Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
1997-03-31
2001-05-15
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S254200, C435S254220, C435S254230
Reexamination Certificate
active
06232111
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
This is related to U.S. Ser. No. 086,216, filed Jul. 1, 1993, now published as WO 95/01422.
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not applicable.
REFERENCE TO MICROFICHE APPENDIX
Not applicable.
FIELD OF THE INVENTION
Not applicable.
BACKGROUND OF THE INVENTION
Production of compounds of pharmaceutical significance by cultivation of recombinant yeasts is an expanding field of science and commerce. Purified recombinant hepatitis B surface antigen (HBsAg) is used as a vaccine for hepatitis B viral disease and is a well-known example of a pharmaceutically-significant recombinant protein.
Recombinant HBsAg is produced by cultivation of yeast cells in complex or chemically-defined (synthetic) culture media. Generally, complex media contain crude sources of nitrogen such as yeast extract and peptones. Although high yields of cells and crude HBsAg are achieved in these complex culture media, overall performance is frequently variable, and sometimes unacceptably inconsistent. Inconsistencies in fermentation performance adversely affect downstream purification steps and may also increase costs for the purified product.
Regulated expression systems are commonly used for the production of recombinant proteins. One type of regulated system provides tight nutritional control of the production of heterologous protein. This type of system maximizes biomass production and product stability while minimizing the adverse effects of heterologous protein expression on the host cell, e.g., Zabriskie et al.,
Enzyme Microbial Technol
. 8:706-717 (1986).
For convenience, applicants employ a recombinant
S. cerevisiae
strain for the production of Recombivax HB® (a trademark of Merck & Co. Inc.), which strain harbors a plasmid composed of the coding sequence for HBsAg linked to the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter, as well as an origin of replication from the yeast 2&mgr; plasmid, and the LEU2 gene for selection in yeast cells. The strain is an adenine auxotroph, i.e., requires adenine for growth. Other adenine auxotrophs of yeast are typically used as recombinant hosts for heterologous protein expression, for example strains bearing mutations at the ADE 1 or ADE 2 loci. See, e.g., Kniskem, P. et al. in
Expression Systems for Processes for Recombinant DNA Products
(Hatch et al., eds.) ACS Symposium Series No.447 (ch.6) pp.65-75 (1991), and Schultz, L. et al.
Gene
61, 123 (1987).
It would be desirable to identify the component(s) of complex media that affect fermentation performance, especially yields. Advantages of such discoveries would include a more reproducible fermentation process and a more predictable purification process.
Yeast extracts are commonly used in the media for yeast fermentations as the source for vitamins, trace elements and nitrogen nutrient. In many fermentation processes the nutrient which becomes limiting during the course of fermentation is the carbon source. The lot-to-lot variation of yeast extract due to variations in vendor's manufacturing processes dramatically affect recombinant yeast fermentation productivity and consistency, e.g. Recombivax HB® (a trademark of Merck & Co., Inc.) fermentation. The problem was partially solved in the past by the “brute-force” fermentation screening (“use-test”) of new yeast extract lots. As a result, additional manpower and facilities had to be tied up, and sometimes “good” lots could not be secured due to delay in decision while other times “poor” lots were purchased and had to be thrown away. This disadvantage can be overcome by first identifying the critical and varying components in yeast extract that affect Recombivax HB® fermentation, and establishing rapid assay methods for these components. After a sufficiently representative database is built, the analytical results can be used to evaluate whether a particular yeast extract lot is desirable for Recombivax HB® fermentation.
The invention relates to a method to rapidly determine whether a yeast extract lot will be “good” for recombinant yeast fermentations, including that which produces HBsAg (Recombivax HB®), by measuring the contents of critical varying components such as adenine, trehalose and lactic acid. This simple and rapid screening procedure eliminates lots with suboptimal levels of these components and allows in most cases (about 80% of lots) superior and consistent fermentation productivity. The method also enables the improvement of fermentation yield by rational supplementation of those components to “poor” yeast extract lots.
Applicants have identified adenine and two metabolizable carbon sources (trehalose and lactate) as critical components in yeast extract causing fermentation inconsistency. Adenine is required for growth while the slowly metabolized trehalose supplies energy after growth phase for recombinant gene expression in the synthesis of expression product. The rapidly utilized lactate exerts a positive effect indirectly by sparing more ethanol as the carbon source for product synthesis. These effects on growth and production are mutually-dependent. A relatively high level of carbon sources (trehalose plus lactate, >4 g/42 g) and a mid level of adenine (0.06~0.1 g/42 g) are necessary characteristics of a good yeast extract lot for yeast cultivation and crude HBsAg production.
SUMMARY OF THE INVENTION
A method for improving the culture medium useful for the cultivation of recombinant yeasts and the production of recombinant proteins is provided. The medium is particularly useful for the cultivation of recombinant strains of
Saccharomyces cerevisiae
which produce HBsAg.
BRIEF DESCRIPTION OF THE DRAWINGS
Not applicable.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is related to a general fermentation process for the production of recombinant proteins by yeast cells. The process of the present invention is demonstrated with the production of HBsAg by batch fermentation of strains of
Saccharomyces cerevisiae
transformed with a plasmid comprising the gene for HBsAg. As will be appreciated by one of ordinary skill in the art, the process of the present invention has a more general application to cultivation of other strains of
S. cerevisiae
and the production of other recombinant products and is not limited to HBsAg.
In general, yeast batch fermentation in complex medium is either a growth-limited process or a carbon source-limited process, depending on the adenine and trehalose/lactate contents of the YE (yeast extract) lot used. The concentration of these critical components in YE can vary dramatically due to variations in vendors' manufacturing processes. These inconsistencies contribute to fluctuations in fermentation performance, e.g., the amount of HBsAg produced. The analytical tools for adenine, trehalose and lactate in YE have been developed. Adenine content determines biomass production while carbon source (trehalose plus lactate) content affects antigen (HBsAg) product synthesis, and these two effects are related to each other. A mid-level adenine (0.06~0.1 g/42 g YE) and a high level trehalose plus lactate (>4 g/42 g YE) are the necessary requirements for a good lot, provided that the concentration of lactate does not exceed about 4.0 g/42 g YE. Concentrations of lactate exceeding about 4.0 g/42 g YE will cause significant change in fermentation pH profile. Many poor lots are improved by rational supplementation of adenine or trehalose or lactate or their combination.
In this invention, there is provided a method for improving culture medium with limiting carbon source for a recombinant yeast prototroph, comprising the steps of:
a) providing a quantity of a given lot of yeast extract to be tested;
b) measuring the concentrations of trehalose and lactate;
c) adjusting the concentration of trehalose plus lactate to more than or equal to about 4.0 g/42 g of yeast extract, provided that the concentration of lactate is less than or equal to about 4.0 g/42 g yeast extract.
In one embodiment of this invention, there is provided a method for improvin
Greasham Randolph L.
Zhang Jinyou
Marx Irene
Merck & Co. , Inc.
Tribble Jack L.
Yablonsky Michael D.
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