U.S. patent application number 10/632414 was filed with the patent office on 2005-04-14 for method of purifying preproinsulin.
This patent application is currently assigned to Aventis Pharma Deutschland GmbH. Invention is credited to Blumenstock, Hans, Havenith, Chantalle, Thurow, Horst.
Application Number | 20050080000 10/632414 |
Document ID | / |
Family ID | 30128614 |
Filed Date | 2005-04-14 |
United States Patent
Application |
20050080000 |
Kind Code |
A1 |
Thurow, Horst ; et
al. |
April 14, 2005 |
Method of purifying preproinsulin
Abstract
The invention relates to a method for the chromatographic
purification of preproinsulins, in which higher molecular weight
substances are removed from an aqueous solution of preproinsulin by
a first chromatography on an anion exchanger in flow-through mode
and a subsequent second chromatography on a cation exchanger in
adsorption mode, and to a method for preparing insulins, which
includes the method for preparing preproinsulins.
Inventors: |
Thurow, Horst; (Hambach,
DE) ; Blumenstock, Hans; (Hattersheim, DE) ;
Havenith, Chantalle; (Hofheim, DE) |
Correspondence
Address: |
ROSS J. OEHLER
AVENTIS PHARMACEUTICALS INC.
ROUTE 202-206
MAIL CODE: D303A
BRIDGEWATER
NJ
08807
US
|
Assignee: |
Aventis Pharma Deutschland
GmbH
Frankfurt am Main Germany
DE
D-65926
|
Family ID: |
30128614 |
Appl. No.: |
10/632414 |
Filed: |
August 1, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60433726 |
Dec 16, 2002 |
|
|
|
Current U.S.
Class: |
514/6.3 ;
514/5.9; 514/7.3; 530/303 |
Current CPC
Class: |
C07K 14/62 20130101 |
Class at
Publication: |
514/003 ;
530/303 |
International
Class: |
A61K 038/28; C07K
014/62 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 1, 2002 |
DE |
10235168.6-43 |
Jul 18, 2003 |
WO |
PCT/EP03/07820 |
Claims
We claim:
1. A method for the chromatographic purification of preproinsulin
of the formula 1, 3wherein X a) is a genetically encodable amino
acid residue or b) is a peptide having from 2 to 35 amino acid
residues, which starts and ends with in each case a basic amino
acid residue, in particular Arg, and which, if it consists of more
than 3 amino acid residues, starts and ends with in each case two
basic amino acid residues, in particular Arg and/or Lys, R.sup.1 a)
is hydrogen, b) is a genetically encodable amino acid residue or c)
is a peptide having from 2 to 15 amino acid residues, R.sup.2 is a
genetically encodable amino acid residue, and and the residues
A1-A20 correspond to the amino acid sequence of the A chain of
human insulin or of an insulin analog and the residues B1-B30
correspond to the amino acid sequence of the B chain of human
insulin or of an insulin analog; wherein said method for
chromatographic purification of preproinsulin comprises: removing
higher molecular weight substances from an aqueous solution of said
preproinsulin by means of a first chromatography on an anion
exchanger in flow-through mode and a subsequent second
chromatography on a cation exchanger in adsorption mode.
2. A method for the chromatographic purification of the genetically
engineered preproinsulin of formula 1 of claim 1, wherein said
preproinsulin has the following amino acid sequence:
Ala-Thr-Thr-Ser-Thr-Gly-Asn-Ser-Ala-Arg-Phe-Val-Asn-Gln-His-Leu-Cys-Gly-S-
er-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-
-Pro-Lys-Thr-Arg-Arg-Glu-Ala-Glu-Asp-Pro-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-G-
ly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-
-Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-A-
sn-Tyr-Cys-Asn (SEQ ID NO: 2).
3. A method for the chromatographic purification of the genetically
engineered preproinsulin of formula 1 of claim 1, wherein said
preproinsulin has the following amino acid sequence:
Ala-Thr-Thr-Ser-Thr-Gly-Asn-Ser-Ala-Arg-Phe-Val-Asn-Gln-His-Leu-Cys-Gly-S-
er-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-
-Pro-Lys-Thr-Arg-Arg-Glu-Ala-Glu-Asp-Pro-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-G-
ly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-
-Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-A-
sn-Tyr-Cys-Gly (SEQ ID NO: 3).
4. A method for the chromatographic purification of the genetically
engineered preproinsulin of formula 1 of claim 1, wherein said
preproinsulin has the following amino acid sequence:
Ala-Thr-Thr-Ser-Thr-Gly-Asn-Ser-Ala-Arg-Phe-Val-Lys-Gln-His-Leu-Cys-Gly-S-
er-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-
-Pro-Glu-Thr-Arg-Asp-Val-Pro-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-S-
er-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg-Gly-Ile-Val-Glu-Gln-
-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn
(SEQ ID NO: 4).
5. Use of the method of claim 1 to separate foreign substances from
said aqueous solution of preproinsulin which induce insulin
denaturation.
6. The method of claim 1 wherein said second chromatography is
carried out at a pH of from 3.0 to 5.5.
7. The method of claim 1 wherein said second chromatography is
carried out under a pressure of from 1 to 30 bar.
8. A method for preparing insulin by expressing nonfolded
preproinsulin, comprising the steps of: a) fermentation of
genetically modified microorganisms which express nonfolded
preproinsulin, b) harvesting the microorganisms and cell
disruption, c) isolating the inclusion bodies containing
undissolved, nonfolded preproinsulin, d) dissolving the
preproinsulin with correct folding of the peptide chain and
simultaneous closure of the disulfide bridges to give
preproinsulin, and subsequently carrying out the chromatographic
purification method of claim 1, e) enzymic cleavage of
preproinsulin to give human insulin, f) purification of human
insulin, g) crystallization of human insulin and drying.
Description
[0001] Approximately 12 million people worldwide suffer from type 1
diabetes mellitus which is characterized by an insufficient
indigenous production of the hormone insulin. Substitution of the
lack of endocrine insulin secretion by applying insulin
preparations is the only possible form of therapy for this type of
diabetes mellitus.
[0002] Insulin preparations are pharmaceutical preparations whose
active substance is the hormone insulin. Here, insulin analogs and
insulin derivatives are used in addition to naturally occurring
insulins.
[0003] Human insulin which is produced in the human pancreas is a
polypeptide comprising 51 amino acid residues which divide into two
peptide chains: the A chain having 21 amino acid residues and the B
chain having 30 amino acid residues. The sequence of the amino acid
residues in both peptide chains has been genetically determined and
is known. Both chains are connected to one another by two disulfide
bridges. In addition, the A chain also contains an intrachain
disulfide bridge.
[0004] Insulin analogs differ from human insulin by substitution of
at least one amino acid residue and/or addition or removal of at
least one amino acid residue. Insulin analogs may either occur
naturally in species other than humans or may have been prepared
artificially. Insulin derivatives contain chemically modified amino
acid residues which contain, for example, additional ester or amido
groups but otherwise show the human or an analog amino acid
sequence.
[0005] Normally, insulin analogs or insulin derivatives exhibit an
altered action kinetics compared to unmodified human insulin.
[0006] For some years, human insulin and the insulin analogs or
insulin derivatives have been prepared by recombinant DNA
technology. In industrial methods, for example, first an
appropriate precursor of the formula 1, the preproinsulin (PPI), is
prepared from which human insulin or the insulin analogs are
prepared by enzymic cleavage. For example, a genetic method for
preparing human insulin comprises the following method steps:
[0007] a) Fermentation of the genetically modified
microorganisms,
[0008] b) Harvesting said microorganisms and cell disruption,
[0009] c) Isolating the inclusion bodies containing the undissolved
fusion protein,
[0010] d) Dissolving said fusion protein with correct folding of
the peptide chain and with simultaneous closure of the disulfide
bridges to give preproinsulin,
[0011] e) Enzymic cleavage of preproinsulin to give human
insulin,
[0012] f) Purification of human insulin,
[0013] g) Crystallization of human insulin and drying of the
obtained product.
[0014] When preparing an insulin analog, the amino acid sequence
(of the A and B chains) in the appropriate regions of preproinsulin
has already been predetermined. Enzymic cleavage of the various
preproinsulins is carried out using proteases such as, for example,
the enzyme trypsin and in addition, if necessary, the enzyme
carboxypeptidase B.
[0015] The preproinsulin is a protein of the formula 1, 1
[0016] in which
[0017] X a) is a genetically encodable amino acid residue or
[0018] b) is a peptide having from 2 to 35 amino acid residues,
which starts and ends with in each case a basic amino acid residue,
in particular Arg, and which, if it consists of more than 3 amino
acid residues, starts and ends with in each case two basic amino
acid residues, in particular Arg and/or Lys,
[0019] R.sup.1 a) is hydrogen,
[0020] b) is a genetically encodable amino acid residue or
[0021] c) is a peptide having from 2 to 15 amino acid residues,
[0022] R.sup.2 is a genetically encodable amino acid residue,
and
[0023] and the residues A1-A20 correspond to the amino acid
sequence of the A chain of human insulin or of an insulin analog
and the residues B1-B30 correspond to the amino acid sequence of
the B chain of human insulin or of an insulin analog.
[0024] The preproinsulin is preferably a protein of the formula 1
in which
[0025] X is a peptide having 35 amino acid residues with the
C-chain sequence of human insulin or simian insulin or is a peptide
having 29 amino acids of the sequence:
[0026]
Arg-Asp-Val-Pro-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-
-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg (SEQ ID NO: 1)
[0027] R.sup.1 is a peptide having from 2 to 15 amino acid
residues, whose carboxyl-terminal amino acid residue is Arg,
[0028] R.sup.2 is the amino acid residue Asn or Gly,
[0029] and the residues A1-A20 correspond to the amino acid
sequence of the A chain of human insulin and the residues B1-B30
correspond to the amino acid sequence of the B chain of human
insulin or of an insulin analog in which Lys replaces Asn in
position B3 and Glu replaces Lys in position B29.
[0030] The process stage--dissolving the fusion protein with
correct folding of the peptide chain and with simultaneous closure
of the disulfide bridges to give preproinsulin--produces, in
addition to the desired monomeric preproinsulin, also polymeric
forms of preproinsulin in a competing reaction. Said polymeric
preproinsulins can be detected, owing to their higher molecular
weight, by HPLC-GPC analysis or by the method of dynamic light
scattering. In order to repress this undesired competing reaction,
the initial concentration of the fusion protein needs to be as low
as possible (De Bernadez et al., Meth. Enzym. 309:217, 1999). In
practice, this process stage produces preproinsulin at a
concentration of from approx. 0.5 to 1 g/l, with approx. 40% of
higher molecular weight proportions being found in addition. The
higher molecular weight proportions include the polymeric
preproinsulins.
[0031] Surprisingly, it was found within the framework of the
present invention that the polymeric forms of preproinsulins
adversely affect the stability of the insulins in the subsequent
process stages by inducing the denaturation of the native insulins.
It is known that, during the denaturation reaction chain, a first
reversible step produces, from the dissolved monomeric insulin
molecules, linear aggregates in which physical adhesive forces hold
together the repeated units. An irreversible subsequent reaction
produces, from the dissolved aggregates, stable insoluble aggregate
bundles (fibrils) which in turn induce the denaturation of native
insulins in an autocatalytic process. These insoluble insulin
fibers are not only biologically inactive but may also cause
blockage of injection needles during application of the
pharmaceutical insulin preparations. In addition, they are also
held responsible for immunological incompatibility reactions which
can occasionally occur during therapy with insulin preparations (J.
Brange et al, J. Pharm. Sc. 1997, 86, 517-525; R. E. Ratner et al.,
Diabetes, 39, 728-733,1990).
[0032] In the subsequent part of the insulin preparation process,
preproinsulin is converted to human insulin with the aid of the
enzymes trypsin and carboxypeptidase B (see Kemmler, W., Peterson,
J. D., and Steiner, D. F., J. Biol. Chem., 246 (1971) 6786-6791).
Here, the linker peptide between the A and B chains (X in the
formula 1) and the pre part at the amino end of the B chain
(R.sup.1 in the formula 1) are removed. The enzymic reaction with
trypsin cleaves not only those peptide bonds whose cleavage
produces human insulin but also, in a competing reaction, other
peptide bonds whose cleavage produces a plurality of undesired
byproducts. The formation of de-Thr insulin due to additional
cleavage between amino acid residues B29 and B30 in formula 1 (see
EP 0 264 250 B1) is particularly undesired. The removal of this
byproduct in the subsequent purification stages results in large
losses of product. In order to repress this undesired side
reaction, the initial concentration of preproinsulin needs to be as
high as possible, i.e. in the range from 8-25 g/l, corresponding to
1-3 mM (see EP 0 264 250 B1). This requirement contrasts with the
requirement mentioned in the last but one paragraph.
[0033] From the above, it is evident that it is advantageous to
introduce, between production of preproinsulin and cleavage of
preproinsulin to insulin, an additional process step which removes
the polymeric preproinsulins as completely as possible and, at the
same time, increases the concentration of monomeric preproinsulin
as much as possible. An additional condition is the need to ensure
a very high yield in this process step.
[0034] It has therefore been proposed (EP 0 600 372 B1) to
concentrate preproinsulin on a hydrophobic adsorber resin. The
applicant was able to show in his own experiments that, although a
high concentration factor of F=10-15 can be achieved, there is
virtually no removal of polymeric preproinsulins. Another proposal
(D. F. Steiner et al., Diabetes, 17 (1968), 725-736) mentions
chromatographic purification of preproinsulin with the aid of an
ion exchanger resin. In our own experiments, we were only able to
achieve a concentration factor of F=5 and a removal of the higher
molecular weight proportions to approx. 5%, using an anion
exchanger resin. Although using a cation exchanger resin removed
the higher molecular weight proportions to approx. 1%, the binding
capacity of the resin for preproinsulin proved to be
unsatisfactory.
[0035] Surprisingly, it was then found that the combination of a
chromatography on an anion exchanger resin in flow-through mode
with an immediately following chromatography on a cation exchanger
resin in adsorption mode provided distinctly superior results. The
present invention therefore relates to a method for effectively
removing the higher molecular weight substances from an aqueous
solution of preproinsulin with simultaneous high concentration of
the monomeric preproinsulin.
[0036] According to the invention, a diluted aqueous solution of a
preproinsulin, as is produced during the preparation process of
insulin, is pumped at pH 7.0 to 9.0, preferably at pH 7.5 to 8.5,
and a conductivity of from 5 to 7 mS/cm through a precolumn packed
with an anion exchanger resin, for example Source 30 Q. In this
case, the monomeric preproinsulin is not bound to the resin but
runs through the column together with the permeate. In contrast,
the majority of the higher molecular weight substances, including
the polymeric preproinsulins, is adsorbed to the resin and thus
removed from preproinsulin. The permeate from this precolumn, which
contains the substance of interest, is adjusted in line to pH 3.0
to 5.5, preferably to pH 4.0 to 5.0, using hydrochloric acid and
then pumped directly onto a second column packed with a cation
exchanger resin, for example Source 30 S. Preproinsulin adsorbs to
this resin and impurities are washed out of the column together
with the permeate. Preproinsulin is desorbed with the aid of an
elution buffer containing sodium chloride at a linearly increasing
concentration of from 1 to 20 g/l, preferably 2.5 to 15.0 g/l. The
purified preproinsulin is collected in a main fraction, whereas
further impurities are removed in a prefraction and a postfraction.
In the main fraction which contained >90% of the initial amount
of preproinsulin, a concentration of from 15 to 20 g/l was measured
(concentration factor F=20-25). The higher molecular weight
substances were removed to a proportion of <0.1%. The
preproinsulin purified in this way can be isolated from the
solution intermediately by crystallization or the solution can be
fed directly to the enzymic cleavage process stage.
[0037] The present invention thus relates to a method for the
chromatographic purification of preproinsulin of the formula 1,
2
[0038] in which
[0039] X a) is a genetically encodable amino acid residue or
[0040] b) is a peptide having from 2 to 35 amino acid residues,
which starts and ends with in each case a basic amino acid residue,
in particular Arg, and which, if it consists of more than 3 amino
acid residues, starts and ends with in each case two basic amino
acid residues, in particular Arg and/or Lys,
[0041] R.sup.1 a) is hydrogen,
[0042] b) is a genetically encodable amino acid residue or
[0043] c) is a peptide having from 2 to 15 amino acid residues,
[0044] R.sup.2 is a genetically encodable amino acid residue,
and
[0045] and the residues A1-A20 correspond to the amino acid
sequence of the A chain of human insulin or of an insulin analog
and the residues B1-B30 correspond to the amino acid sequence of
the B chain of human insulin or of an insulin analog;
[0046] in which method higher molecular weight substances are
removed from an aqueous solution of said preproinsulin by means of
a first chromatography on an anion exchanger in flow-through mode
and a subsequent second chromatography on a cation exchanger in
adsorption mode;
[0047] wherein said preproinsulin can have the following amino acid
sequence:
[0048]
Ala-Thr-Thr-Ser-Thr-Gly-Asn-Ser-Ala-Arg-Phe-Val-Asn-Gln-His-Leu-Cys-
-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-T-
yr-Thr-Pro-Lys-Thr-Arg-Arg-Glu-Ala-Glu-Asp-Pro-Gln-Val-Gly-Gln-Val-Glu-Leu-
-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-G-
ln-Lys-Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-
-Glu-Asn-Tyr-Cys-Asn; (SEQ ID NO: 2)
[0049]
Ala-Thr-Thr-Ser-Thr-Gly-Asn-Ser-Ala-Arg-Phe-Val-Asn-Gln-His-Leu-Cys-
-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-T-
yr-Thr-Pro-Lys-Thr-Arg-Arg-Glu-Ala-Glu-Asp-Pro-Gln-Val-Gly-Gln-Val-Glu-Leu-
-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-G-
ln-Lys-Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-
-Glu-Asn-Tyr-Cys-Gly; (SEQ ID NO: 3)
[0050]
Ala-Thr-Thr-Ser-Thr-Gly-Asn-Ser-Ala-Arg-Phe-Val-Lys-Gln-His-Leu-Cys-
-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-T-
yr-Thr-Pro-Glu-Thr-Arg-Asp-Val-Pro-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-
-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg-Gly-Ile-Val-G-
lu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn
(SEQ ID NO: 4).
[0051] The invention further relates to a method as described above
for separating foreign substances from the solutions of
preproinsulins which induce insulin denaturation.
[0052] The invention further relates to a method as described
above, wherein the second chromatography is carried out at a pH of
from 3.0 to 5.5.
[0053] The invention further relates to a method as described
above, wherein the second chromatography is carried out under a
pressure of from 1 to 30 bar.
[0054] The invention further relates to a method for preparing
insulin by expressing nonfolded preproinsulin, comprising the
steps:
[0055] a) fermentation of genetically modified microorganisms which
express nonfolded preproinsulin,
[0056] b) harvesting the microorganisms and cell disruption,
[0057] c) isolating the inclusion bodies containing undissolved,
nonfolded preproinsulin,
[0058] d) dissolving the preproinsulin with correct folding of the
peptide chain and simultaneous closure of the disulfide bridges to
give preproinsulin, and subsequently running a method for
chromatographic purification of preproinsulin of the formula 1 as
described above,
[0059] e) enzymic cleavage of preproinsulin to give human
insulin,
[0060] f) purification of human insulin,
[0061] g) crystallization of human insulin and drying.
[0062] The contents of all references cited herein are hereby
incorporated in their entirety by reference.
[0063] The above disclosure generally describes the present
invention. A more complete understanding can be obtained by
reference to the following specific examples which are provided
herein for purposes of illustration only, and are not intended to
limit the scope of the invention.
EXAMPLES
[0064] The starting solution for the purification of various
preproinsulins of the formula 1, described in the following
examples 1 to 3, was prepared in the known manner (EP 0 489 780 and
EP 0 600 372) as follows, according to the abovementioned process
stages a, b, c and d:
[0065] During fermentation of the microorganisms (process stage a),
the E. coli cells formed inclusion bodies which contained the
fusion protein having the amino acid sequence of the preproinsulin.
After finishing the fermentation, the cells are isolated by
centrifugation and disrupted by means of the usual high-pressure
homogenization (process stage b). The insoluble inclusion bodies
released in the process were isolated by centrifugation and washed
with water in the centrifuge (process stage c). In the subsequent
process stage d, the fusion protein inclusion bodies were dissolved
in an 8 M guanidine hydrochloride solution at pH 10.8. After
diluting with water and adding cysteine hydrochloride, the fusion
protein was folded with closure of the 3 disulfide bridges at pH
10.8 and 4.degree. C. to give preproinsulin of the formula 1. The
solution was then adjusted to pH 5 using 10% strength hydrochloric
acid, as a result of which foreign proteins were precipitated which
were removed by centrifugation. The supernatant after
centrifugation contained 0.6 to 0.8 g/l monomeric preproinsulin.
The purity of preproinsulin, as determined by HPLC-RP analysis, was
approx. 65% by area. HPLC-GPC analysis determined a proportion of
approx. 45% by area higher molecular weight impurities.
1 HPLC-RP analysis Column: LiChroCART 250-4 from Merck (Superspher
100-RP18e) Instrument: Waters 2690 Software: Waters Millenium
Gradient: A: 25% by volume acetonitrile, 0.3 M NaCl in 0.05 M
phosphate buffer pH 2.5 B: 65% by volume acetonitrile, 0.05 M NaCl
in 0.05 M phosphate buffer pH 2.5 The gradient is characterized by
the following amounts of buffer B according to the corresponding
run times: 0 min 4.0%; 20 min 17.0%; 30 min 37.0%; 40 min 4.0%
Temperature: 35.degree. C. Loading volume: 10 .mu.l Total run time:
55 min Flow rate: 1.0 ml/min Detection: 214 nm (Waters 2487)
[0066] In order to determine the preproinsulin content in the
loading solution, the peak area of preproinsulin in the analyzed
sample was divided by the corresponding peak area of a standard
substance. In order to determine the degree of purity, the peak
area of preproinsulin was divided by the sum of the peak areas of
all elutable substances in the analyzed sample.
2 HPLC-GPC analysis Column: 2 columns in series, stainless steel L
= 300 mm; ID = 7.8 mm Instrument: pump: Waters 510/autosampler:
Wisp 717 Software: Waters Millenium Stationary phase: Shodex
Protein KW 802.5 120-7 diol Separation limits: 2 000 to 80 000
dalton Mobile phase: 30% by volume acetonitrile, 3.5 M acetic acid,
pH 3.0 adjusted with aqueous ammonia Gradient: isocratic
Temperature: room temperature Loading volume: 100 .mu.l Total run
time: 65 min Flow rate: 0.5 ml/min Detection: 276 nm (Waters
2487)
[0067] In order to determine the proportion of higher molecular
substances, the peak areas of all higher molecular substances which
were eluted prior to monomeric preproinsulin were divided by the
sum of the peak areas of all elutable substances. The retention
time for monomeric preproinsulin was determined using a standard
substance.
Example 1
[0068] After completion of the abovementioned process stages a, b,
c and d, a solution of preproinsulin having the following amino
acid sequence was obtained from the appropriately genetically
modified E.coli cells:
[0069]
Ala-Thr-Thr-Ser-Thr-Gly-Asn-Ser-Ala-Arg-Phe-Val-Asn-Gln-His-Leu-Cys-
-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-T-
yr-Thr-Pro-Lys-Thr-Arg-Arg-Glu-Ala-Glu-Asp-Pro-Gln-Val-Gly-Gln-Val-Glu-Leu-
-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-G-
ln-Lys-Arg-Gly-lle-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-
-Glu-Asn-Tyr-Cys-Asn (SEQ ID NO: 2)
[0070] Said preproinsulin corresponds to the formula 1, in
which
3 X is a peptide chain having 35 amino acid residues with the
sequence of simian C peptide, R1 is a peptide chain having 10 amino
acid residues of the sequence:
Ala-Thr-Thr-Ser-Thr-Gly-Asn-Ser-Ala-Arg (SEQ ID NO: 5) R2 is the
amino acid residue Asn (identical to A21 of the A chain of human
insulin) A1-A20 is the peptide chain having the sequence (only A1
to A20) of the A chain of human insulin B1-B30 is the peptide chain
having the sequence of the B chain of human insulin.
[0071] The preproinsulin solution was purified using an apparatus
which comprised primarily two chromatography columns arranged in
series and a stirred vessel arranged in between. The stirred vessel
was used to change the pH of the solution in line between the two
columns.
[0072] In the first chromatography column (manufacturer: Pharmacia,
diameter: 5 cm), a gel bed (bed height: 14 cm, bed volume: 275 ml)
was prepared using the anion exchanger resin DEAE-Sepharose fast
flow (manufacturer: Pharmacia Biotech; Prod. No. 17-0709-05). The
column was operated from top to bottom and at atmospheric pressure
of 1 bar. The flow rate was 2 000 ml/h. A multiway valve, a loading
pump (Ismatec MV) and a bubble trap were installed upstream of the
column. The following solutions were pumped onto the column
successively via the multiway valve:
[0073] 8.1 l of loading solution,
[0074] 2.3 l of displacement buffer,
[0075] 1.4 l of washing buffer,
[0076] 1.4 l of regenerating solution,
[0077] 2 l of equilibration buffer.
[0078] A UV probe (275 nm, with data recording) and another
multiway valve were installed downstream of the column. Via the
second multiway valve, approx. 10.2 l of permeate fraction were
conducted into the abovementioned stirred vessel and, subsequently,
approx. 1 l of washing fraction was conducted into a collecting
vessel. The remaining permeates were discharged into the biological
waste channel via the multiway valve.
[0079] The anion exchange chromatography was operated in
flow-through mode, i.e. the conditions (pH 8.3; conductivity=6.1
mS/cm) were chosen such that the valuable substance preproinsulin
was not bound to the gel but washed through the column together
with the permeate during product application. In contrast,
contaminations were adsorbed to the gel and removed with the
washing buffer and the regenerating solution.
[0080] The solutions used had the following composition:
4 Starting solution for column 1: Starting solution (supernatant
8.0 l from centrifugation) Sodium chloride solution, 25% 100 ml
12.5 ml/l strength (w/w) Sodium hydroxide solution, approx. 4.5 ml
0.6 ml/l 10% (w/w) pH 8.3 Conductivity 6.1 mS/cm Temperature
approx. 5.degree. C. Purified water 1 l Tris(hydroxymethyl) 4.0 g/l
aminomethane Sodium chloride 2.5 g/l Hydrochloric acid, approx. 2.5
ml/l 25% strength (w/w) pH 8.0 Conductivity approx. 5.7 mS/cm
Temperature room temperature Purified water 1 l Tris(hydroxymethyl)
5.0 g/l aminomethane Sodium chloride 15 g/l Hydrochloric acid,
approx. 3 ml/l 25% strength (w/w) pH 8.0 Conductivity approx. 24
mS/cm Temperature room temperature Regenerating solution for
columns 1 and 2: Purified water 0.91 L Sodium chloride 40 g 40 g/l
Sodium hydroxide solution 33% 0.09 l 1 mol/l strength (w/w)
Equilibration buffer for column 1: Purified water 1 l
Tris(hydroxymethyl) 5.0 g/l aminomethane Sodium chloride 2.0 g/l
Hydrochloric acid, approx. 3 ml/l 25% strength (w/w) pH 8.0
Conductivity approx. 5.1 mS/cm Temperature room temperature
[0081] The permeate fraction containing the valuable substance
preproinsulin and the washing fraction containing the majority of
the higher molecular weight impurities were collected at the column
outlet:
5 1. approx. 10.2 l permeate fraction (at start of loading
solution, from UV value 20% (ascending) to UV value 35%
(descending), during product displacement) 2. approx. 1 l washing
fraction (during loading of washing buffer, from UV value 30%
(ascending slope) to UV value 40% (descending))
[0082] All other permeates were discharged into the biological
waste channel.
[0083] FIG. 1 depicts the UV diagram measured at the outlet of
column 1.
[0084] The permeate fraction of the first column was adjusted to pH
3.5 with 90% strength lactic acid inline in the intermediate vessel
(nominal volume: 4 l, with stirrer, pH probe and inlet tube) and
then pumped directly onto the second chromatography column.
[0085] In the second chromatography column (manufacturer:
Pharmacia, diameter: 5 cm), a gel bed (bed height: 10.5 cm, bed
volume: 206 ml) was prepared using the cation exchanger resin
Source 30 S (manufacturer: Pharmacia Biotech; Prod. No.
17-1273-04). The column was operated from top to bottom and at
atmospheric pressure of 1 bar. The flow rate was likewise 2 000
ml/h. A multiway valve, a loading pump and a bubble trap were
installed upstream of the column. The following solutions were
pumped onto the column successively via the multiway valve:
[0086] 10.2 l loading solution (=permeate fraction of column 1,
adjusted to pH 3.5)
[0087] 0.5 l displacement buffer
[0088] 3.0 l elution buffer A/B (equal amounts of A and B)
[0089] 2.3 l regenerating solution
[0090] 2 l equilibration buffer
[0091] A UV probe (275 nm, with data recording) and another
multiway valve were installed downstream of the column. Approx. 1 l
of the main fraction was conducted via the second multiway valve
into a collecting vessel. The remaining permeates were discharged
via the multiway valve into the biological waste channel.
[0092] The cation exchanger chromatography was operated in
adsorption mode, i.e. the valuable substance preproinsulin was
adsorbed to the gel during product application and (after
displacing the loading solution) desorbed again using the elution
buffer A/B. In order to achieve an optimal purification effect, a
linearly increasing sodium chloride gradient was applied in the
elution buffer.
[0093] The solutions used had the following composition:
6 Loading solution for column 2: Permeate fraction of column 1
approx. 10.2 l Lactic acid, 90% strength 14.3 ml 1.4 ml/l pH 3.5
Conductivity approx. 6.3 mS/cm Temperature approx. 5.degree. C.
Displacement buffer for column 2: Purified water 1 l Lactic acid,
90% strength 8.3 ml 0.1 mol/l Sodium chloride 2.5 g 2.5 g/l Sodium
hydroxide solution, approx. 8 ml 10% strength (w/w) pH 3.5
Conductivity approx. 8 mS/cm Temperature room temperature Elution
buffer A for column 2: The elution buffer A is identical to the
displacement buffer for column 2. Elution buffer B for column 2:
Purified water 1 l Lactic acid, 90% strength 8.3 ml 0.1 mol/l
Sodium chloride 15.0 g 15.0 g/l Sodium hydroxide solution, approx.
7 ml 10% strength (w/w) pH 3.5 Conductivity approx. 25 mS/cm
Temperature room temperature Regeneration solution for columns 1
and 2: Purified water 0.91 l Sodium chloride 40 g 40 g/l Sodium
hydroxide solution, 0.09 l 1 mol/l 33% strength (w/w) Equilibrium
buffer for column 2: Purified water 1 l Lactic acid, 90% strength
8.3 g 0.1 mol/l Sodium chloride 2.9 g/l Sodium hydroxide solution
approx. 9 ml 10% strength (w/w) pH 3.5 Conductivity approx. 8.5
mS/cm Temperature room temperature
[0094] The main fraction which contained the valuable substance
preproinsulin was collected at the column outlet:
7 approx. 1.0 l main fraction (during elution, from UV value 65%
(ascending) to UV value 76% (descending))
[0095] All other permeates were discharged into the biological
waste channel.
[0096] FIG. 2 depicts the UV diagram measured at the outlet of
column 2.
[0097] In the purified solution (main fraction column 2), 15 g/l
preproinsulin with a degree of purity of 89% by area were measured
(HPLC-RP analysis). The yield was 91%, based on the amount of
preproinsulin in the starting solution. Higher molecular
proportions of 0.2% by area were determined by HPLC-GPC
analysis.
Example 2
[0098] After completion of the abovementioned process stages a, b,
c and d, a solution of preproinsulin having the following amino
acid sequence is obtained from the appropriately genetically
modified E. coli cells:
[0099]
Ala-Thr-Thr-Ser-Thr-Gly-Asn-Ser-Ala-Arg-Phe-Val-Asn-Gln-His-Leu-Cys-
-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-T-
yr-Thr-Pro-Lys-Thr-Arg-Arg-Glu-Ala-Glu-Asp-Pro-Gln-Val-Gly-Gln-Val-Glu-Leu-
-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-G-
ln-Lys-Arg-Gly-lle-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-
-Glu-Asn-Tyr-Cys-Gly (SEQ ID NO: 3)
[0100] Said preproinsulin corresponds to the formula 1, in
which
8 X is a peptide chain having 35 amino acid residues with the
sequence of simian C peptide, R1 is a peptide chain having 10 amino
acid residues of the sequence:
Ala-Thr-Thr-Ser-Thr-Gly-Asn-Ser-Ala-Arg (SEQ ID NO: 5) R2 is the
amino acid residue Gly A1-A20 is the peptide chain having the
sequence (only A1 to A20) of the A chain of human insulin B1-B30 is
the peptide chain having the sequence of the B chain of human
insulin.
[0101] The preproinsulin solution was purified by again using an
apparatus which comprised primarily two chromatography columns
arranged in series and a stirred vessel arranged in between. The
stirred vessel was used to change the pH of the solution inline
between the two columns. The apparatuses for the second
chromatography stage were designed for pressure stability.
[0102] The chromatography on column 1 and the pH-switching in the
intermediate vessel were carried out as described in example 1 so
that the description and the values will not be repeated here.
[0103] In the second chromatography column (manufacturer: Prochrom,
diameter: 5 cm, material: stainless steel), a gel bed (bed height:
10 cm, bed volume: 196 ml) was prepared using the cationic
exchanger Source 30 S (manufacturer: Pharmacia Biotech; prod. No.:
17-1273-04). The column was operated from top to bottom and at a
working pressure of 10 bar. The flow rate was 3 500 ml/h. A
multiway valve, a loading pump (manufacturer: Besta; type: HD2-300)
were installed upstream of the column. The following solutions were
pumped onto the column successively via the multiway valve:
[0104] 10.2 l loading solution (=permeate fraction of column 1,
adjusted to pH 4.6)
[0105] 0.5 l displacement buffer
[0106] 3.0 l elution buffer A/B (equal amounts of A and B)
[0107] 2.3 l regenerating solution
[0108] 2 l equilibration buffer
[0109] A UV probe (275 nm, with data recording) and another
multiway valve were installed downstream of the column. The main
fraction containing the purified preproinsulin was conducted via
the second multiway valve into a collecting vessel. The remaining
permeates were discharged via the multiway valve into the
biological waste channel.
[0110] The cation exchanger chromatography was operated in
adsorption mode, i.e. the valuable substance preproinsulin was
adsorbed to the gel during product application and (after
displacing the loading solution) desorbed again using the elution
buffer A/B. In order to achieve an optimal purification effect, a
linearly increasing sodium chloride gradient was applied in the
elution buffer.
[0111] The solutions used had the following composition:
9 Loading solution for column 2: Permeate fraction of column 1
approx. 10.2 l Lactic acid, 90% strength 12.2 ml 1.2 ml/l pH 4.6
Conductivity approx. 6.7 mS/cm Temperature approx. 5.degree. C.
Displacement buffer for column 2: Purified water 1 l Lactic acid,
90% strength 8.3 ml 0.1 mol/l Sodium chloride 2.5 g 2.5 g/l Sodium
hydroxide solution, approx. 27 ml 10% strength (w/w) pH 4.6
Conductivity approx. 8 mS/cm Temperature room temperature Elution
buffer A for column 2: The elution buffer A is identical to the
displacement buffer for column 2. Elution buffer B for column 2:
Purified water 1 l Lactic acid, 90% strength 8.3 ml 0.1 mol/l
Sodium chloride 15.0 g 15.0 g/l Sodium hydroxide solution, approx.
27 ml 10% strength (w/w) pH 4.6 Conductivity approx. 25 mS/cm
Temperature room temperature Regeneration solution for columns 1
and 2: Purified water 0.91 l Sodium chloride 40 g 40 g/l Sodium
hydroxide solution, 0.09 l 1 mol/l 33% strength (w/w) Equilibrium
buffer for column 2: Purified water 1 l Lactic acid, 90% strength
8.3 g 0.1 mol/l Sodium chloride 2.9 g/l Sodium hydroxide solution
approx. 26 ml 10% strength (w/w) pH 4.6 Conductivity approx. 8.7
mS/cm Temperature room temperature
[0112] The main fraction which contained the valuable substance
preproinsulin was collected at the column outlet:
10 approx. 0.9 l main fraction (during elution, from UV value 65%
(ascending) to UV value 76% (descending))
[0113] All other permeates were discharged into the biological
waste channel. In the purified solution (main fraction from column
2), 17 g/l preproinsulin with a degree of purity of 93% by area
were measured (HPLC-RP analysis). The yield was 92%, based on the
amount of preproinsulin in the starting solution. Higher molecular
weight proportions of <0.1% by area were determined by HPLC-GPC
analysis.
Example 3
[0114] After completion of the abovementioned process stages a, b,
c and d, a solution of preproinsulin having the following amino
acid sequence is obtained from the appropriately genetically
modified E.coli cells:
[0115]
Ala-Thr-Thr-Ser-Thr-Gly-Asn-Ser-Ala-Arg-Phe-Val-Lys-Gln-His-Leu-Cys-
-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-T-
yr-Thr-Pro-Glu-Thr-Arg-Asp-Val-Pro-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-
-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg-Gly-lle-Val-G-
lu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn
(SEQ ID NO: 4)
[0116] Said preproinsulin corresponds to the formula 1, where
11 X is a peptide chain having 29 amino acid residues with the
sequence: Arg-Asp-Val-Pro-Gln-Val-Glu-Leu-Gly--Gly--
Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-
Ser-Leu-Gln-Lys-Arg (SEQ ID NO: 1) R1 is a peptide chain having 10
amino acid residues with the sequence: Ala-Thr--Thr-Ser-Thr-Gly-A-
sn-Ser-Ala-Arg (SEQ ID NO: 5), R2 is the amino acid residue Asn
(A21 of the A chain of human insulin), A1-A20 is a peptide chain
with the sequence (only A1 to A20) of the A chain of human insulin,
B1-B30 [lacuna] peptide chain with a sequence similar to the B
chain of human insulin, i.e. with Lys replacing Val in position B3
and Glu replacing Lys in position B29.
[0117] The preproinsulin solution was purified using the same
apparatus used in example 1.
[0118] This time, the anion exchanger resin Source 30 Q
(manufacturer: Pharmacia Biotech; Prod.-No.: 17-1275-04) was used
for the chromatography on column 1. Regeneration of this gel
required twice the amount of regenerating solution compared to
examples 1 and 2. The remaining parameters of the first
chromatography, such as composition and volumes of the solutions,
were the same as those described in examples 1 and 2.
[0119] Likewise, the pH-switching in the intermediate vessel was
carried out as described in example 1.
[0120] The second chromatography was this time carried out with a
working pressure of 15 bar. All other parameters of the second
chromatography were identical to those described in example 2.
[0121] In the purified solution (main fraction from column 2), 17
g/l preproinsulin with a degree of purity of 92.5% by area were
measured (HPLC-RP analysis). The yield was 91%, based on the amount
of preproinsulin in the starting solution. Higher molecular weight
proportions of <0.1% by area were determined by HPLC-GPC
analysis.
[0122] Denaturation Assay
[0123] The denaturation assay (table 1) shows that the higher
molecular weight, polymeric forms of preproinsulins, as produced
during the folding reaction, can induce denaturation of native
insulin.
[0124] In the denaturation assay, native insulin glargine, an
Aventis Deutschland GmbH product, which is obtained after enzymic
cleavage of the preproinsulin described in example 2, was
crystallized. Surprisingly, we were able to show in all experiments
that, under the conditions of insulin glargine crystallization (pH
6.1 and 26.degree. C.), denaturation of native insulin occurs when
substances which can induce insulin denaturation are added to the
crystallization mixture.
[0125] A standard solution of the following composition was
prepared for the crystallization mixtures:
12 Insulin glargine 5 g/l Citric acid 5.2 mmol/l Zinc chloride 3
mmol/l Sodium chloride 0.5 g/l n-Propanol 7% (v/v) Purified water
to 500 ml using 1 N hydrochloric acid, pH 3
[0126] The solution was filtered through a membrane filter with a
pore width of 0.1 .mu.m.
[0127] In the denaturation assay, this acidic standard solution was
admixed with solutions of the various assay substances: the washing
fraction of column 1 which contained the removed polymeric forms of
preproinsulins at a concentration of 5 g/l or the main fraction of
column 2 which contained purified preproinsulin at a concentration
of 15 and, respectively, 17 g/l. For further proof that the
phenomena observed were caused by insulin denaturation, 10 ml of a
0.1% strength aqueous stock solution of Poloxamer 171 were added.
Poloxamer 171 is known to be able to suppress insulin denaturation
at hydrophobic interfaces (H. Thurow and K. Geisen, Diabetologia
(1984) 27, 212-218 and EP 0 018 609).
[0128] The solutions were then heated to 26.degree. C. and adjusted
to pH 6.1 with 10% strength sodium hydroxide solution with
stirring, resulting in the precipitation of amorphous insulin. The
amorphous suspension was stirred at 26.degree. C. for 50 hours.
After this time, all mixtures contained insulin crystals.
[0129] The mixtures were analyzed by evaluating samples under the
microscope, looking for the appearance of amorphous particles
(veils) in the background or between the insulin crystals. In
addition, each mixture was divided into two parts of approximately
the same size. The first part was introduced into a 250 ml
measuring cylinder in order to investigate the sedimentation
behavior, and after leaving the mixtures at room temperature for 60
min, sediment volume and supernatant clarity were evaluated. The
second part was adjusted to pH 3 with 1 N hydrochloric acid, and,
after the insulin crystals had dissolved, the clarity of the
resulting solution was evaluated.
[0130] Table 1 shows the result of the denaturation assay. In the
control samples 174 A and 188 A without addition of the polymer
fraction, no denaturation had been observed. Under the microscope,
crystals were visible against a clear background. After 60 minutes,
the crystals had sedimented, resulting in a compact sediment and a
clear supernatant. After dissolving the crystals at pH 3, a clear
solution had been produced. In contrast, the samples 174 B, 188 B
and 174 C, in the case of which 1 ml and, respectively, 5 ml of
polymer fraction had been added to the crystallization mixture,
showed a distinct denaturation of insulin glargine. Under the
microscope, an amorphous veil was visible between the crystals. In
the sedimentation assay, voluminous sediments having a sediment
volume of from 50 to 90 ml (from 250 ml of crystal suspension) had
been produced. After redissolving the crystals at pH 3, more or
less opaque amorphous suspensions had been produced. In the
presence of 20 ppm of Poloxamer 171, no denaturation had been
observed with the addition of 1 ml of polymer solution (188 C). In
the assay mixtures 174 D, 174 E and 174 F which had been admixed
with purified preproinsulins (main fraction of column 2 from
examples 1, 2 and 3), likewise no denaturation of insulin glargine
was observed.
[0131] Similar results, not shown here, were obtained in an
analogous denaturation assay in which human insulin was
crystallized.
13TABLE 1 Influence of higher molecular weight impurities on
insulin glargine denaturation Microscopic image Crystallization
background appearance Appearance after Sedimentation mixture
Additions to the (between rhombohedral dissolving the crystals
behavior of No. crystallization mixture crystals) at pH 3 crystal
suspension 174 A none clear clear +++ 174 B 1 ml of washing
fraction, column 1* amorphous particles slightly opaque +-- 174 C 5
ml of washing fraction, column 1* more amorphous particles opaque
--- 174 D 1 ml of main fraction, example 1** clear clear +++ 174 E
1 ml of main fraction, example 2*** clear clear +++ 174 F 1 ml of
main fraction, example 3**** clear clear +++ 188 A none clear clear
+++ 188 B 1 ml of washing fraction, column 1* amorphous particles
opaque --- 188 C 1 ml of washing fraction, column 1* occasional
amorphous particles clear +++ and 20 ppm Poloxamer 171
Crystallization mixtures of in each case 500 ml with 2 500 mg of
insulin glargine (=5 g/l). +++ = After approx. 60 min, 3 to 5 ml of
sediment from 250 ml of suspension. --- = After approx. 60 min, up
to 90 ml of sediment from 250 ml of suspension. *The washing
fraction of column 1 from example 2 contains 5 g/l protein. **The
main fraction of column 2 from example 1 contains 15 g/l
preproinsulin ***The main fraction of column 2 from example 2
contains 17 g/l preproinsulin ****The main fraction of column 2
from example 3 contained 17 g/
[0132]
Sequence CWU 1
1
4 1 29 PRT Artificial Sequence Description of Artificial
SequenceC-Peptid 1 Arg Asp Val Pro Gln Val Glu Leu Gly Gly Gly Pro
Gly Ala Gly Ser 1 5 10 15 Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu
Gln Lys Arg 20 25 2 96 PRT Artificial Sequence Description of
Artificial Sequence Preproinsulin I 2 Ala Thr Thr Ser Thr Gly Asn
Ser Ala Arg Phe Val Asn Gln His Leu 1 5 10 15 Cys Gly Ser His Leu
Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg 20 25 30 Gly Phe Phe
Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Pro Gln 35 40 45 Val
Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln 50 55
60 Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val Glu Gln
65 70 75 80 Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr
Cys Asn 85 90 95 3 96 PRT Artificial Sequence Description of
Artificial Sequence Preproinsulin II 3 Ala Thr Thr Ser Thr Gly Asn
Ser Ala Arg Phe Val Asn Gln His Leu 1 5 10 15 Cys Gly Ser His Leu
Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg 20 25 30 Gly Phe Phe
Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Pro Gln 35 40 45 Val
Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln 50 55
60 Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val Glu Gln
65 70 75 80 Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr
Cys Gly 85 90 95 4 90 PRT Artificial Sequence Description of
Artificial Sequence Preproinsulin III 4 Ala Thr Thr Ser Thr Gly Asn
Ser Ala Arg Phe Val Lys Gln His Leu 1 5 10 15 Cys Gly Ser His Leu
Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg 20 25 30 Gly Phe Phe
Tyr Thr Pro Glu Thr Arg Asp Val Pro Gln Val Glu Leu 35 40 45 Gly
Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly 50 55
60 Ser Leu Gln Lys Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys
65 70 75 80 Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn 85 90
* * * * *