Intact and middle-down CIEF of commercial therapeutic monoclonal antibody products under non-denaturing conditions.

ABSTRACT: A two-step CIEF with chemical mobilization was developed for charge profiling of the therapeutic mAb rituximab under non-denaturing separation conditions. CIEF of the intact mAb was combined with a middle-down approach analyzing Fc/2 and F(ab´)₂ fragments after digest with a commercial cysteine protease (IdeS). CIEF methods were optimized separately for the intact mAb and its fragments due to their divergent pIs. Best resolution was achieved by combining Pharmalyte (PL) 8-10.5 with PL 3-10 for variants of intact rituximab and of F(ab´)₂ fragments, respectively, whereas PL 6.7-7.7 in combination with PL 3-10 was used for Fc/2 variants. Charge heterogeneity in Fc/2 dominates over F(ab´)₂ . In addition, a copy product of rituximab, and adalimumab were analyzed

SUBMITTER: Schmailzl J 

PROVIDER: S-EPMC7317833 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature