biostudies-literatureUnknown13(1)Cao Mno fundingFragmentation is a well-characterized degradation pathway of therapeutic antibodies and is usually monitored by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS). Although fragments due to cleavage in C_H2 domains linked by intrachain disulfide bonds are common and can be detected by reduced reversed-phase - liquid chromatography mass spectrometry (RP-LCMS) and reduced CE-SDS methods, their separation in nonreduced CE-SDS (nrCE-SDS) has not been reported but speculated as comigrating with intact IgG. A shoulder peak in nrCE-SDS was observed in the stability samples of an IgG-like bispecific antibody and was determined to be mainly caused by fragments from clipping at the C-terminus of leucine (L)306 or L309 (EU numbering) in the C_H2 domain of both heavy chains (HCs) and, to a lesser degree, at the C-terminus of L182 in the C_H1 domain of the knob HC. Subunit LCMS analysis verified that the crystallizable fragment contained variants with one or multiple mass additions of ~18 Da due to clipping. Further investigation revealed that C_H2 clippings at L306 and L309 were largely due to proteolytic activity, and cleavages were present at various levels in all in-house IgG1 and IgG4 molecules studied. Our study shows that C_H2 domain cleavages, with complementary fragments still linked by intrachain disulfide, can be electrophoretically resolved as a front shoulder of the main peak in nrCE-SDS. Given the high occurrence of C_H2 cleavages in antibodies, these findings will have broad applicability and could help manufacturers of therapeutic antibodies in process improvement, product characterization, investigations, formulation stability, and stability comparability studies.mAbs1981806https://www.ebi.ac.uk/biostudies/studies/S-EPMC8565840biostudies-literatureIdentification of a CE-SDS shoulder peak as disulfide-linked fragments from common C_H2 cleavages in IgGs and IgG-like bispecific antibodies.PMC8565840Cao MHunter AMa JParthemore CChen XKilby GJiao YKorman SfalseIdentification of a CE-SDS shoulder peak as disulfide-linked fragments from common C_H2 cleavages in IgGs and IgG-like bispecific antibodies.Fragmentation is a well-characterized degradation pathway of therapeutic antibodies and is usually monitored by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS). Although fragments due to cleavage in C_H2 domains linked by intrachain disulfide bonds are common and can be detected by reduced reversed-phase - liquid chromatography mass spectrometry (RP-LCMS) and reduced CE-SDS methods, their separation in nonreduced CE-SDS (nrCE-SDS) has not been reported but speculated as comigrating with intact IgG. A shoulder peak in nrCE-SDS was observed in the stability samples of an IgG-like bispecific antibody and was determined to be mainly caused by fragments from clipping at the C-terminus of leucine (L)306 or L309 (EU numbering) in the C_H2 domain of both heavy chains (HCs) and, to a lesser degree, at the C-terminus of L182 in the C_H1 domain of the knob HC. Subunit LCMS analysis verified that the crystallizable fragment contained variants with one or multiple mass additions of ~18 Da due to clipping. Further investigation revealed that C_H2 clippings at L306 and L309 were largely due to proteolytic activity, and cleavages were present at various levels in all in-house IgG1 and IgG4 molecules studied. Our study shows that C_H2 domain cleavages, with complementary fragments still linked by intrachain disulfide, can be electrophoretically resolved as a front shoulder of the main peak in nrCE-SDS. Given the high occurrence of C_H2 cleavages in antibodies, these findings will have broad applicability and could help manufacturers of therapeutic antibodies in process improvement, product characterization, investigations, formulation stability, and stability comparability studies.2021-01-01T00:00:00Z2021 Jan-Dec2024-02-15T11:38:02.661Z2022-02-11T12:29:19.383ZS-EPMC85658403471934210.1080/19420862.2021.1981806