The primary production of an infectious recombinant Herpes Simplex Virus vaccine.
The production and extracellular release of a recombinant Herpes Simplex Virus (type 2) from monolayers of infected complementing Vero cells (CR2) are addressed. Growth and virus production conditions are identified that provide adequate virus titers with cell seeding densities and viral multiplicities of infection that could be reasonably handled in manufacturing. Harvesting by sonication of cell monolayers is shown to give the highest recovery of infectious virus (to 2.5 x 10(6) pfu/mL) but leads to process stream contamination by cellular proteins through the rupturing of cells (to 28 pg protein/pfu). By comparison, freeze-thaw cycles and osmotic rupture by hypotonic saline or glycerol shock procedures yield only low virus recovery (typically <10% of that by sonication), and are accompanied by yet higher levels of protein contamination (up to 30-fold higher pg protein/pfu). Addition of the polyanionic polymers, heparin or dextran sulphate to a harvest using either hypotonic saline, glycerol shock or isotonic phosphate buffered saline increased the yield of infectious virus in the supernatant. By contrast, addition of polycationic poly-L-lysine resulted in negligible increase in the supernatant virus titer. The highest virus titers (4.7 x 10(7) pfu/mL) were achieved following treatment of roller bottle cultured cells displaying a high cytopathic effect with heparin at 50 microg/mL for at least 3 h post harvest. This procedure also gave the lowest levels of protein contamination (<2 pg protein/pfu). The fivefold lower yield of infectious virus from cultures displaying a low cytopathic effect (<70% CPE) indicates the importance of cell physiological state at harvest.
O'Keeffe
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Department of Chemical Engineering & Applied Chemistry, Aston University, Aston Triangle, Birmingham, United Kingdom.
Johnston
M D
MD
Slater
N K
NK
D000818:Animals; D001709:Biotechnology; D002522:Chlorocebus aethiops; D018258:Herpesvirus 2, Human / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D008854:Microscopy, Electron; D014614:Vaccines, Synthetic / Q000096:biosynthesis* / Q000302:isolation & purification; D014709:Vero Cells; D014765:Viral Vaccines / Q000096:biosynthesis* / Q000302:isolation & purification; D014776:Virus Cultivation
D014614:Vaccines, Synthetic; D014765:Viral Vaccines