{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"omics_type":["Unknown"],"volume":["13(1)"],"submitter":["Cao M"],"funding":["no funding"],"pubmed_abstract":["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<sub>H</sub>2 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<sub>H</sub>2 domain of both heavy chains (HCs) and, to a lesser degree, at the C-terminus of L182 in the C<sub>H</sub>1 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<sub>H</sub>2 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<sub>H</sub>2 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<sub>H</sub>2 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."],"journal":["mAbs"],"pagination":["1981806"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC8565840"],"repository":["biostudies-literature"],"pubmed_title":["Identification of a CE-SDS shoulder peak as disulfide-linked fragments from common C<sub>H</sub>2 cleavages in IgGs and IgG-like bispecific antibodies."],"pmcid":["PMC8565840"],"pubmed_authors":["Cao M","Hunter A","Ma J","Parthemore C","Chen X","Kilby G","Jiao Y","Korman S"],"additional_accession":[]},"is_claimable":false,"name":"Identification of a CE-SDS shoulder peak as disulfide-linked fragments from common C<sub>H</sub>2 cleavages in IgGs and IgG-like bispecific antibodies.","description":"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<sub>H</sub>2 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<sub>H</sub>2 domain of both heavy chains (HCs) and, to a lesser degree, at the C-terminus of L182 in the C<sub>H</sub>1 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<sub>H</sub>2 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<sub>H</sub>2 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<sub>H</sub>2 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.","dates":{"release":"2021-01-01T00:00:00Z","publication":"2021 Jan-Dec","modification":"2024-02-15T11:38:02.661Z","creation":"2022-02-11T12:29:19.383Z"},"accession":"S-EPMC8565840","cross_references":{"pubmed":["34719342"],"doi":["10.1080/19420862.2021.1981806"]}}