Industrial application of impurity flocculation to streamline antibody purification processes

Abstract

Flocculation technologies offer significant benefits to industrial mammalian cell culture processes, including increased clarification efficiency, impurity removal and process simplification. In this paper, the application and performance of flocculation technologies employed in the harvest process of monoclonal antibody Chinese hamster ovary cell culture are reviewed. Attention has been placed on technologies enhancing the removal of cells, cellular debris and reduction in impurities while maintaining the antibody in the product stream. Many flocculants are systematically evaluated with respect to their mechanism of action, impact on downstream processing and product quality, and potential disadvantages. Practical considerations and future directions for application of flocculation in antibody manufacturing are discussed.

Polymer‐mediated flocculation of transient CHO cultures as a simple, high throughput method to facilitate antibody discovery

“…Of these host cell proteins (HCP), ∼15% are highly acidic while <25% are basic (pI > 7). SmP, a previously described flocculation agent, is a cationic polymer and should therefore bind to basic proteins (representing approximately one quarter of all CHO host cell proteins). However, the hydrophobic portion of the polymer is designed to bind to neutral HCP (representing more than half of all host cell proteins).…”

“…These challenges include the efficient removal of whole cells and cellular debris, HCP, and host DNA while maintaining high monomeric protein yields . One specific polymer namely partially benzylated poly(allylamine) (PAAm‐Bn), also termed Smart Polymer (SmP), has recently been shown to simultaneously clear cell and cellular debris, reduce HCP, HMW aggregates, and host DNA from CHO cell culture . SmP is a salt‐tolerant, cationic polymer substituted with hydrophobic residues that undergoes a soluble‐to‐insoluble transition when exposed to multivalent anions such as phosphate.…”

“…SmP is a salt‐tolerant, cationic polymer substituted with hydrophobic residues that undergoes a soluble‐to‐insoluble transition when exposed to multivalent anions such as phosphate. Residual SmP levels post Protein A purification were reported to be <0.1 ppm, eliminating the need for additional ion exchange chromatography for excess SmP removal . Other known flocculants that have been used for mammalian cell culture include PDADMAC, chitosan, and polyethyleneimine .…”

“…In this study, we coupled our newly developed transient transfection expression platform based on a CHO K1SV GS‐KO host cell line with a recently described process of adding SmP to cell culture as part of the harvest process . After extensive screening and optimization, we developed a high throughput harvest process scalable from 4 mL to 2 L. The addition of SmP as part of the cell harvest process, before Protein A purification, greatly improved final product quality.…”

Continuous cell flocculation for recombinant antibody harvesting

“…In their informative review, Felo et al . summarize the flocculation methods reported in antibody production by CHO cells . While these prior publications have investigated some applications of batch flocculation for CHO cells, no study has yet included comprehensive and wide screening, or expansion of the technology for use in continuous production.…”