Charge Heterogeneity Analysis of Antibody Subunits Generated by FragIT

Charge heterogeneity of monoclonal antibodies is an important critical quality attribute that requires close monitoring due to its potential impact on antibody efficacy and immunogenicity. Since the heterogeneity is mostly caused by post translational modifications such as C-terminal lysine clipping, deamidation, glycation, sialic acid or adduct formation, these modifications can pose significant challenges to the analytical scientists.


In a recently published article by Jaag et al., scientists from the Institute of Pharmaceutical Sciences at University at Tübingen, Germany, present a new approach to charge variant analysis at the intact and sub-unit level by 2D-LC separation. In this workflow, the first-dimension separation is based on strong-cation chromatography (SCX) and the second-dimension separation is based on desalting reversed phase liquid chromatography (RP-HPLC) which enables combination with the mass analysis by mass spectrometric (MS) detection.


The analysis at the subunit level was achieved by digesting humanized IgG1K with FragIT MicroSpin columns from Genovis. These columns contain immobilized FABRICATOR enzyme which digests the antibody below the hinge and generates two fragments, Fc/2 and F(ab´)2. By further reduction with TCEP, three subunits were generated: Fc/2, Fd and the light chain.


The three subunits allowed for a fast separation by RP-HPLC. All three fragments were separated within 30 seconds. Further on, the Fd fragment resulted in two peaks which suggests different intrachain disulphide forms or D (P) clipping. As the aim of the analysis was to achieve a separation of as many charge variants as possible, the subunit level analysis enabled the extraction of detailed information about the molecule. Moreover, the main glycoforms were successfully identified using MS and middle-up approach by 2D-LC-ESI-MS analysis. In comparison to the intact mAb analysis, the 2D -LC method also contributed to orthogonal selectivity for the separation of fragments.


Jaag, S. Shirokikh, M. and Lämmerhofer, M., 2020. Charge variant analysis of protein-based biopharmaceuticals using two-dimensional liquid chromatography hyphenated to mass spectrometry. Journal of Chromatography A, 1636, p.461786.




Generation of F(ab’)2 and Fc/2 fragments.

FragIT kit
Generation and purification of F(ab’)2 and Fc/2 fragments.