Optimized conditions for the production of recombinant amphotropic retroviral vector preparations

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Abstract
The production and stability of recombinant retroviral vectors was examined at various temperatures. The two studied recombinant retroviral vectors, based on different packaging cell lines, exhibited a four-fold increased half-life at 32°C as compared to 37°C. Surprisingly, this increased stability at 32°C was only observed within a very narrow temperature window. At 30°C and 34°C, retroviral vector half-lives were quite similar to that at 37°C. Regardless of the vector half-life, retroviral vectors accumulated in the culture medium for a period of 48 h before an equilibrium was reached between retroviral vector production and decay. Maximal accumulated recombinant retroviral titers were five- to ten-fold increased after a medium incubation at 32°C as compared to 37°C. Furthermore, multiple cycles of freezing and thawing of retroviral vector supernatants hardly affected the recombinant retroviral vector titer, independent of the presence of serum. This knowledge on characteristics of recombinant retroviral vectors has practical implications for the manufacturing of these viruses for clinical gene therapy protocols.

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