WO1990000176A1 - Improved process for purifying insulin - Google Patents
Improved process for purifying insulin Download PDFInfo
- Publication number
- WO1990000176A1 WO1990000176A1 PCT/BR1989/000003 BR8900003W WO9000176A1 WO 1990000176 A1 WO1990000176 A1 WO 1990000176A1 BR 8900003 W BR8900003 W BR 8900003W WO 9000176 A1 WO9000176 A1 WO 9000176A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- human insulin
- insulin
- ion exchange
- process according
- productivity
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
- C07K14/625—Extraction from natural sources
Definitions
- the present invention relates to an improved ion exchange 5.
- Insulin was discovered in 1921 by two Canadian scientists, Frederick Banting and Charles Best. It is an essential drug 10. in the treatment of the IDDM and is an important adjuvant in the treatment of NIDDM.
- Joslin et al (1922) and Karr et al (1931) made the first ob servations of allergy related to insulin.
- Marble et al(1949) and Berson et al(1959) described insulin resistance and lipo 15. distrophy and related them to an immunological phenomena.
- Fineberg et al(1983) have also proved through clinical test that the treatment with human insulin can lead to a smaller formation of anti-insulin antibodies than the treatment with bovine or porcine insulins.
- Insulin is extracted in a 50-70% (v/v) alcohol solution, acidified with sulfuric acid or phosphoric acid. The alcohol is then removed through evapo ration. When salt is added, insulin concentrates through 15. precipitation. An isoeletric precipitation and an alkaline crystallization follow.
- human insulin intermediate is firstly obtained. Then, the trypsin is separated by gel filtration chromatography and the human insulin intermediate 25. is purified by ion exchange chromatography. The human insu lin intermediate is converted, by a chemical reaction, into human insulin, which is crystallized in the presence of zinc ions.
- the human insulin is finally purified through ion exchange 30. chromatography and crystallized in the presence of zinc ions.
- the European Patent Application 0017933 describes a method for the purification of the human insulin derivative through ion exchange chromatography, in DEAE-Sephadex A- 25 35. and tris-7-M urea buffer.
- the World Intellectual Property Organization's WO 83/01704 describes a process for the purification of human insulin ester through ion exchange chromatography in DEAE-Cellulose and tris-urea buffer.
- the process of the present invention is more adequate to industrial production because with higher flow rates, 30. cleaning up and regeneration "in situ", a better chro ato- graphic cycle is achieved, with a substantial increase in the daily production of insulin per column volume.
- the ion exchange steps can be carried out 35. as follows :
- the human insulin or human insulin intermediate is eluted after 5.0 or 3.5 column volumes, by using buffers with diffe rent concentrations of ammonium chloride and sodium chloride.
- the chromatography is carried out at a maximum temperature of 30 degrees with flow rates up to 2.0cm/min.
- the gel After elution of the human insulin intermediate or human in sulin, the gel is washed in the column itself with 1.5 to 2.5 column volumes with a buffer containing a higher concen 15. tration of sodium chloride. The system thus becomes ready for another chro atographic cycle.
- the pools corresponding to the purified product are precipi 20. tated or crystallized in the presence of zinc ions for later processing or final packaging.
- the protein recovery in the ion exchange steps is 75%-80% of the starting material.
- Figure 1 shows an elution profile from an ion exchange chr graphy of the human insulin intermediate on Q-Sepharose Fast
- Figure 2 shows an elution profile from an ion exchange chr graphy of the human insulin on Q-Sepharose Fast Flow.
- a ( 5 x 22 cm ) column was packed with Q-Sepharose Fast Flow and equilibrated with approximately 1100 ml of a 0.06M NH C1, 50% ethanol, pH 8.0, buffer. 5.
- a human insulin intermediate precipitate from a Sephadex G-50 chromatography was dissolved in 90 ml 8M urea at a 22.2 mg/ml concentration. The pH was adjusted to 8.0 with 1M NH ⁇ OH and the insoluble material removed by filtration. The filtrate was applied to the column and the protein elu.
- the 276 nm profile was determined and the fractions corres . ponding to the human insulin intermediate peak were combined
- the column was washed with 1100 ml of 0.1M NH/jCl, 0.8M NaCl,50% ethanol, pH 8.0 buffer and equilibrated with a 0.06 NHzjCh50% ethanol, pH 8.0 buffer, being ready for another purification cycle.
- a human insulin intermediate isoeletric precipitate from a Sephadex G-50 chromatography was dissolved in 1350 ml of 8M urea at a 44.4 mg/ml concentration. The pH was adjusted to 8.0 with 1M NH/0H and the solution clarified by filtra tion. This material was then applied to a Q-Sepharose Fast
- Porcine insulin was not detected by HPLC analysis of the PLJ rified human insulin intermediate, and the chromatographic
- a 5 x 22 cm column was packed with Q-Sepharose Fast Flow and equilibrated with 1100 ml of 0.06M NH CI, 50% ethanol,pH 8.0 buffer. 2.0 grams of human insulin crystals obtained after 5. deblocking of human insulin intermediate were dissolved in 90 ml of 0.04M NH CI, 50% ethanol, 2 mg/ml EDTA, pH 8.0 buf fer. The pH was adjusted to 8.0 with 1M NH CI and the solu . tion clarified by filtration. The elution was carried out with 1300 ml of the equilibrium buffer followed by 860 ml of
- the recovery at this steps was 82 % and the obtained highly purified human insulin had a content higher than 98% when analysed by HPLC.
- the co_ lumn was washed with 430 ml of 0.1M MH CI, 0.8M NaCl, 50% ethanol, pH 8.0 buffer, and equilibrated with 0.06M NH CL,
- the protein recovery was 80% and, highly purified human insu lin was obtained with an insulin content higher than 99% when analysed by HPLC.
Abstract
It is described an improved process for the large scale production of highly purified human insulin crystals using at the ion exchange chromatography of the human insulin intermediate and of the human insulin, gels of a high chemical and physical stability and thus achieving an increase of flow rate and of the productivity, defined as the amount of insulin processed per gel volume per day.
Description
IMPROVED PROCESS FOR PURIFYING INSULIN
TECHNICAL FIELD
The present invention relates to an improved ion exchange 5. chromatography process for the large scale production of highly purified semi-synthetic human insulin crystals.
BACKGROUND ART
Insulin was discovered in 1921 by two Canadian scientists, Frederick Banting and Charles Best. It is an essential drug 10. in the treatment of the IDDM and is an important adjuvant in the treatment of NIDDM.
Joslin et al (1922) and Karr et al (1931) made the first ob servations of allergy related to insulin. Marble et al(1949) and Berson et al(1959) described insulin resistance and lipo 15. distrophy and related them to an immunological phenomena.
The introduction of purification techniques such as gel fil tration and ion exchange chromatography ensured the produc tion of highly purified insulins. Human insulin has been pro duced by fermentation of genetic engineered microorganisms 20. or by enzymatic convertion of porcine insulin. After that an adequate treatment could then be offered to the diabetic pa tient.
Schlichtkrul1 et al (1972) showed that diabetic patients treated with insulin purified by gel filtration in Sephadex 25. G-50-F and in ion exchange chromatography in QAE-Sephadex A- did not present significant antibody formation and had in sulin levels in the serum within normal limits.
Chance et al (1976) showed that insulin preparations puri fied by a combination of gel filtration in Sephadex G-50-F 30. and ion exchange in DEAE-Cellulose, presented significative improvements in the treatment of allergy and lipoatrophy re lated to insulin.
In 1983, at International Symposium on Human Insulin ( Diabe tes Care, vol.6, suppl. 1, March-April) it was demonstrated 35. that human insulin is as potent and effective as monocompone
porcine insulin, capable of having immunological advantages.
Chamovitz et al (1983) have demonstrated that, among studied diabetics, 19% developed IgE antibodies specific to bovine insulin, 17% developed them to porcine insulin and only 6% 5. developed them to human insulin.
Fineberg et al(1983), have also proved through clinical test that the treatment with human insulin can lead to a smaller formation of anti-insulin antibodies than the treatment with bovine or porcine insulins.
10. According to Bodansky and Bodansky(198*4), the desire to avoid aggressive chemicals and to follow Nature in the use of reversible enzymatic reactions, has led to the utilization of proteolytic enzymes for the formation of the peptide bond. Significant progress has been made in the selection of the
15. conditions (enzymes, pH,temperature,reactions mediums, etc.), which favor synthesis in relation to hydrolysis.In this sens the carrying-out of enzymatic reactions in organic medium b Butler and Reithel(1977) and Homandberg et al(1978) was ver important.
20. From the study of enzymatic reactions in organic medium, wit a consequent balance of synthesis rates and hydrolysis, series of papers have been published on enzymatic conversio of porcine insulin into human insulin.
Morihara et al (Nature vol.280,August 1979) reacted porcin 25. insulin with carboxypeptidase A, thus obtaining des-B-30 ins lin which, after purification, was bonded to a threonine e ter in a trypsin-catalysed reaction, in organic medium.
Later, Jonczyk and Gattner (Hoppe-Seyler's Z. Physiol. Chem. Dec. 1981), Rose et al (Bioche .J.,1983,211:671-76), arkusse 30. (Methods Diabetes, Res. Vol.l - part A, 1984),Van Hondenhove (3rd.Conference on the Recovery of Bioproducts-Uppsala-Swede May 11-16,1986), presented papers on the enzymatic conversio in organic medium, in only one step, of porcine insulin int a human insulin intermediate.
35. According to Trends in Diabetics Therapy and Management (Geo ge Street Publications, 1986) the demand for insulin crystal in 1986 equalled 5600 kg, with a possible growth t
approximately 7750 Kg in 1990. According to the same publi cation, human insulin corresponds to 20-25% of the. current insulin market, and a significant increase of the demand for human insulin for 1986-1990 with a concomitant decrease 5. in the demand for porcine insulins is expected,
In virtue of its importance for the adequate treatment of diabetes and its high annual consumption volume, the requi rement for effective methods, with low losses and high cap city, for the large scale production of purified human insu. 10. lin crystals has become stronger.
From frozen hog pancreases,insulin is extracted in a 50-70% (v/v) alcohol solution, acidified with sulfuric acid or phosphoric acid. The alcohol is then removed through evapo ration. When salt is added, insulin concentrates through 15. precipitation. An isoeletric precipitation and an alkaline crystallization follow.
The crystals thus obtained are submitted to a trypsin cata lysed reaction. Because of its incomplete nature, a mixture of insulin, porcine insulin and insulin derivatives is 20. obtained. The purification of human insulin from such a mixture is very difficult in large scale.
To circunvent such a situations human insulin intermediate is firstly obtained. Then, the trypsin is separated by gel filtration chromatography and the human insulin intermediate 25. is purified by ion exchange chromatography. The human insu lin intermediate is converted, by a chemical reaction, into human insulin, which is crystallized in the presence of zinc ions.
The human insulin is finally purified through ion exchange 30. chromatography and crystallized in the presence of zinc ions.
The European Patent Application 0017933 describes a method for the purification of the human insulin derivative through ion exchange chromatography, in DEAE-Sephadex A- 25 35. and tris-7-M urea buffer.
The World Intellectual Property Organization's WO 83/01704 describes a process for the purification of human insulin
ester through ion exchange chromatography in DEAE-Cellulose and tris-urea buffer.
In both W083/02772 and DE 3129404 Al human insulin prepared by enzymatic conversion of porcine insulin is purified 5. through gel filtration chromatography in Sephadex G-50 F.
The aim of the methods described by the above mentioned pa tents is to obtain purified human insulin crystals, which is achieved in a higher or lower degree. Nevertheless, in all these patents, mechanically fragile and easily 10. compressible gels are used,which produce unstable bed in chromatography and are destroyed when exposed to high chemical concentration.
These characteristics lead to low flow rates, a difficult cleaning up and gel regeneration, column packing and re- 15. packing in each chromatography. Therefore, they imply low output chromatographies and thus, even when putting out a product of the desired quality, they are not adequate for a large scale production of purified human insulin.
DISCLOSURE OF THE INVENTION
20. It has now been discovered that the utilization of gels with a high physical and chemical stability of the Sepharose Fast Flow type produced by Pharmacia LKB Biotechnology Uppsala-Sweden, for the purification of insulin through ion exchange, will solve the problems faced by the users
25. of the technique by the adequacy of essential parameters to the chromatography such as flow rate, regeneration and cleaning up of the gel.
The process of the present invention is more adequate to industrial production because with higher flow rates, 30. cleaning up and regeneration "in situ", a better chro ato- graphic cycle is achieved, with a substantial increase in the daily production of insulin per column volume.
In the process for obtaining highly purified human insulin crystals, the ion exchange steps can be carried out 35. as follows :
Up to 15.0 mg of either human insulin intermediate or human
insulin crystals per mililitre of gel are dissolved in a con centrated urea solution or ammonium chloride buffer in an 01 ganic solvent-water (40% to 60% (v/v)) solution and applied in a column with a minimum height of 10 cm, containing gel 5. with a high physical and chemical stability of the Sepharose Fast Flow type (Pharmacia LKB Biotechnology - Uppsala-Sweden
The human insulin or human insulin intermediate is eluted after 5.0 or 3.5 column volumes, by using buffers with diffe rent concentrations of ammonium chloride and sodium chloride.
10. The chromatography is carried out at a maximum temperature of 30 degrees with flow rates up to 2.0cm/min.
After elution of the human insulin intermediate or human in sulin, the gel is washed in the column itself with 1.5 to 2.5 column volumes with a buffer containing a higher concen 15. tration of sodium chloride. The system thus becomes ready for another chro atographic cycle.
In either case, elution is controlled by absorvance at 276 nm of collected fractions.
The pools corresponding to the purified product are precipi 20. tated or crystallized in the presence of zinc ions for later processing or final packaging.
The protein recovery in the ion exchange steps is 75%-80% of the starting material.
When analysed by HPLC (High Performance Liquid Chromatography) 25. in 27.25% acetonitrile in 0.2M Phosphate Buffer in an Aquapor column, the human insulin intermediate presents no porcine in sulin contamination, and the human insulin presents a content of approximately 99%.
Using the ion exchange chromatography data from this inven 30. vention, we can calculate the Productivity, defined as the amount of protein (Human insulin or human insulin intermedia te) processed per day per unit of gel volume.
Productivity - purification of the human insulin intermediate or human insulin through chromatography in Sepharose Fast 35. Flow.
DIMENSIONS OF COLUMN SYSTEM A SYSTEM B
Height 10.0 cm 30.0 cm Diameter 5.0 cm 5.0 cm Sectional area 19.6 sq. cm 19.6 sq. cm
5. Volume 196.3 cubic cm 5888.0 cubic cm
Flow Rate - 2.0 cm per minute
Load - 15 mg protein/cubic cm gel System A = 2.94 g System B = 8.82 g
10. 1 day - 1440 minutes 1 cycle - 8,5 volumes of column System A = 1668.6 cubic cm System B = 4988.0 cubic cm Chromatographic Flow Rate = flow rate x sectional area = 2,0
15. x 19.6 sq. cm = 39.2 cubic cm/mi
Eluent Volume/Day = flow rate x day = 39.2 cubic cm/ x 1440 min = 56448 cubic cm Number of Cycles/Day = eluent volume/day : cycle volume System A = 56448 cubic cm : 1668.
20. cubic cm = 33.8
System B = 56448 cubic cm : 4998. cubic cm = 11.3
Daily Processing = number of cycles/day x load/cycl System A = 33.8 x 2.94 = 99.4 g
25. System B = 11.3 x 9.82 = 99.4 g
Productivity = daily processing : volume of co System A = 506 mg insulin/day x gel
System B = 169 mg insulin/day x
30. gel.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings,
Figure 1 shows an elution profile from an ion exchange chr graphy of the human insulin intermediate on Q-Sepharose Fast
35. Figure 2 shows an elution profile from an ion exchange chr graphy of the human insulin on Q-Sepharose Fast Flow.
EXAMPLE 1
A ( 5 x 22 cm ) column was packed with Q-Sepharose Fast Flow and equilibrated with approximately 1100 ml of a 0.06M NH C1, 50% ethanol, pH 8.0, buffer. 5. A human insulin intermediate precipitate from a Sephadex G-50 chromatography was dissolved in 90 ml 8M urea at a 22.2 mg/ml concentration.The pH was adjusted to 8.0 with 1M NH^OH and the insoluble material removed by filtration. The filtrate was applied to the column and the protein elu.
10. ted with 1500 ml of buffer at a 0.51 cm/min flow rate and 25 degrees.temperature. Fractions of 25 ml each were collee. ted at a 2 to 8 degrees temperature.
The 276 nm profile was determined and the fractions corres. ponding to the human insulin intermediate peak were combined
15. and precipitated in the presence of zinc ions. The precipita. te was collected by centrifugation. The recovery at this step was around 80%. Porcine insulin was not detected by ana. lysis of the precipitate in HPLC using 27.25% acetonitrile in a 0.2M phosphate buffer and an Aquapore RP 300 column.
20. After elution of the human insulin intermediate, the column was washed with 1100 ml of 0.1M NH/jCl, 0.8M NaCl,50% ethanol, pH 8.0 buffer and equilibrated with a 0.06 NHzjCh50% ethanol, pH 8.0 buffer, being ready for another purification cycle.
EXAMPLE 2
25. A human insulin intermediate isoeletric precipitate from a Sephadex G-50 chromatography was dissolved in 1350 ml of 8M urea at a 44.4 mg/ml concentration. The pH was adjusted to 8.0 with 1M NH/0H and the solution clarified by filtra tion. This material was then applied to a Q-Sepharose Fast
30. Flow 30 x 25.2 cm column. The chromatographic conditions,but flow rate of 1.1 cm/min and fractions of 3.3 L, were the same as in example 1.
Porcine insulin was not detected by HPLC analysis of the PLJ rified human insulin intermediate, and the chromatographic
35, recovery was
EXAMPLE 3
A 5 x 22 cm column was packed with Q-Sepharose Fast Flow and equilibrated with 1100 ml of 0.06M NH CI, 50% ethanol,pH 8.0 buffer. 2.0 grams of human insulin crystals obtained after 5. deblocking of human insulin intermediate were dissolved in 90 ml of 0.04M NH CI, 50% ethanol, 2 mg/ml EDTA, pH 8.0 buf fer. The pH was adjusted to 8.0 with 1M NH CI and the solu. tion clarified by filtration. The elution was carried out with 1300 ml of the equilibrium buffer followed by 860 ml of
10. the 0.08M NH CI, 50% ethanol, pH 8.0 buffer. The flow rate was 0.51 cm/min at 25 degrees and 25 ml fractions were col lected at 2-8 degrees. The 276 nm profile was determined and the fractions corresponding to the purified human insulin peak combined and precipitated in the presence of zinc ions.
15. The recovery at this steps was 82 % and the obtained highly purified human insulin had a content higher than 98% when analysed by HPLC. After elution of the human insulin, the co_ lumn was washed with 430 ml of 0.1M MH CI, 0.8M NaCl, 50% ethanol, pH 8.0 buffer, and equilibrated with 0.06M NH CL,
20. 50% ethanol, pH 8.0 buffer.
EXAMPLE 4
60 gms of human insulin crystals obtained after deblocking of human insulin intermediate were dissolved in 1350 ml of 0.04M NH CI, 50% ethanol, 2 mg/ml EDTA, pH 8.0 buffer, and
25. the solution was clarified by filtration. This solution was then applied to a 30x25.2 cm column, packed with Q-Sepharo se Fast Flow. The chromatographic conditions, but flow rate of 1.1 cm/min and fractions of 3.3 L, were the same as in example 3.
30, The protein recovery was 80% and, highly purified human insu lin was obtained with an insulin content higher than 99% when analysed by HPLC.
Claims
1. An improved process for large scale production of highly purified human insulin crystals, characterized by using at the ion exchange chromatography of the human insulin 5, intermediate and of the human insulin, gels with a high chemical and physical stability in a water-organic sol vent buffer, and flow rates up to 2.0 cm/min and a mini mum column height of 10 cm, with a significant improve ment of the Productivity and of the chromatographic ope 10. rational cycle.
2. The process according to claim 1, characterized by using gels of the Sepharose Fast Flow type (Pharmacia LKB Bio technology - Uppsala-Sweden).
3. The process according to claim 1, characterized by the 15, fact that the Productivity at the ion exchange chromato graphic steps of the human insulin intermediate and/or human insulin is equal to 506 mg insulin per ml of gel per day for a 10 cm high column and a 2.0 cm/min flow rate.
20.
4. The process according to claim 1, characterized by the fact that the Productivity at the ion exchange chromato graphic steps of the human insulin intermediate and/or human insulin is equal to 169 mg insulin per ml of gel per day for a 30 cm high column and a 2.0 cm/min flow
30. rate.
5. The process according to claim 1, characterized by using ethanol as the organic solvent.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| BR8803345A BR8803345A (en) | 1988-06-30 | 1988-06-30 | PERFECT PROCESS FOR WIDE-SCALE PRODUCTION OF PURIFIED HUMAN INSULIN CRYSTALS |
| BRPI8803345 | 1988-06-30 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1990000176A1 true WO1990000176A1 (en) | 1990-01-11 |
Family
ID=4045111
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/BR1989/000003 WO1990000176A1 (en) | 1988-06-30 | 1989-03-02 | Improved process for purifying insulin |
Country Status (2)
| Country | Link |
|---|---|
| BR (1) | BR8803345A (en) |
| WO (1) | WO1990000176A1 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000001727A1 (en) * | 1998-06-30 | 2000-01-13 | Novo Nordisk A/S | Seeding crystals for the preparation of peptides or proteins |
| WO2000055184A1 (en) * | 1999-03-15 | 2000-09-21 | Novo Nordisk A/S | Ion exchange chromatography of proteins and peptides with an organic modifier in the elution step |
| US6566490B1 (en) | 1998-06-30 | 2003-05-20 | Novo Nordisk A/S | Seeding crystals for the preparation of peptides or proteins |
| US7749955B2 (en) | 2003-08-21 | 2010-07-06 | Novo Nordisk A/S | Separation of polypeptides comprising a racemized amino acid |
| US8067554B2 (en) | 1999-03-15 | 2011-11-29 | Novo Nordisk A/S | Ion exchange chromatography of GLP-1, analogs and derivatives thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3907676A (en) * | 1970-07-28 | 1975-09-23 | Novo Terapeutisk Labor As | Process for purifying insulin |
| WO1986005497A1 (en) * | 1985-03-15 | 1986-09-25 | Nordisk Gentofte A/S | Novel insulin derivatives and pharmaceutical preparations containing these derivatives |
| GB2173503A (en) * | 1985-04-12 | 1986-10-15 | Berlin Chemie Veb | Purification of insulin |
-
1988
- 1988-06-30 BR BR8803345A patent/BR8803345A/en unknown
-
1989
- 1989-03-02 WO PCT/BR1989/000003 patent/WO1990000176A1/en unknown
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3907676A (en) * | 1970-07-28 | 1975-09-23 | Novo Terapeutisk Labor As | Process for purifying insulin |
| WO1986005497A1 (en) * | 1985-03-15 | 1986-09-25 | Nordisk Gentofte A/S | Novel insulin derivatives and pharmaceutical preparations containing these derivatives |
| GB2173503A (en) * | 1985-04-12 | 1986-10-15 | Berlin Chemie Veb | Purification of insulin |
Non-Patent Citations (1)
| Title |
|---|
| Pharmacia, September 1986, FPMG 50-01-329, (SE), "FPLC: media and column guide. High performance seperation of biomolecules", pages 6,7,17,18 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000001727A1 (en) * | 1998-06-30 | 2000-01-13 | Novo Nordisk A/S | Seeding crystals for the preparation of peptides or proteins |
| US6566490B1 (en) | 1998-06-30 | 2003-05-20 | Novo Nordisk A/S | Seeding crystals for the preparation of peptides or proteins |
| WO2000055184A1 (en) * | 1999-03-15 | 2000-09-21 | Novo Nordisk A/S | Ion exchange chromatography of proteins and peptides with an organic modifier in the elution step |
| US6451987B1 (en) | 1999-03-15 | 2002-09-17 | Novo Nordisk A/S | Ion exchange chromatography of proteins and peptides |
| US7276590B1 (en) | 1999-03-15 | 2007-10-02 | Novo Nordisk A/S | Ion exchange chromatography of proteins and peptides |
| US8067554B2 (en) | 1999-03-15 | 2011-11-29 | Novo Nordisk A/S | Ion exchange chromatography of GLP-1, analogs and derivatives thereof |
| US7749955B2 (en) | 2003-08-21 | 2010-07-06 | Novo Nordisk A/S | Separation of polypeptides comprising a racemized amino acid |
Also Published As
| Publication number | Publication date |
|---|---|
| BR8803345A (en) | 1990-02-13 |
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