Improvement of Cell-Freezing Technologies and Disposable Bioreactors: Toward a Fully Closed USP Process?

Pharmaceutical Discovery, Development and Manufacturing Forum
AIChE Annual Meeting
November 6, 2013 - 3:15pm-3:45pm

Biotech processes still contain a number of open and at risk transfers steps. Cell culture steps from one frozen vial to containers such as T-flasks or shake flasks is usually one of these open phases. This critical expansion step may take several days or even weeks and delay development or production timeline due to contamination. By coupling traditional technologies (bags) and new technologies (disposable bags , cases and bioreactor , Aseptic Transfer Cap) , a study on bags and freezing conditions has been performed. We have evaluated the freezing/thawing of cells in bags without DMSO removal to allow fully closed operations from thawing to the final 1250L Bioreactor inoculation. A first trial on 7 different CHO cell lines was performed to demonstrate if DMSO removal was necessary. Growth and viability were compared to the reference vial during 25 days after thawing. The best parameters have then been applied to different cell lines and media. The first part of this study demonstrated that we succeeded to freeze mammalian cells in bags with direct thawing and transfer in closed culture containers. With this technique , cell amplification timelines were reduced and the risk of cross contamination eliminated. This first improvement was combined to single-use bioreactors that are now commonly used for process development and as seeding or production bioreactors. Disposable bioreactors have a number of advantages such as absence of cleaning , no need for sterilization , easy and fast validation , reduced labor time and increased flexibility. If the benefits associated to these equipments have been well demonstrated on more than a decade , only a few data on their scalability are published. During the period 2010-2012 , we performed a study in order to evaluate the performances of disposable bioreactors at various scale form 3L to 200L. The evaluation was performed both for seeding application and for clinical material production. A fed-batch process producing a highly glycosylated molecule was performed in different types of disposable bioreactors. The data generated allowed to conclude on the scalability and the comparability of these bioreactors. Several clinical runs at 200L and 1250L scale were performed to ensure a meaningful comparison. These process performances were also compared to glass and stainless steel bioreactors of different sizes ranging from 3.6L and 1250L. The quality of the molecule together with the molecule titer and the cell growth was compared between the different single use technologies. This study demonstrated the benefits of using disposable equipment in several key areas. The comparison of stainless steel , glass and disposable equipment showed how comparable they are. Finally , coupling cell freezing in bag and disposable bioreactors allowed us to develop a fully closed USP process. We will extensively discuss the final set up from a technical as well as financial and organizational point of view emphasizing the various savings associated (labour , expense , training…) with single use systems and closed processes.

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