WO1994009028A1 - Process for purifying recombinant growth factor - Google Patents
Process for purifying recombinant growth factor Download PDFInfo
- Publication number
- WO1994009028A1 WO1994009028A1 PCT/US1993/009883 US9309883W WO9409028A1 WO 1994009028 A1 WO1994009028 A1 WO 1994009028A1 US 9309883 W US9309883 W US 9309883W WO 9409028 A1 WO9409028 A1 WO 9409028A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- rafgf
- nacl
- growth factor
- resin
- ion exchange
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 17
- 239000003102 growth factor Substances 0.000 title claims description 5
- 230000001580 bacterial effect Effects 0.000 claims abstract description 9
- 102000003971 Fibroblast Growth Factor 1 Human genes 0.000 claims abstract description 5
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 claims abstract description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 52
- 239000011780 sodium chloride Substances 0.000 claims description 26
- 239000011347 resin Substances 0.000 claims description 15
- 229920005989 resin Polymers 0.000 claims description 15
- 239000003456 ion exchange resin Substances 0.000 claims description 11
- 229920003303 ion-exchange polymer Polymers 0.000 claims description 11
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 9
- 239000006166 lysate Substances 0.000 claims description 6
- 239000013592 cell lysate Substances 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 12
- 108090000623 proteins and genes Proteins 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 101100120045 Bos taurus FGF1 gene Proteins 0.000 description 9
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 description 8
- 150000003839 salts Chemical class 0.000 description 8
- 239000002953 phosphate buffered saline Substances 0.000 description 7
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 6
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 6
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 6
- 229960002897 heparin Drugs 0.000 description 5
- 229920000669 heparin Polymers 0.000 description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 4
- 235000011130 ammonium sulphate Nutrition 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 238000004255 ion exchange chromatography Methods 0.000 description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 1
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 230000006334 disulfide bridging Effects 0.000 description 1
- 229940126864 fibroblast growth factor Drugs 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 239000002628 heparin derivative Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 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
- C07K14/475—Growth factors; Growth regulators
- C07K14/50—Fibroblast growth factor [FGF]
- C07K14/501—Fibroblast growth factor [FGF] acidic FGF [aFGF]
Definitions
- Figure 1 shows the effect of salt on the sorption of recombinant acidic fibroblast growth factor to ion exchange resins.
- the invention is a process for purifying recombinant fibroblast growth factor (rAFGF) from transformed bacterial cells. process is rapid and produces purified rAFGF that is substantially nucleic acids.
- rAFGF recombinant fibroblast growth factor
- Naturally-occurring AFGF has been purified to homo from bovine brain through a complex purification procedure (U. S. 4,444,760). Since the supply of natural AFGF is limited, alternati methods of producing large amounts of AFGF have been develope These methods include the use of recombinant DNA techniques.
- AFGF a cloned AFGF gene in bacterial cells
- large amounts o rAFGF may be readily produced.
- Recombinant AFGF has been p by a variety of procdures, such as those described in EPO 293,593 EPO 408,146 A2.
- heterologous protein Since the gene encoding the heterologous is usually expressed in a foreign host, the heterologous protein ma properties that differ from the native protein. Differences may inc variations in glycosylation patterns, disulfide bonding and tertiary structure. Consequently, purification procedures may require conv
- ion exchangers bind proteins at Jow salt strength, (0.2 M) and release proteins at high salt strength.
- the process of the present invention includes an unconventional type of ion exchange chromatography, in which rAFGF is bound to an ion exchange resin at high salt strength. Under these conditions the binding of contaminating proteins and nucleic acids is minimized. The bound rAFGF is then readily purified.
- a process for recovering substantially pure recombinant p arcoidviicde fidb.roblast growth factor from transformed bacterial cells is
- the invention is a process for purifying recombinant acidic fibroblast growth factor (rAFGF) from transformed bacterial cells.
- the 0 process is rapid and produces purified rAFGF that is substantially free of nucleic acids.
- Recombinant AFGF is obtained using standard methodology such as that described in EPO 259,953. Transformed cells are grown, and recombinant AFGF is obtained by collection of the cell culture fluid or by disruption of washed cells.
- the soluble rAFGF may be diluted to minimize its association with impurities that could inhibit the binding of rAFGF to chromatographic resins.
- Capture, concentration and initial purification of crude rAFGF is carried out by ion exchange chromatography.
- the crude rAFGF is diluted with a solution of between about 0.2 M NaCl and about 2 M NaCl.
- the diluted rAFGF is loaded onto an ion exchange resin.
- the rAFGF that binds to the ion exchange resin is washed with about 0.9 M NaCl to remove impurities such as nucleic acids and proteins.
- the bound rAFGF is eluted with about 1.6 M NaCl.
- the eluted rAFGF is further purified by heparin affinity or heparin analog affinity chromatography. Recombinant AFGF that binds to the affinity resin is washed with about 1 M NaCl to remove impurities. The rAFGF is eluted from the affinity resin with about 1.6 M NaCl.
- Recombinant AFGF as produced by transformed bacterial cells may be a heterogeneous preparation containing both fiill-length and truncated forms of rAFGF.
- the fiill-length and truncated forms copurify through the heparin affinity chromatography step.
- the full-length form may be separated from the truncated forms by hydrophobic interaction chromatography (HIC).
- HIC may be carried out by conventional liquid chromatography or by high performancy liquid chromatography (HPLC) using a variety of resins; preferably the resin is a butyl-agarose resin.
- the rAFGF that is eluted from the heparin affinity resin is diluted with ammonium sulfate (final concentration about 1.6 M) and applied to an HIC resin that has been equilibrated in guanidine and NaCl.
- the rAFGF is eluted with a reverse salt gradient of about 2 M to about 1 M ammonium sulfate in phosphate-buffered saline.
- the truncated forms of rAFGF elute ahead of the full-length rAFGF.
- An aliquot (500 mL) of clarified cell lysate was thawed and divided into five 100 mL samples. The five samples were used to determine lie effects of salts on the sorption of rAFGF to an ion exchange resin.
- One sample was diluted with PBS so that the final concentration of NaCl was about 0.13 M.
- a second sample was diluted with aqueous NaCl so that the final concentration of NaCl was about 0.6 M.
- a third sample was diluted with aqueous NaCl so that the final concentration of NaCl was about 0.8 M.
- a fourth sample was diluted with aqueous NaCl so that the final concentration of NaCl was about 1.0 M.
- a fifth sample was diluted with aqueous MgCl2 in PBS so that the final concentration of MgCl2 was about 0.5 M.
- a bacterial cell paste containing crude rAFGF is suspended in PBS, and the cells are disrupted to yield a cell lysate containing crude rAFGF.
- the lysate is clarified, and aqueous NaCl is added to the supematant to a final concentration of about 0.6 M.
- the diluted lysate is loaded onto an ion exchange resin so that the rAFGF binds to the resin.
- the bound rAFGF is washed with a solution of about 0.9 M NaCl.
- the bound rAFGF is eluted with a solution of about 1.6 M NaCl.
- the eluted rAFGF is mixed with aqueous NaCl so that the final concentration of NaCl is about 0.7 M.
- the diluted rAFGF is loaded onto a heparin affinity resin so that the rAFGF binds to the heparin resin.
- the bound rAFGF is washed with about 0.6 M NaCl and is then eluted with about 1.6 M NaCl.
- the eluted rAFGF is further purified by diluting in ammonium sulfate and then loading onto a HIC resin so that the rAFGF binds to the HIC resin.
- the bound rAFGF is washed with about 0.8 M ammonium sulfate and is then eluted with about 0.05 M sodium phosphate.
- the eluted rAFGF is dialyzed and concentrated.
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
A process for recovering substantially pure recombinant acidic fibroblast growth factor from transformed bacterial cells is provided.
Description
- 1 -
TΓΓ E OF THE INVENTION
PROCESS FOR PURIFYING RECOMBINANT GROWTH FAC
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the effect of salt on the sorption of recombinant acidic fibroblast growth factor to ion exchange resins.
BACKGROUND OF THE INVENTION
This is a continuation-in-part of U. S. S. N.07/961,23
10 October 15, 1992, now pending.
The invention is a process for purifying recombinant fibroblast growth factor (rAFGF) from transformed bacterial cells. process is rapid and produces purified rAFGF that is substantially nucleic acids.
15 Naturally-occurring AFGF has been purified to homo from bovine brain through a complex purification procedure (U. S. 4,444,760). Since the supply of natural AFGF is limited, alternati methods of producing large amounts of AFGF have been develope These methods include the use of recombinant DNA techniques.
20 expressing a cloned AFGF gene in bacterial cells, large amounts o rAFGF may be readily produced. Recombinant AFGF has been p by a variety of procdures, such as those described in EPO 293,593 EPO 408,146 A2.
There may be difficulties associated with the recombi
25 production of proteins. Since the gene encoding the heterologous is usually expressed in a foreign host, the heterologous protein ma properties that differ from the native protein. Differences may inc variations in glycosylation patterns, disulfide bonding and tertiary structure. Consequently, purification procedures may require conv
30 of the heterologous protein to a form more similar to the native pr
Several processes have been developed to extract rAF and convert it into a protein having the characteristics of natural A These processes share common drawbacks and are time-consumin cumbersome and are often low-yielding.
- 2 -
Many of these processes use ion exchange chromatography. Generally, ion exchangers bind proteins at Jow salt strength, (0.2 M) and release proteins at high salt strength.
The process of the present invention includes an unconventional type of ion exchange chromatography, in which rAFGF is bound to an ion exchange resin at high salt strength. Under these conditions the binding of contaminating proteins and nucleic acids is minimized. The bound rAFGF is then readily purified.
10 SUMMA Y OF THE INVENTION
A process for recovering substantially pure recombinant p arcoidviicde fidb.roblast growth factor from transformed bacterial cells is
15
DETAILED DESCRIPTION OF THE INVENTION
The invention is a process for purifying recombinant acidic fibroblast growth factor (rAFGF) from transformed bacterial cells. The 0 process is rapid and produces purified rAFGF that is substantially free of nucleic acids. Recombinant AFGF is obtained using standard methodology such as that described in EPO 259,953. Transformed cells are grown, and recombinant AFGF is obtained by collection of the cell culture fluid or by disruption of washed cells. The soluble rAFGF may be diluted to minimize its association with impurities that could inhibit the binding of rAFGF to chromatographic resins.
Capture, concentration and initial purification of crude rAFGF is carried out by ion exchange chromatography. Preferably, the crude rAFGF is diluted with a solution of between about 0.2 M NaCl and about 2 M NaCl. The diluted rAFGF is loaded onto an ion exchange resin. The rAFGF that binds to the ion exchange resin is washed with about 0.9 M NaCl to remove impurities such as nucleic acids and proteins. The bound rAFGF is eluted with about 1.6 M NaCl.
The eluted rAFGF is further purified by heparin affinity or heparin analog affinity chromatography. Recombinant AFGF that binds
to the affinity resin is washed with about 1 M NaCl to remove impurities. The rAFGF is eluted from the affinity resin with about 1.6 M NaCl.
Recombinant AFGF as produced by transformed bacterial cells may be a heterogeneous preparation containing both fiill-length and truncated forms of rAFGF. The fiill-length and truncated forms copurify through the heparin affinity chromatography step. The full-length form may be separated from the truncated forms by hydrophobic interaction chromatography (HIC). HIC may be carried out by conventional liquid chromatography or by high performancy liquid chromatography (HPLC) using a variety of resins; preferably the resin is a butyl-agarose resin.
The rAFGF that is eluted from the heparin affinity resin is diluted with ammonium sulfate (final concentration about 1.6 M) and applied to an HIC resin that has been equilibrated in guanidine and NaCl. The rAFGF is eluted with a reverse salt gradient of about 2 M to about 1 M ammonium sulfate in phosphate-buffered saline. The truncated forms of rAFGF elute ahead of the full-length rAFGF.
The following examples illustrate the present invention without, however, limiting its scope.
EXAMPLE 1
Effect of salts on rate of uptake of rAFGF bv ion exchange resin A bacterial cell paste containing crude rAFGF was suspended in phosphate-buffered saline. The cells were disrupted to yield a cell lysate containing about 1.4 mg/mL crude rAFGF. The lysate was clarified by centrifugation, and the supernatant was divided into aliquots and then frozen at -70°C.
An aliquot (500 mL) of clarified cell lysate was thawed and divided into five 100 mL samples. The five samples were used to determine lie effects of salts on the sorption of rAFGF to an ion exchange resin. One sample was diluted with PBS so that the final concentration of NaCl was about 0.13 M. A second sample was diluted with aqueous NaCl so that the final concentration of NaCl was about 0.6 M. A third sample was diluted with aqueous NaCl so that the final
concentration of NaCl was about 0.8 M. A fourth sample was diluted with aqueous NaCl so that the final concentration of NaCl was about 1.0 M. A fifth sample was diluted with aqueous MgCl2 in PBS so that the final concentration of MgCl2 was about 0.5 M.
An ion exchange resin (here SP-Spherodex) was sluπϊed, washed with PBS and resuspended in PBS. Aliquots (10 mL) of the resin slurry were added to each of the five samples. Changes in the concentration of soluble rAFGF in the samples was determined periodically by reverse phase HPLC.
The results (Figure 1) were unanticipated. Ion exchange resins are routinely used to bind proteins at low concentrations of salt (<0.2 M). The binding of rAFGF to an ion exchange resin in the presence of high concentrations of NaCl or MgCl2 was surprising.
EXAMPLE 2
Purification of rAFGF
A bacterial cell paste containing crude rAFGF is suspended in PBS, and the cells are disrupted to yield a cell lysate containing crude rAFGF. The lysate is clarified, and aqueous NaCl is added to the supematant to a final concentration of about 0.6 M. The diluted lysate is loaded onto an ion exchange resin so that the rAFGF binds to the resin. The bound rAFGF is washed with a solution of about 0.9 M NaCl. The bound rAFGF is eluted with a solution of about 1.6 M NaCl.
The eluted rAFGF is mixed with aqueous NaCl so that the final concentration of NaCl is about 0.7 M. The diluted rAFGF is loaded onto a heparin affinity resin so that the rAFGF binds to the heparin resin. The bound rAFGF is washed with about 0.6 M NaCl and is then eluted with about 1.6 M NaCl.
The eluted rAFGF is further purified by diluting in ammonium sulfate and then loading onto a HIC resin so that the rAFGF binds to the HIC resin. The bound rAFGF is washed with about 0.8 M ammonium sulfate and is then eluted with about 0.05 M sodium phosphate. The eluted rAFGF is dialyzed and concentrated.
Claims
1. A process for purifying recombinant acidic fibroblast growth factor from a bacterial cell lysate containing crude recombinant acidic fibroblast growth factor comprising:
(a) diluting the lysate with aqueous NaCl such that the concentration of NaCl in the diluted lysate is about 0.6 M;
(b) mixing the diluted lysate with an ion exchange resin, thus permitting the crude growth factor to bind to the resin;
(c) washing the bound growth factor with about 0.6 M NaCl; and
(d) eluting the bound growth factor with about 1.6 M NaCl.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU53613/94A AU5361394A (en) | 1992-10-15 | 1993-10-14 | Process for purifying recombinant growth factor |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US96123492A | 1992-10-15 | 1992-10-15 | |
| US961,234 | 1992-10-15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1994009028A1 true WO1994009028A1 (en) | 1994-04-28 |
Family
ID=25504218
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1993/009883 WO1994009028A1 (en) | 1992-10-15 | 1993-10-14 | Process for purifying recombinant growth factor |
Country Status (2)
| Country | Link |
|---|---|
| AU (1) | AU5361394A (en) |
| WO (1) | WO1994009028A1 (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4511502A (en) * | 1982-12-22 | 1985-04-16 | Genentech, Inc. | Purification and activity assurance of precipitated heterologous proteins |
| EP0408146A2 (en) * | 1989-07-13 | 1991-01-16 | Merck & Co. Inc. | Method for the purification of therapeutically active recombinant acidic fibroblast growth factor |
| US5136025A (en) * | 1990-04-04 | 1992-08-04 | California Biotechnology Inc. | Method to purify basic fibroblast growth factor |
-
1993
- 1993-10-14 AU AU53613/94A patent/AU5361394A/en not_active Abandoned
- 1993-10-14 WO PCT/US1993/009883 patent/WO1994009028A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4511502A (en) * | 1982-12-22 | 1985-04-16 | Genentech, Inc. | Purification and activity assurance of precipitated heterologous proteins |
| EP0408146A2 (en) * | 1989-07-13 | 1991-01-16 | Merck & Co. Inc. | Method for the purification of therapeutically active recombinant acidic fibroblast growth factor |
| US5136025A (en) * | 1990-04-04 | 1992-08-04 | California Biotechnology Inc. | Method to purify basic fibroblast growth factor |
Non-Patent Citations (1)
| Title |
|---|
| BIOTECHNOLOGY, Vol. 6, issued October 1988, HOESS et al., "Recovery of Soluble, Biologically Active Recombinant Proteins from Total Bacterial Lysates Using Ion Exchange Resin", pages 1214-1217. * |
Also Published As
| Publication number | Publication date |
|---|---|
| AU5361394A (en) | 1994-05-09 |
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