Development of a generic transient transfection process at 100 L scale.

Abstract:

:We have developed a generic transient transfection process at 100 L scale, using HEK293-EBNA cells and PEI as the transfection reagent for the production of recombinant IgG. The process, including large-scale plasmid preparation, expression at bioreactor scale, capture, purification and, if necessary, endotoxin removal allows reproducible production of more than 0.5 g IgG for in vitro and in vivo studies. We compared the performance of two HEK cell lines, investigated the effect of conditioned medium, optimized the DNA:PEI ratio and implemented a feed strategy to prolong the culture time to increase product yield. The transient transfection protocol developed enables a closed process from seeding culture to protein capture. The challenge of performing a medium exchange before transfection at large scale is solved by applying a continuous centrifugation step between the seeding bioreactor and the production bioreactor. After 7-8 days the harvest and capture is performed in a one-step operation using a Streamline expanded bed chromatography system. Following a polishing step the purified antibody is transferred to the final formulation buffer. The method has shown to be reproducible at 10, 50, and 100 L scale expressing between 5 and 8 mg L(-1) IgG.

journal_name

Cytotechnology

journal_title

Cytotechnology

authors

Tuvesson O,Uhe C,Rozkov A,Lüllau E

doi

10.1007/s10616-008-9135-2

subject

Has Abstract

pub_date

2008-02-01 00:00:00

pages

123-36

issue

2

eissn

0920-9069

issn

1573-0778

journal_volume

56

pub_type

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