title: Identification and characterization of monoclonal antibody fragments cleaved at the complementarity determining region using orthogonal analytical methods.
abstract: Sodium dodecyl sulfate capillary electrophoresis (CE-SDS) has been widely used to separate and quantify monoclonal antibody (mAb) fragments and impurities. Two fragments with different masses smaller than monomer were observed in the CE-SDS analysis of a recombinant IgG1 mAb treated at 40°C for 28days. Reversed phase liquid chromatography mass spectrometry (RP-LC-MS) and trypsin digestion peptide mapping followed by LC-MS/MS analysis were employed to conclusively demonstrate that these antibody fragments were the degradation products derived from a Ser-Ser peptide bond cleavage at the heavy chain complementarity determining region (CDR). The total percentage of the cleaved antibody heavy chain was increased from 1.1% in the control sample to 4.6% in the forced degradation sample stored at 40°C for 28days by using CE-SDS analysis. Native size exclusion liquid chromatography (SEC-HPLC) was used to analyze the fragment profile of this treated mAb but did not reveal fragments smaller than the monomer. However, a small fragment was detected using denaturing size exclusion liquid chromatography (dSEC-HPLC) and was confirmed to be the N-terminal fragment of the heavy chain cleaved at the CDR region using RP-LC-MS analysis. These results suggested that this N-terminal fragment may be closely associated with the rest of the cleaved antibody molecule through non-covalent bond interactions. Moreover, when dSEC-HPLC and RP-LC-MS were used to analyze the aggregate and monomer samples individually collected from SEC-HPLC, both samples contained the same amount of the cleaved N-terminal fragment. These results suggested that the CDR region cleaved fragments likely not be involved in aggregates formation of this antibody. This case study reiterated that a combination of orthogonal analytical methods plays a crucial role in comprehensively analyzing and characterizing antibody fragmentation.
authors.affiliation: Sunshine Lake Pharma Co., Ltd, Dongguan 523867, China. Electronic address: liwenhua@hecpharm.com.
authors.forename: Wenhua
authors.initials: W
authors.lastname: Li
authors.affiliation: Institute of Biomedicine & National Engineering Research Center of Genetic Medicine, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Yang
authors.affiliation: Sunshine Lake Pharma Co., Ltd, Dongguan 523867, China.
authors.forename: Dongmei
authors.initials: D
authors.lastname: Zhou
authors.affiliation: Sunshine Lake Pharma Co., Ltd, Dongguan 523867, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Xu
authors.affiliation: Sunshine Lake Pharma Co., Ltd, Dongguan 523867, China.
authors.forename: Wenjia
authors.initials: W
authors.lastname: Li
authors.affiliation: Sunshine Lake Pharma Co., Ltd, Dongguan 523867, China. Electronic address: wenchensuen@hecpharm.com.
authors.forename: Wen-Chen
authors.initials: WC
authors.lastname: Suen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D016466:CHO Cells; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods; D022801:Complementarity Determining Regions / Q000737:chemistry*; D003412:Cricetulus; D019075:Electrophoresis, Capillary / Q000379:methods; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D010449:Peptide Mapping / Q000379:methods; D066329:Protein Aggregates; D011994:Recombinant Proteins / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D066329:Protein Aggregates; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate
keywords: CDR region; Capillary electrophoresis; Fragmentation; Liquid chromatography mass spectrometry; Monoclonal antibody; Size exclusion chromatography