Characterization On Critical Quality Attribute Of The Structure Heterogeneous Of Monoclonal Antibody Drug

Unlike small molecule drugs,antibody drugs not only have large molecular mass,complex structure,but also bear glycosylation or other post-translational modifications,resulting in highly heterogeneous structures(heterogeneity).New requirements and challenges are existed for the characterization and quality control of monoclonal antibody drugs.Firstly,the primary structure analysis was carried out using the mAb reference provided by the China Institute of Metrology,and the necessary analysis items were simultaneously confirmed using mAb from the National Institute of Standards and Technology(NIST8671).First,based on liquid chromatography-mass spectrometry,we established a complete process for molecular weight analysis from the intact molecular level.An analytical method for peptide peak assignment and post-translational modification was further established.Based on chromatographic technique and full-column imaging capillary isoelectric focusing technique,an analytical separation method for charge isomers was established.The results showed that this control antibody was subjected to C-terminal lysine knockdown compared to the conventional antibody.Its intact molecule carries five N-glycan types,of which GOF/GOF and G0F/G1F are the main glycoforms.We performed multi-condition trypsin digestion on the standard mAb by HPLC-MS/MS.The chromatographic peak for peptide mapping was successfully assigned.Further nanoLC-MS/MS of the peptides were performed to find modifications.Accurate assignment of disulfide binds,glycopeptides,and other post-translational modifications were achieved.The isoelectric point distribution was measured by "Whole Column Imaging Detection-Capillary Isoelectric Focusing"(WCID-cIEF),which ranged from 8.21 to 8.74.Then the SCX-HPLC was used to detect the charge heterogeneity.The basic peak content was 1.6%,the acid peak content was 27.1%,and the main peak content was 71.3%.This was in good consistent with the WCID-cIEF results.Secondly,we have performed an in-depth study on the influencing factors of charge isomers.Based on the established method for the separation and collection of charge isomers in the previous section.With a low sample consumption,the difference between the charge isomers of IgGl antibodies was explored from the intact protein level(molecular weight distribution)firstly.The difference was reflected in the distribution of glycoforms and the modification of deamidation.The study further went deeply into the peptide level,and detected five major modifications,and detected the deamidation modification(2DeamidationN/Q)affected the charge isomers,and confirmed the Deamidation modification in molecular weight analysis.Glycopeptide information was extracted from the peptide mapping,with the identificaiton of ten glycoforms.The glycoform distribution of each charge isomer was found to be non-uniform,further confirmed the molecular weight detection results.Finally,we analyzed the effect of glycopeptides on the charge isomers from the level of glycopeptides.We analyzed the glycopeptide information upon HILIC enrichment and identified 24 glycoforms(14 more than the peptide mapping method).We revealed that sialic acid modification is a critical influencing factor,which was only detected in the acid peak,and its content decreased with the decrease of acidity.However,sialic acid modification was not found in peptide map analysis and molecular weight analysis.This result illustrated the value of comprehensively analyzing the charge isomers of antibodies.This research provides a reference approach for the quality control of biopharmaceutical analysis.