title: Rapid characterization of biotherapeutic proteins by size-exclusion chromatography coupled to native mass spectrometry.
abstract: High-molecular weight aggregates such as antibody dimers and other side products derived from incorrect light or heavy chain association typically represent critical product-related impurities for bispecific antibody formats. In this study, an approach employing ultra-pressure liquid chromatography size-exclusion separation combined with native electrospray ionization mass spectrometry for the simultaneous formation, identification and quantification of size variants in recombinant antibodies was developed. Samples exposed to storage and elevated temperature(s) enabled the identification of various bispecific antibody size variants. This test system hence allowed us to study the variants formed during formulation and bio-process development, and can thus be transferred to quality control units for routine in-process control and release analytics. In addition, native SEC-UV/MS not only facilitates the detailed analysis of low-abundant and non-covalent size variants during process characterization/validation studies, but is also essential for the SEC-UV method validation prior to admission to the market.
authors.affiliation: a Pharma Technical Development Analytics, Roche Diagnostics GmbH, Nonnenwald 2 , 82377 Penzberg , Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Haberger
authors.affiliation: a Pharma Technical Development Analytics, Roche Diagnostics GmbH, Nonnenwald 2 , 82377 Penzberg , Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Leiss
authors.affiliation: a Pharma Technical Development Analytics, Roche Diagnostics GmbH, Nonnenwald 2 , 82377 Penzberg , Germany.
authors.forename: Anna-Katharina
authors.initials: AK
authors.lastname: Heidenreich
authors.affiliation: a Pharma Technical Development Analytics, Roche Diagnostics GmbH, Nonnenwald 2 , 82377 Penzberg , Germany.
authors.forename: Oxana
authors.initials: O
authors.lastname: Pester
authors.affiliation: a Pharma Technical Development Analytics, Roche Diagnostics GmbH, Nonnenwald 2 , 82377 Penzberg , Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Hafenmair
authors.affiliation: a Pharma Technical Development Analytics, Roche Diagnostics GmbH, Nonnenwald 2 , 82377 Penzberg , Germany.
authors.forename: Michaela
authors.initials: M
authors.lastname: Hook
authors.affiliation: a Pharma Technical Development Analytics, Roche Diagnostics GmbH, Nonnenwald 2 , 82377 Penzberg , Germany.
authors.forename: Lea
authors.initials: L
authors.lastname: Bonnington
authors.affiliation: a Pharma Technical Development Analytics, Roche Diagnostics GmbH, Nonnenwald 2 , 82377 Penzberg , Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Wegele
authors.affiliation: a Pharma Technical Development Analytics, Roche Diagnostics GmbH, Nonnenwald 2 , 82377 Penzberg , Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Haindl
authors.affiliation: a Pharma Technical Development Analytics, Roche Diagnostics GmbH, Nonnenwald 2 , 82377 Penzberg , Germany.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Reusch
authors.affiliation: a Pharma Technical Development Analytics, Roche Diagnostics GmbH, Nonnenwald 2 , 82377 Penzberg , Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Bulau
mesh_terms: D018033:Antibodies, Bispecific / Q000032:analysis / Q000737:chemistry*; D002850:Chromatography, Gel; D066329:Protein Aggregates
chemical_list: D018033:Antibodies, Bispecific; D066329:Protein Aggregates
keywords: Aggregation; bispecific antibodies; critical quality attributes; fragmentation; native mass spectrometry; size-exclusion chromatography; ultra high performance liquid chromatography