The use of chitosan as a flocculant in mammalian cell culture dramatically improves clarification throughput without adversely impacting monoclonal antibody recovery.
Flocculants have been employed for many years as aides in the clarification of wastewater, chemicals and food. Flocculants aggregate and agglutinate fine particles resulting in their settling from the liquid phase and a reduction in solution turbidity. These materials have not been widely used in the clarification of mammalian cell culture harvest. In this paper we examined chitosan as a flocculent of cells and cell particulates in NS0 culture harvest and the subsequent further clarification of this material by continuous flow centrifugation followed by depth and absolute filtration. Chitosan is an ideal flocculant for biotechnology applications as it is produced from non-mammalian sources (typically arthropod shells) and is also available in a highly purified form that is low in heavy metals, volatile organics and microbial materials. Chitosan is a polymer of deacetylated chitin. The deacetylation imparts limited solubility on insoluble chitin and the amino groups on the polymer result in a polycationic material at acidic and neutral pH that can interact with polyanions, such as DNA and cell culture debris (typically negatively charged). Likely the interaction of chitosan with cell culture particulate forms a germinal center for further interaction and agglomeration of particulates thereby reducing the solubility of these materials resulting in their settling out into the solid phase. Chitosan improved the clarification throughput six to seven folds without a deleterious effect on monoclonal antibody recovery or purity. The procedure for utilizing chitosan is facile, easily implemented, and highly effective in improving material clarity and increasing material throughput.
Riske
Frank
F
Purification Development, Genzyme Corporation, Framingham, MA 01701, USA. frank.riske@genzyme.com
Schroeder
James
J
Belliveau
Julie
J
Kang
Xuezhen
X
Kutzko
Joseph
J
Menon
Manoj K
MK
D019342:Acetic Acid / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000302:isolation & purification; D019149:Bioreactors; D018929:Cell Culture Techniques; D002460:Cell Line; D048271:Chitosan / Q000737:chemistry*; D005427:Flocculation; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D048271:Chitosan; D019342:Acetic Acid