U.S. patent application number 11/018736 was filed with the patent office on 2005-06-09 for crystallisation of a glp-1 analogue.
Invention is credited to Arentsen, Anne Charlotte.
Application Number | 20050124542 11/018736 |
Document ID | / |
Family ID | 28457452 |
Filed Date | 2005-06-09 |
United States Patent
Application |
20050124542 |
Kind Code |
A1 |
Arentsen, Anne Charlotte |
June 9, 2005 |
Crystallisation of a GLP-1 analogue
Abstract
Crystals of glucagon-like peptide-1 (GLP-1) and GLP-1 analogues,
and processes for preparation of crystals of GLP-1 and GLP-1
analogues.
Inventors: |
Arentsen, Anne Charlotte;
(Holte, DK) |
Correspondence
Address: |
NOVO NORDISK, INC.
PATENT DEPARTMENT
100 COLLEGE ROAD WEST
PRINCETON
NJ
08540
US
|
Family ID: |
28457452 |
Appl. No.: |
11/018736 |
Filed: |
December 21, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11018736 |
Dec 21, 2004 |
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09769692 |
Jan 25, 2001 |
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6844321 |
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60183300 |
Feb 17, 2000 |
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Current U.S.
Class: |
514/11.7 ;
514/7.2; 530/399 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 2299/00 20130101; C07K 14/605 20130101 |
Class at
Publication: |
514/012 ;
530/399 |
International
Class: |
C07K 014/605 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 31, 2000 |
DK |
PA 2000 00156 |
Claims
We claim:
1. A process for producing crystals of a GLP-1 analogue comprising:
a) preparing an aqueous solution comprising a GLP-1 analogue, a
salt, and an organic solvent, and b) isolation of the crystals
after formation.
2. The process according to claim 1 wherein in step a) adjusting pH
to pl-4<pH<pl, or to pl<pH<pl+4, wherein pl is the
isoelectric point of the GLP-1 analogue.
3. The process according to claim 1 wherein the crystals are needle
shaped crystals of a GLP-1 analogue.
4. The process according to claim 1 wherein the crystals has a
length of at least 0.5 .mu.m.
5. The process according to claim 1 wherein the GLP-1 analogue in
the aqueous solution has a purity of less than 95%, as measured by
HPLC.
6. The process according to claim 1 wherein the GLP-1 analogue in
the aqueous solution is present in a concentration of at least 0.5
mg/ml.
7. The process according to claim 1 wherein the GLP-1 analogue is
selected from non-synthetic GLP-1 analogues.
8. The process according to claim 1 wherein the GLP-1 analogue is
Arg.sup.34GLP-1 (7-37) or Arg.sup.26GLP-1 (7-37).
9. The process according to claim 1 wherein the salt is present in
a concentration of at least 25 mM.
10. The process according to claim 1 wherein the organic solvent is
present in a concentration of from 0.5 to 50% (vol/vol).
11. The process according to claim 1 wherein the organic solvent is
selected from C.sub.1-6alkanol, C.sub.16-alkenol, C.sub.1-6alkynol,
urea, guanidine, C.sub.1-6alkanoic acid, ketone, DMSO,
C.sub.2-4-glycol, C.sub.3-7-polyalcohol including sugars, or
mixtures thereof.
12. A method for producing a GLP-1 analogue or a GLP-1 analogue
whereto is attached a lipophilic substituent comprising the steps:
a) preparing an aqueous solution comprising a GLP-1 analogue, a
salt, and an organic solvent, and b) isolation of the crystals
after formation.
13. Crystals of a GLP-1 analogue optainable by the method according
to claim 12.
14. Needle shaped crystals of a GLP-1 analogue.
15. A pharmaceutical composition comprising needle shaped crystals
of a GLP-1 analogue and a pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
09/769,692 filed on Jan. 25, 2001, now pending and claims priority
under 35 U.S.C. 119 of Danish application no. PA 2000 00156 filed
Jan. 31, 2000; and of U.S. provisional application No. 60/183,300
filed on Feb. 17, 2002; the contents of which are fully
incorporated herein by reference.
[0002] The present invention relates to novel crystals of GLP-1 and
analogues thereof, such as needle shaped crystals, and processes
for the preparation of crystals of GLP-1 and analogues thereof.
BACKGROUND
[0003] The hormones regulating insulin secretion belong to the
so-called enteroinsular axis, designating a group of hormones,
released from the gastrointestinal mucosa in response to the
presence and absorption of nutrients in the gut, which promote an
early and potentiated release of insulin. The enhancing effect on
insulin secretion, the so-called incretin effect, is probably
essential for a normal glucose tolerance. Many of the
gastrointestinal hormones, including gastrin and secretin
(cholecystokinin is not insulinotropic in man), are insulinotropic,
but the only physiologically important ones, those that are
responsible for the incretin effect, are the glucose-dependent
insulinotropic polypeptide, GIP, and glucagon-like peptide-1
(GLP-1). Because of its insulinotropic effect, GIP, isolated in
1973 (1) immediately attracted considerable interest among
diabetologists. However, numerous investigations carried out during
the following years clearly indicated that a defective secretion of
GIP was not involved in the pathogenesis of insulin-dependent
diabetes mellitus (IDDM) or non-insulin-dependent diabetes mellitus
(NIDDM) (2). Furthermore, as an insulinotropic hormone, GIP was
found to be almost ineffective in NIDDM (2). The other incretin
hormone, GLP-1 is the most potent insulinotropic substance known
(3). Unlike GIP, it is surprisingly effective in stimulating
insulin secretion in NIDDM patients. In addition, and in contrast
to the other insulinotropic hormones (perhaps with the exception of
secretin) it also potently inhibits glucagon secretion. Because of
these actions it has pronounced blood glucose lowering effects
particularly in patients with NIDDM.
DESCRIPTION OF THE INVENTION
[0004] Human GLP-1 is a 37 amino acid residue peptide originating
from preproglucagon which is synthesised i.a. in the L-cells in the
distal ileum, in the pancreas and in the brain. Processing of
preproglucagon to give GLP-1(7-36)amide, GLP-1(7-37) and GLP-2
occurs mainly in the L-cells. A simple system is used to describe
fragments and analogues of this peptide. Thus, for example,
Gly.sup.8-GLP-1 (7-37) designates a fragment of GLP-1 formally
derived from GLP-1 by deleting the amino acid residues Nos. 1 to 6
and substituting the naturally occurring amino acid residue in
position 8 (Ala) by Gly. Similarly, Lys.sup.34(N.sup..epsilon.-
-tetradecanoyl)-GLP-1(7-37) designates GLP-1(7-37) wherein the
.epsilon.-amino group of the Lys residue in position 34 has been
tetradecanoylated.
[0005] GLP-1 and analogues thereof can be produced by a method
which comprises culturing or fermenting a host cell containing a
DNA sequence encoding the GLP-1 analogue and capable of expressing
said analogue in a suitable nutrient medium under conditions
permitting the expression of the peptide, after which the resulting
GLP-1 analogue is recovered from the culture or fermentation
broth.
[0006] The implementation of a crystallisation step in the
manufacturing process for the preparation of a GLP-1 analogue
resulted in removal of coloured compounds (coloured impurities)
from the fermentation broth, reduction of yeast host cell proteins,
such as Saccharomyces cerevisiae proteins (SCP) as well as removal
of water, and low loss of the GLP-1 analog from the mother
liquor.
[0007] The GLP-1 analog was then re-dissolved and further purified
by conventional High Performance Cation Exchange Chromatography
(HP-CIEC) and Reverse Phase High Performance Liquid Chromatography
(RP-HPLC) followed by a second crystallisation step at the pl of
the GLP-1 analogue. Hereafter, the analogue was acylated, e.g. as
disclosed in WO 98/08871, and the resulting solution containing
mono-acylated GLP-1 analogue was further purified by conventional
RP-HPLC and finally precipitated at pl of the mono-acylated GLP-1
analogue.
[0008] Thus, the resulting crystals are an important and useful
intermediate product in the manufacturing process for preparing a
GLP-1 analogue and for preparing a mono-acylated GLP-1 analogue.
The resulting crystals of the GLP-1 analogue are also useful in the
preparation of a pharmaceutical composition, such as an injectable
drug, comprising the crystals and a pharmaceutically acceptable
carrier.
[0009] Accordingly, the present invention relates to a process for
producing crystals of a GLP-1 analogue comprising:
[0010] a) preparing an aqueous solution comprising a GLP-1
analogue, a salt, and an organic solvent, and
[0011] b) isolating the crystals after formation.
[0012] The GLP-1 analogue to be crystallized in step a) is either
substantially pure or is impure. The purity can be measured by
analytical HPLC and/or capillary electrophoresis.
[0013] In step a) a buffer may optionally be added to said
solution. Usually it is most convenient to add a buffer to the
solution, such as any buffer including but not limited to: citrate
buffers, phosphate buffers, tris buffers, bis-Tris buffer, borate
buffers, lactate buffers, glycyl glycin buffers, arginine buffers,
carbonate buffers, acetate buffers, glutamate buffers, ammonium
buffers, glycin buffers, alkylamine buffers, aminoethyl alcohol
buffers, ethylenediamine buffers, tri-ethanol amine, imidazole
buffers, pyridine buffers and barbiturate buffers and mixtures
thereof. The concentration of buffer added is easily decided by the
skilled practitioner using his common general knowledge, but will
usually be in the range from 0 mM to 100 mM, such as 0.5 mM to 50
mM, e.g. 5-10 mM.
[0014] Further in step a) pH of the solution may be adjusted by
means of an acid or base to keep it constant or it may vary,
provided that pl of the GLP-1 analogue is not reached. Usually it
is most convenient to adjust pH with an acid, e.g. HCl, if pH of
the aqueous solution is above the isoelectric point (hereinafter
pl) of the GLP-1 analogue, or with a base, e.g. NaOH, if pH of the
aqueous solution is below the pl of the GLP-1 analogue. The pH of
the solution is easily decided by the skilled practitioner using
his common general knowledge, but will usually be within a certain
range from the pl of the GLP-1 analogue, such as between about pl-4
(id est, pl minus 4) and pl or between about pl and pl+4 (id est pl
plus 4), depending on whether the aqueous solution of the GLP-1
analogue is below the pl or above the pl. The pH of the aqueous
solution may be adjusted relatively far from the pl of the GLP-1
analogue, such as at about pl-4 or pl+4 and then, optionally, by
step or linear gradient, be driven towards the pl until formation
of crystals occur, in particular needle shaped crystals. In case of
the GLP-1 analogue being Arg.sup.34GLP-1(7-37), having a pl of
about 5.4, it is preferred to prepare an aqueous solution of
Arg.sup.34GLP-1(7-37) above pl, preferably at about pH 8.5-9.5, and
then adjust the pH to about pH 6-7 with an acid, e.g. HCl. In one
embodiment of the invention the pH is adjusted so that
pl-4<pH<pl, preferably pl-2<pH<pl. In another
embodiment of the invention the pH is adjusted so that
pl<pH<pl+4, preferably pl<pH<pl+2.
[0015] Further in step a) an excipient may optionally be added that
influence the stability or solubility of the GLP-1 analogue.
[0016] In one embodiment of the invention the crystals are needle
shaped crystals of a GLP-1 analogue having a length of at least 0.5
.mu.m. In another embodiment of the invention the crystals are
needle shaped crystals of a GLP-1 analogue having a length of at
least 2 .mu.m. In a further embodiment of the invention the
crystals are needle shaped crystals of a GLP-1 analogue having a
length of at least 8 .mu.m. In a further embodiment of the
invention the crystals are needle shaped crystals of a GLP-1
analogue having a length of 0.5 .mu.m to 50 .mu.m, such as 2 .mu.m
to 50 .mu.m, e.g. 2 .mu.m to 30 .mu.m. In a further embodiment of
the invention the crystals are needle shaped crystals of a GLP-1
analogue having a length of 8 .mu.m to 50 .mu.m.
[0017] In another embodiment of the invention the GLP-1 analogue to
be crystallized has a purity of less than 98%, as measured by HPLC.
In a further embodiment of the invention the GLP-1 analogue to be
crystallized has a purity of less than 95%, such as less than 90%,
as measured by HPLC.
[0018] In another embodiment of the invention the GLP-1 analogue to
be crystallized has a purity of more than 20%, such as more than
30%, as measured by HPLC. In a further embodiment of the invention
the GLP-1 analogue to be crystallized has a purity between 20% and
90%, such as a purity between 20% and 50%, as measured by HPLC.
[0019] In a further embodiment of the invention the GLP-1 analogue
in the aqueous solution is present in a concentration of at least
0.5 mg/ml.
[0020] In a further embodiment of the invention the GLP-1 analogue
in the aqueous solution is present in a concentration of from 0.5
mg/ml to 20 mg/ml, such as 2-10 mg/ml.
[0021] In a further embodiment of the invention the GLP-1 analogue
is selected from non-synthetic GLP-1 analogues.
[0022] In a further embodiment of the invention the GLP-1 analogue
is selected from the Thr.sup.8, Met.sup.8, Gly.sup.8 and Val.sup.8
analogues of GLP-1(7-37) and GLP-1(7-36) amide, more preferred the
Gly.sup.8 and Val.sup.8 analogues of GLP-1(7-37) and GLP-1(7-36)
amide, most preferred the Val.sup.8 analogues of GLP-1(7-37) and
GLP-1(7-36) amide.
[0023] In a further embodiment of the invention the GLP-1 analogue
has the formula II:
1 (SEQ ID: NO 1) 7 8 9 10 11 12 13 14 15 16 17
His-Xaa-Xaa-Gly-Xaa-Phe-Thr-Xaa-Asp-Xaa-Xaa- 18 19 20 21 22 23 24
25 26 27 28 Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Phe- 29 30 31
32 33 34 35 36 37 38 Ile-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-X- aa 39
40 41 42 43 44 45 Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa (II)
[0024] wherein
[0025] Xaa at position 8 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu,
Asp, Met, or Lys,
[0026] Xaa at position 9 is Glu, Asp, or Lys,
[0027] Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, Ile, Val,
Glu, Asp, or Lys,
[0028] Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, lie, Val,
Glu, Asp, or Lys,
[0029] Xaa at position 16 is Val, Ala, Gly, Ser, Thr, Leu, Ile,
Tyr, Glu, Asp, or Lys,
[0030] Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, Ile, Val,
Glu, Asp, or Lys,
[0031] Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, Ile, Val,
Glu, Asp, or Lys,
[0032] Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, or Lys,
[0033] Xaa at position 20 is Leu, Ala, Gly, Ser, Thr, Leu, Ile,
Val, Glu, Asp, or Lys,
[0034] Xaa at position 21 is Glu, Asp, or Lys,
[0035] Xaa at position 22 is Gly, Ala, Ser, Thr, Leu, Ile, Val,
Glu, Asp, or Lys,
[0036] Xaa at position 23 is Gin, Asn, Arg, Glu, Asp, or Lys,
[0037] Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, Ile, Val,
Arg, Glu, Asp, or Lys,
[0038] Xaa at position 25 is Ala, Gly, Ser, Thr, Leu, Ile, Val,
Glu, Asp, or Lys,
[0039] Xaa at position 26 is Lys, Arg, Gin, Glu, Asp, or His,
[0040] Xaa at position 27 is Glu, Asp, or Lys,
[0041] Xaa at position 30 is Ala, Gly, Ser, Thr, Leu, Ile, Val,
Glu, Asp, or Lys,
[0042] Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys,
[0043] Xaa at position 32 is Leu, Gly, Ala, Ser, Thr, Ile, Val,
Glu, Asp, or Lys,
[0044] Xaa at position 33 is Val, Gly, Ala, Ser, Thr, Leu, Ile,
Glu, Asp, or Lys,
[0045] Xaa at position 34 is Lys, Arg, Glu, Asp, or His,
[0046] Xaa at position 35 is Gly, Ala, Ser, Thr, Leu; Ile, Val,
Glu, Asp, or Lys,
[0047] Xaa at position 36 is Arg, Lys, Glu, Asp, or His,
[0048] Xaa at position 37 is Gly, Ala, Ser, Thr, Leu, Ile, Val,
Glu, Asp, or Lys, or is deleted,
[0049] Xaa at position 38 is Arg, Lys, Glu, Asp, or His, or is
deleted,
[0050] Xaa at position 39 is Arg, Lys, Glu, Asp, or His, or is
deleted,
[0051] Xaa at position 40 is Asp, Glu, or Lys, or is deleted,
[0052] Xaa at position 41 is Phe, Trp, Tyr, Glu, Asp, or Lys, or is
deleted,
[0053] Xaa at position 42 is Pro, Lys, Glu, or Asp, or is
deleted,
[0054] Xaa at position 43 is Glu, Asp, or Lys, or is deleted,
[0055] Xaa at position 44 is Glu, Asp, or Lys, or is deleted,
and
[0056] Xaa at position 45 is Val, Glu, Asp, or Lys, or is deleted,
or
[0057] (a) a C-1-6-ester thereof, (b) amide, C-1-6-alkylamide, or
C-1-6-dialkylamide thereof and/or (c) a pharmaceutically acceptable
salt thereof, provided that
[0058] (i) when the amino acid at position 37, 38, 39, 40, 41, 42,
43 or 44 is deleted, then each amino acid downstream of the amino
acid is also deleted.
[0059] In a further embodiment of the GLP-1 analogue of formula II,
the amino acids at positions 37-45 are absent.
[0060] In another embodiment of the GLP-1 analogue of formula II,
the amino acids at positions 38-45 are absent.
[0061] In another embodiment of the GLP-1 analogue of formula II,
the amino acids at positions 39-45 are absent.
[0062] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 8 is Ala, Gly, Ser, Thr, Met, or Val.
[0063] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 8 is Gly, Thr, Met, or Val.
[0064] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 8 is Val.
[0065] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 9 is Glu.
[0066] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 11 is Thr.
[0067] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 14 is Ser.
[0068] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 16 is Val.
[0069] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 17 is Ser.
[0070] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 18 is Ser, Lys, Glu, or Asp.
[0071] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 19 is Tyr, Lys, Glu, or Asp.
[0072] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 20 is Leu, Lys, Glu, or Asp.
[0073] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 21 is Glu, Lys, or Asp.
[0074] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 22 is Gly, Glu, Asp, or Lys.
[0075] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 23 is Gin, Glu, Asp, or Lys.
[0076] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 24 is Ala, Glu, Asp, or Lys.
[0077] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 25 is Ala, Glu, Asp, or Lys.
[0078] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 26 is Lys, Glu, Asp, or Arg.
[0079] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 27 is Glu, Asp, or Lys.
[0080] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 30 is Ala, Glu, Asp, or Lys.
[0081] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 31 is Trp, Glu, Asp, or Lys.
[0082] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 32 is Leu, Glu, Asp, or Lys.
[0083] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 33 is Val, Glu, Asp, or Lys.
[0084] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 34 is Lys, Arg, Glu, or Asp.
[0085] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 35 is Gly, Glu, Asp, or Lys.
[0086] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 36 is Arg, Lys, Glu, or Asp.
[0087] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 37 is Gly, Glu, Asp, or Lys.
[0088] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 38 is Arg, or Lys, or is deleted.
[0089] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 39 is deleted.
[0090] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 40 is deleted.
[0091] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 41 is deleted.
[0092] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 42 is deleted.
[0093] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 43 is deleted.
[0094] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 44 is deleted.
[0095] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 45 is deleted.
[0096] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 26 is Arg, each of Xaa at positions 37-45 is
deleted, and each of the other Xaa is the amino acid in native
GLP-1 (7-36).
[0097] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 26 is Arg, each of Xaa at positions 38-45 is
deleted, and each of the other Xaa is the amino acid in native
GLP-1 (7-37).
[0098] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 26 is Arg, each of Xaa at positions 39-45 is
deleted, and each of the other Xaa is the amino acid in native
GLP-1 (7-38).
[0099] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 34 is Arg, each of Xaa at positions 37-45 is
deleted, and each of the other Xaa is the amino acid in native
GLP-1 (7-36).
[0100] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 34 is Arg, each of Xaa at positions 38-45 is
deleted, and each of the other Xaa is the amino acid in native
GLP-1 (7-37).
[0101] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 34 is Arg, each of Xaa at positions 39-45 is
deleted, and each of the other Xaa is the amino acid in native
GLP-1 (7-38).
[0102] In another embodiment of the GLP-1 analogue of formula II,
Xaa at positions 26 and 34 is Arg, Xaa at position 36 is Lys, each
of Xaa at positions 37-45 is deleted, and each of the other Xaa is
the amino acid in native GLP-1 (7-36).
[0103] In another embodiment of the GLP-1 analogue of formula II,
Xaa at positions 26 and 34 is Arg, Xaa at position 36 is Lys, each
of Xaa at positions 38-45 is deleted, and each of the other Xaa is
the amino acid in native GLP-1 (7-37).
[0104] In another embodiment of the GLP-1 analogue of formula II,
Xaa at positions 26 and 34 is Arg, Xaa at position 36 is Lys, each
of Xaa at positions 39-45 is deleted, and each of the other Xaa is
the amino acid in native GLP-1 (7-38).
[0105] In another embodiment of the GLP-1 analogue of formula II,
Xaa at positions 26 and 34 is Arg, Xaa at position 38 is Lys, each
of Xaa at positions 39-45 is deleted, and each of the other Xaa is
the amino acid in native GLP-1 (7-38).
[0106] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 8 is Thr, Ser, Gly, or Val, Xaa at position 37 is
Glu, Xaa at position 36 is Lys, each of Xaa at positions 38-45 is
deleted, and each of the other Xaa is the amino acid in native
GLP-1 (7-37).
[0107] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 8 is Thr, Ser, Gly, or Val, Xaa at position 37 is
Glu, Xaa at position 36 is Lys, each of Xaa at positions 39-45 is
deleted, and each of the other Xaa is the amino acid in native
GLP-1 (7-38).
[0108] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 8 is Thr, Ser, Gly or Val, Xaa at position 37 is
Glu, Xaa at position 38 is Lys, each of Xaa at positions 39-45 is
deleted, and each of the other Xaa is the amino acid in native
GLP-1 (7-38).
[0109] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 18, 23 or 27 is Lys, and Xaa at positions 26 and 34
is Arg, each of Xaa at positions 37-45 is deleted, and each of the
other Xaa is the amino acid in native GLP-1 (7-36).
[0110] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 18, 23 or 27 is Lys, and Xaa at positions 26 and 34
is Arg, each of Xaa at positions 38-45 is deleted, and each of the
other Xaa is the amino acid in native GLP-1(7-37).
[0111] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 18, 23 or 27 is Lys, and Xaa at positions 26 and 34
is Arg, each of Xaa at positions 39-45 is deleted, and each of the
other Xaa is the amino acid in native GLP-1 (7-38).
[0112] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 8 is Thr, Ser, Gly, or Val, Xaa at position 18, 23
or 27 is Lys, and Xaa at position 26 and 34 is Arg, each of Xaa at
positions 37-45 is deleted, and each of the other Xaa is the amino
acid in native GLP-1(7-36).
[0113] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 8 is Thr, Ser, Gly, or Val, Xaa at position 18, 23
or 27 is Lys, and Xaa at position 26 and 34 is Arg, each of Xaa at
positions 38-45 is deleted, and each of the other Xaa is the amino
acid in native GLP-1(7-37).
[0114] In another embodiment of the GLP-1 analogue of formula II,
Xaa at position 8 is Thr, Ser, Gly, or Val, Xaa at position 18, 23
or 27 is Lys, and Xaa at position 26 and 34 is Arg, each of Xaa at
positions 39-45 is deleted, and each of the other Xaa is the amino
acid in native GLP-1(7-38).
[0115] Such GLP-1 analogues of formula II includes, but is not
limited to, Arg.sup.26-GLP-1(7-37); Arg.sup.34-GLP-1(7-37);
Lys.sup.36-GLP-1(7-37); Arg.sup.26,34Lys.sup.36-GLP-1(7-37);
Arg.sup.26,34Lys.sup.38GLP-1(7-38);
Arg.sup.26,34Lys.sup.39-GLP-1(7-39);
Arg.sup.26,34Lys.sup.40-GLP-1(7-40);
Arg.sup.26Lys.sup.36-GLP-1(7-37); Arg 34Lys.sup.36-GLP-1(7-37);
Arg.sup.26Lys.sup.39-GLP-1(7-39); Arg.sup.34Lys.sup.40-GLP-1(7-40);
Arg.sup.26,34Lys.sup.36,39-GLP-1(7-39);
Arg.sup.26,34Lys.sup.36,40-GLP-1(- 7-40);
Gly.sup.8Arg.sup.26-GLP-1(7-37); Gly.sup.8Arg.sup.34-GLP-1(7-37);
Val.sup.8-GLP-1(7-37); Thr.sup.8-GLP-1(7-37);
Gly.sup.8-GLP-1(7-37); Met.sup.8-GLP-1(7-37);
Gly.sup.3Lys.sup.36-GLP-1(7-37);
Gly.sup.8Arg.sup.26,34Lys.sup.36-GLP-1(7-37);
Gly.sup.8Arg.sup.26,34Lys.s- up.39-GLP-1(7-39);
Gly.sup.8Arg.sup.26,34Lys.sup.40-GLP-1(7-40);
Gly.sup.8Arg.sup.26Lys.sup.36-GLP-1(7-37);
Gly.sup.8Arg.sup.34Lys.sup.36-- GLP-1(7-37);
Gly.sup.8Arg.sup.26Lys.sup.39-GLP-1(7-39);
Gly.sup.8Arg.sup.34Lys.sup.40-GLP-1(7-40);
Gly.sup.8Arg.sup.26,34Lys.sup.- 36,39-GLP-1(7-39);
Gly.sup.8Arg.sup.26,34Lys.sup.36,40-GLP-1(7-40); Arg
.sup.26,34Lys.sup.38GLP-1(7-38);
Arg.sup.26,34Lys.sup.39GLP-1(7-39);
Arg.sup.26,34Lys.sup.40GLP-1(7-40);
Arg.sup.26,34Lys.sup.41GLP-1(7-41);
Arg.sup.26,34Lys.sup.42GLP-1(7-42);
Arg.sup.26,34Lys.sup.43GLP-1(7-43);
Arg.sup.26Lys.sup.44GLP-1(7-44);
Arg.sup.26,34Lys.sup.45GLP-1(7-45);
Arg.sup.26,34Lys.sup.38GLP-1(1-38);
Arg.sup.26,34Lys.sup.39GLP-1(1-39);
Arg.sup.26,34Lys.sup.40GLP-1(1-40);
Arg.sup.26,34Lys.sup.41GLP-1(1-41);
Arg.sup.26,34Lys.sup.42GLP-1(1-42);
Arg.sup.26,34Lys.sup.43GLP-1(1-43);
Arg.sup.26,34Lys.sup.44GLP-1(1-44);
Arg.sup.26,34Lys.sup.45GLP-1(1-45);
Arg.sup.26,34Lys.sup.38GLP-1(2-38); Arg
.sup.26,34Lys.sup.39GLP-1(2-39);
Arg.sup.26,34Lys.sup.40GLP-1(2-40);
Arg.sup.26,34Lys.sup.41GLP-1(2-41); Arg.sup.26,34
Lys.sup.42GLP-1(2-42); Arg.sup.26,34Lys.sup.43GLP-1(2-43);
Arg.sup.26,34Lys.sup.44GLP-1(2-44);
Arg.sup.26,34Lys.sup.45GLP-1(2-45);
Arg.sup.26,34Lys.sup.38GLP-1(3-38);
Arg.sup.26,34Lys.sup.39GLP-1(3-39);
Arg.sup.26,34Lys.sup.40GLP-1(3-40);
Arg.sup.26,34Lys.sup.41GLP-1(3-41);
Arg.sup.26,34Lys.sup.42GLP-1(3-42);
Arg.sup.26,34Lys.sup.43GLP-1(3-43);
Arg.sup.26,34Lys.sup.44GLP-1(3-44);
Arg.sup.26,34Lys.sup.45GLP-1(345);
Arg.sup.26,34Lys.sup.38GLP-1(4-38);
Arg.sup.26,34Lys.sup.39GLP-1(4-39);
Arg.sup.26,34Lys.sup.40GLP-1(4-40);
Arg.sup.26,34Lys.sup.41GLP-1(4-41);
Arg.sup.26,34Lys.sup.42GLP-1(4-42);
Arg.sup.26,34Lys.sup.43GLP-1(4-43);
Arg.sup.26,34Lys.sup.44GLP-1(4-44);
Arg.sup.26,34Lys.sup.45GLP-1(4-45);
Arg.sup.26,34Lys.sup.38GLP-1(5-38);
Arg.sup.26,34Lys.sup.39GLP-1(5-39);
Arg.sup.26,34Lys.sup.40GLP-1(5-40);
Arg.sup.26,34Lys.sup.41GLP-1(5-41);
Arg.sup.26,34Lys.sup.42GLP-1(5-42);
Arg.sup.26,34Lys.sup.43GLP-1(5-43);
Arg.sup.26,34Lys.sup.44GLP-1(5-44);
Arg.sup.26,34Lys.sup.45GLP-1(5-45);
Arg.sup.26,34Lys.sup.38GLP-1(6-38);
Arg.sup.26,34Lys.sup.39GLP-1(6-39);
Arg.sup.26,34Lys.sup.40GLP-1(6-40);
Arg.sup.26,34Lys.sup.41GLP-1(6-41);
Arg.sup.26,34Lys.sup.42GLP-1(6-42);
Arg.sup.26,34Lys.sup.43GLP-1(6-43);
Arg.sup.26,34Lys.sup.44GLP-1(6-44); Arg .sup.26,34Lys
.sup.45GLP-1(6-45); Arg.sup.26Lys.sup.38GLP-1(1-38);
Arg.sup.34Lys.sup.38GLP-1(1-38);
Arg.sup.26,34Lys.sup.36,38GLP-1(1-38);
Arg.sup.26Lys.sup.38GLP-1(7-38); Arg.sup.34Lys.sup.38GLP-1(7-38);
Arg.sup.26,34Lys.sup.36,38GLP-1(7-38);
Arg.sup.26,34Lys.sup.38GLP-1(7-38);
Arg.sup.26Lys.sup.39GLP-1(1-39); Arg.sup.34Lys.sup.39GLP-1(1-39);
Arg.sup.26,34Lys.sup.36,39GLP-1(1-39);
Arg.sup.26Lys.sup.39GLP-1(7-39); Arg.sup.34Lys.sup.39GLP-1(7-39)
and Arg.sup.26,34Lys.sup.36,39GLP-1(7-39). Each one of these
specific GLP-1 analogues constitutes an alternative embodiment of
the invention.
[0116] In a further embodiment of the invention the GLP-1 analogue
has the formula III
A-HN-GLP-1(8-B)-X (III)
[0117] wherein
[0118] A is: 1
[0119] wherein R.sup.1, R.sup.2 and R.sup.3 are independently H,
lower alkyl having 1 to 6 carbon atoms, optionally substituted
phenyl, NH.sub.2, NH--CO-(lower alkyl), --OH, lower alkoxy having 1
to 6 carbon atoms, halogen, SO.sub.2-- (lower alkyl) or CF.sub.3,
said phenyl is optionally substituted with at least one group
selected from NH.sub.2, --OH, lower alkyl or lower alkoxy having
1-6 carbon atoms, halogen, SO.sub.2-(lower alkyl), NH--CO-(lower
alkyl) or CF.sub.3, or R.sup.1 and R.sup.2 may together form a
bond;
[0120] Y is a five or six membered ring system selected from the
group consisting of: 2
[0121] wherein Z is N, O or S, said ring system is optionally
substituted with one or more functional groups selected from the
group consisting of NH.sub.2, NO.sub.2, OH, C.sub.1-4 alkyl,
C.sub.1-4 alkoxy, halogen (Cl, Br, F, I), CF.sub.3 and aryl;
[0122] B is an integer in the range of 35-45; and
[0123] X is --OH, --NH.sub.2, or a C.sub.1-4 alkyl amide or
C.sub.1-4 dialkyl amide group;
[0124] or an analogue thereof.
[0125] Such GLP-1 analogues of formula III includes, but is not
limited to
[0126] Arg.sup.26-GLP-1(7-37); Arg.sup.34-GLP-1(7-37);
Lys.sup.36-GLP-1(7-37); Arg.sup.26,34Lys.sup.36-GLP-1(7-37);
Arg.sup.26,34Lys.sup.38GLP-1(7-38); Arg.sup.26,34Lys.sup.39-GLP-1
(7-39); Arg.sup.26,34Lys.sup.40-GLP-1 (7-40);
Arg.sup.26Lys.sup.36-GLP-1(7-37); Arg.sup.34Lys.sup.36-GLP-1(7-37);
Arg.sup.26Lys.sup.39-GLP-1(7-39); Arg.sup.34Lys.sup.40-GLP-1(7-40);
Arg.sup.26,34Lys.sup.36,39-GLP-1 (7-39);
Arg.sup.26,34Lys.sup.3640-GLP-1(7-40); Gly.sup.8Arg.sup.26-GLP-1(-
7-37); Gly.sup.8Arg.sup.34-GLP-1(7-37); Gly.sup.8Lys.sup.36-GLP-1
(7-37); Gly.sup.8Arg.sup.26,34Lys.sup.36-GLP-1(7-37);
Gly.sup.8Arg.sup.26,34Lys.s- up.39-GLP-1(7-39);
Gly.sup.8Arg.sup.26,34Lys.sup.40-GLP-1 (7-40);
Gly.sup.8Arg.sup.26Lys.sup.36-GLP-1(7-37);
Gly.sup.8Arg.sup.34Lys.sup.36-- GLP-1(7-37);
Gly.sup.8Arg.sup.26Lys.sup.39-GLP-1(7-39);
Gly.sup.8Arg.sup.34Lys.sup.40-GLP-1 (740);
Gly.sup.8Arg.sup.26,34Lys.sup.- 36,39-GLP-1(7-39); or
[0127] Gly.sup.8Arg.sup.26,34Lys.sup.3640-GLP-1(7-40). Each one of
these specific GLP-1 analogues constitutes an alternative
embodiment of the invention.
[0128] In a further embodiment of the invention the GLP-1 analogue
has the formula IV
A-GLP-1(19-B)-X (IV)
[0129] wherein
[0130] A is a peptide comprising the amino acid residues of
GLP-1(8-18) or a fragment thereof;
[0131] B is an integer in the range of 35-45; and
[0132] X is H, --NH.sub.2, or a C.sub.1-6 alkyl amide or C.sub.1-6
dialkyl amide group; or an analogue thereof.
[0133] In an embodiment of the GLP-1 analogue of formula IV, A is a
peptide selected from the group consisting of GLP-1 (8-18),
GLP-1(9-18), GLP-1(10-18), GLP-1(11-18), GLP-1(12-18),
GLP-1(13-18), GLP-1(14-18), GLP-1(15-18), GLP-1(16-18),
GLP-1(17-18) and GLP-1(18). Preferably, A is GLP-1(8-18),
GLP-1(9-18), GLP-1(10-18), GLP-1(11-18) or GLP-1(12-18), and B is
36, 37 or 38. Most preferably, A is GLP-1(8-18).
[0134] In a further embodiment of the GLP-1 analogue of formula IV,
B is 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44. In a more preferred
embodiment, B is 36. In another more preferred embodiment. B is 37.
In another more preferred embodiment, B is 38.
[0135] Such GLP-1 analogues of formula IV includes, but is not
limited to
[0136] Arg.sup.26-GLP-1 (8-37); Arg.sup.34-GLP-1 (8-37);
Lys.sup.36-GLP-1 (8-37); Arg.sup.26,34Lys.sup.36-GLP-1 (8-37);
Arg.sup.26,34Lys.sup.33GLP-- 1(838); Arg.sup.26,34Lys.sup.39-GLP-1
(8-39); Arg.sup.26,34Lys.sup.40-GLP-- 1 (8-40);
Arg.sup.26Lys.sup.36-GLP-1(8-37); Arg.sup.34Lys.sup.36-GLP-1(8-3-
7); Arg.sup.26Lys.sup.39-GLP-1(8-39);
Arg.sup.34Lys.sup.40-GLP-1(8-40); Arg.sup.26,34Lys.sup.36,39-GLP-1
(8-39); Arg.sup.26,34Lys.sup.36,40GLP-1(- 840);
Gly.sup.8Arg.sup.26-GLP-1(8-37); Gly.sup.3Arg.sup.34-GLP-1(8-37);
Gly.sup.8Lys.sup.36-GLP-1(8-37);
Gly.sup.8Arg.sup.26,34Lys.sup.36-GLP-1(8- -37); Gly.sup.8Arg
.sup.26,34Lys.sup.39-GLP-1(8-39);
Gly.sup.8Arg.sup.26,34Lys.sup.40-GLP-1 (8-40);
Gly.sup.8Arg.sup.26Lys.sup- .36-GLP-1(8-37);
Gly.sup.8Arg.sup.34Lys.sup.36-GLP-1(8-37);
Gly.sup.8Arg.sup.26Lys.sup.39-GLP-1(8-39);
Gly.sup.8Arg.sup.34Lys.sup.40-- GLP-1(8-40);
Gly.sup.8Arg.sup.26,34Lys.sup.36,39-GLP-1(8-39); or
[0137] Gly.sup.8Arg.sup.26,34Lys.sup.36,40-GLP-1(8-40). Each one of
these specific GLP-1 analogues constitutes an alternative
embodiment of the invention.
[0138] In a further embodiment of the invention the GLP-1 analogue
is Arg.sup.26GLP-1(7-37).
[0139] In a further embodiment of the invention the GLP-1 analogue
is Arg.sup.34GLP-1 (7-37).
[0140] In a further embodiment of the invention the salt is present
in a concentration of at least 25 mM. In a further embodiment of
the invention the salt is present in a concentration of from 25 mM
to 800 mM, such as 100 to 200 mM. The salt may be added to the
aqueous solution comprising the GLP-1 analogue all at once or as a
step or linear gradient.
[0141] In a further embodiment of the invention the salt is
selected from inorganic salts. Such inorganic salts, includes but
is not limited to chlorides, bromides, fluorides, iodides,
sulphates or nitrates with ammonium, alkaline metals or earth
alkaline metals, or mixtures thereof, e.g. NaCl, KCl, NH.sub.4Cl,
CaCl.sub.2, sodium sulphate, ammonium nitrate, potassium sulphate,
ammonium sulphate, or mixtures thereof.
[0142] In a further embodiment of the invention the salt is
selected from organic salts. Such organic salts, includes but is
not limited to acetates, citrates or tartrates with ammonium,
alkaline metals or earth alkaline metals, or mixtures thereof, e.g.
sodium acetate, potassium acetate, ammonium acetate, sodium
citrate, potassium citrate, potassium tartrate, ammonium citrate,
calcium acetate or mixtures thereof.
[0143] In a further embodiment of the invention the organic solvent
is present in a concentration of at least 0.5% (vol/vol). In a
further embodiment of the invention the organic solvent is present
in a concentration of from 0.5 to 50% (vol/vol), such as 1 to 15%
(vol/vol). The organic solvent may be added to the aqueous solution
comprising the GLP-1 analogue all at once or as a step or linear
gradient.
[0144] In a further embodiment of the invention the organic solvent
is selected from C.sub.1-6 alkanol, C.sub.1-6alkenol,
C.sub.1-6alkynol, urea, guanidine, C.sub.1-6-alkanoic acid, ketone,
DMSO, C.sub.2-6-glycol, C.sub.3-7-polyalcohol including sugars, or
mixtures thereof. Each of these solvents constitutes an individual
embodiment of the invention.
[0145] In a further embodiment of the invention the organic solvent
is selected from C.sub.1-6 alkanol, ketone or C.sub.3-7-polyalcohol
including sugars.
[0146] In a further embodiment of the invention the organic solvent
is selected from methanol, ethanol, n-propanol, allyl alcohol,
n-butanol, n-pentanol, n-hexanol, 2-propanol, tert-butyl alcohol,
1,2-ethanediol, 1,2-propanediol, 2-methyl-2,4-pentanediol,
glycerol, methylethyl ketone or acetone. Each of these solvents
constitutes an individual embodiment of the invention. The organic
solvent is preferably selected from ethanol, glycerol or
acetone.
[0147] After preparation of the solution comprising a GLP-1
analogue, it is normally placed at ambient temperature, and the
crystals will start to form after a while. After formation the
crystals are isolated from the mother liquor. The temperature of
the solution is easily decided by the skilled practitioner using
his common general knowledge, and he may decide to place the
solution at a constant temperature, or optionally place the
solution at one temperature and then by step or linear gradient
move the temperatur to a lower temperature.
[0148] In a further embodiment of the invention the solution is
placed at a temperature from about -10.degree. C. to +40.degree. C.
In further embodiments of the invention the solution is placed at a
temperature from -5.degree. C. to 40.degree. C., -2.degree. C. to
40.degree. C., -1.degree. C. to 40.degree. C., 4.degree. C. to
37.degree. C., or 20 to 25.degree. C. Each of these ranges
constitutes an individual embodiment of the invention.
[0149] Formation of the crystals may start after 10-60 minutes
although it may take a shorther or longer period of time. After
formation of the crystals they may be isolated from the mother
liquor by filtration, decantation, centrifugation or other means
known to the skilled practitioner.
[0150] The present invention also relates to crystals, preferably
needle shaped crystals, of a GLP-1 analogue optainable by the
process comprising:
[0151] a) preparing an aqueous solution comprising a GLP-1
analogue, a salt, and an organic solvent, and
[0152] b) isolation of the crystals after formation.
[0153] The present invention also relates to needle shaped crystals
of a GLP-1 analogue having a length of at least 0.5 .mu.m. In
another embodiment of the invention the crystals are needle shaped
crystals of a GLP-1 analogue having a length of at least 2 .mu.m.
In a further embodiment of the invention the crystals are needle
shaped crystals of a GLP-1 analogue having a length of 0.5 .mu.m to
50 .mu.m, such as 2 .mu.m to 50 .mu.m, e.g. 2 .mu.m to 30 .mu.m. In
a further embodiment of the invention the crystals are needle
shaped crystals of a GLP-1 analogue having a length of at least 8
.mu.m. In a further embodiment of the invention the crystals are
needle shaped crystals of a GLP-1 analogue having a length of 8
.mu.m to 50 .mu.m.
[0154] In a further aspect the present invention relates to a
pharmaceutical composition comprising crystals, preferably needle
shaped crystals, of a GLP-1 analogue together with a
pharmaceutically acceptable carrier or excipient.
[0155] The GLP-1 analogues can be produced by a method which
comprises culturing or fermenting a host cell containing a DNA
sequence encoding the GLP-1 analogue and capable of expressing said
analogue in a suitable nutrient medium under conditions permitting
the expression of the peptide, after which the resulting GLP-1
analogue is recovered from the culture or fermentation broth.
Hereinafter, culturing will be used to cover both culturing and
fermenting and the like.
[0156] The medium used to culture the cells may be any conventional
medium suitable for growing the host cells, such as minimal or
complex media containing appropriate supplements. Suitable media
are available from commercial suppliers or may be prepared
according to published recipes (e.g. in catalogues of the American
Type Culture Collection). The GLP-1 analogue produced by the cells
may then be recovered from the culture medium by conventional
procedures including, optionally lysis of cells, separating the
host cells from the medium by centrifugation or filtration,
precipitating the proteinaceous components of the supernatant or
filtrate by means of a salt, e.g. ammonium sulphate, purification
by conventional purification techniques, such as chromatographic
techniques, if necessary, purification by ion exchange
chromatography according to the present invention, and
subsequently, subjecting to analytical tests, e.g. PAGE, IEF, if
necessary, subjecting to further purification, if necessary, and
isolation of the pure GLP-1 analogue.
[0157] The DNA sequence encoding the GLP-1 analogue may suitably be
of genomic or cDNA origin, for instance obtained by preparing a
genomic or cDNA library and screening for DNA sequences coding for
all or part of the GLP-1 analogue by hybridisation using synthetic
oligonucleotide probes in accordance with standard techniques (see,
for example, Sambrook, J, Fritsch, E F and Maniatis, T, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
New York, 1989). The DNA sequence encoding the GLP-1 analogue may
also be prepared synthetically by established standard methods,
e.g. the phosphoamidite method described by Beaucage and Caruthers,
Tetrahedron Letters 22 (1981), 1859-1869, or the method described
by Matthes et al., EMBO Journal 3 (1984), 801-805. The DNA sequence
may also be prepared by polymerase chain reaction using specific
primers, for instance as described in U.S. Pat. No. 4,683,202 or
Saiki et al., Science 239 (1988), 487-491.
[0158] The DNA sequence may be inserted into any vector which may
conveniently be subjected to recombinant DNA procedures, and the
choice of vector will often depend on the host cell into which it
is to be introduced. Thus, the vector may be an autonomously
replicating vector, i.e. a vector which exists as an
extrachromosomal entity, the replication of which is independent of
chromosomal replication, e.g. a plasmid. Alternatively, the vector
may be one which, when introduced into a host cell, is integrated
into the host cell genome and replicated together with the
chromosome(s) into which it has been integrated.
[0159] The vector is preferably an expression vector in which the
DNA sequence encoding the GLP-1 analogue is operably linked to
additional segments required for transcription of the DNA, such as
a promoter. The promoter may be any DNA sequence which shows
transcriptional activity in the host cell of choice and may be
derived from genes encoding proteins either homologous or
heterologous to the host cell. Examples of suitable promoters for
directing the transcription of the DNA encoding the GLP-1 analogue
in a variety of host cells are well known in the art, cf. for
instance Sambrook et al., supra.
[0160] The DNA sequence encoding the GLP-1 analogue may also, if
necessary, be operably connected to a suitable terminator,
polyadenylation signals, transcriptional enhancer sequences, and
translational enhancer sequences. The recombinant vector may
further comprise a DNA sequence enabling the vector to replicate in
the host cell in question.
[0161] The vector may also comprise a selectable marker, e.g. a
gene the product of which complements a defect in the host cell or
one which confers resistance to a drug, e.g. ampicillin, kanamycin,
tetracyclin, chloramphenicol, neomycin, hygromycin or
methotrexate.
[0162] To direct a GLP-1 analogue into the secretory pathway of the
host cells, a secretory signal sequence (also known as a leader
sequence, prepro sequence or pre sequence) may be provided in the
recombinant vector. The secretory signal sequence is joined to the
DNA sequence encoding the GLP-1 analogue in the correct reading
frame. Secretory signal sequences are commonly positioned 5' to the
DNA sequence encoding the GLP-1 analogue. The secretory signal
sequence may be that normally associated with the GLP-1 analogue or
may be from a gene encoding another secreted protein.
[0163] The procedures used to ligate the DNA sequences coding for
the GLP-1 analogue, the promoter and optionally the terminator
and/or secretory signal sequence, respectively, and to insert them
into suitable vectors containing the information necessary for
replication, are well known to persons skilled in the art (cf., for
instance, Sambrook et al., supra).
[0164] The host cell into which the DNA sequence or the recombinant
vector is introduced may be any cell which is capable of producing
the GLP-1 analogue and includes bacteria, vira, e.g. baculo virus,
yeast, fungi, insect cells and higher eukaryotic cells. Examples of
suitable host cells well known and used in the art are, without
limitation, E coli, Saccharomyces cerevisiae, or mammalian BHK or
CHO cell lines.
[0165] Some of the GLP-1 analogue, can be produced according to
conventional organic peptide synthetic chemistry. The resulting
synthetic mixture may then be chemically modified, e.g. by
alkylation, acylation, ester formation or amide formation or the
like, and purified, or purified as it is and then modified
chemically.
[0166] Usually, the fermentation broth comprising the GLP-1
analogue will also contain amino acids, small peptides, large
peptides, unrelated proteins, reactants, cell debris, host cell
proteins, endotoxins, and/or vira depending on whether recombinant
DNA techniques and/or chemical modification techniques have been
used or whether organic peptide synthetic chemistry techniques have
been used.
[0167] Thus, any method, such as an industrial method, for
producing a GLP-1 analogue, which includes a crystallization step
according to the present invention is also an aspect of the present
application.
[0168] Accordingly, the present invention relates in a further
aspect to a method for producing a GLP-1 analogue or a GLP-1
analogue whereto is attached a lipophilic substituent
comprising:
[0169] a) expressing the GLP-1 analogue in a host cell, such as
yeast,
[0170] b) precipitating the analogue at its pl,
[0171] c) preparing an aqueous solution comprising the GLP-1
analogue, a salt, and an organic solvent, and
[0172] d) isolation of the crystals after formation, and
[0173] e) further purification, optionally followed by repetition
of steps b to e, to isolation of crystals of the GLP-1 analogue,
and
[0174] f) optionally acylation of the GLP-1 analogue, optionally
followed by purification.
[0175] In a further aspect the present invention relates to a
process for producing crystals of a GLP-1 analogue comprising:
[0176] a) preparing an aqueous solution comprising a GLP-1
analogue, and a salt, and
[0177] b) isolation of the crystals after formation.
[0178] Another invention relates to a process for producing
crystals of a GLP-1 analogue comprising:
[0179] a) preparing an aqueous solution comprising a GLP-1
analogue, and a salt, and
[0180] b) isolation of the crystals after formation. Anyone of the
above embodiments is also intended to represent embodiments of this
invention where possible.
[0181] The term "an organic solvent", as used herein, is intended
to include any organic solvent which do not denature the GLP-1
analogue. The organic solvent includes but is not limited to
C.sub.1-6-alkanol, C.sub.1-6-alkenol or C.sub.1-6-alkynol, urea,
guanidine, or C.sub.1-6alkanoic acid, such as acetic acid, ketone,
such as acetone, dimethylsulphoxide (DMSO), polymeric solvents,
C.sub.2-6-glycol, C.sub.3-7-polyalcohol including sugars, or
mixtures thereof.
[0182] The term "C.sub.1-6alkanol", "C.sub.1-6alkenol" or
"C.sub.1-6alkynol", as used herein, alone or in combination is
intended to include those C.sub.1-6alkane, C.sub.1-6alkene or
C.sub.1-6alkyne groups of the designated length in either a linear
or branched or cyclic configuration whereto is linked a hydroxyl
(--OH) (cf. Morrison & Boyd, Organic Chemistry, 4.sup.th ed).
Examples of linear alcohols are methanol, ethanol, n-propanol,
allyl alcohol, n-butanol, n-pentanol and n-hexanol. Examples of
branched alcohols are 2-propanol and tert-butyl alcohol. Examples
of cyclic alcohols are cyclo propyl alcohol and
2-cyclohexen-1-ol.
[0183] The term "C.sub.1-6alkanoic acid", as used herein, is
intended to include a group of the formula R'COOH wherein R' is H
or C.sub.1-5alkyl, such as acetic, propionic, butyric,
.alpha.-methylbutyric, or valeric acid (cf. Morrison & Boyd,
Organic Chemistry, 4.sup.th ed).
[0184] The term "C.sub.1-12-alkyl", as used herein, is intended to
include a branched or straight alkyl group having from one to 12
carbon atoms. Typical C.sub.1-12-alkyl groups are C.sub.1-5-alkyl
groups which include, but are not limited to, methyl, ethyl,
n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl,
pentyl, iso-pentyl, and the like (cf. Morrison & Boyd, Organic
Chemistry, 4.sup.th ed).
[0185] The term "C.sub.2-6-glycol", as used herein, is intended to
include a C.sub.2-6-alkane containing two hydroxyl groups on
different carbon atoms which may be adjacent or not. A typical
C.sub.2-6-glycol include, but is not limited to 1,2-ethanediol,
1,2-propanediol, or 2-methyl-2,4-pentanediol (cf. Morrison &
Boyd, Organic Chemistry, 4.sup.th ed).
[0186] The term "C.sub.2-4-alkane", as used herein, is intended to
include a branched or straight alkane group having from two to six
carbon atoms. Typical C.sub.2-6-alkane groups include, but are not
limited to ethane, propane, iso-propane, butane, iso-butane,
pentane, hexane and the like (cf. Morrison & Boyd, Organic
Chemistry, 4.sup.th ed).
[0187] The term "C.sub.3-7-polyalcohol including sugars", as used
herein, is intended to include a group of the formula
HOCH.sub.2(CHOH).sub.nCH.su- b.2OH wherein n is an integer from
1-5, and monosaccharides such as glycerol, mannose, glucose (cf.
Morrison & Boyd, Organic Chemistry, 4.sup.th ed).
[0188] The term "a GLP-1 analogue", as used herein, is intended to
designate GLP-1 (7-37), GLP-1 (7-36) amide as well as analogues,
fragments, homologues, and derivatives thereof, which are capable
of being produced by conventional recombinant DNA techniques as
well as conventional synthetic methods. Such GLP-1 analogues
include but are not limited to native glucagon-like peptide-1, for
instance such peptide fragments which comprises GLP-1 (7-37) and
functional derivatives thereof as disclosed in WO 87/06941; such
peptide fragments which comprise GLP-1 (7-36) and functional
derivatives thereof as disclosed in WO 90/11296; such analogues of
the active GLP-1 peptides 7-34, 7-35, 7-36, and 7-37 as disclosed
in WO 91/11457; such N-terminal truncated fragments of GLP-1 as
disclosed in EP 0699686-A2; such GLP-1 analogues and derivatives
that include an N-terminal imidazole group as disclosed in EP
0708179-A2; and such exendins as disclosed in WO 9746584 and U.S.
Pat. No. 5,424,286.
[0189] The term "exendins", as used herein, is intended to
designate exendin as well as analogs, derivatives, and fragments
thereof, e.g. exendin-3 and -4. Examples of exendin as well as
analogs, derivatives, and fragments thereof to be included within
the present invention are those disclosed in WO 9746584 and U.S.
Pat. No. 5,424,286. U.S. Pat. No. 5,424,286 describes a method for
stimulating insulin release with exendin polypeptide(s). The
exendin polypeptides disclosed include
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGX; wherein X.dbd.P or Y, and
HX1X2GTFITSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS; wherein X1.times.2=SD
(exendin-3) or GE (exendin-4)). The exendin-3 and -4 and fragments
are useful in treatment of diabetes mellitus (types I or II) and
prevention of hyperglycaemia. They normalise hyperglycaemia through
glucose-dependent, insulin-independent and insulin-dependent
mechanisms. Exendin-4 is specific for exendin receptors, i.e. it
does not interact with vasoactive intestinal peptide receptors. WO
9746584 describes truncated versions of exendin peptide(s) for
treating diabetes. The disclosed peptides increase secretion and
biosynthesis of insulin, but reduce those of glucagon. The
truncated peptides can be made more economically than full length
versions.
[0190] The term "crystals" as used herein, is intended to designate
crystals of any shape, such as single needle shaped crystals (which
is the same as needle-like crystals), single irregular shaped
crystals, single oblong crystals as well as clusters of two or more
crystals and mixtures thereof (cf. "Preparation and analysis of
protein crystals" by A. McPherson).
[0191] The term "non-synthetic GLP-1 analogues" as used herein, is
intended to designate a GLP-1 analogue which comprises only
naturally occurring amino acid residues and is capable of being
produced by recombinant DNA techniques or expressed by organisms,
e.g. microorganisms.
[0192] The term "ketone" as used herein, is intended to designate a
compound of the formula R.sup.1--CO--R.sup.2 wherein R.sup.1 and
R.sup.2 are independently of each other selected from
C.sub.1-12-alkyl, preferably C.sub.1-5-alkyl (cf. Morrison &
Boyd, Organic Chemistry, 4.sup.th ed).
[0193] The term "polymeric solvents" as used herein, is intended to
comprise poly(acrylic acid), carboxymethylcellulose, poly(ethylene
glycol), poly(propylene glycol), poly(vinyl alcohol),
poly(vinylpyrrolidone) and the like (cf. "Preparation and analysis
of protein crystals" by A. McPherson).
[0194] The term "analogues" as used herein, is intended to
designate a peptide wherein one or more amino acid residues of the
parent peptide have been substituted by another amino acid residue
and/or wherein one or more amino acid residues of the parent
peptide have been deleted and/or wherein one or more amino acid
residues have been added to the parent peptide. Such addition can
take place either at the N-terminal end or at the C-terminal end of
the parent peptide or both.
[0195] The term "derivatives" as used herein, is intended to
designate a peptide in which one or more of the amino acid residues
of the parent peptide have been chemically modified, e.g. by
alkylation, acylation, ester formation or amide formation or the
like.
[0196] The term "a salt" as used herein, is intended to include any
organic or inorganic salt, including but not limited to NaCl, KCl,
NH.sub.4Cl, CaCl.sub.2, sodium acetate, potassium acetate, ammonium
acetate, sodium citrate, potassium citrate, ammonium citrate,
sodium sulphate, potassium sulphate, ammonium sulphate, calcium
acetate or mixtures thereof (cf. Remington's Pharmaceutical
Sciences, Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, or
Remington: The Science and Practice of Pharmacy, 19th Edition
(1995), or handbooks from Amersham-Pharmacia Biotech).
[0197] The term "a buffer" as used herein, is intended to include
any buffer including but not limited to: citrate buffers, phosphate
buffers, tris buffers, bis-Tris buffer, borate buffers, lactate
buffers, glycyl glycin buffers, arginine buffers, carbonate
buffers, acetate buffers, glutamate buffers, ammonium buffers,
glycin buffers, alkylamine buffers, aminoethyl alcohol buffers,
ethylenediamine buffers, tri-ethanol amine, imidazole buffers,
pyridine buffers and barbiturate buffers and mixtures thereof (cf.
Remington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing
Co., Easton, Pa., 1990, or Remington: The Science and Practice of
Pharmacy, 19th Edition (1995), or handbooks from Amersham-Pharmacia
Biotech).
[0198] The present invention is further illustrated by the
following examples which, however, are not to be construed as
limiting the scope of protection. The features disclosed in the
foregoing description and in the following examples may, both
separately and in any combination thereof, be material for
realising the invention in diverse forms thereof.
EXPERIMENTAL PART
[0199] Crystallisation of Arg.sup.34GLP1.sub.(7-37) in the
Manufacturing Process for Preparing a Mono Acylated GLP-1
Analogue
[0200] Arg.sup.34GLP1.sub.(7-37) was expressed in yeast, that is
Saccharomyces cerevisiae (Sacch. cerevisiae), by conventional
recombinant DNA technology. The fermentation broth was purified by
a conventional reverse phase capture step followed by a first
precipitation step (precipitate (A)) at the iso-electric point (pl)
of Arg.sup.34GLP1.sub.(7-37). Arg.sup.34GLP1.sub.(7-37) was then
redissolved and further purified by conventional High Performance
Cation Exchange Chromatography (HP-CIEC) and Reverse Phase High
Performance Liquid Chromatography (RP-HPLC) followed by a second
precipitation step (precipitate (B)) at the pl of
Arg.sup.34GLP1.sub.(7-37).
[0201] Then Arg.sup.34GLP1.sub.(7-37) was acylated, e.g. as
disclosed in WO 98/08871, and the resulting solution containing
mono-acylated Arg.sup.34GLP1.sub.(7-37) was further purified by
conventional RP-HPLC and finally precipitated at pl of
mono-acylated Arg.sup.34GLP1.sub.(7-37)- .
[0202] The implementation of a crystallisation step in the
manufacturing process for the preparation of mono-acylated
Arg.sup.34GLP1.sub.(7-37) results in removal of coloured compounds
from the fermentation broth, reduction of Saccharomyces cerevisiae
proteins (host cell proteins) (SCP) as well as removal of water,
and low loss of Arg.sup.34GLP1.sub.(7-37) from the mother
liquor.
[0203] Crystallisation of Arg.sup.34GLP1.sub.(7-37) from
precipitate (A) and precipitate (B) has been performed. An overview
of the experiments is given in the following section along with a
description of the crystallisation procedure.
[0204] General Procedure:
[0205] The precipitate (A) from the first precipitation step (or
the precipitate (B) from the second precipitation step) was
suspended in water and pH was adjusted to pH 8.5-9.5 with NaOH by
which the precipitate dissolved. The Arg.sup.34GLP1.sub.(7-37)
concentration (referred to as GLP1 conc. in the tables) of the
stock solution was measured by analytical RP-HPLC (Analytical
procedure 427-AF006.D02: Purity and Concentration of GLP-1 (Inger
Bastholm, 1999)). Then salt, organic solvent and buffer compound
was added to the desired concentration and the solution was
adjusted to the specified pH with HCl, then gently swirled and
placed at the specified temperature (scale: 3-5 ml). Formation of
crystals started to occur after 10-60 minutes and after 16-18 hr
the crystal morphology was studied in microscope (Microscope BX50
from Olympus). For quantification a portion of the mother liquor
was removed and centrifuged. The Arg.sup.34GLP1.sub.(7-37) content
was measured by analytical RP-HPLC. The loss by crystallisation was
calculated by division of the supernatant content by the content in
the starting material.
[0206] N.A.=Not Assessed
2 Example GLP1 Loss precipitate A conc. Salt Solvent Buffer pH
Temp. Crystal morphology/size [%] 1 3.4 mg/ml 75 Mm NaCl None 6 mM
bis-Tris 6.5 20-25.degree. C. Amorphous precipitate. 45.6 2 " 75 Mm
NaCl 5% ethanol 6 mM bis-Tris 6.5 20-25.degree. C. Irregular
shaped. 13.5 3 " 75 mM NaCl 10% ethanol 6 mM bis-Tris 6.5
20-25.degree. C. Irregular shaped. 12.4 4 " 75 mM NaCl 15% ethanol
6 mM bis-Tris 6.5 20-25.degree. C. Amorphous precipitate. 13.0 5 "
150 mM NaCl None 6 mM bis-Tris 6.5 20-25.degree. C. Irregular
shaped. 13.4 6 " 150 mM NaCl 5% ethanol 6 mM bis-Tris 6.5
20-25.degree. C. Irregular shaped, few needle 11.2 shaped (ca. 5
.mu.m). 7 " 150 mM NaCl 10% ethanol 6 mM bis-Tris 6.5 20-25.degree.
C. Needle shaped (5-15 .mu.m). 9.1 8 " 150 mM NaCl 15% ethanol 6 mM
bis-Tris 6.5 20-25.degree. C. Amorphous precipitation. 11.3 9 3.4
mg/ml 150 mM NaCl 10% ethanol 6 mM bis-Tris 6.6 20-25.degree. C.
Needle shaped. 5.7 10 " 200 mM NaCl 10% ethanol 6 mM bis-Tris 6.6
20-25.degree. C. Needle shaped. 5.9 11 " 250 mM NaCl 10% ethanol 6
mM bis-Tris 6.5 20-25.degree. C. Needle shaped, clusters. 5.7 12 "
300 mM NaCl 10% ethanol 6 mM bis-Tris 6.6 20-25.degree. C. Needle
shaped, clusters. 5.1 13 5.6 mg/ml 150 mM NaCl 2.5% acetone 5 mM
bis-Tris 6.5 20-25.degree. C. Small, irregular shaped. 8.4 14 " 150
mM NaCl 5% acetone 5 mM bis-Tris 6.5 20-25.degree. C. Small, oblong
crystals 8.2 (ca. 5 .mu.m). 15 " 150 mM NaCl 10% acetone 5 mM
bis-Tris 6.5 20-25.degree. C. Irregular shaped, many 8.9 needle
shaped (7-9 .mu.m). 16 " 150 mM NaCl 2.5% glycerol 5 mM bis-Tris
6.5 20-25.degree. C. Small, irregular shaped 8.6 (1-2 .mu.m). 17 "
150 mM NaCl 5% glycerol 5 mM bis-Tris 6.5 20-25.degree. C. Small,
irregular shaped. 9.5 (2-3 .mu.m). 18 " 150 mM NaCl 10% glycerol 5
mM bis-Tris 6.5 20-25.degree. C. Small, irregular shaped 8.0 (1-2
.mu.m). Example GLP1 Loss Precipitate B conc. Salt Solvent Buffer
pH Temp. Crystal morphology/size [%] 19 4 mg/ml 75 mM NaCl 5%
ethanol 5 mM bis-Tris 6.5 20-25.degree. C. Needle shaped. 4.0 20 "
75 mM NaCl 5% ethanol 5 mM bis-Tris 6.5 4.degree. C. Needle shaped.
6.5 21 " 75 mM NaCl 10% ethanol 5 mM bis-Tris 6.5 20-25.degree. C.
Needle shaped. 4.3 22 " 75 mM NaCl 10% ethanol 5 mM bis-Tris 6.5
4.degree. C. Needle shaped. 4.5 23 " 75 mM NaCl 15% ethanol 5 mM
bis-Tris 6.5 20-25.degree. C. Needle shaped. 5.3 24 " 75 mM NaCl
15% ethanol 5 mM bis-Tris 6.5 4.degree. C. Needle shaped. 6.0 25 "
100 mM NaCl None 5 mM bis-Tris 6.5 20-25.degree. C. Needle shaped.
3.8 26 " 100 mM NaCl None 5 mM bis-Tris 6.5 4.degree. C. Amorphous.
7.0 27 " 100 mM NaCl 5% ethanol 5 mM bis-Tris 6.5 20-25.degree. C.
Needle shaped. N.A. 28 " 100 mM NaCl 5% ethanol 5 mM bis-Tris 6.5
4.degree. C. Irregular shaped. 6.0 29 " 100 mM NaCl 10% ethanol 5
mM bis-Tris 6.5 20-25.degree. C. Needle shaped. 3.3 30 " 100 mM
NaCl 10% ethanol 5 mM bis-Tris 6.5 4.degree. C. Needle shaped. 4.0
31 " 100 mM NaCl 15% ethanol 5 mM bis-Tris 6.5 20-25.degree. C.
Needle shaped. 4.0 32 " 100 mM NaCl 15% ethanol 5 mM bis-Tris 6.5
4.degree. C. Needle shaped, clusters. 1.3 33 " 200 mM NaCl None 5
mM bis-Tris 6.5 20-25.degree. C. Granular, few needle 2.8 shaped.
34 " 200 mM NaCl None 5 mM bis-Tris 6.5 4.degree. C. Amorphous
precipitation. 2.8 35 " 200 mM NaCl 10% ethanol 5 mM bis-Tris 6.5
20-25.degree. C. Needle shaped. 2.8 36 " 200 mM NaCl 10% ethanol 5
mM bis-Tris 6.5 4.degree. C. Amorphous, few needle 2.0 shaped. 37 "
300 mM NaCl None 5 mM bis-Tris 6.5 20-25.degree. C. Granular, few
needle 2.3 shaped. 38 " 300 mM NaCl None 5 mM bis-Tris 6.5
4.degree. C. Amorphous. 2.0 39 " 300 mM NaCl 10% ethanol 5 mM
bis-Tris 6.5 20-25.degree. C. Needle shaped. 2.5 40 " 300 mM NaCl
10% ethanol 5 mM bis-Tris 6.5 4.degree. C. Amorphous precipitation,
2.0 few needle shaped. 41 3.3 mg/ml 100 mM NaCl 2% ethanol 5 mM
bis-Tris 6.5 20-25.degree. C. Needle shaped (ca. 7 .mu.m). N.A. 42
3.2 mg/ml 100 mM NaCl 5% ethanol 5 mM bis-Tris 6.5 20-25.degree. C.
Needle shaped (7-11 .mu.m). 4.9 43 3.2 mg/ml 100 mM NaCl 7% ethanol
5 mM bis-Tris 6.5 20-25.degree. C. Needle shaped (7-11 .mu.m). 4.9
44 3.1 mg/ml 100 mM NaCl 10% ethanol 5 mM bis-Tris 6.5
20-25.degree. C. Needle shaped (7-9 .mu.m). 5.5 45 2.9 mg/ml 100 mM
NaCl 15% ethanol 5 mM bis-Tris 6.5 20-25.degree. C. Needle shaped
(7-9 .mu.m) N.A. clusters. 46 3.2 mg/ml 200 mM NaCl 2% ethanol 5 mM
bis-Tris 6.5 20-25.degree. C. Needle shaped (ca. 7 .mu.m). N.A. 47
3.1 mg/ml 200 mM NaCl 5% ethanol 5 mM bis-Tris 6.5 20-25.degree. C.
Needle shaped (7-11 .mu.m). 4.7 48 3.0 mg/ml 200 mM NaCl 7% ethanol
5 mM bis-Tris 6.5 20-25.degree. C. Needle shaped (7-11 .mu.m). 5.2
49 2.9 mg/ml 200 mM NaCl 10% ethanol 5 mM bis-Tris 6.5
20-25.degree. C. Needle shaped (7-11 .mu.m). 6.2 50 2.8 mg/ml 200
mM NaCl 15% ethanol 5 mM bis-Tris 6.5 20-25.degree. C. Needle
shaped (7-9 .mu.m), N.A. clusters. 51 2.6 mg/ml 75 mM NaCl 7%
ethanol 5 mM bis-Tris 6.5 20-25.degree. C. Needle shaped (5-6
.mu.m). 5.8 52 2.3 mg/ml 300 mM NaCl 7% ethanol 5 mM bis-Tris 6.5
20-25.degree. C. Needle shaped (9-12 .mu.m). 5.8 53 3.4 mg/ml 100
mM KCl 7% ethanol 5 mM bis-Tris 6.5 20-25.degree. C. Needle shaped
(4-9 .mu.m). 4.8 54 3.2 mg/ml 200 mM KCl 7% ethanol 5 mM bis-Tris
6.5 20-25.degree. C. Needle shaped (4-9 .mu.m), 4.7 55 3.4 mg/ml
100 mM (NH.sub.4).sub.2SO.sub.4 7% ethanol 5 mM bis-Tris 6.5
20-25.degree. C. Needle shaped (7-9 .mu.m). 5.1 56 3.2 mg/ml 200 mM
(NH.sub.4).sub.2SO.sub.4 7% ethanol 5 mM bis-Tris 6.5 20-25.degree.
C. Needle shaped (4-7 .mu.m). 5.4 57 0.5 mg/ml 200 mM NaCl 7%
ethanol 5 mM bis-Tris 6.5 20-25.degree. C. Few needle shaped. 22.5
58 0.6 mg/ml 200 mM NaCl 7% ethanol 5 mM bis-Tris 6.5 20-25.degree.
C. Few needle shaped. 18.8 59 1.0 mg/ml 200 mM NaCl 7% ethanol 5 mM
bis-Tris 6.5 20-25.degree. C. Few needle shaped. 11.7 60 1.4 mg/ml
200 mM NaCl 7% ethanol 5 mM bis-Tris 6.5 20-25.degree. C. Needle
shaped. 8.8 61 2.0 mg/ml 200 mM NaCl 7% ethanol 5 mM bis-Tris 6.5
20-25.degree. C. Needle shaped. 4.5 62 3.0 mg/ml 200 mM NaCl 7%
ethanol 5 mM bis-Tris 6.5 20-25.degree. C. Needle shaped. 4.7 63
5.0 mg/ml 200 mM NaCl 7% ethanol 5 mM bis-Tris 6.5 20-25.degree. C.
Irregular shaped. 3.0 64 5.8 mg/ml 200 mM NaCl 7% ethanol 5 mM
bis-Tris 6.5 20-25.degree. C. Amorphous precipitation. 3.2 65 3.2
mg/ml 200 mM NaCl 7% ethanol 5 mM bis-Tris 6.0 20-25.degree. C.
Needle shaped. 2.1 66 " 200 mM NaCl 7% ethanol 5 mM bis-Tris 6.2
20-25.degree. C. Needle shaped. 2.8 67 " 200 mM NaCl 7% ethanol 5
mM bis-Tris 6.6 20-25.degree. C. Needle shaped. 4.0 68 " 200 mM
NaCl 7% ethanol 5 mM bis-Tris 7.0 20-25.degree. C. Needle shaped.
5.5 69 " 200 mM NaCl 7% ethanol 5 mM bis-Tris 7.2 20-25.degree. C.
Needle shaped. 6.6
[0207]
Sequence CWU 1
1
1 1 39 PRT Homo sapiens VARIANT (1)..(39) Xaa = any amino acid 1
His Xaa Xaa Gly Xaa Phe Thr Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Phe Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35
* * * * *