
drug solution. Thus, a solid-in-oil-in-water process can
be used to produce microspheres with high drug loading
15 (greater then 10~) with low to moderate initial bursts.
The solid drug formulation used for microencapsulation
must be stable in organic solvents and it must have a small
size (1-5 ~.m) relative to the microspheres (30-100 ~tzn) to
permit high loading and low burst of the drug. For protein
20 formulations, one method of obtaining small dried solids is
spray drying. A recent report by Mummenthaler et al.,
Pharm. Res. 11(1):12-20 (1994) describes the process of
spray drying rhGH formulations. Since rhGH is easily
denatured by surface interactions such as air-liquid
interfaces, the spray drying of rhGH must be performed with
surfactants in the rhGH formulation. However, as noted
above, the presence of some surfactants can have a negative
effect on the stability of rhGH in methylene chloride. '
Spray dried rhGH formulations with different surfactants
and trehalose, which were above observed to be the best for
stabilization of the aqueous rhGH formulations, were tested
for stability in methylene chloride and ethyl acetate.
Spray dried rhGH was prepared from each of the
formulations listed in Table VII. These formulations were
sprayed at 5 mL/min with an inlet temperature of 90° C, an
air nozzle flow rate of 600 L/hr, and a drying air rate of
36,000 L/hr. The spray dried rhGH was then collected from
the filter and the cyclone units of the spray drier. The
final solid usually was approximately 5 ~tm in diameter.
~1~4~.~4
~O 94119020 , PCT/US94/01666
21
The spray dried rhGH powder was then tested for
stability by treatment with either ethyl actate or
methylene chloride. A spray dried powder mass. equivalent
to 10 mg of rhGH was added to 2 mL of the organic solvent
in a 3 cc glass test tube. The suspension was next
homogenized at 10,000 rpm for 30 sec with a microfine
homogenization tip. After mixing, 20 ~L of the homogeneous
suspension was added to 980 '..1.L of 5 mM KP04, pH 8 to
extract out the protein. The extracted protein
concentration was determined by absorbance at 278 nm and
the sample was also analyzed by size exclusion
chromatography. As shown in Table VII, the formulation
without surfactant had the greatest extent of aggregation
when treated with methylene chloride. This aggregation was
likely the result of the surface denaturation of rhGH
during the drying process as previously observed for spray
drying of rhGH. By adding either Tween~ 20 or PEG (3350
MW) to the formulation, the amount of aggregation for the
methylene chloride treated samples was reduced, but the
overall recovery yield was still low and the monomer
content was much less than 90~. In contrast, if the same
spray dried rhGH formulations containing surfactant were
treated with ethyl acetate, the amount of aggregation was
neglible and complete recovery of monomeric rhGH was
achieved. Therefore, spray dried rhGH formulations
consisting of trehalose and either Tween~ 20 or PEG (3350
MW) were stable in ethyl acetate but did not protect the
protein from denaturation in methylene chloride.
WO 94/19020 2 ~ ~ ~ 2 2 PCT/US94/01666
Table VII
Stability of spray dried solid rhGH formulations in
methylene chloride and ethyl acetate.
Soluble '
Protein
% % %
Fo rmu 18t 10 n RecoveryaReooverybLarge % %
(Total) (Soluble)Aggreg Dimers Monomer
Methylene Chloride
Tests
mg/mL rhGH -- -- 12 . $ . 75 .
3 8 4
3.75 m /mL trehalose
10 mg/ml rhGH 56.5 65.2 1.9 13.3 78.1
2.5 mg/mL trehalose
0.2~ Tween~ 20
10 mg/mL rhGH 50.7 56.7 3.9 12.3 77.7
2.5 mg/mL trehalose
0.2~ PEG (3350 Mw)
Ethyl ACetBte Tests
10 mg/mL rhGH 111.7 126.8 0.9 0.0 99.2
2.5 mg/mL trehalose
0.2~ Tween~ 20
10 mg/mL rhGH 114.3 125.5 1.1 0.0 98.9
2.5 mg/mL trehalose
0.2 ~ PEG (3350
MW)
5.0 mg/mL rhGH 106.8 110.4 0.3 3.0 96.7
1.25 mg/mL trehalose
0.2~ Tween~ 20
The total recovery of protein was defined as the amount of protein
extracted into buffer after treatment in the organic solvent divided
by the calculated amount of protein added to the extraction buffer
(0.02 mL x 5 mg/mL).
10 b SEC-HPLC results for protein extracted into buffer after treatment
with organic solvent. The percent recovery of soluble protein was
defined as the ratio of the concentrations from the total peak area
of the sample and a reference standard times 100. The control and
sample rhGH concentrations were determined by absorbance at 278 nm.
Example V
tabilitv of Sprat/ Freeze-Dried rhGH Formulations in
Methvlene Chloride and Ethvl Acetate.
Spray drying at high temperatures can have a
detrimental effect on the protein and it produces protein
particles which are often hollow spheres (Mummenthaler et
al., Pharm. Res. 11(1):12-20 (1994). In addition, it is ,
difficult to collect the small particles (1-5 ~..~m) required
for microencapsulation and the overall yield of these ,
particles is usually very low (less than 50~). An
alternative to high temperature spray drying is spray
freeze-drying. Spray freeze-drying of rhGH formulations
results in a fine particles (2-3 ~,tm) that readily break
apart into very small solids (less than 1 ~tm). This type
~~54164 '
. ~ .:,
~O 94/19020 , PCT/US94/01666
23
of solid formulation is preferred for microencapsulation in
a polymer matrix since it can provide a high loading (able
to pack more solid into 30-100 ),tm microspheres) of
homogeneously dispersed solid protein (reduced burst due to
fine suspension).
Spray freeze-drying of rhGH was performed with the
formulations listed in Table VIII. Again, a surfactant was
required to stabilize rhGH during the spraying process but
other proteins which are not easily denatured by surface
interactions would probably not require tine use of a
surfactant. The spray freeze-dried rhGH was prepared by
pumping the formulation at 5 mL/min and operating the air
nozzle at 600 L/hr as used for high temperature spray
drying (Mummenthaler et al., Pharm. Res. 11(1):12-20 1994).
The solutions were sprayed into an open metal tray of
liquid nitrogen. After spraying, the tray was placed in a
prechilled lyophilizer set at -30° C. The liquid nitrogen
was allowed to evaporate and the protein was then
lyophilized (primary drying: -30° C, 100 mTorr, 52 hrs;
secondary drying: 5° C, 100 mTorr, 18 hrs). The final
powder was then removed and placed in sealed glass vials
prior to use.
The spray freeze-dried rhGH powder was then tested for
stability by treatment with ethyl actate and methylene
chloride. A spray freeze-dried powder mass equivalent to
10 mg of rhGH was added to 2 mL of the organic solvent in a
3 cc glass test tube. The suspension was next homogenized
at 10,000 rpm for 30 sec with a microfine homogenization
tip. After mixing, 20 ~1.L of the homogeneous suspension was
added to 980 ~1L of 5 mM KP04, pH 8 to extract out the
protein. The extracted protein concentration was
determined by absorbance at 278 nm and the sample was also
analyzed by size exclusion chromatography. As shown in
Table VIII, the spray freeze-dried formulation containing
PEG was more stable in methylene chloride than the
formulation containing Tween~ 20 as observed above with the
aqueous formulations. However, both formulations did not
yield high recovery of monomeric rhGH. lnThen these same
formulations were treated with ethyl acetate, complete
recovery of monomeric protein was achieved with both
WO 94/19020 PCT/US94/01666
24
formulations. The trehalose in the formulations provided
stabilization against organic solvent denaturation (ethyl
acetate) while the surfactants stabilized the protein
against surface denaturation during spray freeze-drying.
Thus, spray freeze dried formulations containing both '
trehalose and a surfactant will yield complete recovery of
rhGH from ethyl acetate.
Table VIII
Stability of spray freeze-dried solid rhGH
formulations in methyleae chloride and ethyl
acetate.
Soluble
Protein
% % %
FO rmulat iOn RecoveryaRecoveryb Larga % %
(Total) (Soluble) ~9gregDimers Monomer
Meth lane Chloride
Tests
5 mg/mL rhGH 37.2 34.0 6.2 8.3 85.5
1.25 mg/mL trehalose
0.2~ Tween~ 20
5 mg/ml rhGH 68.8 66.8 2.3 15.8 78.8
1.25 mg/mL trehalose
0.2~ PEG (3350
MW)
Eth 1 Acetate Tests
5 mg/mL rhGH 94.6 117.7 0.5 0.9 98.7
1.25 mg/mL trehalose
0.2~ Tween~ 20
5 mg/ml rhGH 97.7 104.7 0.6 0.0 99.4
1.25 mg/mL trehalose
0.2~ PEG (3350
MW)
a The total recovery of protein was defined as the amount of protein
extracted into buffer after treatment in the organic solvent divided
by the calculated amount of protein added to the extraction buffer
(0.02 mL x 5 mg/mL).
SEC-HPLC results for protein extracted into buffer after treatment
with organic solvent. The percent recovery of soluble protein was
2 0 defined as the ratio of the concentrations from the total peak area
of the sample and a reference standard times 100. Control and
sample rhGH concentrations were determined by absorbence at 278 nm.
