U.S. patent application number 17/419899 was filed with the patent office on 2022-09-29 for collagenase formulations and methods of producing the same.
The applicant listed for this patent is ENDO GLOBAL AESTHETICS LIMITED. Invention is credited to Karunakar SUKURU.
Application Number | 20220305094 17/419899 |
Document ID | / |
Family ID | 1000006458963 |
Filed Date | 2022-09-29 |
United States Patent
Application |
20220305094 |
Kind Code |
A1 |
SUKURU; Karunakar |
September 29, 2022 |
COLLAGENASE FORMULATIONS AND METHODS OF PRODUCING THE SAME
Abstract
Disclosed herein are improved collagenase-containing
formulations and methods of preparing the same. The
collagenase-containing formulations comprise a collagenase, about
30 mM to about 240 mM of a disaccharide, about 50 mM to about 800
mM of mannitol, and about 6 mM to about 10 mM of a Tris-HCl.
Lyophilized and reconstituted formulations are also provided.
Inventors: |
SUKURU; Karunakar; (Garnet
Valley, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ENDO GLOBAL AESTHETICS LIMITED |
Dublin |
|
IE |
|
|
Family ID: |
1000006458963 |
Appl. No.: |
17/419899 |
Filed: |
January 3, 2020 |
PCT Filed: |
January 3, 2020 |
PCT NO: |
PCT/US2020/012202 |
371 Date: |
June 30, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62788916 |
Jan 6, 2019 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/4886 20130101;
A61P 17/00 20180101; C12Y 304/24007 20130101; C12Y 304/24003
20130101; A61K 31/7016 20130101 |
International
Class: |
A61K 38/48 20060101
A61K038/48; A61K 31/7016 20060101 A61K031/7016; A61P 17/00 20060101
A61P017/00 |
Claims
1. A formulation comprising: a collagenase; about 30 mM to about
240 mM of a disaccharide; about 50 mM to about 800 mM of mannitol;
and about 6 mM to about 10 mM of a Tris-HCl.
2. The formulation of claim 1, wherein the collagenase comprises a
collagenase I.
3. The formulation of claim 2, wherein the collagenase I comprises
the amino acid sequence of SEQ ID NO: 1.
4. The formulation of claim 1, wherein the collagenase comprises a
collagenase II.
5. The formulation of claim 4, wherein the collagenase II comprises
the amino acid sequence of SEQ ID NO: 2.
6. The formulation of claim 1, wherein the collagenase comprises a
mixture of collagenase I and collagenase II.
7. The formulation of claim 6, wherein the collagenase I comprises
the amino acid sequence of SEQ ID NO: 1 and the collagenase II
comprises the amino acid sequence of SEQ ID NO: 2.
8. The formulation of claim 7, wherein the collagenase is
collagenase Clostridium histolyticum (CCH).
9. The formulation of claim 1, wherein the disaccharide comprises
sucrose or trehalose.
10. The formulation of claim 1, wherein the pH of the formulation
is about 7.8 to about 8.8.
11. The formulation of claim 1, wherein the formulation comprises:
CCH; about 60 mM sucrose; about 225 mM mannitol; and about 10 mM
Tris-HCl, wherein the formulation has a pH of about 8.5.
12. The formulation of claim 1, further comprising a surfactant
comprising polysorbate 20, polysorbate 80, or poloxamer 188.
13. The formulation of claim 12, comprising from about 0.01% to
about 2% of the surfactant.
14. The formulation of claim 13, comprising about 0.02% of the
surfactant.
15. The formulation of claim 1, wherein the formulation is
liquid.
16. A lyophilized formulation comprising: a collagenase; a
disaccharide; mannitol; and Tris-HCl.
17. The lyophilized formulation of claim 16, wherein the
collagenase comprises a collagenase I.
18. The lyophilized formulation of claim 17, wherein the
collagenase I comprises the amino acid sequence of SEQ ID NO:
1.
19. The lyophilized formulation of claim 16, wherein the
collagenase comprises a collagenase II.
20. The lyophilized formulation of claim 19, wherein the
collagenase II comprises the amino acid sequence of SEQ ID NO:
2.
21. The lyophilized formulation of claim 16, wherein the
collagenase comprises a mixture of collagenase I and collagenase
II.
22. The lyophilized formulation of claim 21, wherein the
collagenase I comprises the amino acid sequence of SEQ ID NO: 1 and
the collagenase II comprises the amino acid sequence of SEQ ID NO:
2.
23. The lyophilized formulation of claim 21 or 22, wherein the
collagenase is collagenase Clostridium histolyticum (CCH).
24. The lyophilized formulation of claim 16, wherein the
disaccharide comprises sucrose or trehalose.
25. The lyophilized formulation of claim 16, wherein, prior to
lyophilization, the formulation comprises: CCH; 60 mM sucrose; 225
mM mannitol; and 10 mM Tris-HCl, and having a pH of about 8.5.
26. The lyophilized formulation of claim 16, wherein the
lyophilized formulation is stable at pressures above 380
.mu.bar.
27. The lyophilized formulation of claim 26, wherein the
lyophilized formulation is stable at a pressure of about 4000
.mu.bar.
28. The lyophilized formulation of claim 16, wherein the
lyophilized formulation is stable at: (a) 2-8.degree. C. for at
least 36 months; (b) 25.degree. C./60% relative humidity for at
least 36 months; (c) 40.degree. C./75% relative humidity for at
least 6 months; or (d) any combination of (a) to (c).
29. The lyophilized formulation of claim 16, wherein the
lyophilized formulation is formed by a method comprising: freezing
the formulation at a temperature between about -25.degree. C. and
-55.degree. C. to form a frozen formulation; and drying the frozen
formulation at a temperature between about 25.degree. C. and about
50.degree. C. to form the lyophilized formulation.
30. The lyophilized formulation of claim 29, wherein the
lyophilized formulation is formed by a method comprising: freezing
the formulation at a single temperature between about -25.degree.
C. and -55.degree. C. to form a frozen formulation; and drying the
frozen formulation at a single temperature between about 25.degree.
C. and about 50.degree. C. to form the lyophilized formulation.
31. The lyophilized formulation of claim 16, wherein the
lyophilized formulation is formed by a lyophilization method that
is performed for less than 72 hours.
32. The lyophilized formulation of claim 16, wherein the
lyophilized formulation is formed by a lyophilization method that
is performed at a pressure of between about 380 .mu.bar to about
4000 .mu.bar.
33. The lyophilized formulation of claim 16, in a unit-dose vial,
multi-dose vial, cartridge, or syringe.
34. A reconstituted formulation comprising: a collagenase; a
disaccharide; mannitol; Tris-HCl; calcium chloride; and sodium
chloride.
35. The reconstituted formulation of claim 34, wherein the
collagenase is collagenase Clostridium histolyticum (CCH).
36. The reconstituted formulation of claim 35, wherein the
disaccharide comprises sucrose or trehalose.
37. The reconstituted formulation of claim 34, wherein the
reconstituted formulation is isotonic to human blood.
38. A kit comprising: a container comprising the lyophilized
formulation of claim 16; and a container comprising a sterile
diluent comprising calcium chloride and sodium chloride.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is the U.S. National Stage Application of
International Patent Application No. PCT/US2020/012202, filed Jan.
3, 2020, which claims priority to U.S. Provisional Application No.
62/788,916, filed Jan. 6, 2019, the disclosure of each of which are
hereby incorporated by reference in their entirety.
REFERENCE TO A SEQUENCE LISTING
[0002] This application includes a Sequence Listing submitted
electronically as a text file named
"117326_000001_Sequence_Listing.txt", created on Jan. 3, 2020 with
a size of 17,447 bytes. The Sequence Listing is incorporated by
reference herein.
FIELD OF THE INVENTION
[0003] Disclosed herein are collagenase-comprising formulations
with improved stability and storage properties.
BACKGROUND OF THE INVENTION
[0004] XIAFLEX.RTM. (collagenase from Clostridium histolyticum
(CCH)) is currently approved for the treatment of Dupuytren's
Contracture (DC) and Peyronie's Disease (PD). The currently
approved XIAFLEX.RTM. formulation is supplied as a lyophilized cake
containing 0.9 mg CCH in 3 CC vials along with a diluent vial. The
current XIAFLEX.RTM. formulation (pre-lyophilization) has a
lyophilization cycle time of about 72 hours in vials. Efficient
lyophilization is required for shelf life and enzyme stability.
SUMMARY OF THE INVENTION
[0005] Disclosed herein are formulations comprising: a collagenase;
about 30 mM to about 240 mM of a disaccharide; about 50 mM to about
800 mM of mannitol; and about 6 mM to about 10 mM of a
Tris-HCl.
[0006] Also provided herein are lyophilized formulations
comprising: a collagenase; a disaccharide; mannitol; and
Tris-HCl.
[0007] Reconstituted formulations comprising: a collagenase; a
disaccharide; mannitol; Tris-HCl; calcium chloride; and sodium
chloride are also disclosed.
[0008] Kits are also provided, wherein the kits comprise: a
container comprising any of the disclosed lyophilized formulations;
and a container comprising a sterile diluent comprising calcium
chloride and sodium chloride.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The summary, as well as the following detailed description,
is further understood when read in conjunction with the appended
drawings. For the purpose of illustrating the disclosed
formulations, there are shown in the drawings exemplary embodiments
of the formulations; however, the formulations are not limited to
the specific embodiments disclosed. In the drawings:
[0010] FIG. 1 illustrates the effect of pH on protein interactions
in exemplary formulations comprising trehalose, mannitol, and
various collagenases.
[0011] FIG. 2A and FIG. 2B illustrate an exemplary hydrogen
peroxide challenges analyzing the effect of pH and excipients on
turbidity. NTU--Nephelometric Turbidity Units; PS--Polysorbate;
T--Trehalose, S--Sucrose, M--Mannitol; H.sub.2O.sub.2--Hydrogen
peroxide; 7.5, 8.0, and 8.5 refers to formulation pH.
[0012] FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F, FIG.
3G, FIG. 3H, FIG. 3I, FIG. 3J, FIG. 3K, FIG. 3L, FIG. 3M, FIG. 3N,
FIG. 3O, FIG. 3P, FIG. 3Q, and FIG. 3R illustrate scanning electron
microscopy (SEM) images of cakes from various lyophilized
formulations (Va: 0.93 mg/ml CCH; 60 mM Sucrose; 112.5 mM Mannitol;
10 mM Tris/HCl buffer pH 8.5; Vb: 0.93 mg/ml CCH; 60 mM Sucrose;
225 mM Mannitol; 10 mM Tris/HCl buffer pH 8.5; and Vc: 0.93 mg/ml
CCH; 60 mM Sucrose; 337.5 mM Mannitol; 10 mM Tris/HCl buffer pH
8.5).
[0013] FIG. 4A, FIG. 4B, and FIG. 4C illustrate images of cakes
from various lyophilized formulations under a pressure of 128
.mu.bar (FIG. 4A), 380 .mu.bar (FIG. 4B), and 1030 .mu.bar (FIG.
4C).
[0014] FIG. 5A and FIG. 5B illustrate images of cakes from various
lyophilized formulations under a pressure of 128 .mu.bar (FIG. 5A)
and 4000 .mu.bar (FIG. 5B).
[0015] FIG. 6 illustrates the moisture content over time in various
lyophilized formulations.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0016] The disclosed formulations may be understood more readily by
reference to the following detailed description taken in connection
with the accompanying figures, which form a part of this
disclosure. It is to be understood that the disclosed formulations
are not limited to the specific formulations described and/or shown
herein, and that the terminology used herein is for the purpose of
describing particular embodiments by way of example only and is not
intended to be limiting of the claimed formulations.
[0017] Unless specifically stated otherwise, any description as to
a possible mechanism or mode of action or reason for improvement is
meant to be illustrative only, and the disclosed formulations are
not to be constrained by the correctness or incorrectness of any
such suggested mechanism or mode of action or reason for
improvement.
[0018] Throughout this text, the descriptions refer to formulations
and methods of preparing the formulations. Where the disclosure
describes or claims a feature or embodiment associated with a
formulation, such a feature or embodiment is equally applicable to
the methods of forming the formulation. Likewise, where the
disclosure describes or claims a feature or embodiment associated
with a method of forming a formulation, such a feature or
embodiment is equally applicable to the formulation.
[0019] Where a range of numerical values is recited or established
herein, the range includes the endpoints thereof and all the
individual integers and fractions within the range, and also
includes each of the narrower ranges therein formed by all the
various possible combinations of those endpoints and internal
integers and fractions to form subgroups of the larger group of
values within the stated range to the same extent as if each of
those narrower ranges was explicitly recited. Where a range of
numerical values is stated herein as being greater than a stated
value, the range is nevertheless finite and is bounded on its upper
end by a value that is operable within the context of the invention
as described herein. Where a range of numerical values is stated
herein as being less than a stated value, the range is nevertheless
bounded on its lower end by a non-zero value. It is not intended
that the scope of the disclosure be limited to the specific values
recited when defining a range. All ranges are inclusive and
combinable.
[0020] When values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. Reference to a particular numerical value
includes at least that particular value, unless the context clearly
dictates otherwise.
[0021] It is to be appreciated that certain features of the
disclosed formulations which are, for clarity, described herein in
the context of separate embodiments, may also be provided in
combination in a single embodiment. Conversely, various features of
the disclosed formulations that are, for brevity, described in the
context of a single embodiment, may also be provided separately or
in any subcombination.
[0022] As used herein, the singular forms "a," "an," and "the"
include the plural.
[0023] Various terms relating to aspects of the description are
used throughout the specification and claims. Such terms are to be
given their ordinary meaning in the art unless otherwise indicated.
Other specifically defined terms are to be construed in a manner
consistent with the definitions provided herein.
[0024] The term "comprising" is intended to include examples
encompassed by the terms "consisting essentially of" and
"consisting of"'; similarly, the term "consisting essentially of"
is intended to include examples encompassed by the term "consisting
of."
[0025] The following abbreviations are used herein: collagenase
Clostridium histolyticum (CCH), United States Pharmacopeia (USP),
Nephelometric Turbidity Units (NTU), Polysorbate (PS), Hydrogen
peroxide (H.sub.2O.sub.2).
[0026] Provided herein are formulations comprising, or consisting
of:
[0027] a collagenase;
[0028] about 30 mM to about 240 mM of a disaccharide;
[0029] about 50 mM to about 800 mM of mannitol; and
[0030] about 6 mM to about 10 mM of a Tris-HCl.
[0031] The formulations can contain between about 0.2 mg/ml to
about 50 mg/ml of collagenase. For example, the lyophilized
formulation can contain about 0.2 mg/ml, about 0.3 mg/ml, about 0.4
mg/ml, about 0.6 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1
mg/ml, about 1.2 mg/ml, about 1.4 mg/ml, about 1.6 mg/ml, about 1.8
mg/ml, about 2 mg/ml, about 2.5 mg/ml, about 3 mg/ml, about 3.5
mg/ml, about 4 mg/ml, about 4.5 mg/ml, about 5 mg/ml, about 10
mg/ml, about 15 mg/ml, about 20 mg/ml, about 25 mg/ml, about 30
mg/ml, about 35 mg/ml, about 40 mg/ml, about 45 mg/ml, or about 50
mg/ml of collagenase.
[0032] As used herein, "collagenase" refers to any of the
following: (a) collagenase (including mutants) having activity as
defined by EC 3.4.24.3
(www.brenda-enzymes.org/enzyme.php?ecno=3.4.24.3 (accessed Jul. 3,
2019); (b) collagenase produced by fermentation of Clostridium
histolyticum (also known as Hathewaya histolytica); (c) CCH (as
described herein); (d) collagenase having at least 50% sequence
alignment with collagenase I (also referred as class I collagenase)
as determined by BLAST; (e) collagenase having at least 50%
sequence alignment with collagenase II (also referred as class II
collagenase) as determined by BLAST; (f) collagenase produced by
fermentation of other source organisms (i.e., non-Clostridium
histolyticum), e.g., mammalian, crustacean, fungal, bacterial or
microbial collagenase; (g) collagenase obtained by recombinant
techniques; (h) collagenase with a molecular mass from about 65 kDa
to about 130 kDa; (i) collagenase designated as collagenase I or
collagenase II; (j) mixtures of collagenase I and II; (k)
collagenase from strain JCM 1403 (ATCC 19401) or derivatives
thereof; (l) collagenase from strain ATCC 21000 or derivatives
thereof; (m) collagenase from ATCC 69334 or derivatives thereof;
(n) collagenase from C. perfringens; (o) collagenase from Vibrio
alginolyticus; (p) collagenase from Streptomyces; (q) collagenase
from Pseudomonas; (r) collagenase from Achromobacter iophagus; (s)
collagenase described by Worthington Biochemical Corp.
(www.Worthington-biochem.com; "Product Highlights"); (t)
collagenase described by Sigma-Aldrich (www.sigma-aldrich.com); (u)
collagenase having one or more of the following characteristics:
[0033] V.sub.max (min.sup.-1) of about 0.08 to 7.70 (SRC assay), or
about 0.3 to 30.5 (GPA assay); [0034] K.sub.M, of about 4.1 to 410
nM (SRC assay), or about 0.03 to 3.1 mM (GPA assay); [0035]
K.sub.cat (sec.sup.-1) of about 1.1 to 107 (SRC assay), or about 93
to 9,179 (GPA assay); [0036] 1/K.sub.cat, microseconds of about 376
to 37,222 (SRC assay), or about 4 to 428 (GPA assay); or [0037]
K.sub.cat/K.sub.M, mM.sup.-1 sec.sup.-1 of about 5,140 to 508,814
(SRC assay), or about 60 to 5,934 (GPA assay); (v) collagenase
described by Nordmark Arzneimittel GmbH & Co. KG; (w)
collagenase from strain 004; or (x) equivalents or mixtures of any
of the foregoing. Non-limiting examples of collagenases that may be
used in the disclosure herein are described in U.S. Pat. Nos.
7,811,560, 9,757,435, 9,744,138, and Int'l Pub. No.
WO2012/125948.
[0038] In some embodiments the collagenase can comprise a
collagenase I. A suitable collagenase I includes, for example, a
collagenase I comprising an amino acid sequence that is 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 1. In some aspects, the
collagenase I comprises the amino acid sequence of SEQ ID NO:
1.
[0039] In some embodiments, the collagenase can comprise a
collagenase II. A suitable collagenase II includes, for example, a
collagenase II comprising an amino acid sequence that is 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 2. In some aspects, the
collagenase II comprises the amino acid sequence of SEQ ID NO:
2.
TABLE-US-00001 TABLE 1 Sequences Collagenase I
IANTNSEKYDFEYLNGLSYTELTNLIKNIKWNQINGLFNYSTGSQKFFG (SEQ ID NO: 1)
DKNRVQAIINALQESGRTYTANDMKGIETFTEVLRAGFYLGYYNDGLSY
LNDRNFQDKCIPAMIAIQKNPNFKLGTAVQDEVITSLGKLIGNASANAE
VVNNCVPVLKQFRENLNQYAPDYVKGTAVNELIKGIEFDFSGAAYEKDV
KTMPWYGKIDPFINELKALGLYGNITSATEWASDVGIYYLSKFGLYSTN
RNDIVQSLEKAVDMYKYGKIAFVAMERITWDYDGIGSNGKKVDHDKFLD
DAEKHYLPKTYTFDNGTFIIRAGEKVSEEKIKRLYWASREVKSQFHRVV
GNDKALEVGNADDVLTMKIFNSPEEYKFNTNINGVSTDNGGLYIEPRGT
FYTYERTPQQSIFSLEELFRHEYTHYLQARYLVDGLWGQGPFYEKNRLT
WFDEGTAEFFAGSTRTSGVLPRKSILGYLAKDKVDHRYSLKKTLNSGYD
DSDWMFYNYGFAVAHYLYEKDMPTFIKMNKAILNTDVKSYDEIIKKLSD
DANKNTEYQNHIQELADKYQGAGIPLVSDDYLKDHGYKKASEVYSEISK
AASLTNTSVTAEKSQYFNTFTLRGTYTGETSKGEFKDWDEMSKKLDGTL
ESLAKNSWSGYKTLTAYFTNYRVTSDNKVQYDVVFHGVLTDNADISNNK
APIAKVTGPSTGAVGRNIEFSGKDSKDEDGKIVSYDWDFGDGATSRGKN
SVHAYKKTGTYNVTLKVTDDKGATATESFTIElKNEDTTTPITKEMEPN
DDIKEANGPIVEGVTVKGDLNGSDDADTFYFDVKEDGDVTIELPYSGSS
NFTWLVYKEGDDQNHIASGIDKNNSKVGTFKATKGRHYVFIYKHDSASN
ISYSLNIKGLGNEKLKEKENNDSSDKATVIPNFNTTMQGSLLGDDSRDY
YSFEVKEEGEVNIELDKKDEFGVTWTLHPESNINDRITYGQVDGNKVSN
KVKLRPGKYYLLVYKYSGSGNYELRVNK Collagenase II
AVDKNNATAAVQNESKRYTVSYLKTLNYYDLVDLLVKTEIENLPDLFQY (SEQ ID NO: 2)
SSDAKEFYGNKTRMSFIMDEIGRRAPQYTEIDHKGIPTLVEVVRAGFYL
GFHNKELNEINKRSFKERVIPSILAIQKNPNFKLGTEVQDKIVSATGLL
AGNETAPPEVVNNFTPIIQDCIKNMDRYALDDLKSKALFNVLAAPTYDI
TEYLRATKEKPENTPWYGKIDGFINELKKLALYGKINDNNSWIIDNGIY
HIAPLGKLHSNNKIGIETLTEVMKIYPYLSMQHLQSADQIERHYDSKDA
EGNKIPLDKFKKEGKEKYCPKTYTFDDGKVIIKAGARVEEEKVKRLYWA
SKEVNSQFFRVYGIDKPLEEGNPDDILTMVIYNSPEEYKLNSVLYGYDT
NNGGMYIEPDGTFFTYERKAEESTYTLEELFRHEYTHYLQGRYAVPGQW
GRTKLYDNDRLTWYEEGGAELFAGSTRTSGILPRKSIVSNIHNTTRNNR
YKLSDTVHSKYGASFEFYNYACMFMDYMYNKDMGILNKLNDLAKNNDVD
GYDNYIRDLSSNHALNDKYQDHMQERIDNYENLTVPFVADDYLVRHAYK
NPNEIYSEISEVAKLKDAKSEVKKSQYFSTFTLRGSYTGGASKGKLEDQ
KAMNKFIDDSLKKLDTYSWSGYKTLTAYFTNYKVDSSNRVTYDVVFHGY
LPNEGDSKNSLPYGKINGTYKGTEKEKIKFSSEGSFDPDGKIVSYEWDF
GDGNKSNEENPEHSYDKVGTYTVKLKVTDDKGESSVSTTTAEIKDLSEN
KLPVIYMHVPKSGALNQKVVFYGKGTYDPDGSIAGYQWDFGDGSDFSSE
QNPSHVYTKKGEYTVTLRVMDSSGQMSEKTMKIKITDPVYPIGTEKEPN
NSKETASGPIVPGIPVSGTIENTSDQDYFYFDVITPGEVKIDINKLGYG
GATWVVYDENNNAVSYATDDGQNLSGKFKADKPGRYYIHLYMFNGSYMP YRINIEGSVGR
[0040] In some embodiments, the collagenase can comprise a mixture
of collagenase I and collagenase II. The collagenase can comprise,
for example, a mixture of a collagenase I comprising an amino acid
sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% identical to the amino acid sequence of SEQ ID NO: 1 and a
collagenase II comprising an amino acid sequence that is 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 2. In some aspects, the
collagenase comprises a mixture of the collagenase I comprising the
amino acid sequence of SEQ ID NO: 1 and the collagenase II
comprising the amino acid sequence of SEQ ID NO: 2. Suitable
mixtures of the collagenase I and collagenase II include, for
example, a collagenase I:collagenase II mass ratio of 0.1:1,
0.25:1, 0.5:1, 0.75:1, 1:1, 1.1:1, 1.25:1, 1.5:1, 1.75:1, 2:1,
1:0.1, 1:0.25, 1:0.5; 1:0.75, 1:1.1, 1:1.25, 1:1.5, 1:1.75, or 1:2.
Each of the collagenase I and collagenase II may have a purity of
at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100% as measured by, for example, reverse phase HPLC.
[0041] In some embodiments, the collagenase can comprise
collagenase Clostridium histolyticum (CCH). "CCH," as used herein,
refers to collagenase Clostridium histolyticum containing a mixture
of collagenase I (SEQ ID NO: 1) and collagenase II (SEQ ID NO: 2)
in an approximate 1:1 mass ratio. CCH is obtained by the
fermentation of Clostridium histolyticum (also known as Hathewaya
histolytica).
[0042] Suitable disaccharides include those that: [0043] Stabilize
proteins; [0044] Protect proteins during both freezing and
dehydration; [0045] Inhibit lyophilization-induced unfolding;
[0046] Are non-reducing; and/or [0047] Tend to remain amorphous
during lyophilization.
[0048] In some embodiments, the disaccharide comprises sucrose or
trehalose. In some aspects, the formulation comprises: a
collagenase; about 30 mM to about 240 mM sucrose; about 50 mM to
about 800 mM of mannitol; and about 6 mM to about 10 mM of a
Tris-HCl. In some aspects, the formulation comprises: a
collagenase; about 30 mM to about 240 mM of trehalose; about 50 mM
to about 800 mM of mannitol; and about 6 mM to about 10 mM of a
Tris-HCl.
[0049] The disaccharide can be present at a concentration of about
30 mM to about 240 mM, about 60 mM to about 240 mM, about 90 mM to
about 240 mM, about 120 mM to about 240 mM, about 150 mM to about
240 mM, about 180 mM to about 240 mM, about 210 mM to about 240 mM,
about 30 mM to about 210 mM, about 30 mM to about 180 mM, about 30
mM to about 150 mM, about 30 mM to about 120 mM, about 30 mM to
about 90 mM, or about 30 mM to about 60 mM. The disaccharide can be
present at a concentration of about 30 mM, 60 mM, 90 mM, 120 mM,
150 mM, 180 mM, 210 mM, or 240 mM.
[0050] The mannitol can be present at a concentration of about 50
mM to about 800 mM, about 100 mM to about 800 mM, about 150 mM to
about 800 mM, about 200 mM to about 800 mM, about 250 mM to about
800 mM, about 300 mM to about 800 mM, about 350 mM to about 800 mM,
about 400 mM to about 800 mM, about 450 mM to about 800 mM, about
500 mM to about 800 mM, about 550 mM to about 800 mM, about 600 mM
to about 800 mM, about 650 mM to about 800 mM, about 700 mM to
about 800 mM, about 750 mM to about 800 mM, about 50 mM to about
750 mM, about 50 mM to about 700 mM, about 50 mM to about 650 mM,
about 50 mM to about 600 mM, about 50 mM to about 550 mM, about 50
mM to about 500 mM, about 50 mM to about 450 mM, about 50 mM to
about 400 mM, about 50 mM to about 350 mM, about 50 mM to about 300
mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, about
50 mM to about 150 mM, or about 50 mM to about 100 mM. The mannitol
can be present at a concentration of about 50 mM, 100 mM, 150 mM,
200 mM, 225 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550
mM, 600 mM, 650 mM, 700 mM, 750 mM, or 800 mM.
[0051] The pH of the formulation can be about 7.8 to about 8.8. The
pH can be about 7.8, about 7.9, about 8.0, about 8.1, about 8.2,
about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, or about
8.8.
[0052] The formulation can comprise: CCH; about 60 mM sucrose;
about 225 mM mannitol; and about 10 mM Tris-HCl, wherein the
formulation has a pH of about 8.5. The formulation can comprise:
about 0.9 mg CCH/ml; about 60 mM sucrose; about 225 mM mannitol;
and about 10 mM Tris-HCl, wherein the formulation has a pH of about
8.5.
[0053] The formulation can consist of: CCH; about 60 mM sucrose;
about 225 mM mannitol; and about 10 mM Tris-HCl, wherein the
formulation has a pH of about 8.5. The formulation can consist of:
about 0.9 mg CCH/ml; about 60 mM sucrose; about 225 mM mannitol;
and about 10 mM Tris-HCl, wherein the formulation has a pH of about
8.5.
[0054] The disclosed formulation can further comprise a surfactant.
Suitable surfactants include, for example, polysorbate 20,
polysorbate 80, or poloxamer 188. The surfactant can be present at
a concentration of about 0.01% to about 2%, about 0.05% to about
2%, about 0.1% to about 2%, about 0.15% to about 2%, about 0.2% to
about 2%, about 0.25% to about 2%, about 0.3% to about 2%, about
0.4% to about 2%, about 0.5% to about 2%, about 1% to about 2%,
about 1.5% to about 2%, about 0.01% to about 1.5%, about 0.01% to
about 1%, about 0.01% to about 0.5%, about 0.01% to about 0.1%, or
about 0.01% to about 0.05%. The surfactant can be present at a
concentration of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%,
0.2%, 0.5%, 1%, 1.5%, or 2%. In some embodiments, the formulation
further comprises polysorbate 20 at a concentration of about 0.02%.
In some embodiments, the formulation further comprises polysorbate
80 at a concentration of about 0.02%. In some embodiments, the
formulation further comprises poloxamer 80 at a concentration of
about 0.02%.
[0055] The above formulations can be liquid.
[0056] The disclosed formulations can be lyophilized under more
aggressive conditions compared to previous collagenase-containing
formulations, such as XIAFLEX.RTM.. For example, the disclosed
formulations can be lyophilized in shorter times, using higher
pressures, and/or with fewer drying steps (e.g., single temperature
drying), resulting in a lyophilized formulation that exhibits
increased stability and that maintains acceptable collagenase
activity upon reconstitution. As shown herein, the pH and mannitol
lead to the formation of a more robust formulation that can
subsequently undergo more aggressive lyophilization.
[0057] Also provided herein are lyophilized formulations. The
lyophilized formulations can be formed by the lyophilization of any
of the above formulations. In some embodiments, the lyophilized
formulations comprise, or consist of: [0058] a collagenase; [0059]
a disaccharide; [0060] mannitol; and [0061] Tris-HCl.
[0062] The lyophilized formulation can contain between about 0.2 mg
to about 50 mg of collagenase. For example, the lyophilized
formulation can contain about 0.2 mg, 0.4 mg, 0.6 mg, 0.8 mg, 0.9
mg, 1 mg, 1.2 mg, 1.4 mg, 1.6 mg, 1.8 mg, 2 mg, 2.5 mg, 3 mg, 3.5
mg, 4 mg, 4.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg,
40 mg, 45 mg, or 50 mg of collagenase.
[0063] The collagenase can comprises a collagenase I. A suitable
collagenase I includes, for example, a collagenase I comprising an
amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% identical to the amino acid sequence of SEQ ID
NO: 1. In some embodiments, the collagenase I comprises the amino
acid sequence of SEQ ID NO: 1.
[0064] The collagenase can comprise a collagenase II. A suitable
collagenase II includes, for example, a collagenase II comprising
an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ
ID NO: 2. In some embodiments, the collagenase II comprises the
amino acid sequence of SEQ ID NO: 2.
[0065] The collagenase can comprise a mixture of collagenase I and
collagenase II. The collagenase can comprise, for example, a
mixture of a collagenase I comprising an amino acid sequence that
is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
identical to the amino acid sequence of SEQ ID NO: 1 and a
collagenase II comprising an amino acid sequence that is 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 2. In some embodiments, the
collagenase comprises a mixture of the collagenase I comprising the
amino acid sequence of SEQ ID NO: 1 and the collagenase II
comprising the amino acid sequence of SEQ ID NO: 2. Suitable
mixtures of the collagenase I and collagenase II include, for
example, a collagenase I:collagenase II mass ratio of 0.1:1,
0.25:1, 0.5:1, 0.75:1, 1:1, 1.1:1, 1.25:1, 1.5:1, 1.75:1, 2:1,
1:0.1, 1:0.25, 1:0.5; 1:0.75, 1:1.1, 1:1.25, 1:1.5, 1:1.75, or 1:2.
In some embodiments, the collagenase is collagenase Clostridium
histolyticum (CCH).
[0066] Each of the collagenase I and collagenase II may have a
purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% as measured by, for example, reverse phase
HPLC.
[0067] Suitable disaccharides include, for example, sucrose or
trehalose. In some embodiments, the lyophilized formulation
comprises, or consists of, a collagenase, sucrose, mannitol, and
Tris-HCl. In some embodiments, the lyophilized formulation
comprises, or consists of, a collagenase, trehalose, mannitol, and
Tris-HCl.
[0068] The lyophilized formulations can be in a unit-dose vial,
multi-dose vial, cartridge, or syringe. The lyophilized formulation
can contain between about 0.2 mg to about 50 mg of collagenase. For
example, the lyophilized formulation can contain about 0.2 mg, 0.4
mg, 0.6 mg, 0.8 mg, 0.9 mg, 1 mg, 1.2 mg, 1.4 mg, 1.6 mg, 1.8 mg, 2
mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 10 mg, 15 mg, 20 mg,
25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg of collagenase. The
vial, cartridge, or syringe can have a volume of 2 mL to 50 mL,
such as 5 mL, 7.5 mL, 10 mL, 15 mL, 20 mL, 30 mL, 40 mL, or 50 mL.
The vial, cartridge, or syringe can contain between about 0.2 mg to
about 50 mg of collagenase. For example, the vial, cartridge, or
syringe can contain about 0.2 mg, 0.4 mg, 0.6 mg, 0.8 mg, 1 mg, 1.2
mg, 1.4 mg, 1.6 mg, 1.8 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5
mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg,
or 50 mg of collagenase. The vial, cartridge, or syringe can
contain between about 0.2 mg to about 50 mg of the lyophilized
formulation. For example, the vial, cartridge, or syringe can
contain about 0.2 mg, 0.4 mg, 0.6 mg, 0.8 mg, 1 mg, 1.2 mg, 1.4 mg,
1.6 mg, 1.8 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 10
mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg of
the lyophilized formulation.
[0069] Prior to lyophilization, the formulation can comprise, or
consist of: a collagenase; about 30 mM to about 240 mM of a
disaccharide; about 50 mM to about 800 mM of mannitol; and about 6
mM to about 10 mM of a Tris-HCl. Prior to lyophilization, the
formulation can comprise, or consist of: about 0.9 mg
collagenase/ml; about 30 mM to about 240 mM of a disaccharide;
about 50 mM to about 800 mM of mannitol; and about 6 mM to about 10
mM of a Tris-HCl. Prior to lyophilization, the formulation can
comprise, or consist of: CCH; 60 mM sucrose; 225 mM mannitol; 10 mM
Tris-HCl, and have a pH of about 8.5. In some embodiments, prior to
lyophilization, the formulation can comprise, or consist of: about
0.9 mg CCH/ml; 60 mM sucrose; 225 mM mannitol; 10 mM Tris-HCl, and
have a pH of about 8.5.
[0070] The disclosed lyophilized formulations have increased
stability compared to previous collagenase-containing formulations,
such as XIAFLEX.RTM.. For example, the disclosed lyophilized
formulations are stable at pressures above 380 .mu.bar, above 400
.mu.bar, above 450 .mu.bar, above 500 .mu.bar, above 550 .mu.bar,
above 600 .mu.bar, above 650 .mu.bar, above 700 .mu.bar, above 750
.mu.bar, above 800 .mu.bar, above 850 .mu.bar, above 900 .mu.bar,
above 950 .mu.bar, above 1000 .mu.bar, above 1500 .mu.bar, above
2000 .mu.bar, above 2500 .mu.bar, above 3000 .mu.bar, above 3500
.mu.bar, or above 4000 .mu.bar. In some embodiments, the
lyophilized formulation is stable at a pressure of about 4000
.mu.bar.
[0071] The disclosed lyophilized formulations also exhibit improved
shelf life and storage conditions compared to previous
collagenase-containing formulations. For example, the disclosed
lyophilized formulations exhibit an extended shelf life at low
temperatures, such as 2-8.degree. C., and at elevated temperatures,
such as room temperature (40.degree. C./75% relative humidity). The
disclosed lyophilized formulation can be stable at, for example:
[0072] (a) 2-8.degree. C. for at least 36 months; [0073] (b)
25.degree. C./60% relative humidity for at least 36 months; [0074]
(c) 40.degree. C./75% relative humidity for at least 6 months; or
[0075] (d) any combination of (a) to (c).
[0076] The disclosed lyophilized formulation can be formed by a
method comprising: freezing the formulation at a temperature
between about -25.degree. C. and -55.degree. C. to form a frozen
formulation; and drying the frozen formulation at a temperature
between about 25.degree. C. and about 50.degree. C. to form the
lyophilized formulation. Suitable temperatures for the freezing
step include about -25.degree. C., -30.degree. C., -35.degree. C.,
-40.degree. C., -45.degree. C., -50.degree. C., or -55.degree. C.
Suitable temperatures for the drying step include about 25.degree.
C., 30.degree. C., 35.degree. C., 40.degree. C., 45.degree. C., or
50.degree. C.
[0077] It has been shown that the lyophilized formulations can be
formed using a single temperature freezing step and a single
temperature drying step. For example, the lyophilized formulation
can be formed by a method comprising: freezing the formulation at a
single temperature between about -25.degree. C. and -55.degree. C.
to form a frozen formulation; and drying the frozen formulation at
a single temperature between about 25.degree. C. and about
50.degree. C. to form the lyophilized formulation. The single
temperature freezing step can be performed at a temperature between
about -25.degree. C. and about -55.degree. C. For example, the
single temperature freezing step can be performed at about
-25.degree. C., -30.degree. C., -35.degree. C., -40.degree.
C.-45.degree. C., -50.degree. C., or -55.degree. C. The single
temperature drying step can be performed at a temperature between
about 25.degree. C. and about 50.degree. C. For example, the single
temperature drying step can be performed at about 25.degree. C.,
30.degree. C., 35.degree. C., 40.degree. C., 45.degree. C., or
50.degree. C. When the method is performed with a single
temperature freezing step and a single temperature drying step, the
method may further comprise a "ramp up" step between the freezing
and drying to allow the lyophilizer to reach the suitable drying
temperature.
[0078] The disclosed lyophilized formulations can be formed by a
lyophilization method that is much faster than the lyophilization
method used to form other collagenase-containing lyophilized
formulations. The lyophilized formulation can be formed by a
lyophilization method that is performed for less than 72 hours. In
some embodiments, the methods can be performed for less than 30
hours. In some embodiments, the methods can be performed for less
than 18 hours. In some embodiments, the method can be performed for
about 15 hours to about 25 hours.
[0079] The disclosed lyophilized formulations can be formed by a
lyophilization method that uses a much higher pressure than the
lyophilization method used to form other collagenase-containing
lyophilized formulations. The lyophilized formulation can be formed
by a lyophilization method that is performed at a pressure of
between about 380 .mu.bar to about 4000 .mu.bar. The disclosed
methods can be performed at a pressure of between about 500 .mu.bar
to about 4000 .mu.bar, between about 750 .mu.bar to about 4000
.mu.bar, between about 1000 .mu.bar to about 4000 .mu.bar. The
disclosed methods can be performed at 380 .mu.bar, 500 .mu.bar, 750
.mu.bar, 1000 .mu.bar, 1500 .mu.bar, 2000 .mu.bar, 2500 .mu.bar,
3000 .mu.bar, 3500 .mu.bar, or 4000 .mu.bar.
[0080] The disclosed lyophilized formulations, once reconstituted,
can be used to treat or reduce collagen-mediated conditions,
including the severity of cellulite (also known as edematous
fibrosclerotic panniculopathy (EFP)), Dupuytren's contracture (DC)
with a palpable cord, or Peyronie's disease (PD) with a palpable
plaque and curvature deformity of at least 30 degrees.
[0081] Also provided herein are reconstituted formulations
comprising, or consisting of: a collagenase; a disaccharide;
mannitol; Tris-HCl; calcium chloride; and sodium chloride.
[0082] The collagenase can comprise a collagenase I. A suitable
collagenase I includes, for example, a collagenase I comprising an
amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% identical to the amino acid sequence of SEQ ID
NO: 1. In some embodiments, the collagenase I comprises the amino
acid sequence of SEQ ID NO: 1. The collagenase can comprise a
collagenase II. A suitable collagenase II includes, for example, a
collagenase II comprising an amino acid sequence that is 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 2. In some embodiments, the
collagenase II comprises the amino acid sequence of SEQ ID NO: 2.
The collagenase can comprise a mixture of collagenase I and
collagenase II. The collagenase can comprise, for example, a
mixture of a collagenase I comprising an amino acid sequence that
is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
identical to the amino acid sequence of SEQ ID NO: 1 and a
collagenase II comprising an amino acid sequence that is 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 2. In some embodiments, the
collagenase comprises a mixture of the collagenase I comprising the
amino acid sequence of SEQ ID NO: 1 and the collagenase II
comprising the amino acid sequence of SEQ ID NO: 2. Suitable
mixtures of the collagenase I and collagenase II include, for
example, a collagenase I:collagenase II mass ratio of 0.1:1,
0.25:1, 0.5:1, 0.75:1, 1:1, 1.1:1, 1.25:1, 1.5:1, 1.75:1, 2:1,
1:0.1, 1:0.25, 1:0.5; 1:0.75, 1:1.1, 1:1.25, 1:1.5, 1:1.75, or 1:2.
In some embodiments, the collagenase is collagenase Clostridium
histolyticum (CCH).
[0083] Each of the collagenase I and collagenase II may have a
purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% as measured by, for example, reverse phase
HPLC.
[0084] Suitable disaccharides include, for example, sucrose or
trehalose. In some embodiments, the reconstituted formulation
comprises, or consists of, a collagenase, sucrose, mannitol,
Tris-HCl, calcium chloride, and sodium chloride. In some
embodiments, the lyophilized formulation comprises, or consists of,
a collagenase, trehalose, mannitol, Tris-HCl, calcium chloride, and
sodium chloride.
[0085] Suitable amounts of calcium chloride and sodium chloride
include those that enable the reconstituted formulation to be
isotonic to human blood. In some embodiments, the reconstituted
formulation comprises about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%,
0.06%, 0.07%, 0.08%, 0.09%, 0.1%, or greater than 0.1% of calcium
chloride. In some embodiments, the reconstituted formulation
comprises about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%,
0.9%, 1%, or greater than 1% of sodium chloride.
[0086] The reconstituted formulation can also comprise water for
injection (WFI).
[0087] The disclosed reconstituted formulations can be used to
treat or reduce collagen-mediated conditions, including the
severity of cellulite (also known as edematous fibrosclerotic
panniculopathy (EFP)), Dupuytren's contracture (DC) with a palpable
cord, or Peyronie's disease (PD) with a palpable plaque and
curvature deformity of at least 30 degrees.
[0088] The reconstituted formulations can comprise about 0.01 mg to
about 50 mg of the collagenase in a single or divided dose. The
reconstituted formulation can comprise, for example, about 0.05 mg
to about 15 mg, about 0.10 mg to about 10 mg, about 0.15 mg to
about 5 mg, about 0.20 mg to about 3 mg, or about 0.25 mg to about
2 mg of the collagenase in a single or divided dose. The
reconstituted formulation can comprise, for example, about 0.05 mg,
about 0.10 mg, about 0.15 mg, about 0.20 mg, about 0.25 mg, about
0.30 mg, about 0.35 mg, about 0.40 mg, about 0.45 mg, about 0.50
mg, about 0.55 mg, about 0.60 mg, about 0.65 mg, about 0.70 mg,
about 0.75 mg, about 0.80 mg, about 0.85 mg, about 0.90 mg, about
0.95 mg, about 1.00 mg, 1.05 mg, about 1.10 mg, about 1.15 mg,
about 1.20 mg, about 1.25 mg, about 1.30 mg, about 1.35 mg, about
1.40 mg, about 1.45 mg, about 1.50 mg, about 1.55 mg, about 1.60
mg, about 1.65 mg, about 1.70 mg, about 1.75 mg, about 1.80 mg,
about 1.85 mg, about 1.90 mg, about 1.95 mg, about 2.00 mg, 2.05
mg, about 2.10 mg, about 2.15 mg, about 2.20 mg, about 2.25 mg,
about 2.30 mg, about 2.35 mg, about 2.40 mg, about 2.45 mg, about
2.50 mg, about 2.55 mg, about 2.60 mg, about 2.65 mg, about 2.70
mg, about 2.75 mg, about 2.80 mg, about 2.85 mg, about 2.90 mg,
about 2.95 mg, about 3.00 mg, 3.05 mg, about 3.10 mg, about 3.15
mg, about 3.20 mg, about 3.25 mg, about 3.30 mg, about 3.35 mg,
about 3.40 mg, about 3.45 mg, about 3.50 mg, about 3.55 mg, about
3.60 mg, about 3.65 mg, about 3.70 mg, about 3.75 mg, about 3.80
mg, about 3.85 mg, about 3.90 mg, about 3.95 mg, about 4.00 mg,
4.05 mg, about 4.10 mg, about 4.15 mg, about 4.20 mg, about 4.25
mg, about 4.30 mg, about 4.35 mg, about 4.40 mg, about 4.45 mg,
about 4.50 mg, about 4.55 mg, about 4.60 mg, about 4.65 mg, about
4.70 mg, about 4.75 mg, about 4.80 mg, about 4.85 mg, about 4.90
mg, about 4.95 mg, about 5.00 mg, 5.05 mg, about 5.10 mg, about
5.15 mg, about 5.20 mg, about 5.25 mg, about 5.30 mg, about 5.35
mg, about 5.40 mg, about 5.45 mg, about 5.50 mg, about 5.55 mg,
about 5.60 mg, about 5.65 mg, about 5.70 mg, about 5.75 mg, about
5.80 mg, about 5.85 mg, about 5.90 mg, about 5.95 mg, about 6.00
mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30
mg, about 35 mg, about 40 mg, about 45 mg, or about 50 mg of the
collagenase.
[0089] The reconstituted formulation can have a total volume of
about 0.1 mL to about 50 mL. For example, the reconstituted
formulation can have a total volume of about 0.1 mL, 0.2 mL, 0.3
mL, 0.4 mL, 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL, 3 mL, 3.5 mL, 4 mL,
4.5 mL, 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45
mL, or 50 mL.
[0090] Kits comprising the disclosed lyophilized formulations and a
sterile diluent are also provided. The kits can comprise: a
container comprising any of the disclosed lyophilized formulations;
and a container comprising a sterile diluent comprising calcium
chloride and sodium chloride.
[0091] Suitable containers for the lyophilized formulation and/or
the sterile diluent include, for example, a vial, cartridge, or
syringe. The vial can be a unit-dose vial or a multi-dose vial.
Suitable container sizes include, for example, 2 mL to 50 mL
containers, such as 5 mL, 7.5 mL, 10 mL, 15 mL, 20 mL, 30 mL, 40
mL, or 50 mL.
[0092] The container comprising the disclosed lyophilized
formulation can comprise between about 0.2 mg to about 50 mg
collagenase. For example, the container can comprise about 0.2 mg,
0.4 mg, 0.6 mg, 0.8 mg, 1 mg, 1.2 mg, 1.4 mg, 1.6 mg, 1.8 mg, 2 mg,
2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25
mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg collagenase. The container
comprising the disclosed lyophilized formulation can comprise
between about 0.2 mg to about 50 mg of the lyophilized formulation.
For example, the container can comprise about 0.2 mg, 0.4 mg, 0.6
mg, 0.8 mg, 1 mg, 1.2 mg, 1.4 mg, 1.6 mg, 1.8 mg, 2 mg, 2.5 mg, 3
mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg,
35 mg, 40 mg, 45 mg, or 50 mg of the lyophilized formulation.
[0093] The container comprising the sterile diluent can comprise an
amount of sterile diluent that, upon reconstitution of the
lyophilized formulation, results in a solution that is isotonic to
human blood. In some embodiments, the sterile diluent comprises
about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%,
0.09%, 0.1%, or greater than 0.1% of calcium chloride. In some
embodiments, the sterile diluent comprises about 0.1%, 0.2%, 0.3%,
0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, or greater than 1% of
sodium chloride.
[0094] The volume of the sterile diluent can be about 0.1 mL to
about 50 mL. For example, the volume of the sterile diluent can be
about 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 1 mL, 1.5 mL, 2 mL,
2.5 mL, 3 mL, 3.5 mL, 4 mL, 4.5 mL, 5 mL, 10 mL, 15 mL, 20 mL, 25
mL, 30 mL, 35 mL, 40 mL, 45 mL, or 50 mL.
[0095] Provided herein are methods of lyophilizing any of the
disclosed formulations, the methods comprising: freezing the
formulation at a temperature between about -25.degree. C. and
-55.degree. C. to form a frozen formulation; and drying the frozen
formulation at a temperature between about 25.degree. C. and about
50.degree. C. to form the lyophilized formulation.
[0096] In some embodiments, the freezing is performed at a single
temperature and the drying is performed at a single temperature. In
such embodiments, the methods may further comprise a "ramp up" step
between the freezing and drying to allow the lyophilizer to reach
the suitable drying temperature. The single temperature freezing
step can be performed at a temperature between about -25.degree. C.
and about -55.degree. C. For example, the single temperature
freezing step can be performed at about -25.degree. C., -30.degree.
C., -35.degree. C., -40.degree. C., -45.degree. C., -50.degree. C.,
or -55.degree. C. The single temperature drying step can be
performed at a temperature between about 25.degree. C. and about
50.degree. C. For example, the single temperature drying step can
be performed at about 25.degree. C., 30.degree. C., 35.degree. C.,
40.degree. C., 45.degree. C., or 50.degree. C.
[0097] The disclosed methods can be performed for less than 72
hours. In some embodiments, the methods can be performed for less
than 30 hours. In some embodiments, the methods can be performed
for less than 18 hours. In some embodiments, the method can be
performed for about 15 hours to about 25 hours.
[0098] The disclosed methods can be performed at a pressure of
between about 128 .mu.bar to about 4000 .mu.bar, between about 380
.mu.bar to about 4000 .mu.bar, between about 500 .mu.bar to about
4000 .mu.bar, between about 750 .mu.bar to about 4000 .mu.bar,
between about 1000 .mu.bar to about 4000 .mu.bar. The disclosed
methods can be performed at 380 .mu.bar, 500 .mu.bar, 750 .mu.bar,
1000 .mu.bar, 1500 .mu.bar, 2000 .mu.bar, 2500 .mu.bar, 3000
.mu.bar, 3500 .mu.bar, or 4000 .mu.bar.
EXAMPLES
[0099] The following examples are provided to further describe some
of the embodiments disclosed herein. The examples are intended to
illustrate, not to limit, the disclosed embodiments.
[0100] The XIAFLEX.RTM. formulation (CCH, 10 mM Tris/HCl pH 8.0, 60
mM sucrose) has a relatively long lyophilization cycle time of 72
hours. An objective of the below studies was to achieve a more
efficient lyophilization process with reduced cycle time and
improved yields.
[0101] The following criteria were considered during formulation
and lyophilization process optimizations:
Formulation:
[0102] pH that optimizes thermodynamic stability of protein; [0103]
Trehalose or sucrose as cryoprotectant/lyoprotectant and to
stabilize the protein; [0104] Addition of a bulking/isotonicity
agent (e.g., mannitol); and [0105] Utilization of a nonionic
surfactant to reduce protein aggregation, if necessary.
Lyophilization Process:
[0105] [0106] Inhibition of protein unfolding during freezing and
drying; [0107] Glass transition temperature of the product to
exceed the planned storage temperature; [0108] Ability to maintain
a relatively low water content; and [0109] An elegant cake
structure.
[0110] As a part of the work to identify an optimal formulation
composition for lyophilization, several different solution
formulations ("pre-lyophilization") containing various combinations
of excipients such as sucrose, trehalose, and mannitol with and
without surfactant at different levels were evaluated. In addition,
untreated and siliconized (baked on) vials were also evaluated.
Formulation Robustness Studies--Study 1
[0111] The purpose of these studies was to challenge two
formulation variants by freeze/thaw testing, sheer stress, thermal
stress, peroxide stress and hydrophobic surface contact in
comparison to the original XIAFLEX.RTM. formulation. The following
formulations were exposed to thermal/shear stress and freeze/thaw
stress test: [0112] Variant A (V.sub.A): CCH, 10 mM Tris/HCl pH
8.5, 60 mM trehalose, 225 mM mannitol [0113] Variant B (V.sub.B):
CCH, 10 mM Tris/HCl pH 8.0, 60 mM trehalose, 225 mM mannitol
[0114] Original XIAFLEX.RTM. drug substance formulation (Or): CCH,
10 mM Tris/HCl pH 8.0, 60 mM sucrose
[0115] Standard glass vials and siliconized glass vials were used
for the test to investigate the influence of hydrophobic surfaces
on the protein stability. The formulations were supplemented with
two types of surfactants (polysorbate 20 and poloxamer 188) to
examine the influence of surfactants on the stability of the
formulations during the challenge testing. To force oxidative
stress, the formulations were challenged in the presence of a
strong oxidizing agent (hydrogen peroxide). In total, a matrix of
21 variants were used for the challenge testing.
Experimental Design
[0116] CCH was formulated in three different formulation variants.
The formulation variants were exposed to thermal stress with
agitation and freeze/thaw stress to challenge the formulation
candidates. Hydrogen peroxide was added to the samples to force
oxidative stress to the proteins. Each sample was analyzed after
stress exposure by turbidity measurements to monitor the formation
of aggregates.
Treatment of Glass Vials and Stoppers
[0117] Vials were washed in a laboratory dishwasher with purified
water. Afterwards, the vials were dried and heat-treated at
300.degree. C. for 2 hours for depyrogenization/sterilization.
Stoppers were autoclaved at 2 bar and 121.degree. C. for 20 min in
sterilization bags and dried for 8 hours at 80.degree. C.
Sample Preparation
[0118] First XIAFLEX.RTM. drug substance was dialyzed against the
formulation variants A or B. The dialysis was accomplished in three
independent dialysis steps to achieve a quantitative buffer
exchange. 60 ml of the XIAFLEX.RTM. drug substance was dialyzed
against formulation variant A and 40 ml of the XIAFLEX.RTM. drug
substance was dialyzed against formulation variant B. XIAFLEX.RTM.
drug substance was transferred into two preconditioned (in dialysis
buffer) Slide-A-Lyzer.TM. cassettes (Thermo Scientific, Rockford,
USA). Filled Slide-A-Lyzer.TM.cassettes were incubated in 2000 ml
or 1000 ml of the target buffer for 2 hours before a first buffer
change (2000 ml/1000 ml) was performed. After dialysis for two
additional hours, the buffer was changed a second time (2000 ml for
both formulation variants) to finalize the dialysis overnight. The
protein sample was removed from the Slide-A-Lyzer.TM. cassettes and
the concentration was adjusted to 1 mg/ml by dilution.
Addition of Detergents and Hydrogen Peroxide
[0119] After dialysis, the samples were supplemented with
polysorbate 20, poloxamer 188, hydrogen peroxide, and combinations
thereof by adding stock solutions (10% w/w polysorbate 20, 10% w/w
poloxamer 188, 30% w/w H.sub.2O.sub.2). Variants of the original
XIAFLEX.RTM. formulation were prepared by adding the detergents or
hydrogen peroxide directly to the bulk solution. The composition of
each variant is shown in Table 2:
TABLE-US-00002 TABLE 2 Formulation variants for the challenge
testing +PS20 +P188 +H.sub.2O.sub.2 # Sample code container 0.02%
(0.1%) (0.1%) 1 V.sub.A1 glass type 1 2 V.sub.A2 glass type 1 X 3
V.sub.A3 glass type 1 X 4 V.sub.A1s silicon, glass 5 V.sub.A2s
silicon, glass X 6 V.sub.A3s silicon, glass X 7 V.sub.A4s silicon,
glass X 8 V.sub.A5s silicon, glass X X 9 V.sub.A6s silicon, glass X
X 10 V.sub.B1s silicon, glass 11 V.sub.B2s silicon, glass X 12
V.sub.B3s silicon, glass X 13 V.sub.B4s silicon, glass X 14
V.sub.B5s silicon, glass X X 15 V.sub.B6s silicon, glass X X 16
Or1s silicon, glass 17 Or2s silicon, glass X 18 Or3s silicon, glass
X 19 Or4s silicon, glass X 20 Or5s silicon, glass X X 21 Or6s
silicon, glass X X
[0120] Each formulation was sterile filtrated under laminar flow
and exposed to thermal/agitation and freeze/thaw stress.
Freeze/Thaw Testing
[0121] The freeze/thaw stability of the formulations was tested by
running 3 freeze/thaw cycles in total. Siliconized 6R glass vials
or 2R standard glass vials were filled with 1.0 ml of each
formulation. 3 vials were prepared for each variant. Liquid samples
were frozen from 25.degree. C. to -30.degree. C. within 55 min at a
controlled freezing rate and warmed up again to room temperature
within 55 min at a controlled heating rate. For an adequate
temperature control, the samples were loaded into a pilot freeze
dryer. After each freeze/thaw cycle the turbidity of the samples
was determined. For the turbidity measurement, 1 ml of each sample
was filled in single-use turbidity cuvettes and analyzed. After
analyzing, the liquid was returned to the glass vial and the
experiment was continued.
Thermal Stress Test
[0122] Test samples of all formulations were stressed at 40.degree.
C. for 4 days under agitation (200 rpm) in 2R vials (1 ml fill
volume). Turbidity of the stressed samples was analyzed
afterwards.
Turbidity Measurement
[0123] The turbidity of the samples was determined using a 2100AN
turbidity meter (Hach Lange, Dusseldorf, Germany) according to the
European Pharmacopeia. The system was calibrated as follows: [0124]
Hydrazine sulphate solution: Dissolved 1.0 g of hydrazine sulphate
in purified water and diluted to 100.0 ml with the same solvent.
Allowed to stand for 4-6 h. [0125] Hexamethylene tetramine
solution: Dissolved 2.5 g of hexamethylene tetramine in 25.0 ml
purified water in a 100 ml glass-toppered volumetric flask. [0126]
Primary opalescent suspension: Added 25.0 ml hydrazine sulphate
solution to the hexamethylene tetramine solution in the volumetric
flask. Mixed and allowed to stand for 24 h. [0127] Standard of
opalescence: Diluted 15.0 ml of the primary opalescent suspension
to 1000.0 ml with purified water. This suspension was freshly
prepared (stored for 24 h at most). [0128] Reference suspensions:
Prepared the reference suspensions according to Table 3.
TABLE-US-00003 [0128] TABLE 3 Preparation of the reference solution
for the calibration of the turbidity meter. I II II IV Standard of
opalescence 5.00 mL 10.0 mL 30.0 mL 50.0 mL Purified water 95.0 mL
90.0 mL 70.0 mL 50.0 mL
Results and Discussion
[0129] Table 4 displays the result of the freeze/thaw testing. None
of the variants showed an increase in turbidity with increasing
number of freeze/thaw cycles. The enzymes seemed to be stable
against freeze/thaw stress. The addition of surfactants or hydrogen
peroxide showed no influence on the turbidity of the samples.
[0130] Table 5 shows the results of the thermal stress at
40.degree. C. and agitation at 200 rpm. Variants containing
hydrogen peroxide showed a clear increase of turbidity (as shown by
the increased nephelometric turbidity units (NTU)) after thermal
stress. In variants V.sub.A and variants V.sub.B (mannitol is
present in both formulations) the resulting turbidity was
remarkably lower (decreased NTU) than in the original XIAFLEX.RTM.
formulation variant ("Or"--without mannitol). The beneficial effect
of mannitol is believed to be caused by its radical scavenger
properties.
TABLE-US-00004 TABLE 4 Results for the freeze/thaw challenge test +
PS20 + P188 + H.sub.2O.sub.2 Turbidity [NTU] # Sample code
container 0.02% (0.1%) (0.1%) T0 1x F/T 2x F/T 3x F/T 1 V.sub.A1
glass type 1 3.2 3.2 2.8 2.5 2 V.sub.A2 glass type 1 X 3.3 2.7 2.3
2.7 3 V.sub.A3 glass type 1 X 2.6 2.2 2.3 2.9 4 V.sub.A1s silicon,
glass 3.2 4.2 2.9 1.8 5 V.sub.A2s silicon, glass X 3.3 2.7 2.5 2.3
6 V.sub.A3s silicon, glass X 2.6 2.1 2.1 2.5 7 V.sub.A4s silicon,
glass X 2.9 3.5 2.6 3.0 8 V.sub.A5s silicon, glass X X 2.0 2.3 2.2
2.6 9 V.sub.A6s silicon, glass X X 2.6 2.5 2.5 2.5 10 V.sub.B1s
silicon, glass 1.9 2.5 2.4 2.7 11 V.sub.B2s silicon, glass X 2.3
2.2 2.6 2.8 12 V.sub.B3s silicon, glass X 3.0 2.5 2.6 3.1 13
V.sub.B4s silicon, glass X 2.0 2.0 2.6 3.1 14 V.sub.B5s silicon,
glass X X 2.2 1.9 2.1 2.5 15 V.sub.B6s silicon, glass X X 2.1 2.3
2.9 2.5 16 Or1s silicon, glass 2.3 1.9 1.9 3.0 17 Or2s silicon,
glass X 2.7 2.4 2.5 2.9 18 Or3s silicon, glass X 2.2 2.9 2.4 2.7 19
Or4s silicon, glass X 2.0 2.8 2.7 3.0 20 Or5s silicon, glass X X
2.5 2.8 2.2 3.0 21 Or6s silicon, glass X X 1.8 3.2 2.3 2.4
TABLE-US-00005 TABLE 5 Results of the thermal challenge test
Turbidity [NTU] Sam- 4 days ple + PS20 + P188 + H.sub.2O.sub.2
40.degree. C. + # code container 0.02% (0.1%) (0.1%) T0 200 rpm 1
V.sub.A1 glass type 1 3.2 3.1 2 V.sub.A2 glass type 1 X 3.3 2.5 3
V.sub.A3 glass type 1 X 2.6 2.4 4 V.sub.A1s silicon, glass 3.2 2.6
5 V.sub.A2s silicon, glass X 3.3 2.5 6 V.sub.A3s silicon, glass X
2.6 2.6 7 V.sub.A4s silicon, glass X 2.9 7.0 8 V.sub.A5s silicon,
glass X X 2.0 6.2 9 V.sub.A6s silicon, glass X X 2.6 6.3 10
V.sub.B1s silicon, glass 1.9 2.7 11 V.sub.B2s silicon, glass X 2.3
2.3 12 V.sub.B3s silicon, glass X 3.0 1.9 13 V.sub.B4s silicon,
glass X 2.0 24.2 14 V.sub.B5s silicon, glass X X 2.2 24.5 15
V.sub.B6s silicon, glass X X 2.1 25.8 16 Or1s silicon, glass 2.3
2.1 17 Or2s silicon, glass X 2.7 2.5 18 Or3s silicon, glass X 2.2
2.9 19 Or4s silicon, glass X 2.0 44.8 20 Or5s silicon, glass X X
2.5 46.5 21 Or6s silicon, glass X X 1.8 54.7
Conclusions
[0131] The increase of turbidity differed significantly between
Variant V.sub.A and V.sub.B. It is believed that higher repulsive
interactions between the molecules at pH 8.5 compared to pH 8.0
reduced the tendency for aggregate formation caused by the
oxidative stress.
[0132] The addition of surfactants showed neither a positive nor a
negative effect on the turbidity of the samples. The hydrophobic
surface of siliconized glass vials had no impact on the turbidity
of the samples after thermal stress.
[0133] The samples were analyzed by turbidity measurements to
monitor the formation of aggregates. The turbidity of the samples
did not increase by freeze/thaw cycling. The formulations seemed to
be stable against the formation of aggregates under freeze/thaw
stress (up to 3 cycles). Hydrophobic surface contact as well as
addition of hydrogen peroxide had no influence on the turbidity of
the samples either with or without surfactants during freeze/thaw
stress. Under thermal stress, a clear increase in turbidity (as
measured by NTU) under forced oxidative stress was observed
(addition of hydrogen peroxide). Variants containing mannitol and
having higher repulsive interactions between the molecules
exhibited a less pronounced increase in turbidity under forced
oxidative stress. The stability of the formulations against
oxidative stress was higher in the order from variant A>variant
B>original XIAFLEX.RTM. formulation. The presence of detergents
under forced oxidative stress had no influence on the turbidity of
the samples. Without oxidative stress, all samples remained clear
during thermal/agitation stress. Hydrophobic surface contact had no
influence on the turbidity of the samples either with or without
detergents during thermal/agitation stress.
Formulation Robustness Studies--Study 2
[0134] Twelve (12) formulation variants with different pH values
and mannitol concentrations were investigated by CG-MALS and
nanoDSC. Each variant was additionally exposed to thermal stress
with agitation and to freeze/thaw stress testing. To force
oxidative stress, each variant was supplemented with 0.1%
H.sub.2O.sub.2. The objectives of this study were to: [0135]
Characterize the molecular interactions of the two protein species
collagenase I and collagenase II in various formulation variants by
means of CG-MALS and nano DSC; and [0136] Test the stability of
XIAFLEX.RTM. and the additional formulation variants against
freeze/thaw and thermal stress.
[0137] The following CCH formulations were prepared and tested:
TABLE-US-00006 TABLE 6 Formulations Tris Mannitol Sucrose # pH mM
mM mM 1 7.5 10 0 60 2 8.0 10 0 60 3 8.5 10 0 60 4 7.5 10 112.5 60 5
8.0 10 112.5 60 6 8.5 10 112.5 60 7 7.5 10 225 60 8 8.0 10 225 60 9
8.5 10 225 60 10 7.5 10 337.5 60 11 8.0 10 337.5 60 12 8.5 10 337.5
60
[0138] Collagenase I and collagenase II was dialyzed against the
corresponding formulation buffer. After dialysis, the sample
solutions were further diluted. Colloidal stability and
thermodynamic stability of collagenase I, collagenase II, and the
mixture was determined by CG-MALS and nanoDSC, respectively. The
stability of the proteins in each formulation variant was
additionally investigated in a stress test (freeze/thaw and
thermal/shear stress) study. To force oxidative stress during the
stability test, each formulation variant was examined in the
presence and absence of 0.1% H.sub.2O.sub.2 (sub-variant without
hydrogen peroxide are marked with a; sub-variant with hydrogen
peroxide are marked with b).
Dialysis
[0139] The dialysis of collagenase I and collagenase II was
accomplished in three independent dialysis steps to achieve a
quantitative buffer exchange. 14 ml of the collagenase I
intermediate sample and 14 ml of the collagenase II intermediate
sample was transferred into two preconditioned (in dialysis buffer)
Slide-A-Lyzer.TM. cassettes (Thermo Scientific, Rockford, USA).
Filled Slide-A-Lyzer.TM. cassettes were incubated in 1000 ml of the
target buffer for 2 hours before a first buffer change (1000 ml)
was performed. After dialysis for two further hours the buffer was
changed a second time (1000 ml) to finalize the dialysis overnight.
The protein sample was removed from the Slide-A-Lyzer.TM. cassettes
and processed further. Each dialysis step represents a 1/35 fold
buffer exchange leading to a calculated buffer exchange factor of
approx. 2.times.10.sup.5 in total.
Nano Differential Scanning calorimetry
[0140] Differential Scanning calorimetry (DSC) is a technique used
to assess the stability of a protein in its native form by
measuring the heat change associated with the molecule's thermal
denaturation when heated at a constant rate. A protein in solution
is in equilibrium between its native (folded) and denatured
(unfolded) conformations. Native proteins respond to heating by
unfolding (thermal denaturation) at a characteristic temperature (T
onset). The more intrinsically stable the biopolymer, the higher
the onset temperature of the unfolding transition. DSC measures the
enthalpy of unfolding that results from heat-induced denaturation
and can elucidate factors that contribute to the folding and
stability of native biomolecules. These include hydrophobic
interactions, hydrogen bonding, conformational entropy, and the
physical environment. The following experimental method was
performed: [0141] Mode: Scanning [0142] Temperature parameters:
Lower: 20.degree. C. [0143] Upper: 100.degree. C. [0144] Rate:
1.degree. C./min heating and cooling [0145] Equilibration: 600 s
[0146] Pressure parameters: Manual, 3.0 atm [0147] Data interval: 1
s
[0148] A buffer scan was conducted prior to each sample run to
generate a baseline.
Sample Preparation
[0149] Dialyzed samples were diluted with the corresponding
formulation buffer to 1 mg/ml. The corresponding dialysis buffer
was used as buffer scan and buffer reference.
CG-MALS
[0150] Interactions between protein molecules in solution were
characterized by analyzing changes in their light scattering
behavior at different concentrations by calculating the second
virial coefficient (A2). A2 is a measure of protein-protein
interactions in solution. Negative A2 values indicate attractive
protein-protein interactions while positive values indicate
repulsive protein interactions. A protein solution is "colloidally
unstable" when the A2 values are negative. A higher A2 value
indicates greater repulsion, which is indicative of less protein
interaction and less potential for protein aggregation and better
stability. A2 values for various formulations were determined by
CG-MALS and used as measures of non-specific protein-protein
interactions
[0151] The apparent weight average molecular weight (Mw.sub.app) is
determined for each step in the concentration gradient by analyzing
the light scattering and concentration data. Significant
interactions between macromolecules manifest as changes in
Mw.sub.app vs. concentration. A.sub.2 calculation was conducted via
Zimm plot analysis by an extrapolation to 0 mg/ml concentration
according to formula I below:
K * .times. c R .function. ( .theta. , c ) = 1 M w .times. P
.function. ( .theta. ) + A 2 .times. c ( Formula .times. I )
##EQU00001##
wherein: [0152] R(.theta.,c): excess Rayleigh ratio of the solution
as a function of scattering angle .theta. and concentration c. It
is directly proportional to the intensity of the excess light
scattered by the solute and the light scattered by the pure
solvent. [0153] Mw: Weight average molecular weight. [0154] A2: 2nd
virial coefficient [0155] c: Concentration [0156] K*: Optical
constant (4p.sup.2(dn/dc).sup.2n.sub.0.sup.2/N.sub.al.sub.o.sup.4)
[0157] P(.theta.): describes the angular dependence of the
scattered light, and can be related to the rms Radius.
Sample Preparation
[0158] Dialyzed samples were used undiluted for CG-MALS
measurements. The corresponding dialysis buffer was used as diluent
for the CG-MALS experiment. The samples and the buffer were passed
over a 0.1 .mu.m filter. Before loading the samples into the
CG-MALS system the exact concentration of the samples was
determined by UV-absorption measurement. The concentration was used
to calculate the concentrations of each gradient step.
Sample Measurement
[0159] A Calypso II CG-MALS system was used to supply the MALS
detector with the concentration gradient of the analyte. The
samples were loaded on syringe pump 1 and syringe pump 2 of the
system and the dialysis buffer on syringe pump 3. The CG-MALS
measurement consisted of three steps. In the first step, a
concentration gradient of sample 1 ranging from 10% to 100% was
applied to determine the self-virial coefficient of sample 1 in the
formulation. The second step consisted of a cross-over gradient, in
which the concentration of sample 1 was reduced from 90% to 10%
while the concentration of sample 2 was increased from 10% to 90%.
This step was conducted to determine the cross-virial coefficient.
In the third step, a concentration gradient of sample 2 ranging
from 100% to 10% was applied to determine the self-virial
coefficient of sample 2 in the formulation. At each gradient step
0.7 ml of sample was injected into the MALS detector. The resulted
light scattering signal was recorded over a time period of 180 sec.
Multi component Zimm plot analysis with fixed molecular weight was
performed with Calypso software version 2.1.5.
UV Measurement
[0160] The concentration of collagenase I and collagenase II in
solution was determined using an 8452A UV spectrometer (Agilent
Technologies, Santa Clara, USA). Samples were measured at a
concentration of about 3 mg/ml using plastic cuvettes with an
optical path thickness of 0.2 cm. The concentration was calculated
according to Lambert-Beer's law using an extinction coefficient of
1.52 ml/(mg*cm) for collagenase I and 1.48 ml/(mg*cm) for
collagenase II, respectively.
Freeze/Thaw Testing
[0161] The freeze/thaw stability of the formulations was tested by
running 3 freeze/thaw cycles in total. Dialyzed collagenase I and
dialyzed collagenase II was mixed to exhibit a solution containing
0.5 mg/ml collagenase I and 0.5 mg/ml collagenase II.
[0162] 2R glass vials were filled with 1.0 ml of each formulation.
3 vials were prepared for each variant. Liquid samples were frozen
from 25.degree. C. to -30.degree. C. within 55 min at a controlled
freezing rate and warmed up again to room temperature within 55 min
at a controlled heating rate. For an adequate temperature control
the samples were loaded into a pilot freeze dryer. After each
freeze/thaw cycle, the turbidity of the samples was determined. For
the turbidity measurement, 1 ml of each sample was filled in
single-use turbidity cuvettes and analyzed. After analyzing, the
liquid was returned to the glass vial and the experiment was
continued.
Thermal Stress Test
[0163] Dialyzed collagenase I and dialyzed collagenase II was mixed
to exhibit a solution containing 0.5 mg/ml collagenase I and 0.5
mg/ml collagenase II. Test samples of all formulations were
stressed at 40.degree. C. for 4 days under agitation (200 rpm) in
2R vials (1 ml fill volume). Turbidity of the stressed samples was
analyzed afterwards.
Turbidity Measurement
[0164] The turbidity of the samples was determined as described
above.
Results and Discussion
[0165] Table 7 shows the results of the CG-MALS and nanoDSC
measurements of each formulation variant.
[0166] Colloidal stability--The pH value of the formulation showed
a strong impact on the colloidal stability of collagenase I,
collagenase II, and its mixture. Strongest repulsive interactions
were observed at pH 8.5. At pH 7.5, the repulsive interactions were
lower. At pH 7.5, collagenase I showed attractive interactions when
mannitol was not present in the formulation and the repulsive
interactions become stronger with higher concentrations of
mannitol. At more basic pH values, the effect of mannitol became
subordinated.
[0167] Thermodynamic stability--collagenase I showed a slight pH
dependency of the onset temperature of unfolding. Higher T onset
values were observed at pH 7.5 compared to pH 8.5. Mannitol showed
slight and concentration dependent positive effect on the
thermodynamic stability of collagenase I. The thermodynamic
stability of collagenase II was independent from the investigated
pH values. The presence of mannitol seemed to be beneficial for the
thermodynamic stability of collagenase II.
TABLE-US-00007 TABLE 7 Results of the CG-MALS and nanoDSC analysis
of the additional formulations A2 10.sup.-4 mol*m /g.sup.2 Nano DSC
AUX-1 Tonset [.degree. C.] Tm [.degree. C.] Tris Mannitol Sucrose
AUX- <> AUX- AUX- AUX- AUX- # pH mM mM mM AUX-1 II AUX-II 1
II 1 II 1 7.5 10 0 60 -0.32 0.51 0.19 47.1 51.2 51.3 54.8 2 8.0 10
0 60 0.03 0.91 0.65 46.9 51.7 51.8 55.1 3 8.5 10 0 60 1.93 1.52
1.68 46.4 51.0 51.7 55.0 4 7.5 10 112.5 60 0.24 0.32 0.51 49.1 51.3
52.6 55.6 5 8.0 10 112.5 60 0.99 1.25 0.96 47.2 51.6 51.9 55.4 6
8.5 10 112.5 60 2.33 1.95 1.75 47.0 51.9 52.0 55.6 7 7.5 10 225 60
0.76 1.69 0.94 49.5 53.0 53.2 56.2 8 8.0 10 225 60 1.93 1.77 1.24
48.8 53.2 53.2 56.2 9 8.5 10 225 60 2.22 1.97 1.43 48.8 53.0 53.0
56.0 10 7.5 10 337.5 60 1.58 1.79 0.91 49.9 52.6 53.4 56.4 11 8.0
10 337.5 60 2.04 1.62 0.82 48.8 53.9 53.1 56.3 12 8.5 10 337.5 60
1.69 2.03 1.33 49.0 53.9 53.3 56.3
[0168] Table 8 shows the turbidity values of the samples during the
subsequent freeze thaw cycles.
TABLE-US-00008 TABLE 8 Turbidity values of the formulations with
subsequent freeze/thaw cycles. Tris Man- nitol Su- crose
H.sub.2O.sub.2 Turbidity [NTU] # pH mM mM mM % T0 1x F/T 2x F/T 3x
F/T 1a 7.5 10 0 60 0 2.1 3.0 2.8 2.6 2a 8.0 10 0 60 0 2.2 2.2 3.5
2.2 3a 8.5 10 0 60 0 2.8 1.8 3.8 2.1 4a 7.5 10 112.5 60 0 2.4 2.5
3.4 2.1 5a 8.0 10 112.5 60 0 3.0 2.6 3.0 2.3 6a 8.5 10 112.5 60 0
2.6 1.9 3.5 2.5 7a 7.5 10 225 60 0 1.8 2.8 3.6 2.5 8a 8.0 10 225 60
0 2.5 3.2 2.2 2.4 9a 8.5 10 225 60 0 2.7 3.0 3.0 2.5 10a 7.5 10
337.5 60 0 2.8 2.8 3.0 2.3 11a 8.0 10 337.5 60 0 2.6 3.0 3.4 3.3
12a 8.5 10 337.5 60 0 2.5 2.9 3.0 2.2 1b 7.5 10 0 60 0.1 1.9 2.2
3.3 2.6 2b 8.0 10 0 60 0.1 2.6 2.6 2.1 3.1 3b 8.5 10 0 60 0.1 2.6
1.8 2.1 1.8 4b 7.5 10 112.5 60 0.1 2.8 2.8 3.6 2.9 5b 8.0 10 112.5
60 0.1 1.9 2.8 3.3 2.3 6b 8.5 10 112.5 60 0.1 2.2 2.2 2.1 2.6 7b
7.5 10 225 60 0.1 2.3 2.6 2.1 2.9 8b 8.0 10 225 60 0.1 2.6 2.2 3.7
2.4 9b 8.5 10 225 60 0.1 1.9 2.9 2.5 3.6 10b 7.5 10 337.5 60 0.1
2.2 2.7 1.9 3.1 11b 8.0 10 337.5 60 0.1 2.9 2.9 3.5 2.7 12b 8.5 10
337.5 60 0.1 2.0 1.8 2.2 1.8
[0169] None of the variants showed an increase of turbidity after
the freeze/thaw testing. The presence of hydrogen peroxide was well
tolerated in each formulation under freeze/thaw stress.
[0170] Table 9 shows the turbidity values of each formulation
before and after thermal stress.
TABLE-US-00009 TABLE 9 Turbidity values before and after thermal
stress. Turbidity [NTU] 4 days/ Tris Mannitol Sucrose
H.sub.2O.sub.2 200 rpm/ # pH mM mM mM % T0 40.degree. C. 1a 7.5 10
0 60 0 2.1 2.3 2a 8.0 10 0 60 0 2.2 2.3 3a 8.5 10 0 60 0 2.8 2.4 4a
7.5 10 112.5 60 0 2.4 2.9 5a 8.0 10 112.5 60 0 3.0 2.4 6a 8.5 10
112.5 60 0 2.6 2.7 7a 7.5 10 225 60 0 1.8 2.8 8a 8.0 10 225 60 0
2.5 2.7 9a 8.5 10 225 60 0 2.7 2.6 10a 7.5 10 337.5 60 0 2.8 2.8
11a 8.0 10 337.5 60 0 2.6 2.7 12a 8.5 10 337.5 60 0 2.5 2.7 1b 7.5
10 0 60 0.1 1.9 388.0 2b 8.0 10 0 60 0.1 2.6 34.0 3b 8.5 10 0 60
0.1 2.6 10.4 4b 7.5 10 112.5 60 0.1 2.8 252.0 5b 8.0 10 112.5 60
0.1 1.9 22.7 6b 8.5 10 112.5 60 0.1 2.2 7.5 7b 7.5 10 225 60 0.1
2.3 336.0 8b 8.0 10 225 60 0.1 2.6 19.6 9b 8.5 10 225 60 0.1 1.9
7.0 10b 7.5 10 337.5 60 0.1 2.2 196.0 11b 8.0 10 337.5 60 0.1 2.9
20.7 12b 8.5 10 337.5 60 0.1 2.0 6.6
[0171] In the absence of oxygen radicals, the turbidity remained
constant in all investigated formulation variants. In the presence
of hydrogen peroxide, the turbidity of the samples increased
strongly. This indicated that oxidative stress induced the
formation of aggregates. More basic pH of 8.5 stabilized the
proteins against aggregation induced by oxidative stress. Mannitol
showed a further beneficial effect on the turbidity of the samples
after thermal stress in the presence of hydrogen peroxide. Mannitol
seemed to act as a radical scavenger improving the stability of the
proteins against oxidative stress.
Formulation Robustness Studies--Study 3
[0172] The following study utilized sucrose and trehalose at
concentrations of 60 mM and mannitol at 225 mM. Several
concentrations of surfactant were evaluated and the concentration
used for a given sample is noted in the data tables below. The
effect of pH on various formulations pre-lyophilization was
evaluated as a part of the formulation optimization.
[0173] The robustness of the proposed formulations as solutions was
investigated by subjecting them to the following stresses: [0174]
Three freeze-thaw cycles (-30.degree. C. to 25.degree. C.); and/or
[0175] Shaking at 200 rpm at 40.degree. C. for four days
[0176] The stressed samples were then tested for turbidity and the
key product quality attributes were assessed using the following
methods: [0177] Protein concentration by UV.sub.A280 [0178] Mass
and Ratio of collagenase I and collagenase II by Reverse
Phase--High Performance Liquid Chromatography (discussed herein)
[0179] Soluble Rat Tail Collagen (SRC) Enzyme Activity Assay Method
(collagenase I; discussed herein) [0180] Glycyl-L-prolyl-L-alanine
(GPA) Enzyme Activity Assay Method (collagenase II; discussed
herein)
Results
[0181] Turbidity: Data generated for freeze/thaw cycling are
presented in Table 10.
TABLE-US-00010 TABLE 10 Effect of Freeze-Thaw Cycling on Product
Turbidity Turbidity [NTU] Sample Description Freeze-Thaw Bulking
Cycle Stabilizer Agent Surfactant pH Container T.sub.0 1 2 3
Trehalose Mannitol None 8.5 Glass type 1 3.2 3.2 2.8 2.5 Trehalose
Mannitol Polysorbate 8.5 Glass type1 3.3 2.7 2.3 2.7 20.sup.a
Trehalose Mannitol None 8.5 Siliconized 3.2 4.2 2.9 1.8 glass
Trehalose Mannitol Polysorbate 8.5 Siliconized 3.3 2.7 2.5 2.3
20.sup.a glass Trehalose Mannitol None 8.0 Siliconized 1.9 2.5 2.4
2.7 glass Trehalose Mannitol Polysorbate 8.0 Siliconized 2.3 2.2
2.6 2.8 20.sup.a glass Sucrose None None 8.0 Siliconized 2.3 1.9
1.9 3.0 (control) glass Sucrose None Polysorbate 8.0 Siliconized
2.7 2.4 2.5 2.9 20.sup.a glass .sup.a0.02%
[0182] These results demonstrated that multiple freeze-thaw cycles
do not affect protein aggregation as indicated by the consistent
turbidity values for any of the formulation/container combinations
tested. The results indicated that the tested excipients were
appropriate for further evaluation.
[0183] Data generated for shaking/exposure to high temperatures are
presented in Table 11.
TABLE-US-00011 TABLE 11 Effect of Heat/Shaking on Product Turbidity
Sample Description Turbidity [NTU] Bulking After Stabilizer Agent
Surfactant pH Container T.sub.0 Mixing.sup.a Trehalose Mannitol
None 8.5 Glass type 1 3.2 3.1 Trehalose Mannitol Polysorbate 8.5
Glass type1 3.3 2.5 20.sup.b Trehalose Mannitol None 8.5
Siliconized glass 3.2 2.6 Trehalose Mannitol Polysorbate 8.5
Siliconized 3.3 2.5 20.sup.b glass Trehalose Mannitol None 8.0
Siliconized glass 1.9 2.7 Trehalose Mannitol Polysorbate 8.0
Siliconized 2.3 2.3 20.sup.b glass Sucrose None None 8.0
Siliconized 2.3 2.1 (Control) glass Sucrose None Polysorbate 8.0
Siliconized 2.7 2.5 20.sup.a glass .sup.aAfter mixing at 40.degree.
C. and 200 rpm for 4 days .sup.b0.02%
[0184] These results demonstrated that heat/shaking did not affect
protein aggregation as indicated by the consistent turbidity values
for any of the formulation/container combinations tested. The
results indicate that the tested excipients were appropriate for
further evaluation.
[0185] A second study was performed to evaluate the effect(s) of
various concentrations of polysorbate 20 and higher pH (8.8) on
product turbidity under stressed conditions. The results for this
study are presented in Table 12.
TABLE-US-00012 TABLE 12 Effect of Surfactant Concentration on
Product Turbidity Under Stress Conditions Sample Description
Turbidity [NTU] Bulking Freeze Thaw Cycle After Stabilizer Agent
Surfactant pH T.sub.0 1 2 3 Shaking.sup.a Trehalose Mannitol
Polysorbate 8.5 2.1 2.3 2.5 1.9 2.0 20.sup.c Trehalose Mannitol
None 8.8 2.2 2.6 2.3 2.6 2.7 None Mannitol Polysorbate 8.5 2.3 1.9
1.9 2.6 2.0 20.sup.b Trehalose None Polysorbate 8.5 1.8 2.3 2.0 2.5
1.8 20.sup.b Trehalose None None 8.5 2.4 2.2 2.0 2.7 2.4
.sup.aAfter shaking at 40.degree. C. and 200 rpm for 4 days
.sup.b0.02% .sup.c0.1%
[0186] These data demonstrated that the surfactant levels studied
(0.02% and 0.1%) and pH values of 8.5 and 8.8 have no negative
impact on protein aggregation as indicated by consistent turbidity
values upon freeze/thaw cycling or upon heat/shaking. The results
indicated that thermodynamically stable formulations and use of
polysorbate 20 as a surfactant in the concentration range studied
can be considered. Further, the data also indicated that the use of
trehalose or mannitol alone or in combination has no negative
impact on the protein aggregation as measured by sample
turbidity.
[0187] Key Product Quality Attribute Indicators--Data generated to
assess the impact of freeze/thaw cycling for 3 cycles from
-30.degree. C. to 25.degree. C. and of thermal stress/shaking of
40.degree. C. and 200 rpm for 4 days are presented in Tables 13 and
14, respectively.
TABLE-US-00013 TABLE 13 Effect of Freeze/Thaw Cycles on Key Product
Quality Attributes Results Sample Description Mass Ratio Bulking UV
A.sub.280 Coll. II Coll. I SRC GPA Stabilizer Agent Surfactant pH
Vial (mg/mL) (.mu.g/mL) (.mu.g/mL) Ratio (Coll. I).sup.a (Coll.
II).sup.a Trehalose Mannitol None 8.5 Glass type1 1.0 483 488 1.0
1.04 1.05 Trehalose Mannitol Polysorbate 20 8.5 Glass type1 1.0 508
518 1.0 1.12 1.08 Trehalose Mannitol None 8.5 Siliconized 1.0 511
501 1.0 0.99 1.06 Trehalose Mannitol Polysorbate 20 8.5 Siliconized
1.0 504 516 1.0 1.03 1.09 Trehalose Mannitol None 8.0 Siliconized
1.0 525 510 1.0 1.03 1.02 Trehalose Mannitol Polysorbate 20 8.0
Siliconized 1.0 531 541 1.0 1.01 1.09 Sucrose None None 8.0
Siliconized 1.0 503 487 1.0 1.04 1.01 Sucrose None Polysorbate 20
8.0 Siliconized 1.0 513 524 1.0 1.06 1.08 Trehalose Mannitol
Polysorbate 20.sup.b 8.5 Siliconized 1.0 531 482 0.9 1.03 1.04
Trehalose Mannitol None 8.8 Siliconized 0.9 509 474 0.9 1.03 0.97
None Mannitol Polysorbate 20.sup.c 8.5 Siliconized 1.0 539 487 0.9
1.03 1.13 Trehalose None Polysorbate 20.sup.c 8.5 Siliconized 1.0
558 507 0.9 1.05 1.09 Trehalose None None 8.5 Siliconized 1.0 537
498 0.9 1.06 1.04 .sup.aRelative to Reference .sup.b0.1%
.sup.c0.02% Coll. I = collagenase I; Coll. II = collagenase II
TABLE-US-00014 TABLE 14 Effect of 40.degree. C. Temperature and
Shaking at 200 rpm (4 Days) on Key Product Quality Attributes
Result Sample Description Mass Ratio Bulking UV A.sub.280 Coll. II
Coll. I SRC GPA Stabilizer Agent Surfactant pH Vial (mg/mL)
(.mu.g/mL) (.mu.g/mL) Ratio (Coll. I).sup.a (Coll. II).sup.a
Trehalose Mannitol None 8.5 Glass type1 1.0 474 474 1.0 0.96 0.95
Trehalose Mannitol Polysorbate 20 8.5 Glass type1 1.0 458 475 1.0
0.95 0.99 Trehalose Mannitol None 8.5 Siliconized 1.0 439 459 1.0
0.95 0.94 Trehalose Mannitol Polysorbate 20 8.5 Siliconized 1.0 431
468 0.9 0.94 0.97 Trehalose Mannitol None 8.0 Siliconized 1.0 462
475 1.0 0.95 0.97 Trehalose Mannitol Polysorbate 20 8.0 Siliconized
1.0 444 485 0.9 0.94 0.99 Sucrose None None 8.0 Siliconized 1.0 421
454 0.9 0.85 1.00 Sucrose None Polysorbate 20 8.0 Siliconized 1.0
414 464 0.9 0.90 1.02 Trehalose Mannitol Polysorbate 20.sup.b 8.5
Siliconized 1.0 502 506 1.0 0.96 0.98 Trehalose Mannitol None 8.8
Siliconized 0.9 472 501 1.1 1.00 1.03 None Mannitol Polysorbate
20.sup.c 8.5 Siliconized 1.0 511 512 1.0 0.93 1.00 Trehalose None
Polysorbate 20.sup.c 8.5 Siliconized 1.0 524 521 1.0 0.96 0.99
Trehalose None None 8.5 Siliconized 1.0 491 513 1.0 0.98 1.00
.sup.aRelative to Reference .sup.b0.1% .sup.c0.02% Coll. I =
collagenase I; Coll. II = collagenase II
[0188] These data confirmed that the modification of the
formulation with the three tested excipients at various pH levels
has no adverse effect on the protein stability/concentration and
enzymatic activity and that these components are all appropriate
for further evaluation in the lyophilized formulations. pH values
in the range tested also did not demonstrate adverse effect on
product quality.
Formulation Robustness Studies--Study 4
[0189] Various formulations were also characterized by
Composition-Gradient Multi-Angle Light Scattering (CG-MALS) to
assess self- and hetero-protein interactions and Nano Differential
Scanning calorimetry (DSC) to assess protein unfolding. CG-MALS and
nanoDSC were performed as described above.
[0190] A study was also conducted to determine if hydrogen peroxide
as an oxidizing agent impacts the formulations being studied
(solutions pre-lyophilization) based on turbidity values.
Results
[0191] CG-MALS--Results are presented in Table 15.
TABLE-US-00015 TABLE 15 Effect of Composition and pH on Protein
Interactions by CG-MALS CG-MALS Variable Parameters A2 [10.sup.-4
mol*mL/g.sup.2] Sodium Coll. I Chloride Trehalose Mannitol Sucrose
Coll. I Coll. II Coll. II pH (mM) (mM) (mM) (mM) (self) (self)
(hetero) 7.5 None 60 0 0 0.25 0.45 0.43 8.0 None 60 0 0 1.01 0.93
0.94 8.5 None 60 0 0 2.16 1.97 1.83 7.5 100 60 0 0 0.31 0.42 0.44
8.5 100 60 0 0 0.85 0.54 0.60 7.5 None 120 0 0 0.30 0.43 0.40 8.5
None 120 0 0 2.24 1.90 1.85 8.5 50 120 0 0 0.47 0.28 0.35 7.5 100
120 0 0 0.26 0.17 0.20 8.5 100 120 0 0 0.56 0.27 0.34 8.5 None 60
225 0 2.15 1.77 1.79 8.5 None 0 225 60 2.00 1.77 1.84 Coll I =
collagenase I; Coll. II = collagenase II
[0192] For a given formulation composition, the highest repulsive
interaction was observed at pH 8.5 within and across the proteins
(collagenase I & collagenase II) as illustrated in FIG. 1 for
the trehalose-containing formulations.
[0193] Trehalose, sucrose, and mannitol also do not adversely
affect the repulsive forces and are therefore were appropriate
potential components of the formulation.
[0194] Nano-DSC--The variables studied using Nano-DSC to assess
protein unfolding (pH, type and concentration of excipient) are
presented in Table 16.
TABLE-US-00016 TABLE 16 Effect of Composition and pH on Onset
Temperature (T.sub.onset) for Protein Unfolding and Protein
Unfolding Temperature (T.sub.m) Variable Parameters Sodium Nano DSC
Chloride Trehalose Mannitol Sucrose T.sub.onset [.degree. C.]
T.sub.m [.degree. C.] pH (mM) (mM) (mM) (mM) Coll. I Coll. II Coll.
I Coll. II 7.5 None 60 None None 46.8 52.7 52.8 55.5 8.0 None 60
None None 46.3 52.0 52.1 55.3 8.5 None 60 None None 45.9 51.2 51.8
55.1 7.5 100 60 None None 48.8 51.0 53.3 53.8 8.5 100 60 None None
49.0 50.3 52.3 53.0 7.5 None 120 None None 48.0 52.2 52.6 55.7 8.5
None 120 None None 45.9 51.5 52.0 55.3 8.5 50 120 None None 48.5
49.1 52.6 53.8 7.5 100 120 None None 48.7 50.4 53.8 54.1 8.5 100
120 None None 48.4 48.9 52.9 53.3 8.5 None 60 225 None 46.4 51.8
52.3 55.8 8.5 None None 225 60 45.3 51.4 52.5 55.9 Coll I =
collagenase I; Coll. II = collagenase II
[0195] The results demonstrated that the various formulation
components studied had no impact on the onset temperature
(T.sub.onset) for protein unfolding and protein melting temperature
(Tm). Furthermore, T.sub.onset data indicated that a temperature of
40.degree. C. can be used successfully during the secondary drying
step of the lyophilization process without affecting the protein
stability.
[0196] Hydrogen Peroxide Challenge--Hydrogen peroxide was used to
challenge the formulations in a short term study (FIG. 2A and FIG.
2B). Trehalose-containing formulations (with or without mannitol)
and sucrose-containing formulations (with and without mannitol)
showed significantly lower turbidity at pH 8.5 compared to the same
formulations at pH 8.0 and pH 7.5 after exposure to hydrogen
peroxide. Lower turbidity values are indicative of a stable
formulation with low protein aggregation. These results were in
line with the high A2 values (high repulsive interaction) seen with
formulation pH at 8.5 indicating the potential for improved
stability. The results also indicated that, upon exposure to
hydrogen peroxide, trehalose and mannitol-containing formulations
with or without polysorbate (pH 8.0 or 8.5) showed significantly
lower turbidity values compared to sucrose-containing formulations
with no mannitol at pH 8.0. The presence of a surfactant had no
positive or negative impact on turbidity.
Conclusion
[0197] Based on the results of the formulation optimization work,
which identified optimal qualitative and quantitative excipient
composition, the following formulations (pre-lyophilization) were
taken forward for evaluation of the lyophilization cycle: [0198]
CCH and sucrose with mannitol with or without polysorbate 20 at pH
8.5 [0199] CCH and trehalose with mannitol with or without
polysorbate 20 at pH 8.5
[0200] Note that the lowest concentration of polysorbate 20 studied
(0.02%) was used for future analyses as the higher concentration
offered no benefit.
[0201] The lyophilization cycle development for both formulation
variants was conducted in 5 mL vials. Table 17 provides specifics
on the formulations that were evaluated.
TABLE-US-00017 TABLE 17 Lyophilization Trial Formulation Samples
Description (solution, pre-lyophilization) Experimental Bulking
Formulation CCH.sup.a Stabilizer Agent Surfactant Buffer 1.sup.b
0.9 mg/mL 60 mM None None 10 mM Sucrose Tris/HCl pH 8.0 2.sup. 60
mM 225 mM None 10 mM Sucrose Mannitol Tris/HCl pH 8.5 3.sup. 60 mM
225 mM 0.02% 10 mM Sucrose Mannitol Polysorbate Tris/HCl 20 pH 8.5
4.sup. 60 mM 225 mM None 10 mM Trehalose Mannitol Tris/HCl pH 8.5
5.sup. 60 mM 225 mM 0.02% 10 mM Trehalose Mannitol Polysorbate
Tris/HCl 20 pH 8.5 .sup.aCollagenase clostridium histolyticum
.sup.bControl (approved XIAFLEX .RTM. formulation)
Lyophilization Cycle Optimization--Study 1 Varying Mannitol
Concentration
[0202] The objective of this work was to study the effect of
varying ratios of mannitol with fixed concentration of sucrose in
the absence of surfactant on the lyophilization process and cycle
time. The following experimental variants were prepared along with
placebo Lyo samples:
TABLE-US-00018 TABLE 18 Compositions of the formulations Fill
Variant # Packaging system Formulation volume Va 5 ml glass vial
0.93 mg/ml CCH; 60 mM 1 ml Sucrose; 112.5 mM Mannitol; 10 mM
Tris/HCl buffer pH 8.5 Vb 5 ml glass vial 0.93 mg/ml CCH; 60 mM 1
ml Sucrose; 225 mM Mannitol; 10 mM Tris/HCl buffer pH 8.5 Vc 5 ml
glass vial 0.93 mg/ml CCH; 60 mM 1 ml Sucrose; 337.5 mM Mannitol;
10 mM Tris/HCl buffer pH 8.5 Placebo 5 ml glass vial 60 mM Sucrose;
337.5 mM 1 ml Mannitol; 10 mM Tris/HCI buffer pH 8.5
[0203] An intermediate lyophilization cycle ("Lyo cycle") was used
to freeze dry the products (with a goal of about 36 hrs total cycle
time). Provided below is information on the pilot freeze dryer used
in these experiments: [0204] Manufacturer: Hof Sonderanlagenbau
(Lohra, Germany) [0205] Shelf area: 0.5 m.sup.2 [0206] Ice
condenser capacity: 10 kg [0207] Adjustable ice condenser
temperature [0208] In-vial temperature recording [0209]
Differential pressure measurement [0210] Connectable radiation
cage
Preparation of Packaging Materials
[0211] Lyophilization stoppers were autoclaved at 121.degree. C.
for 15 min and dried for 8 hours at 105.degree. C. Vials were
rinsed with purified water and depyrogenized at 300.degree. C. for
2 hours.
[0212] The formulation variants were prepared by dialysis. The
dialysis of XIAFLEX.RTM. drug substance was accomplished in three
independent dialysis steps to achieve a quantitative buffer
exchange. 125 ml of the XIAFLEX.RTM. drug substance was dialyzed
against variant a, b and c. XIAFLEX.RTM. drug substance was
transferred into two preconditioned (in dialysis buffer) dialysis
tubes. Filled dialysis tubes were incubated in 1 L of the target
buffer for 2 hours before a first buffer change (1 L) was
performed. After dialysis for two additional hours the buffer was
changed a second time (2 L) to finalize the dialysis overnight. The
protein sample was removed from the dialysis tube and the
concentration was adjusted to 0.93 mg/ml (.+-.10%) by dilution
(concentration was checked by UV 280 nm). As a control,
XIAFLEX.RTM. drug substance was used without dialysis.
Filtration and Filling
[0213] The lyophilization solutions were passed over a 0.22 .mu.m
filter before filling. 1 ml of the corresponding lyophilization
solution was filled into the vial.
Lyophilization
[0214] The filled vials with lyophilization-stoppers attached to
the vials in "lyo-position" were loaded into the pilot freeze
dryer. About 125 vials per formulation and 100 placebo vials of Vb
were loaded. The lyophilization cycle was run with the following
parameters: [0215] Freezing temperature (shelf): -50.degree. C.
[0216] Primary drying temperature (shelf): -10.degree. C. [0217]
Secondary drying temperature (shelf): 40.degree. C. [0218]
Pressure: 0.25 mbar [0219] Cumulative time: approximately 41
hours
[0220] To control product temperature during freeze drying, thermo
couples were inserted into product vials. Pressure was controlled
during lyophilization by using a Pirani-pressure sensor. Pressure
regulation was managed via vacuum and dosing valve (nitrogen
injection). Vials were closed at a pressure of 750 mbar under
nitrogen atmosphere.
Karl-Fischer Titration
[0221] The content of one vial of the corresponding lyophilizate
was weighed into a glass vial which was sealed with a crimp cap.
The sample was transferred into the oven of the Karl-Fischer
coulometer (756/774; Metrohm) which was heated to 100.degree. C.
The septum of the cap was penetrated by an injection needle, and
the generated water vapor was directly transferred into the
titration chamber of the Karl-Fischer coulometer via dry nitrogen.
Measurement was repeated one time. Empty glass vials were used for
blank correction.
Crystal Water Analysis
[0222] The oven sample processor 774 enables a unique temperature
ramping method in Karl Fischer titration. The sample was heated up
by a defined heating rate and the released water was directly
transferred into the titration chamber of the Karl-Fischer
coulometer. By recording the generated water and the water drift
(.mu.g water/min) depending on the oven temperature, specified
events where water was released (e.g. the release of hydrate water)
can be detected. 50 to 100 mg of the lyophilizates was weighted
into an empty 6R type 1 glass vial and closed with an
alu-crimp-cab. The sample was transferred into the oven of the
sample processor. There the sample is heated up by a defined
temperature ramp from 50.degree. C. to 140.degree. C. in 45 min
(2.degree. C./min). The ramp was concluded at 140.degree. C. to
exclude undesired Maillard-reactions.
Visual Appearance
[0223] Lyophilizates were removed from the vial by carefully
breaking the glass vial and the lyophilization cake was cut
vertically to screen its inner layer for collapse zones.
Scanning Electron Microscopy
[0224] Lyophilizates were analyzed by SEM to evaluate their
microstructure.
[0225] Lyophilizates were cut, and the vertical cross sections and
top/bottom surfaces were analyzed via SEM at 50.times. and
150.times. magnification.
Reconstitution
[0226] Vials were reconstituted with 4 ml of a solution containing
0.03% calcium chloride and 0.66% sodium chloride. Time for complete
dissolution of the lyophilizates was recorded.
Nano Differential Scanning Calorimetry
[0227] Nano DSC was performed as described above.
Sample Preparation
[0228] Lyophilization solutions and reconstituted lyophilizates
were analyzed at a concentration of 0.93 mg CCH/ml. The
corresponding buffer was used as buffer scan and buffer
reference.
Results
[0229] The digital data acquisition proved that the freeze drying
process was conducted as intended. As indicated by Pirani to
conductivity pressure sensor difference, sublimation of the batch
was finished after about 15 hours of total lyophilization time. The
end of sublimation of each different sub-lot was indicated by the
steep increase of the product temperature during primary
drying.
[0230] The primary drying step was terminated after about 17 hours
of total lyophilization time. After 18 hours of secondary drying
one vial of each variant was removed from the freeze dryer and the
residual moisture level was determined by Karl-Fischer titration
while the secondary drying step was prolonged for the rest of the
batch. The Karl-Fischer analysis revealed that the residual
moisture level was already below the desired value. Thus, the rest
of the batch was unloaded immediately after receiving these
results. Total duration of secondary drying was 19 hours.
[0231] Visual appearance--All samples showed excellent macroscopic
appearance without any signs of cake defects (data not shown).
[0232] Reconstitution behavior--Lyophilizates of all sub-lots
showed quick and spontaneous dissolution within seconds (<30
sec.)
[0233] Residual moisture--Table 19 shows the residual moisture
levels of the samples measured by Karl-Fischer titration.
TABLE-US-00019 TABLE 19 Residual Moisture (n = 3) Sample Residual
Moisture [%] Va 0.16 0.16 0.17 Vb 0.24 0.22 0.27 Vc 0.16 0.13
0.16
[0234] Scanning Electron Microscopy (SEM)--SEM analysis of the lyo
cakes did not reveal any signs of collapse nor other cake defects
(See FIG. 3A-FIG. 3R). The inner structure of all formulation
variants was comparable. The top surface of variant Va (lowest
amount of sucrose) had a more open-porous structure and showed less
skin formation than variants Vb and Vc. The bottom surface of
variant Vc looked more dense then the bottom surface of the other
formulations.
[0235] Nano DSC--Table 20 summarizes the obtained results.
TABLE-US-00020 TABLE 20 Results of the nano DSC Analysis Before and
After Lyophilization Before lyophilization After lyophilization Tm
Tonset .DELTA.H Tm Tonset .DELTA.H Variant [.degree. C.] [.degree.
C.] [kJ/mol] [.degree. C.] [.degree. C.] [kJ/mol] Va 54.9 47.9 1116
55.4 48.3 1140 Vb 55.4 48.2 1165 55.7 48.6 1183 Vc 56.1 49.2 1207
56.2 49.2 1124
[0236] To demonstrate the precision of the unfolding temperature
and denaturation enthalpy, variant Vc (before lyophilization) was
analyzed six times. The samples were derived from one sample
preparation, which eliminated any concentration uncertainty. The
difference between the results for T onset and Tm was below 1%
whereas variation of the enthalpy was more than 7%, which is well
in line with the specifications of the instrument (TA instruments
states a uncertainty of 5% for the enthalpy of unfolding for
lysozyme) (data not shown). It can be concluded that all
formulations were equal before and after lyophilization within the
error tolerances of the method in respect to unfolding temperature
and transition enthalpy, indicating that the lyophilization process
conditions used had no negative impact on the CCH. Increasing
amounts of mannitol seem to result in an increased thermodynamic
stability expressed by slightly higher values of Tm and T
onset.
Conclusion
[0237] The observations and all of the data collected above
indicate that the lyophilized formulations of CCH can be
manufactured using a range of mannitol concentrations in presence
of sucrose in Tris buffer. This work also identified more moderate
lyophilization conditions leading to a cycle time of about 40
hrs.
Lyophilization Cycle Optimization--Study 2 Fixed Mannitol
Concentration
[0238] The objective of this work was to update the lyophilization
cycle to result in a shorter process time and more efficient and
robust process.
Experimental Design
[0239] A laboratory scale lyophilization trial with the four
experimental formulation variants along with the XIAFLEX.RTM.
formulation as a control was completed using 5 cc vials. These
variants are listed in Table 21 below.
TABLE-US-00021 TABLE 21 Lyophilization Trial Formulation Samples
Description (solution, pre-lyophilization) Experimental Bulking
Formulation CCH.sup.a Stabilizer Agent Surfactant Buffer 1.sup.b
0.9 mg/mL 60 mM None None 10 mM Sucrose Tris/HCl pH 8.0 2.sup. 60
mM 225 mM None 10 mM Sucrose Mannitol Tris/HCl pH 8.5 3.sup. 60 mM
225 mM 0.02% 10 mM Sucrose Mannitol Polysorbate Tris/HCl 20 pH 8.5
4.sup. 60 mM 225 mM None 10 mM Trehalose Mannitol Tris/HCl pH 8.5
5.sup. 60 mM 225 mM 0.02% 10 mM Trehalose Mannitol Polysorbate
Tris/HCl 20 pH 8.5 .sup.aCollagenase clostridium histolyticum
.sup.bControl (approved XIAFLEX .RTM. formulation)
[0240] To prepare experimental formulations #2-#5, formulation #1
was buffer exchanged using dialysis to introduce the new excipients
(trehalose, mannitol, and polysorbate 20) into the formulation in
Tris buffer, pH 8.5.
[0241] Initial lyophilization cycle development work was conducted
using lyocycle conditions that are slightly more aggressive than
the conditions used for the XIAFLEX.RTM. formulation in terms of
drying temperatures, pressures, and times. Secondary drying was
conducted at 40.degree. C. to facilitate efficient drying of the
product and achieve a target moisture of less than 0.5%.
Comparative details of the XIAFLEX.RTM. process and the
experimental lyophilization process are outlined in Table 22
below.
TABLE-US-00022 TABLE 22 Comparison of XIAFLEX .RTM. (0.9 mg
CCH/vial) and Experimental Lyocycle Process (0.92 mg CCH/vial)
Experimental XIAFLEX .RTM. Lyophilization Process Process Freezing
temperature -50.degree. C. -50.degree. C. Primary drying
temperature -37.degree. C. -10.degree. C. Secondary drying
temperature 30.degree. C. 40.degree. C. Pressure 40 microns 188
millitorr Cumulative time (approximate) 70.5 hours 57.5 hours
Results and Conclusion
[0242] The lyophilized cakes for all formulations rapidly dissolved
in the diluent with a reconstitution time of less than 10 seconds
and had moisture contents (KF) of less than 0.4%.
[0243] At shortened experimental cycle time, the XIAFLEX.RTM. cake
appeared shrunken compared to the experimental formulation
variants, which all showed a well formed and robust cake. All of
the experimental formulations were tested for various
characteristics including protein concentration, collagenase I and
collagenase II mass composition, and biologic activity. The test
results for all of the formulations aligned with the XIAFLEX.RTM.
formulation.
[0244] Experimental formulations #3 and #5 were selected for
further testing. These formulations were used to conduct informal
short term stability studies at 5.degree. C. and at accelerated
storage conditions of 25.degree. C./60% RH. Samples were analyzed
per approved test methods. All results generated for both
formulations were in line with historical data for the XIAFLEX.RTM.
formulation, indicating that the formulation changes being studied
do not adversely affect product quality (data not shown). As noted
previously, the currently approved limits may be revised based on a
statistical analysis of data generated for the experimental
formulations.
[0245] In summary, formulation optimization development work and
data generated to date indicated that the two studied protein
stabilizers (sucrose, trehalose), the addition of a bulking agent
(mannitol), and a surfactant (polysorbate 20), do not adversely
affect product quality and that these formulations would be
suitable to pursue further.
Lyophilization Cycle Optimization--Study 3 Pressure Analysis
Study Objective
[0246] Pressure tests were performed to facilitate identification
of the optimal lyophilization process conditions. This test set out
to identify the maximum tolerable pressure and other reliable
process parameters for freezing, primary drying, and secondary
drying during lyophilization under process relevant conditions to
realize a fast and robust lyophilization cycle.
Experimental Design
[0247] Three formulation variants in 5 cc vials were used as
provided in Table 23.
TABLE-US-00023 TABLE 23 Lyophilization Trial Formulation Samples
Description (solution, pre-lyophilization) Experimental Bulking
Formulation CCH.sup.a Stabilizer Agent Surfactant Buffer 1.sup.b
0.9 60 mM None None 10 mM mg/mL Sucrose Tris/HCl pH 8.0 3.sup. 60
mM 225 mM 0.02% 10 mM Sucrose Mannitol Polysorbate Tris/HCl pH 20
8.5 5.sup. 60 mM 225 mM 0.02% 10 mM Trehalose Mannitol Polysorbate
Tris/HCl pH 20 8.5 .sup.aCollagenase clostridium histolyticum
.sup.bControl (approved XIAFLEX .RTM. formulation)
[0248] To identify the pressure for the sublimation phase, two
pressure tests were performed at different shelf temperatures and
different pressure steps. Sample vials filled with the experimental
formulations were loaded onto one freeze dryer shelf (middle
position). After freezing, the chamber pressure was set to initial
value of 128 .mu.bar and shelf temperature was increased to an
initial value (-10.degree. C. in test 1/+10.degree. C. in test 2).
Lyophilization was allowed to run for a period of time to generate
a small volume of lyophilizate above the ice interface. Chamber
pressure was then increased step wise (e.g., 380 .mu.bar, 1030
.mu.bar etc.) and the sample vials were video monitored for cake
collapse or other visual adverse effects. The shelf temperature was
set to -10.degree. C. to achieve a moderate energy input.
[0249] Pictures of the vials at the end of each pressure step are
presented in FIG. 4A-FIG. 4C. As shown in those figures, the
Control (#1, XIAFLEX.RTM. in the vial shown on the far right) cake
collapsed with increasing pressure. The maximum tolerable pressure
for the XIAFLEX.RTM. formulation was therefore between 128 .mu.bar
and 380 .mu.bar, equivalent to an ice interface temperature of
between -40.degree. C. and -30.degree. C. Experimental Formulations
#3 and #5 remained intact over the entire pressure range
investigated.
[0250] A second pressure test was conducted, in which experimental
formulations #3 and #5 were further investigated. The pressure
range was expanded to 4 mbar. The temperature of the shelf during
the test was set to +10.degree. C. to provide sufficient energy
input for enabling efficient sublimation at this pressure
range.
[0251] The results are provided in FIG. 5A and FIG. 5B. No
collapsed cakes or any other defects was observed over the entire
pressure range investigated. Based on these findings, it was
concluded that formulations containing mannitol can be freeze dried
at a much higher chamber pressure, which will result in more
efficient and robust lyophilization process with a shorter
lyophilization cycle time.
[0252] This initial lyophilization cycle development work supported
the use of mannitol to pursue a more efficient and robust
lyophilization cycle.
Lyophilization Cycle Optimization--Study 4 Further Optimization
[0253] Based on the results of the pressure tests above,
optimization of the lyophilization parameters was performed for
experimental formulations #3 and #5 (data not shown).
[0254] As the pressure tests demonstrated that the
mannitol-containing formulations can be freeze dried at chamber
pressures up to 4 mbar with no risk of structural collapse during
sublimation, the lyocycles for the optimization trials were run
using a chamber pressure of 1 mbar to take advantage of high
sublimation rates.
[0255] Two experimental lyocycle runs were performed to identify
the basic parameters for a robust and efficient freeze drying
cycle. Samples were analyzed post-lyophilization for visual
appearance, residual moisture, physical stability, and by scanning
electron microscopy (SEM).
[0256] Run #1: Utilized experimental formulation #3 only. Results
from this run indicated that a higher temperature during secondary
drying is required to reach a residual moisture level below 0.5%.
Results for other physical attribute tests were acceptable (data
not shown). A summary of the lyophilization parameters used are
presented below:
[0257] Freezing temperature (shelf): -50.degree. C.
[0258] Drying temperature (sublimation and secondary drying)
(shelf): 35.degree. C. [0259] Pressure: 1 mbar [0260] Cumulative
time: approximately 21.5 hours
[0261] Run #2: Utilized experimental formulation #3 only. This run
identified adequate conditions for drying using 40.degree. C. All
results were acceptable (data not shown). A summary of the
lyophilization parameters used are presented below: [0262] Freezing
temperature (shelf): -50.degree. C. [0263] Drying temperature
(sublimation and secondary drying) (shelf): 40.degree. C. [0264]
Pressure: 1 mbar [0265] Cumulative time: approximately 21.5
hours
Conclusions
[0266] The lyocycle optimization studies identified basic
parameters for an efficient freeze drying cycle. All tests for the
physical attributes of the lyophilized cakes were acceptable.
Stability Studies--Long Term Stability Studies
[0267] Six process validation lots of the CCH formulation (1:1
mixture of collagenase I and collagenase II at a concentration of 1
mg/mL in 10 mM Tris, 60 mM sucrose, 225 mM mannitol, pH 8.5) were
manufactured and placed on stability. One early formulation
development lot, which utilized the same formulation but a lower
fill quantity in a comparable container closure system, was
manufactured and placed on stability.
[0268] The 6 process validation lots were monitored on long-term
stability. Storage conditions included long-term stability at
various storage condition (2-8.degree. C.; 25.degree. C./60%
relative humidity (RH); and 40.degree. C./75% RH) to support
product shelf life as well as short-term stability studies at
various storage conditions to support evaluation of potential
temperature excursions during shipping or storage. Data for an
early formulation development lot, which utilized the same
formulation but a lower fill quantity (0.46 mg) in a comparable
container closure system, was also included.
[0269] A summary of the studies along with available stability data
is provided in Table 24.
TABLE-US-00024 TABLE 24 CCH Formulation Stability Study Summary
Fill Quantity Length of Available (mg Lot Study Data CCH/vial)
Number Storage Condition (months) (months) 0.46 003H16 5.degree. C.
.+-. 3.degree. C. 36 36 25.degree. C. .+-. 2.degree. C./ 36 36 60%
.+-. 5% RH 40.degree. C. .+-. 2.degree. C./ 6 6 75% .+-. 5% RH 0.92
307984 5.degree. C. .+-. 3.degree. C. 36 24 25.degree. C. .+-.
2.degree. C./ 36 24 60% .+-. 5% RH 40.degree. C. .+-. 2.degree. C./
6 6 75% .+-. 5% RH 307985 5.degree. C. .+-. 3.degree. C. 36 24
25.degree. C. .+-. 2.degree. C./ 36 24 60% .+-. 5% RH 40.degree. C.
.+-. 2.degree. C./ 6 6 75% .+-. 5% RH 313877 5.degree. C. .+-.
3.degree. C. 36 18 25.degree. C. .+-. 2.degree. C./ 36 18 60% .+-.
5% RH 40.degree. C. .+-. 2.degree. C./ 6 6 75% .+-. 5% RH 1.84
320971 5.degree. C. .+-. 3.degree. C. 36 12 25.degree. C. .+-.
2.degree. C./ 36 12 60% .+-. 5% RH 40.degree. C. .+-. 2.degree. C./
6 6 75% .+-. 5% RH 320972 5.degree. C. .+-. 3.degree. C. 36 12
25.degree. C. .+-. 2.degree. C./ 36 12 60% .+-. 5% RH 40.degree. C.
.+-. 2.degree. C./ 6 6 75% .+-. 5% RH 320973 5.degree. C. .+-.
3.degree. C. 36 12 25.degree. C. .+-. 2.degree. C./ 36 12 60% .+-.
5% RH 40.degree. C. .+-. 2.degree. C./ 6 6 75% .+-. 5% RH
[0270] Stability batches were tested for appearance (pre- and
post-reconstitution), reconstitution time, osmolality, pH,
concentration by UV.sub.A280, quantitative sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE), reverse-phase high
performance liquid chromatography (RP-HPLC) [purity], mass and
ratio by composition by RP-HPLC, size-exclusion high-performance
liquid chromatography (SEC-HPLC), soluble rat tail collagen (SRC)
assay for collagenase I potency, glycyl-L-prolyl-L-alanine (GPA)
assay for collagenase II potency, moisture, particulates,
endotoxin, and container closure integrity helium leak.
Results and Analysis
[0271] Assessments of the stability results along with a trending
analysis for each test at various storage condition (2-8.degree.
C.; 25.degree. C./60% relative humidity (RH); and 40.degree. C./75%
RH), as applicable, are outlined below:
[0272] Appearance (Pre- and Post-Reconstitution)--The results for
appearance (pre- and post-reconstitution) at the storage conditions
tested showed no significant changes or unexpected trends (data not
shown).
[0273] Reconstitution Time--The results for reconstitution time at
the storage conditions tested showed no significant changes or
unexpected trends (data not shown).
[0274] Osmolality--The results for osmolality at the storage
conditions tested showed no significant changes or unexpected
trends (data not shown).
[0275] pH--The results for pH at the storage conditions tested
showed no significant changes or unexpected trends (data not
shown).
[0276] Concentration by UV.sub.A280--The results for concentration
by UV.sub.A280 at the storage conditions tested showed no
significant changes or unexpected trends (data not shown).
[0277] Quantitative SDS-PAGE--The results for quantitative SDS-PAGE
at the storage conditions tested showed no significant changes or
unexpected trends (data not shown).
[0278] RP-HPLC (Purity)--The results for RP-HPLC (purity) at the
storage conditions tested showed no significant changes or
unexpected trends (data not shown).
[0279] Mass and Ratio Composition by RP-HPLC--The results for mass
and ratio by composition RP-HPLC at the storage conditions tested
showed no significant changes or unexpected trends (data not
shown).
[0280] SEC-HPLC--The results for SEC-HPLC at the storage conditions
tested showed no significant changes or unexpected trends (data not
shown).
[0281] SRC Assay for collagenase I Potency--The results for SRC
assay for collagenase I potency at the storage conditions tested
showed no significant changes or unexpected trends (data not
shown).
[0282] GPA Assay for collagenase II Potency--The results for GPA
assay for collagenase II potency at the storage conditions tested
showed no significant changes or unexpected trends (data not
shown).
[0283] Moisture--As shown in FIG. 6, at 25.degree. C./60% relative
humidity, the mannitol-containing formulations showed less moisture
than the XIAFLEX.RTM. formulation, indicating the improved
stability of the mannitol-containing formulations.
[0284] Particulates--The results for particulates at the storage
conditions tested showed no significant changes or unexpected
trends (data not shown).
[0285] Container Closure Integrity/Helium Leak--The results for
container closure integrity/helium leak showed no significant
changes or unexpected trends (data not shown).
Photostability Study
[0286] A study was conducted to determine photostability of the CCH
formulation following exposure to 1.2 million lux hours of cool,
white light followed by 200 watt hours/square meter (W/m.sup.2) of
UV energy. Samples were exposed to 8.00 kilolux of cool, white
light for 150 hours, followed by exposure to 10.00 W/m.sup.2 UV
light for 20 hours. All results following this exposure were
comparable to an unexposed control (data not shown).
Reconstitution Stability Study
[0287] To demonstrate compatibility of the lyophilized CCH
formulation with the sterile diluent used for reconstitution and to
generate stability data for the reconstituted product under
potential in-use conditions, several reconstitution stability
studies were executed. The lyophilized CCH formulation was
reconstituted with sterile diluent and subjected to analysis at
specified time points following storage at specified temperatures.
Table 25 summarizes the reconstitution stability studies
performed.
TABLE-US-00025 TABLE 25 Summary of Reconstitution Stability Studies
Fill Quantity (mg CCH/vial) Description of Study 0.92 Temperature
Cycling: Following reconstitution with diluent and storage at
25.degree. C./60% RH for 24 hours, then at 2.degree. C.-8.degree.
C. for 96 hours and then at 25.degree. C./60% RH for an additional
24 hours (testing at T0 and end of study) 25.degree. C./60% RH for
24 hours: Following reconstitution with diluent and storage at
25.degree. C./60% RH for 24 hours (testing at T0 and end of study)
2.degree. C.-8.degree. C. for 120 hours: Following reconstitution
with diluent and storage at 2.degree. C.-8.degree. C. for 120 hours
(testing at T0, 24 hours, 96 hours, and 120 hours) 1.84 Temperature
Cycling: Following reconstitution with diluent and storage at
25.degree. C./60% RH for 24 hours, then at 2.degree. C.-8.degree.
C. for 96 hours and then at 25.degree. C./60% RH for an additional
24 hours (testing at T0 and end of study) 25.degree. C./60% RH for
24 hours: Following reconstitution with diluent and storage at
25.degree. C./60% RH for 24 hours (testing at T0 and end of study)
2.degree. C.-8.degree. C. for 120 hours: Following reconstitution
with diluent and storage at 2.degree. C.-8.degree. C. for 120 hours
(testing at T0, 24 hours, 96 hours and 120 hours)
[0288] All results at time zero and subsequent stability time
points were comparable and no trends in the data noted (data not
shown). These studies utilized microplate versions of the SRC and
GPA test methods for determination of potency as these are higher
throughput and quicker turnaround than the cuvette-based test
methods; for each, the overall analytical approach of the
microplate test method is the same as the cuvette test method.
These results demonstrated compatibility of the lyophilized CCH
formulation and the sterile diluent as well as demonstrated the
stability of the reconstituted CCH formulation under potential
conditions of use.
Conclusion
[0289] Available stability data for the 6 process validation lots
of CCH formulation demonstrated stability of the material through
at least the 18-month pull point at both 5.degree. C. and
25.degree. C./60% RH storage conditions, as well as through the
6-month pull point at the 40.degree. C./75% RH storage condition.
Additionally, available data for a formulation development lot of
the CCH formulation in a smaller vial demonstrated stability
through the 24-month pull point at both the 5.degree. C. and
25.degree. C./60% RH storage conditions as well as through the
6-month pull point at the 40.degree. C./75% RH storage
condition.
[0290] The CCH formulation demonstrated acceptable photostability
as all results generated following exposure were comparable to a
control (unexposed) sample.
[0291] Following reconstitution with sterile diluent, CCH
formulation showed acceptable stability for up to 24 hours when
stored at 25.degree. C./60% RH and for up 120 hours when stored at
5.degree. C. Reconstituted CCH formulation also demonstrated
acceptable stability when stored at 25.degree. C./60% RH for 24
hours, then at 2.degree. C. to 8.degree. C. for 96 hours and then
at 25.degree. C./60% RH for an additional 24 hours.
[0292] Future annual stability studies will be conducted and
include storage at 5.degree. C..+-.3.degree. C. and 25.degree.
C..+-.2.degree. C./60%.+-.5% RH conditions. Material at both
storage conditions will be evaluated through the proposed 36-month
shelf life.
[0293] Those skilled in the art will appreciate that numerous
changes and modifications can be made to the preferred embodiments
of the invention and that such changes and modifications can be
made without departing from the spirit of the invention. It is,
therefore, intended that the appended claims cover all such
equivalent variations as fall within the true spirit and scope of
the invention.
[0294] The disclosures of each patent, patent application, and
publication cited or described in this document are hereby
incorporated herein by reference, in its entirety.
EMBODIMENTS
[0295] The following list of embodiments is intended to complement,
rather than displace or supersede, the previous descriptions.
[0296] Embodiment 1. A formulation comprising: [0297] a
collagenase; [0298] about 30 mM to about 240 mM of a disaccharide;
[0299] about 50 mM to about 800 mM of mannitol; and [0300] about 6
mM to about 10 mM of a Tris-HCl. [0301] Embodiment 2. The
formulation of embodiment 1, wherein the collagenase comprises a
collagenase I. [0302] Embodiment 3. The formulation of embodiment
2, wherein the collagenase I comprises the amino acid sequence of
SEQ ID NO: 1. [0303] Embodiment 4. The formulation of embodiment 1,
wherein the collagenase comprises a collagenase II. [0304]
Embodiment 5. The formulation of embodiment 4, wherein the
collagenase II comprises the amino acid sequence of SEQ ID NO: 2.
[0305] Embodiment 6. The formulation of embodiment 1, wherein the
collagenase comprises a mixture of collagenase I and collagenase
II. [0306] Embodiment 7. The formulation of embodiment 6, wherein
the collagenase I comprises the amino acid sequence of SEQ ID NO: 1
and the collagenase II comprises the amino acid sequence of SEQ ID
NO: 2. [0307] Embodiment 8. The formulation of embodiment 6 or 7,
wherein the collagenase is collagenase Clostridium histolyticum
(CCH). [0308] Embodiment 9. The formulation of any one of the
previous embodiments, wherein the disaccharide comprises sucrose or
trehalose. [0309] Embodiment 10. The formulation of any one of the
previous embodiments, wherein the pH of the formulation is about
7.8 to about 8.8. [0310] Embodiment 11. The formulation of any one
of the previous embodiments, wherein the formulation comprises:
[0311] CCH; [0312] about 60 mM sucrose; [0313] about 225 mM
mannitol; and [0314] about 10 mM Tris-HCl, [0315] wherein the
formulation has a pH of about 8.5. [0316] Embodiment 12. The
formulation of any one of the previous embodiments, further
comprising a surfactant comprising polysorbate 20, polysorbate 80,
or poloxamer 188. [0317] Embodiment 13. The formulation of
embodiment 12, comprising from about 0.01% to about 2% of the
surfactant. [0318] Embodiment 14. The formulation of embodiment 13,
comprising about 0.02% of the surfactant. [0319] Embodiment 15. The
formulation of any one of the previous embodiments, wherein the
formulation is liquid. [0320] Embodiment 16. A lyophilized
formulation comprising: [0321] a collagenase; [0322] a
disaccharide; [0323] mannitol; and [0324] Tris-HCl. [0325]
Embodiment 17. The lyophilized formulation of embodiment 16,
wherein the collagenase comprises a collagenase I. [0326]
Embodiment 18. The lyophilized formulation of embodiment 17,
wherein the collagenase I comprises the amino acid sequence of SEQ
ID NO: 1. [0327] Embodiment 19. The lyophilized formulation of
embodiment 16, wherein the collagenase comprises a collagenase II.
[0328] Embodiment 20. The lyophilized formulation of embodiment 19,
wherein the collagenase II comprises the amino acid sequence of SEQ
ID NO: 2. [0329] Embodiment 21. The lyophilized formulation of
embodiment 16, wherein the collagenase comprises a mixture of
collagenase I and collagenase II. [0330] Embodiment 22. The
lyophilized formulation of embodiment 21, wherein the collagenase I
comprises the amino acid sequence of SEQ ID NO: 1 and the
collagenase II comprises the amino acid sequence of SEQ ID NO: 2.
[0331] Embodiment 23. The lyophilized formulation of embodiment 21
or 22, wherein the collagenase is collagenase Clostridium
histolyticum (CCH). [0332] Embodiment 24. The lyophilized
formulation of any one of embodiments 16-23, wherein the
disaccharide comprises sucrose or trehalose. [0333] Embodiment 25.
The lyophilized formulation of any one of embodiments 16-24,
wherein, prior to lyophilization, the formulation comprises: [0334]
CCH; [0335] 60 mM sucrose; [0336] 225 mM mannitol; and [0337] 10 mM
Tris-HCl, and [0338] having a pH of about 8.5. [0339] Embodiment
26. The lyophilized formulation of any one of embodiments 16-25,
wherein the lyophilized formulation is stable at pressures above
380 .mu.bar. [0340] Embodiment 27. The lyophilized formulation of
embodiment 26, wherein the lyophilized formulation is stable at a
pressure of about 4000 .mu.bar. [0341] Embodiment 28. The
lyophilized formulation of any one of embodiments 16-27, wherein
the lyophilized formulation is stable at: [0342] (e) 2-8.degree. C.
for at least 36 months; [0343] (f) 25.degree. C./60% relative
humidity for at least 36 months; [0344] (g) 40.degree. C./75%
relative humidity for at least 6 months; or [0345] (h) any
combination of (a) to (c). [0346] Embodiment 29. The lyophilized
formulation of any one of embodiments 16-28, wherein the
lyophilized formulation is formed by a method comprising: [0347]
freezing the formulation at a temperature between about -25.degree.
C. and -55.degree. C. to form a frozen formulation; and [0348]
drying the frozen formulation at a temperature between about
25.degree. C. and about 50.degree. C. to form the lyophilized
formulation. [0349] Embodiment 30. The lyophilized formulation of
embodiment 29, wherein the lyophilized formulation is formed by a
method comprising: [0350] freezing the formulation at a single
temperature between about -25.degree. C. and -55.degree. C. to form
a frozen formulation; and [0351] drying the frozen formulation at a
single temperature between about 25.degree. C. and about 50.degree.
C. to form the lyophilized formulation. [0352] Embodiment 31. The
lyophilized formulation of any one of embodiments 16-30, wherein
the lyophilized formulation is formed by a lyophilization method
that is performed for less than 72 hours. [0353] Embodiment 32. The
lyophilized formulation of any one of embodiments 16-31, wherein
the lyophilized formulation is formed by a lyophilization method
that is performed at a pressure of between about 380 .mu.bar to
about 4000 .mu.bar. [0354] Embodiment 33. The lyophilized
formulation of any one of embodiments 16-32, in a unit-dose vial,
multi-dose vial, cartridge, or syringe. [0355] Embodiment 34. A
reconstituted formulation comprising: [0356] a collagenase; [0357]
a disaccharide; [0358] mannitol; [0359] Tris-HCl; [0360] calcium
chloride; and [0361] sodium chloride. [0362] Embodiment 35. The
reconstituted formulation of embodiment 34, wherein the collagenase
is collagenase Clostridium histolyticum (CCH). [0363] Embodiment
36. The reconstituted formulation of embodiment 34 or 35, wherein
the disaccharide comprises sucrose or trehalose. [0364] Embodiment
37. The reconstituted formulation of any one of embodiments 34-36,
wherein the reconstituted formulation is isotonic to human blood.
[0365] Embodiment 38. A kit comprising: [0366] a container
comprising the lyophilized formulation of any one of embodiments
16-32; and [0367] a container comprising a sterile diluent
comprising calcium chloride and sodium chloride.
Sequence CWU 1
1
211008PRTClostridium histolyticum 1Ile Ala Asn Thr Asn Ser Glu Lys
Tyr Asp Phe Glu Tyr Leu Asn Gly1 5 10 15Leu Ser Tyr Thr Glu Leu Thr
Asn Leu Ile Lys Asn Ile Lys Trp Asn 20 25 30Gln Ile Asn Gly Leu Phe
Asn Tyr Ser Thr Gly Ser Gln Lys Phe Phe 35 40 45Gly Asp Lys Asn Arg
Val Gln Ala Ile Ile Asn Ala Leu Gln Glu Ser 50 55 60Gly Arg Thr Tyr
Thr Ala Asn Asp Met Lys Gly Ile Glu Thr Phe Thr65 70 75 80Glu Val
Leu Arg Ala Gly Phe Tyr Leu Gly Tyr Tyr Asn Asp Gly Leu 85 90 95Ser
Tyr Leu Asn Asp Arg Asn Phe Gln Asp Lys Cys Ile Pro Ala Met 100 105
110Ile Ala Ile Gln Lys Asn Pro Asn Phe Lys Leu Gly Thr Ala Val Gln
115 120 125Asp Glu Val Ile Thr Ser Leu Gly Lys Leu Ile Gly Asn Ala
Ser Ala 130 135 140Asn Ala Glu Val Val Asn Asn Cys Val Pro Val Leu
Lys Gln Phe Arg145 150 155 160Glu Asn Leu Asn Gln Tyr Ala Pro Asp
Tyr Val Lys Gly Thr Ala Val 165 170 175Asn Glu Leu Ile Lys Gly Ile
Glu Phe Asp Phe Ser Gly Ala Ala Tyr 180 185 190Glu Lys Asp Val Lys
Thr Met Pro Trp Tyr Gly Lys Ile Asp Pro Phe 195 200 205Ile Asn Glu
Leu Lys Ala Leu Gly Leu Tyr Gly Asn Ile Thr Ser Ala 210 215 220Thr
Glu Trp Ala Ser Asp Val Gly Ile Tyr Tyr Leu Ser Lys Phe Gly225 230
235 240Leu Tyr Ser Thr Asn Arg Asn Asp Ile Val Gln Ser Leu Glu Lys
Ala 245 250 255Val Asp Met Tyr Lys Tyr Gly Lys Ile Ala Phe Val Ala
Met Glu Arg 260 265 270Ile Thr Trp Asp Tyr Asp Gly Ile Gly Ser Asn
Gly Lys Lys Val Asp 275 280 285His Asp Lys Phe Leu Asp Asp Ala Glu
Lys His Tyr Leu Pro Lys Thr 290 295 300Tyr Thr Phe Asp Asn Gly Thr
Phe Ile Ile Arg Ala Gly Glu Lys Val305 310 315 320Ser Glu Glu Lys
Ile Lys Arg Leu Tyr Trp Ala Ser Arg Glu Val Lys 325 330 335Ser Gln
Phe His Arg Val Val Gly Asn Asp Lys Ala Leu Glu Val Gly 340 345
350Asn Ala Asp Asp Val Leu Thr Met Lys Ile Phe Asn Ser Pro Glu Glu
355 360 365Tyr Lys Phe Asn Thr Asn Ile Asn Gly Val Ser Thr Asp Asn
Gly Gly 370 375 380Leu Tyr Ile Glu Pro Arg Gly Thr Phe Tyr Thr Tyr
Glu Arg Thr Pro385 390 395 400Gln Gln Ser Ile Phe Ser Leu Glu Glu
Leu Phe Arg His Glu Tyr Thr 405 410 415His Tyr Leu Gln Ala Arg Tyr
Leu Val Asp Gly Leu Trp Gly Gln Gly 420 425 430Pro Phe Tyr Glu Lys
Asn Arg Leu Thr Trp Phe Asp Glu Gly Thr Ala 435 440 445Glu Phe Phe
Ala Gly Ser Thr Arg Thr Ser Gly Val Leu Pro Arg Lys 450 455 460Ser
Ile Leu Gly Tyr Leu Ala Lys Asp Lys Val Asp His Arg Tyr Ser465 470
475 480Leu Lys Lys Thr Leu Asn Ser Gly Tyr Asp Asp Ser Asp Trp Met
Phe 485 490 495Tyr Asn Tyr Gly Phe Ala Val Ala His Tyr Leu Tyr Glu
Lys Asp Met 500 505 510Pro Thr Phe Ile Lys Met Asn Lys Ala Ile Leu
Asn Thr Asp Val Lys 515 520 525Ser Tyr Asp Glu Ile Ile Lys Lys Leu
Ser Asp Asp Ala Asn Lys Asn 530 535 540Thr Glu Tyr Gln Asn His Ile
Gln Glu Leu Ala Asp Lys Tyr Gln Gly545 550 555 560Ala Gly Ile Pro
Leu Val Ser Asp Asp Tyr Leu Lys Asp His Gly Tyr 565 570 575Lys Lys
Ala Ser Glu Val Tyr Ser Glu Ile Ser Lys Ala Ala Ser Leu 580 585
590Thr Asn Thr Ser Val Thr Ala Glu Lys Ser Gln Tyr Phe Asn Thr Phe
595 600 605Thr Leu Arg Gly Thr Tyr Thr Gly Glu Thr Ser Lys Gly Glu
Phe Lys 610 615 620Asp Trp Asp Glu Met Ser Lys Lys Leu Asp Gly Thr
Leu Glu Ser Leu625 630 635 640Ala Lys Asn Ser Trp Ser Gly Tyr Lys
Thr Leu Thr Ala Tyr Phe Thr 645 650 655Asn Tyr Arg Val Thr Ser Asp
Asn Lys Val Gln Tyr Asp Val Val Phe 660 665 670His Gly Val Leu Thr
Asp Asn Ala Asp Ile Ser Asn Asn Lys Ala Pro 675 680 685Ile Ala Lys
Val Thr Gly Pro Ser Thr Gly Ala Val Gly Arg Asn Ile 690 695 700Glu
Phe Ser Gly Lys Asp Ser Lys Asp Glu Asp Gly Lys Ile Val Ser705 710
715 720Tyr Asp Trp Asp Phe Gly Asp Gly Ala Thr Ser Arg Gly Lys Asn
Ser 725 730 735Val His Ala Tyr Lys Lys Thr Gly Thr Tyr Asn Val Thr
Leu Lys Val 740 745 750Thr Asp Asp Lys Gly Ala Thr Ala Thr Glu Ser
Phe Thr Ile Glu Ile 755 760 765Lys Asn Glu Asp Thr Thr Thr Pro Ile
Thr Lys Glu Met Glu Pro Asn 770 775 780Asp Asp Ile Lys Glu Ala Asn
Gly Pro Ile Val Glu Gly Val Thr Val785 790 795 800Lys Gly Asp Leu
Asn Gly Ser Asp Asp Ala Asp Thr Phe Tyr Phe Asp 805 810 815Val Lys
Glu Asp Gly Asp Val Thr Ile Glu Leu Pro Tyr Ser Gly Ser 820 825
830Ser Asn Phe Thr Trp Leu Val Tyr Lys Glu Gly Asp Asp Gln Asn His
835 840 845Ile Ala Ser Gly Ile Asp Lys Asn Asn Ser Lys Val Gly Thr
Phe Lys 850 855 860Ala Thr Lys Gly Arg His Tyr Val Phe Ile Tyr Lys
His Asp Ser Ala865 870 875 880Ser Asn Ile Ser Tyr Ser Leu Asn Ile
Lys Gly Leu Gly Asn Glu Lys 885 890 895Leu Lys Glu Lys Glu Asn Asn
Asp Ser Ser Asp Lys Ala Thr Val Ile 900 905 910Pro Asn Phe Asn Thr
Thr Met Gln Gly Ser Leu Leu Gly Asp Asp Ser 915 920 925Arg Asp Tyr
Tyr Ser Phe Glu Val Lys Glu Glu Gly Glu Val Asn Ile 930 935 940Glu
Leu Asp Lys Lys Asp Glu Phe Gly Val Thr Trp Thr Leu His Pro945 950
955 960Glu Ser Asn Ile Asn Asp Arg Ile Thr Tyr Gly Gln Val Asp Gly
Asn 965 970 975Lys Val Ser Asn Lys Val Lys Leu Arg Pro Gly Lys Tyr
Tyr Leu Leu 980 985 990Val Tyr Lys Tyr Ser Gly Ser Gly Asn Tyr Glu
Leu Arg Val Asn Lys 995 1000 10052991PRTClostridium histolyticum
2Ala Val Asp Lys Asn Asn Ala Thr Ala Ala Val Gln Asn Glu Ser Lys1 5
10 15Arg Tyr Thr Val Ser Tyr Leu Lys Thr Leu Asn Tyr Tyr Asp Leu
Val 20 25 30Asp Leu Leu Val Lys Thr Glu Ile Glu Asn Leu Pro Asp Leu
Phe Gln 35 40 45Tyr Ser Ser Asp Ala Lys Glu Phe Tyr Gly Asn Lys Thr
Arg Met Ser 50 55 60Phe Ile Met Asp Glu Ile Gly Arg Arg Ala Pro Gln
Tyr Thr Glu Ile65 70 75 80Asp His Lys Gly Ile Pro Thr Leu Val Glu
Val Val Arg Ala Gly Phe 85 90 95Tyr Leu Gly Phe His Asn Lys Glu Leu
Asn Glu Ile Asn Lys Arg Ser 100 105 110Phe Lys Glu Arg Val Ile Pro
Ser Ile Leu Ala Ile Gln Lys Asn Pro 115 120 125Asn Phe Lys Leu Gly
Thr Glu Val Gln Asp Lys Ile Val Ser Ala Thr 130 135 140Gly Leu Leu
Ala Gly Asn Glu Thr Ala Pro Pro Glu Val Val Asn Asn145 150 155
160Phe Thr Pro Ile Ile Gln Asp Cys Ile Lys Asn Met Asp Arg Tyr Ala
165 170 175Leu Asp Asp Leu Lys Ser Lys Ala Leu Phe Asn Val Leu Ala
Ala Pro 180 185 190Thr Tyr Asp Ile Thr Glu Tyr Leu Arg Ala Thr Lys
Glu Lys Pro Glu 195 200 205Asn Thr Pro Trp Tyr Gly Lys Ile Asp Gly
Phe Ile Asn Glu Leu Lys 210 215 220Lys Leu Ala Leu Tyr Gly Lys Ile
Asn Asp Asn Asn Ser Trp Ile Ile225 230 235 240Asp Asn Gly Ile Tyr
His Ile Ala Pro Leu Gly Lys Leu His Ser Asn 245 250 255Asn Lys Ile
Gly Ile Glu Thr Leu Thr Glu Val Met Lys Ile Tyr Pro 260 265 270Tyr
Leu Ser Met Gln His Leu Gln Ser Ala Asp Gln Ile Glu Arg His 275 280
285Tyr Asp Ser Lys Asp Ala Glu Gly Asn Lys Ile Pro Leu Asp Lys Phe
290 295 300Lys Lys Glu Gly Lys Glu Lys Tyr Cys Pro Lys Thr Tyr Thr
Phe Asp305 310 315 320Asp Gly Lys Val Ile Ile Lys Ala Gly Ala Arg
Val Glu Glu Glu Lys 325 330 335Val Lys Arg Leu Tyr Trp Ala Ser Lys
Glu Val Asn Ser Gln Phe Phe 340 345 350Arg Val Tyr Gly Ile Asp Lys
Pro Leu Glu Glu Gly Asn Pro Asp Asp 355 360 365Ile Leu Thr Met Val
Ile Tyr Asn Ser Pro Glu Glu Tyr Lys Leu Asn 370 375 380Ser Val Leu
Tyr Gly Tyr Asp Thr Asn Asn Gly Gly Met Tyr Ile Glu385 390 395
400Pro Asp Gly Thr Phe Phe Thr Tyr Glu Arg Lys Ala Glu Glu Ser Thr
405 410 415Tyr Thr Leu Glu Glu Leu Phe Arg His Glu Tyr Thr His Tyr
Leu Gln 420 425 430Gly Arg Tyr Ala Val Pro Gly Gln Trp Gly Arg Thr
Lys Leu Tyr Asp 435 440 445Asn Asp Arg Leu Thr Trp Tyr Glu Glu Gly
Gly Ala Glu Leu Phe Ala 450 455 460Gly Ser Thr Arg Thr Ser Gly Ile
Leu Pro Arg Lys Ser Ile Val Ser465 470 475 480Asn Ile His Asn Thr
Thr Arg Asn Asn Arg Tyr Lys Leu Ser Asp Thr 485 490 495Val His Ser
Lys Tyr Gly Ala Ser Phe Glu Phe Tyr Asn Tyr Ala Cys 500 505 510Met
Phe Met Asp Tyr Met Tyr Asn Lys Asp Met Gly Ile Leu Asn Lys 515 520
525Leu Asn Asp Leu Ala Lys Asn Asn Asp Val Asp Gly Tyr Asp Asn Tyr
530 535 540Ile Arg Asp Leu Ser Ser Asn His Ala Leu Asn Asp Lys Tyr
Gln Asp545 550 555 560His Met Gln Glu Arg Ile Asp Asn Tyr Glu Asn
Leu Thr Val Pro Phe 565 570 575Val Ala Asp Asp Tyr Leu Val Arg His
Ala Tyr Lys Asn Pro Asn Glu 580 585 590Ile Tyr Ser Glu Ile Ser Glu
Val Ala Lys Leu Lys Asp Ala Lys Ser 595 600 605Glu Val Lys Lys Ser
Gln Tyr Phe Ser Thr Phe Thr Leu Arg Gly Ser 610 615 620Tyr Thr Gly
Gly Ala Ser Lys Gly Lys Leu Glu Asp Gln Lys Ala Met625 630 635
640Asn Lys Phe Ile Asp Asp Ser Leu Lys Lys Leu Asp Thr Tyr Ser Trp
645 650 655Ser Gly Tyr Lys Thr Leu Thr Ala Tyr Phe Thr Asn Tyr Lys
Val Asp 660 665 670Ser Ser Asn Arg Val Thr Tyr Asp Val Val Phe His
Gly Tyr Leu Pro 675 680 685Asn Glu Gly Asp Ser Lys Asn Ser Leu Pro
Tyr Gly Lys Ile Asn Gly 690 695 700Thr Tyr Lys Gly Thr Glu Lys Glu
Lys Ile Lys Phe Ser Ser Glu Gly705 710 715 720Ser Phe Asp Pro Asp
Gly Lys Ile Val Ser Tyr Glu Trp Asp Phe Gly 725 730 735Asp Gly Asn
Lys Ser Asn Glu Glu Asn Pro Glu His Ser Tyr Asp Lys 740 745 750Val
Gly Thr Tyr Thr Val Lys Leu Lys Val Thr Asp Asp Lys Gly Glu 755 760
765Ser Ser Val Ser Thr Thr Thr Ala Glu Ile Lys Asp Leu Ser Glu Asn
770 775 780Lys Leu Pro Val Ile Tyr Met His Val Pro Lys Ser Gly Ala
Leu Asn785 790 795 800Gln Lys Val Val Phe Tyr Gly Lys Gly Thr Tyr
Asp Pro Asp Gly Ser 805 810 815Ile Ala Gly Tyr Gln Trp Asp Phe Gly
Asp Gly Ser Asp Phe Ser Ser 820 825 830Glu Gln Asn Pro Ser His Val
Tyr Thr Lys Lys Gly Glu Tyr Thr Val 835 840 845Thr Leu Arg Val Met
Asp Ser Ser Gly Gln Met Ser Glu Lys Thr Met 850 855 860Lys Ile Lys
Ile Thr Asp Pro Val Tyr Pro Ile Gly Thr Glu Lys Glu865 870 875
880Pro Asn Asn Ser Lys Glu Thr Ala Ser Gly Pro Ile Val Pro Gly Ile
885 890 895Pro Val Ser Gly Thr Ile Glu Asn Thr Ser Asp Gln Asp Tyr
Phe Tyr 900 905 910Phe Asp Val Ile Thr Pro Gly Glu Val Lys Ile Asp
Ile Asn Lys Leu 915 920 925Gly Tyr Gly Gly Ala Thr Trp Val Val Tyr
Asp Glu Asn Asn Asn Ala 930 935 940Val Ser Tyr Ala Thr Asp Asp Gly
Gln Asn Leu Ser Gly Lys Phe Lys945 950 955 960Ala Asp Lys Pro Gly
Arg Tyr Tyr Ile His Leu Tyr Met Phe Asn Gly 965 970 975Ser Tyr Met
Pro Tyr Arg Ile Asn Ile Glu Gly Ser Val Gly Arg 980 985 990
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