Combination of SDS‐PAGE and intact mass analysis for rapid determination of heterogeneities in monoclonal antibody therapeutics

Abstract

mAbs are currently mainstream in biopharmaceuticals, and their market has been growing due to their high target specificity. Characterization of heterogeneities in mAbs is performed to secure their quality and safety by physicochemical analyses. However, they require time-consuming task, which often strain the resources of drug development in pharmaceuticals. Rapid and direct method to determine the heterogeneities should be a powerful tool for pharmaceutical analysis. Considering the advantages of electrophoresis and MS, this study addresses the combination of SDS-PAGE and intact mass analysis, which provides direct, rapid, and orthogonal determination of heterogeneities in mAb therapeutics. mAb therapeutics that migrated in SDS-PAGE were recovered from gel by treatment with SDC-containing buffer. Usage of SDC-containing buffer as extraction solvent and ethanol-based staining solution enhanced the recovery of intact IgG from SDS-PAGE gels. Recovery of mAbs reached more than 86% with 0.2% SD. The heterogeneities, especially N-glycan variants in the recovered mAb therapeutics, were clearly determined by intact mass analysis. We believe that the study is important in pharmaceuticals‧ perspective since orthogonal combination of gel electrophoresis and intact mass analysis should be pivotal role for rapid and precise characterization of mAbs.

Mass spectrometry for quality control of bispecific antibodies after SDS‐PAGE in‐gel digestion

“…Maybe the fast translation of proteins leads to reduced accuracy in protein folding or in some cases even to its interruption. patterns can easily be identified (Ladwig et al, 2017;Wang et al, 2018;Yamada et al, 2017). Our results underline the importance of integrating several analytical methods for quality control during GMP production.…”

“…A corresponding analysis of (b), (c), and (d) is presented in the tables below. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns can easily be identified (Ladwig et al, 2017;Wang et al, 2018;Yamada et al, 2017). Our results underline the importance of integrating several analytical methods for quality control during GMP production.…”

“…Thus, we were able to show how limitations of SDS‐PAGE analysis can be overcome with additional MS analysis. In general, MS is a highly sensitive tool, which can help to validate the GMP production process of bsAb, as impurities or heterogeneities in glycosylation patterns can easily be identified (Ladwig et al, 2017; Wang et al, 2018; Yamada et al, 2017). Our results underline the importance of integrating several analytical methods for quality control during GMP production.…”

A comparative study of CE‐SDS, SDS‐PAGE, and Simple Western: Influences of sample preparation on molecular weight determination of proteins

“…The investigation of a therapeutic IgG antibody, namely Matuzumab (lc-M 23.63 kDa, hc-M 49.66 kDa, see Patent WO 2009/04 3490 A1 [37]), was included for the comparability to previous studies [10,12,[46][47][48][49][50][51][52][53]. Typically, SDS-PAGE and CE-SDS were compared in terms of purity and size heterogeneity assays, to examine the variety in analyzing antibodies.…”

Insights from capillary electrophoresis approaches for characterization of monoclonal antibodies and antibody drug conjugates in the period 2016–2018

“…Concerning the analysis of mAb glycosylations, chemical modification based on 8-Aminopyrene-1,3,6-trisulfonic-acid (APTS) reaction allows the derivation of fluorophoric group and the addition of three negative charges to the glycan moiety, which is considered today as one of the reference method [23,[25][26][27][28]. However recently, other dye reagents like 2-aminobenzoic acid (2-AA) (excitation: 325 nm emission: 405 nm) [29] and Teal™ (excitation and emission: same as APTS) [28], providing respectively one and three negative charges, have been reported for glycan analysis. These methods revealed excellent precision and accuracy.…”