A new seed-train expansion method for recombinant mammalian cell lines

A new approach has been developed and used to minimize the time and more carefully monitor and control the seed-train expansion process of recombinant mammalian cell lines. The process uses 50 or 100 ml cryo-bags that contain frozen cells at high cell densities of 20 x 10(6) ml(-1) (100 ml bags) or 40 x 10(6) cells ml(-1) (50 ml bags). The frozen bag cell suspension is thawed and transferred directly into a bioreactor that has been modified such that pH, DO and temperature can be controlled at the initial volume of two liters (the working volume eventually increases to 12 l). The successful use of these cryo-bags and the modified 'inoculation' bioreactor to initiate a new seed train expansion of rBHK or rCHO cells is described herein. The interval between cell thawing and the accumulation of sufficient cell mass to inoculate a production reactor is reduced by at least 25 to 30 days compared to the conventional method that begins with the thaw of 1-2 ml cryo-vials. This 'one-step' technology leads to a much more consistent scale-up by reducing manual operations and avoiding subjective decisions during the scale-up phase. The cell metabolic rates and product integrity were similar to the control experiments. Furthermore, it was found that it is not necessary to include a wash step to remove DMSO prior to the inoculation.