In-Depth Characterization of mAb Charge Variants by On-Line Multidimensional Liquid Chromatography-Mass Spectrometry

In-depthcharacterization of charge heterogeneity is a pivotalstep desired in the therapeutics antibody development. To this end,a novel on-line multidimensional liquid chromatography-mass spectrometry(MDLC-MS) method for charge variant characterization was developedto dig out potential risks on safety and efficacy. This method implemented96-well plate fractionation and on-column preconcentration by multi-injection,thereby facilitating detection of charged species at low abundance.Eleven charge variants of mAb-A were preliminarily characterized by2DLC-(CEX x RP-C-4)-MS. TRVHS and RVHS signal peptidevariants of mAb-A were found in basic peaks of the CEX profile. Theresults supported process development in a timely manner, and thesignal peptide-containing variants with potential immunogenicity weresuccessfully removed by an optimized purification process. The retainedseven charge variants of mAb-A were further characterized by 4DLC-(CEXx RP-C-4 x TrypsinxRP-C-18)-MS.Post-translational modifications including deamidation, cyclizationof N-terminal glutamine, C-terminal lysine truncation as well as prolineamidation, and methionine oxidation were identified, and their potentialrisks were evaluated. Biological activity of the seven charge variantswas evaluated by 2DLC (CEX x Fc & gamma;RIIIa). Increased Fc & gamma;RIIIareceptor binding affinity was observed in the acidic variants. TheMDLC-MS detection can be completed in 72 h with 1.25 mg of mAb, demonstratingto be sample-economic, time-effective, and labor-saving. It provideda powerful and timely tool for charge variant characterization andmet the aggressive timeline desired for antibody development.

Automated summary

A novel MDLC-MS method was developed for charge variant characterization in therapeutic antibody development, aiming to discover potential risks on safety and efficacy. The method enabled detection of charged species at low abundance by implementing 96-well plate fractionation and on-column preconcentration. The charge variants of mAb-A were characterized, and post-translational modifications were identified and evaluated for their potential risks. The MDLC-MS detection demonstrated sample-economic, time-effective, and labor-saving features, providing a powerful and timely tool for charge variant characterization in antibody development.