Clip Formation in the Complementarity Determining Region of Bevacizumab Lowers Monomer Stability and Affinity for Both FcRn and FcγR: A Comprehensive Characterization of the Clipped Variant Including its Higher Order Structure

The presence of monoclonal antibody (mAb) fragments in pharmaceutical mAb products is a critical quality attribute and should be controlled for safety. Several mAb fragments derived from clip formation in the complementarity determining regions (CDRs), as well as from cleavage in the hinge region, h...

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Bibliographic Details
Published in:Journal of pharmaceutical sciences 2022-12, Vol.111 (12), p.3243-3250
Main Authors: Atsumi, Yuriko, Yamada, Ayumi, Kojima, Yuka, Yagi, Yuki, Nishimura, Koichiro, Wakamatsu, Kaori
Format: Article
Language:English
Subjects:
Antibody(s)
Bevacizumab - chemistry
Biopharmaceutical characterization
Complementarity Determining Regions
Protein binding
Protein structure(s)
Receptors, IgG
Separation science
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Summary:The presence of monoclonal antibody (mAb) fragments in pharmaceutical mAb products is a critical quality attribute and should be controlled for safety. Several mAb fragments derived from clip formation in the complementarity determining regions (CDRs), as well as from cleavage in the hinge region, have been reported. However, the properties of CDR-clipped variants are not fully understood because of difficulties in separating them from intact mAbs under non-denaturing conditions due to similarities in size. We have established a method for separating CDR-clipped variants under non-denaturing conditions using an appropriate size exclusion chromatography column.1 In this report, we provide a comprehensive characterization of a CDR-clipped variant from bevacizumab. The variant exhibited a lower pI, a higher tendency to form dimers, and a lower affinity for both neonatal Fc receptor (FcRn) and Fcγ receptor (FcγR). The effects of clip formation in CDR H3 on the higher order structure were analyzed by hydrogen/deuterium exchange mass spectrometry, and the observed changes in the structures of the VH, CH2, and VL domains were in agreement with the lowered affinity for antigen, FcRn, and Fcγ receptors. These findings suggest that clip formation in the CDR may affect the efficacy, safety, and pharmacokinetics of pharmaceutical mAbs.
ISSN:0022-3549
1520-6017
DOI:10.1016/j.xphs.2022.08.024