CA2402562A1 - A process for the production of human interferon alpha from genetically engineered yeast - Google Patents
A process for the production of human interferon alpha from genetically engineered yeast Download PDFInfo
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- 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
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/56—IFN-alpha
Abstract
A process for the production of physiologically-active human interferon alpha from genetically engineered yeast, Pichia pastoris, comprising digesting, with an enzyme to produce a linearized plasmid, a plasmid having a promoter and operationally linked to a human interferon alpha gene in the absence of a fusion region; transforming Pichia pastoris cells with the linearized plasmid by homologous recombination to form Pichia pastoris clones; screening the Pichia pastoris clones for high interferon alpha expression to find a high interferon-yielding Pichia pastoris clone; growing the high interferon-yielding Pichia pastoris clone; and purifying physiologically-active human interferon alpha protein from the high interferon-yielding Pichia pastoris clones.
Description
A PROCESS FOR THE PRODUCTION OF HUMAN INTERFERON ALPHA FROM
GENETICALLY ENGINEERED YEAST
CLAIM OF PRIORITY
This application claims priority to Indian Patent Application No. 826/MAS/98, filed March 19, 1999.
REFERENCE TO CTTATIONS
Complete citations to the cited references can be found in the Bibliography preceding the claims.
FIELD OF THE INVENTION
The invention relates to a process for the production of human interferon alpha from genetically engineered yeast. More particularly the invention relates to the cloning and expression of human interferon alpha gene in the methylotropic yeast, Pichia pastoris and a process for purification of the said protein.
DESCRIPTION OF THE RELATED ART
Interferon, the body's most rapidly produced defense against viruses, is a protein secreted by the body cells when they are exposed to viruses, bacteria, and different types of macromolecules. The secreted interferon then stimulates surrounding cells to produce other proteins, which in turn may regulate viral multiplication, the immune response, cell growth, and other cell functions. There are three classes of human interferons:
(i) interferon alpha, which is secreted by leukocytes, (ii) interferon beta, which is secreted by fibroblasts, (iii) interferon gamma, which is secreted by lymphocytes.
Interferon a and (3 have been referred to as type I interferon and interferon y has been referred to as type II interferon. Human interferon a proteins generally contain 165 or 166 amino acids and have molecular weights ranging from 17,000 - 20,000 daltons, as determined by SDS-PAGE.
Interferon a has been used for the treatment of various viral and cancer related diseases, for example to treat hepatitis B, C and D viral infections and cancer diseases like hairy cell leukemia, AmS-related Kaposi's sarcoma, chronic myelogenous leukemia, and renal cell carcinoma.
The human leukocyte interferon alpha is produced either from human cell lines grown in tissue culture or through human leukocytes collected from blood donors.
Horawitz, et al. 1982, S US patents 4680261, 5503828, 5391713, 4732683, 4696899, 5789551 and European patent EP0945463 . These processes are laborious, tedious and time consuming. The medium employed is costly and the yields of purified material obtained are low. There is a risk from contamination of the blood used for the preparation of leukocytes by an unidentified infectious agent.
With the advent of recombinant DNA technology, it has been possible to clone the human interferon alpha gene in microorganisms and produce sufficient quantities of human interferon alpha from these microorganisms. Stahelin, et al. 1981, Ho, et al., 1989, Yang, et al. 1992, Tarnowski, et al. 1986, Thatcher, et al. 1986, Tiute, et al. 1982, US patents 5710027, 5661009, 4765903, 5196323, 4315852, 4845032, 4530901 and European patents EP 0032134 and EP
0679718 describe the process for production of human interferon alpha from recombinant E. coli and Saccharomyces cerevisiae. Although expression and purification of human interferon alpha in E. coli overcame the problems and potential risks associated with the production from natural sources, it has its own drawbacks. The expressed protein in some cases is not correctly processed. The purified protein should be free of bacterial endotoxins.
Additional purification steps are required to remove the endotoxins. Accordingly, the process employed comprises multiple chromatographic steps and thus is time consuming. These processes are difficult to scale up, a prerequisite for large scale production. The yields of recombinant human interferon alpha expressed in Saccharomyces is low. Thus there exists a need for expressing human interferon alpha in a suitable host and a purification process which is simple, efficient and easily scalable.
Methylotropic yeast Pichia pastoris is increasingly becoming popular as a protein expression system. Pichia has the following advantages: first, extremely high yields of intra cellular proteins; second, ease of fermentation to high cell density; third, genetic stability and scale-up without loss of yield; and fourth, no endotoxin contamination.
Thus, the drawbacks associated with E. coli expression and purification of recombinant interferon can be overcome by cloning and expressing the human interferon alpha in the methylotrophic yeast Pichia pastoris. Accordingly, the aim of the invention is to clone and express the human interferon alpha gene in Pichia pastoris. Another object of the present invention is to develop an efficient purification process which can easily be scaled up for the recombinant human interferon alpha expressed in Pichia pastoris.
GENETICALLY ENGINEERED YEAST
CLAIM OF PRIORITY
This application claims priority to Indian Patent Application No. 826/MAS/98, filed March 19, 1999.
REFERENCE TO CTTATIONS
Complete citations to the cited references can be found in the Bibliography preceding the claims.
FIELD OF THE INVENTION
The invention relates to a process for the production of human interferon alpha from genetically engineered yeast. More particularly the invention relates to the cloning and expression of human interferon alpha gene in the methylotropic yeast, Pichia pastoris and a process for purification of the said protein.
DESCRIPTION OF THE RELATED ART
Interferon, the body's most rapidly produced defense against viruses, is a protein secreted by the body cells when they are exposed to viruses, bacteria, and different types of macromolecules. The secreted interferon then stimulates surrounding cells to produce other proteins, which in turn may regulate viral multiplication, the immune response, cell growth, and other cell functions. There are three classes of human interferons:
(i) interferon alpha, which is secreted by leukocytes, (ii) interferon beta, which is secreted by fibroblasts, (iii) interferon gamma, which is secreted by lymphocytes.
Interferon a and (3 have been referred to as type I interferon and interferon y has been referred to as type II interferon. Human interferon a proteins generally contain 165 or 166 amino acids and have molecular weights ranging from 17,000 - 20,000 daltons, as determined by SDS-PAGE.
Interferon a has been used for the treatment of various viral and cancer related diseases, for example to treat hepatitis B, C and D viral infections and cancer diseases like hairy cell leukemia, AmS-related Kaposi's sarcoma, chronic myelogenous leukemia, and renal cell carcinoma.
The human leukocyte interferon alpha is produced either from human cell lines grown in tissue culture or through human leukocytes collected from blood donors.
Horawitz, et al. 1982, S US patents 4680261, 5503828, 5391713, 4732683, 4696899, 5789551 and European patent EP0945463 . These processes are laborious, tedious and time consuming. The medium employed is costly and the yields of purified material obtained are low. There is a risk from contamination of the blood used for the preparation of leukocytes by an unidentified infectious agent.
With the advent of recombinant DNA technology, it has been possible to clone the human interferon alpha gene in microorganisms and produce sufficient quantities of human interferon alpha from these microorganisms. Stahelin, et al. 1981, Ho, et al., 1989, Yang, et al. 1992, Tarnowski, et al. 1986, Thatcher, et al. 1986, Tiute, et al. 1982, US patents 5710027, 5661009, 4765903, 5196323, 4315852, 4845032, 4530901 and European patents EP 0032134 and EP
0679718 describe the process for production of human interferon alpha from recombinant E. coli and Saccharomyces cerevisiae. Although expression and purification of human interferon alpha in E. coli overcame the problems and potential risks associated with the production from natural sources, it has its own drawbacks. The expressed protein in some cases is not correctly processed. The purified protein should be free of bacterial endotoxins.
Additional purification steps are required to remove the endotoxins. Accordingly, the process employed comprises multiple chromatographic steps and thus is time consuming. These processes are difficult to scale up, a prerequisite for large scale production. The yields of recombinant human interferon alpha expressed in Saccharomyces is low. Thus there exists a need for expressing human interferon alpha in a suitable host and a purification process which is simple, efficient and easily scalable.
Methylotropic yeast Pichia pastoris is increasingly becoming popular as a protein expression system. Pichia has the following advantages: first, extremely high yields of intra cellular proteins; second, ease of fermentation to high cell density; third, genetic stability and scale-up without loss of yield; and fourth, no endotoxin contamination.
Thus, the drawbacks associated with E. coli expression and purification of recombinant interferon can be overcome by cloning and expressing the human interferon alpha in the methylotrophic yeast Pichia pastoris. Accordingly, the aim of the invention is to clone and express the human interferon alpha gene in Pichia pastoris. Another object of the present invention is to develop an efficient purification process which can easily be scaled up for the recombinant human interferon alpha expressed in Pichia pastoris.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an illustration diagramming the interferon clone development.
FIG. 2 is a flowchart outlining a first preferred embodiment of the downstream processing and purification of interferon alpha.
FIG. 3 is a flowchart outlining a second preferred embodiment of the downstream processing and purification of interferon alpha.
DETAILED DESCRIPTION
This invention provides a process for the production of physiologically-active human interferon alpha from genetically engineered yeast. The process has the following steps. A
plasmid having a promoter and operationally linked to a human interferon alpha gene in the absence of a fi.~sion region is digested with an enzyme (preferably Note to produce a linearized plasmid. Pichia pastoris cells are transformed with the linearized plasmid by homologous recombination to form Pichia pastoris clones. The Pichia pastoris clones are screened for high interferon alpha expression to find a high interferon-yielding Pichia pastoris clone. The high interferon-yieldingPichiapastoris clone is grown. Physiologically-active human interferon alpha protein is purified from the high interferon-yielding Pichia pastoris clones.
Preferably, the plasmid is constructed by cloning human interferon alpha gene into a plasmid pHIL-D2 containing an AOX1 promoter. Other promoters, such as GAP, MOX, FMD, ADH, LAC4, XPR2, LEU2, GAM1, PGK1, GAL7, GADPH, CYC1, and CUP1, are known and will work with similar success. E. coli is transformed with the plasmid pHII,-D2 containing the cloned human interferon alpha gene. The transformed E. coli is then screened for a recombinant clone carrying the interferon alpha gene in proper orientation with respect to the AOXl promoter in plasmid pHIL-D2. The pHIi,-D2 plasmid is available commercially from Invitrogen Corporation (Carlsbad, California, US) and has been described in their catalog. The transformed Pichia pastoris clone that expresses human interferon alpha was deposited at the American Type Culture Collection, 10801 University Blvd., Manassas, Virginia 20110-2209, US, on February 3, 2000, and is available under accession number PTA-1276.
Preferably, the production of human interferon alpha from genetically engineered yeast uses the following steps:
1. cloning human interferon alpha gene into plasmid pHIL-D2;
2. transforming E. coli with the plasmid pHIL-D2 containing the cloned human interferon gene;
3. screening the transformed E. coli for a recombinant clone carrying the interferon alpha gene in proper orientation with respect to AOX1 promoter in the plasmid pHIL-D2;
FIG. 1 is an illustration diagramming the interferon clone development.
FIG. 2 is a flowchart outlining a first preferred embodiment of the downstream processing and purification of interferon alpha.
FIG. 3 is a flowchart outlining a second preferred embodiment of the downstream processing and purification of interferon alpha.
DETAILED DESCRIPTION
This invention provides a process for the production of physiologically-active human interferon alpha from genetically engineered yeast. The process has the following steps. A
plasmid having a promoter and operationally linked to a human interferon alpha gene in the absence of a fi.~sion region is digested with an enzyme (preferably Note to produce a linearized plasmid. Pichia pastoris cells are transformed with the linearized plasmid by homologous recombination to form Pichia pastoris clones. The Pichia pastoris clones are screened for high interferon alpha expression to find a high interferon-yielding Pichia pastoris clone. The high interferon-yieldingPichiapastoris clone is grown. Physiologically-active human interferon alpha protein is purified from the high interferon-yielding Pichia pastoris clones.
Preferably, the plasmid is constructed by cloning human interferon alpha gene into a plasmid pHIL-D2 containing an AOX1 promoter. Other promoters, such as GAP, MOX, FMD, ADH, LAC4, XPR2, LEU2, GAM1, PGK1, GAL7, GADPH, CYC1, and CUP1, are known and will work with similar success. E. coli is transformed with the plasmid pHII,-D2 containing the cloned human interferon alpha gene. The transformed E. coli is then screened for a recombinant clone carrying the interferon alpha gene in proper orientation with respect to the AOXl promoter in plasmid pHIL-D2. The pHIi,-D2 plasmid is available commercially from Invitrogen Corporation (Carlsbad, California, US) and has been described in their catalog. The transformed Pichia pastoris clone that expresses human interferon alpha was deposited at the American Type Culture Collection, 10801 University Blvd., Manassas, Virginia 20110-2209, US, on February 3, 2000, and is available under accession number PTA-1276.
Preferably, the production of human interferon alpha from genetically engineered yeast uses the following steps:
1. cloning human interferon alpha gene into plasmid pHIL-D2;
2. transforming E. coli with the plasmid pHIL-D2 containing the cloned human interferon gene;
3. screening the transformed E. coli for a recombinant clone carrying the interferon alpha gene in proper orientation with respect to AOX1 promoter in the plasmid pHIL-D2;
4. digesting the plasmid pHIL-D2 in E. coli and Notl enzyme to get a Notl fragment of pHIL-D2.
5. transforming the Pichia pastoris cells with the Notl fragment of pHIL-D2 harboring interferon alpha gene by homologous recombination;
6. screening the Pichia pastoris clones for interferon alpha expression and confirmation of nucleotide sequence of interferon alpha gene from a high interferon-yielding clone;
7. growing the high yielding Pichia pastoris clone in a fermentor under optimal conditions as herein described;
8. washing the Pichia pastoris cells obtained from the fermentor with a buffer, as herein described;
9. breaking the Pichia pastoris cells with glass beads in a bead mill in the presence of protease inhibitor, as herein described;
10. adding protein solubilizing agent, as herein described to final concentration of 4-8 M and stirnng for 2-10 hours at 200 - 300 rpm and 4-7°C with or without centrifugation;
11. diluting the extract of the above step 10-30 fold with buffer, as herein described followed by clarification either by centrifugation or filtration and with or without concentration of the extract;
12. adjusting the pH of the above extract with a buffer, as herein described to pH
3-5 followed by centrifizgation or filtration;
3-5 followed by centrifizgation or filtration;
13. adsorbing the above extract on cation exchange column of "SP-SEPHAROSE" (Pharmacia Fine Chemicals, New Market, New Jersey, US) and eluting the protein containing human interferon alpha with alkali chloride;
14. adjusting the pH ofthe above eluted sample containing human interferon alpha to neutral pH and adsorbing it on immuno-affinity column containing monoclonal antibodies against human interferon alpha coupled to a matrix, as herein described and eluting interferon alpha at pH below 4.0; and 15. diafiltering followed by sterile filtering the eluted interferon alpha.
The preferred conditions for growing said high yielding Pichia pastoris clone in a fermentor are pH 5.0, 28-30°C, and 500-1500 rpm for 2 days and inducing it with methanol for 48 hours.
The preferred buffer used to wash the Pichia pastoris cells obtained from the fermentor is sodium phosphate buffer of molarity 25-100 mM and pH 6.5-8, 1-5 mM
ethylenediaminetetraacetic acid (EDTA).
The preferred protease inhibitor used during breaking in the bead mill is 0.5 -2.0 mM phenylmethylsulfonyl fluoride (PMSF).
The preferred protein solubilizing agents used are guanidine chloride or urea at a concentration of 4-8 M.
The preferred bufferused forthe dilution ofthe extract 10-30 fold is Tris-HCl 100 mM, urea 0-1 M, pH 6.5-8Ø
The clarification is preferably carried out by centrifugation or filtration and concentration is preferably by ultra-filtration.
The concentrated sample is diluted with citrate buffer (25-100 mM, pH 4-5) followed by centrifugation and filtration.
The clarification is either by centrifugation or filtration and without concentration and the pH of the extract is adjusted with a citrate buffer (1-2 M, pH 2-5) The buffer used in adjusting the pH of the extract to pH 3-5 preferably is a citrate, either 25-100 mM or 1-2 M, pH 2-5 depending upon the volume of the extract.
The alkali chloride for eluting the protein containing human interferon alpha preferably is sodium chloride.
The preferred matrix used is one that is an affinity support for coupling of ligands via primary amines. A most preferred matrix is "AFFI-GEL-10" available from BIO-RAD, Hercules, California, US. Interferon alpha is preferably eluted at pH 2-4.
The invention will now be described with reference to the following flow diagrams and the examples:
Referring to FIG. 1, which outlines the steps for the cloning of human interferon alpha gene in Pichia pastoris, the human interferon alpha gene is amplified (preferably by PCR) and digested with EcoRI. pHIL-D2 plasmid carrying the AOX1 promoter is linearized by digesting with EcoRI. The interferon alpha-gene is ligated into the digested pHIL-D2. E.
coli cells are transformed with pHIL-D2-IFN plasmid. The E. coli transformants are screened for a recombinant in which IFN alpha gene is in the correct orientation with respect to the AOX1 promoter present in pHIL-D2 plasmid. Pichia pastoris is transformed with the Notl digested pHIi,- D2-IFN plasmid. This results in the integration of IFN gene into yeast genome by homologous recombination. Recombinants are selected by their ability to grow on minimal medium. Recombinants are screened for intracellular expression of human alpha interferon.
Pichia pastoris clone expressing interferon alpha is grown in a fermentor in a minimal medium, pH 5.0, 28-30°C, 500 - 1200 rpm for Z days and induced with methanol for 48 hours.
Referring to FIG. 2, which outlines the steps for a first preferred method of downstream processing and purification of human interferon alpha from fermentation onwards, the fermentor culture is harvested and the cells are washed with lysis buffer, 25 mM sodium phosphate buffer pH 8.0, 2 mM EDTA. The washed cells are broken with glass beads in a bead mill in the presence of 1 mM PMSF.-To the broken cell extract, solid guanidine chloride is added to a final concentration of 7 M and stirred at 200 - 300 rpm for 4-6 hours with or without centrifugation. The extract of the above step is diluted twenty times with a buffer, 25 mM Tris-HCl pH 7.5 containing 1-10 p.M PMSF and clarified by centrifizgation or filtration. The clarified extract is concentrated 10 fold by ultra filtration. The 10-fold concentrated extract is diluted again 10 times with 50 mM citrate buffer pH 4.0 containing 1 p,M PMSF. The citrate diluted extract is clarified by centrifugation or filtration and concentrated 10 fold by ultra-filtration. The above concentrated extract is subjected to cation exchange chromatography on SP-sepharose and eluted with a gradient of NaCI.
The pH of the above eluted IFN fraction is adjusted to 7.0, and the fraction is loaded onto an immuno-affinity column containing monoclonal antibodies coupled to an AFFI
GEL-10 matrix (BIO-RAD, Hercules, California, US). Pure interferon is eluted with 0.2 M acetic acid and 0.15 M NaCI. The eluted interferon is diafiltered and sterile filtered.
The human interferon alpha gene is amplified and cloned in the same manner as described for Example 1.
Referring to FIG. 3, which outlines the steps for a second preferred method of downstream processing and purification of human interferon alpha from fermentation onwards, the fermentor culture is harvested and the cells are washed with lysis buffer;
25 mM sodium phosphate buffer; and 2 mM EDTA, pH 8Ø The washed cells are broken with glass beads in a bead mill in the presence of 1 mM PMSF. To the broken cell extract solid guanidine chloride is added to a final concentration of 7 M and stirred at 200-300 rpm for 4-6 hours with or without centrifi~gation. The extract of the above step is diluted twenty times with a buffer, 25 mM Tris-HCl pH 7.5 containing 1- 10 pM PMSF, and clarified by centrifugation or filtration. The pH of the above clarified extract is brought down to 4 with 1-2 M citrate, pH 2-4.
The above pH-adjusted extract is subjected to cation exchange chromatography on SP-sepharose and eluted with a gradient of NaCI. The pH of the above-eluted fraction containing IFN is adjusted to 7.0 and loaded onto an immuno-affinity column containing monoclonal antibodies coupled to an AFFI-GEL-10 matrix. Pure interferon is eluted with 0.2 M acetic acid 0.15 M NaCI. The eluted interferon is diafiltered and sterile filtered.
Table 1 details the specific activity and the yield of purified recombinant Interferon alpha.
Biological activity of interferon alpha was determined by viral cytopathic effect reduction assay.
Madin Darby Bovine Kidney (MDBK) cells and vesicular stomatitis virus (VSV) were used in the assay. The assay was calibrated with an international reference standard obtained from National Institute for Biological Standards and Control, U.K. Data is presented for 3 batches of purified Interferon alpha.
SPECIFIC
ACTIVITY
AND
YIELD
OF
PURIFIED
INTERFERON
ALPHA
S. Batch No. Avera a s ecific Total units/liter No. activi 1 0399 4.4 x 10g IU/m 600 x 10g IU
2 0499 4.66 x 10g IU/m 620 x l Og IU
3 0599 5.2 x 10g IU/mg 560 x 10g ICT
The above process for the production of interferon alpha from genetically engineered yeast has several advantages over earlier processes, which also used recombinant DNA
technology. First, Pichia pastoris can be grown to very high cell densities, and the interferon gene can be expressed using a strong alcohol-oxidase promoter so that high yields of the recombinant human interferon alpha can be obtained. Furthermore, methanol is an inexpensive inducer. Second, because the interferon gene is stably integrated into the yeast genome by homologous recombination, there is no need to use an antibiotic to maintain the plasmid. Third, the purification process employed is simple, efficient, and results in high recovery of the expressed protein. Fourth, the process can be scaled up easily for large-scale purification of human interferon alpha. Finally, because yeast is a eukaryote, it can provide a more suitable environment for the folding of the eukaryotic interferon protein. Perhaps, it is for this reason that the interferon produced by the above process was found to give much higher specific activity than those reported earlier for interferon purified from E. coli.
Thus the subject process for the production of recombinant human interferon alpha from the genetically engineered yeast Pichia pastoris is simple, efficient and easily scalable for large scale production. The yield and specific activity of purified interferon alpha is higher than those reported from other systems.
It is understood that the invention is not confined to the particular construction and arrangement of parts herein illustrated and described, but embraces such modified forms thereof as come within the scope of the following claims.
BIBLIOGRAPHY
PATENTS CITED
EP 4530901 July, 1985 Weissmann EP 0043980 January, 1982 Goeddel, et al.
US 4680261 July, 1987 Nobuhara, et al.
US 5503828 April, 1996 Testa, et al.
US 5391713 February, 1995 Borg, et al.
US 4732683 March, 1988 Georgiades, et al.
US 4696899 September, 1987 Toth, et al.
US 5789551 August, 1998 Pestka EP 0945463 September, 1999 Attalla , et al.
US 5710027 January, 1998 Hauptmann, et al.
US 5661009 August, 1997 Stabinsky EP 0032134 July, 1981 Weissmann US 105629 August, 1988 D'Andrea, et al.
US 5196323 March, 1993 Bodo Gerhard, et al.
US 4315852 February, 1982 Leibowitz, et al.
EP 0679718 November, 1995 Ettlin, et al.
US 4845032 July, 1989 Obermeier OTHER REFERENCES
1. Staehelin, T., et al., 1981, Purification of recombinant human leukocyte interferon with monoclonal antibodies, Methods in Enrymology, Vol. 78, SOS-512.
S 2. Ho, L. J., et al., 1989, Production of human leukocyte interferon in E
coli by control of growth rate in fed-batch fermentation, Biotechnology Letters, Vol. 11, 695-698.
3. Yang, X. M., et al., 1992, Production of recombinant human interferon alpha by E. coli using a computer controlled cultivation process, Journal ofBiotechnology, Vol.
23, 291-301.
4. Gwynne, D. L, et al. 1987, Genetically engineered secretion of active human interferon and a bacterial endoglucanase from Aspergillus nidulans, Biotechnology, Vol.
5, 713-719.
5. Tiute, M. F., et al., 1982, Regulated high efficiency expression of human interferon alpha in Saccharomyces cerevisiae, The EMBO Journal, Vol. 1, 603-608.
6. Horowitz, B., 1986, Large scale production and recovery of human leukocyte interferon from peripheral blood leukocytes, Methods in Enzymology, Vol. 119, 39-47.
7. Tarnowski, J. S., et al., 1986, Large scale purification of recombinant human leukocyte interferon, Methods in Enzymology, Vol. 199, 153-165.
8. Thatcher, D. R., et al., 1986, Purification of recombinant human IFN-a2, Methods in Enzymology, Vol. 119, 166-177.
9. Sudbert, P. E., 1996, The expression of recombinant proteins in yeasts, Current Opinion in Biotechnology, Vol. 7, 517-524 10. Romanos, M., 1998, Advances in the use of Pichia pastoris for high level gene expression, Current Opinion in Biotechnology, Vol. 6, 527-533.
The preferred conditions for growing said high yielding Pichia pastoris clone in a fermentor are pH 5.0, 28-30°C, and 500-1500 rpm for 2 days and inducing it with methanol for 48 hours.
The preferred buffer used to wash the Pichia pastoris cells obtained from the fermentor is sodium phosphate buffer of molarity 25-100 mM and pH 6.5-8, 1-5 mM
ethylenediaminetetraacetic acid (EDTA).
The preferred protease inhibitor used during breaking in the bead mill is 0.5 -2.0 mM phenylmethylsulfonyl fluoride (PMSF).
The preferred protein solubilizing agents used are guanidine chloride or urea at a concentration of 4-8 M.
The preferred bufferused forthe dilution ofthe extract 10-30 fold is Tris-HCl 100 mM, urea 0-1 M, pH 6.5-8Ø
The clarification is preferably carried out by centrifugation or filtration and concentration is preferably by ultra-filtration.
The concentrated sample is diluted with citrate buffer (25-100 mM, pH 4-5) followed by centrifugation and filtration.
The clarification is either by centrifugation or filtration and without concentration and the pH of the extract is adjusted with a citrate buffer (1-2 M, pH 2-5) The buffer used in adjusting the pH of the extract to pH 3-5 preferably is a citrate, either 25-100 mM or 1-2 M, pH 2-5 depending upon the volume of the extract.
The alkali chloride for eluting the protein containing human interferon alpha preferably is sodium chloride.
The preferred matrix used is one that is an affinity support for coupling of ligands via primary amines. A most preferred matrix is "AFFI-GEL-10" available from BIO-RAD, Hercules, California, US. Interferon alpha is preferably eluted at pH 2-4.
The invention will now be described with reference to the following flow diagrams and the examples:
Referring to FIG. 1, which outlines the steps for the cloning of human interferon alpha gene in Pichia pastoris, the human interferon alpha gene is amplified (preferably by PCR) and digested with EcoRI. pHIL-D2 plasmid carrying the AOX1 promoter is linearized by digesting with EcoRI. The interferon alpha-gene is ligated into the digested pHIL-D2. E.
coli cells are transformed with pHIL-D2-IFN plasmid. The E. coli transformants are screened for a recombinant in which IFN alpha gene is in the correct orientation with respect to the AOX1 promoter present in pHIL-D2 plasmid. Pichia pastoris is transformed with the Notl digested pHIi,- D2-IFN plasmid. This results in the integration of IFN gene into yeast genome by homologous recombination. Recombinants are selected by their ability to grow on minimal medium. Recombinants are screened for intracellular expression of human alpha interferon.
Pichia pastoris clone expressing interferon alpha is grown in a fermentor in a minimal medium, pH 5.0, 28-30°C, 500 - 1200 rpm for Z days and induced with methanol for 48 hours.
Referring to FIG. 2, which outlines the steps for a first preferred method of downstream processing and purification of human interferon alpha from fermentation onwards, the fermentor culture is harvested and the cells are washed with lysis buffer, 25 mM sodium phosphate buffer pH 8.0, 2 mM EDTA. The washed cells are broken with glass beads in a bead mill in the presence of 1 mM PMSF.-To the broken cell extract, solid guanidine chloride is added to a final concentration of 7 M and stirred at 200 - 300 rpm for 4-6 hours with or without centrifugation. The extract of the above step is diluted twenty times with a buffer, 25 mM Tris-HCl pH 7.5 containing 1-10 p.M PMSF and clarified by centrifizgation or filtration. The clarified extract is concentrated 10 fold by ultra filtration. The 10-fold concentrated extract is diluted again 10 times with 50 mM citrate buffer pH 4.0 containing 1 p,M PMSF. The citrate diluted extract is clarified by centrifugation or filtration and concentrated 10 fold by ultra-filtration. The above concentrated extract is subjected to cation exchange chromatography on SP-sepharose and eluted with a gradient of NaCI.
The pH of the above eluted IFN fraction is adjusted to 7.0, and the fraction is loaded onto an immuno-affinity column containing monoclonal antibodies coupled to an AFFI
GEL-10 matrix (BIO-RAD, Hercules, California, US). Pure interferon is eluted with 0.2 M acetic acid and 0.15 M NaCI. The eluted interferon is diafiltered and sterile filtered.
The human interferon alpha gene is amplified and cloned in the same manner as described for Example 1.
Referring to FIG. 3, which outlines the steps for a second preferred method of downstream processing and purification of human interferon alpha from fermentation onwards, the fermentor culture is harvested and the cells are washed with lysis buffer;
25 mM sodium phosphate buffer; and 2 mM EDTA, pH 8Ø The washed cells are broken with glass beads in a bead mill in the presence of 1 mM PMSF. To the broken cell extract solid guanidine chloride is added to a final concentration of 7 M and stirred at 200-300 rpm for 4-6 hours with or without centrifi~gation. The extract of the above step is diluted twenty times with a buffer, 25 mM Tris-HCl pH 7.5 containing 1- 10 pM PMSF, and clarified by centrifugation or filtration. The pH of the above clarified extract is brought down to 4 with 1-2 M citrate, pH 2-4.
The above pH-adjusted extract is subjected to cation exchange chromatography on SP-sepharose and eluted with a gradient of NaCI. The pH of the above-eluted fraction containing IFN is adjusted to 7.0 and loaded onto an immuno-affinity column containing monoclonal antibodies coupled to an AFFI-GEL-10 matrix. Pure interferon is eluted with 0.2 M acetic acid 0.15 M NaCI. The eluted interferon is diafiltered and sterile filtered.
Table 1 details the specific activity and the yield of purified recombinant Interferon alpha.
Biological activity of interferon alpha was determined by viral cytopathic effect reduction assay.
Madin Darby Bovine Kidney (MDBK) cells and vesicular stomatitis virus (VSV) were used in the assay. The assay was calibrated with an international reference standard obtained from National Institute for Biological Standards and Control, U.K. Data is presented for 3 batches of purified Interferon alpha.
SPECIFIC
ACTIVITY
AND
YIELD
OF
PURIFIED
INTERFERON
ALPHA
S. Batch No. Avera a s ecific Total units/liter No. activi 1 0399 4.4 x 10g IU/m 600 x 10g IU
2 0499 4.66 x 10g IU/m 620 x l Og IU
3 0599 5.2 x 10g IU/mg 560 x 10g ICT
The above process for the production of interferon alpha from genetically engineered yeast has several advantages over earlier processes, which also used recombinant DNA
technology. First, Pichia pastoris can be grown to very high cell densities, and the interferon gene can be expressed using a strong alcohol-oxidase promoter so that high yields of the recombinant human interferon alpha can be obtained. Furthermore, methanol is an inexpensive inducer. Second, because the interferon gene is stably integrated into the yeast genome by homologous recombination, there is no need to use an antibiotic to maintain the plasmid. Third, the purification process employed is simple, efficient, and results in high recovery of the expressed protein. Fourth, the process can be scaled up easily for large-scale purification of human interferon alpha. Finally, because yeast is a eukaryote, it can provide a more suitable environment for the folding of the eukaryotic interferon protein. Perhaps, it is for this reason that the interferon produced by the above process was found to give much higher specific activity than those reported earlier for interferon purified from E. coli.
Thus the subject process for the production of recombinant human interferon alpha from the genetically engineered yeast Pichia pastoris is simple, efficient and easily scalable for large scale production. The yield and specific activity of purified interferon alpha is higher than those reported from other systems.
It is understood that the invention is not confined to the particular construction and arrangement of parts herein illustrated and described, but embraces such modified forms thereof as come within the scope of the following claims.
BIBLIOGRAPHY
PATENTS CITED
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EP 0679718 November, 1995 Ettlin, et al.
US 4845032 July, 1989 Obermeier OTHER REFERENCES
1. Staehelin, T., et al., 1981, Purification of recombinant human leukocyte interferon with monoclonal antibodies, Methods in Enrymology, Vol. 78, SOS-512.
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8. Thatcher, D. R., et al., 1986, Purification of recombinant human IFN-a2, Methods in Enzymology, Vol. 119, 166-177.
9. Sudbert, P. E., 1996, The expression of recombinant proteins in yeasts, Current Opinion in Biotechnology, Vol. 7, 517-524 10. Romanos, M., 1998, Advances in the use of Pichia pastoris for high level gene expression, Current Opinion in Biotechnology, Vol. 6, 527-533.
Claims (22)
1. A process for the production of physiologically-active human interferon alpha from genetically engineered yeast comprising:
a. digesting, with an enzyme to produce a linearized plasmid, a plasmid having a promoter and operationally linked to a human interferon alpha gene in the absence of a fusion region.
b. transforming Pichia pastoris cells with the linearized plasmid by homologous recombination to form Pichia pastoris clones;
c. screening the Pichia pastoris clones for high interferon alpha expression to find a high interferon-yielding Pichia pastoris clone;
d. growing the high interferon-yielding Pichia pastoris clone; and e. purifying physiologically-active human interferon alpha protein from the high interferon-yielding Pichia pastoris clones.
a. digesting, with an enzyme to produce a linearized plasmid, a plasmid having a promoter and operationally linked to a human interferon alpha gene in the absence of a fusion region.
b. transforming Pichia pastoris cells with the linearized plasmid by homologous recombination to form Pichia pastoris clones;
c. screening the Pichia pastoris clones for high interferon alpha expression to find a high interferon-yielding Pichia pastoris clone;
d. growing the high interferon-yielding Pichia pastoris clone; and e. purifying physiologically-active human interferon alpha protein from the high interferon-yielding Pichia pastoris clones.
2. The process of claim 1, wherein in step (a) the plasmid digested is constructed by the following steps: cloning human interferon alpha gene into a plasmid pHIL-D2 containing an AOX1 promoter; transforming E. coli with the plasmid pHIL-D2 containing the cloned human interferon alpha gene; and screening the transformed E.
coli for a recombinant clone carrying the interferon alpha gene in proper orientation with respect to the AOX1 promoter in plasmid pHIL,-D2.
coli for a recombinant clone carrying the interferon alpha gene in proper orientation with respect to the AOX1 promoter in plasmid pHIL,-D2.
3. The process of claim 1, wherein in step (a) the digesting comprises digesting with a NotI
enzyme.
enzyme.
4. The process of claim 1, wherein in step (d) the growing comprises growing the high interferon-yielding Pichia pastoris clones in a fermentor at pH 5.0, 28-30°C, and stirring at 500-1200 rpm for 2 days; and further comprising inducing the high interferon-yielding Pichia pastoris clones with methanol for 48 hours.
5. The process of claim 1, wherein in step (e) the purifying comprises:
i. washing the high interferon-yielding Pichia pastoris clones with a buffer;
ii. breaking the high interferon-yielding Pichia pastoris clones;
iii. adding a protein solubilizing agent to the high interferon-yielding Pichia pastoris to form an extract;
iv. diluting the extract with a buffer, then clarifying the diluted extract;
v. adjusting the pH of the extract with a buffer to pH 3-5, then centrifuging or filtering the extract;
vi. adsorbing the extract onto a cation exchange column and eluting the physiologically-active human interferon alpha protein;
vii. adjusting the pH of the physiologically-active human interferon alpha protein to neutral pH; and adsorbing it onto an immunoaffinity column containing monoclonal antibodies against human interferon alpha, the monoclonal antibody being coupled to a matrix; and eluting pure physiologically-active interferon alpha protein at a pH below 4.0; and viii. diafiltering; then ix. sterile filtering the eluted physiologically-active interferon alpha protein.
i. washing the high interferon-yielding Pichia pastoris clones with a buffer;
ii. breaking the high interferon-yielding Pichia pastoris clones;
iii. adding a protein solubilizing agent to the high interferon-yielding Pichia pastoris to form an extract;
iv. diluting the extract with a buffer, then clarifying the diluted extract;
v. adjusting the pH of the extract with a buffer to pH 3-5, then centrifuging or filtering the extract;
vi. adsorbing the extract onto a cation exchange column and eluting the physiologically-active human interferon alpha protein;
vii. adjusting the pH of the physiologically-active human interferon alpha protein to neutral pH; and adsorbing it onto an immunoaffinity column containing monoclonal antibodies against human interferon alpha, the monoclonal antibody being coupled to a matrix; and eluting pure physiologically-active interferon alpha protein at a pH below 4.0; and viii. diafiltering; then ix. sterile filtering the eluted physiologically-active interferon alpha protein.
6. The process of claim 5, wherein in step (ii) the breaking comprises breaking with glass beads in a bead mill in the presence of a protease inhibitor.
7. The process of claim 6, wherein in step (ii) the protease inhibitor comprises 0.5-2.0 mM phenylmethylsulfonyl fluoride.
8. The process of claim 5, wherein in step (i) the buffer comprises 25-100 mM
sodium phosphate buffer, pH 6.5-8Ø
sodium phosphate buffer, pH 6.5-8Ø
9. The process of claim 5, wherein in step (iii) the adding comprises adding the protein solubilizing agent to final concentration of 4-8 M, and stirring for 2-10 hours at 200-300 rpm, at 4-7°C.
10. The process of claim 9 wherein in step (iii), the protein solubilizing agent comprises 4-8 M guanidine chloride or urea.
11. The process of claim 5, wherein in step (iv), the diluting comprises diluting 10-30 fold;
and the buffer comprises 25-100 mM Tris-HCl; urea 0-1M, pH 6.5-8.0; and the clarifying comprises centrifuging or filtering.
and the buffer comprises 25-100 mM Tris-HCl; urea 0-1M, pH 6.5-8.0; and the clarifying comprises centrifuging or filtering.
12. The process of claim 11, further comprising concentrating the extract by ultra-filtrating.
13. The process of claim 5, wherein in step (iv) the clarifying comprises either centrifuging or filtering.
14. The process of claim 5, wherein in step (v) the adjusting the pH of the extract with a buffer comprises adjusting with a citrate buffer, pH 2-5, either at 25-100 mM
or 1-2 M.
or 1-2 M.
15. The process of claim 5, wherein in step (vi) the adsorbing comprises adsorbing the extract onto a cation exchange column.
16. The process of claim 15, further comprising diluting the concentrated extract with a 25-100 mM citrate buffer, pH 4-5; then centrifuging or filtering.
17. The process of claim 5, wherein in step (vi) the eluting comprises eluting with an alkali chloride.
18. The process of claim 17, wherein in step (vi) the eluting comprises eluting with a sodium chloride.
19. The process of claim 5, wherein in step (vii) the adsorbing comprises adsorbing it onto an immunoaffinity column containing monoclonal antibodies against human interferon alpha, the monoclonal antibody being coupled to a matrix, the matrix being an affinity support for coupling of ligands via primary amines.
20. The process of claim 5, wherein in step (vii) the eluting comprises eluting pure physiologically-active interferon alpha protein at a pH between 2 and 4.
21. A process for the production of physiologically-active human interferon alpha from genetically engineered yeast comprising:
a. cloning human interferon alpha gene in the absence of a fusion region into a pHIL-D2 plasmid containing an AOX1 promoter;
b. transforming E. coli with the pHIL-D2 plasmid containing the cloned human interferon alpha gene;
c. screening the transformed E. coli for a recombinant clone carrying the interferon alpha gene in proper orientation with respect to the AOX1 promoter in the pHIL-D2 plasmid;
d. digesting the pHIL-D2 plasmid with an enzyme to produce a fragment of pHIL-D2;
e. transforming Pichia pastoris cells with the fragment of pHIL-D2, harbouring interferon alpha gene, by homologous recombination to form Pichia pastoris clones;
f. screening the Pichia pastoris clones for high interferon alpha expression to find a high interferon-yielding Pichia pastoris clone;
g. growing the high interferon-yielding Pichia pastoris clone;
h. washing the high interferon-yielding Pichia pastoris clones with a buffer;
i. breaking the high interferon-yielding Pichia pastoris clones;
j. adding a protein solubilizing agent to the interferon-yielding Pichia pastoris to form an extract;
k. diluting the extract with a buffer, then clarifying the diluted extract;
l. adjusting the pH of the extract with a buffer to pH 3-5, then centrifuging or filtering the extract;
m. adsorbing the extract onto a cation exchange column and eluting a protein mixture containing physiologically-active human interferon alpha;
n. adjusting the pH of the protein mixture containing physiologically-active human interferon alpha to neutral pH; and adsorbing it onto an immunoaffinity column containing monoclonal antibodies against human interferon alpha, the monoclonal antibody being coupled to a matrix; and eluting pure physiologically-active interferon alpha protein at a pH below 4.0; and o. diafiltering; then sterile filtering the eluted physiologically-active interferon alpha protein.
a. cloning human interferon alpha gene in the absence of a fusion region into a pHIL-D2 plasmid containing an AOX1 promoter;
b. transforming E. coli with the pHIL-D2 plasmid containing the cloned human interferon alpha gene;
c. screening the transformed E. coli for a recombinant clone carrying the interferon alpha gene in proper orientation with respect to the AOX1 promoter in the pHIL-D2 plasmid;
d. digesting the pHIL-D2 plasmid with an enzyme to produce a fragment of pHIL-D2;
e. transforming Pichia pastoris cells with the fragment of pHIL-D2, harbouring interferon alpha gene, by homologous recombination to form Pichia pastoris clones;
f. screening the Pichia pastoris clones for high interferon alpha expression to find a high interferon-yielding Pichia pastoris clone;
g. growing the high interferon-yielding Pichia pastoris clone;
h. washing the high interferon-yielding Pichia pastoris clones with a buffer;
i. breaking the high interferon-yielding Pichia pastoris clones;
j. adding a protein solubilizing agent to the interferon-yielding Pichia pastoris to form an extract;
k. diluting the extract with a buffer, then clarifying the diluted extract;
l. adjusting the pH of the extract with a buffer to pH 3-5, then centrifuging or filtering the extract;
m. adsorbing the extract onto a cation exchange column and eluting a protein mixture containing physiologically-active human interferon alpha;
n. adjusting the pH of the protein mixture containing physiologically-active human interferon alpha to neutral pH; and adsorbing it onto an immunoaffinity column containing monoclonal antibodies against human interferon alpha, the monoclonal antibody being coupled to a matrix; and eluting pure physiologically-active interferon alpha protein at a pH below 4.0; and o. diafiltering; then sterile filtering the eluted physiologically-active interferon alpha protein.
22. A recombinant human interferon made from the method of claim 1.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/IB2000/000339 WO2001068827A1 (en) | 2000-03-16 | 2000-03-16 | A process for the production of human interferon alpha from genetically engineered yeast |
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|---|---|
| CA2402562A1 true CA2402562A1 (en) | 2001-09-20 |
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| EP (1) | EP1272624A4 (en) |
| JP (1) | JP2003526365A (en) |
| CN (1) | CN1452658A (en) |
| AU (1) | AU2000231863A1 (en) |
| CA (1) | CA2402562A1 (en) |
| WO (1) | WO2001068827A1 (en) |
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|---|---|---|---|---|
| US20060223145A1 (en) * | 2002-11-01 | 2006-10-05 | Lohray Braj B | Method for producing recombinant human interferon alpha 2b polypeptide in pichia pastoris |
| AU2008201682B2 (en) * | 2004-02-02 | 2011-02-24 | Ambrx, Inc. | Modified human interferon polypeptides and their uses |
| NZ586034A (en) | 2004-02-02 | 2011-10-28 | Ambrx Inc | Modified human growth hormone polypeptides and their uses |
| CU24158B1 (en) * | 2012-09-18 | 2016-03-30 | Ct De Ingeniería Genética Y Biotecnología | 1-KESTOSA OBTAINING METHOD |
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| JPH06209763A (en) * | 1993-01-13 | 1994-08-02 | Green Cross Corp:The | Mutant |
-
2000
- 2000-03-16 AU AU2000231863A patent/AU2000231863A1/en not_active Abandoned
- 2000-03-16 CN CN00819549.8A patent/CN1452658A/en active Pending
- 2000-03-16 EP EP00909584A patent/EP1272624A4/en not_active Withdrawn
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| JP2003526365A (en) | 2003-09-09 |
| AU2000231863A1 (en) | 2001-09-24 |
| WO2001068827A1 (en) | 2001-09-20 |
| EP1272624A1 (en) | 2003-01-08 |
| CN1452658A (en) | 2003-10-29 |
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