WO2016005903A4 - A process for obtaining exendin-4 - Google Patents
A process for obtaining exendin-4 Download PDFInfo
- Publication number
- WO2016005903A4 WO2016005903A4 PCT/IB2015/055133 IB2015055133W WO2016005903A4 WO 2016005903 A4 WO2016005903 A4 WO 2016005903A4 IB 2015055133 W IB2015055133 W IB 2015055133W WO 2016005903 A4 WO2016005903 A4 WO 2016005903A4
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- exendin
- cell
- protein
- group
- subjecting
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract 25
- 108010011459 Exenatide Proteins 0.000 title claims abstract 21
- JUFFVKRROAPVBI-PVOYSMBESA-N chembl1210015 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)N[C@H]1[C@@H]([C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO[C@]3(O[C@@H](C[C@H](O)[C@H](O)CO)[C@H](NC(C)=O)[C@@H](O)C3)C(O)=O)O2)O)[C@@H](CO)O1)NC(C)=O)C(=O)NCC(=O)NCC(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 JUFFVKRROAPVBI-PVOYSMBESA-N 0.000 title claims abstract 21
- 229960001519 exenatide Drugs 0.000 title claims abstract 21
- 238000004587 chromatography analysis Methods 0.000 claims abstract 5
- 108090000623 proteins and genes Proteins 0.000 claims 11
- 102000004169 proteins and genes Human genes 0.000 claims 9
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims 6
- 238000001914 filtration Methods 0.000 claims 5
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims 4
- 238000001042 affinity chromatography Methods 0.000 claims 4
- 239000013592 cell lysate Substances 0.000 claims 4
- 238000005352 clarification Methods 0.000 claims 4
- 238000009295 crossflow filtration Methods 0.000 claims 4
- 230000029087 digestion Effects 0.000 claims 4
- 230000002934 lysing effect Effects 0.000 claims 4
- 239000012537 formulation buffer Substances 0.000 claims 3
- 238000011146 sterile filtration Methods 0.000 claims 3
- 108010013369 Enteropeptidase Proteins 0.000 claims 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims 2
- 235000011054 acetic acid Nutrition 0.000 claims 2
- 238000005119 centrifugation Methods 0.000 claims 2
- 238000012258 culturing Methods 0.000 claims 2
- 238000011026 diafiltration Methods 0.000 claims 2
- 239000002552 dosage form Substances 0.000 claims 2
- 238000000855 fermentation Methods 0.000 claims 2
- 230000004151 fermentation Effects 0.000 claims 2
- 108020001507 fusion proteins Proteins 0.000 claims 2
- 102000037865 fusion proteins Human genes 0.000 claims 2
- RLSSMJSEOOYNOY-UHFFFAOYSA-N m-cresol Chemical compound CC1=CC=CC(O)=C1 RLSSMJSEOOYNOY-UHFFFAOYSA-N 0.000 claims 2
- 235000010355 mannitol Nutrition 0.000 claims 2
- 229940100630 metacresol Drugs 0.000 claims 2
- 238000001471 micro-filtration Methods 0.000 claims 2
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims 2
- 239000001632 sodium acetate Substances 0.000 claims 2
- 235000017281 sodium acetate Nutrition 0.000 claims 2
- 230000001131 transforming effect Effects 0.000 claims 2
- 238000000108 ultra-filtration Methods 0.000 claims 2
- 241000588724 Escherichia coli Species 0.000 claims 1
- 238000004520 electroporation Methods 0.000 claims 1
- 239000001963 growth medium Substances 0.000 claims 1
- 230000035939 shock Effects 0.000 claims 1
- 239000000126 substance Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 238000000746 purification Methods 0.000 abstract 1
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
- C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Endocrinology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Vascular Medicine (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present disclosure provides a process for preparing Exendin-4 from high cell density employing specific steps to obtain purified Exendin-4. The process produces Exendin-4 in higher purity and yield, employing chromatographic and non-chromatographic techniques in the process. The present disclosure also provides for a process for purification of Exendin-4.
Claims
AMENDED CLAIMS
received by the International Bureau on 01 march 2016 (01.03.2016)
A process for obtaining Exendin-4, said process comprising acts of:
a) obtaining vector comprising gene for Exendin-4 set forth in SEQ ID No. 1 and transforming host cell with the vector to obtain transformed cell;
b) culturing the transformed cell in culture medium and subjecting to fermentation;
c) lysing the cell to obtain crude Exendin-4 and subjecting cell lysate to clarification to obtain clarified sample;
d) subjecting the clarified sample to affinity chromatography and tangential flow filtration to obtain protein;
e) digestion of the protein; and subjecting the digested protein to chromatography and filtration to obtain purified Exendin-4.
The process as claimed in claim 1, wherein the gene sequence for the Exendin-4 includes solubility or affinity tag or combination thereof; and the Exendin-4 is expressed as a fusion protein.
The process as claimed in claim 1, wherein the host cell is E.coli; and the vector is selected from group comprising pET-32a and pET24.
The process as claimed in claim 1, wherein the transforming is carried out by technique selected from group comprising Heat shock method, electroporation and chemical method or any combinations thereof; and the culturing is carried out at temperature ranging from about 25°C to about 40°C, preferably about 37°C, for time duration ranging from about 1 hour to about 20 hours, preferably about 17 hours, at pH ranging from about 6.8 to about 7.4, preferably about 7.2.
The process as claimed in claim 1, wherein the fermentation is carried out at temperature ranging from about 35°C to about 40°C, preferably about 37°C, for time duration ranging from about 11 to 13 hours, at pH ranging from about 6.5 to 7.5.
6. The process as claimed in claim 1, wherein the lysing of cell is carried out by subjecting the cell to high pressure Homogenizer or Microfluidizer or French Press, or any combinations thereof; and wherein the clarification of the cell lysate is carried out by techniques selected from group comprising centrifugation, tangential flow filtration and microfiltration; or any combinations thereof.
7. The process as claimed in claim 1, wherein the digestion of protein is with enterokinase enzyme; and the chromatography of step c) is selected from group comprising affinity chromatography and reverse phase chromatography, or combination thereof; and the filtration is selected from group comprising ultrafiltration, diafiltration and sterile filtration, or any combinations thereof.
8. The process as claimed in claim 1, wherein the Exendin - 4 obtained by the process has a purity of at least 97%, preferably about 99%; and the Exendin-4 is formulated into dosage form in formulation buffer comprising component selected from group of sodium acetate, acetic acid, D-Mannitol and Meta-cresol or any combinations thereof.
9. A process of purifying Exendin-4, said method comprising acts of:
a) lysing cell comprising Exendin-4 protein and subjecting the cell lysate to clarification to obtain clarified sample;
b) subjecting the clarified sample to affinity chromatography and tangential flow filtration;
c) digestion of protein; and subjecting the digested protein to chromatography and filtration to obtain purified Exendin-4.
10. The process as claimed in claim 9, wherein the lysing of cell is carried out by subjecting the cell to high pressure Homogenizer or French Press or Microfluidizer, or any combinations thereof; and the clarification of the cell lysate is carried out by techniques selected from group comprising centrifugation, tangential flow filtration and microfiltration; or any combinations thereof.
11. The process as claimed in claim 9, wherein the digestion of protein is with enterokinase enzyme; and the chromatography of step c) is selected from group comprising affinity chromatography and reverse phase chromatography, or
combination thereof; and the filtration is selected from group comprising ultrafiltration, diafiltration and sterile filtration, or any combinations thereof.
12. The process as claimed in claim 9, wherein the protein is fusion protein; and the filtration of step c) is followed by sterile filtration.
13. Exendin-4 as set forth in SEQ ID No. 1, wherein the Exendin-4 is formulated into dosage form in formulation buffer.
14. The Exendin-4 as claimed in claim 13, wherein the Exendin-4 is obtained by the process as claimed in claim 1; the Exendin-4 has purity of at least 97%, preferably about 99%; and the formulation buffer comprises component selected from group of sodium acetate, acetic acid, D-Mannitol and Meta-cresol or any combinations thereof.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IN3042/CHE/2014 | 2014-07-08 | ||
| IN3042CH2014 | 2014-07-08 |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| WO2016005903A2 WO2016005903A2 (en) | 2016-01-14 |
| WO2016005903A3 WO2016005903A3 (en) | 2016-03-10 |
| WO2016005903A4 true WO2016005903A4 (en) | 2016-04-28 |
Family
ID=53969386
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/IB2015/055133 WO2016005903A2 (en) | 2014-07-08 | 2015-07-07 | A process for obtaining exendin-4 |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2016005903A2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI871300B (en) * | 2019-01-28 | 2025-02-01 | 美商安進公司 | A continuous manufacturing process for biologics manufacturing by integration of drug substance and drug product processes |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2307038A4 (en) * | 2008-06-27 | 2013-03-27 | Univ Duke | THERAPEUTIC AGENTS COMPRISING ELASTINE-LIKE PEPTIDES |
| CN101665799A (en) * | 2009-06-29 | 2010-03-10 | 华东师范大学 | Recombination preparation method and application of Exendin-4 derivative |
-
2015
- 2015-07-07 WO PCT/IB2015/055133 patent/WO2016005903A2/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| WO2016005903A2 (en) | 2016-01-14 |
| WO2016005903A3 (en) | 2016-03-10 |
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