U.S. patent application number 13/270136 was filed with the patent office on 2012-04-12 for human growth hormone formulations.
This patent application is currently assigned to Althea Technologies, Inc.. Invention is credited to Sujit K. Basu, Lawrence Bush, Wen-Li Chung, Sergey Pechenov.
Application Number | 20120088724 13/270136 |
Document ID | / |
Family ID | 39402753 |
Filed Date | 2012-04-12 |
United States Patent
Application |
20120088724 |
Kind Code |
A1 |
Chung; Wen-Li ; et
al. |
April 12, 2012 |
Human Growth Hormone Formulations
Abstract
Formulations containing complexed human growth hormone crystals
are described. Also described are needleless injection systems for
crystalline proteins.
Inventors: |
Chung; Wen-Li; (San Mateo,
CA) ; Bush; Lawrence; (Seattle, WA) ;
Pechenov; Sergey; (Malden, MA) ; Basu; Sujit K.;
(Newton, MA) |
Assignee: |
Althea Technologies, Inc.
San Diego
CA
|
Family ID: |
39402753 |
Appl. No.: |
13/270136 |
Filed: |
October 10, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12519720 |
May 4, 2010 |
8071544 |
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PCT/US2007/087417 |
Dec 13, 2007 |
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13270136 |
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60870605 |
Dec 18, 2006 |
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Current U.S.
Class: |
514/11.4 ;
206/438 |
Current CPC
Class: |
A61P 5/06 20180101; C07K
14/61 20130101; A61K 9/0021 20130101; A61K 47/34 20130101; A61P
5/00 20180101; A61K 47/6921 20170801; A61K 9/0019 20130101; A61K
38/27 20130101; A61K 47/645 20170801 |
Class at
Publication: |
514/11.4 ;
206/438 |
International
Class: |
A61K 38/27 20060101
A61K038/27; A61B 19/02 20060101 A61B019/02; A61P 5/06 20060101
A61P005/06 |
Claims
1.-26. (canceled)
27. A hGH preparation comprising: poly-arginine complexed
recombinant human growth hormone or human growth hormone derivative
(hGH) crystals; a buffer at a pH between about 6.1 to about 6.8; a
sodium salt; and a suspending agent.
28.-29. (canceled)
30. A human growth hormone (hGH) preparation comprising: a
suspension of hGH crystals coated with a polyelectrolyte; a
biologically compatible buffer having a pH range from about 5.0 to
about 8.0; a crystallinity promoting agent in an amount sufficient
to maintain crystallinity of said hGH crystals; and a tonicity
modifier allowing for total osmolality of said preparation in the
range of about 250 mOsm/kg to about 450 mOsm/kg.
31. The hGH preparation of claim 30, further comprising a
preservative.
32. The hGH preparation of claim 31, wherein said preservative is
phenol.
33. The hGH preparation of claim 32, wherein said phenol is at a
concentration of 0.2-0.3% w/v.
34. The hGH preparation of claim 30, further comprising a chemical
stabilizer.
35. The hGH preparation of claim 30, wherein said a biologically
compatible buffer has a buffer salt concentration in the range of 1
to 150 mM.
36. The hGH preparation of claim 35, wherein said buffer salt is
selected from the group consisting of acetate, triethanolamine,
imidazole, phosphate, citrate, and Tris-HCl.
37. The hGH preparation of claim 30, wherein said biologically
compatible buffer comprises at least one of sodium phosphate and
sodium citrate.
38. The hGH preparation of claim 30, wherein said biologically
compatible buffer comprises sodium phosphate.
39. The hGH preparation of claim 30, wherein said crystallinity
promoting agent comprises at least one of Sodium acetate and
polyethylene glycol (PEG).
40. The hGH preparation of claim 30, wherein said crystallinity
promoting agent comprises PEG at about 1% w/v to about 25% w/v.
41. The hGH preparation of claim 30, wherein said crystallinity
promoting agent comprises phenol.
42.-48. (canceled)
49. The preparation of claim 30, further comprising hyaluronic
acid.
50.-125. (canceled)
126. The hGH preparation of claim 30, wherein said crystallinity
promoting agent comprises a suspending agent at about 2.5% to about
20%.
127. The hGH preparation of claim 126, wherein said suspending
agent comprises one or more of PEG, mannitol, glycine and
sucrose.
128. The hGH preparation of claim 30, wherein said polyelectrolyte
is at least one of polyarginine, polylysine, and polyornithine.
129. The hGH preparation of claim 30, wherein said tonicity
modifier is selected from the group consisting of a neutral salt,
sodium chloride, sodium acetate, Tris-HCL, a buffer salt outside of
its working buffering pH range, a salt of an amino acid in the pH
range outside its buffering pH range, glycine sodium salt, a
polyol, and a polyethylene glycol.
130. The hGH preparation of claim 30, wherein the crystalline hGH
concentration is about 5 mg/ml to about 100 mg/mL.
131. The hGH preparation of claim 30, wherein the preparation is
disposed in a container containing at least one dose.
132. The hGH preparation of claim 27, comprising said suspending
agent at about 2.5% to about 20%.
133. The hGH preparation of claim 27, wherein said suspending agent
comprises one or more of PEG, mannitol, glycine and sucrose.
134. The hGH preparation of claim 27, wherein said buffer is a
phosphate buffer.
135. The hGH preparation of claim 27, wherein said sodium salt
comprises at least one of sodium chloride and sodium acetate at
60-200 mM.
136. The hGH preparation of claim 27, wherein the crystalline hGH
concentration is about 5 mg/ml to about 100 mg/mL.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Application Ser.
No. 60/870,605, filed on Dec. 18, 2006, the content of which is
hereby incorporated by reference in its entirety.
TECHNICAL FIELD
[0002] This disclosure relates to formulations of protein
therapeutics, and more particularly to formulations of human Growth
Hormone.
BACKGROUND
[0003] Somatotropin or growth hormone ("GH") is a mammalian protein
comprising a class of tropic hormones synthesized and secreted in
the brain by the major gland of the endocrine system, the
adenohypophysis. The secretion of GH and other tropic hormones by
the adenohypophysis regulates the activity of cells in other
endocrine glands and tissues throughout the body. Specifically, GH
is secreted by somatotrophs of the anterior pituitary gland and
functions to stimulate the liver and other tissues to synthesize
and secrete IGF-1, a protein that controls cell division, regulates
metabolic processes and exists in a free state or binds to one of
six other proteins designated as IGFBP-1 through 6. The secretion
process itself is modulated by opposing actions of somatoliberin
(promoting GH release) and somatostatin (inhibiting GH
release).
[0004] Human growth hormone ("hGH") serves as a critical hormone in
the regulation of cell and organ growth and in physiological
function upon various stages of aging. For example, overproduction
of hGH results in gigantism in children and acromegaly in adults,
whereas under-production leads to dwarfism in children [Mauras et
al., J. Clin. Endocrinology and Metabolism, 85(10), 3653-3660
(2000); Frindik et al., Hormone Research, 51(1), 15-19 (1999);
Leger et al., J. Clin. Endocrinology and Metabolism, 83(10),
3512-3516 (1998)], Turner's Syndrome (females only) [Bramswig,
Endocrine, 15(1), 5-13 (2001); Pasquino et al., Hormone Research,
46(6), 269-272 (1996)] and chronic renal insufficiency [Carroll et
al., Trends in Endocrinology and Metabolism, 11(6), 231-238 (2000);
Ueland et al., J. Clin. Endocrinology and Metabolism, 87(6),
2760-2763 (2002); Simpson et al., Growth Hormone & IGF
Research, 12, 1-33 (2002)]. In adults, hGH deficiency can affect
metabolic processing of proteins, carbohydrates, lipids, minerals
and connective tissue and can result in muscle, bone or skin
atrophy [Mehls and Haas, Growth Hormone & IGF Research,
Supplement B, S31-S37 (2000); Fine et al., J. Pediatrics, 136(3),
376-382 (2000); Motoyama et al., Clin. Exp. Nephrology, 2(2),
162-165 (1998)]. Other hGH deficiency disorders characterized by
growth failure include AIDS wasting syndrome [Hirschfeld, Hormone
Research, 46, 215-221 (1996); Tritos et al., Am. J. Medicine,
105(1), 44-57 (1998); Mulligan et al., J. Parenteral and Enteral
Nutrition, 23(6), S202-S209 (1999); Torres and Cadman, BioDrugs,
14(2), 83-91 (2000)] and Prader-Willi syndrome [Ritzen, Hormone
Research, 56(5-6), 208 (2002); Eiholzer et al., Eur. J. Pediatrics,
157(5), 368-377 (1998)].
[0005] To date, treatment regimens for hGH deficiency in humans
focus primarily on subcutaneous injection of purified hGH made by
recombinant DNA technology. The therapeutic is packaged as either a
solution in a cartridge or a lyophilized powder requiring
reconstitution at the time of use. The frequency of injection
varies depending on the disease being treated and the commercially
available product being used.
[0006] The use of subcutaneous administration as a rapid delivery
route for hGH is necessitated by the inherent instability of the
protein in solution. That instability results from cleavage of
critical intramolecular crosslinks at specific positions within the
amino acid sequence of the protein, which in turn disrupts the
essential three-dimensional structure recognized by and associated
with cellular surfaces in the patient. The mechanism for hGH
cleavage or degradation is orchestrated primarily by oxidation of
methionine residues or deamidation of aspartic acid residues upon
dissolution, thereby rendering the protein inactive.
SUMMARY
[0007] The present disclosure provides formulations of crystalline
hGH with improved stability. It further provides methods of
preparing and methods of treatment utilizing such formulations.
[0008] The disclosure also describes crystalline protein
formulations that can be administered to subjects with a needleless
(jet) injection system, and methods of using such formulations.
[0009] In some aspects, the disclosure provides an hGH preparation
including one or more, and preferably all, of: polyelectrolyte
(preferably, poly-arginine); complexed recombinant human growth
hormone or human growth hormone derivative (hGH) crystals; a buffer
(e.g., salt buffer); a sodium salt having a sodium ion
concentration range of 60 to 200 mM; and a suspending agent.
[0010] In some embodiments, the suspending agent is polyethylene
glycol (e.g., PEG 3350, 6000, or 8000).
[0011] In some embodiments, the suspending agent is polyethylene
glycol from about 2.5 to about 20% w/v.
[0012] In some embodiments, the hGH or hGH derivative is stable for
a predetermined length of time and/or at a predetermined condition.
In some embodiments, the length of time is at least 6, 9, 10, 12,
14, 16, 17, 18, 20, 22, or 24 months.
[0013] In some embodiments, the length of time is at least about
12, about 18, about 24, about 30 or about 36 months.
[0014] In some embodiments, the pH of the preparation is pH 6 to
7.
[0015] In some embodiments, preparation includes a phosphate buffer
and the pH of the preparation has a pH of 6 to 7.
[0016] In some embodiments, the concentration of the poly-arginine
complexed hGH crystal is from about 5 mg/mL to about 50 mg/mL. In
some preferred embodiments, the concentration is between about 20
and about 30 mg/ml.
[0017] In some embodiments, the poly-arginine complexed hGH
crystals have a particle size distribution of about 2 to about 100
.mu.m (e.g., about 5 to about 20 .mu.m, e.g., about 3 to about 15
.mu.m).
[0018] In some embodiments, the ratio of hGH to polyarginine is
from about 3 to about 15, e.g., from about 5 to about 8.
[0019] In some embodiments, the hGH preparation is disposed in a
container with no head space or head space of up to 10 mm in a
cylinder type siliconized container.
[0020] In some embodiments, the hGH preparation is disposed in a
container containing multiple doses.
[0021] In some embodiments, the hGH preparation is disposed in a
container containing a single dose.
[0022] In some embodiments, the container comprises a closure of
either Teflon coated stoppers or rubber formulation 4432.
[0023] In some embodiments, the hGH preparation is disposed in a
container with a fill volume of 0.2 mL to 1.0 mL.
[0024] In some embodiments, the preparation is disposed in a
delivery device, e.g., a syringe suitable for subcutaneous
injection, e.g., a pre-filled syringe.
[0025] In some embodiments, the preparation is disposed in a
syringe having a needle of 29 gauge or finer.
[0026] In some embodiments, the preparation is disposed in a
needle-free injector.
[0027] In some embodiments, the hGH is present at about 5 to about
100 mg/ml, about 10 to about 50 mg/l, or about 20 to about 30
mg/ml.
[0028] In some embodiments, the preparation also contains an
antimicrobial agent, e.g., phenol or m-cresol.
[0029] In some embodiments, the preparation also contains
hyaluronic acid.
[0030] In some embodiments, the preparation is free of polymers
other than a suspension agent.
[0031] In some embodiments, the preparation is free of histidine
buffer.
[0032] In some embodiments, the preparation also contains a
preservative. In preferred embodiments, wherein the preservative is
phenol or m-cresol. In more preferred embodiments, the preservative
is phenol.
[0033] In some aspects, the disclosure features an hGH preparation
that contains: poly-arginine complexed recombinant human growth
hormone or human growth hormone derivative (hGH) crystals at 5-50
mg/ml; phosphate buffer at pH of 6.1-6.8; sodium chloride or sodium
acetate at 60-200 mM; 2.5-20% polyethylene glycol, e.g., 6000 or
8000; that is disposed in a siliconized prefiled syringe with no
more than 10 mm head space; and a volume of 0.2-1.0 ml.
[0034] In some embodiments, the syringe has a needle of 29 gauge or
finer.
[0035] In some embodiments, the preparation is disposed in a
needle-free injector.
[0036] In some aspects, the disclosure features an hGH preparation
that contains: an hGH preparation containing one or more of, and
preferably all, of: a suspension of hGH crystals coated with
polyelectrolyte, e.g., polyagininc, wherein the crystalline hGH
concentration is at 5-100 mg/mL, 10-50 mg/mL, or 20 to 30 mg/mL; a
biologically compatible buffer maintaining crystallinity of the
complex, preferably with pH range 5.0 to 8.0; a crystallinity
promoting agent which maintains or increases crystallinity of said
complex; and a tonicity modifier allowing for total osmolality of
said compositions in the range of 250-450 mOsm/kg, 270-350 mOsm/kg,
or 280-330 mOsm/kg.
[0037] In some embodiments, the hGH preparation includes a
preservative, e.g., phenol. In some embodiments, the phenol is at a
concentration of about 0.25% or about 0.2 to about 0.3% w/v.
[0038] In some embodiments, the hGH preparation includes a chemical
stabilizer, e.g., methionine or EDTA.
[0039] In some embodiments, the biologically compatible buffer has
a buffer salt concentration in the range of about 1 to about 150
mM, about 2 to about 50 mM, or about 10 mM.
[0040] In some embodiments, the buffer salt is selected from the
group that includes (consists of) acetate, triethanolamine,
imidazole, phosphate, citrate, and Tris-HCl. In other embodiments,
the buffer salt is selected from the group that includes phosphate,
glycine, histidine, citrate, acetate, and Tris.
[0041] In some embodiments, the biologically compatible buffer is
sodium phosphate, e.g., in the pH range of 5.8 to 7.0 or 6.0 to
6.5.
[0042] In some embodiments, the biologically compatible buffer
comprises sodium phosphate and sodium citrate buffers, e.g., 2 mM
Na citrate 8 mM Na phosphate.
[0043] In some embodiments, the crystallinity promoting agent which
maintains or increases crystallinity of said complex contains Na
acetate, or polyethylene glycol e.g., PEG 3350-PEG 8000.
[0044] In some embodiments, the crystallinity promoting agent which
maintains or increases crystallinity of said complex comprises PEG
at about 1 to about 25 w/v %, e.g., about 2 to about 10 w/v %; or
about 4 to about 6 w/v %.
[0045] In some embodiments, the crystallinity promoting agent which
maintains or increases crystallinity of said complex contains
phenol, e.g., at a concentration of about 0.1 to about 0.5%
w/v.
[0046] In some embodiments, the tonicity modifier is selected from
the group consisting of a neutral salt(s), sodium chloride, sodium
acetate, Tris-HCl, a buffer salt outside of its working buffering
pH range, a salt(s) of an amino acid(s) in the pH range outside its
buffering pH range, glycine sodium salt, a polyol (such as mannitol
or sorbitol), and a polyethylene glycol.
[0047] In some embodiments, the hGH preparation is disposed in a
container containing multiple doses.
[0048] In some embodiments, the hGH preparation is disposed in a
container containing a single dose.
[0049] In some embodiments, the container comprises a closure of
either Teflon coated stoppers or rubber formulation 4432.
[0050] In some embodiments, the hGH preparation is disposed in a
container with a fill volume of 0.2 mL to 1.0 mL.
[0051] In some embodiments, the hGH preparation is disposed in a
delivery device, e.g., a syringe, suitable for subcutaneous
injection, e.g., a pre-filled syringe.
[0052] In some embodiments, the hGH preparation is disposed in a
needle-free injector.
[0053] In some embodiments, the hGH preparation contains hyaluronic
acid.
[0054] In some aspects, the disclosure features a method of making
an hGH preparation comprising combining the components of a
preparation described herein. For example, combining an hGH
preparation including one or more, and preferably all, of:
polyelectrolyte (preferably, poly-arginine); complexed recombinant
human growth hormone or human growth hormone derivative (hGH)
crystals; a buffer (e.g., salt buffer); a sodium salt having a
sodium ion concentration range of 60 to 200 mM; and a suspending
agent. As another example, combining poly-arginine complexed
recombinant human growth hormone or human growth hormone derivative
(hGH) crystals at 5-50 mg/ml; phosphate buffer at pH of 6.1-6.8;
sodium chloride or sodium acetate at 60-200 mM; 2.5-20%
polyethylene glycol, e.g., 6000 or 8000.
[0055] In some aspects, the disclosure features a method of
packaging a hGH preparation including disposing the components of a
preparation described herein in a container to a fill volume of
0.2-1.0 ml wherein said container is a syringe, e.g., with no more
than 10 mm head space and is preferably suitable for subcutaneous
injection, to the patient.
[0056] In some aspects, the disclosure features a method of
packaging a hGH preparation including disposing the components of
the preparation of claim 1 or claim 26 in a container to a fill
volume which includes multiple dosages of 0.2-1.0 ml.
[0057] In some aspects, the disclosure features a method of
delivering a hGH preparation to a patient. The method includes
[0058] providing a hGH preparation described herein and
[0059] administering, e.g., by injection, no more than once every
2, 3, 4, 5, 6, 7, 8, 9, or 10 days.
[0060] In some aspects, the disclosure features a method of
providing a hGH preparation, or information about a hGH
preparation, to a party, e.g., a distributor, physician, pharmacy,
hospital, HMO, wholesaler, retailer, government, patient or health
care provider. The method includes comprising:
[0061] instructing the party that a hGH preparation containing the
components of a preparation described herein is stable for a
predetermined length of time and/or at a predetermined
condition.
[0062] In some embodiments, the length of time is at least 6, 9,
10, 12, 14, 16, 17, 18, 20, 22, or 24 months.
[0063] In some embodiments, the length of time is at least 12, 18,
24, 30 or 36 months.
[0064] In some embodiments, the condition is temperature, e.g.,
2-8.degree. C.
[0065] In some embodiments, the condition is room temperature,
e.g., about 25.degree. C.
[0066] In some aspects, the disclosure features a method of
satisfying a standard, e.g., a standard imposed by a government
agency, e.g., the FDA, e.g., a release specification or a label
specification, or pharmaceutical compendium. The method includes:
[0067] providing evidence that, or asserting that, a hGH
preparation, e.g., an hGH preparation described herein, is stable
for a predetermined length of time and/or at a predetermined
condition.
[0068] In some embodiments, the length of time is at least 6, 9,
10, 12, 14, 16, 17, 18, 20, 22, or 24 months.
[0069] In some embodiments, the length of time is at least 12, 18,
24, 30 or 36 months.
[0070] In some embodiments, the condition is temperature, e.g.,
2-8.degree. C.
[0071] In some embodiments, the condition is room temperature,
e.g., about 25.degree. C.
[0072] In some aspects, the disclosure features a needle-free
injector having disposed therein a preparation of crystallized
protein.
[0073] In some embodiments, the preparation is a solution of
crystals.
[0074] In some embodiments, the crystals are cross-linked.
[0075] In some embodiments, the crystals are complexed with a
polyelectrolyte, e.g., polyarginine.
[0076] In some embodiments, the preparation is of hGH or an hGH
derivative.
[0077] In some embodiments, the preparation is an hGH or an hGH
derivative preparation described herein.
[0078] In some embodiments, the preparation is not a vaccine.
[0079] In some embodiments, the crystals in the preparation have an
average largest dimension of 0.5, 0.33, or 0.25 or less than the
diameter of the delivery orifice of the injector.
[0080] In some embodiments, the preparation further includes
hyaluronic acid.
[0081] In some embodiments, the concentration of protein in said
preparation is less than about 100 mg/ml or is between about 2 and
about 50 mg/ml.
[0082] In some aspects, the disclosure features a method of
delivering a crystallized protein preparation to a subject. The
method includes supplying a crystallized protein preparation
disposed in a needle-free injector and injecting the preparation
into the subject.
[0083] In some embodiments, the preparation is a solution of
crystals.
[0084] In some embodiments, the said crystals are cross-linked.
[0085] In some embodiments, the crystals are complexed with a
polyelectrolyte, e.g., polyarginine.
[0086] In some embodiments, the preparation is of hGH or an hGH
derivative.
[0087] In some embodiments, the preparation is an hGH or an hGH
derivative preparation described herein.
[0088] In some embodiments, the preparation is not a vaccine.
[0089] In some embodiments, the crystals in said preparation have
an average largest dimension of 0.5, 0.33, or 0.25 or less than the
diameter of the delivery orifice of the injector.
[0090] In some embodiments, the preparation further includes
hyaluronic acid.
[0091] In some embodiments, the concentration of protein in said
preparation is less than about 100 mg/ml or is between about 2 and
about 50 mg/ml.
[0092] In some aspects, the disclosure features a lyophilized
preparation of hGH or an hGH derivative, wherein upon resuspension,
the preparation provides a suspension that contains: hGH or hGH
derivative crystal suspension (e.g., poly-arginine complexed hGH
crystal suspension), a buffer, a salt, and polyethylene glycol.
[0093] In some embodiments, the crystal suspension is present in an
amount between about 20 to about 50 mg/ml.
[0094] In some embodiments, the buffer comprises Tris.
[0095] In some embodiments, the buffer comprises phosphate. In some
embodiments, the buffer further includes Tris or histidine.
[0096] In some embodiments, the pH of the suspension is between
about 7 and about 9.
[0097] In some embodiments, the salt comprises sodium chloride or
sodium acetate. In some embodiments, the salt is present in an
amount between about 50 mM to about 100 mM.
[0098] In some embodiments, the polyethylene glycol comprises
polyethylene glycol 3350, 6000 or 8000. In some embodiments, the
polyethylene glycol is present in an amount between about 2.5% and
about 10%.
[0099] In some embodiments, the suspension comprises: about 20 to
about 50 mg/mL hGH crystal suspension; Tris or a combination buffer
of phosphate with either Tris or histidine; at a pH between about 7
and about 9; sodium chloride or sodium acetate in an amount between
about 50 mM to about 100 mM; and polyethylene glycol 3350, 6000 or
8000 in an amount between about 2.5% and about 10%.
[0100] In some embodiments, the preparation also contains a
preservative. In some embodiments, the preservative is phenol,
m-cresol, chlorobutanol, or benzyl alcohol (e.g., preferably
phenol, m-cresol, or chlorobutanol, more preferably phenol).
[0101] In some embodiments, the suspension is disposed in a
siliconized vials or coated surface container closure.
[0102] In some aspects, the disclosure features a process for
producing lyophilized crystalline hGH. The method includes: [0103]
a primary drying cycle of about -30.degree. C. to about 10.degree.
C.
[0104] In some embodiments, the process further includes a 2nd
drying cycle of about 20.degree. C. to about 40.degree. C.
[0105] In some embodiments, the disclosure features a lyophilized
crystalline hGH product produced by a process described herein.
[0106] In some aspects, the disclosure features a lyophilized
preparation of hGH containing 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 100 mM Na Acetate, 5% PEG6000, pH 7.5.
[0107] In some aspects, the disclosure features a lyophilized
preparation of hGH consisting of 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 100 mM Na Acetate, 5% PEG6000, pH 7.5.
[0108] In some aspects, the disclosure features a lyophilized
preparation of hGH containing 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 0.2% HA, 100 mM Na Acetate, 5% PEG6000, pH
7.5.
[0109] In some aspects, the disclosure features a lyophilized
preparation of hGH consisting of 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 0.2% HA, 100 mM Na Acetate, 5% PEG6000, pH
7.5.
[0110] In some aspects, the disclosure features a lyophilized
preparation of hGH containing 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 5% PEG6000, pH 7.0.
[0111] In some aspects, the disclosure features a lyophilized
preparation of hGH consisting of 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 5% PEG6000, pH 7.0.
[0112] In some aspects, the disclosure features a lyophilized
preparation of hGH containing 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 5% PEG6000, pH 7.5.
[0113] In some aspects, the disclosure features a lyophilized
preparation of hGH consisting of 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 5% PEG6000, pH 7.5.
[0114] In some aspects, the disclosure features a lyophilized
preparation of hGH containing 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 5% PEG8000, pH 7.5.
[0115] In some aspects, the disclosure features a lyophilized
preparation of hGH consisting of 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 5% PEG8000, pH 7.5.
[0116] In some aspects, the disclosure features a lyophilized
preparation of hGH containing 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 5% PEG3350, pH 7.5.
[0117] In some aspects, the disclosure features a lyophilized
preparation of hGH consisting of 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 5% PEG3350, pH 7.5.
[0118] In some aspects, the disclosure features a lyophilized
preparation of hGH containing 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 100 mM NaCl, 5% PEG6000, pH 7.5.
[0119] In some aspects, the disclosure features a lyophilized
preparation of hGH consisting of 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 100 mM NaCl, 5% PEG6000, pH 7.5.
[0120] In some aspects, the disclosure features a lyophilized
preparation of hGH containing 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 100 mM NaCl, 5% PEG8000, pH 7.5.
[0121] In some aspects, the disclosure features a lyophilized
preparation of hGH consisting of 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 100 mM NaCl, 5% PEG8000, pH 7.5.
[0122] In some aspects, the disclosure features a lyophilized
preparation of hGH containing 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 100 mM NaCl, 5% PEG3350, pH 7.5.
[0123] In some aspects, the disclosure features a lyophilized
preparation of hGH consisting of 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 100 mM NaCl, 5% PEG3350, pH 7.5.
[0124] In some aspects, the disclosure features a lyophilized
preparation of hGH containing 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM histidine, 100 mM NaCl, 5% PEG6000, pH 7.0.
[0125] In some aspects, the disclosure features a lyophilized
preparation of hGH consisting of 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM histidine, 100 mM NaCl, 5% PEG6000, pH 7.0.
[0126] In some aspects, the disclosure features a lyophilized
preparation of hGH containing 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 1% sucrose, 5% PEG6000, pH 7.5.
[0127] In some aspects, the disclosure features a lyophilized
preparation of hGH consisting of 25 mg/mL poly-Arg complexed hGH
crystals, 25 mM Tris, 1% sucrose, 5% PEG6000, pH 7.5.
[0128] In some aspects, the disclosure features a resuspended
lyophilate described herein.
[0129] In some aspects, the disclosure features a method of
delivering a hGH preparation to a patient. The method includes:
providing a lyophilized hGH preparation described herein, and
[0130] administering the preparation (e.g., after resuspending the
lyophilate), e.g., by injection, to said patient, e.g., no more
than once every 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.
[0131] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs.
Although methods and materials similar or equivalent to those
described herein can be used in the practicing or testing of the
present disclosure, suitable materials and methods are described
below.
[0132] All cited publications, patent applications, patents, and
other references mentioned herein are incorporated by reference in
their entirety. In case of conflict, the present specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and not intended to be
limiting.
[0133] The details of one or more embodiments of the disclosure are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the disclosure will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF DRAWINGS
[0134] FIG. 1 is a graph showing the correlation of pH with the
percentage of degradation by CEX: 3 month 25.degree. C. data was
used for the correlation.
[0135] FIG. 2 is a graph showing that formulations containing
sodium salt (higher ionic strength) have less degradation than
formulations without salt by CEX. Stability data of one week at
40.degree. C.
[0136] FIG. 3 is a graph showing the percentage of degradation for
excipient screening, excipient screening by CEX after two weeks at
40.degree. C.
[0137] FIG. 4 is a graph showing the percentage of degradation
(excipient) for various suspending agents at pH 6.8 by CEX. All
formulations contained the following components: phosphate, NaCl at
pH 6.8.
[0138] FIG. 5 is a graph showing the effects of salt species and
concentration on degradation by CEX. All formulations contained the
following components phosphate with either PEG6000 or PEG8000.
[0139] FIG. 6 is a graph showing percentage of degradation versus
PEG concentration by CEX (10% PEG results not shown).
[0140] FIGS. 7A, 7B, 7C, and 7D are a series of graphs showing a
recovered hGH concentration comparison. FIG. 7A shows the results
obtained with a 1 ml deliver volume subjected to invert treatment;
FIG. 7B shows the results obtained with a 1 ml deliver volume
subjected to swirl treatment; FIG. 7C shows the results obtained
with a 0.2 ml deliver volume subjected to invert treatment; FIG. 7D
shows the results obtained with a 0.2 ml deliver volume subjected
to swirl treatment.
[0141] FIGS. 8A, 8B, 8C, and 8D are a series of graphs showing a
percentage of weight recovery comparison. FIG. 8A shows the results
obtained with a 1 ml deliver volume subjected to invert treatment;
FIG. 8B shows the results obtained with a 1 ml deliver volume
subjected to swirl treatment; FIG. 8C shows the results obtained
with a 0.2 ml deliver volume subjected to invert treatment; FIG. 8D
shows the results obtained with a 0.2 ml deliver volume subjected
to swirl treatment.
[0142] FIG. 9 is a graph showing the overlay of particle size
distribution in an ALTU-238 sample originally free of crystalline
aggregates, after first, and after second injections with a
BioJector 2000 injection system.
[0143] FIG. 10 is a graph showing the overlay of particle size
distribution in HA-ALTU-238 sample prior to injection, after first,
and after second injections with BioJector 2000 injection
system.
[0144] FIGS. 11A and B are graphs showing the overlay of volume-(A)
and number-(B) based particle size distribution of BC-Lipase (20
.mu.m) samples before injection and after injections through BD
syringe with preattached 30 G.times.1/2'' needle, BioJector 2000,
and VetJet systems.
[0145] FIGS. 12A and B are graphs showing the overlay of volume-(A)
and number-(B) based particle size distribution of BC-Lipase (30
.mu.m) samples before injection, and after injections through BD
syringe with preattached 30 G.times.1/2'' needle, BioJector 2000
and VetJet systems.
[0146] FIGS. 13A and B are graphs showing the overlay of volume-(A)
and number-(B) based particle size distribution of BC-Lipase (80
.mu.m) samples before injection, and after injections with
BioJector 2000, through BD syringe with preattached 30
G.times.1/2'' needle, and syringe with 25 G.times.5/8'' needle.
[0147] FIGS. 14A and B are graphs showing the overlay of volume-(A)
and number-(B) based particle size distribution of Glutaryl
Alcalase samples before injection, and after injections with the
use of BioJector 2000, through BD syringe with preattached 30
G.times.1/2'' needle, and 25 G.times.5/8'' and 18 G.times.1''
needles.
[0148] FIG. 15 is a graph showing the effect of PEG concentrations
on the amount of dissolved hGH in the supernatant. The base
formulation was Tris buffer at pH 7.5
[0149] FIG. 16 is a graph showing particle size distributions for
samples generated by different lyo cycles.
DETAILED DESCRIPTION
Overview: Crystalline hGH Stability
[0150] Over time, crystalline hGH suspensions can develop soluble
protein aggregates, the hGH can undergo oxidation, deamidation,
chemical degradation, and crystal form changes, and the hGH can
dissolve in solution.
[0151] The disclosure provides crystalline hGH suspensions, e.g.,
poly-arginine complexed hGH crystals suspensions, that have
increased stability. Embodiments described herein include one or
more measures to address one or more stability issues. As examples,
various factors have been addressed to increase the stability of
crystalline hGH formulations, e.g., protein concentration, buffer
selection, salt selection and concentration, pH, choice and
concentration of suspending agents, storage container selection,
choice and concentration of preservatives, and fill volume. One,
two, three, four, or all of these factors can be altered or
controlled to increase the stability of a protein of interest.
[0152] As one example of a liquid suspension of crystalline hGH
with improved stability, the suspension can have one or more of the
following properties: contain about 5-50 mg/ml hGH crystal
suspension; contain a phosphate-containing buffer; contained in a
pH range of about 6.1 to about 6.8; contain sodium chloride or
sodium acetate at a concentration of about 60 mM to about 200 mM;
contain polyethylene glycol 6000 or 8000 at about 2.5% to about
20%; be aliquoted at a fill volume of about 0.2 to about 1 ml; be
disposed in a siliconized prefilled syringe with limited head space
(e.g., no head space to about 10 mm head space) or in another
cylinder type of container or device with limited head space.
[0153] As one example, the disclosure provides a formulation of
crystalline hGH suspension that is stable for 18 to 24 months at
5.+-.3.degree. C. (refrigerated) and at least one month at room
temperature 25.+-.2.degree. C. (in use) conditions.
[0154] Formulations described herein, e.g., liquid formulations
containing a crystalline protein (e.g., poly-Arg complexed
crystalline hGH), have increased stability. E.g., upon storage in a
container, at a temperature of 2-8.degree. C. for a period of up to
3, 6, 9, 12, or 24 months (or in some embodiments longer), a
protein in the composition will retain at least 50, 75, 85, 90, 95,
or 100% of the stability it had prior to storage (e.g., the protein
can retain about 77% of the stability at 25.degree. C. over a
period of three months). Stability, as used herein, includes
parameters such as protein structure (e.g., minimizing or
preventing changes in protein structure, e.g., protein aggregation
or protein degradation) and/or efficacy of the protein, e.g.,
therapeutic efficacy (e.g., ability to cause an increase in body
weight).
[0155] As used herein, the term "increased stability" refers to a
decrease (e.g., about 5%, about 10%, about 15%, about 20%, about
25%, about 30%, about 35%, about 40%, about 45%, about 50%, about
55%, about 60%, about 65%, about 70%, about 75%, about 80%, about
85%, about 90%, about 95%, about 96%, about 97%, about 98%, or
about 99% decrease) in the amount of protein deamidation, protein
oxidation, protein aggregation, protein dissolution, or therapeutic
efficacy over a fixed period of time under testing or fixed storage
conditions
[0156] Human Growth Hormone
[0157] The term "growth hormone (GH)" refers generally to growth
hormones secreted by the pituitary gland in mammals. Although not
an exhaustive list, examples of mammals include human, apes,
monkey, rat, pig, dog, rabbit, cat, cow, horse, mouse, rat and
goat. According to a preferred embodiment of this disclosure, the
mammal is a human.
[0158] "Human growth hormone (hGH)" denotes a protein having an
amino acid sequence, structure and function characteristic of
native human growth hormone. As used herein, human growth hormone
(hGH) also includes any isoform of native human growth hormone,
including but not limited to, isoforms with molecular masses of 5,
17, 20, 22, 24, 36 and 45 kDa (Haro et al., J. Chromatography B,
720, 39-47 (1998)). Thus, the term hGH includes the 191 amino acid
sequence of native hGH, somatotropin, and the 192 amino acid
sequence containing an N-terminal methionine (Met-hGH) and somatrem
(U.S. Pat. Nos. 4,342,832 and 5,633,352). hGH may be obtained by
isolation and purification from a biological source or by
recombinant DNA methods. If made by recombinant DNA methodology,
hGH is denoted as recombinant human growth hormone (rhGH). Met-hGH
is typically prepared by recombinant DNA methodology.
[0159] The term "human growth hormone derivative" refers to a
protein that differs by at least about 1% but not by more than
about 20% from the amino acid sequence of the 191 amino acid
sequence of hGH or the 192 amino acid-sequence of Met-hGH. For
example, the derivative can differ by about 1% to about 20%, about
2% to about 15%, or about 5% to about 10% from the 191 amino acid
sequence of hGH or the 192 amino acid-sequence of Met-hGH; the
protein can differ by about 1%, about 2%, about 3%, about 4%, about
51%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%,
about 12%, about 13%, about 14%, or about 15% from the 191 amino
acid sequence of hGH or the 192 amino acid-sequence of Met-hGH. The
differences between the derivative and the 191 amino acid hGH or
the 192 amino acid Met-hGH amino acid sequence can be one or more
substitutions (e.g., conservative or non conservative
substitutions), deletions, additions (e.g., insertions or amino- or
carboxy-terminal additions)), modifications, or combinations
thereof. In some embodiments, the derivative maintains a biological
activity and/or a chemical and/or physical property of the 191
amino acid hGH or the 192 amino acid Met-hGH amino acid sequence.
Likewise, in some embodiments, a formulation containing a
derivative (e.g., a formulation of poly-Arg complexed crystalline
hGH derivative) possesses a chemical and/or physical property of a
similarly-prepared formulation containing the 191 amino acid hGH or
the 192 amino acid Met-hGH amino acid sequence (e.g., a formulation
of poly-Arg complexed crystalline hGH).
[0160] In various embodiments of the present disclosure, human
growth hormone derivatives comprise organic cations of hGH or
Met-hGH, substitution, deletion and insertion variants of
biologically synthesized hGH or Met-hGH proteins,
post-translationally modified hGH and Met-hGH proteins, including
deamidation, phosphorylation, glycoslylation, acetylation,
aggregation and enzymatic cleavage reactions [Haro et al., J.
Chromatography B, 720, 39-47 (1998)], chemically modified hGH or
Met-hGH proteins derived from biological sources, polypeptide
analogs and chemically synthesized peptides containing amino acid
sequences analogous to those of hGH or Met-hGH.
[0161] Methods used to prepare hGH or Met-hGH include isolation
from a biological source, recombinant DNA methodology, synthetic
chemical routes or combinations thereof. To date, genes that encode
for different DNA sequences of hGH include hGH-N and hGH-V [Haro et
al., J. Chromatography B, 720, 39-47 (1998); Bennani-Baiti et al.,
Genomics, 29, 647-652 (1995)].
[0162] The term "valency" is defined as an element's ability to
combine with other elements and which is dictated by the number of
electrons in the outermost shell of the atom and expressed as the
number of atoms of hydrogen (or any other standard univalent
element) capable of uniting with (or replacing) its atoms
[Webster's New World Dictionary of Science, Lindley, D. and Moore
T. H., Eds., Macmillan, New York, N.Y., 1998]. The terms
"monovalent cation" and "divalent cation" refer to ions carrying a
positive charge that have either a valence state of one or two,
respectively. Cations having different valence states can be
organic or inorganic in nature. Examples of monovalent inorganic
cations include ammonium (NH.sub.4.sup.+) and Group I elements of
the periodic table (H.sup.+, Li.sup.+, Na.sup.+, K.sup.+,
Rb.sup.30, Cs.sup.+, and Fr.sup.+) and divalent inorganic cations
include Group II elements (Be.sup.2+, Mg.sup.2+, Ca.sup.2+,
Sr.sup.2+, Ba.sup.2+, Mn.sup.2+, Co.sup.2+, Ni.sup.2+, Cu.sup.2+,
Zn.sup.2+, Cd.sup.2+, Mo.sup.2+ and Ra.sup.2+).
[0163] "Organic cation crystal of human growth hormone or a human
growth hormone derivative" refers to human growth hormone that has
been crystallized in the presence of an organic cation. The term
"organic cation" refers to a positively charged atom or group of
atoms that contain carbon. Examples of organic cations include
quaternary ammonium cations, tetraethylammonium (TEA),
tributylmethylammonium (TBuMA), procainamide ethobromide (PAEB),
azidoprocainamide methoiodide (APM), d-tubocurarine, metocurine
vecuronium, rocuronium, 1-methyl-4-phenylpyridinium, choline and
N-(4,4-axo-n-pentyl)-21-deoxyajm-alinium (APDA).
[0164] hGH is commercially available in lyophilized form and is
typically produced by recombinant DNA methods. Crystallization of
hGH can be accomplished by preparing a buffered solution of hGH,
purifying and/or desalting, dialyzing and concentrating the
solution and adding a monovalent or divalent cation or salt to the
solution. The latter step results in the formation of an organic or
inorganic cation bound to hGH.
[0165] One preferred embodiment of this disclosure relates to
monovalent cation crystals of hGH or an hGH derivative. In a more
preferred embodiment, the monovalent cation is selected from the
group consisting of: lithium, sodium, potassium and ammonium. In a
most preferred embodiment, the monovalent cation is sodium. In a
most preferred embodiment, human growth hormone or a human growth
hormone derivative comprises from about 1 to about 500 monovalent
cation molecules per monomer or monomer chain of human growth
hormone or human growth hormone derivative.
[0166] The term "monovalent cation salt" includes both inorganic
and organic counterions or molecules that from an ionic bond with
the monovalent ion. In a preferred embodiment, the monovalent
cation salt is a sodium salt. In a more preferred embodiment, the
sodium salt is selected from the group consisting of sodium
citrate, sodium phosphate and sodium acetate. In a most preferred
embodiment, the sodium salt is sodium acetate.
[0167] In one preferred embodiment, crystalline hGH or crystalline
hGH derivative complexed with protamine is provided. Likewise, in
another preferred embodiment, crystalline hGH or crystalline hGH
derivative complexed with polyarginine is provided.
[0168] A crystallization and complexation process for hGH to
produce long-acting hGH has been developed, and this product has
demonstrated a controlled release profile in clinical trials (see
US application 2004-0209804, WO 2004/060310, WO 2004/060920).
Poly-arginine (poly-Arg) complexation results in an improved
controlled release profile. This drug delivery method is designed
for fewer injections without the use of polymers or fusion proteins
to offer a more user-friendly treatment alternative for patients.
The uniqueness of this crystallization technology is that little or
no increase of soluble aggregates is observed since limited free
hGH is present in the supernatant.
[0169] For example, poly-Arg crystalline hGH can be prepared as
follows:
[0170] A frozen bulk feed solution of soluble
recombinantly-produced hGH (rhGH) is obtained, e.g., derived from
E. coli (Novartis) or from yeast (Lucky Gold). Approximately 3.5 ml
(12 mg/ml rhGH in Tris-HCl (10 mM, pH 8.0)) of thawed rhGH feed
solution is purified using a 10DG-desalting column supplied by
Biorad. Prior to sample loading, the column is conditioned by
washing the column with 30 ml of Tris-HCl (10 mM, pH 8.0). The rhGH
sample is then loaded and allowed to enter the column by gravity.
After discarding the first three ml of eluant, another 5.0 ml of 10
mM Tris-HCl pH 8.0 is then added. 4.5 ml of the desalted rhGH is
eluted and collected. Concentration by centrifugation is then
performed using a Millipore concentrator (MWCO 10,000) at 3500 rpm
for 20-30 min. The concentration of hGH is in the range of 30 mg/ml
as measured by absorbance at 280 nm/0.813 (1 mg/ml hGH A280=0.813
absorbance units). Crystals are grown by adding 1M Tris-HCl (pH
8.6), 50% PEG-6000 and 1M Ca-acetate to the rhGH 30 mg/ml stock
preparation so that a final concentration of 15 mg/ml rhGH, 100 mM
Tris-HCl (pH 8.6), 2% (v/v) PEG-6000 and 85 mM Ca-acetate is
obtained. The solution is then mixed gently and incubated at
33.degree. C. for 12-16 hours. Needle-like-crystals are obtained
ranging in length from approximately 2 to 25 .mu.m. After
extracting the supernatant and centrifuging and pelleting the
crystals, crystallization yield is measured, e.g., the yield can be
greater than 85%. The crystals can also be formed at temperatures
between 33.degree. C. and 15.degree. C. but require increased
crystallization time and may have a reduced yield. After
crystallization, yield is determined, calcium rhGH crystals are
re-suspended in a formulation vehicle (e.g., 5 mM CaOAc, 100 mM
Tris-HCl (pH 8.6), 6% PEG-6000, and 4.2 mg/ml polyarginine) so that
a final concentration of 21 mg/ml or 25 mg/ml of calcium rhGH
crystals is achieved. The protein to additive ratio for rhGH to
polyarginine was 5:1 (mg:mg). These ratios are calculated to be
mole ratios of approximately 1:0.587 for rhGH:polyarginine. The
above rhGH pellets are homogenously re-suspended in the appropriate
mother liquor and incubated overnight at 2-8.degree. C. before
being centrifuged to obtain a condensed pellet. The supernatants
are removed and the pellets can be re-suspended in the same mother
liquor without ionic additive and can be stored at 4.degree. C.
[0171] A further preferred embodiment of this disclosure includes
monovalent or divalent crystals of hGH or an hGH derivative
complexed or co-crystallized with protamine or polyarginine. More
preferably, the crystals are sodium crystals complexed or
co-crystallized with protamine or polyarginine.
[0172] The soluble form of hGH may be characterized by a variety of
methods, including reversed phase high performance liquid
chromatography (RP-HPLC), size exclusion chromatography high
performance liquid chromatography (SEC-HPLC) and hydrophobic
interaction chromatography (HIC) (Wu et al., J. Chromatography,
500, 595-606 (1990); "Hormone Drugs", FDA publication, (1982)). On
the other hand, the crystalline form of hGH may be characterized by
optical microscopy and X-ray diffraction. In general, the
conditions of crystallization will determine the shape of a protein
crystal, i.e., a shape selected from the group consisting of
spheres, needles, rods, plates (hexagonals and squares), rhomboids,
cubes, bipyramids and prisms.
[0173] Crystals of hGH or an hGH derivative according to this
disclosure form rod-like or needle-like morphologies when imaged
with optical microscopy. In one embodiment, crystals of hGH or an
hGH derivative form rods or needles that are between about 0.1 and
about 200 .mu.m in length. In a preferred embodiment, crystals of
hGH or an hGH derivative form rods or needles that are between
about 3 and about 100 .mu.m in length. In a more preferred
embodiment, crystals of hGH or an hGH derivative form rods or
needles that are between about 10 and about 25 .mu.m in length.
[0174] ALTU-238 is a ready to use suspension of hGH crystals
complexed with poly-L-Arginine in a formulation vehicle that is
long-acting (e.g., provides week-long release of hGH after
subcutaneous administration through a 29-gauge or finer needle).
The formulation of ALTU-238 is 25 mg/mL rhGH, 5 mg/mL poly-Arg, 25
mM Tris, 100 mM Na Acetate, 5% w/v PEG 6000 pH 7.5, which can be
used, e.g., as a standard for comparison studies to assess the
stability of the formulations described herein.
[0175] Two formulations, liquid and lyophilized, can be prepared
with poly-L-Arginine complexed with hGH crystals. Descriptions of
each are provided.
Liquid Suspension Formulation
[0176] Formulation Vehicle
[0177] The combination of pH, buffer, salt, suspending agent,
hyaluronic acid ("HA") and/or preservative in which the poly-Arg
complexed hGH crystals are suspended can be referred to as the
"formulation vehicle." Each of the components of the vehicle can be
varied separately or in combination and optimized to result in a
suspension of complexed hGH crystals with desired properties, e.g.,
increased stability.
[0178] pH
[0179] The pH of a suspension can be a major factor affecting the
stability of hGH complexed crystals (a suspension of
poly-Arg-complexed hGH crystals). For example, the pH of the
suspension can affect the deamidation and oxidation of the hGH
complexed crystals. In some embodiments, a pH above about 7.0 or
7.5 (depending on other formulation conditions) can cause increases
in both protein deamidation (FIG. 1) and oxidation. Deamidation can
be measured, e.g., by cation exchange HPLC (CEX-HPLC). Oxidation
can be measured, e.g., by reverse phase HPLC (RP-HPLC). pH below
about 5.5 or about 5.0 can lead to protein turbidity, e.g.,
depending on other properties of the suspension. Turbidity can be
measured, e.g., by UV absorbance readings at 320 nm.
[0180] In some embodiments, the pH of the suspension is between
about 6 and about 7.5. In preferred embodiments, the pH is between
6.1 and 6.8. For example, the pH can be about 6.1, about 6.3, about
6.5, or about 6.8.
[0181] One can test a candidate pH by providing a poly-Arg
complexed crystalline rhGH suspension that contains 25 mg/mL rhGH,
5 mg/mL poly-Arg, 25 mM Tris, 100 mM Na Acetate, 5% w/v PEG 6000 at
a candidate pH value. The stability of the suspension at the
candidate pH, measured, e.g., as a percent oxidation or
degradation, at a predetermined time is compared with one or more
standards. For example, a suitable standard would be a suspension
similar to the test conditions except that the pH of the suspension
is not adjusted, e.g., the pH of the standard can be 7.5. The
stabilities of the test (the suspension adjusted to the candidate
pH) and standard (the pH is not adjusted) suspensions are compared.
Suitability can be shown by the test suspension increasing
stability (e.g., as measured by a smaller amount of oxidation or
deamidation) as compared with the standard. Another standard can be
a suspension similar to the test suspension except that in place of
the candidate pH, the suspension has another pH described herein,
for example, pH 6.5. Suitability can be shown by the suspension at
the candidate pH having comparable or better effects on stability
than the suspension at pH 6.5.
[0182] Buffer
[0183] The buffer used to make a suspension can be a major factor
affecting the stability of hGH complexed crystals (a suspension of
poly-Arg-complexed hGH crystals). For example, the buffer of the
suspension can affect the crystal size and turbidity (e.g., due to
insoluble aggregates) of the suspension of hGH complexed crystals.
Turbidity can be measured, e.g., by UV absorbance readings at 320
nm. Crystal size can be evaluated, e.g., by microscopy and by laser
diffraction particle size counter.
[0184] In some embodiments, the buffer can be phosphate buffer,
glycine buffer, histidine buffer, citrate buffer, acetate buffer,
or Tris buffer. In preferred embodiments, the buffer is histidine
buffer, citrate buffer, or Tris buffer. In more preferred
embodiments, the buffer is phosphate buffer. The concentration of
the buffer can also be varied, e.g., the concentration of the
buffer components, e.g., phosphate, glycine, histidine, citrate,
acetate, or Tris can be varied.
[0185] One can test a candidate buffer by providing a poly-Arg
complexed crystalline rhGH suspension that contains 25 mg/mL rhGH,
5 mg/mL poly-Arg, 100 mM Na Acetate, 5% w/v PEG 6000 at pH 6.5 in a
test buffer. The stability of the suspension in the candidate
buffer, measured, e.g., as the amount of turbidity, at a
predetermined time is compared with one or more standards. For
example, a suitable standard would be a composition similar to the
test conditions except that the buffer of the composition is a
buffer described herein, e.g., the buffer of the standard can be
phosphate buffer. The stabilities of the test (the suspension in
the test buffer) and standard (the suspension in phosphate buffer)
suspensions are compared. Suitability can be shown by the test
suspension increasing stability (e.g., as measured by a smaller
amount of turbidity) as compared with the standard. Another
standard can be a suspension similar to the test suspension except
that in place of the candidate buffer, the suspension has another
buffer described herein, for example, Tris buffer. Suitability can
be shown by the suspension in the candidate buffer having
comparable or better effects on stability than the suspension in
phosphate buffer. As another example, the concentration of the
buffer can also be tested, e.g., the stability (e.g., turbidity) of
a suspension in Tris at a test concentration, e.g., 50 mM, can be
compared to a suspension in Tris at a standard concentration
described herein, e.g., 25 mM. Suitability can be shown by the
suspension in the candidate concentration having comparable or
better effects on stability than the suspension in standard
concentration.
[0186] Salt
[0187] The salt used in a suspension can be a major factor
affecting the stability of hGH complexed crystals (a suspension of
poly-Arg-complexed hGH crystals). For example, the salt content in
the suspension can affect the deamidation rate and/or levels in the
suspension of hGH complexed crystals. As an example, higher salt
content (e.g., increasing ionic strength) can lead to less
deamidation. Deamidation can be measured, e.g., by cation exchange
HPLC (CEX-HPLC).
[0188] In some embodiments, the salt can be a sodium salt such as
sodium chloride or sodium acetate. In preferred embodiments, sodium
chloride is used in the suspension. The salt concentration can also
be varied, e.g., the concentration can be from about 1 mM to about
200 mM, e.g., from about 60 mM to about 200 mM.
[0189] One can test a candidate buffer by providing a poly-Arg
complexed crystalline rhGH suspension that contains a test salt, 25
mg/mL rhGH, 5 mg/mL poly-Arg, 5% w/v PEG 6000 at pH 6.5 in
phosphate buffer. The stability of the suspension with the
candidate salt, measured, e.g., as the amount of deamidation, at a
predetermined time is compared with one or more standards. For
example, a suitable standard would be a composition similar to the
test conditions except that the salt of the composition is a salt
described herein, e.g., the salt of the standard can be sodium
chloride. The stabilities of the test (the suspension with the test
salt) and standard (the suspension with sodium chloride)
suspensions are compared. Suitability can be shown by the test
suspension increasing stability (e.g., as measured by a smaller
amount of deamidation) as compared with the standard. Another
standard can be a suspension similar to the test suspension except
that in place of the candidate salt, the suspension has another
salt described herein, for example, sodium acetate. Suitability can
be shown by the suspension with the candidate salt having
comparable or better effects on stability than the suspension with
sodium acetate. As another example, the concentration of the given
salt can also be tested, e.g., the stability (e.g., deamidation) of
a suspension with sodium chloride at a test concentration, e.g., 20
mM, can be compared to a suspension with sodium chloride at a
standard concentration described herein, e.g., 60 mM. Suitability
can be shown by the suspension with the candidate concentration
having comparable or better effects on stability than the
suspension in standard concentration.
[0190] Suspending Agent
[0191] The suspending agent used in a suspension can be a major
factor affecting the stability of hGH complexed crystals (a
suspension of poly-Arg-complexed hGH crystals). For example, the
suspending agent in the suspension can affect the rate of chemical
degradation and/or deamidation rate and/or levels in the suspension
of hGH complexed crystals. No or low amounts of suspending agent
can lead to caking of the suspension. Chemical degradation can be
measured, e.g., by RP-HPLC. Deamidation can be measured, e.g., by
cation exchange HPLC (CEX-HPLC).
[0192] In some embodiments, the suspending agent can be a
polyethylene glycol, mannitol, glycine, or sucrose. In preferred
embodiments, the suspending agent can be a polyethylene glycol,
mannitol, or glycine. In more preferred embodiments, the suspending
agent is a polyethylene glycol such as PEG3350, PEG6000, or
PEG8000. The amount of the suspending agent can also be varied,
e.g., the amount can be from about 2.5% to about 20%.
[0193] One can test a candidate suspending agent by providing a
poly-Arg complexed crystalline rhGH suspension that contains a test
suspending agent, 25 mg/mL rhGH, 5 mg/mL poly-Arg, 100 mM Na
Acetate, at pH 6.5 in phosphate buffer. The stability of the
suspension with the candidate suspending agent, measured, e.g., as
the amount of deamidation, at a predetermined time is compared with
one or more standards. For example, a suitable standard would be a
composition similar to the test conditions except that the
suspending agent of the composition is a suspending agent described
herein, e.g., the standard can be PEG8000. The stabilities of the
test (the suspension with the test suspending agent) and standard
(the suspension with PEG8000) suspensions are compared. Suitability
can be shown by the test suspension increasing stability (e.g., as
measured by a smaller amount of deamidation) as compared with the
standard. Another standard can be a suspension similar to the test
suspension except that in place of the candidate suspending agent,
the suspension has another suspending agent described herein, for
example, PEG6000. Suitability can be shown by the suspension with
the candidate suspending agent having comparable or better effects
on stability than the suspension with PEG6000. As another example,
the concentration of the given suspending agent can also be tested,
e.g., the stability (e.g., deamidation) of a suspension with PEG at
a test amount, e.g., 25%, can be compared to a suspension with PEG
at a standard concentration described herein, e.g., 5%. Suitability
can be shown by the suspension with the candidate amount having
comparable or better effects on stability than the suspension in
standard amount.
[0194] hGH Concentration
[0195] The concentration of poly-Arg complexed crystals in a
suspension can be a another factor affecting the stability of hGH
complexed crystals (a suspension of poly-Arg-complexed hGH
crystals). For example, at higher concentrations, the suspension
may become thick. Protein stability can be assayed by RP-HPLC for
oxidation levels or rates. Stability can also be measured by cation
exchange HPLC (CEX-HPLC) for deamidation levels or rates.
[0196] In some embodiments, the concentration can be about 5 to
about 50 mg/ml. In preferred embodiments, the concentration is
about 20 to about 30 mg/ml.
[0197] One can test a candidate concentration by providing a
poly-Arg complexed crystalline rhGH suspension that contains a test
concentration of rhGH, 5 mg/mL poly-Arg, 100 mM Na Acetate, 5% w/v
PEG 6000 at pH 6.5 in phosphate buffer. The stability of the
suspension at the candidate concentration, measured, e.g., as the
amount of deamidation, at a predetermined time is compared with one
or more standards. For example, a suitable standard would be a
composition similar to the test conditions except that the
concentration of the composition is a concentration described
herein, e.g., about 35 mg/ml. The stabilities of the test (the
suspension at the test concentration) and standard (the suspension
at 25 mg/ml) suspensions are compared. Suitability can be shown by
the suspension at the candidate concentration having comparable or
better effects on stability (e.g., as measured by a smaller amount
of deamidation) as compared with the standard.
[0198] hGH to Poly-Arg Ratio
[0199] The ratio of hGH to poly-Arg in the complexed crystals in a
suspension can be a another factor affecting the stability of the
crystals. For example, it is possible that at lower hGH to poly-Arg
ratios, the degradation level may be higher than at higher hGH to
poly-Arg ratios (e.g., ration of 7). Degradation can be measured,
e.g., by reverse phase HPLC (RP-HPLC).
[0200] In some embodiments, the hGH to poly-Arg ratio can be about
3 to about 15. In preferred embodiments, the ratio is about 7 to
about 11.
[0201] One can test a candidate ratio by providing a poly-Arg
complexed crystalline rhGH suspension that contains test amounts of
poly-Arg to alter the hGH to poly-Arg ratio, 25 mg/ml rhGH, 100 mM
Na Acetate, 5% w/v PEG 6000 at pH 6.5 in phosphate buffer. The
stability of the suspension at the candidate ratio, measured, e.g.,
as the amount of oxidation, at a predetermined time is compared
with one or more standards. For example, a suitable standard would
be a composition similar to the test conditions except that the
ratio of hGH to poly-Arg of the composition is a ratio described
herein, e.g., 5 (i.e., the poly-Arg is present at a concentration
of 5 mg/ml). The stabilities of the test (the suspension at the
test ration) and standard (the suspension with a ratio of 5)
suspensions are compared. Suitability can be shown by the
suspension at the candidate ratio having comparable or better
effects on stability (e.g., as measured by lower oxidation levels)
as compared with the standard.
[0202] Storage Container and Container Closure
[0203] The choice of container used to store a suspension and/or
the choice of container closure can affect the stability of hGH
complexed crystals (a suspension of poly-Arg-complexed hGH
crystals). For example, the choice of container and/or closure can
affect dose consistency or product loss. For example, crystals can
accumulate in the in the bottle neck of the vial or accumulate on
the closure (e.g., on the walls of the closure). Further, the shape
of the container and/or the closure can also affect dose
consistency. Dose consistency can be measured, e.g., by measuring
the amount of recovered hGH concentration or by measuring the
extractable weight, e.g., after agitating the samples.
[0204] In some embodiments, vials and prefilled syringes made with
various materials, e.g., siliconized, plastic (e.g., CZ) resin, or
polypropylene vials are used. In some embodiments, the closure can
be a Teflon coated stopper (e.g., a Diekyo Fluorotec stopper) or a
rubber stopper, e.g., a butyl rubber stopper, e.g., West 4432/50.
In preferred embodiments, siliconized containers, especially
cylindrical siliconized or coated-surface containers, are used. In
more preferred embodiments, siliconized prefilled syringes are
used. In preferred embodiments, Teflon or butyl rubber stoppers are
used as the closures.
[0205] One can test a candidate container by providing a poly-Arg
complexed crystalline rhGH suspension that contains 25 mg/ml rhGH,
5 mg/ml poly-Arg, 100 mM Na Acetate, 5% w/v PEG 6000 at pH 6.5 in
phosphate buffer in a test container. The stability of the
suspension in the candidate container, measured, e.g., as the
amount of extractable weight, at a predetermined time is compared
with one or more standards. For example, a suitable standard would
be the composition stored in a container described herein, e.g., a
prefilled syringe. The stabilities of the suspension in the
candidate container and the standard container are compared.
Suitability can be shown by the suspension in the candidate
container having comparable or better effects on stability (e.g.,
as measured by weight recovery) as compared with the standard.
[0206] Candidate container closures can be evaluated in a like
manner.
[0207] Head Space
[0208] Finally, the head space in the container can also affect
protein stability. For example, the head space of container closure
can have a significant impact on delivered dose consistency.
[0209] In some embodiments, prefilled syringes with stake needles
are preferred because of the limited dead volume (range from 2 to 5
.mu.L) for hGH complexed crystals or any protein crystals products.
There is no bottle neck for the prefilled syringes; also, the head
space is much smaller and can be controlled by the stopper
placement; the plunger can push almost all of the crystal out of
the syringe barrel (except the dead volume). Other containers,
e.g., vials, can also be used. In preferred embodiments, minimal or
no head space is present in the container closure. Companies can
fill material with no head space. Limited head space is preferred
for container closures used for protein crystals for dose
consistency. In a most preferred embodiment, prefilled syringes
with stake needles and a head space of less than 10 mm are more
suitable for protein crystal parenteral products.
[0210] Preservative
[0211] Preservatives, e.g., anti-microbial preservatives, can be
used in the preparation of multi-dose pharmaceutical formulations.
The optional addition of and the concentration of a preservative in
a formulation of poly-Arg complexed crystals in suspension can be a
factor affecting the stability of hGH complexed crystals (a
suspension of poly-Arg-complexed hGH crystals). For example, the
presence of a preservative may affect the stability, crystal
structure, and/or efficacy (e.g., release profile) of the complexed
hGH. Crystal structure can be assayed by microscopy. Release
profile can be measured by assaying the concentration of hGH in the
formulation supernatant or calculating the percent of free hGH in
the formulation.
[0212] In some embodiments, the preservative can be phenol,
meta-cresol, benzyl alcohol, methyl paraben, clorobutanol. In
preferred embodiments, the preservative is phenol or m-cresol. In
more preferred embodiments, the preservative is phenol. The amount
of the preservative can also be varied, e.g., the amount preferably
can be from about 0.1% to about 1%.
[0213] One can test a candidate preservative by providing a
poly-Arg complexed crystalline rhGH suspension that contains the
candidate preservative, 25 mg/ml rhGH, 5 mg/mL poly-Arg, 100 mM Na
Acetate, 5% w/v PEG 6000 at pH 6.5 in phosphate buffer. The
stability of the suspension with the candidate preservative,
measured, e.g., as the concentration of hGH in the formulation
supernatant, at a predetermined time is compared with one or more
standards. For example, a suitable standard would be a composition
similar to the test conditions except that no preservative is
present. The stabilities of the test (the suspension with the test
the concentration of hGH in the formulation supernatant) and
standard (the suspension without a preservative) suspensions are
compared. Suitability can be shown by the suspension with the
candidate preservative having comparable or better effects on
stability (e.g., as measured by a smaller amount of hGH in the
supernatant) as compared with the standard. As another example, the
concentration of the given preservative can also be tested, e.g.,
the stability of a suspension with phenol at a test amount, e.g.,
2%, can be compared to a suspension with phenol at a standard
concentration described herein, e.g., 0.5%. Suitability can be
shown by the suspension with the candidate amount having comparable
or better effects on stability than the suspension in standard
amount.
[0214] Hyaluronic Acid
[0215] Hyaluronic acid ("HA") can also be included in the
formulations described herein. The present disclosure contemplates
the use of both HA and salts thereof. For example, HA may
neutralize excess crystal charge, thereby reducing potential
injection site reactions that may otherwise be caused by the
administration of a formulation of complexed hGH crystals. Other
advantages can include: contributing to a sustained release profile
of complexed crystals after administration to a subject (e.g.,
human), allowing injection with fine gauged (e.g., very fine
gauged, e.g., 30-gauge) needles, preserving crystallinity and
integrity of complex over time. In some embodiments, the use of HA
does not alter the hGH release profile or in vivo efficacy.
[0216] In preferred embodiments, HA is present in the formulation
in an amount between about 0.01% and 0.5% (w/v), more preferably
about 0.2% (w/v).
[0217] One can test a candidate concentration of HA by providing a
poly-Arg complexed crystalline rhGH suspension that contains a test
concentration of HA, 25 mg/ml rhGH, 5 mg/mL poly-Arg, 100 mM Na
Acetate, 5% w/v PEG 6000 at pH 6.5 in phosphate buffer. The
stability of the suspension at the candidate concentration,
measured, e.g., as the dissolution rate (e.g., measured by size
exclusion chromatography), at a predetermined time is compared with
one or more standards. For example, a suitable standard would be a
composition similar to the test conditions except that the HA
concentration of the composition is a concentration described
herein, e.g., about 0.2% (w/v). The stabilities of the test (the
suspension at the test concentration) and standard (the suspension
with 0.2% HA)) suspensions are compared. Suitability can be shown
by the suspension at the candidate concentration having comparable
or better effects on stability (e.g., as measured by no increase in
dissolution rate) as compared with the standard.
Lyophilized Formulation
[0218] Lyophilized Formulations of hGH Crystals
[0219] Lyophilized hGH or hGH derivative protein formulations
(e.g., complexed with poly-Arg) (e.g., after reconstitution of a
lyophilized preparation) can increase the stability of the hGH
formulation. For example, the lyophilized formulations described
herein are predicted to achieve about 24 months or longer chemical
stability at refrigerated or room temperatures.
[0220] Factors that can affect the stability of the formulation
include: the buffer, pH, salt, suspending agent, preservative, and
choice of storage container. One, two, three, four, five, or all of
these factors can be altered or controlled to increase the
stability of a protein of interest.
[0221] For example, as described herein, lyophilized formulations
of poly-Arg complexed hGH crystals preferably contain one or more
of the following: prepared using a buffer (preferably Tris or
combination of histidine and phosphate) at the pH range from 7 to
9, a salt (preferably sodium chloride or sodium acetate), and a
suspending agent (preferable polyethylene glycol 6000 or 8000). In
some formulations, a preservative is also included. The container
closure systems that are compatible with these formulations include
siliconized or coated vials, such as Schott type I plus coated
glass vials.
[0222] These components and amounts are suitable for the
formulations prior to lyophilization and also for the formulations
after the lyophilized hGH is reconstituted. For example, a given
volume of a pre lyophilization formulation can have a given
composition, e.g., 25 mg/mL poly-Arg complexed hGH crystals, 25 mM
Tris, 5% PEG8000, pH 7.5. To reconstitute the crystals post
lyophilization, a volume of liquid (e.g., saline or water,
preferably water) that is equivalent to the volume of the
formulation prior to lyophilization is added to reconstitute the
lyophilized preparation. Because the same volume was used, the post
lyophilization preparation will have the same or substantially the
same composition as the formulation prior to lyophilization.
Depending on what liquid was used to reconstitute the preparation,
additional components may exist in the post lyophilization
preparation (e.g., if saline was used for reconstitution instead of
water, the salt concentration may be increased). Optionally, the
amount of liquid added for reconstitution can be adjusted to
account for pre lyophilization volume contributed by the protein
crystals. Thus, a volume of liquid is added to bring the total
volume of the post lyophilization formulation to the total volume
pre lyophilization. As another alternative, a different (e.g.,
smaller or larger volume) of liquid can be used for reconstitution
to dilute (if a larger volume is used) or concentrate (if a smaller
volume is used) the components of the formulation.
[0223] Optimization of these factors and suitability determinations
can be performed as described above.
[0224] Generally, lyophilization (also known as freeze drying) is a
dehydration process typically used to preserve a perishable
material or make the material more convenient for transport. Freeze
drying works by freezing the material and then reducing the
surrounding pressure and adding enough heat to allow the frozen
water in the material to sublime directly from the solid phase to
gas. Products, e.g., proteins or protein crystals, can be
lyophilized to make them more stable, or easier to dissolve in
water for subsequent use.
[0225] A description of a typical lyophilization are as follows.
There are three stages in the complete lyophilization process:
Freezing, Primary Drying, and Secondary Drying.
[0226] Freezing: The freezing process consists of freezing the
material. This is often done by placing the material in a
freeze-drying flask and rotating the flask in a bath of dry ice and
methanol, or liquid nitrogen. On a larger-scale, freezing is
usually done using a freeze-drying machine. It is important to
freeze the material at a temperature below the eutectic point of
the material. Since the eutectic point occurs at the lowest
temperature where the solid and liquid phase of the material can
coexist, freezing the material at a temperature below this point
ensures that sublimation rather than melting will occur in the
following steps.
[0227] Primary Drying: During the primary drying phase, the
pressure is lowered and enough heat is supplied to the material for
the water to sublimate. The amount of heat necessary can be
calculated using the sublimating molecules' latent heat of
sublimation. In this initial drying phase, about 98% of the water
in the material is sublimated. This phase may be slow, because if
too much heat is added the material's structure could be
altered.
[0228] In this phase, pressure is controlled through the
application of partial vacuum. The vacuum speeds sublimation making
it useful as a deliberate drying process. Furthermore, a cold
condenser chamber and/or condenser plates provide a surface(s) for
the water vapor to re-solidify on. This condenser plays no role in
keeping the material frozen; rather, it prevents water vapor from
reaching the vacuum pump, which could degrade the pump's
performance. Condenser temperatures are often below 10.degree. C.,
e.g., -30.degree. C. or -50.degree. C., e.g., in ranges below these
temperatures.
[0229] Secondary Drying The secondary drying phase aims to
sublimate the water molecules that are adsorped during the freezing
process, since the mobile water molecules were sublimated in the
primary drying phase. This part of the freeze-drying process is
governed by the material's adsorption isotherms. In this phase, the
temperature is raised even higher than in the primary drying phase
to break any physico-chemical interactions that have formed between
the water molecules and the frozen material. Usually the pressure
is also lowered in this stage to encourage sublimation. However,
there are products that benefit from increased pressure as
well.
[0230] After the freeze drying process is complete, the vacuum is
usually broken with an inert gas, such as nitrogen, before the
material is sealed.
[0231] Properties of Freeze-dried Products: If a freeze-dried
substance is sealed to prevent the reabsorption of moisture, the
substance may be stored at room temperature without refrigeration,
the process can increase the shelf life of pharmaceuticals.
[0232] Lyophilization also causes less damage to the substance than
other dehydration methods using higher temperatures. Lyophilization
does not usually cause shrinkage or toughening of the material
being dried.
[0233] Lyophilized products can be rehydrated (reconstituted) much
more quickly and easily because it leaves microscopic pores. The
pores are created by the ice crystals that sublimate, leaving gaps
or pores in its place. This is especially important when it comes
to pharmaceutical uses.
[0234] Analytical Methods
[0235] Formulation development can be assessed by the following
characteristics: storage stability at various temperatures,
container-closure compatibility as a function of dose consistency,
potential stickiness, ease of administration of product, and loss
to container closure. The stability test methods and their
application are listed in Table 1 below:
TABLE-US-00001 TABLE 1 Stability test methods and their purposes
TEST METHODS APPLICATION pH pH maintenance Appearance Suspension
description Particle Size Distribution Physical stability Crystal
Morphology Physical stability Purity by Reverse Phase Chemical
degradation (oxidation) Chromatography (RP-HPLC) Purity by Size
Exclusion Soluble aggregates Chromatography (SEC) Poly(L-arginine)
Total Content Facilitate controlled release by Reverse Phase
Chromatography Free Poly(L-arginine) and hGH Physical stability
Content in Supernatant by Reverse Phase Chromatography Absorbance
at 280 nm Protein Concentration Undissolved crystals/aggregates
Insoluble aggregates by Absorbance at 320 nm Cation Exchange HPLC
Chemical degradation (deamidation) Crystal Dissolution hGH product
release profile
[0236] Chemical Degradation
[0237] Arrhenius-Plot kinetics can be employed to predict the rate
of chemical degradation at 2-8.degree. C. using short-term
stability data, e.g., generated from samples that are stressed at
higher storage temperatures such as 50.degree. C., 40.degree. C.,
35.degree. C., 30.degree. C. and 25.degree. C. Extrapolated
2-8.degree. C. data can be used to establish a proposed shelf life
or expiration date.
[0238] Arrhenius stability prediction can be used to test candidate
formulations. The Arrhenius stress model is a common life stress
relationship utilized in accelerated testing. It has been widely
used when the stimulus or acceleration variable (such as
deamidation, oxidation, etc) is thermal (i.e., temperature). It is
derived from the Arrhenius reaction rate equation proposed by the
Swedish physical chemist Svandte Arrhenius. The degradation rate at
5.degree. C. can be predicted using stability results from several
elevated temperatures. For example, samples of poly-Arg (pR)
complexed crystals in prefilled syringes with Fluoroteck stoppers
are placed at 2-8.degree. C., 25.degree. C., 30.degree. C.,
35.degree. C., 40.degree. C. and 50.degree. C. Based on the
reported data, the Arrhenius equation is applied and degradation
rate at 5.degree. C. is predicted. The results can be compared to
the real stability of a known/standard lot that was analyzed by the
same methods.
[0239] In addition, formulations can be tested by a dissolution
profile assay, using a standard hGH formulation for comparison.
[0240] The following are examples of analytical methods that can be
carried out to evaluate formulations containing crystalline
hGH.
[0241] hGH Content
[0242] The hGH content of a suspension containing hGH crystals
complexed with poly-Arg can be determined by UV spectrophotometry.
The suspension of hGH crystals complexed with poly-Arg is dissolved
in 50 mM glycine, pH 2.6 and absorbance at 280 nm is measured. The
concentration of hGH is calculated by multiplying the measured
absorbance value by the dilution factor, and then dividing by the
absorption coefficient for rhGH, 0.75 mLmg-1cm-1.
[0243] Turbidity
[0244] The turbidity of the suspension of hGH crystals complexed
with poly-Arg can be determined by UV spectrophotometry. The
suspension of hGH crystals complexed with poly-Arg is dissolved in
50 mM glycine, pH 2.6 and absorbance at 320 nm is measured.
[0245] Particle Size Distribution
[0246] Particle size distribution can be evaluated to assess the
consistency of the manufacturing process for a suspension
containing hGH crystals complexed with poly-Arg. Particle size
distribution can be determined by laser diffraction using a Coulter
LS 230 Particle Size Analyzer (Coulter Corp., Miami, Fla.) with
micro volume module. The sample is diluted in sample buffer or
1.times.PBS or the formulation vehicle to achieve an operation
range of 8 to 12% obscuration. Each sample is analyzed in
triplicate and data analysis is performed using the Fraunhofer
optical model. The volume representing the cumulative distribution
limits for 10% (dl10), 50% (median) and 90% (dl90) are reported.
The test method can be performed for product stability testing as
well (i.e., to assess the potential for particle distribution
change upon storage).
[0247] RP-HPLC (Percentage of Oxidation, and Poly-L-Arginine
Content)
[0248] Same mobile phases and column can be used for these
analyses. The method for poly L arginine (poly-Arg) quantitation
can be carried out at 214 nm and hGH at 280 nm. The amounts of poly
L arginine, and hGH are used to calculate and report free hGH to
supernatant and total poly-L-arginine content. The hGH/poly-Arg
ratio (mass-to-mass) can be calculated using total hGH
concentration derived from OD280 using an extinction coefficient of
0.75. Quantitation of poly-L-arginine can be determined by using a
calibration curve of poly-L-arginine standards. The concentration
of poly L-arginine in the test sample can be determined based on
the linear regression line of poly-L-arginine standards. A C5
Supelco Discovery Bio Wide Pore column, 5 cm.times.4.6 mm, 3 .mu.m
particle size, 300 A pore size, can be used. hGH samples can be
prepared for HPLC analysis by dissolution with a 50 mM glycine
buffer, pH 2.6. The elution of rhGH is carried out at a flow rate
of 1.0 mL/min with a mobile phase gradient formed by mobile phase A
(water with 0.1% trifluoroacetic acid) and mobile phase B
(acetonitrile with 0.1% trifluoroacetic acid) as follows: 0-2 min
held at 95% A-5% B, 2-8 min linear change to 50% A-50% B, 8-20 min
linear change to 30% A-70% B, 20-22 min linear change to 10% A-90%
B, 22-25 min held at 10% A-90% B, followed by return to the initial
conditions of 95% A-5% B. The peak areas of oxidized rhGH are
calculated and compared against that of non oxidized rhGH. Purity
is calculated as the fractional peak area of the oxidized peak
relative to the total area of all rhGH related peaks.
[0249] Soluble Aggregation (% High Molecular Weight)
[0250] High molecular weight soluble aggregates of rhGH can be
separated from rhGH monomer by size exclusion HPLC. A Phenomenex
BioSep-SEC-S 2000 7.8 mm I.D..times.60 cm column can be used. hGH
samples are prepared for HPLC analysis by dissolution with a 50 mM
glycine buffer of pH 2.6. The elution of rhGH is carried out
isocratically at a flow rate of 0.4 mL/min with a mobile phase
consisting of 60 mM sodium phosphate, 3% isopropanol, pH 7.0.
Detection of rhGH monomer and aggregates is carried out at 280 nm.
The peak areas of aggregated rhGH are measured and compared against
that of rhGH monomer peak. The peak before the main peak of rhGH is
the aggregated species of rhGH. Percent aggregate is calculated as
the fractional peak area of the aggregated peak relative to the
total area of all rhGH protein peaks.
[0251] Deamidation and Other Degradations
[0252] Deamidated rhGH peaks can be separated from non-deamidated
rhGH by Cation Exchange HPLC (CEX-HPLC). A PolyLC PolySULFOETHYL A,
4.6 mm.times.50 mm, 5 .mu.m, 300 .ANG. column can be used. hGH
samples are prepared for HPLC analysis by dissolution with a 50 mM
glycine buffer, pH 2.6. The elution of rhGH is carried out at a
flow rate of 1.0 mL/min with a mobile phase gradient of increasing
salt concentration formed by mobile phase A (50 mM sodium acetate,
pH 4.6) and mobile phase B (50 mM sodium acetate, 250 mM sodium
chloride, pH 4.6) as follows: Start at 100% A-0% B, 0 5 min linear
change to 80% A-20% B, 5-40 min linear change to 40% A-60% B, 4-45
min linear change to 0% A-100% B, and return to initial conditions
(100% A) in 5 min. Detection of deamidated and non-deamidated rhGH
was carried out at 280 nm. The peaks before the main rhGH peak in
the stressed sample are presumably deamidated and degraded rhGH
species. The peak areas of deamidated rhGH are calculated and
compared against that of non-deamidated rhGH. Purity is calculated
as the fractional peak area of the degraded peak relative to the
total area of all rhGH related protein peaks.
[0253] Dissolution Profile
[0254] A dissolution test for hGH crystals is developed to monitor
the relevant parameters in effecting dissolution of poly L
arginine-coated hGH crystals in a suitable buffer. Citrate, pH 5.0
can be used as the dissolution buffer. Dissolution rate of the
complexed hGH is measured under the condition of fixed mg of hGH
crystals in citrate pH 5.0 medium at constant agitation speed at
37.degree. C. An initial dispersion is made followed by
centrifugation to remove the supernatant, then replenished with new
medium that is similar to physiological condition. The procedure is
repeated every 15 minutes up to one hour, then the dissolution rate
is calculated.
[0255] Dissolution rate may also be measured by UV absorbance or
RP-HPLC (e.g., Agilent Technologies).
[0256] Bioactivity and pK Profile
[0257] The species used for in-vivo evaluation can be Rattus
norvegicus (Wistar Rats, hypophysectomized). Treatment groups are
designed to determine the comparability of crystalline hGH with a
commercial hGH product at a matched weekly dose level. The diluted
test articles are slowly injected subcutaneously in the
thoraco-lumbar region on either side of the spine. The needle is
directed away from the spine and inserted up to the hub before any
material was injected. The site of injection is shaved and marked
up to 3 days prior to dosing and thereafter as required to
facilitate injection. All doses are administered using a
30-gauge.times.8 mm needle attached to a 300 .mu.L syringe (BD part
number BD 320438). Each single unit marking on the syringe is
equivalent to 10 .mu.L. Diluted Control and Test articles are
carefully inverted in order to ensure suspension or solution
uniformity without causing foaming prior to withdrawal into the
syringe. The experiments provide a way to compare the bioactivity
of hGH formulations when administered to hypophysectomized male
Wistar rats. The selection of the control hGH formulation against
which comparisons are made can be based, in part, on
previously-established efficacy in a standard rat weight gain
assay. Test or control material is administered to
hypophysectomized male Wistar rats (9 rats/group) by subcutaneous
(SC) injection. For example, growth of hypophysectomized rats after
receiving a single SC injection of 5.6 mg/kg of a control
formulation and a test formulation can be compared. As another
example, daily SC injections of 0.8 mg/kg of a soluble commercial
rhGH (e.g., Nutropin AQ) for 7 days (i.e. 5.6 mg/kg/week) can be
used as a control. Body weights are measured weekly prior to the
start of dosing and daily from Day -7 (seven days prior to dosing)
until the end of the observation period on Day 8. In addition,
blood can be withdrawn for blood level hGH determination for pK
profile assay in a separate study. The blood hGH level can be
determined by ELISA.
[0258] Pharmaceutical Formulations
[0259] The crystalline protein formulations described herein, e.g.,
poly-Arg complexed hGH crystals, can be formulated into
pharmaceutical formulations for therapeutic use, e.g., to treat a
subject suffering from an hGH insufficiency or deficiency.
[0260] Crystals of human growth hormone or a human growth hormone
derivative can be combined with any pharmaceutically acceptable
excipient. According to this disclosure, a "pharmaceutically
acceptable excipient" is an excipient that acts as a filler or a
combination of fillers used in pharmaceutical compositions.
Preferred excipients included in this category are: 1) amino acids,
such as glycine, arginine, aspartic acid, glutamic acid, lysine,
asparagine, glutamine, proline; 2) carbohydrates, e.g.,
monosaccharides such as glucose, fructose, galactose, mannose,
arabinose, xylose, ribose; 3) disaccharides, such as lactose,
trehalose, maltose, sucrose; 4) polysaccharides, such as
maltodextrins, dextrans, starch., glycogen; 5) alditols, such as
mannitol, xylitol, lactitol, sorbitol; 6) glucuronic acid,
galacturonic acid; 7) cyclodextrins, such as methyl cyclodextrin,
hydroxypropyl-.beta.-cyclodextrin and alike; 8) inorganic
molecules, such as sodium chloride, potassium chloride, magnesium
chloride, phosphates of sodium and potassium, boric acid, ammonium
carbonate and ammonium phosphate; 9) organic molecules, such as
acetates, citrate, ascorbate, lactate; 10) emulsifying or
solubilizing/stabilizing agents like acacia, diethanolamine,
glyceryl monostearate, lecithin, monoethanolamine, oleic acid,
oleyl alcohol, poloxamer, polysorbates, sodium lauryl sulfate,
stearic acid, sorbitan monolaurate, sorbitan monostearate, and
other sorbitan derivatives, polyoxyl derivatives, wax,
polyoxyethylene derivatives, sorbitan derivatives; and 11)
viscosity increasing reagents like, agar, alginic acid and its
salts, guar gum, pectin, polyvinyl alcohol, polyethylene oxide,
cellulose and its derivatives propylene carbonate, polyethylene
glycol, hexylene glycol, tyloxapol. Salts of such compounds may
also be used. A further preferred group of excipients includes
sucrose, trehalose, lactose, sorbitol, lactitol, mannitol,
inositol, salts of sodium and potassium, such as acetate,
phosphates, citrates and borate, glycine, arginine, polyethylene
oxide, polyvinyl alcohol, polyethylene glycol, hexylene glycol,
methoxy polyethylene glycol, gelatin,
hydroxypropyl-.beta.-cyclodextrin, polylysine and polyarginine.
[0261] In some embodiments of this disclosure, the excipient is
selected from the group consisting of: amino acids, salts,
alcohols, carbohydrates, proteins, lipids, surfactants, polymers,
polyamino acids and mixtures thereof. In some preferred
embodiments, the excipient is selected from the group consisting
of: protamine, polyvinylalcohol, cyclodextrins, dextrans, calcium
gluconate, polyamino acids, such as polyarginine, polylysine and
polyglutamate, polyethylene glycol, dendrimers, polyorthinine,
polyethyleneimine, chitosan and mixtures thereof. In more preferred
embodiments, the excipient is selected from the group consisting
of: protamine, polyarginine, polyethylene glycol and mixtures
thereof.
[0262] Crystals of human growth hormone or a human growth hormone
derivative according to this disclosure can also be combined with a
carrier or excipient, a substance that, when added to a
therapeutic, speeds or improves its action (see, e.g., The On-Line
Medical Dictionary at http://cancerweb.ncl.ac.uk/omd/index.html).
Examples of carriers or excipients include, for example, buffer
substances, such as phosphates, glycine, sorbic acid, potassium
sorbate, partial glyceride mixtures of saturated vegetable fatty
acids, waters, salts or electrolytes, such as protamine sulfate,
disodium hydrogen phosphate, sodium chloride, zinc slats, colloidal
silica, magnesium, trisilicate, cellulose-based substances and
polyethylene glycol. Carriers or excipients for gel base forms may
include, for example, sodium carboxymethylcellulose, polyacrylates,
polyoxyethylene-polyoxypropylene-block copolymers, polyethylene
glycol and wood wax alcohols.
[0263] In yet other preferred embodiments, the excipient is
protamine. Furthermore, crystals of hGH or an hGH derivative and
protamine are present in an hGH:protamine ratio of about 5:1 to
about 1:10 (w/w). That ratio may also range between about 10:1 to
about 20:1 (w/w). Most preferably, that ratio ranges between about
12:1 to about 15:1 (w/w). According to alternate embodiments, that
ratio is between about 3:1 and about 1:10 (w/w). In other
embodiments, that ratio is between about 5:1 and about 40:1 (w/w).
And, in a further embodiment, that ratio is about 5:1 (w/w).
[0264] In another aspect, the pharmaceutically acceptable excipient
is selected from the group consisting of polyamino acids, including
polylysine, polyarginine and polyglutamate. In preferred
embodiments, the excipient is polylysine. In a more preferred
embodiment, polylysine has a molecular weight between about 1,500
and about 8,000 kD. In other embodiments, the crystals of hGH or an
hGH derivative and polylysine are present in an hGH:polylysine
ratio of about 5:1 to about 40:1 (w/w). That ratio may also range
between about 10:1 to about 20:1 (w/w). Most preferably, that ratio
ranges between about 12:1 to about 15:1 (w/w). According to
alternate embodiments, that ratio is about 5:1 to about 1:50 (w/w).
In further embodiments, that ratio is about 5:1 (w/w).
[0265] In yet other preferred embodiments, the excipient is
polyarginine. In more preferred embodiments, polyarginine has a
molecular weight between about 15,000 and about 60,000 kD. In other
embodiments, the crystals of hGH or an hGH derivative and
polyarginine are present in an hGH:polyarginine ratio of about 5:1
to about 40:1 (w/w). That ratio may also range between about 10:1
to about 20:1 (w/w). Most preferably, that ratio ranges between
about 12:1 to about 3:1 (w/w). According to alternate embodiments,
that ratio is about 5:1 to about 1:50 (w/w). In other embodiments,
that ratio is between about 12:1 and about 15:1 (w/w). In further
embodiments, that ratio is about 5:1 (w/w).
[0266] Other embodiments of the disclosure include an injectable
crystalline suspension comprising about 20 mg/ml of crystals of hGH
or an hGH derivative. The suspension is characterized by easy
resuspendability, slow sedimentation, and a time action profile of
about 7 days. It may be injected once weekly, using a 30 gauge
syringe and providing an 80% level of effective loading. The
suspension is substantially pure, as reflected by parameters of
0.02% aggregation (SE-HPLC) and 2.3% related proteins (RP-HPLC).
This purity is maintained for at least about 4 months under
refrigerated conditions.
[0267] Other embodiments of the disclosure relate to a crystal of
hGH or an hGH derivative which is characterized as having delayed
dissolution behavior when introduced into an individual, as
compared to that of conventional soluble hGH or hGH formulations.
According to this disclosure, dissolution of crystals of hGH or an
hGH derivative is characterized by either in vitro or in vivo
dissolution parameters. For example, in vitro dissolution is
described as the concentration of soluble hGH (expressed as a
percentage of total or mg of total hGH or hGH derivative crystals
originally present) obtained per 15 minutes or per wash step in a
sequential dissolution process. In other embodiments, crystals of
hGH or an hGH derivative are characterized by an in vitro
dissolution rate of between about 2 and about 16% of said crystal
per wash step upon exposure to a dissolution buffer (50 mM HEPES
(pH 7.2), 140 mM NaCl, 10 mM KCl and 0.02% (v/v) NaN.sub.3) at a
temperature of 37.degree. C., wherein the concentration of hGH or
an hGH derivative is present in solution at a concentration of
about 2 mg/ml. In another embodiment, crystals of hGH or an hGH
derivative are characterized by an in vitro dissolution rate of
between about 0.04 to about 0.32 mg of said crystal per wash step
in a sequential dissolution process. On the other hand, in vivo
dissolution is described by serum levels of hGH in a mammal over
time after a single injection of hGH into the mammal.
[0268] Therapeutic Use and Dosage
[0269] In mammals, GH stimulates tissues to synthesize and secrete
IGF-1, a protein that, in turn, plays a role in cell division and
metabolic processes. As will be appreciated by those of skill in
the art, serum hGH and IGF-1 levels are dependent on many factors,
including physiological and treatment-related factors. Such factors
include, but are not limited to: physiological factors, such as:
birth age and bone age, sex, body weight, developmental stage
(e.g., increased level at puberty) and treatment-related factors,
such as dose, rate (kinetics) of dosing and route of
administration, and patient diagnosis and medical history. Also,
those of skill in the art will appreciate that different hGH and
IGF-1 levels may be beneficial, both from the standpoint of safety
and efficacy, for different patient populations.
[0270] Adults or children suffering from a variety of hGH
insufficiencies, disease states or syndromes may be treated by
various regimens of exogenously delivered hGH using hGH crystals or
hGH derivative crystals according to this disclosure. For example,
an endocrinologist may initiate therapy using a dose of about 0.2
mg/kg/week for a child, increasing the dose to about 0.3 mg/kg/week
after several weeks or months of treatment, with the dose being
further increased to about 0.7 mg/kg/week around puberty. As will
be appreciated by those of skill in the art, the level of such
exogenously delivered hGH dosed to adults or children requiring hGH
delivery is also dependent upon the existing physiological level or
concentrations of hGH.
[0271] Dosage regimens for hGH in adults or children are often
expressed in terms of mg/kg or International Units (IU/kg). Such
regimens are generally scheduled for either a day or a week, i.e.,
mg/kg/day or mg/kg/week. With such considerations in mind,
according to one embodiment of this disclosure, a single
administration of crystals of hGH or an hGH derivative, or a
composition comprising such crystals, for example, a single weekly
administration of about 9 mg per 30 kg child, provides an in vivo
hGH serum concentration of greater than about 10 ng/ml on days 1
and 2 post-administration, greater than about 5 ng/ml on days 3 and
4 post-administration and about 0.3 ng/ml on day 5 to day 7
post-administration. Alternatively, a single administration of
crystals of hGH or an hGH derivative, or a composition comprising
such crystals, provides an in vivo hGH serum concentration of about
0.3 ng/ml to about 2,500 ng/ml hGH, preferably about 0.5 ng/ml to
about 1,000 ng/ml hGH, most preferably about 1 ng/ml to about 100
ng/ml hGH for between about 0.5 hours and about 40 days
post-administration in said mammal, preferably for between about
0.5 hours and any one of about 10 days, 7 days or 1 day
post-administration. Similarly, a single administration of crystals
of hGH or an hGH derivative, or a composition comprising such
crystals, provides an in vivo serum concentration of above about 2
ng/ml hGH, preferably above about 5 ng/ml hGH, most preferably
above about 10 ng/ml hGH for between about 0.5 hours to about 40
days post-administration in said mammal, preferably for any one of
about 10, 7 or 1 days post-administration. In a more preferred
embodiment of this disclosure, a single administration of crystals
of hGH or an hGH derivative, or a composition comprising such
crystals, provides an in vivo serum concentration of greater than
about 0.3 ng/ml hGH for between about 0.5 hours and about 40 days
in a mammal, preferably for any one period of any one of about 10,
7 or 1 days post-administration. According to one embodiment of
this disclosure, a single weekly administration of crystals of hGH
or an hGH derivative, or a composition comprising such crystals,
provides an in vivo hGH serum concentration of greater than about
10 ng/ml hGH on days 1 and 2 post-administration, greater than
about 5 ng/ml hGH on days 3 and 4 post-administration and above
about 0.3 ng/ml hGH on day 5 to day 7 post-administration. In a
further embodiment, a single administration of crystals of hGH or
an hGH derivative, or a composition comprising such crystals,
provides an in vivo serum concentration of greater than about 0.3
ng/ml hGH for between about 0.5 hours and about 10 days
post-administration.
[0272] According to other embodiments of this disclosure, a single
administration of crystals of hGH or an hGH derivative, or a
composition comprising such crystals, provides an in vivo IGF-1
serum elevation over baseline IGF-1 level prior to said
administration of greater than 50 ng/ml from about 10 hours to
about 72 hours post-administration and between about 0.5 ng/ml to
about 50 ng/ml from about 72 hours to about 15 days
post-administration, preferably about 10 days post-administration.
Alternatively, a single administration of crystals of hGH or an hGH
derivative, or a composition comprising such crystals, provides an
in vivo IGF-1 serum elevation of about 5 ng/ml to about 2,500
ng/ml, preferably about 100 ng/ml to about 1,000 ng/ml, for about
0.5 hours to about 40 days post-administration, preferably about 7
days post-administration. Alternatively, a single administration of
crystals of hGH or an hGH derivative, or a composition comprising
such crystals, according to the present disclosure may provide an
in vivo IGF-1 serum elevation of above about 50 ng/ml, preferably
above about 100 ng/ml, for about 0.5 hours to about 40 days
post-administration, preferably about 7 days post-administration.
According to one embodiment of this disclosure, a single
administration of crystals of hGH or an hGH derivative, or a
composition comprising such crystals, provides an in vivo IGF-1
serum elevation over baseline IGF-1 level prior to said
administration of greater than about 50 ng/ml from about 10 hours
to about 72 hours post-administration and between about 0.5 ng/ml
to about 50 ng/ml from about 72 hours to about 15 days
post-administration or 72 hours to about 10 days
post-administration.
[0273] According to this disclosure, a single administration is
defined as between about 0.01 mg/kg/week to about 100 mg/kg/week
hGH crystals or hGH derivative crystals, or a composition
comprising such crystals, wherein the volume of the administration
is between 0.1 ml and about 1.5 ml. For example, pediatric growth
hormone deficiency may be dosed with hGH crystals or hGH derivative
crystals, or a composition comprising such crystals, at about 0.3
mg/kg/week, e.g., about 9 mg for a 30 kg child. Turner syndrome may
be dosed with hGH crystals or hGH derivative crystals, or a
composition comprising such crystals, at about 0.375 mg/kg/week,
e.g., about 11.25 mg for a 30 kg child. Additionally, adult growth
hormone deficiency may be dosed with hGH at about 0.2 mg/kg/week,
e.g., about 16 mg for a 80 kg adult. AIDS wasting disease may be
dosed with hGH at 6 mg/day, e.g., 42 mg/week.
[0274] In yet other embodiments, crystals of hGH or an hGH
derivative, or composition comprising such crystals, display a
relative bioavailability similar to that of soluble hGH in a
mammal. The crystals according to this disclosure have a relative
bioavailability of at least 50% or greater compared to that of
soluble hGH, delivered by the same route (e.g., subcutaneous or
intramuscular injection), wherein said bioavailability is measured
by the area under curve (AUC) of total in vivo hGH serum
concentration for said soluble hGH and said crystal. Crystals of
hGH or an hGH derivative are thus characterized by an advantageous
in vivo dissolution rate.
[0275] The present disclosure further provides methods of
administering crystals of hGH or an hGH derivative to a mammal
having a disorder associated with human growth hormone deficiency
or insufficiency or which is ameliorated by treatment with hGH. The
method comprises the step of administering to the mammal a
therapeutically effective amount of a crystal of hGH or an hGH
derivative. Alternatively, the method comprises the step of
administering to the mammal an effective amount of a composition
comprising crystals of hGH or an hGH derivative alone or with an
excipient. Various embodiments of crystals of hGH or an hGH
derivative according to this disclosure are: calcium crystals,
monovalent crystals, protamine crystals or polyarginine crystals of
hGH or an hGH derivative. Such crystals, or compositions comprising
them, may be administered by a time regimen of about once every
three days, about once a week, about once every two weeks or about
once every month.
[0276] Disorders related to hGH insufficiency or deficiency that
may be treated according to this disclosure include, but are not
limited to: adult growth hormone deficiency, pediatric growth
hormone deficiency, Prader-Willi syndrome, Turner syndrome, short
bowel syndrome, chronic renal insufficiency, idiopathic short
stature, dwarfism, hypopituitary dwarfism, bone regeneration,
female infertility, intrauterine growth retardation, AIDS-related
cachexia, Crohn's disease, burns, as well as other genetic and
metabolic disorders. In one embodiment of this disclosure, the
disorder is pediatric growth hormone deficiency and treatment
results in annualized growth velocity of between about 7 cm and
about 11 cm in the child undergoing treatment.
[0277] In another embodiment, a calcium crystal of hGH or an hGH
derivative may serve as a useful adjunct for bone therapy, as well
as treatment of human growth hormone deficiency in a mammal.
[0278] The present disclosure also provides methods for inducing
weight gain in a mammal, comprising the step of administering to
said mammal a therapeutically effective amount of crystals of hGH
or an hGH derivative. Alternatively, such methods comprise the step
of administering to said mammal a therapeutically effective amount
of a composition comprising crystals of hGH or an hGH derivative
and an excipient. In one embodiment of such methods, the weight
gain induced in a hypophysectomized rat is between about 5% and
about 40% after administration of said crystals by injection once a
week.
[0279] Crystals of hGH, crystals of an hGH derivative or
compositions comprising them alone, or with an excipient, may be
administered alone, or as part of a pharmaceutical, therapeutic or
prophylactic preparation. They may be administered by any
conventional administration route including, for example,
parenteral, oral, pulmonary, nasal, aural, anal, dermal, ocular,
intravenous, intramuscular, intraarterial, intraperitoneal,
mucosal, sublingual, subcutaneous, transdermal, topical, buccal or
intracranial routes.
[0280] In some embodiments, crystals of hGH or an hGH derivative,
or compositions (e.g., formulations containing crystalline hGH
suspensions) comprising them, with or without an excipient, are
administered by oral route or parenteral route. In preferred
embodiments, crystals of hGH or an hGH derivative, or compositions
comprising them, with or without an excipient, are administered by
subcutaneous or intramuscular route.
[0281] In preferred embodiments, the crystals or compositions of
this disclosure, are administered by subcutaneous route, using a
needle having a gauge greater than or equal to 27. In one
embodiment of this disclosure, the needle gauge may be equal to 30.
The crystals or compositions may be administered from a pre-filled
syringe or a meta dose infusion pump. Alternatively, they may be
administered by needle-free injection.
[0282] This disclosure advantageously permits sustained release of
hGH into a mammal. In one embodiment, the crystals or compositions
according to this disclosure are administered about once a week. In
another embodiment, the crystals or compositions according to this
disclosure are administered about once every two weeks. In yet
another embodiment, the crystals or compositions according to this
disclosure are administered about once every month. It will be
appreciated by those of skill in the art that the specific
treatment regimen will depend upon factors such as the disease to
be treated, the age and weight of the patient to be treated,
general physical condition of the patient and judgment of the
treating physician.
[0283] Kits
[0284] Kits containing a formulation containing poly-Arg complexed
crystalline hGH and instructions for use, and optionally, a device
for administering the formulation are also part of the present
disclosure. The formulation can be in a suitable container (e.g.,
vial, prefilled syringe, etc). The instructions can take any form,
e.g., a pamphlet or sheet, or a world wide web address to a site
where instructions are provided. The instructions can include,
e.g., instructions for storage and/or administration.
Overview: Needle-Less Injection Systems for Crystalline Protein
Formulations
[0285] This disclosure further relates to the field of crystalline
suspensions of therapeutic proteins for use with needle-free (jet)
injectors for administration to subjects. The disclosure also
features methods of preparing formulations of crystalline proteins
that are suitable for needless injection.
[0286] Needless injection devices help to increase subject
compliance by improving ease of drug administration; decreasing
injection time, and possibly reducing pain upon injection. In some
aspects, crystalline protein formulations have advantages over
soluble formulations. Crystallization can extend the release time
of a protein, thereby decreasing the frequency of administration,
and possibly increasing subject compliance. As one example, a
crystalline insulin formulation is available. By using
crystallization technology, it was possible to extend release time
of insulin, thereby decreasing the frequency of drug administration
and increasing subject compliance.
[0287] Needle-free options may also help to an increase subject
compliance with crystalline drugs, as needle phobia has been
reported in about 5-10% of the patient population. Further, it
would be beneficial for subjects to have multiple delivery options
for administration of crystalline formulations (e.g., parenteral
formulations), including a needle-free option.
[0288] Another example of an application for needle-free injectors
in conjunction with crystalline protein formulation is the
veterinary market. It is possible to use crystalline formulation
providing extended drug release in combination with jet injectors
to improve animal care compliance. Compliance can be increased in
part because of decreased frequency of administrations, shorter
time required for injection, and decreased pain upon injection.
[0289] A needle-free jet injector is a type of medical injecting
syringe that uses a high-pressure narrow jet of the injection
liquid instead of a hypodermic needle to penetrate the epidermis.
It is powered by compressed air or gas, either by a pressure hose
from a big cylinder, or from a built-in gas cartridge or small
cylinder. Some are multi-shot, and some are one-shot.
[0290] Needle-free injection systems are known. Examples include:
BioJector 2000, VetJet, Vitajet, JET2000, MIT-V jet injector,
LectraJet, Akra Dermojet, and Med-E-Jet injector.
[0291] A jet injector, e.g., BioJector needle-free (jet) injection
system, can be used to study the feasibility of using jet injectors
with crystalline protein formulations. BioJector's needle-free
injection technology works by forcing liquid medication at high
speed through a tiny orifice held against the skin. This creates a
fine stream of high-pressure medication that penetrates the skin,
depositing the medication in the tissue beneath.
[0292] BioJector 2000: The BioJector 2000 consists of two
components: a hand-held, reusable jet injector and a sterile,
single-use, disposable plastic syringe. The BioJector 2000 uses
disposable carbon dioxide cartridges as a power source. The carbon
dioxide gas provides consistent, reliable pressure on the plunger
of the disposable syringe, thereby propelling the medication into
the tissue.
[0293] The second component of the system, the BioJector single-use
disposable syringe, consists of a plastic, needle-free, variable
dose syringe. The body of the syringe is transparent and has
graduated markings to aid accurate filling. There are five
different BioJector syringes, each of which is intended for a
different injection depth or body type.
[0294] VetJet and Vitajet: The VetJet is a modified Vitajet for use
in the veterinary market. VetJet is also composed of two
components, a portable injector unit and a disposable syringe.
VetJet is powered by a spring.
[0295] Needle-free injectors have been in use for administration of
liquid non-crystalline protein formulations. A possible reason for
absence of reported case of needle-free device for suspensions
crystalline protein is that protein crystals represent particulates
in suspension which could make injection via needle-free device
difficult or which could cause physical changes to crystalline
formulation, thereby undermining therapeutic action of the
formulation.
[0296] Vaccines are examples of suspensions of antigens formulated
with an adjuvant (e.g., aluminum hydroxide). Needle-free jet
injectors have been used to administer vaccines. In contrast,
crystalline protein formulations have different mode of actions,
different purpose, different requirements for integrity of
formulations, and different suspension properties as compared to
vaccine formulations.
[0297] The standard approach of administration of soluble and
crystalline therapeutic parenteral formulations is with hypodermic
needs. Pain upon injection is associated with the size of the
needle and the amount of time that the needle is held at the
injection site of the subject. Larger needle gauge and increased
time of injection are both associated with increased pain at the
injection site caused by the needle. Because jet injectors have
comparatively small orifice size (Table 2) and injection time is
very short, pain upon injection is significantly less compared to
the needle injection systems.
[0298] However, the use of needle free (jet) injectors for
therapeutic suspensions (e.g., suspensions of crystalline protein)
requires additional considerations. As described herein, the use of
needle-free injectors for delivery of crystalline protein
suspension formulations was evaluated. Becton Dickinson needles of
30 to 18 gauges were used for comparison in the study. Needle G30
was regular wall and G25, 21 and 18 needles were thin-wall.
Comparison of needle inner and outer diameters represent in Table
2.
TABLE-US-00002 TABLE 2 Orifice sizes of jet injection systems and
inner diameters of needles used in the study Orifice or Needle
Needle Injection system I.D., .mu.m O.D., .mu.m BioJector 2000
system with Size 2 syringe 1S021 114 114*.sup., 1 VetJet system
with 0.0062'' nozzle syringe Ref # 157 157*.sup., 2 K7000 30 G
needle 160 310 25 G needle 310 510 21 G needle 590 820 18 G needle
980 1270 *O.D. for jet injectors equals I.D of jet injector
orifice; .sup.1Equivalent needle gauge is 36 .sup.2Equivalent
needle gauge is 34
[0299] The capability of crystalline suspension to be administered
using needle-free jet injectors was assessed. Limitations on
crystalline suspensions that could be used in needle-free injectors
exist. Surprisingly, it was determined that formulations containing
crystalline suspensions that are appropriately formulated and
selected can be administered using needle-free (jet) injection
systems.
[0300] Advantages of the disclosure include: [0301] use of
needle-free (jet) injection devices for administration of
crystalline protein suspensions formulations; [0302] retaining
therapeutic action of said crystalline formulations; and [0303]
reduced pain upon injection when using needle-free (jet)
injectors.
[0304] As demonstrated herein, factors that affect the ability to
use needless injectors include: [0305] the sedimentation rate of
the crystalline protein and [0306] the size of the particles (i.e.,
crystals).
[0307] For example, if the sedimentation rate of the crystalline
protein is high, after injection, a portion of the protein may
remain in the injector and not be delivered to the subject. As a
result, the subject may not receive the required dosage of the
protein. If the largest dimension of the crystals is too large,
e.g., larger than the orifice, or in some cases more than 1/3 the
diameter of the orifice, the crystals may clog the orifice, the
crystals may not successfully load into the injection module,
and/or the crystals may be damaged by the interaction with the
orifice border (e.g., altering crystal morphology or size).
[0308] Needle-free injection can be used to administer any
formulation containing a crystalline protein suspension, if certain
conditions are satisfied. For example, if a crystalline protein
suspension has a low sedimentation rate, e.g., the majority of the
protein is delivered to the subject and does not remain in the
injector (e.g., even after a delay between preparing the syringe
and administering to a subject and/or independent of the position
in which the injector is held (e.g., upside down, horizontal)), it
may be suitable for needless administration.
[0309] Likewise, if the particle size of the crystals is smaller
than the orifice, and ideally, if the particle size (e.g., the
largest dimension of the particle) is about or less than 1/3 the
diameter of the orifice, the crystalline protein may be suitable
for needless administration.
[0310] If a formulation of crystalline protein does not appear to
be well-suited for needless injection, e.g., based on an evaluation
of the sedimentation rate and/or particle size of the crystals, or
based on an attempt (e.g., unsuccessful attempt) to use needless
injection to administer the crystalline protein, the properties of
the formulation can be optimized (e.g., to decrease the crystal
size or decrease the sedimentation rate, without decreasing the
efficacy of the protein by an unacceptable amount), e.g., by
varying the pH, buffer, salt concentration, suspending agent,
preservative, etc., to make the formulation suitable for use with a
needless injection system.
[0311] Particle Size
[0312] The particle size of crystalline protein can be measured,
e.g., by laser diffraction (e.g., using a Coulter LS 230 Particle
Size Analyzer (Coulter Corp., Miami, Fla.) with micro volume
module). If the particle size (e.g., the largest dimension) is less
than the size or the orifice, e.g., less than about 1/2,
particularly if the size is less than about 1/3 the diameter of the
orifice, the formulation may be suited for needless injection.
[0313] Suitability of a crystalline protein with a given particle
size for needless injection can be tested, e.g., by injecting a
crystalline protein formulation with a needle-free injector (e.g.,
BioJector 2000) and observing particle size after first and
repeated injections and comparing the particle sizes post-injection
to the particle size prior to injection. Particle size can be
measured using a Coulter LS 230 particle sizer prior to injection,
and after first and repeated injections. Fraunhofer optical model
can be used to analyze data. The particle size can be verified with
microscopic observation. Microscopic observation can also be used
to detect possible changes in crystal morphology. If minimal or no
changes in particle size are observed and the majority of the dose
is delivered from the injector, the crystalline protein may be
suitable for needless injection.
[0314] If a crystalline protein does not perform well in this
analysis, i.e., the particle size is larger than the orifice, the
formulation vehicle can be optimized, or the crystals themselves
(e.g., the crystals may be complexed with an agent (e.g.,
polycation or polyamino acid e.g., poly-Arg), or the ratio of the
complexing agent to protein may be varied) to affect the crystal
size may be optimized.
[0315] Sedimentation Rate
[0316] The sedimentation rate can affect the suitability of a
crystalline protein for needless injection. If the sedimentation
rate is high, the dose delivered to a subject may be inconsistent.
High crystalline suspension sedimentation rate may be defined as a
rate that allows for dose redistribution in a needle-free device
during the time required for dose preparation and administration,
e.g., about 90% redistribution after an hour incubation of the
device in a horizontal position. Low sedimentation rate may be
defined as a rate that allows for a preparation and administration
of dose without substantial dose redistribution. In one embodiment,
a high sedimentation rate may be defined as the rate allowing for
less than 90% dose injection upon incubation of needle-free device
in horizontal position. In some embodiments, low sedimentation rate
may be defined as rate allowing for uniform dose (e.g., minimal or
no dose re-distribution; e.g., less than about 35%, about 30%,
about 25%, about 20%, about 15%, about 10%, about 9%, about 8%,
about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about
1%) after more than one hour incubation in any position.
[0317] For example, the sedimentation rate can be measured
indirectly. As one example, a suspension is held for three minutes
in a horizontal, vertical upright or upside down position. If
substantial (e.g., about 80%) dose redistribution is observed or
measured, e.g., after 10 or 20 minutes incubation time, the
sedimentation rate is viewed as high. As another example, a
suspension incubated for one hour in various positions with no dose
redistribution is viewed as having a low sedimentation rate.
[0318] Suitability of a crystalline protein for needless injection
based on its sedimentation rate can be evaluated, e.g., by loading
a suspension into a BioJector syringe and injecting the suspension
either immediately after loading or injecting after a hold period
of one hour in horizontal, vertical upright, and upside down
positions. The hold samples are compared with the sample injected
immediately after loading into the jet injector's syringe. The
after-injection content of the protein that was injected and the
amount remaining inside the syringe are determined and expressed as
percentages, considering as 100% the material that was loaded into
syringe. If there are no or minor differences observed in dose
distribution between the immediately-injected sample and the
injected samples that had been held prior to injection, the
crystalline protein may be suitable for needless injection.
[0319] If a crystalline protein does not perform well in this
analysis, i.e., the delivered dose decreased greatly for the
samples held prior to injection, the formulation vehicle can be
optimized, or the crystals themselves (e.g., the crystals may be
complexed with an agent (e.g., polycation or poly amino acid, e.g.,
poly-Arg), or the ratio of the complexing agent to protein may be
varied) to affect the crystal size, or the concentration of the
protein may be optimized.
[0320] Methods of Use
[0321] Needless injection systems can be used to administer a
formulation containing a crystalline protein to a subject. For
example, the subject can be a patient in need of treatment with the
crystalline protein, e.g., the crystalline protein can be
crystalline hGH and the patient may suffer from an hGH deficiency.
The dose and dose schedule for administration are determined based
on a variety of factors. Such factors include, but are not limited
to, physiological factors, such as birth age and bone age, sex,
body weight, developmental stage (e.g., increased level at puberty)
and treatment-related factors, such as dose, rate (kinetics) of
dosing and route of administration, and patient diagnosis and
medical history.
[0322] Kits
[0323] Kits containing a formulation containing a crystalline
protein and instructions for use in needless injection, and
optionally, a device for needless injection are also part of the
present disclosure. The formulation can be in a suitable container
(e.g., vial, prefilled syringe, etc). The instructions can take any
form, e.g., a pamphlet or sheet, or a world wide web address to a
site where instructions are provided. The instructions can include,
e.g., instructions for storage and/or administration.
[0324] Analytical Methods
[0325] Analytical methods that can be utilized in assessing the
suitability of crystalline proteins for needless injection include
the following.
[0326] Injection
[0327] Crystalline formulations are injected with the use BioJector
2000 system with 1S021 syringe and VetJet system with 0.0062''
nozzle syringe Ref # K7000, BD 0.5 cc syringe with preattached 30
G.times.1/2'' needle (BD Part #328466), through 30 G.times.1/2''
(BD Part #305106), 25 G.times.5/8'' (BD Part #305122), 18
G.times.11/2'' (BD Part #305196) attached with luer-lock to 1 mL
syringe (BD Part #309628). 0.25 mL Samples are tested in
duplicates. BioJector 2000 and VetJet systems are used according to
BioJector's recommendations.
[0328] SE-HPLC
[0329] To determine purity using the SE-HPLC (size-exclusion HPLC)
method, a Phenomenex BioSep SEC-S-2000 column is used. Ten
microliters of 2 mg/mL concentration sample is injected into the
column. The running buffer is composed of 3% IPA and 60 mM sodium
phosphate, pH 7.0. The flow rate is set to 0.6 mL/min and the run
time is 30 minutes. Detection is performed at 214 nm. The resulting
chromatogram is manually integrated. Percent purity (monomer) was
calculated based on peak area.
[0330] RP-HPLC
[0331] RP-HPLC is used for purity testing. To determine the purity
of a protein using the RP-HPLC method, a C5 Supelco Discovery Bio
Wide Pore Column, 5 cm.times.4.6 mm, 3 .mu.m particle size, 30 nm
Pore size is used. The column thermostat temperature is set to
37.degree. C. Ten microliters of sample at 2 mg/mL concentration is
injected. The elution is carried out at a flow rate of 1.0 mL/min
with a gradient system present by mobile phase A (0.1% TFA in
water) and mobile phase B (0.8% TFA in MeCN). The gradient system
is changed from 5% B to 50% B from 0-2.5 min., then 50% to 70% B in
2.5-15.5 min. then, 70% to 90% in 15.5 to 17 min., immediately
following this 5% B is re-established. A 3 min post-time is held
before the start of the next run. Detection is performed at 214 nm.
The resulting chromatogram is manually integrated. Percent purity
is calculated based on peak area.
[0332] SCX-HPLC (Strong Cation Exchange) Chromatography
[0333] The SCX-HPLC method is used to measure deamidation specific
in stability testing. To determine the purity of protein using the
IEX-HPLC method, a PolyLC (NEST Group cat #P054SE0503) column was
used. The column thermostat temperature was set to 30.degree. C.
Twenty microliters of sample at 2 mg/mL concentration injected. The
elution is carried out at a flow rate of 1 mL/min with a gradient
system present by mobile phase A (50 mM Na Acetate, pH 4.6) and
mobile phase B (50 mM Na Acetate, pH 4.6 250 mM NaCl). The gradient
system is changed from 0% B to 20% B from 0 to 5 min., then 20% to
70% B in 5 to 25 min. then, 70% to 100% in 25 to 25.1 min. 100% B
is kept until 27 min. following 27.1 min, 0% B is re-established
for a 5 min. post-time. Detection is performed at 214 nm and 280
nm. The resulting chromatogram was manually integrated. Percent
purity was calculated based on peak area.
[0334] Particle Size Distribution (PSD)
[0335] PSD is determined by laser diffraction using a Coulter LS
230 Particle Size Analyzer (Coulter Corp., Miami, Fla.) with micro
volume module. The sample is diluted in sample buffer to achieve an
operation range of 8% to 12% obscuration. Data analysis is
performed using the Fraunhofer optical model. Size in .mu.m of
particle fraction representing cumulative volume distribution
limits of 10% (dl10), 50% (median) and 90% (dl90) is reported.
[0336] 3-pH Dissolution Test
[0337] This test is used to assess changes in dissolution profiles
in protein samples.
[0338] Particle Surface Charge
[0339] Particle Surface Charge is determined using Zetasizer Nano-Z
(Malvern Instruments Ltd. UK). Running buffer was 20 mM Tris, pH
7.5. Zeta Potential Transfer Standard is used to calibrate the
instrument (Malvern catalog #DTS1050). For each sample, 12 .mu.l
crystalline aliquot (concentration between 10 to 30 mg/ml) is added
to 588 .mu.l running buffer, and three measurements are taken at
room temperature.
[0340] Microscopic Appearance
[0341] The microscopic appearance of the crystalline test article
is assessed to detect any relevant changes in the test article due
to injections.
EXAMPLES
Example 1
[0342] A crystallization and complexation process for hGH to
produce long-acting hGH with a controlled release profile has been
developed (US pub. app. no. 2004/0209804). The poly-Arg
complexation is critical in contribution of the controlled release
profile because the bare hGH crystals perform the same as the daily
injection product in the market. Poly-Arg complexed hGH crystals or
hGH derivative, or compositions or formulations comprising them,
have several advantages including: the capability of once per week
delivery, ready to use crystalline suspension form, safety,
efficacy, purity, stability and syringeability over a period of
time. The complexed hGH drug delivery method is designed for fewer
injections without the use of polymers or fusion proteins and to
offer a more user-friendly treatment alternative for subjects. The
uniqueness of this crystallization technology is that no increase
of soluble aggregates is observed because limited free hGH is
present in the supernatant.
[0343] Formulation development studies were conducted to evaluate
the effect of pH and buffer species. Based on the results,
excipients and ionic strength were then screened. Acceptable
formulation development was assessed by the following
characteristics: storage stability at various temperatures,
container-closure compatibility as a function of dose consistency,
potential stickiness, ease of administration of product, and loss
to container closure. The stability test methods and their
application are listed in Table 1.
[0344] Several factors such as hGH to pR ratio, particle size
distribution, were critical in the release profile upon injection.
Changes in these factors during stability of ALTU-238 were studied.
In addition, dissolution profile assays were used to assess the
controlled release profiles, as the improved formulations need to
demonstrate comparable release profiles as the earlier
formulation.
[0345] Both glass vials and prefilled syringes were evaluated
during the development phase as potential container closure
configurations for the finished drug product. Additional
development studies evaluated siliconized vs. non-glass vials
and/or syringes.
[0346] A fixed protein fill concentration covering a delivered
volume ranging from 0.2 mL to 1.0 mL was evaluated.
[0347] Arrhenius-Plot kinetics were employed to predict the rate of
chemical degradation at 2-8.degree. C. using short-term stability
data generated from samples that were stressed at higher storage
temperatures such as 50.degree. C., 40.degree. C., 35.degree. C.,
30.degree. C. and 25.degree. C. Extrapolated 2-8.degree. C. data
was used to establish the proposed shelf life or expiration
date.
[0348] In addition, selected formulations were tested with rat
hypox model to evaluate the controlled release profile, using the
clinical trial formulation for comparison.
[0349] As described herein, preferred liquid suspension protein
formulations of complexed hGH are prepared using a buffer
(preferably phosphate) at the pH range from 6 to 7, a salt
(preferably sodium chloride or sodium acetate), and a suspending
agent (preferable polyethylene glycol 6000 or 8000). The container
closure systems that are compatible with these formulations are
siliconized prefilled syringes with a head space less than 10 mm or
vials with no head space.
[0350] The results indicate that chemical degradation and crystal
form changes are considerations for ALTU-238 stability, the extent
of which is affected by the buffer species, ionic strength and pH.
pH is a major factor affecting the stability of hGH complexed
crystals for chemical degradation, increasing pH>7.0 increases
deamidation. As demonstrated, pH has a significant effect on
deamidation (FIG. 1). Preferably, the pH should be in the range
from pH 6.1 to 7.2, more preferably in the range of pH 6.1 to
6.8.
[0351] Table 3 shows OD.sub.320 data of the dissolved crystals in
glycine buffer and indicates that insoluble aggregates were formed.
The histidine pH 5.0 and 5.5 samples were very turbid after the
addition of dissolution buffer. This turbidity could be filtered
out by a 0.45 .mu.m filer indicating insoluble aggregates were
formed. Additionally, histidine containing samples at pH 6.0, 6.5
and 7.0 also had higher OD.sub.320 compared to other phosphate
buffers at the same pHs. It implies the presence of insoluble
aggregates or changes of the crystal form resulting in
non-dissolvable complexed crystals in glycine buffer. These results
also indicate phosphate is a preferable buffer compared to
histidine at the same pH ranges. Histidine is not preferred for
higher level of light scattering due to undissolved crystals or
insoluble aggregates.
TABLE-US-00003 TABLE 3 Summary of turbidity by OD.sub.320 of the
dissolved crystals in glycine, pH 2.6 buffer for the stability
samples 1 week at Formula Time 0 40.degree. C. Phosphate, pH 6.5
0.014 0.016 Phosphate, pH 7.0 0.008 0.014 Phosphate, pH 7.5 0.011
0.016 Histidine, pH 5.0 0.009 4.204 Histidine, pH 5.5 0.010 0.278
Histidine, pH 6.0 0.010 0.063 Histidine, pH 6.5 0.009 0.053
Histidine, pH 7.0 0.005 0.046 Control ALTU-28 standard 0.034
0.020
[0352] The crystal sizes of these histidine and citrate containing
samples were smaller than the others that suggest that the crystals
were dissolving. Higher solubility is observed in citrate buffers
(Table 4). The solubility of hGH complexed crystals is less than
0.1 mg/mL in phosphate buffer.
TABLE-US-00004 TABLE 4 Solubility in citrate buffer at various pH.
Solubility in citrate pH buffer mg/mL hGH 7 0.9 6 1.7 5.5 1.6 The
hGH complexed crystals have higher solubility in citrate buffer
[0353] Therefore, histidine and citrate are not preferred buffers.
The solubility of hGH complexed crystals in phosphate buffer at
various pH was less than 0.1 mg/mL. The preferred formulation uses
a phosphate salt buffer species for poly-Arg complexed hGH or an
hGH derivative crystal.
[0354] FIG. 2 shows the stability results of poly-Arg complexed hGH
crystals at various pH values in the presence of NaCl to assess the
effect of ionic strength. The figure demonstrates that increasing
ionic strength does influence the rate of deamidation. With higher
salt content, lower deamidation and oxidation level were observed
after storage at 40.degree. C.
[0355] Polyethylene glycol ("PEG") is the suspending agent used in
previous formulations. Alternative suspending agents such as
PEG3350, PEG8000, mannitol and sucrose were compared. In addition,
effect of replacing salt with glycine on deamidation rate or
oxidation was evaluated. Phosphate buffer at pH 6.8 was used for
this study. FIGS. 3 to 5 show the results. The formulations
containing NaCl appear to have the least amount of deamidation. No
significant differences are observed in chemical degradation for
different salts (sodium acetate or sodium chloride) or suspending
agents (PEG3350, PEG6000 or PEG8000) at the same pH of 6.8 (FIGS. 3
to 5). No significant differences were observed for the oxidation
level among the tested formulation at the same pH.
[0356] In one embodiment, the preferred salt to be used for hGH
complexed crystals is sodium acetate or sodium chloride. In a more
preferred embodiment, the salt concentration for poly-Arg complexed
crystals of hGH is 60 mM to 200 mM sodium salt.
[0357] The concentration range of polyethylene glycol (2.5%, 5%,
10% and 20%) was also established. With no suspending agent,
potential caking could be problematic. FIG. 6 shows the results of
deamidation, and no difference in stability is observed. In
addition, all samples could be resuspended within one minute with
shaking. In one embodiment, preferred PEG concentration to be used
for hGH complexed crystals is between 2.5% to 20%.
[0358] These experiments also establish the hGH to poly-Arg ratios
desired for the complexed crystals. The targeted ratio was between
3 to 11 for various lots of complexed protein. At the hGH/poly-Arg
ratio of 3, the oxidation level is significantly higher than those
at ratios of 7 and 15 at the elevated temperature. All samples used
the same formulation containing of Tris, sodium acetate and PEG6000
at pH 7.5. However, no significant difference was observed for
deamidation level.
[0359] The in vitro dissolution profile was performed on selected
time point samples to evaluate any changes during the test period.
The results indicated that there was no significant difference in
the dissolution profiles among all tested hGH/poly-Arg ratios
(Table 5). The preferred hGH/pR ratio is between 5 to 15.
TABLE-US-00005 TABLE 5 Summary of the in-vitro dissolution profile
TARGETED 3 MONTHS 5.degree. C. RATE HGH/PR ratio mg of hGH
released/hr 3 0.30 7 0.28 11 0.23
[0360] Crystals of hGH or an hGH derivative according to this
disclosure form shining rod-like or needle-like morphologies when
imaged with optical microscopy and SEM. In some embodiments,
crystals of hGH or an hGH derivative form needles that are between
about 0.1 and about 200 .mu.m in length. In preferred embodiments,
crystals of hGH or an hGH derivative form needles that are between
about 2 and about 100 .mu.m in length. In more preferred
embodiments, crystals of hGH or an hGH derivative form needles that
are between about 2 and about 25 .mu.m in length for 50% mean
particle size distribution.
[0361] The concentration of the poly-Arg complexed hGH crystals was
also defined. The stability data ranging from 5 to 50 mg/mL of
poly-Arg complexed hGH crystals demonstrated that there are no
observed differences in stability. Based on the results, the free
hGH and poly-Arg in the supernatant are comparable for all tested
concentrations. However, at the 50 mg/mL level, the suspension was
very thick and required more strength to eject the product.
Therefore, in some embodiments hGH complexed crystal concentrations
are 5 to 50 mg/mL. In preferred embodiments, the concentration
should be between 20 to 30 mg/mL poly-Arg complexed hGH
crystals.
[0362] The selection of container closure for hGH crystals focused
on dose consistency. Vials and prefilled syringes that were made
with various material siliconized and CZ resin were compared. Two
different types of treatment, invert or swirl 10 times, were
applied respectively to the vials/syringes for each configuration
once per day. The extractable weight and ALTU-238 concentration
were recorded at each time point (Day 1, Day 3, Day 5 and Day 7).
The loss of product to prefilled syringes is much less than the
vials by this agitation study due to the head space. FIGS. 7A-7D
and 8A-D summarize the results.
[0363] Noticeable crystals were accumulated in the bottle neck of
the vials, and crystals were stuck to the walls of container
closure. Therefore, both the shape and head space of container
closure impact delivered dose consistency. The prefilled syringes
with stake needles appear to be most ideal for the limited dead
volume (range from 2 to 5 .mu.L) for hGH complexed crystals or any
protein crystal products. There is no bottle neck for the prefilled
syringes; the head space is also much smaller and can be controlled
by the stopper placement; the plunger can push almost all of the
crystal out of the syringe barrel (except the dead volume).
However, these can be resolved if no head space is present in the
container closure. Companies (e.g., Hyauron)) do fill material with
no head space. In conclusion, for dose consistency, limited or no
head space is preferred for container closures used for protein
crystals. Prefilled syringes/stake needles and a head space of less
than 10 mm are more suitable for protein crystal parenteral
products.
[0364] Stoppers of Teflon coated (i.e. Diekyo Fluorotec) and 4432
formulation stoppers were tested as closures for pR complexed hGH
crystals. The data indicate that both stoppers have no
compatibility issues.
[0365] The fill volume, e.g., 0.2 to 1.0 mL, for poly-Arg coated
hGH crystals was also examined. There are no differences among all
tested fill volumes. hGH complexed crystals preferably have a fill
volume between 0.2 mL to 1.0 mL.
[0366] Lead formulations were identified for Arrhenius stability
prediction. The Arrhenius stress model (or relationship) has been
widely used when the stimulus or acceleration variable (such as
deamidation, oxidation, etc.) is thermal (i.e., temperature). The
degradation rate at 5.degree. C. can be predicted using stability
results from several elevated temperatures. Samples of the poly-Arg
complexed crystals in prefilled syringes with Fluoroteck stoppers
were placed at 2-8.degree. C., 25.degree. C., 30.degree. C.,
35.degree. C., 40.degree. C. and 50.degree. C. Based on the data
reported, the Arrhenius equation was applied and degradation rate
at 5.degree. C. was predicted. The results were compared to the
real stability of a previous lot that was analyzed by the same
methods. In addition, the real time stability of the lead
formulation was listed for comparison. It is estimated that and
.about.7% degradation increase after 24 months storage at 5.degree.
C. for the ALTU-238 by CEX (Tables 6 and 7).
TABLE-US-00006 TABLE 6 The prediction of percentage of deamidation
of the dissolved crystals by CEX #17 PI, 100 MM PI, 200 MM Tris, Na
NACL, NACL, Acetate .degree. C. 1/T PEG6000 PEG6000 lot 1 lot 2 lot
3 PEG6000 50.degree. C. 0.00310 -3.29 -3.32 -3.34 -3.27 -3.26 -3.16
40.degree. C. 0.00319 -4.00 -4.05 -4.06 -4.06 -4.01 -3.80
35.degree. C. 0.00325 -4.78 -4.87 -4.92 -4.86 -4.78 -4.57
30.degree. C. 0.00330 -5.41 -5.46 -5.46 -5.36 -5.27 -5.13
25.degree. C. 0.00336 -5.73 -5.73 -5.55 -5.37 -4.96 -4.92 5.degree.
C. 0.00360 -9.56 -8.45 -8.17 -7.19 -6.80 -7.26 Predicted LnK for
5.degree. C. 0.00360 -9.25 -8.40 -8.13 -7.32 -6.89 -7.23 Predicted
% deamidation 0.000096 0.000225 0.000295 0.000665 0.001022 0.000727
Rate/day at 5.degree. C. Corr. Coeff. 0.979 0.993 0.988 0.972 0.946
0.972 Assumed 10% to start with, 17.0% 26.4% 31.6% 58.6% 84.6%
63.1% after 24 months
TABLE-US-00007 TABLE 7 Comparison of the predicted and actual
percentage degradation results by CEX Lead Formulation in
phosphate, sodium chloride and PEG6000 6 months 6 months Time 0 at
5.degree. C. at 5.degree. C. Actual Actual Predicted 12% 11% 14%
Previous formulation in Tris, sodium acetate and PEG6000 15 months
15 months Time 0 at 5.degree. C. at 5.degree. C. Assumed Actual
Predicted 10% 29% 43%
[0367] The dissolution profile of the formulations defined based on
this disclosure was compared to the one used in the previous
analyses by both dispersed dissolution profile assay and rat hypox
study model. The results (Table 8) indicate that comparable
dissolution rate and rat PD and PK profiles were attained.
Therefore, the formulations defined herein can provide a controlled
release profile of once per week injection for human therapy.
TABLE-US-00008 TABLE 8 Dispersed dissolution profile results from
different study and time points. stability #1 #2 Correlation time
Mg hGH/hr Mg hGH/hr coefficient point released released (r) 1 Month
at 0.21 0.19 0.83 25.degree. C. 3 Months at 0.25 0.24 5.degree. C.
3 Months at 0.18 0.17 25.degree. C. 3 Months at 0.23 0.22 5.degree.
C. 4 Months at 0.20 0.23 5.degree. C. 2 Months at 0.25 0.26
5.degree. C. Average 0.22 0.22 RSD 13% 15% The correlation
coefficient was calculated.
[0368] No significant difference in the PD profile was observed
between the previous and present formulations of ALTU-238 in a rat
hypophysectomized model. No significant difference in body weight
gain as well as pK profile was observed between the daily rhGH
control, previous and present formulations in this
hypophysectomized rat model. Mean body weight changes were 18%,
18%, and 16%, respectively.
Example 2
[0369] Studies were performed to prepare a target formulation of a
poly-Arg complexed crystalline hGH suspension that is stable for
about 18 to about 24 months at 5.+-.3.degree. C. (refrigerated) and
at least one week at room temperature 25.+-.2.degree. C. (in use)
conditions.
[0370] Both the stability of the hGH molecule and system stability
were considered. Instability of molecule includes chemical
degradation and aggregation. System stability should provide
stability for 1-week long release profile and stability of the
suspension including crystal aggregation, sedimentation and surface
adsorption.
[0371] An array of analytical methods were used to study
degradation in the complexed hGH formulations. Chemical stability
or formation of hGH-related impurities was monitored with RP-HPLC
and IEX-HPLC assays. Aggregation was monitored using SE-HPLC
assays. Changes in particle size distribution were studied with a
laser light scattering technique. Changes in suspension appearance
was observed visually.
[0372] Control stability measurements were based on five
representative batches of previous formulations. For example,
regarding chemical stability, the stability of a previous
formulation is 8 to 12 months at 2-8.degree. C. and 1 to 3 months
at room temperature.
[0373] Regarding aggregation, the projected stability is more than
three years at 2-8.degree. C. and 1 to 3 years at room temperature.
The previous suspension is stable with respect to particle size
distribution, appearance and sedimentation over a two year
observation period.
[0374] Chemical instability in hGH molecules can include
deamidation, formation of succinimide intermediates, oxidation, and
clipping. The optimizations described herein help to increase hGH
chemical stability in complexed hGH formulations.
[0375] Degradation of hGH may be process induced degradation and/or
formulation induced degradation. Formulation derived degradation
may be a consequence of using a suboptimal formulation vehicle
composition and the components and characteristics of the vehicle,
such as pH, buffer, salt, suspending agent, preservative, and
contaminants derived from these components.
[0376] During these studies, conditions influencing hGH stability
in crystalline hGH suspensions coated with poly-Arginine were
studied. Formulation vehicles allowing for increased hGH stability
were identified. The formulations were similar to previous
formulations in terms of therapeutic action.
[0377] An experiment was performed to study the influence of pH and
buffer composition on hGH chemical stability in formulations. hGH
crystals complexed to poly-Arginine were resuspended in formulation
vehicles listed in Table 9. hGH concentration in the samples was 25
mg/mL and poly-Arginine content was about 5 mg/ml. Samples were
stored at 5.+-.3.degree. C. and 25.+-.2.degree. C. conditions.
TABLE-US-00009 TABLE 9 Compositions of formulation vehicles (pH
screen) Lot # Composition pH 1 25 mM Tris, 100 mM NaAc, 5% PEG 6000
8.0 Previous 25 mM Tris, 100 mM NaAc, 5% PEG 6000 7.5 formulation
(ALTU-238; Control) 3 25 mM Tris, 100 mM NaAc, 5% PEG 6000 7.0 -4
10 mM Histidine, 25 mM Tris, 100 mM NaAc, 5% 7.0 PEG 6000 5 10 mM
Histidine, 25 mM Tris, 100 mM NaAc, 5% 6.5 PEG 6000 6 10 mM
Histidine, 25 mM Tris, 100 mM NaAc, 5% 6.0 PEG 6000 7 25 mM Tris,
100 mM NaAc, 5% PEG 6000 5.5 8 25 mM Tris, 100 mM NaAc, 5% PEG 6000
5.0
[0378] At the beginning of study, purity of the control sample was
determined (Table 10). After 1 month storage at room temperature,
samples were analyzed with reverse phase (RP-HPLC) and anion
exchange (IEX-HPLC) HPLC (IEX-HPLC results are shown in Table
10).
TABLE-US-00010 TABLE 10 Time 0 characterization of control sample
Buffer RP-HPLC, IEX-HPLC, Lot # pH component % % Control 7.5 Tris
95.99 94.53
TABLE-US-00011 TABLE 11 Characterization of samples stored 1 month
at 25 .+-. 2.degree. C. Buffer RP-HPLC, IEX-HPLC, Lot # pH
component % % 1 8.0 Tris 90.59 78.01 Control 7.5 Tris 92.53 80.70 3
7.0 Tris 91.78 86.33 4 7.0 Histidine 82.03 85.31 5 6.5 Histidine
79.00 88.70 6 6.0 Histidine 69.00 88.96 7 5.5 Acetate 81.81
90.89
[0379] To our surprise, based on RP-HPLC stability data, buffer
components influenced chemical stability of the formulations.
Surprisingly, histidine buffer was found to cause significantly
faster chemical degradation in formulations. Thus, the buffer
component has a strong influence on hGH chemical degradation as
analyzed by RP-HPLC. In the range of explored buffers under the
test conditions, the optimal pH was 7.5 with decreasing stability
on either extreme of this value.
[0380] Interestingly, according to IEX-HPLC, deamidation is
strongly influenced by the pH of the formulation vehicle. Higher pH
promotes deamidation. With decreasing pH, deamidation rate
decreased significantly (Table 11).
[0381] Based on data from this example, overall hGH chemical
degradation in complexed hGH formulations was found to be
influenced by buffer component, composition of formulation vehicle,
and pH. Based on these data, further screens were performed to vary
these parameters in order to get increased chemical stability of
hGH protein while maintaining other parameters of the hGH
formulation.
[0382] In order to screen for an improved stability formulation,
additional formulation vehicles were tested (Tables 12-16). hGH
crystals complexed to poly-Arginine were resuspended in formulation
vehicles to yield an hGH concentration of 25 mg/mL. Stability was
studied after samples were stored at 5.+-.3.degree. C. and
25.+-.2.degree. C. conditions.
TABLE-US-00012 TABLE 12 Compositions of formulation vehicles (pH
screen) Lot # Composition pH 9 10 mM Histidine, 25 mM Tris, 100 mM
NaAc, 5% PEG 7.5 6000 10 10 mM Histidine, 25 mM Tris, 100 mM NaAc,
5% PEG 6.5 6000 11 10 mM Histidine, 25 mM Tris, 100 mM NaAc, 5% PEG
6.0 6000 12 10 mM Na Phosphate, 25 mM Tris, 100 mM NaAc, 5% 7.5 PEG
6000 13 10 mM Na Phosphate, 25 mM Tris, 100 mM NaAc, 5% 6.5 PEG
6000 14 10 mM Na Phosphate, 25 mM Tris, 100 mM NaAc, 5% 6.0 PEG
6000 15 10 mM Ethanolamine, 25 mM Tris, 100 mM NaAc, 5% 7.5 PEG
6000 6 10 mM Triethanolamine, 25 mM Tris, 100 mM NaAc, 5% 7.5 PEG
6000 17 10 mM Triethanolamine, 25 mM Tris, 100 mM NaAc, 5% 6.8 PEG
6000 18 10 mM Ethanolamine, 25 mM Tris, 100 mM NaAc, 5% 6.5 PEG
6000 19 10 mM Ethanolamine, 25 mM Tris, 100 mM NaAc, 5% 6.0 PEG
6000
TABLE-US-00013 TABLE 13 Compositions of formulation vehicles in
additional pH screen Lot # Composition pH 21 10 mM Ethanolamine, 25
mM Tris, 100 mM NaAc, 5% 7.0 PEG 6000 22 10 mM Triethanolamine, 25
mM Tris, 100 mM NaAc, 5% 7.0 PEG 6000 23 30 mM Triethanolamine, 100
mM NaAc, 5% PEG 6000 6.8 24 50 mM Triethanolamine, 100 mM NaAc, 5%
PEG 6000 6.8 25 50 mM Triethanolamine, 100 mM NaAc 6.8 26 50 mM
Triethanolamine, 100 mM NaCl 6.8 27 50 mM Triethanolamine, 4%
Mannitol 6.8
TABLE-US-00014 TABLE 14 Compositions of formulation vehicles Lot #
Composition pH 55-01 10 mM Histidine, 0.3% Poloxamer 188, 0.3%
Phenol, 4% 6.0 Mannitol 55-2 10 mM Histidine, 0.3% Poloxamer 188,
0.3% Phenol, 4% 6.5 Mannitol 55-3 10 mM Histidine, 0.3% Poloxamer
188, 0.3% Phenol, 4% 7.0 Mannitol 55-4 10 mM Histidine, 0.3%
Poloxamer 188, 0.3% Phenol, 4% 7.4 Mannitol
TABLE-US-00015 TABLE 15 Compositions of formulation vehicles #
Composition pH 55-01n 10 mM Na Citrate, 0.2% w/v Polysorbate 20,
0.25% w/v 6.0 Phenol, 150 mM NaCl pH 6.0* 55-2n 10 mM Na Citrate,
0.25% w/v Phenol, 150 mM NaCl pH 6.0 6.0 55-3n 10 mM Na Citrate,
0.2% w/v Polysorbate 20, 150 mM 6.0 NaCl 55-4n 10 mM Na Citrate,
150 mM NaCl 6.0 55-5n 10 mM Na Citrate, 75 mM NaCl, 75 mM NaAc 6.0
55-6n 10 mM Na Citrate, 150 mM NaAc 6.0 55-7n 10 mM Na Citrate, 125
mM NaCl, 5% PEG6000 6.0 55-8n 10 mM Na Citrate, 150 mM NaAc, 5%
PEG6000 6.0 55-9n 10 mM Na Citrate, 25 mM Tris 125 mM NaAc, 5% PEG
6.0 6000 55-10n 10 mM Na Citrate, 25 mM Tris 125 mM NaAc, 5% PEG
7.5 6000 55-11n 10 mM Na Citrate, 25 mM Tris 125 mM NaCl, 5% PEG
7.5 6000 55-12n 25 mM Tris, 100 mM NaCl, 5% PEG 6000 7.5 55-13n 25
mM Tris 125mM NaCl 7.5 55-14n 25 mM Tris, 100 mM Na Ac 7.5 55-15n
25 mM Tris, 100 mM NaAc, 0.2% w/v Polysorbate 20, 7.5 0.25% w/v
Phenol, 5% PEG 6000
TABLE-US-00016 TABLE 16 Compositions of formulation vehicles in
additional pH screen # Composition pH 57-01 25 mM Imidazole, 100 mM
Na Acetate, 5% w/v 6.5 PEG6000 57-02 25 mM Imidazole, 100 mM Na
Acetate, 5% w/v 7.0 PEG6000 57-03 25 mM Imidazole, 100 mM Na
Acetate, 5% w/v 7.5 PEG6000 57-04 10 mM Methionine, 25 mM Tris, 100
mM Na Acetate, 7.5 5% w/v PEG6000 57-05 10 mM EDTA, 25 mM Tris, 100
mM Na Acetate, 5% w/v 7.5 PEG6000 57-06 25 mM Tris, 100 mM Na
Acetate, 2% w/v PEG6000 7.5 57-07 25 mM Tris, 100 mM Na Acetate, 2%
w/v PEG3350 7.5 57-08 50 mM Triethanolamine, 25 mM Tris, 100 mM Na
7.5 Acetate, 2% w/v PEG3350 57-09 50 mM Triethanolamine, 100 mM
NaCl, 2% w/v 7.5 PEG3350 57-10 25 mM Triethanolamine, 10 mM
Methionine, 100 mM 6.8 NaCl, 2% w/v PEG3350
[0383] Based on results from 1 and 3 months stability time-points,
preferred stability formulations were identified (Table 17).
Preferred stability formulation selections were also based on
crystallinity, poly-Arg content, and dissolution properties.
TABLE-US-00017 TABLE 17 Optimum stability formulations Lot #
Composition pH 02 (Control) 25 mM Tris, 100 mM Na Acetate, 5% w/v
PEG6000 7.5 13 10 mM Na Phosphate, 25 mM Tris, 100 mM NaAc, 6.5 5%
PEG 6000 23 30 mM Triethanolamine, 100 mM Na Acetate, 5% w/v 6.8
PEG6000 24 50 mM Triethanolamine, 100 mM Na Acetate, 5% w/v 6.8
PEG6000 55-01 10 mM Histidine, 0.3% Poloxamer 188, 0.3% Phenol, 6.0
4% Mannitol 55-01N 10 mM Na Citrate, 0.2% Tween 20, 0.25% Phenol,
6.0 150 mM NaCl 57-01 25 mM Imidazole, 100 mM Na Acetate, 5% w/v
6.5 PEG6000 57-10 25 mM Triethanolamine, 10 mM Methionine, 100 mM
6.8 NaCl, 2% w/v PEG3350
[0384] At 6 months time-point samples stored at refrigerated and
room temperature conditions analyzed (Table 19 and 20). Purity
compared to control sample 02 which represent composition identical
to the control formulation. Time 0 purity results of control sample
described in Table 18.
TABLE-US-00018 TABLE 18 Time 0 characterization of control sample
RP-HPLC IEX-HPLC %, Main %, %, Main %, # peak Impurities peak
Deamidated 02 (Control) 95.99 4.01 94.53 5.47
TABLE-US-00019 TABLE 19 Characterization of sample stored 6 months
at 5 .+-. 3.degree. C. RP-HPLC IEX-HPLC %, Main %, (Total %, Main
%, Lot # peak Impurities peak Deamidated 02 (Control) 90.62 9.38
87.67 12.33 13 92.85 7.15 92.39 7.61 23 93.27 6.73 90.59 9.41 24
93.20 6.80 90.74 9.26 55-01 91.57 8.43 91.24 8.76 55-01N 93.29 6.71
93.51 6.49 57-01 92.55 7.45 90.64 9.36 57-10 92.88 7.12 90.37
9.63
TABLE-US-00020 TABLE 20 Characterization of sample stored 6 months
at 25 .+-. 2.degree. C. RP-HPLC IEX-HPLC %, Main %, Total %, Main
%, Lot # peak Impurities peak Deamidated 02 (Control) 72.18 27.82
49.27 50.73 13 78.36 21.64 65.26 34.74 23 79.12 20.88 60.76 39.24
24 79.51 20.49 60.58 39.42 55-01 71.45 28.55 59.88 40.12 55-01N
80.11 19.89 76.79 23.21 57-01 79.01 20.99 56.34 43.66 57-10 72.18
27.82 49.27 50.73
[0385] Methodology of estimation of degradation rates and selection
of optimum formulations: Degradation rates were used to compare
formulations and identify optimum stability formulations.
Degradation rates are expressed as rate of accumulation of hGH
related substances. Rates are calculated as the difference between
degradation product content at Time 0 and 6 months divided by
number of months (6 months in current case) stored at refrigerated
condition.
[0386] Percent of impurity is determined as the content of all
peaks besides the main peak on respective HPLC chromatograms.
Chemical degradation other than deamidation was determined by
RP-HPLC. Deamidation was determined by IEX-HPLC.
[0387] Degradation rates were calculated as the difference between
the content of degradation products in the sample at given
time-point (6 months) and at the beginning of the study divided by
duration of observation period (6 months). The chemical degradation
rate was expressed through combining degradation rates obtained
with RP-HPLC and IEX-HPLC. Formulations with lowest degradation
rates are the optimum formulations. Degradation rates in samples
stored 6 months at 5.+-.3.degree. C. are given in Table 21.
TABLE-US-00021 TABLE 21 Degradation rates in samples stored 6
months at 5 .+-. 3.degree. C. RP-HPLC, IEX-HPLC, Degradation rate,
Lot # %/Month %/Month %/month 02--Control 0.90 1.14 2.0 13 0.52
0.36 0.9 23 0.45 0.66 1.1 24 0.47 0.63 1.1 55-01 0.74 0.55 1.3
55-01N 0.45 0.17 0.6 57-01 0.57 0.65 1.2 57-10 0.52 0.69 1.2
[0388] In order to compare stability, purity of historical previous
batches were determined (Table 22) and degradation rates were
calculated (Table 23). Degradation rates in historical batches were
compared to the control sample and to the new formulations.
TABLE-US-00022 TABLE 22 Characterization of historical Phase I
ALTU-238 batches stored at 5 .+-. 3.degree. C. Months stored at T0
RP- RP- T0 IEX- IEX- Lot # 5.degree. C. HPLC, % HPLC, % HPLC, %
HPLC,% 204-35-01 14.0 96.00 89.66 95.00 80.00 161-29-2 23.0 97.00
83.51 95.00 70.00 05-021-001 12.0 96.00 88.49 95.00 77.52 161-93-08
16.5 98.50 78.41 95.00 61.34
TABLE-US-00023 TABLE 23 Degradation rates and shelf-life of
historical batches stored at 5 .+-. 3.degree. C. RP-HPLC, IEX-HPLC,
Degradation rate, Lot # %/Month %/Month %/month 204-35-01 0.45 1.07
1.5 161-29-2 0.59 1.09 1.7 05-021-001 0.63 1.48 2.1 161-93-08 1.22
2.04 3.3
[0389] Surprisingly, degradation rates in the optimized
formulations were significantly lower than in the control sample
and in the historical batches. In current screens several
formulations were identified with greatly decreased degradation
rates. Two preferred improved stability formulations were
identified: #13 (10 mM Na2HPO4, 25 mM Tris, 100 MM NaAc, 5% PEG
6000, pH 6.5) and #55-01N (10 mM Na Citrate, 0.2% Tween20, 0.25%
Phenol, 150 mM NaCl pH 6.0).
[0390] Estimation of Room Temperature Degradation Rates
[0391] Analysis of the stability of the new formulations at room
temperature yielded the same improved stability formulation #55-01N
(Table 24). Other formulations identified as optimal at
refrigerated storage conditions had better stability than the
control formulation when stored at room temperature.
TABLE-US-00024 TABLE 24 Degradation rates and shelf-life of samples
stored 6 months at 25 .+-. 2.degree. C. RP-HPLC, IEX-HPLC,
Degradation rate, Lot # %/Month %/Month %/month 02--control 3.97
7.54 11.51 13 2.94 4.88 7.82 23 2.81 5.63 8.44 24 2.75 5.66 8.41
55-01 4.09 5.78 9.87 55-01N 2.65 2.96 5.61 -57-10 2.83 6.37
9.20
[0392] Experiments were performed to see how the new formulation
vehicles that provide improved chemical stability influence hGH
aggregation in suspensions of hGH crystals complexed to
poly-Arginine. hGH crystals complexed to poly-Arginine were
resuspended in optimized stability formulation vehicles.
Aggregation was studied using SE-HPLC after samples were stored at
5.+-.3.degree. C.
TABLE-US-00025 TABLE 25 hGH aggregation samples stored 6 months at
5 .+-. 3.degree. C. SE- SE- Aggre- HPLC, HPLC, gation % at % at 6
rate, %/ Lot # Formulation vehicle time 0 months month 333-3-01 25
mM Tris, 100 mM NaAc, 5% 99.94 99.57 0.06 PEG 6000 pH 7.5--Control
333-3-02 10 mM Na Citrate, 0.2% 99.94 99.64 0.05 Tween20, 0.25%
Phenol, 150 mM NaCl pH 6.0--FV in the sample 55-01N 333-3-13 10 mM
Na.sub.2HPO.sub.4, 25 mM Tris, 99.94 99.79 0.025 100 mM NaAc, 5%
PEG 6000, pH 6.5 FV in the sample 13
[0393] To our surprise, the new formulation vehicles did not
increase aggregate formation rate in suspensions of hGH crystals
complexed to poly-Arginine. Indeed, for some of the optimized
stability formulations, aggregated hGH formation rate decreased two
fold as compared control sample (Table 25).
[0394] In Vivo Results: Hypophysectomized Rat Study
[0395] These experiments were performed to determine whether the
therapeutic action of poly-Arginine complexed hGH crystals in
optimized stability formulations vehicles is equivalent to the hGH
release profile of the control formulation. No significant
difference in the therapeutic action was observed between a control
lot and an optimized stability formulation Lot #299-35-06 (10 mM Na
Citrate, 0.2% Tween20, 0.25% Phenol, 150 mM NaCl pH 6.0) in
hypophysectomized male Wistar rats.
[0396] The efficacy confirmation in the standard rat weight gain
assay is an important step for the clinical and commercial
development of an hGH formulation. Test or control material was
administered to hypophysectomized male Wistar rats (9 rats/group)
by SC (subcutaneous) injection. The study compared the growth of
hypophysectomized rats after receiving a single SC injection of 5.6
mg/kg of single-dose and multi-dose control and new formulations or
daily SC injections of 0.8 mg/kg of soluble commercial rhGH
(Nutropin AQ) for 7 days (i.e. 5.6 mg/kg/week). Body weights were
measured weekly prior the start of dosing and daily from Day -7
(seven days prior dosing) until the end of observation period on
day 8. No significant difference in body weight gains were observed
between the daily rhGH injection formulation, the control, and the
optimized stability formulation in this hypophysectomized rat
model; mean body weight changes were 18.+-.3%, 18.+-.2%, and
16.+-.4%, respectively.
[0397] Conclusions
[0398] These studies have identified formulation vehicles for
suspensions of hGH crystals coated with poly-Arginine in
formulation vehicles. The formulations: [0399] a) provide improved
stability [0400] b) maintain crystallinity in the complex
comparable to the control formulation [0401] c) allow
administration through 29 or finer gauge needle
[0402] These formulations provide: [0403] a) suspension of hGH
crystals coated with polyelectrolyte where crystalline hGH
concentration in the range of 5-100 mg/mL, preferably 10-50 mg/mL,
more preferably in the range of 20 to 30 mg/mL. [0404] b)
suspension in formulation vehicles comprised of biologically
compatible buffers that maintain crystallinity of the hGH complexes
with pH in the range of 5.0 to 8.0. Buffer salt concentration in
the range of 1 to 150 mM concentration. Preferably, 2-50 mM; more
preferably 10 mM concentration. The examples of buffer salts
include: acetate, triethanolamine, imidazole, phosphate, citrate,
Tris-HCl. A preferred buffer is sodium phosphate in the pH range of
5.8 to 7.0, preferably 6.0 to 6.5. A combination of buffers, e.g.,
sodium phosphate and sodium citrate buffers, can be used, for
example, 2 mM Na citrate 8 mM Na phosphate. [0405] c) Components
maintaining or increasing crystallinity of the hGH complex. As
example is a neutral salt such as Na Acetate or polyethylene glycol
(PEG 3350-PEG 8000). Preferred PEG concentrations range from 1-25
w/v %, more preferred 2-10 w/v %; more preferred 4-6 w/v %. Phenol
can be used at concentrations of 0.1-0.5%. [0406] d) Tonicity
modifiers allowing for total osmolality of the compositions in the
range of 250-450 mOsm/kg, preferably 270-350 mOsm/kg, more
preferably 280-330 mOsm/kg. Examples include neutral salts such as
sodium chloride, sodium acetate, Tris-HCl. Buffer salt outside of
their working buffering pH range could be used as neutral salts. A
preferred salt is sodium acetate or sodium chloride. Salts of amino
acids could be used as neutral salts in the pH ranges outside their
buffering pH range. Preferred salt of amino acids is glycine sodium
salt. Polyols as mannitol, sorbitol also could be used as tonicity
modifier. Polyethylene glycols could be used as tonicity modifiers.
[0407] e) Optionally, a preservative, e.g., phenol at a
concentration of about 0.25% w/v. Preferred phenol concentration
ranges are between 0.2-0.3%. [0408] f) Optionally components
increasing chemical stability of said formulation such as
methionine, EDTA, etc.
[0409] Specific Examples of Lead Formulation Vehicles:
10 mM Na Phosphate, 25 mM Tris, 100 mM NaAc, 5% PEG 6000, pH
6.5
10 mM Na Phosphate, 0.25% Phenol, 150 mM NaCl, 5% PEG 6000 pH
6.0
10 mM Na Citrate, 0.2% Tween 20, 0.25% Phenol, 150 mM NaCl, pH
6.0
30 mM Triethanolamine, 100 mM Na Acetate, 5% PEG6000 pH 6.8
25 mM Triethanolamine, 10 mM Methionine, 100 mM NaCl, 2% PEG3350 pH
6.8
25 mM Imidazole, 100 mM Na Acetate, 5% PEG6000 pH 6.5
[0410] Specific Examples of Potentially Beneficial Formulations
Vehicles:
2 mM Na citrate, 8 mM Na phosphate, 120 mM NaCl, 0.3% Phenol pH 6.0
10 mM Na Phosphate, 100 mM Glycine, 0.3% phenol, pH 6.0 10 mM Na
Phosphate, 100 mM NaCl, 0.3% phenol, pH 6.0 10 mM Na Phosphate, 100
mM Na acetate, 5% PEG 6000, pH 6.0
10 mM Na Phosphate, 100 mM NaCl, 5% PEG 6000, pH 6.0
10 mM Na Phosphate, 100 mM NaCl, 5% PEG 6000, pH 6.5
[0411] 2 mM Na citrate, 8 mM Na phosphate, 120 mM NaCl, 0.3% Phenol
pH 5.4 10 mM Na phosphate, 100 mM NaCl, 10% w/v PEG3350 pH 6.0 10
mM Na citrate, 80 mM Na Glycine, 10% w/v PEG6000 pH 5.4 10 mM Na
acetate, 120 mM NaCl, 10% w/v PEG6000 pH 5.6
[0412] Preparations of Said Formulations: Protocol
[0413] (1) Production of poly-Arginine complexed hGH crystal, 25
mg/ml (Govardhan et al., 2004: WO 2004/060310)
[0414] (2) Centrifugation of step 1 product, 3,5000 rpm, 15 min,
4.degree. C.
[0415] (3) Remove supernatant, record volume.
[0416] (4) Add same volume formulation vehicle.
[0417] (5) Resuspend the pellet.
[0418] (6) Repeat step 3, then add volume of formulation vehicle to
get desired hGH concentration upon resuspension and resuspend.
[0419] (7) Store the final suspension at 2-8.degree. C.
[0420] Samples are analyzed with gradient low pH RP-HPLC method to
monitor chemical degradation excluding deamidation; deamidation is
detected with use of cation exchange HPLC; finally chemical
degradation is expressed through combining impurity numbers
obtained with RP-HPLC and IEX-HPLC. SE-HPLC is used to monitor
aggregation. Ratio of bound to free poly-Arginine calculated based
on RP-HPLC determined concentration of total and supernatant
located poly-Arginine. Crystallinity determined as ratio of hGH
concentration in supernatant to the hGH concentration in
crystalline phase of formulation by RP-HPLC calibration.
[0421] Sample preparation for HPLC analysis: Crystals dissolved to
perform RP-, IEX- and SE-HPLC analyses. Crystals are centrifuged
and supernatant removed; crystalline pellet is dissolved in acetic
acid water solution with pH 2.8 in order to give 2 mg/mL
concentration. After 2 minutes of incubation, solutions are
centrifuged 4 minutes at 12000 rpm. Samples are then analyzed using
HPLC.
[0422] RP-HPLC: RP-HPLC is used to determine the purity of hGH
using RP-HPLC method a C5 Supelco Discovery Bio Wide Pore Column, 5
cm.times.4.6 mm, 3 .mu.m particle size, 30 nm Pore size is used.
The column thermostat temperature is set to 37.degree. C. 10 .mu.L
of sample at 2 mg/mL concentration is injected. The elution is
carried out at a flow rate of 1.0 mL/min with a gradient system
present by mobile phase A (0.1% TFA in water) and mobile phase B
(0.1% TFA in MeCN). The gradient system is changed from 5% B to 50%
B from 0-2.5 min., then 50% to 70% B in 2.5-15.5 min. then, 70% to
90% in 15.5 to 17 min. Immediately following, 5% B is
re-established for a 3 min. post-time. Detection is performed at
214 nm. The resulting chromatogram is manually integrated. Percent
purity is calculated based on peak area.
[0423] IEX-HPLC Weak Anion Exchange Chromatography: WAX-hGH method
is used as deamidation specific measure in stability testing at
Time 0 point. To determine the purity of hGH using the WAX method,
a PolyMA WAX (Supelco cat #59602-U) is used. The column thermostat
temperature is set to 37.degree. C. 54 of sample at 2 mg/mL
concentration is injected. The elution is carried out at a flow
rate of 0.5 mL/min with a gradient system present by mobile phase A
(50 mM TRIS, pH 8.0) and mobile phase B (50 mM TRIS, pH 8.0 500 mM
NaCl). The gradient system is changed from 5% B to 30% B from 0-30
min., then 30% to 50% B in 30 to 40 min. then, 50% to 95% in 40 to
42 min. 95% B is kept until 45 min. following, 50% B is
re-established for a 3 min. post-time. Detection is performed at
214 nm and 280 nm. The resulting chromatogram is manually
integrated. Percent purity is calculated based on peak area.
[0424] IEX-HPLC Strong Cation Exchange Chromatography: SCX-hGH
method is used as deamidation specific in stability testing. To
determine the purity of hGH using the IEX-HPLC method, a PolyLC
(NEST Group cat # P054SE0503) is used. The column thermostat
temperature is set to 30.degree. C. 20 .mu.L of sample at 2 mg/mL
concentration is injected. The elution is carried out at a flow
rate of 1 mL/min with a gradient system present by mobile phase A
(50 mM Na Acetate, pH 4.6) and mobile phase B (50 mM Na Acetate, pH
4.6 250 mM NaCl). The gradient system is changed from 0% B to 20% B
from 0 to 5 min., then 20% to 70% B in 5 to 25 min. then, 70% to
100% in 25 to 25.1 min. 100% B is kept until 27 min. following 27.1
min, 0% B is re-established for a 5 min. post-time. Detection is
performed at 214 nm and 280 nm. The resulting chromatogram is
manually integrated. Percent purity is calculated based on peak
area.
[0425] SE-HPLC: used to determine aggregate content in samples.
SE-HPLC Phenomenex BioSep SEC-S-2000 column is used. 10 .mu.l of 2
mg/mL concentration sample is injected into the column. The running
buffer is composed of 3% IPA and 60 mM sodium phosphate, pH 7.0.
The flow rate is set to 0.6 mL/min and the run time is 30 min.
Detection is performed at 214 nm. The resulting chromatogram is
manually integrated. Percent purity is calculated based on peak
area.
[0426] Crystallinity: Crystallinity is calculated as ratio of hGH
concentration (based on RP-HPLC calibration) in supernatant to the
total hGH concentration in sample.
Crystallinity=((C.sub.hGH total-Ch.sub.GH supernatant)/C.sub.hGH
total)).times.100%
[0427] Ratio of Bound to Free Poly-Arginine: ratio is calculated
based on RP-HPLC determined (based on poly-Arginine calibration)
concentration of total and supernatant located poly-Arginine.
Poly-Argininity=((C.sub.poly-R total -C.sub.poly-R
supernatant)/C.sub.poly-R total)).times.100%
[0428] Visual Appearance: visual appearance of the test articles
assessed visually to detect any relevant changes in the test
article.
Example 3
[0429] This disclosure relates to the field of sustained release
multi-dose suspensions of hGH crystals complexed with
poly-electrolyte.
[0430] Some liquid formulations of human growth hormone are
multi-dose formulations. They contain antimicrobial preservatives
which allow for the use formulation in the single vial for multiple
administrations. hGH solutions also could be filled in the
cartridge which contain antimicrobial preservatives that allow for
multiple administration of hGH. It is worth to mention that despite
being multi-dose, these liquid formulations require once per day
administration to achieve pharmacological effect. It will greatly
benefit patients, especially children, if hGH could be formulated
as sustained release product, e.g., a once per week injection
product. It will greatly benefit patients also if multi-dose
formulations of a once-weekly product were available on the market.
Addition of antimicrobial preservatives is a known way of
preparation of multi-dose pharmaceutical formulations.
[0431] Polyelectrolyte complexation with hGH crystal has been shown
to be effective in vivo (e.g., administered through 29 G needle) to
sustain the hGH release to 5-6 days, and stimulate the elevation of
IGF-1 above baseline values for at least 7 days.
[0432] In these studies, performed to increase chemical stability
of hGH formulations, crystalline hGH complexed to poly-Arginine was
suspended in buffer solutions containing sodium citrate, or sodium
phosphate buffers and also containing detergents, salts, and
antimicrobial preservative.
[0433] Surprisingly, the addition of antimicrobial preservatives
does not have adverse effects on crystallinity of hGH complexed to
poly-Arginine; crystallinity was maintained in suspensions.
[0434] Interestingly, citrate buffer led to increases of free
(soluble) hGH content in suspension of hGH crystals complexed to
poly-Arginine--an indication of crystal dissolution in formulation
which can lead to significant shortening of release profile. In
contrast, the addition of phosphate buffer did not increase the
content of free hGH in formulation and later in vivo animal studies
confirmed equivalence to the release profile of a previously known
formulation of complexed hGH.
[0435] Another part of experiment was to put hGH not complexed
(bare crystals) to poly-Arginine into various buffer solutions.
These crystals were readily soluble in said formulations. This
experiment showed that bare hGH crystals could not maintain
crystallinity in the multi-dose formulation vehicles studied here.
By contrast, rhGH crystals complexed with polyarginine could be
used for the purpose of multi-dose formulations using different
antimicrobial preservatives.
[0436] This experiment was conducted to assess whether the addition
of antimicrobial preservative phenol affects the content of free
hGH in formulations. As a control, a previously known (control)
formulation was studied. Three more samples were prepared by
resuspending pellet of hGH crystals complexed with poly-Arginine
into citrate buffer-based formulation vehicles with salt, with or
without surfactant and phenol.
TABLE-US-00026 TABLE 26 Free hGH content in samples stored for 1
month at 5 .+-. 3.degree. C. C hGH % super- of natant, free Lot#
Sample ID Buffer Composition mg/mL hGH 333-3-01 Control 25 mM Tris,
100 mM Na 0 0 complexed hGH Acetate, 5% w/v PEG6000 pH 7.5 333-3-02
Complexed hGH 10 mM Na Citrate, 1.6 6.4 in phenol 0.2% Tween 20,
containing buffer 0.25% Phenol, 150 mM NaCl pH 6.0 333-3-07
Complexed hGH 10 mM Na Citrate, 1.2 4.6 in phenol 0.25% Phenol, 150
containing buffer mM NaCl pH 6.0 without surfactant 333-3-08
Complexed hGH 10 mM Na Citrate, 4.3 17.2 in similar buffer 0.2%
Tween 20, 150 without phenol mM NaCl pH 6.0
[0437] Results from this study showed that the addition of phenol
does not have adverse effects on the content of free hGH both in a
control formulation as well as to citrate buffer-based solutions
containing surfactant and salt (Table 26). Surprisingly, the
phenol-containing formulation showed a lower concentration of free
hGH in suspension and the surfactant (Tween)-containing formulation
showed a higher concentration of free hGH in suspension.
[0438] This experiment was performed to assess the influence of
citrate and phosphate buffers on the content of free hGH in
suspensions of hGH crystals complexed with poly-Arginine. As a
control, a previously known hGH (control) formulation was used. Two
more samples were prepared by resuspending pellet of hGH crystals
complexed to poly-Arginine into citrate or phosphate buffer-based
formulation vehicles containing also surfactant, salt and
phenol.
TABLE-US-00027 TABLE 27 Free hGH content in samples stored for 1
month at 5 .+-. 3.degree. C. C hGH % super- of natant, free Lot#
Sample ID Buffer Composition mg/mL hGH 333-3-01 Control 25 mM Tris,
100 mM Na 0 0 complexed hGH Acetate, 5% w/v PEG6000 pH 7.5 333-3-02
Complexed hGH 10 mM Na Citrate, 1.6 6.4 in citrate 0.2% Tween 20,
0.25% based buffer Phenol, 150 mM NaCl pH 6.0 333-3-05 Complexed
hGH 10 mM Na Phosphate, 0.2 0.6 in phosphate 0.2% Tween 20, 0.25%
based buffer Phenol, 150 mM NaCl pH 6.0
[0439] Results from this experiment showed that control formulation
has a very minor content of free (soluble) hGH, indicating the
integrity of hGH crystals complexed with poly-Arginine (Table 27).
Surprisingly, the content of free (soluble) hGH was higher when hGH
crystals complexed with poly-Arginine were transferred in
citrate-based buffer, indicating partial dissolution of hGH
crystals. This crystals dissolution can lead to significant
shortening of in vivo hGH release profile. In contrast, when hGH
crystals complexed with poly-Arginine were transferred into
phosphate-based buffer, the content of free (soluble) hGH was an
order of magnitude lower than in citrate-based buffer. Thus, use of
phosphate-based buffers as formulation vehicles for hGH crystals
complexed with poly-Arginine could provide an advantage in terms of
maintaining once-weekly release profiles seen with the control
formulation.
[0440] Multi-dose formulations that would maintain crystallinity of
hGH crystals complexed with poly-Arginine in suspension preferably
contain phosphate buffer, phenol as a preservative, and a tonicity
modifying component.
[0441] An experiment was undertaken to demonstrate influence of
citrate and phosphate buffers on content of free hGH in suspensions
of hGH crystals not complexed with poly-Arginine (i.e., bare hGH
crystals). As control, the base formulation vehicle from the
previous (control) complexed hGH formulation was used. Two more
samples were prepared by resuspending pellet of bare crystals into
citrate or phosphate buffer-based formulation vehicles containing
also surfactant, salt, and phenol.
TABLE-US-00028 TABLE 28 Free hGH content in samples stored for 1
month at 5 .+-. 3.degree. C. C hGH % super- of natant, free Lot#
Sample ID Buffer Composition mg/mL hGH 249-54-02 control 25 mM
Tris, 100 mM Na 0 0 Acetate, 5% w/v PEG6000 pH 7.5 333-48-05 Bare
hGH 25 mM Tris, 100 mM Na 9 35 crystals in Acetate, 5% w/v base
control PEG6000 pH 7.5 formulation vehicle 333-48-07 Bare hGH 10 mM
Na Citrate, 0.2% 27 100 crystals in Tween 20, 0.25% Phenol, citrate
150 mM NaCl pH 6.0 based buffer 333-48-08 Bare hGH 10 mM Na
Phosphate, 0.2% 27 100 crystals in Tween 20, 0.25% Phenol,
phosphate 150 mM NaCl pH 6.0 based buffer
[0442] Results from this experiment showed that base control
formulation vehicle allows partial dissolution of bare hGH crystals
(Table 28). Surprisingly, bare hGH crystals were readily soluble in
citrate or phosphate-based buffers that also contained surfactant,
salt and preservative. Dissolution of crystals in formulation can
lead to significant shortening of in vivo hGH release profile.
[0443] The next experiment was performed to demonstrate the
influence different preservatives at different concentrations on
the content of free hGH and poly-Arginine in suspensions of hGH
crystals complexed with poly-Arginine in the control formulation
vehicle or in sodium phosphate low pH formulation vehicles. As a
control, formulation vehicles without the addition of preservatives
were used. The content of free hGH and poly-Arginine in the
supernatants of samples was analyzed after one month incubation at
room temperature.
TABLE-US-00029 TABLE 29 Free hGH content in samples stored for 1
month at room temperature in low pH sodium phosphate based buffer C
poly- C hGH Arginine % of free supernatant, % of free supernatant,
poly- Lot# Formulation vehicle composition mg/mL hGH mg/mL Arginine
333-52-01 Control low pH FV--10 mM 0.0043 0.02 0.1097 3.05 NaHPO4,
100 mM NaCl, 5% PEG6000, pH 6.0 333-52-02 0.5% phenol in control
low pH 0.0081 0.04 0.2304 6.40 FV 333-52-03 0.2% phenol in control
low pH 0.0266 0.12 0.1813 5.04 FV 333-52-04 0.1% phenol in control
low pH 0.0248 0.11 0.1047 2.91 FV 333-52-05 1.5% benzyl alcohol in
control 0.0184 0.08 0.2336 6.49 low pH FV 333-52-06 0.75% benzyl
alcohol in control 0.0248 0.11 0.2352 6.53 low pH FV 333-52-07 0.1%
benzyl alcohol in control 0.0183 0.08 0.1848 5.13 low pH FV
333-52-08 0.1% methyl paraben in control 0.0145 0.06 0.1222 3.39
low pH FV 333-52-09 0.05% methyl paraben in control 0.0325 0.14
0.0743 2.06 FV 333-52-10 0.02% methyl paraben in control 0.0367
0.16 0.0498 1.38 low pH FV 333-52-11 0.3% m-cresol in control low
pH 0.0097 0.04 0.1138 3.16 FV 333-52-12 0.2% m-cresol in control
low pH 0.0057 0.03 0.1007 2.80 FV 333-52-13 0.1% m-cresol in
control low pH 0.0114 0.05 0.1086 3.02 FV 333-52-14 0.5%
clorobutanol in control low 0.0132 0.06 0.1919 5.33 pH FV 333-52-15
0.3% clorobutanol in control low 0.0211 0.09 0.1302 3.62 pH FV
333-52-16 0.1% clorobutanol in control low 0.0331 0.15 0.1187 3.30
pH FV
TABLE-US-00030 TABLE 30 Free hGH content in samples stored for 1
month at room temperature in control formulation vehicle C poly- C
hGH Arginine % of free supernatant, % of free supernatant, poly-
Lot# Formulation vehicle composition mg/mL hGH mg/mL Arginine
333-52-22 Control vehicle--25 mM 0.0038 0.02 0.1147 3.19 Tris, 100
mM Na Acetate, 5% w/v PEG 6000, pH 7.5 333-52-17 0.5% phenol in
control vehicle 0.0026 0.01 0.3223 8.95 333-52-18 1.5% benzyl
alcohol in control 0.0085 0.04 0.3343 9.29 vehicle 333-52-19 0.1%
methyl paraben in control 0.0051 0.02 0.1186 3.30 vehicle 333-52-20
0.3% m-cresol in control vehicle 0.0023 0.01 0.1562 4.34 333-52-21
0.5% clorobutanol in control 0.0036 0.02 0.3169 8.80 vehicle
[0444] Results from this experiment showed that addition of
antimicrobial preservatives does not significantly change the
amount of free hGH and poly-Arginine in suspensions of hGH crystals
complexed with poly-Arginine in 10 mM NaHPO.sub.4, 100 mM NaCl, 5%
PEG6000, pH 6.0 formulation vehicle (Tables 29 and 30). In the
examples where 1.5% benzyl alcohol and 0.5% clorobutanol were added
to the control formulation vehicle, an increase in the amount of
free poly-Arginine in supernatants was observed. The increase of
free poly-Arginine however did not cause an increase of free hGH
concentrations.
[0445] This experiment was performed to demonstrate the influence
of different preservatives at different concentrations on chemical
stability of hGH in suspensions of hGH crystals complexed with
poly-Arginine in the control formulation vehicle and in sodium
phosphate low pH formulation vehicle. As a control, control and
sodium phosphate low pH formulation vehicles without addition of
preservatives were used. Increases in content of hGH degradation
products were analyzed after one month incubation at room
temperature.
TABLE-US-00031 TABLE 31 Accumulation of hGH degradation products in
samples stored for 1 month at room temperature in low pH sodium
phosphate based buffer Increase Increase in hGH in hGH impurities,
impurities, according to according to Lot# Buffer Composition
RP-HPLC % IEX-HPLC % 333-52-01 Control low pH FV--10 4.48 7.94 mM
NaHPO4, 100 mM NaCl, 5% PEG6000, pH 6.0 333-52-02 0.5% phenol in
control low 3.76 4.16 pH FV 333-52-03 0.2% phenol in control low
4.08 3.35 pH FV 333-52-04 0.1% phenol in control low 4.15 4.69 pH
FV 333-52-05 1.5% benzyl alcohol in 5.23 2.37 control low pH FV
333-52-06 0.75% benzyl alcohol in 5.06 3.45 control low pH FV
333-52-07 0.1% benzyl alcohol in 6.95 4.34 control low pH FV
333-52-08 0.1% methyl paraben in 5.30 3.85 control low pH FV
333-52-09 0.05% methyl paraben in 3.95 4.47 control FV 333-52-10
0.02% methyl paraben in 5.14 5.10 control low pH FV 333-52-11 0.3%
m-cresol in control low 10.09 1.89 pHFV 333-52-12 0.2% m-cresol in
control low 5.14 1.84 pHFV 333-52-13 0.1% m-cresol in control low
4.53 4.04 pH FV 333-52-14 0.5% clorobutanol in control 9.16 1.55
low pH FV 333-52-15 0.3% clorobutanol in control 7.36 1.97 low pH
FV 333-52-16 0.1% clorobutanol in control 5.14 6.08 low pH FV
TABLE-US-00032 TABLE 32 Accumulation of hGH degradation products in
samples stored for 1 month at room temperature in control
formulation vehicle Increase Increase in hGH in hGH impurities,
impurities, according to according to Lot# Buffer Composition
RP-HPLC % IEX-HPLC % 333-52-22 Control vehicle 4.66 9.09 25 mM
Tris, 100 mM Na Acetate, 5% w/v PEG 6000, pH 7.5 333-52-17 0.5%
phenol in control 5.96 7.91 vehicle 333-52-18 1.5% benzyl alcohol
in 4.70 6.55 control vehicle 333-52-19 0.1% methyl paraben in 5.10
8.41 control vehicle 333-52-20 0.3% m-cresol in control 7.47 7.30
vehicle 333-52-21 0.5% clorobutanol in control 20.78 4.82
vehicle
[0446] Surprisingly, the addition of antimicrobial preservatives
can increase chemical stability of hGH in suspensions of hGH
crystals complexed with poly-Arginine in 10 mM NaHPO.sub.4, 100 mM
NaCl, 5% PEG6000, pH 6.0 formulation vehicle (Tables 31 and 32).
This improvement was not as pronounced in the case of clorobutanol.
Also, the addition of 0.5% of clorobutanol to base ALTU-238
formulations decreased chemical stability of hGH. Thus,
clorobutanol is not a preferred antimicrobial preservative for use
with formulations containing complexed hGH.
[0447] This experiment was performed to assess the influence of
antimicrobial preservatives on the morphology of hGH crystals
complexed with poly-Arginine in different formulation vehicles. As
a control, control and sodium phosphate low pH formulation vehicles
without addition of preservatives were used. Microscopic
observation performed after one month incubation at room
temperature. Microscopic appearance of hGH crystals coated with
poly-Arginine in suspensions of control formulation vehicle without
antimicrobial preservatives, with % 0.5 of Phenol, and with 0.3% of
meta-cresol (right) was examined. Microscopic appearance of hGH
crystals coated with poly-Arginine in suspensions of low pH
phosphate based formulation vehicle (10 mM NaHPO4, 100 mM NaCl, 5%
PEG6000, pH 6.0) without antimicrobial preservatives and with 0.2%
of phenol and with 0.2% of meta-cresol was also examined.
[0448] Surprisingly, the addition of antimicrobial preservatives
had no adverse influence on morphology of hGH crystals complexed
with poly-Arginine.
[0449] In Vivo Results: Hypophysectomized Rat Study
[0450] This experiment was performed to assess whether the release
profile of poly-Arginine complexed hGH crystals in multi-dose
formulation vehicles (with preservative) is equivalent to hGH
release profile of single-dose control hGH formulation. No
significant difference in the pharmacodynamic profile was observed
between a single-dose formulation of complexed hGH in control
buffer without preservative and a multi-dose formulation complexed
hGH in control buffer with phenol in hypophysectomized male Wistar
rats.
[0451] The efficacy confirmation in the standard rat weight gain
assay is an important step for the clinical and commercial
development of an hGH formulation. Test or control material was
administered to hypophysectomized male Wistar rats (9 rats/group)
by SC injection. The study compared the growth of hypophysectomized
rats after receiving a single SC injection of 5.6 mg/kg of
single-dose and multi-dose (with preservative) hGH formulation in
control buffers or daily SC injections of 0.8 mg/kg of soluble
commercial rhGH (Nutropin AQ) for 7 days (i.e., 5.6 mg/kg/week).
Body weights were measured weekly prior the start of dosing and
daily from Day -7 (seven days prior dosing) until the end of
observation period on day 8. No significant difference in body
weight gains were observed between the daily rhGH control,
single-dose and multi-dose formulations in this hypophysectomized
rat model; mean body weight changes were 18.+-.3%, 18.+-.2%, and
16.+-.4%, respectively.
[0452] In Vivo Results: Hypophysectomized Rat Study
[0453] This experiment was performed to assess whether the release
profile of poly-Arginine complexed hGH crystals in multi-dose
formulation vehicles (with preservative) is equivalent to hGH
release profile of single-dose control hGH formulation. No
significant difference in the PD profile was observed between a
single-dose formulation of complexed hGH in control buffer without
preservative and a multi-dose formulation complexed hGH in control
buffer with phenol in hypophysectomized male Wistar rats. Test or
control material was administered to hypophysectomized male Wistar
rats (9 rats/group) by SC injection. The study compared the growth
of hypophysectomized rats after receiving a single SC injection of
5.6 mg/kg of single-dose and multi-dose formulations. Body weights
were measured weekly prior the start of dosing and daily from Day
-7 (seven days prior dosing) until the end of observation period on
day 8. No significant difference in body weight gains were observed
between the single-dose and multi-dose formulations in this
hypophysectomized rat model; mean body weight changes were 15.+-.2%
and 16.+-.1% (mean.+-.SE), respectively.
[0454] Preparations of Said Formulations: Protocol: [0455] (1)
Production of poly-Arginine complexed hGH crystal, 25 mg/ml
(Govardhan et al., 2004-WO 2004/060310) [0456] (2) Centrifugation
of step 1 product, 3,5000 rpm, 15 min, 4.degree. C. [0457] (3)
Remove supernatant, record volume. [0458] (4) Add same volume
multi-dose formulation vehicle. [0459] (5) Resuspend the pellet.
[0460] (6) Repeat step 3, then add volume of formulation vehicle to
get desired hGH concentration upon resuspension and resuspend.
[0461] (7) Store the final suspension at 2-8.degree. C.
[0462] Analytical Methods
[0463] RP-HPLC is used to detect all hGH related impurities but
deamidated hGH (Deamidation is determined with use of IEX-HPLC, see
below) performed on C5 Supelco Discovery Bio Wide Pore Column, 5
cm.times.4.6 mm, 3 .mu.m particle size, 30 nm Pore size. Thermostat
temperature is 37.degree. C. The elution is carried out with a
gradient system present by mobile phase A (0.1% TFA in H.sub.2O)
and mobile phase B (0.1% TFA in MeCN). Gradient system changed from
5% B to 50% B from 0-2.5 min., then 50% to 70% Bin 2.5-15.5 min.
then, 70% to 90% in 1.5 min., immediately following this 5% B is
re-established. A 3 min post-time is held before the start of the
next run. Flow rate is 1.0 ml/min. Injection volume is 10 .mu.l for
hGH sample at 2 mg/ml concentration. Detection performed at 214 nm.
Percent purity is calculated based on peak area.
[0464] IEX-HPLC is used as deamidation specific assay. To determine
the purity of hGH using the a PolyLC (NEST Group cat # P054SE0503)
is used. The column thermostat temperature is set to 30.degree. C.
20 .mu.L of sample at 2 mg/mL concentration injected. The elution
is carried out at a flow rate of 1 mL/min with a gradient system
present by mobile phase A (50 mM Na Acetate, pH 4.6) and mobile
phase B (50 mM Na Acetate, pH 4.6 250 mM NaCl). The gradient system
is changed from 0% B to 20% B from 0 to 5 min., then 20% to 70% B
in 5 to 25 min. then, 70% to 100% in 25 to 25.1 min. 100% B is kept
until 27 min. following 27.1 min, 0% B is re-established for a 5
min. post-time. Detection is performed at 280 nm. Percent purity is
calculated based on peak area.
Example 4
[0465] These experiments relate to the field of crystalline
suspensions of therapeutic proteins for use with needle free (jet)
injectors for administration. Different devices are utilized for
administering liquid soluble formulations of pharmaceuticals
including needle free (jet) injector devices. These devices help to
increase patient compliance by improving ease of drug
administration; decreasing injection time and possibly reducing
pain upon injection.
[0466] Crystalline protein formulations may have advantage over
soluble formulations. Needle-free options could increase patient
compliance when using crystalline drugs even more. However, needle
free delivery options for crystalline insulin suspension are not
available. Needle phobia is reported in about 5-10% of patient
population. It would be beneficial for patients to have multiple
delivery capabilities for administration of crystalline parenteral
formulations, including needle-free option.
[0467] Another possible application of jet injectors in conjunction
with crystalline protein formulation is in the veterinary market.
Use of crystalline formulations, e.g., in combination with jet
injectors, can provide extended drug release to improve animal care
compliance, e.g., as a result of decreased frequency of
administrations and shorter time required for injection and
decreased pain upon injection.
[0468] Needle-free injectors are in use for administration of
liquid therapeutic protein formulations. A possible reason for
absence of reported cases of needle-free device for crystalline
protein is that protein crystals represent particulates in
suspension, which could make injection via needle-free device
impossible or which could bring physical changes to crystalline
formulation undermining therapeutic action of said formulation.
[0469] The standard approach of administration of soluble and
crystalline therapeutic parenteral formulations is use of
hypodermic needs. Pain upon injection is associated with size of
the needle and time needle is held at the injection site of the
patient. Larger needle gauge and increased time of injection is
associated with increased pain at the injection site caused by the
needle. Since jet injectors have comparatively small orifice size
(Table 2) and injection time is very short, pain upon injection is
significantly less compared to the needle injection systems. Use of
needle free (jet) injectors impose additional requirement on
formulations.
[0470] Herein, the influence of needle-free injectors on
crystalline protein suspension formulation with needle injection
systems was compared. BD needles of 30 to 18 gauges were used as
comparators in the study. Needle G30 was regular wall and G25, 21
and 18 needles were thin-wall. Comparison of needle inner and outer
diameters represent in Table 2.
[0471] A BioJector needle-free (jet) injection system was used to
study the feasibility of using jet injectors with crystalline
protein formulations.
[0472] BioJector 2000: The B-2000 consists of two components: a
hand-held, reusable jet injector and a sterile, single-use,
disposable plastic syringe. The BioJector 2000 injector uses a
disposable carbon dioxide cartridges as a power source. The carbon
dioxide gas provides consistent, reliable pressure on the plunger
of the disposable syringe, thereby propelling the medication into
the tissue.
[0473] The second component of the system, the BioJector single-use
disposable syringe consists of a plastic, needle-free, variable
dose syringe. The body of the syringe is transparent and has
graduated markings to aid accurate filling. There are five
different BioJector syringes, each of which is intended for a
different injection depth or body type.
[0474] VetJet and Vitajet: The VetJet is a modified Vitajet for use
in the veterinary market. VetJet is also composed of two
components, a portable injector unit and a disposable syringe.
VetJet is powered by a spring.
[0475] The capability of crystalline suspensions to be administered
using needle-free jet injectors was explored. Limitations on
crystalline suspension that can be used in needle-free injectors
were identified. However, crystalline formulations that are
appropriately formulated and selected can be administered using
needle-free (jet) injection systems.
[0476] In order to study the feasibility of using crystalline
protein formulations with needle free injector, suspensions of hGH
crystals coated with poly-Arginine is buffer solution containing 25
mg/mL rhGH, 5 mg/mL poly-Arg, 25 mM Tris, 100 mM Na Acetate, 5% w/v
PEG 6000 pH 7.5--ALTU-238--as well as similar formulation
containing 0.2% of hyaluronic acid (HA)--HA-ALTU-238--were used. We
also compared influence of injection using needle-free and
needle-based systems on a soluble commercial hGH formulation (see
Table 33).
TABLE-US-00033 TABLE 33 Soluble and crystalline hGH samples Lot #
Concentration Description 249-67-05 5 mg/mL Commercial soluble hGH
formulation Nutropin AQ.sup.1 249-67-06 25 mg/mL ALTU-238 Phase I
formulation 249-70-04 25 mg/mL Reduced size ALTU-238 249-81-01 25
mg/mL HA-ALTU-238 .sup.1Manufactured and commercialized in the US
by Genentech Inc., South San Francisco, CA.
[0477] Properties of crystalline suspensions of model proteins with
different crystal size and different protein concentrations (see
Table 34) when injected using needle-free as well as needle based
injection systems were also explored.
TABLE-US-00034 TABLE 34 Crystalline protein suspension formulations
sample description Concen- Lot # tration Description Particle size
249-82-01 30 mg/mL BC-Lipase small size hexagonal 16 (20 .mu.m at
crystals Lot #145-75-2 largest dimension) 249-83-01 30 mg/mL
BC-Lipase medium size 30 (45 .mu.m at hexagonal crystals LP-7
largest dimension) 249-84-01 30 mg/mL BC-Lipase big size rhomboidal
32 (70 .times. 40 .times. crystals Lot L-00298-K 15 .mu.m)
249-85-01 40 mg/mL Glutaryl Alcalase big size 42 .mu.m (150 .times.
elongated bipiramidal crystals 30 .times. 30)
[0478] For all the samples viscosities of formulation vehicles were
comparable to the viscosity of water.
[0479] This experiment was performed to study the effects of
different injection systems on aggregation and chemical purity of
hGH in crystalline ALTU-238 suspension. Injections were performed
in triplicate. After injection, crystals were dissolved and soluble
hGH was analyzed with respect to aggregation, as analyzed by
SE-HPLC, and chemical degradation, as analyzed by RP-HPLC and
deamidation specific IEX-HPLC.
TABLE-US-00035 TABLE 35 Aggregation and chemical purity of hGH in
ALTU-238 after injection with different injection systems SE- RP-
IEX- HPLC, HPLC, HPLC, Sample % % % ALTU-238 Lot# 249-67-06 before
99.51 90.72 84.44 injection 249-67-06 after BD syringe with 99.10
91.42 84.85 30G .times. 1/2'' preattached needle 249-67-06 after
BioJector 2000 injector 99.48 91.03 83.54 249-67-06 after VetJet
injector 99.32 91.86 84.22
[0480] There was no significant difference in the content of
monomer or hGH related substances in the control sample and test
samples injected with the use of 30 G.times.1/2'' needle, BioJector
2000 or VetJet injection systems (Table 35). These data support the
conclusion that there was no measured impact of shear at the
molecular level as a result of using any of the tested injection
systems.
[0481] In order to study possible influence of shear stress on
crystalline hGH suspensions, samples prior and after injections
were analyzed for content of free hGH and poly-Arginine in the
supernatants of crystalline hGH suspensions. The control
suspension, ALTU-238, is a previously known suspension of coated or
complexed hGH crystals with poly-Arginine in a formulation vehicle.
Shear stress could change release properties of said formulation.
ALTU-238 contains a typical range of free hGH and poly-Arginine.
Changes of these parameters could indicate adverse influence of
said injection systems on crystals in suspensions and could affect
hGH release profile. Injections were performed in triplicate.
Supernatants of the control sample as well as the samples after
using different injection systems were analyzed for content of hGH
and poly-Arginine. The average content for the three runs of free
hGH and poly-Arginine is listed in the Table 36.
TABLE-US-00036 TABLE 36 hGH and poly-Arginine content in the
supernatants of ALTU-238 samples Sample C hGH sup C p-R sup
ALTU-238 Lot # 249-67-06 0.039 0.264 249-67-06 after BD syringe
with 0.003 0.171 30G .times. 1/2'' preattached needle 249-67-06
after BioJector 2000 injector 0.003 0.163 249-67-06 after VetJet
injector 0.023 0.219
[0482] No increase in the content of free hGH or poly-Arginine as
result of using 30 G.times.1/2'' needle, BioJector 2000 or VetJet
injection systems was observed. These data suggest that the studied
injection systems do not adversely influence the crystalline hGH
suspension.
[0483] To study influence of different injection systems on the
dissolution profile of complexed hGH crystal formulations, an
ALTU-238 control sample and ALTU-238 samples after injection were
analyzed for dissolution in a 3-pH dissolution test. Injections
were performed in duplicate. The average of two runs is presented
in Table 37.
TABLE-US-00037 TABLE 37 3-pH dissolution, average of 2 runs
Dissolu- Dissolu- Dissolu- tion at tion at tion at pH 2.5 pH 5.0 pH
7.0 Sample (%) (%) (%) ALTU-238 Lot # 249-67-06--control 110.00
63.30 24.17 249-67-06 after BD syringe with 101.39 69.66 25.87 30G
.times. 1/2'' preattached needle 249-67-06 after BioJector 2000
injector 101.64 65.83 25.22 249-67-06 after VetJet injector 105.73
67.05 25.17
[0484] No significant differences in hGH dissolution were seen
among the control sample and samples injected with the 30
G.times.1/2'' needle, BioJector 2000, or VetJet injection systems
(Table 37). These data support the feasibility of using needle-free
(jet) injectors for administration of ALTU-238.
[0485] An experiment was conducted to show the effect of different
injection systems on particle surface charge. Zeta potential in
ALTU-238 samples was studied after injection with a BioJector 2000
injector. After the first injection, the sample was collected and
injected repeatedly.
TABLE-US-00038 TABLE 38 Zeta potential of ALTU-238 samples prior
injection, after first and second injection with BioJector 2000
system Sample .xi.-potential, mV ALTU-238 Lot # 249-9-02 prior to
injection 21.8 .+-. 0.8 249-70-05 = 249-9-02 after 1.sup.st
injection with 22.7 .+-. 1.5 BioJector 2000 249-70-06 = 249-9-02
after 2.sup.nd injection with 22.6 .+-. 0.5 BioJector 2000
[0486] There were no significant changes in particle surface charge
after the first and repeated injections with BioJector 2000
compared to the original sample prior to injection (Table 38).
Injection with BioJector 2000 does not change .xi.-potential in
ALTU-238 formulations.
[0487] In this experiment, the influence of BioJector 2000 jet
injections on particle size of crystalline hGH
suspension--ALTU-238--was examined. Particle size was measured
using a Coulter LS 230 particle sizer prior to injection, and after
the first and repeated injections. A Fraunhofer optical model was
used to analyze data. Microscopic observation was used to detect
possible changes in crystal morphology.
TABLE-US-00039 TABLE 39 Particle size distribution in ALTU-238
samples Size .mu.m Size .mu.m Size .mu.m Sample % <10 % <50 %
<90 249-70-04 = 249-9-02 prior injection 1.57 3.50 6.61
249-70-05 = 249-9-02 after 1.sup.st injection 1.61 3.31 6.02 with
BioJector 2000 249-70-06 = 249-9-02 after 2.sup.nd injection 1.71
3.42 6.01 with BioJector 2000
[0488] No significant change in particle size after the first and
repeated injections with BioJector 2000 compared to the original
sample prior to injection (Table 39; FIG. 9). These numbers are
consistent with microscopic observation.
[0489] No reduction in particle size or changes in crystal
morphology were observed after injection of ALTU-238 with any of
injection systems. Similarly, no changes were observed after
repeated injections.
[0490] A suspension of hGH crystals coated with poly-Arginine in a
formulation vehicle containing hyaluronic acid, HA-ALTU-238, was
used to study feasibility of using jet injectors with this
crystalline protein suspension formulations. In this experiment,
the influence of different injection systems on hGH aggregation was
examined. Injections were performed in duplicate. After injection,
crystals were dissolved and soluble hGH was analyzed for
aggregation by SE-HPLC.
TABLE-US-00040 TABLE 40 Monomer protein content Sample SE-HPLC, %
249-75-01 (HA-ALTU-238) 99.83 249-75-01 after BD syringe with 99.83
30G .times. 1/2'' preattached needle 249-75-01 after BioJector 2000
injector 99.61 249-75-01 after VetJet injector 99.83
[0491] There was no significant difference in monomer hGH content
in the control HA-ALTU-238 sample and samples injected with the use
the 30 G.times.1/2'' needle, BioJector 2000, or VetJet injection
systems (Table 40). These data support the conclusion that there
was no measured impact of shear at the molecular level as a result
of using any of the tested injection systems.
[0492] In this experiment, the influence of the BioJector 2000 jet
injection on particle size of a crystalline hGH
suspension--HA-ALTU-238. Particle size was measured using a Coulter
LS 230 particle sizer prior injection, and after first and repeated
injections. A Fraunhofer optical model was used to analyze data.
Microscopic observation was used to detect possible changes in
crystal morphology.
TABLE-US-00041 TABLE 41 Particle size distribution in HA-ALTU-238
sample Size .mu.m Size .mu.m Size .mu.m Sample % <10 % <50 %
<90 249-81-01 = HA-ALTU-238 prior to 2.28 4.87 8.96 injection
249-81-02 = 249-81-01 after 1.sup.st injection 2.27 4.59 8.29 with
BioJector 2000 249-81-03 = 249-81-01 after 2.sup.nd injection 1.96
4.05 7.23 with BioJector 2000 249-81-04 = 249-81-01 after 1.sup.st
injection 1.97 4.23 7.70 with VetJet 249-81-05 = 249-81-01 after
2.sup.nd injection 1.89 4.21 8.30 with VetJet 249-81-06 = 249-81-01
after 1.sup.st injection 1.85 4.12 7.59 through 30G .times. 1/2''
needle 249-81-07 = 249-81-01 after 2.sup.nd injection 1.91 4.05
7.45 through 30G .times. 1/2''needle
[0493] There was no significant change in particle size of sample
after first and repeated injection with all of the injection
systems used comparing to sample prior injection (Table 41). See
also FIG. 10. These numbers are consistent with microscopic
observations. Similar observations were made for ALTU-238
formulations.
[0494] No reduction in particle size or crystal morphology was
observed when an HA-ALTU-238 sample was used for injections. After
repeated injections, there was no further reduction in particle
size as compared to the particle size observed in the sample
originally free of crystalline aggregates. These data indicate
feasibility of using needle-free injectors for administration of
crystalline protein suspension formulations.
[0495] To study the influence of jet injectors on soluble hGH
formulations, Nutropin AQ samples were used. No particulate
formation was observed in the Nutropin AQ sample before injection
or after injection through the 30 G.times.1/2 needle. However,
after injection with the BioJector 2000 and VetJet injectors,
formation of particulate matter in Nutropin AQ sample was observed.
This particulate matter likely formed as a result of caused by the
use of jet injectors. These data indicate that Nutropin AQ
formulation may not be appropriate for needle-free injection
systems and corroborate the notion that appropriate formulation
development may be required for successful use of the needle free
injectors for protein formulations.
[0496] ALTU-238 is a suspension of hGH crystals coated with
poly-Arginine. The crystals have a needle shape of about 10 to 20
.mu.m length and about 1-2 .mu.m thickness. The suspension itself
is stable. In previous studies, no significant sedimentation of
crystals over at least a 6 month storage period was observed.
However, there is an example of commercial insulin suspension
formulations in which crystals tend to settle fast.
[0497] To extend the scope of the studies herein, the influence of
jet injection systems on properties of other crystalline protein
suspensions where crystals had different sizes and/or morphology
and/or sedimentation properties was examined. A commercial insulin
Humulin Ultra formulation was loaded into the BioJector syringe and
injected after a hold period of three minutes in horizontal,
vertical upright or upside down position. These hold samples were
compared to a sample that was injected almost immediately after
loading the formulation into the jet injector's syringe. After
injection, the amount of insulin in the injected part and the
amount remaining inside the syringe were determined and expressed
as a percentage, considering as 100% the material that was loaded
into the syringe.
TABLE-US-00042 TABLE 42 Distribution of insulin in injected part
and remaining in syringe after immediate injection and after 3
minute hold in different positions Sample Injected, % Remaining in
syringe, % Injected immediately 90 10 Hold horizontal 29 71 Hold
vertical upright 61 39 Hold vertical upside down 90 10
[0498] In the sample that was injected immediately, 90% of the dose
was delivered. When injection was delayed and the injector was held
in the horizontal position, the dose delivered was as low as 29% of
that loaded into syringe (Table 42). This could seriously endanger
a patient. The use of jet injectors are not recommended for ad
ministering this insulin formulation. The insulin suspension
formulation is an example of crystalline protein formulations with
high crystal sedimentation rate. Jet injectors may be inappropriate
for use with crystalline protein suspensions with high
sedimentation rates.
[0499] The same experiment was performed with an ALTU-238
formulation. The formulation was loaded into the BioJector's
syringe and injected after a hold period of one hour in the
horizontal, vertical upright or upside down position. These hold
samples were compared to a sample that had been injected almost
immediately after loading the formulation into the jet injector's
syringe. After injection, the amount of hGH in the injected part
and the amount remaining inside the syringe were determined and
expressed as a percentage, considering as 100% the material that
had been loaded into the syringe.
TABLE-US-00043 TABLE 43 Distribution of hGH in injected and
remaining in syringe part after immediate injection and after one
hour hold in different positions Sample Injected, % Remaining in
syringe, % Injected immediately 95 5 Hold horizontal 95 5 Hold
vertical upright 95 5 Hold vertical upside down 95 5
[0500] Unlike with the example testing insulin, there were no
differences observed in dose distribution between the ALTU-238
sample that was immediately injected and the samples that were held
before injection (Table 43). The hold time used for ALTU-238 was
significantly longer.
[0501] No adverse influences of jet injection systems on
aggregation and chemical purity of crystalline hGH in ALTU-238
formulations were observed. Moreover, particle size, morphology,
particle surface charge and release properties did not change as
result of using jet injection systems.
[0502] To extend the scope of the study, the influence of jet
injection systems on the properties of other model crystalline
protein suspensions, where crystals had different size, morphology,
and sedimentation properties, were explored. In this experiment,
the effects of jet injection systems on a suspension of small size
hexagonal Burkholderia Cepacia ("BC") Lipase crystals were
examined. No visual microscopic changes in crystal size or
morphology in suspension were observed for the BC-Lipase crystals
before and after injection.
TABLE-US-00044 TABLE 44 Particle size distribution of BC-Lipase
suspension Size .mu.m Size .mu.m Size .mu.m Sample % <10 %
<50 % <90 249-82-01 4.35 15.89 23.93 249-82-02 after
BioJector 2000 injection 4.05 14.62 21.44 249-82-03 after VetJet
injection 3.92 14.75 21.83 249-82-04 after BD syringe with 4.06
15.14 23.00 30G .times. 1/2'' preattached needle
[0503] There did not appear to be an effect on BC-Lipase particle
size (Table 44, FIGS. 11A and B) or morphology as result of using
any of the tested injection systems. Jet injection systems could be
used for injection of crystalline protein suspension formulations
when crystals are hexagonal with a median particle size of about 20
micrometers.
[0504] The effects of jet injection systems on the properties of
other model crystalline protein suspensions, where crystals had a
larger size and hexagonal morphology were next explored. In this
experiment, the effects of jet injection systems on a suspension of
hexagonal BC-Lipase crystals with a medium particle size of about
30 .mu.m were examined. No changes in crystal morphology upon
injection were detected, and no significant changes in volume based
distribution were detected (Table 45).
TABLE-US-00045 TABLE 45 Particle size distribution Size .mu.m Size
.mu.m Size .mu.m Sample % <10 % <50 % <90 249-83-01 15.32
31.38 45.27 249-83-02 after BioJector 2000 injection 11.69 29.03
43.37 249-83-03 after VetJet injection 13.06 30.14 44.20 249-83-04
after BD syringe with 15.94 32.21 46.20 30G .times. 1/2''
preattached needle
[0505] A significant decrease in number-based particle size
distribution as a result of injection of BC-Lipase crystals through
a BioJector 2000 or a VetJet needle-free (jet) injection systems
was observed (FIGS. 12A and B). This is likely due to the
generation of a great number of small particles with the use of j
et systems but not with a 30 G needle. Although all samples were
easily injected, it should be taken into account that the use of
jet injection systems could change release profiles of formulations
as a result of the increased amount of small size particles.
[0506] The effects of jet injection systems on the properties of
other model crystalline protein suspensions, where crystals were
even larger and had hexagonal morphology, were examined. In this
experiment, the effects of jet injection systems on a suspension of
rhomboidal three-dimensional BC-Lipase crystals with large crystals
with a medium particle size of about 80 .mu.m were examined.
TABLE-US-00046 TABLE 46 Particle size distribution Size .mu.m Size
.mu.m Size .mu.m Sample % <10 % <50 % <90 249-84-01 9.29
32.72 51.49 249-84-02 after BioJector 2000 injection 5.98 26.11
42.65 249-83-04 after BD syringe with 2.08 9.23 32.33 30G .times.
1/2'' preattached needle 249-83-05 after 25G .times. 5/8'' needle
5.25 24.48 43.24
[0507] Significant filtration was observed when trying to draw
samples in injection module. Samples were loaded into the injection
modules through the plunger side. It was not possible to load the
samples into the VetJet system because of device design
specifics.
[0508] When particle size is close to the size of the injection
device orifice, crystalline samples either can not be loaded into
the injection module or undergo destruction due to interaction with
the injection device orifice. In the case of the 30 G needle,
significant reduction in crystal size was observed because of the
filtration effect. The smallest needle gauge the crystalline
suspension was able to pass into and out of was a 25 gauge syringe.
Even in this case, shear stress caused the formation of small
crystalline debris (Table 46, FIGS. 13A and B). Hence, the largest
dimension of the crystals in suspension should have at least about
3 times less size than the diameter of the orifice of j et
injectors in order to be able used with jet injectors.
[0509] In other experiments, the feasibility of using jet injectors
with a model suspension of crystalline Glutaryl Alcalase was
explored. These elongated bipiramidal crystals have a largest
dimension size of about 150 .mu.m with a cross section size of
about 30 .mu.m.
TABLE-US-00047 TABLE 47 Particle size distribution Size .mu.m Size
.mu.m Size .mu.m Sample % <10 % <50 % <90 249-85-01 9.17
41.62 66.68 249-85-02 after BioJector 2000 injection 2.27 29.25
58.11 249-85-04 after BD syringe with 4.97 36.98 59.21 30G .times.
1/2'' preattached needle 249-85-05 after 25G .times. 5/8'' needle
8.39 39.53 61.38 249-85-05 after 18G .times. 1'' needle 8.72 39.00
60.71
[0510] Significant filtration was observed when trying to draw the
samples into the injection modules. Sample was drawn freely into
the syringe only when an 18 G needle was used. Samples were loaded
into the injection module through the plunger side (not including
the 18 G needle example). It was not possible to load the sample
into the VetJet system because of specifics of the device design.
An increase in injection time was observed for the crystalline
Glutaryl Alcalase suspension when the BioJector 2000 was used.
Accumulation of crystalline debris in samples was observed after
using the jet injector and fine gauge needles.
[0511] Despite no measured significant changes in particle size, as
measured by a Coulter LS230 (Table 47), significant decreases in
the number-based particle size distribution (FIGS. 14A and B) were
observed as result of injecting Glutaryl Alcalase crystals through
the BioJector 2000 needle-free injection system and the 30 G
needle. This can be explained by the destruction of crystals when
using the jet system or 30 G needle for injection. When particle
size is close to the size of the injection device orifice,
crystalline samples either can not be loaded into injection module
or undergo destruction because of interactions with the injection
device wall. As the diameter of needles was increased, less
destruction of crystals was observed.
[0512] Method Protocols
[0513] Injection: crystalline formulations are injected using the
BioJector 2000 system with 1S021 syringe and VetJet system with
0.0062'' nozzle syringe Ref # K7000, BD 0.5 cc syringe with
preattached 30 G.times.1/2'' needle (BD Part #328466), through 30
G.times.1/2'' (BD Part #305106), 25 G.times.5/8'' (BD Part
#305122), 18 G.times.11/2'' (BD Part #305196) attached with
luer-lock to 1 mL syringe (BD Part #309628). 0.25 mL Samples are
tested in duplicate. BioJector 2000 and VetJet systems are used
according to BioJector's recommendations.
[0514] SE-HPLC: To determine purity using the SE-HPLC method, a
Phenomenex BioSep SEC-S-2000 column is used. Ten microliters of 2
mg/mL concentration sample is injected into the column. The running
buffer is composed of 3% IPA and 60 mM sodium phosphate, pH 7.0.
The flow rate is set to 0.6 mL/min and the run time is 30 minutes.
Detection is performed at 214 nm. The resulting chromatogram is
manually integrated. Percent purity (monomer) is calculated based
on peak area.
[0515] RP-HPLC: RP-HPLC is used for purity testing. To determine
the purity of hGH using the RP-HPLC method, a C5 Supelco Discovery
Bio Wide Pore Column, 5 cm.times.4.6 mm, 3 .mu.m particle size, 30
nm Pore size is used. The column thermostat temperature was set to
37.degree. C. Ten microliters of sample at 2 mg/mL concentration is
injected. The elution is carried out at a flow rate of 1.0 mL/min
with a gradient system present by mobile phase A (0.1% TFA in
water) and mobile phase B (0.8% TFA in MeCN). Gradient system
changed from 5% B to 50% B from 0-2.5 min., then 50% to 70% B in
2.5-15.5 min. then, 70% to 90% in 15.5 to 17 min., immediately
following this 5% B is re-established. A 3 min post-time is held
before the start of the next run. Detection is performed at 214 nm.
The resulting chromatogram is manually integrated. Percent purity
is calculated based on peak area.
[0516] IEX-HPLC (Strong Cation Exchange) Chromatography: SCX-hGH is
used as deamidation specific in stability testing. To determine the
purity of hGH using the IEX-HPLC method, a PolyLC (NEST Group cat #
P054SE0503) column is used. The column thermostat temperature is
set to 30.degree. C. Twenty microliters of sample at 2 mg/mL
concentration is injected. The elution is carried out at a flow
rate of 1 mL/min with a gradient system present by mobile phase A
(50 mM Na Acetate, pH 4.6) and mobile phase B (50 mM Na Acetate, pH
4.6 250 mM NaCl). The gradient system is changed from 0% B to 20% B
from 0 to 5 min., then 20% to 70% B in 5 to 25 min. then, 70% to
100% in 25 to 25.1 min. 100% B is kept until 27 min. following 27.1
min, 0% B is re-established for a 5 min. post-time. Detection is
performed at 214 nm and 280 nm. The resulting chromatogram is
manually integrated. Percent purity is calculated based on peak
area.
[0517] Particle Size Distribution (PSD): Particle size distribution
is determined by laser diffraction using a Coulter LS 230 Particle
Size Analyzer (Coulter Corp., Miami, Fla.) with micro volume
module. The sample is diluted in Sample buffer (Formulation Vehicle
for Crystalline Protein 3) to achieve an operation range of 8% to
12% obscuration. Data analysis is performed using the Fraunhofer
optical model. Size in .mu.m of particle fraction representing
cumulative volume distribution limits of 10% (dl10), 50% (median)
and 90% (dl90) is reported.
[0518] 3-pH Dissolution Test: A 3-pH dissolution test is used to
assess changes in dissolution profiles in ALTU-238 samples.
[0519] Particle Surface Charge: Particle surface charge is
determined using Zetasizer Nano-Z (Malvern Instruments Ltd. UK).
The running buffer is 20 mM Tris, pH 7.5. Zeta Potential Transfer
Standard is used to calibrate the instrument (Malvern catalog
#DTS1050). For each sample, a 12 .mu.l crystalline aliquot
(concentration between 10 to 30 mg/ml) is added to 588 .mu.l
running buffer, and three measurements are taken at room
temperature.
[0520] Microscopic Appearance: The appearance of the crystalline
test article is assessed by microscopy to detect any relevant
changes in the test article due to injections.
Example 5
[0521] ALTU-238 is a long-acting poly-arginine (poly-Arg) coated
crystalline human growth hormone (somatropin). As a further
development, lyophilized forms and reconstituted lyophilized
formulations were developed. The reconstituted lyophilized
formulations are predicted to achieve 24 months or longer chemical
stability at 2-8.degree. C. after being resuspended.
[0522] An exemplary lyophilized formulation of hGH or an hGH
derivative contains:
[0523] 1. About 20 to about 50 mg/mL hGH crystal suspension
[0524] 2. Tris or a combination buffer of phosphate with either
Tris or histidine as the buffer,
[0525] 3. pH range from 7 to 9
[0526] 4. Salt: sodium chloride or sodium acetate ranging from 50
mM to 100 mM
[0527] 5. Polyethylene glycol 6000 or 8000 as the suspending and
caking agents, ranging from 2.5% to 10%
[0528] 6. Siliconized vials or coated vials such as Schott type I
plus glass vials, or coated surface container closure
[0529] 7. Freezing shelf temperature of -50.degree. C. to
-60.degree. C., the primary drying shelf temperature of -30.degree.
C. to 10.degree. C. and the 2nd drying of 20.degree. C. to
40.degree. C. for the lyo cycle parameter.
[0530] These components and amounts are suitable for the
formulations prior to lyophilization and also for the formulations
after the lyophilized hGH is reconstituted.
[0531] One or more of the test methods listed in Table 48 are used
to evaluate the stability of the reconstituted formulations made
from the lyophilized forms. Details for carrying out the test
methods are known in the art and described herein.
TABLE-US-00048 TABLE 48 Test Methods TEST METHODS APPLICATION pH pH
maintenance Cake and reconstitution appearance Suspension
description Particle Size Distribution Physical stability
Reconstitution time Physical stability Crystal Morphology Physical
stability Purity by Reverse Phase Chemical degradation (oxidation)
Chromatography (RP-HPLC) Purity by Size Exclusion Soluble
aggregates Chromatography (SEC) Poly(L-arginine) Total Content
Facilitate controlled release by Reverse Phase Chromatography Free
Poly(L-arginine) and hGH Physical stability Content in Supernatant
by Reverse Phase Chromatography Absorbance at 280 nm Protein
Concentration Undissolved crystals/aggregates Insoluble aggregates
by Absorbance at 320 nm Cation Exchange HPLC (CEX) Chemical
degradation (deamidation) Crystal Dissolution hGH product release
profile
[0532] The first lyophilization study evaluated the dissolved hGH
in the supernatant and the effect of excipients on the stability
upon lyophilization. The base formulation was Tris buffer at pH
7.5. Various concentrations of PEG; carbohydrates (sucrose,
mannitol, sorbitol and lactose); and formulations with and without
salt were tested. The free hGH in the supernatant is an indication
of the crystal integrity. Thus, the amount of dissolved hGH in the
supernatant was monitored to assure that the controlled release
profiles were unchanged post lyophilization. The result of
dissolved hGH concentrations in the supernatant of the pre
lyophilization (prelyo) sample as shown in FIG. 15 indicates that
the PEG concentration does have an impact on the solubility of the
crystal, and the lower the percentage of PEG, the more crystals are
dissolved in the supernatant. For the experiments shown in the
figure, the base formulation was Tris buffer at pH 7.5. In
addition, sample with no PEG have the highest amount of dissolved
hGH in the supernatant.
[0533] ALTU-238 in water had the appearance of a loose powder,
without a cake appearance after lyophilization. Otherwise, all
samples exhibited reasonable cake formation with slight shrinkage
from the edge of the vial. Upon reconstitution, the poly-Arg
complexed crystals without any excipient settled to the bottom of
the vials due to lack of suspending agents. The formulation
containing a carbohydrate such as mannitol, sucrose, sorbitol or
lactose suspended immediately upon reconstitution but settled down
within 15 minutes, indicating a difference in sedimentation rate
after lyophilization. The starting material or prelyo suspension
was mostly small 2 to 20 .mu.m clusters of tiny rod shape crystals.
However, the crystal sizes appear to be significantly larger for
some formulations post lyophilization. The formulations examined
pre and post lyophilization contained 25 mg/mL Altu-238 in Tris, pH
7.5; mg/mL complexed crystals in Tris, 5% lactose, pH 7.5; or 25
mg/mL complexed crystals in Tris, sodium acetate and 5% PEG6000, pH
7.5. The results indicate that the formulation containing 5% PEG
maintained the crystal morphology post lyophilization.
[0534] The target hGH/pR ratio was 5 to 7.5 for the prelyo
suspension. However, the hGH/poly-Arg ratios of the prelyo
suspension were much higher (.about.20) for the formulation
containing 1% PEG or carbohydrate, which suggests a loss of
poly-Arg coating in these formulations, likely resulting from
crystal dissolving. This was confirmed by the fact that the hGH
concentrations in the supernatant were also higher for the prelyo
suspensions. In addition, the postlyo crystals are clumps for
samples containing 1% PEG or carbohydrate more so than those of 5%
PEG, which again suggests loss of poly-Arg, resulting in crystals
sticking together and thus changing the morphology. Therefore, the
concentration of PEG is critical in stabilizing ALTU-238 during the
lyo process.
[0535] Also, the pH of the reconstituted lyo samples shifts higher
after the lyo process, from pH 7.5 to 8.2 for formulations
containing Tris. To identify the cause, a freeze/thaw study of
these tested formulations was performed in the FTS lyophilizer
using the same freezing rate, 1.degree. C./min, as the lyo cycle.
Then all samples were thawed at room temperature. The results
indicated that the pH shift was only about 0.05 to 0.09 per pH
unit. Therefore, it was the drying process that induced the pH
changes.
[0536] To improve the formulation, the following studies were
focused on the selection of buffer species and buffer
concentrations. The pH was raised to .about.8 for the Tris
containing prelyo suspension because Tris has the maximum buffer
capacity at this pH; and/or adding phosphate as one of the buffer
components because phosphate can buffer at pH 7.5 upon
reconstitution. The idea of selecting histidine as the second
buffer was to prevent pH shifting too low due to the presence of
phosphate during the lyophilization process, resulting in crystal
dissolution. Two fill volumes: 0.5 mL and 1.2 mL, to deliver 0.2 mL
and 1.0 mL of drug product, were evaluated. The three buffer
systems that could achieve our target were formulated in Tris, pH
8.0; histidine with phosphate at pH 7.5. One interesting
observation was that the pH dropped to 8.0 for the sample that was
formulated in Tris, pH 8.6, which confirmed the theory that optima
buffer range for complexed hGH crystals should be around 8. Based
on all of these results, the buffer using Tris at pH 8; and
combinations of two buffers which contain histidine and phosphate
or Tris/phosphate are selected for poly-Arg complexed hGH
crystals.
[0537] Selected formulations were tested for stability, and pH
measurements were obtained after various storage periods. Other
than the initial pH shifts, there were no changes of pH during the
storage period.
[0538] The in-vitro dispersed dissolution rate was tested, and no
significant changes are observed upon storage; the dissolution
rates were comparable to the clinical trial lots.
[0539] The polyethylene glycol concentrations of 2.5%, 5%, and 10%
and the range of 2.5% t 10% were also established. All samples
could be resuspended within one minute. No differences were
observed in oxidation, deamidation, aggregation, or dissolved hGH
in these formulations after 3 months at 2-8.degree. C. or
40.degree. C.
[0540] In addition, no differences in stability were observed for
hGH concentrations ranging from 20 to 50 mg/mL of poly-Arg
complexed hGH crystals upon reconstitution. Based on the results,
the free hGH in the supernatant and dispersed dissolution profile
are comparable for all tested concentrations. In addition, there
are no differences in chemical stability profiles.
[0541] The lyo cycle was optimized using the formulation containing
Tris, sodium acetate, and PEG6000 at pH 7.5. Various lyo parameters
were tested. The freezing DSC thermograms of a PEG containing
sample indicate a thermo event occurred around -20.degree. C. which
is contributed to by PEG. To avoid the complication by PEG,
<-20.degree. C. to product temperatures were targeted for the
selection of the primary drying temperature for the lyo cycle
development. As shown in Table 49, at freezing temperature of
-60.degree. C. or -50.degree. C. and the primary drying shelf
temperature range from -30.degree. to 10.degree. C., all finished
lyo products exhibited reasonable cake formation with a smooth
surface, slight shrinkage from the edge of the vial. No differences
are observed in the cake appearance of lyo cakes from various lyo
cycles. In addition, the measured product temperatures were all
less than -20.degree. C., as desired.
TABLE-US-00049 TABLE 49 Summary of lyophilization cycle parameters.
The vacuum was set at 100 mTorr SET PRIMARY SECONDARY SHELF
FREEZING DRYING DRYING FINAL TEMP SHELF SHELF SHELF TEMP DEW
PRODUCT (.degree. C.) TEMP (.degree. C.) TEMP (.degree. C.)
(.degree. C.) (.degree. C.) TEMP. (.degree. C.) Lyo#6 -30 -60 -30
20 -42.1 -34 Lyo#8 -30 -60 -30 40 -42.1 -34 Lyo#9 -10 -60 -10 40
-40.1 N/A Lyo#11 0 -50 0 30 -43.1 -22 Lyo#10 10 -60 10 40 -42.0
-25
[0542] The particle size distribution was analyzed and the particle
size distributions are listed in FIG. 16. There was no difference
in particle size distribution for all tested cycles.
[0543] In conclusion, the lyo cycles defined for the embodiments
herein are: [0544] a. Freezing rate of 1.degree. C./min [0545] b.
Frozen at -50.degree. C. to -60.degree. C. [0546] c. Primary drying
shelf temperature range from -30.degree. C. to 10.degree. C. [0547]
d. Secondary Drying shelf temperature from 20.degree. C. to
40.degree. C.
[0548] The stability results of one of the lead formulations were
summarized in the table below. All data are comparable when compare
the samples that generated with these two cycles.
[0549] Various container closures for the lyo products were also
compared. The poly-Arg coated crystals quickly and easily stick to
glass vials without silicon oil and coated surface. The crystals
stick to the non-siliconized vial easily, and this stickiness will
be worse for a multi-dose product. Therefore, the container closure
for poly-Arg complexed hGH crystals need to be siliconized or vials
with coated surface such as type I plus coated vials.
[0550] When reconstituted with a diluent containing a preservative,
the reconstituted formulation may be used as a multi-dose
formulation. The advantage of a multi-dose formulation is that it
facilitates ease of use for the patient, reduces waste by allowing
full use of the vial contents, thereby resulting significant
savings.
[0551] Four commonly used preservatives were tested for suitability
with complexed hGH crystals: phenol, m-cresol, chlorobutanol, and
benzyl alcohol. The lyo formulations containing Tris, sodium
acetate, and PEG were reconstituted with water containing one of
these preservatives and the stability of the reconstituted samples
was analyzed. All vials could be reconstituted/resuspended within
10 seconds and no changes of crystal morphology were seen, except
for the sample containing benzyl alcohol which had more clumps.
[0552] The stability of these reconstituted suspensions containing
preservatives was analyzed. The results indicate that all
reconstitution suspensions are stable at 2-8.degree. C. for at
least one month. Therefore, m-cresol, phenol or Chlorobutanol can
be used as a preservative with the lyo formulations.
[0553] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
* * * * *
References