An integrated process for mammalian cell perfusion cultivation and product purification using a dynamic filter.
In the present work, a dynamic filter was employed to develop an integrated perfusion/purification process. A recombinant CHO cell line producing a human anti-HIV IgG was employed in the experiments. In the first part of this work, the dynamic filter was fitted with conventional microfiltration membranes and tested as a new external cell retention device for perfusion cultivations. The filter was connected to a running perfusion bioreactor and operated for approximately 400 h at an average cell concentration of 10 million cells mL(-)(1), whereby cell viability remained above 90% and no problems of sterility were experienced. In the second part of this work, the dynamic filter was employed to simultaneously carry out cell separation and product purification, using membrane adsorbers containing Protein A affinity ligands. An automated system was built, which integrated the features of an automated perfusion bioreactor and of a liquid chromatography system. The IgG was continuously adsorbed onto the affinity membranes and was periodically recovered through elution cycles. After connection of the filter, the system was operated for approximately 300 h, whereby three elution cycles were carried out. No progressive increase in transmembrane pressure was observed, indicating no membrane fouling problems, and the IgG was recovered practically free of contaminants in a 14-fold concentrated form, indicating that the integrated, one-step perfusion/purification process developed during this work is a promising alternative for the production of biologicals derived from mammalian cells.
Castilho
Leda R
LR
GBF-German Research Centre for Biotechnology, Biochemical Engineering Division, Mascheroder Weg 1, D-38124 Braunschweig, Germany. ledacastilho@ieg.com.br
Anspach
F Birger
FB
Deckwer
Wolf-Dieter
WD
D000327:Adsorption; D000818:Animals; D019149:Bioreactors; D001709:Biotechnology / Q000295:instrumentation* / Q000379:methods*; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D006224:Cricetinae; D005374:Filtration / Q000295:instrumentation / Q000379:methods*; D015483:HIV Antibodies / Q000096:biosynthesis / Q000302:isolation & purification; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000302:isolation & purification*; D008567:Membranes, Artificial; D010477:Perfusion / Q000295:instrumentation / Q000379:methods*; D013997:Time Factors
D015483:HIV Antibodies; D007074:Immunoglobulin G; D008567:Membranes, Artificial