Controlled shear affinity filtration (CSAF): A new technology for integration of cell separation and protein isolation from mammalian cell cultures

• Published in: Biotechnology and bioengineering Vol. 78; no. 7; pp. 806 - 814
• Main Authors: Vogel, Jens H., Anspach, Birger, Kroner, Karl-Heinz, Piret, James M., Haynes, Charles A.
• Format: Journal Article
• Language: English
• Published: New York Wiley Subscription Services, Inc., A Wiley Company 30.06.2002; Wiley
• Subjects: Animal cells; Animals; Biological and medical sciences; Biotechnology; Cell Culture Techniques - methods; cell separation; CHO Cells - chemistry; chromatography; Chromatography, Liquid - instrumentation; Chromatography, Liquid - methods; Cricetinae; Equipment Design; Eukaryotic cell cultures; Filtration - instrumentation; Filtration - methods; Fundamental and applied biological sciences. Psychology; Lysine - chemistry; mammalian cell culture; membrane filtration; Membranes, Artificial; Methods. Procedures. Technologies; Miscellaneous; Others; protein isolation; protein purification; Proteins - isolation & purification; rotating disk filter; Sensitivity and Specificity; Tissue Plasminogen Activator - chemistry; Tissue Plasminogen Activator - isolation & purification; Various methods and equipments; Cell culture; Shear; Purification; Serine endopeptidases; Enzyme; Rodentia; In vitro; Chromatography; t-Plasminogen activator; Peptidases; Separation method; Ovary; Vertebrata; Mammalia; Cell line; Adsorption; Batchwise; Animal; Female genital system; Membrane filtration; Hydrolases; Isolation; Recombinant protein; Hamster
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.10262

Controlled shear affinity filtration (CSAF) integrates animal cell separation and product isolation in a single unit operation through the use of a specifically designed rotating disk filter with incorporated membrane chromatography column. Because of the decoupling of shear force and pressure generation and the specific hydrodynamics of the system, shear rates can be easily optimized and precisely controlled to maximize filtration performance while viability of the shear sensitive animal cells is maintained. In this study, the general methodology is demonstrated using the integration of Chinese hamster ovary cell separation and isolation of recombinant tissue plasminogen activator (t‐PA) as a model example. Direct capture of t‐PA from cell culture broth was realized by using custom‐made affinity membranes with lysine as a robust, small molecular weight affinity ligand. Small‐scale t‐PA adsorption experiments, as well as microfiltration experiments, were used to design the integrated CSAF process. A Chinese hamster ovary batch culture was processed with a lab‐scale prototype, yielding 86% of the t‐PA in the concentrated, particle‐free eluate, whereas 95% of the bulk protein was removed. Because the viability of the cells is not significantly affected and high specific flux rates can be achieved, the CSAF technology should also be well suited for continuous perfusion with integrated product isolation. A truly continuous operation could be realized with two systems in tandem configuration. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 806–814, 2002.