title: Development of a novel and efficient cell culture flocculation process using a stimulus responsive polymer to streamline antibody purification processes.
abstract: Recent advances in mammalian cell culture processes have significantly increased product titers, but have also resulted in substantial increases in cell density and cellular debris as well as process and product related impurities. As such, with improvements in titer, corresponding improvements in downstream processing are essential. In this study we have developed an alternative antibody harvest process that incorporates flocculation using a novel stimulus responsive polymer, benzylated poly(allylamine), followed by depth filtration. As tested on multiple antibodies, this process demonstrates high process yield, improved clearance of cells and cell debris, and efficient reduction of aggregates, host cell proteins (HCP) and DNA. A wide operating window was established for this novel flocculation process through design of experiments condition screening and optimization. Residual levels of impurities in the Protein A eluate were achieved that potentially meet requirements of drug substance and thus alleviate the burden for further impurities removal in subsequent chromatography steps. In addition, efficient clearance of residual polymer was demonstrated using a fluorescence tagged polymer in the presence of a stimulus reagent. The mechanism of HCP and aggregates removal during flocculation was also explored. This novel and efficient process can be easily integrated into current mAb purification platforms, and may overcome downstream processing challenges.
authors.affiliation: Bioprocess Sciences, ImClone Systems, a Wholly-Owned Subsidiary of Eli Lilly and Company, 450 East 29th Street, New York, New York, 10016.
authors.forename: Yun Kenneth
authors.initials: YK
authors.lastname: Kang
authors.forename: James
authors.initials: J
authors.lastname: Hamzik
authors.forename: Michael
authors.initials: M
authors.lastname: Felo
authors.forename: Bo
authors.initials: B
authors.lastname: Qi
authors.forename: Julia
authors.initials: J
authors.lastname: Lee
authors.forename: Stanley
authors.initials: S
authors.lastname: Ng
authors.forename: Gregory
authors.initials: G
authors.lastname: Liebisch
authors.forename: Behnam
authors.initials: B
authors.lastname: Shanehsaz
authors.forename: Nripen
authors.initials: N
authors.lastname: Singh
authors.forename: Kris
authors.initials: K
authors.lastname: Persaud
authors.forename: Dale L
authors.initials: DL
authors.lastname: Ludwig
authors.forename: Paul
authors.initials: P
authors.lastname: Balderes
mesh_terms: D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D001688:Biological Products / Q000302:isolation & purification*; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods; D005591:Chemical Fractionation / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D005374:Filtration / Q000379:methods*; D005427:Flocculation; D006801:Humans; D011994:Recombinant Proteins / Q000302:isolation & purification; D013678:Technology, Pharmaceutical / Q000379:methods*
chemical_list: D000906:Antibodies; D001688:Biological Products; D011994:Recombinant Proteins
keywords: HCP; HMW; aggregates; depth filtration; flocculation; harvest; host DNA; hydrophobic interaction; impurity removal; monoclonal antibody; precipitation; purification; residual polymer; salt tolerant; smart polymer; stimulus responsive polymer