title: PDADMAC flocculation of Chinese hamster ovary cells: enabling a centrifuge-less harvest process for monoclonal antibodies.
abstract: High titer (>10 g/L) monoclonal antibody (mAb) cell culture processes are typically achieved by maintaining high viable cell densities over longer culture durations. A corresponding increase in the solids and sub-micron cellular debris particle levels are also observed. This higher burden of solids (≥15%) and sub-micron particles typically exceeds the capabilities of a continuous centrifuge to effectively remove the solids without a substantial loss of product and/or the capacity of the harvest filtration train (depth filter followed by membrane filter) used to clarify the centrate. We discuss here the use of a novel and simple two-polymer flocculation method used to harvest mAb from high cell mass cell culture processes. The addition of the polycationic polymer, poly diallyldimethylammonium chloride (PDADMAC) to the cell culture broth flocculates negatively-charged cells and cellular debris via an ionic interaction mechanism. Incorporation of a non-ionic polymer such as polyethylene glycol (PEG) into the PDADMAC flocculation results in larger flocculated particles with faster settling rate compared to PDADMAC-only flocculation. PDADMAC also flocculates the negatively-charged sub-micron particles to produce a feed stream with a significantly higher harvest filter train throughput compared to a typical centrifuged harvest feed stream. Cell culture process variability such as lactate production, cellular debris and cellular densities were investigated to determine the effect on flocculation. Since PDADMAC is cytotoxic, purification process clearance and toxicity assessment were performed.
authors.affiliation: a Purification Process Development ; Amgen Inc. ; Seattle , WA USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: McNerney
authors.forename: Anne
authors.initials: A
authors.lastname: Thomas
authors.forename: Anna
authors.initials: A
authors.lastname: Senczuk
authors.forename: Krista
authors.initials: K
authors.lastname: Petty
authors.forename: Xiaoyang
authors.initials: X
authors.lastname: Zhao
authors.forename: Rob
authors.initials: R
authors.lastname: Piper
authors.forename: Juliane
authors.initials: J
authors.lastname: Carvalho
authors.forename: Matthew
authors.initials: M
authors.lastname: Hammond
authors.forename: Satin
authors.initials: S
authors.lastname: Sawant
authors.forename: Jeanine
authors.initials: J
authors.lastname: Bussiere
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D016466:CHO Cells; D002498:Centrifugation; D006224:Cricetinae; D003412:Cricetulus; D005427:Flocculation; D006801:Humans; D011092:Polyethylene Glycols / Q000737:chemistry*; D011095:Polyethylenes / Q000737:chemistry*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D011095:Polyethylenes; D000644:Quaternary Ammonium Compounds; C041004:poly-N,N-dimethyl-N,N-diallylammonium chloride; D011092:Polyethylene Glycols; C000595209:polyethylene glycol 1000
keywords: CCF, clarified centrifuged cell culture fluid; CHO, Chinese hamster ovary; DADMAC, diallyldimethylammonium chloride; DF, diafiltration volume; FBRM, focused beam reflectance measurement; HCP, host cell proteins; HI, hemolytic index; IV, intravenous; MF, microfiltration; MW, molecular weight; NTU, Nephelometric Turbidity Unit; PBS, phosphate buffered saline; PCV, packed cell volume; PDADMAC, poly diallyldimethylammonium chloride; PEG, polyethylene glycol; QPCR, quantitative polymerase chain reaction; RBC, red blood cells; TC, total cells; VCD, viable cell density; cytotoxicity; in-vitro hemolysis; in-vivo rodent toxicity; mAb, monoclonal antibody; mammalian cell culture; monoclonal antibody; n-aPA, neutralized acidified Protein A pool; particles/s, particles per second; polycationic flocculation harvest; rcf, relative centrifugal force; reagent clearance; w/v, weight to volume