CA1175745A - Methods of producing hb sag - Google Patents
Methods of producing hb sagInfo
- Publication number
- CA1175745A CA1175745A CA000394009A CA394009A CA1175745A CA 1175745 A CA1175745 A CA 1175745A CA 000394009 A CA000394009 A CA 000394009A CA 394009 A CA394009 A CA 394009A CA 1175745 A CA1175745 A CA 1175745A
- Authority
- CA
- Canada
- Prior art keywords
- temperature
- hbsag
- cells
- hollow fiber
- hepatitis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Saccharide Compounds (AREA)
- Peptides Or Proteins (AREA)
- Low-Molecular Organic Synthesis Reactions Using Catalysts (AREA)
Abstract
METHODS OF PRODUCING HBsAg 16525 ABSTRACT OF THE DISCLOSURE
Hepatitis B surface antigen (HBsAg) is produced in vitro in high titer and purity from tissue cultures of cells that shed HBsAg. The cells are grown on hollow fiber capillary units having a molecular weight cut-off of about 10,000.
Hepatitis B surface antigen (HBsAg) is produced in vitro in high titer and purity from tissue cultures of cells that shed HBsAg. The cells are grown on hollow fiber capillary units having a molecular weight cut-off of about 10,000.
Description
~ ~.7~7~5 METHOD OF PRODUCING HBsAg BACKGROUND OF THE INVENTION
..
Hepatitis B surface antigen (HBsAg) has been shown to be effective as a vaccine against 5 hepatitis B disease. The usual source of this antigen is plasma obtained from donors, e.g., by phasmaphoresis. As a result the supply of plasma containing this antigen is uncertain and expensive as most plasma is free of HBsAg.
It has been known heretofore to grow in vitro on hollow fiber capillary units tissue cultures of cells which shed HBsAg, The hollow fiber units used heretofore have had a molecular weight cut off of 100,000 or greater.
It is an object of the presen~ invention to provide an improved in vitro tissue culture method for preparing HBsAg. Another object is to provide a method for preparing ~Bs~g in higher yield and at a faster rate. These and other objects of the present invention will be apparent from the following description.
~L~75745
..
Hepatitis B surface antigen (HBsAg) has been shown to be effective as a vaccine against 5 hepatitis B disease. The usual source of this antigen is plasma obtained from donors, e.g., by phasmaphoresis. As a result the supply of plasma containing this antigen is uncertain and expensive as most plasma is free of HBsAg.
It has been known heretofore to grow in vitro on hollow fiber capillary units tissue cultures of cells which shed HBsAg, The hollow fiber units used heretofore have had a molecular weight cut off of 100,000 or greater.
It is an object of the presen~ invention to provide an improved in vitro tissue culture method for preparing HBsAg. Another object is to provide a method for preparing ~Bs~g in higher yield and at a faster rate. These and other objects of the present invention will be apparent from the following description.
~L~75745
-2- 16525 SUMMARY OF THE INVENTION
Hepatitis B surface antigen (HBsAg) is produced ln vitro in high titer and purity from tissue cultures of cells that shed HBsAg. The cells are grown on hollow fiber capillary units having a molecular weight cut-off of about 10,000.
DETAILED DESCRIPTION
The present invention relates to a method of producing hepatitis B surface antigen (HBsAg) ln vitro on hollow fiber capillary units having a molecular weight cut-off of about 10,000.
It has now been found that the yield of HBsAg from cells which shed HBsAg is increased markedly and at a faster rate when the cells are grown in cell culture ln vitro on hollow fiber capillary units having a molecular weight cut-off of about 10,000. Higher yields are obtained when the growth cycle has two different elevated temperatures. Each temperature is above room tempera-ture and the first temperature is higher than thesecond temperature. The first temperature is from about 35 to about 38~C while the second temperature is from about 30 to about 3~C. Preferably the first temperature is about 37C and the second temperature is about 32C.
It has been found in addition that higher yields are obtained when caffeine is present in the _ vitro cell culture nutrient medium in an amount effective to improve yield of HBsAg or when the _ vitro cell culture is effected on a permeable membrane such as, for example, a bundle of hollow fiber capillary units. The caffeine may be present in a level at which it exerts a detectable improvement in yield up to a level above which it exerts a toxic effect.
" ~L7S7~;
Hepatitis B surface antigen (HBsAg) is produced ln vitro in high titer and purity from tissue cultures of cells that shed HBsAg. The cells are grown on hollow fiber capillary units having a molecular weight cut-off of about 10,000.
DETAILED DESCRIPTION
The present invention relates to a method of producing hepatitis B surface antigen (HBsAg) ln vitro on hollow fiber capillary units having a molecular weight cut-off of about 10,000.
It has now been found that the yield of HBsAg from cells which shed HBsAg is increased markedly and at a faster rate when the cells are grown in cell culture ln vitro on hollow fiber capillary units having a molecular weight cut-off of about 10,000. Higher yields are obtained when the growth cycle has two different elevated temperatures. Each temperature is above room tempera-ture and the first temperature is higher than thesecond temperature. The first temperature is from about 35 to about 38~C while the second temperature is from about 30 to about 3~C. Preferably the first temperature is about 37C and the second temperature is about 32C.
It has been found in addition that higher yields are obtained when caffeine is present in the _ vitro cell culture nutrient medium in an amount effective to improve yield of HBsAg or when the _ vitro cell culture is effected on a permeable membrane such as, for example, a bundle of hollow fiber capillary units. The caffeine may be present in a level at which it exerts a detectable improvement in yield up to a level above which it exerts a toxic effect.
" ~L7S7~;
-3- 16525 Typically the caffeine is employed at from about O.OOOlM to about 0.007M, preferably at from about O.OOOlM to about 0.0003M.
Any cells which shed HBsAg may be used in the process of the present invention. Examples of such cells are some human hepatomas, high yielding clones from such human hepatomas, and liver cells from hepatitls B infected chimpanzees.
The human hepatoma tissue is grown in vitro in the presence of a nutrient medium. ~y a nutrient medium is meant one which permits growth of cells in vitro. Some specific nutrient media are, for example, Medium 199, Morgan et al., ~roc. Soc. Exp. Biol.
~ Med.; 73:1-8, 1950; Basal Medium Eagle, Eagle Science, 122, 501-504, 1955; Morton, In Vitro, ~Jol. 6, No. 2, pp. 89-108, 1970; Dulbecco's Modified Eagle's Medium, Dulbecco et al., Virology, 8, 396, 1959; Smith et al., J. Virol. 1~, 185-196, 1960; Morton, op. cit.
Minimum Essential Medium (Eagle), Science, 130, 432 (1959) and RPMI Media, Moore et al., J.A.r~.~. 199, 519-524, 1967; Morton, op. cit.
The hollow fiber capillary unit is formed of a plurality of anisotropic hollow fiber membranes which provide a matrix for the culture of cells and organ explants. A bundle of these capillaries forms a thre~ demensional vascular system which permits controlled perfusion of cell aggregates with nutrients, as well as exchange of excreted substances. The capillaries consist of a sponge-like body with a very thin (0.5 - 1 ~m), smooth layer of extremely fine, controlled pore size (approximate range: OoO01 - 0.01 ~ml on the lumen side. From that surface outward, the ~.7~7~5
Any cells which shed HBsAg may be used in the process of the present invention. Examples of such cells are some human hepatomas, high yielding clones from such human hepatomas, and liver cells from hepatitls B infected chimpanzees.
The human hepatoma tissue is grown in vitro in the presence of a nutrient medium. ~y a nutrient medium is meant one which permits growth of cells in vitro. Some specific nutrient media are, for example, Medium 199, Morgan et al., ~roc. Soc. Exp. Biol.
~ Med.; 73:1-8, 1950; Basal Medium Eagle, Eagle Science, 122, 501-504, 1955; Morton, In Vitro, ~Jol. 6, No. 2, pp. 89-108, 1970; Dulbecco's Modified Eagle's Medium, Dulbecco et al., Virology, 8, 396, 1959; Smith et al., J. Virol. 1~, 185-196, 1960; Morton, op. cit.
Minimum Essential Medium (Eagle), Science, 130, 432 (1959) and RPMI Media, Moore et al., J.A.r~.~. 199, 519-524, 1967; Morton, op. cit.
The hollow fiber capillary unit is formed of a plurality of anisotropic hollow fiber membranes which provide a matrix for the culture of cells and organ explants. A bundle of these capillaries forms a thre~ demensional vascular system which permits controlled perfusion of cell aggregates with nutrients, as well as exchange of excreted substances. The capillaries consist of a sponge-like body with a very thin (0.5 - 1 ~m), smooth layer of extremely fine, controlled pore size (approximate range: OoO01 - 0.01 ~ml on the lumen side. From that surface outward, the ~.7~7~5
-4- 16525 pores become increasingly larger 5-10 ~m at the perimeter. This structure provides a unique com-bination of selectively and extremely high permeability to liquids even at very low or zero pressure. The choice of desired membrane "cut-off"
levels offers selective control of macromolecule transport. The open exterior of the capillaries presents a large surface for cell attachment and allows cells to penetrate toward the barrier at the lumen. The anisotropic fiber membranes may be pre-pared as described in V.S. patent 3,615,02~. The bundle of hollow fiber capillary units may be prepared as described in V.S. patent 3,821,087.
While many types of cells have been grown on hollow fiber capillary units, e.g., mouse fibroblasts, human breast and choriocarcinoma, rat pancreas, rat pituitary tumor, rat villus crypt, human hepatocytes, rat hepatoma, monkey kidney, baby hamster ovary, and rat lung, due to the many variables in biologic materials, preparation and operating parameters, specific performance with other types of cells cannot be forecast.
,7St~5 ~5~ 16525 The following examples illustrate the present invention without, however, limiting the same thereto.
EXAMPLE 1 (comparative) A unit of capillary bundles (Vitafibe ~
Amicon hollow-capillary unit 3P10) having a 10,000 molecular weight cut-off point, a 1,000 ml reservoir bottle, a ~eristaltic pump and Silastic tubing (about 2 meters) are autoclaved and assembled under aseptic conditions in the following manner:
RESERVOIR BOTTL
The extracapillary space (2 ml) is charged with a suspension of 7,5 X 106 cells of a freshly harvested human hepatoma cell line (BLC/PRF/5, MacNab et al., Br. J. Cancer (1976) 34, 509-515, a culture of which has been deposited with American Type Culture Collection and given accession number CCL 8024. The unit is placed in a 37C CO2 incubator. Every 30 minutes the hollow fiber unit is turned 180 around its longest axis. After 2 hours Eagle's Minimum Essential Medium (EMEM) containing 10% fetal calf serum, L-glutamine 2 mM, and Neomycin 50 ~g/ml is circulated through the capillary unit at a flow rate of 5 ml/minute. After 2 weeks at 37C the temperature of the incubator is reduced and maintained at 32C
and 10 4M caffeine is added. Cell growth is monitored by glucose utilization. ~ntigen samples are taken from the extracapillary space and assayed by complement fixation. The results are summarized in the following table:
Age of Cell Culture (days) Complement Fixation Titer Elimination of the fetal calf serum does not have any significant effect on titers and facilitates further purification of the HBsAg. Conventional mono-layer tissue culture systems under similar conditions produced only traces of antigen.
EXAM~LE 2 Three units of capillary ~undles (~itafibe Amicon) one 3P10, one P30 and one 3S100 having re-spectively molecular weight cut-off points of 10,000;
30,000 and 100,000 are each charged with a suspension of 6.0 X 106 cells of a freshly harvested higher HBsAg yielding clone of PLC/PRF/5 cells. The units, set-up as described in Example 1, are placed in a 37C incubator. After 2 hours Eagle's Minimum Essential Medium (EMEM) containing 10% fetal calf serum, L-glutamine 2mM, and Neoymcin 50 ~g/ml is circulated through the capillary unit at a flow rate of 5 ml/minute. After 2 weeks at 37C the temperature of the incubator is reduced and maintained at 32C
and 104M caffeine is added. Cell growth is monitored by glucose utilization. Antigen samples are taken from the extracapillary space and assayed by complement fixation. Samples fr~m the circulating fluid are assayed by an enzyme immunoassay (AVS2YMETM, Abbott Labs). The results are summarized in the followin~
table:
~7~7~5 Day10,000 MW 30,000 MW100,000 The unit with 10,000 molecular weight cut-off point proves superior yields although glucose consumption is similar in all three units.
. ..
levels offers selective control of macromolecule transport. The open exterior of the capillaries presents a large surface for cell attachment and allows cells to penetrate toward the barrier at the lumen. The anisotropic fiber membranes may be pre-pared as described in V.S. patent 3,615,02~. The bundle of hollow fiber capillary units may be prepared as described in V.S. patent 3,821,087.
While many types of cells have been grown on hollow fiber capillary units, e.g., mouse fibroblasts, human breast and choriocarcinoma, rat pancreas, rat pituitary tumor, rat villus crypt, human hepatocytes, rat hepatoma, monkey kidney, baby hamster ovary, and rat lung, due to the many variables in biologic materials, preparation and operating parameters, specific performance with other types of cells cannot be forecast.
,7St~5 ~5~ 16525 The following examples illustrate the present invention without, however, limiting the same thereto.
EXAMPLE 1 (comparative) A unit of capillary bundles (Vitafibe ~
Amicon hollow-capillary unit 3P10) having a 10,000 molecular weight cut-off point, a 1,000 ml reservoir bottle, a ~eristaltic pump and Silastic tubing (about 2 meters) are autoclaved and assembled under aseptic conditions in the following manner:
RESERVOIR BOTTL
The extracapillary space (2 ml) is charged with a suspension of 7,5 X 106 cells of a freshly harvested human hepatoma cell line (BLC/PRF/5, MacNab et al., Br. J. Cancer (1976) 34, 509-515, a culture of which has been deposited with American Type Culture Collection and given accession number CCL 8024. The unit is placed in a 37C CO2 incubator. Every 30 minutes the hollow fiber unit is turned 180 around its longest axis. After 2 hours Eagle's Minimum Essential Medium (EMEM) containing 10% fetal calf serum, L-glutamine 2 mM, and Neomycin 50 ~g/ml is circulated through the capillary unit at a flow rate of 5 ml/minute. After 2 weeks at 37C the temperature of the incubator is reduced and maintained at 32C
and 10 4M caffeine is added. Cell growth is monitored by glucose utilization. ~ntigen samples are taken from the extracapillary space and assayed by complement fixation. The results are summarized in the following table:
Age of Cell Culture (days) Complement Fixation Titer Elimination of the fetal calf serum does not have any significant effect on titers and facilitates further purification of the HBsAg. Conventional mono-layer tissue culture systems under similar conditions produced only traces of antigen.
EXAM~LE 2 Three units of capillary ~undles (~itafibe Amicon) one 3P10, one P30 and one 3S100 having re-spectively molecular weight cut-off points of 10,000;
30,000 and 100,000 are each charged with a suspension of 6.0 X 106 cells of a freshly harvested higher HBsAg yielding clone of PLC/PRF/5 cells. The units, set-up as described in Example 1, are placed in a 37C incubator. After 2 hours Eagle's Minimum Essential Medium (EMEM) containing 10% fetal calf serum, L-glutamine 2mM, and Neoymcin 50 ~g/ml is circulated through the capillary unit at a flow rate of 5 ml/minute. After 2 weeks at 37C the temperature of the incubator is reduced and maintained at 32C
and 104M caffeine is added. Cell growth is monitored by glucose utilization. Antigen samples are taken from the extracapillary space and assayed by complement fixation. Samples fr~m the circulating fluid are assayed by an enzyme immunoassay (AVS2YMETM, Abbott Labs). The results are summarized in the followin~
table:
~7~7~5 Day10,000 MW 30,000 MW100,000 The unit with 10,000 molecular weight cut-off point proves superior yields although glucose consumption is similar in all three units.
. ..
Claims (9)
1. A method for preparing hepatitis B
surface antigen which comprises growing cells which shed hepatitis B surface antigen in the presence of a nutrient medium on hollow fiber capillary units having a molecular weight cut-off of about 10,000.
surface antigen which comprises growing cells which shed hepatitis B surface antigen in the presence of a nutrient medium on hollow fiber capillary units having a molecular weight cut-off of about 10,000.
2. A method according to claim 1 wherein the growing is effected with two sequential stages having differing temperatures, each temperature being above room temperature with the first temperature being above the second temperature.
3. A method according to claim 1 wherein the first temperature is from about 35 to about 38°C.
4. A method according to claim 1 wherein the second temperature is from about 30°C to about 34°C.
5. A method according to claim 1 wherein the first temperature is about 37°C and the second temperature is about 32°C.
6. A method according to claim 1 wherein the growing takes place on a permeable membrane.
7. A method according to claim 5 wherein the permeable membrane is a hollow fiber capillary unit.
8. A method according to claim 2 wherein caffeine is present during the second stage.
9. A method according to claim 8 wherein the caffeine is present in an amount from about 0.0001 M
to about 0.0003 M.
to about 0.0003 M.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US06/225,555 US4416986A (en) | 1981-01-16 | 1981-01-16 | Methods of producing HBsAg |
| US225,555 | 1981-01-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1175745A true CA1175745A (en) | 1984-10-09 |
Family
ID=22845332
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000394009A Expired CA1175745A (en) | 1981-01-16 | 1982-01-12 | Methods of producing hb sag |
Country Status (10)
| Country | Link |
|---|---|
| US (1) | US4416986A (en) |
| EP (1) | EP0056761B1 (en) |
| JP (1) | JPS57141294A (en) |
| AT (1) | ATE6402T1 (en) |
| CA (1) | CA1175745A (en) |
| DE (1) | DE3260046D1 (en) |
| DK (1) | DK15382A (en) |
| ES (1) | ES8300473A1 (en) |
| GR (1) | GR74755B (en) |
| PT (1) | PT74246B (en) |
Families Citing this family (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4393133A (en) * | 1980-06-12 | 1983-07-12 | The Wistar Institute Of Anatomy And Biology | Human hepatoma derived cell line, process for preparation thereof, and uses therefor |
| US5223428A (en) * | 1982-12-14 | 1993-06-29 | Baxter International Inc. | Method for in vitro culture of mammalian cells |
| US4742158A (en) * | 1986-04-25 | 1988-05-03 | Merck & Co., Inc. | Purification of hepatitis pre-S antigens by polymerized serum albumin affinity binding |
| US6309635B1 (en) | 1986-11-20 | 2001-10-30 | Children's Medical Center Corp. | Seeding parenchymal cells into compression resistant porous scaffold after vascularizing in vivo |
| US5567612A (en) * | 1986-11-20 | 1996-10-22 | Massachusetts Institute Of Technology | Genitourinary cell-matrix structure for implantation into a human and a method of making |
| CA1340581C (en) * | 1986-11-20 | 1999-06-08 | Joseph P. Vacanti | Chimeric neomorphogenesis of organs by controlled cellular implantation using artificial matrices |
| US6022742A (en) * | 1986-11-26 | 2000-02-08 | Kopf; Henry B. | Culture device and method |
| DE69118690T2 (en) * | 1990-05-16 | 1997-01-09 | Baylor College Medicine | A PERMANENT HUMAN HEPATOGENIC CELL LINE AND ITS USE IN SUPPORTING LIVER FUNCTION |
| WO1993007913A1 (en) * | 1991-10-24 | 1993-04-29 | Children's Medical Center Corporation | Neomorphogenesis of urological structures in vivo from cell culture |
| US5709854A (en) * | 1993-04-30 | 1998-01-20 | Massachusetts Institute Of Technology | Tissue formation by injecting a cell-polymeric solution that gels in vivo |
| US5498537A (en) * | 1994-03-09 | 1996-03-12 | Cellco, Inc. | Serum-free production of packaged viral vector |
| US5716404A (en) * | 1994-12-16 | 1998-02-10 | Massachusetts Institute Of Technology | Breast tissue engineering |
| US6123727A (en) * | 1995-05-01 | 2000-09-26 | Massachusetts Institute Of Technology | Tissue engineered tendons and ligaments |
| US5855610A (en) | 1995-05-19 | 1999-01-05 | Children's Medical Center Corporation | Engineering of strong, pliable tissues |
| US5741685A (en) * | 1995-06-07 | 1998-04-21 | Children's Medical Center Corporation | Parenchymal cells packaged in immunoprotective tissue for implantation |
| US6129761A (en) * | 1995-06-07 | 2000-10-10 | Reprogenesis, Inc. | Injectable hydrogel compositions |
| JP2005521401A (en) * | 2002-03-27 | 2005-07-21 | イミュネックス・コーポレーション | Methods for increasing polypeptide production |
| JP5327793B2 (en) * | 2006-09-26 | 2013-10-30 | 国立大学法人京都大学 | Hepatitis virus propagation method and use thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3883393A (en) * | 1972-05-18 | 1975-05-13 | Us Health Education & Welfare | Cell culture on semi-permeable tubular membranes |
| US4220725A (en) * | 1978-04-03 | 1980-09-02 | United States Of America | Capillary cell culture device |
| CA1164799A (en) * | 1979-09-04 | 1984-04-03 | Paula A. Giesa | Cell culture of hepatitis a virus |
| US4301250A (en) * | 1979-11-02 | 1981-11-17 | Merck & Co., Inc. | Method of producing hepatitis B surface antigen |
| PT72015B (en) * | 1979-11-13 | 1982-06-20 | Merck & Co Inc | Process for preparing "invitro" cultures of hepatitis b virus |
| DE3162469D1 (en) * | 1980-03-27 | 1984-04-12 | Merck & Co Inc | Method of growing hepatitis b surface antigen |
-
1981
- 1981-01-16 US US06/225,555 patent/US4416986A/en not_active Expired - Lifetime
-
1982
- 1982-01-06 PT PT74246A patent/PT74246B/en unknown
- 1982-01-12 CA CA000394009A patent/CA1175745A/en not_active Expired
- 1982-01-14 GR GR67007A patent/GR74755B/el unknown
- 1982-01-14 ES ES508742A patent/ES8300473A1/en not_active Expired
- 1982-01-14 JP JP57003549A patent/JPS57141294A/en active Pending
- 1982-01-15 EP EP82400074A patent/EP0056761B1/en not_active Expired
- 1982-01-15 DE DE8282400074T patent/DE3260046D1/en not_active Expired
- 1982-01-15 AT AT82400074T patent/ATE6402T1/en not_active IP Right Cessation
- 1982-01-15 DK DK15382A patent/DK15382A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| US4416986A (en) | 1983-11-22 |
| DE3260046D1 (en) | 1984-04-05 |
| PT74246A (en) | 1982-02-01 |
| JPS57141294A (en) | 1982-09-01 |
| DK15382A (en) | 1982-07-17 |
| EP0056761A1 (en) | 1982-07-28 |
| ES508742A0 (en) | 1982-11-16 |
| ATE6402T1 (en) | 1984-03-15 |
| GR74755B (en) | 1984-07-11 |
| EP0056761B1 (en) | 1984-02-29 |
| ES8300473A1 (en) | 1982-11-16 |
| PT74246B (en) | 1984-05-29 |
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