Characterization of mAb size heterogeneity originating from a cysteine to tyrosine substitution using denaturing and native LC-MS

Isabel Ruppen; Liesa Verscheure; Isabel Vandenheede; Alexia Ortiz; Iván Sánchez de Melo; Timo Liebig; Pat Sandra; Marie-Elise Beydon; Koen Sandra

doi:10.1016/j.jpba.2023.115743

Characterization of mAb size heterogeneity originating from a cysteine to tyrosine substitution using denaturing and native LC-MS

J Pharm Biomed Anal. 2023 Nov 30:236:115743. doi: 10.1016/j.jpba.2023.115743. Epub 2023 Sep 22.

Authors

Isabel Ruppen¹, Liesa Verscheure², Isabel Vandenheede², Alexia Ortiz², Iván Sánchez de Melo¹, Timo Liebig¹, Pat Sandra², Marie-Elise Beydon¹, Koen Sandra³

Affiliations

¹ mAbxience Research, Manuel Pombo Angulo 28, 28050 Madrid, Spain.
² RIC group, President Kennedypark 26, 8500 Kortrijk, Belgium.
³ RIC group, President Kennedypark 26, 8500 Kortrijk, Belgium. Electronic address: koen.sandra@RIC-group.com.

PMID: 37757547
DOI: 10.1016/j.jpba.2023.115743

Abstract

Upon assessing the comparability between a biosimilar mAb and its reference product by non-reducing CE-SDS, increased levels of a heavy-heavy-light chain (HHL) variant, present as a low molecular weight (LMW) peak, were observed. RPLC-MS applied at top, middle-up and bottom-up level revealed the existence of Cys-to-Tyr substitutions, predominantly at position HC226 involved in connecting LC and HC, explaining the abundant HHL levels. Antigen binding was not impacted by the presence of this size variant suggesting a non-covalent association of Tyr substituted HHL and LC. The latter complex is not maintained in the denaturing conditions associated with CE-SDS and RPLC-MS. Its existence could, nevertheless, be confirmed by native SEC-MS which preserves non-covalent protein interactions during separation and electrospray ionization. Amino acid analysis furthermore demonstrated a depletion of Cys during the fed-batch process indicating that the observed size/sequence variant is not of genetic but rather of metabolic origin. Native SEC-MS showed that supplementing the cell culture medium with Cys halts misincorporation of Tyr and promotes the formation of the desired mAb structure. To the best of our knowledge, Cys-to-Tyr substitutions preventing interchain disulfide bridge formation have not been described earlier. This observation adds to the impressive structural heterogeneity reported to date for mAbs.

Keywords: CE-SDS; Cysteine to tyrosine substitution; LC-MS; Monoclonal antibody; Native MS; Size variant.

Publication types

Review