OPTIMIZED CONDITIONS FOR THE PRODUCTION OF RECOMBINANT AMPHOTROPIC RETROVIRAL VECTOR PREPARATIONS

The production and stability of recombinant retroviral vectors was exa mined at various temperatures. The two studied recombinant retroviral vectors, based on different packaging cell lines, exhibited a four-fol d increased half-life at 32 degrees C as compared to 37 degrees C. Sur prisingly this increased stability, at 32 degrees C was only observed within a very narrow temperature window. At 30 degrees C and 34 degree s C, retroviral vector half-lives were quite similar to that at 37 deg rees C. Regardless of the vector half-life, retroviral vectors accumul ated in the culture medium for a period of 48 h before an equilibrium was reached between retroviral vector production and decay. Maximal ac cumulated recombinant retroviral titers were five- to ten-fold increas ed after a medium incubation, at 32 degrees C as compared to 37 degree s C. Furthermore, multiple cycles of freezing and thawing of retrovira l vector supernatants hardly affected the recombinant retroviral vecto r titer, independent of the presence of serum. This knowledge on chara cteristics of recombinant retroviral vectors has practical implication s for the manufacturing of these viruses for clinical gene therapy pro tocols.