A Middle-up Approach using FabALACTICA for Characterization of Bispecific Antibodies

In recent years, bispecifics have gained popularity due to their therapeutic advantages over conventional IgG’s. In particular, the T-cell bi-specifics have received a great deal of attention due to their potential for improved efficacy. However, because of their complex TCB formats, there are multiple challenges associated with manufacturing and analysis of these type of biomolecules. A number of product and process related side products are formed which require close monitoring and identification. Moreover, the existence of various charge variants is common which can be challenging to fully characterize and understand.

In this study, scientists present the development of a cation-exchange chromatography and native electrospray mass spectrometry (CEC-ESI-MS) method for charge variant analysis. Three different acidic charge variants, one main peak and one basic variant were successfully separated and isolated by preparative scale cation-exchange chromatography. The CEC peak fractions were extensively characterized with mass spectrometric detection (SEC-UV-MS), peptide mapping (LC-MS) and cell-based potency testing. The analysis of data collected confirmed the presence of N and O-glycosylation sites in the acidic region, dimer and trimer variants, deamidation and Fab glycosylation (with and without sialic acid).


Although the mass spectrometry analysis by ESI-MS provided details on the existence of various modifications of the TCB molecule, the intact ESI-MS could not distinguish between Fc galactose and lysine glycation (both modifications have a mass increase of 162 Da). A middle-up approach which was applied instead of the intact analysis, allowed for a differentiation between the modifications in Fc and Fab region. FabALACTICA, a cysteine protease which cleaves human IgG above the hinge was used in this experiment which enabled further assessment of the glycation levels in the main peak fractions (MP) and the acidic peak (AP1). The results showed higher levels of glycation in AP 1 Fab fragment (31%) compared to the MP fragment (15 %). Additionally, this workflow confirmed the assignment of Fab N- and O-glycosylation in all acidic charge variants.


Haberger et al., 2021. Multiattribute Monitoring of Antibody Charge Variants by Cation-Exchange Chromatography Coupled to Native Mass Spectrometry.  Journal of American Society for Mass Spectrometry.


FabALACTICA product box





FabALACTICA – IgG-specific protease digesting human IgG1 at a specific site above the hinge.