Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Culture medium – per se
Patent
1994-12-05
1997-03-04
Smith, Lynette F.
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Culture medium, per se
4242301, 4352351, 435407, 435408, C12N 984, C12N 700, C12N 500, A61K 39245
Patent
active
056078528
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
Varicella zoster virus (VZV) causes chickenpox and zoster (shingles). Chickenpox is a highly contagious disease that occurs in persons with no VZV immunity. More than 90% of the population is exposed during the first two decades of life. The disease is a severe threat to the immunosuppressed and to adults. In many cases, VZV becomes latent in dorsal root ganglion cells. Shingles, a painful chronic condition, occurs when VZV is reactivated from the latent state.
Prevention of chickenpox by vaccination is a desirable goal, and the institution of universal childhood vaccination with a live attenuated varicella vaccine is envisioned. The prior art has reported the propagation of VZV in various cell culture systems and the use of live, attenuated, cell-free VZV as a vaccine. U.S. Pat. No. 3,985,615 describes the production in guinea pig primary embryonic cells of the attenuated Oka strain of VZV, suitable for vaccine use. U.S. Pat. No. 4,008,317 describes the cultivation of a temperature-sensitive mutant of VZV in WI-38 cells for use as a vaccine stablilizer. Compositions useful for the maintainance of viable VZV, such as SPGA, are also known in the art.
The major limitation to commercial production of a VZV vaccine is the yield of cell-free VZV from cell culture systems known in the art. Cell-free VZV yields are improved by about a factor of 5-20 fold by application of the new process of this invention.
Thus, the present invention is a process for the production of a live, attenuated, cell-free VZV vaccine in high yield.
Monolayer cell culture methods known in the art typically yield about 80,000 to 160,000 cells/cm.sup.2 [Mann, Dev. Biol. Stand. 37, 149-152 (1977); Wood and Minor, Biologicals 18, 143-146 (1990)]. Use of monolayer cell cultures for production of viral antigens has been hampered by the restricted density to which these monolayer cultures could be grown.
Attempts at increasing cell culture densities in the past have turned to specialized perfusion culture vessels to increase saturation densities to about 1.times.10.sup.6 cells per cm.sup.2 [Mann, Dev. Biol. Stand. 37, 149-152 (1977)]. The present invention offers a unique means to increase cell yields while using existing cell culture systems.
This invention provides a process whereby attached cells may be grown to much higher densities than was heretofore possible, thus enabling higher yields of viruses cultured on cell monolayers for vaccine production.
SUMMARY OF THE INVENTION
This invention relates to a novel process for monolayer cell culture and a novel composition for use in the process. The process requires use of a rich culture medium, as opposed to a minimal medium, and supplementation of the rich medium with an optimized concentation of lipid. According to the process of this invention, use of a novel growth medium comprising SRFE-2 medium supplemented with between about 0.02-0.4 g/mL of soybean lipid, substantial increases in monolayer cell culture density is achievable. The increased monolayer cell density thus achieved is useful for enhanced production of viral antigens for anti-viral vaccine manufacture.
As applied to the production of a particular vaccine, the present invention is a process for the production of large amounts of a live, attenuated, cell-free VZV vaccine, useful to prevent chickenpox, which comprises optimally propagating VZV in cell culture, and harvesting the virus under conditions which maximize VZV yield and stability. The steps of the optimized process comprise: cells, such as MRC-5, to confluency in monolayer culture, using high culture volumes or rich culture medium, and supplying a non-metabolizable disaccharide, such as sucrose; point of confluency as possible with as high a multiplicity of infection of VZV-infected cells as practical; about 22-96 hours and harvesting at the point of peak VZV production; containing a lysosomotropic agent, such as ammonium chloride or chloroquine, prior to harvesting the VZV infected cells; sclution, and either disrupting the cells immediate
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Friedman Paul A.
Krah David L.
Provost Philip J.
Carty Christine E.
Merch & Co., Inc.
Smith Lynette F.
Tribble Jack L.
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