U.S. patent application number 10/719552 was filed with the patent office on 2004-04-22 for purified insulin composition.
Invention is credited to Bogsnes, Are.
Application Number | 20040077527 10/719552 |
Document ID | / |
Family ID | 8094571 |
Filed Date | 2004-04-22 |
United States Patent
Application |
20040077527 |
Kind Code |
A1 |
Bogsnes, Are |
April 22, 2004 |
Purified insulin composition
Abstract
The invention concerns a process of chromatographically
separating glycosylated proteins from non-glycosylated proteins by
subjecting a solution comprising glycolsylated and non-glycosylated
proteins to chromatography using a Ca.sup.++ containing eluant. By
using this process a fraction comprising non-glycosylated proteins
substantially free from glycosylated proteins is obtained. The
process may be applied to the separation of proteins used in the
medical industry, such as insulin.
Inventors: |
Bogsnes, Are; (Niva,
DK) |
Correspondence
Address: |
NOVO NORDISK PHARMACEUTICALS, INC
100 COLLEGE ROAD WEST
PRINCETON
NY
08540
US
|
Family ID: |
8094571 |
Appl. No.: |
10/719552 |
Filed: |
November 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10719552 |
Nov 21, 2003 |
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09633526 |
Aug 7, 2000 |
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09633526 |
Aug 7, 2000 |
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09290856 |
Apr 13, 1999 |
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6180757 |
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Current U.S.
Class: |
530/350 ;
514/20.9; 514/5.9; 530/303 |
Current CPC
Class: |
C07K 1/20 20130101 |
Class at
Publication: |
514/003 ;
530/303 |
International
Class: |
A61K 038/28 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 15, 1998 |
DK |
0528/98 |
Claims
1. A composition comprising recombinant insulin or an analogue
thereof, wherein (i) said composition comprises about 0.02% to 0.2%
glycosylated insulin or insulin analogue and (ii) said recombinant
insulin or insulin analogue is produced in a yeast cell.
2. A composition as defined in claim 1, wherein said glycosylated
insulin or insulin analogue is mono-glycosylated.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the process of
chromatographically separating glycosylated proteins or protein
precursors from non-glycosylated proteins or protein precursors
using a Ca.sup.++ containing eluant.
BACKGROUND AND DESCRIPTION OF THE INVENTION
[0002] In the manufacture of proteins or protein precursors the
separation of glycosylated proteins from non-glycosylated proteins
represents an independent field of research. The industrial
implications of the present invention relate to the optimisation of
protein purification. It is the purpose of the optimised
purification to achieve an end product to be used commercially
comprising non-glycosylated proteins, substantially free from
glycosylated proteins.
[0003] Proteins or protein precursors may originate from yeast
expression systems. A correlation between a high expression level
and an increase in glycosylated protein precursors has been
observed. As a consequence the need for a more efficient process of
separating the non-glycosylated proteins from the glycosylated
proteins has become even more apparent.
[0004] In the yeast expression system a yeast organism produces
proteins or protein precursors synthesised intracellularly. The
yeast host organism is transformed by an expression vehicle
harbouring DNA encoding the desired protein. The process comprises
preparing a culture of the transformed yeast host organism, growing
the culture and recovering the protein from the culture medium.
[0005] Prior art recovery of the desired protein comprised multiple
steps of purification using the process of chromatography, such as
ion exchange chromatography involving an eluant containing salt. By
using the method of reverse phase high performance liquid
chromatography (RP-HPLC) the procedure of separating
non-glycosylated proteins from glycosylated proteins can be carried
out. The eluant used for the eluation of the test samples is
preferably an organic solvent containing salt, such as KCl.
Although various salts have been applied to the organic solvent in
the prior art the effect of a Ca.sup.++ containing eluant has never
been disclosed.
[0006] In the present context the term eluant is synonymous with
the buffer used for eluation. The present invention provides an
improved purification of the desired protein by using a Ca.sup.++
containing eluant.
SUMMARY OF THE INVENTION
[0007] The present invention provides an improved process of
separating glycosylated proteins from non-glycosylated proteins.
Accordingly the present invention relates to a process of
separating glycosylated proteins from non-glycosylated proteins by
subjecting a solution comprising glycosylated and non-glycosylated
proteins to RP-HPLC (reverse phase high performance liquid
chromatography) using a Ca.sup.++ containing eluant, and obtaining
a fraction comprising non-glycosylated proteins, said fraction
substantially free from glycosylated proteins.
[0008] In the present invention the term protein refers to all
proteins or protein precursors and the term glycosylated proteins
includes glycosylated proteins or protein precursors. In a
preferred embodiment the protein is insulin or an analogue or
precursor thereof.
[0009] Another object of the present invention is a fraction
obtainable by using the process according to the invention
comprising non-glycosylated proteins substantially free from
glycosylated proteins.
DRAWINGS
[0010] The following is a short description of the figures
illustrating the invention. The peaks on the chromatograms are
marked on the figures. The term hydrolysis pool indicates the pool
used for purification.
[0011] FIG. 1 shows the profile of the hydrolysis pool before the
purification using RP-HPLC.
[0012] FIG. 2 shows the profile of the hydrolysis pool after
purification using RP-HPLC.
[0013] FIG. 3 shows the separation profile of the RP-HPLC run using
a buffer system with 100 mM CaCl.sub.2. The peak representing the
glycosylated X14 forms is almost baseline separated.
[0014] FIG. 4 shows the separation profile of the RP-HPLC run using
a buffer system with 50 mM CaCl.sub.2.
[0015] FIG. 5 shows the separation profile of the RP-HPLC run using
a buffer system with 50 mM KCl.
[0016] FIG. 6 shows the separation profile of the RP-HPLC run using
a buffer system with 200 mM KCl.
[0017] FIG. 7 shows the separation profile of the RP-HPLC run using
a buffer system with 100 mM KCl and 25 mM CaCl.sub.2.
[0018] FIG. 8 shows the separation profile of the RP-HPLC run using
a buffer system with 150 mM KCl and 12.5 mM CaCl.sub.2.
[0019] FIG. 9 shows the separation profile of a RP-HPLC run for the
purification of the insulin analogue DesB30 using one buffer system
without CaCl.sub.2 and another buffer system with 20 mM
CaCl.sub.2.
[0020] FIG. 10 shows the separation profile of an analytical
RP-HPLC run for the purification of the insulin analogue DesB30
using one buffer system without CaCl.sub.2 and another buffer
system with 20 mM CaCl.sub.2.
DETAILED DESCRIPTION OF THE INVENTION
[0021] Proteins or protein precursors and analogues thereof may
originate from yeast expression systems. In the manufacturing
process of proteins or protein precursors the use of
chromatographical purification is widespread. The proteins or
protein precursors are often subjected to chemical modifications
and a series of chromatografical purification steps, such as
RP-HPLC, hydrophobe interaction chromatography and ion exchange.
The present invention concerns a purification step in which the use
of chromatography in the separation of glycosylated proteins from
non-glycosylated proteins has proven to be particularly successful.
The process comprises subjecting a solution comprising glycosylated
and non-glycosylated proteins to chromatography using a Ca.sup.++
containing eluant, and thereby obtaining a fraction comprising
non-glycosylated proteins, said fraction substantially free from
glycosylated proteins.
[0022] The principle of protein purification using column
chromatography is based upon differences in the equilibrium between
the stationary and the mobile phase of the proteins to be
separated. Using an appropriate combination of stationary and
mobile phases, the proteins will leave the column at different
intervals.
[0023] The chromatography method may be any column chromatography
method, preferably RP-HPLC, or hydrophobe interaction
chromatography.
[0024] In the present invention the advantage is the increased
purity of the fraction of non-glycosylated proteins. The fraction
of glycosylated proteins may consist of mono-glycosylated proteins
and poly-glycosylated proteins. The present invention provides
means for the improvement of the separation of glycosylated
proteins, such as mono-glycosylated proteins and poly-glycosylated
proteins, from non-glycosylated proteins.
[0025] Another aspect of the present invention is obtaining an
additional fraction comprising glycosylated proteins and
substantially no non-glycosylated proteins.
[0026] Depending on the composition of the starting material the
glycosylated proteins may be mono-glycosylated, or at least a part
of the glycosylated proteins may be di-glycosylated, or at least a
part of the glycosylated proteins may be poly-glycosylated.
[0027] Compared to other chromatography methods, where a Ca.sup.++
containing eluant is not used, the present invention holds the
advantage of a higher productivity due to an increased column load
of approximately 50%. The efficiency of the purification is
influenced by the ligand and by the particle pore size of the
matrix. The particle pore size may vary dependent upon the nature
of the protein to be purified. When the protein is insulin or an
analogue thereof the optimal column matrix is 200 .ANG. and the
column may be loaded up to 150-250 mg/cm.sup.2 when the starting
material is of the given composition described below, and the
length of the column used is as described below.
[0028] The column temperature may be 10-30.degree. C., preferably
18-25.degree. C. Any temperature, lower than the preferred
temperature range may lead to an increase in expenses of
purification by slowing the process down.
[0029] By using methods for the purification of non-glycosylated
proteins described by prior art, it has not yet been possible to
achieve a fraction substantially free from glycosylated proteins
while maintaining such a high productivity. However the
introduction of the present invention has improved the purification
process markedly, and the final fraction comprising
non-glycosylated proteins is substantially free from glycosylated
proteins.
[0030] By using the means of the present invention a fraction
comprising non-glycosylated proteins is obtainable, wherein said
fraction is substantially free from glycosylated proteins, whilst
the productivity level of said fraction is increased.
[0031] The present invention has proved to be especially
advantageous in the separation of glycosylated insulins from
non-glycosylated insulins. Preferably, the concentration of
glycosylated insulins in the fraction of non-glycosylated insulin
is less than 0.2%, such as less than 0.1%. This is approximately a
ten-fold reduction in the concentration of glycosylated insulins
compared to the starting material.
[0032] The starting material for the separation or purification may
be any protein solution comprising non-glycosylated and
glycosylated proteins. The starting material may be the medium
obtained from yeast expression systems directly, or the starting
material may have been subjected to several purification or
chemical modification steps prior to the separation according to
the invention.
[0033] Without being bound by theory it is the general belief that
the interaction of the proteins with Ca.sup.++ leads to a change of
the proteins hydrophobicity. It is the differential change in
hydrophobicity effected by minor molecular differences, such as
sugar or ester groups, that is believed to constitute the very core
of the principle behind the separation of glycosylated proteins
from non-glycosylated proteins or otherwise modified proteins. The
degree to which the proteins change in hydrophobicity upon the
presence of Ca.sup.++ are dependent on the nature of the protein,
such as the presence of glycosyl and/or ester groups.
[0034] Thus, the method according to the present invention may be
used to separate any variants of proteins, wherein the
hydrophobicity of the variants are changed differently by the
addition of Ca.sup.++ in the eluant.
[0035] The application of Ca.sup.++ to an organic solvent-based
eluant greatly enhances separation of glycosylated proteins from
non-glycosylated proteins compared to that of an eluant comprising
K.sup.+ or Na.sup.+ or NH.sub.4.sup.+ alone or in combination. This
fact is due to the selective effect of Ca.sup.++ on the increase in
hydrophobicity of non-glycosylated proteins. In addition to
Ca.sup.++ ions the eluant may comprise cations such as K.sup.+
and/or Na.sup.+ and/or NH.sub.4.sup.+ ions. The Ca.sup.++ ions may
be supplied by any suitable source, such as CaCl.sub.2.
[0036] The present inventor has found that the combination of
Ca.sup.++ and K.sup.+ ions in the eluant improves the
chomatographic properties. In the present invention the preferred
composition of the eluant is a mixture of KCl and CaCl.sub.2 where
the Ca.sup.++ concentration in the eluant is below 300 mM, such as
below 200 mM, preferably below approximately 100 mM, more
preferably approximately 5-50 mM, and a K.sup.+ concentration of
100-300 mM, such as approximately 200 mM. Within the given
concentration ranges the concentration of KCl in the eluant
preferably exceeds the eluants CaCl.sub.2 concentration.
[0037] When Na.sup.+ and/or NH.sub.4.sup.+ ions are present the
Na.sup.+ and/or NH.sub.4.sup.+ concentration in the eluant is
100-300 mM, such as approximately 200 mM.
[0038] The preferred pH value of the eluant is higher than the
isoelectric point for the protein, and the analogues thereof, to be
separated. With respect to insulin, most insulins have an
isoelectric point not below pH 5.0.
[0039] In the present invention the retention volume is between 5
and 10 CV (column volume) in an isocratic eluation.
[0040] The eluant may in principle comprise any organic solvent.
The organic solvent may be ethanol, methanol, isopropanol or
acetonitril. In the present invention the preferred organic solvent
is ethanol having a concentration in the range of 20-30% by
weight.
[0041] The invention further relates to a fraction obtainable by
using the process according to the invention comprising
non-glycosylated proteins, wherein the fraction is substantially
free from glycosylated proteins. The invention is advantageously
used for separating insulins, wherein the fraction may have a
concentration of glycosylated insulins of less than 0.2% by
weight/volume.
[0042] The purified non-glycosylated proteins, in particular
insulins, may find application in any technical field, such as the
medical field, such as the treatment of diabetes.
[0043] Experimentals
[0044] The following is a description of the experiments performed
to improve the separation of non-glycosylated insulins from
glycosylated insulins. By using an eluant containing Ca.sup.++ ions
it was found that the separation is greatly enhanced, and a more
pure end product of non-glycosylated insulins were achieved.
[0045] Materials and Methods
[0046] General Methods
[0047] All experiments were performed on pools containing
glycosylated and non-glycosylated forms of the insulin analogue X14
or the insulin analogue DesB30. X14 has the same a and b chains as
do human insulin, the only diffence being the substitution in
position b28 of proline in human insulin for aspartic acid in X14.
DesB30 has the same a and b chains as do human insulin, the only
diffence being the lack of treonin in position b30.
[0048] All percentages are given as % by weight.
[0049] Examples 1-3 were conducted with X14, and example 4 was
conducted with DesB30.
[0050] Analysis
[0051] Selected pools were fractionated and analysed on RP-HPLC.
The pools were analysed for the main product as well as for
glycosylated variants. Fractions of X14 were also analysed for
ethylester.
[0052] Chromatography
[0053] The chromatography was done on a BioCAD HPLC system from
PerSeptive Biosystems equipped with a 6 or 3 mm flow cell. The
following data were constant for all experiments, except when
specified as a variable in an experiment:
1 Matrix FD* 200 .ANG. C18 15 .mu.m reverse phase particles
Temperature 25.degree. C. Column 250 mm .times. I.D. 10 mm (0.785
cm.sup.2), column volume = 19.6 ml Flow 2.5 cm/min Regeneration
buffer 70% ethanol + 1M acetic acid *FD: Fuji Davison
[0054] Buffer Composition
[0055] General Experimental Conditions
[0056] All chemicals used for buffers were of analytical grade.
Water was of WFI (water for injection) quality. All buffers were
adjusted to pH 7.0 with HCl.
[0057] Process Parameters:
2 Column operation Equilibration 16% ethanol 5 CV* Application
variable Wash 16% ethanol 1 CV Eluation <12 CV Regeneration 5 CV
*(CV = column volume)
EXAMPL 1
[0058] Purification with Buffer Systems Containing CaCl.sub.2
3 Test parameters: Eluation Load FIG. Eluant [EtOH] (mg/cm.sup.2)
No. 2.5 g/kg Tris, 100 mM CaCl.sub.2 28% 30 3 2.5 g/kg Tris, 50 mM
CaCl.sub.2 Approx. 30 4 29%
[0059] Results and Discussion
[0060] CaCl.sub.2 was dramatically different from any other salt
tested (for comparison see Example 2). With 100 mM (FIG. 3), the
peak representing the glycosylated X14 forms was almost baseline
separated. The X14 peak was broad and had a very low preflank
slope, but the postflank was on the other hand very steep. In the
run with 50 mM (FIG. 4) the ethanol concentration during the run
was manually adjusted, and therefore cannot be directly compared,
but nevertheless, it indicates a very similar result.
[0061] It was noteworthy that the hydrophobicity of X14 was
selectively increased by CaCl.sub.2, demonstrated by the higher
ethanol concentration necessary for eluation of X14 compared to KCl
alone (see Example 2 for comparison). Glycosylated X14 forms and
ethylester seemed to be less affected by CaCl.sub.2.
[0062] Thus, the experiments show that CaCl.sub.2 buffers
selectively increase the X14 hydrophobicity, resulting in a
complete separation of the glycosylated X14 forms.
EXAMPLE 2 (COMPARISON)
[0063] Purification with Buffer Systems Containing KCl
[0064] These tests were conducted to show the purification profile
of the processes using KCl without CaCl.sub.2.
4 Test parameters: Eluation Load Eluant [EtOH] (mg/cm.sup.2) FIG.
No. 10 mM Bis-tris, 50 mM KCl 26.6% 30 5 10 mM Bis-tris, 200 mM KCl
26.6% 30 6
[0065] Results and Discussion
[0066] With a KCl concentration of 50 mM (FIG. 5), the preflank of
the eluting peak started with a steep slope followed by a more
steep slope. This indicates the presence of smaller, closely
eluting peaks. Increasing the KCl concentration to 200 mM (FIG. 6)
improved the separation but not to the level of separation using a
Ca.sup.++ containing eluant. These observations were confirmed by
analysis of the fractions.
EXAMPLE 3
[0067] Purification with Buffer Systems Containing KCl and
CaCl.sub.2 Combined
[0068] These tests were performed to examine the influence of a
buffer system combining KCl and CaCl.sub.2 on the simultaneous
separation of ethylester and glycosylated X14 forms.
[0069] Test 1
5 Test parameters: Eluation Load Eluant [EtOH] (mg/cm.sup.2) FIG.
No. 2.5 g/kg Tris, 25 mM CaCl.sub.2, 28% 30 7 100 mM KCl 2.5 g/kg
Tris, 12.5 mM CaCl.sub.2, 28% 30 8 150 mM KCl
[0070] Results and discussion 1
[0071] A mixture of KCl and CaCl.sub.2 (100/25 mM) resulted in a
better separation of the ethylester than with CaCl.sub.2 buffers
alone (FIG. 7). The combination 150/12.5 mM gave an even better
separation of the ethylester (FIG. 8). However, at the same time
the peak representing the glycosylated X14 forms was less
separated, but still much better than with KCl alone. The slow rise
of the preflank slope was less pronounced.
[0072] The experiments show that both KCl and CaCl.sub.2 are
important buffer components. The experiments show that the presence
of Ca.sup.++ in the buffer greatly improves the purification
process. Increasing Ca.sup.2+ concentrations selectively increased
the hydrophobicity of X14 when compared to the ethylester and the
glycosylated X14 forms.
[0073] Test 2
[0074] This experiment was conducted as in test 1 except for the
following changed parameters: 180 mM KCL and 5 mM CaCl.sub.2,
ethanol 27.4% (eluation) and the load was 150 mg/cm.sup.2. The
profile of the pool was examined before and after purification.
[0075] Results and Discussion 2
[0076] By this purification it is possible to obtain a fraction of
non-glycosylated X14 substantially free from glycosylated X14. FIG.
1 shows the profile of the pool before purification. As indicated
the content of X14 in the pool was 89.98%. FIG. 2 shows the profile
of the pool after purification. Here it is demonstrated that the
content of X14 increased to 98.38% after the purification, and the
content of mono-glycosylated X14 decreased significantly from 0.50%
to 0.02%.
EXAMPLE 4
[0077] Purification of DesB30 Using Buffer Systems With or Without
CaCl.sub.2
[0078] These tests were performed to demonstrate the effect of
Ca.sup.++ on the eluation profile of the insulin analogue DesB30.
All experiments were performed at pH: 7.2. The following data were
used:
6 Matrix FD* 200 .ANG. C18 15 .mu.m reverse phase particles
Temperature 22.degree. C. Column 250 mm .times. I.D. 10 mm (0.785
cm.sup.2), column volume = 19.6 ml Flow 3.0 cm/min Regeneration
buffer equilibrated with 16% ethanol, 0.1M citric acid *FD: Fuji
Davison
[0079] Process Parameters
7 Column operation Equilibration 16% ethanol 2.5 CV* Application
variable Wash 16% ethanol 1 CV Eluation <12 CV Regeneration 2 CV
*(CV = column volume)
[0080] Test 1
8 Test parameters: Eluation Load Eluant [EtOH] (mg/cm.sup.2) FIG.
No. 15 mM Tris, 5 mM maleic acid, 27% 190 9 0 mM CaCl.sub.2, 200 mM
KCl 15 mM Tris, 5 mM maleic acid, 27% 190 9 20 mM CaCl.sub.2, 200
mM KCl
[0081] FIG. 9 shows the difference between the eluation profile of
DesB30 when using an eluant containing 20 mM CaCl.sub.2 compared to
using an eluant containing no CaCl.sub.2. It is clear that when
Ca.sup.++ is present in the eluant the separation of
non-glycosylated insulin from glycosylated insulin is improved.
[0082] Test 2
9 Test parameters: Eluation Load Eluant [EtOH] (mg/cm.sup.2) FIG.
No. 15 mM Tris, 5 mM maleic acid, 26.8% 190 10 0 mM CaCl.sub.2, 200
mM KCl 15 mM Tris, 5 mM maleic acid, 27.8% 190 10 20 mM CaCl.sub.2,
200 mM KCl
[0083] Compared to test 1 the ethanol concentration (eluation) was
changed to obtain similar retention.
[0084] FIG. 10 shows the difference between the eluation profile of
DesB30 when using an eluant containing 20 mM CaCl.sub.2 compared to
the profile when using an eluant containing no CaCl.sub.2.
[0085] The pool content was examined before and after eluation.
[0086] Before the eluation there was 1.22% mono-glycosylated DesB30
in the eluation pool. After purification using a Ca.sup.++
containing eluant the amount of mono-glycosylated DesB30 had been
reduced approximately fivefold to 0.21%. When compared to the 1.10%
of mono glycosylated DesB30 left after the eluation using an eluant
containing no Ca.sup.++, it is clear that when Ca.sup.++ is present
in the eluant the separation of non-glycosylated insulin from
glycosylated insulin is improved.
* * * * *