title: Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry.
abstract: An advanced two-dimensional liquid chromatography/mass spectrometry platform was used to quantify individual host cell proteins (HCPs) present at various purification steps for several therapeutic monoclonal antibodies (mAbs) produced in Chinese hamster ovary cells. The methodology produced reproducible identifications and quantifications among replicate analyses consistent with a previously documented individual limit of quantification of ~13 ppm. We were able to track individual HCPs from cell culture fluid to protein A eluate pool to subsequent viral inactivation pool and, in some cases, further downstream. Approximately 500 HCPs were confidently identified in cell culture fluid and this number declined progressively through the purification scheme until no HCPs could be confidently identified in polishing step cation-exchange eluate pools. The protein A eluate pool of nine different mAbs contained widely differing numbers, and total levels, of HCPs, yet the bulk of the total HCP content in each case consisted of a small subset of normally intracellular HCPs highly abundant in cell culture fluid. These observations hint that minimizing cell lysis during cell culture/harvest may be useful in minimizing downstream HCP content. Clusterin and actin are abundant in the protein A eluate pools of most mAbs studied. HCP profiling by this methodology can provide useful information to process developers and lead to the refinement of existing purification platforms.
authors: Product Attribute Sciences, Amgen Inc.; Thousand Oaks, CA USA.
authors: Qingchun
authors: Q
authors: Zhang
authors: Product Attribute Sciences, Amgen Inc.; Thousand Oaks, CA USA.
authors: Andrew M
authors: AM
authors: Goetze
authors: Drug Substance Development, Amgen Inc.; Thousand Oaks, CA USA.
authors: Huanchun
authors: H
authors: Cui
authors: Drug Substance Development, Amgen Inc.; Thousand Oaks, CA USA.
authors: Jenna
authors: J
authors: Wylie
authors: Drug Substance Development, Amgen Inc.; Seattle, WA USA.
authors: Steve
authors: S
authors: Trimble
authors: Drug Substance Development, Amgen Inc.; Thousand Oaks, CA USA.
authors: Art
authors: A
authors: Hewig
authors: Product Attribute Sciences, Amgen Inc.; Thousand Oaks, CA USA.
authors: Gregory C
authors: GC
authors: Flynn
mesh_terms: D000199:Actins / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D001709:Biotechnology / Q000379:methods; D016466:CHO Cells; D002853:Chromatography, Liquid / Q000379:methods; D051152:Clusterin / Q000032:analysis; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media / Q000737:chemistry; D004340:Drug Contamination / Q000517:prevention & control; D013058:Mass Spectrometry / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013205:Staphylococcal Protein A
chemical_list: D000199:Actins; D000911:Antibodies, Monoclonal; D051152:Clusterin; D003470:Culture Media; D011506:Proteins; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A
keywords: Host cell proteins; antibody; mass spectrometry; monoclonal