ABSTRACT
Posttranslational modifications of antibody products affect their stability,charge distribution,and drug activity and are thus a critical quality attribute.The comprehensive mapping of antibody modifications and different charge isomers(CIs)is of utmost importance,but is challenging.We intended to quanti-tatively characterize the posttranslational modification status of CIs of antibody drugs and explore the impact of posttranslational modifications on charge heterogeneity.The CIs of antibodies were fraction-ated by strong cation exchange chromatography and verified by capillary isoelectric focusing-whole column imaging detection,followed by stepwise structural characterization at three levels.First,the differences between CIs were explored at the intact protein level using a top-down mass spectrometry approach;this showed differences in glycoforms and deamidation status.Second,at the peptide level,common modifications of oxidation,deamidation,and glycosylation were identified.Peptide mapping showed nonuniform deamidation and glycoform distribution among CIs.In total,10 N-glycoforms were detected by peptide mapping.Finally,an in-depth analysis of glycan variants of CIs was performed through the detection of enriched glycopeptides.Qualitative and quantitative analyses demonstrated the dynamics of 24 N-glycoforms.The results revealed that sialic acid modification is a critical factor ac-counting for charge heterogeneity,which is otherwise missed in peptide mapping and intact molecular weight analyses.This study demonstrated the importance of the comprehensive analyses of antibody CIs and provides a reference method for the quality control of biopharmaceutical analysis.
ABSTRACT
The Rad1 gene is evolutionarily conserved from yeast to human. The fission yeast Schizosaccharomyces pombe Rad1 ortholog promotes cell survival against DNA damage and is required for G(2)/M checkpoint activation. In this study, mouse embryonic stem (ES) cells with a targeted deletion of Mrad1, the mouse ortholog of this gene, were created to evaluate its function in mammalian cells. Mrad1 (-/-) ES cells were highly sensitive to ultraviolet-light (UV light), hydroxyurea (HU) and gamma rays, and were defective in G(2)/M as well as S/M checkpoints. These data indicate that Mrad1 is required for repairing DNA lesions induced by UV-light, HU and gamma rays, and for mediating G(2)/M and S/M checkpoint controls. We further demonstrated that Mrad1 plays an important role in homologous recombination repair (HRR) in ES cells, but a minor HRR role in differentiated mouse cells.