Study On New Methods Of Structural Characterization And Impurity Analysis Of Three Mab Drugs Including Trastuzumab Base On Mass Spectrometry

Monoclonal antibody(mAb)drugs,as a class of macromolecular glycoprotein drugs,have significant efficacy in the treatment of cancer,autoimmune diseases,virus infection and other diseases,and have a wide prospect of clinical application.They are one of the hot fields in the research of biotherapeutics.The bioprocessing of mAb drugs is complex,involving structural-functional studies,cell line development,upstream and downstream purification,etc.Small changes in the structure of mAbs as well as the types and contents of impurities in drugs may significantly affect the safety and effectiveness of the products.Therefore,the quality of mAbs needs to be analyzed and controlled in every process of bioprocessing.It is very important to establish an efficient,sensitive and accurate method for structural characterization and impurity analysis of mAbs for development,manufacture and audition of mAbs.In this study,based on mass spectrometry technology,an integrated analytical strategy was established to comprehensively characterize the structure of mAbs,including intact mass analysis,middle-down,bottom-up,and released N-glycan analysis.At the intact protein level,a new intact mass analysis method was established.After optimizing the mobile phase,the column of liquid phase separation and MS parameter settings,the established native SEC-MS and denatured RPLC-MS methods were able to achieve accurate molecular weight determination and N-glycosylation analysis of mAbs within 5 min.The established native and denatured intact mass analysis methods were applied to analyze trastuzumab,adalimumab and bevacizumab:(1)a total of 14 glycoforms of trastuzumab were identified,with the molecular weight(MW)ranging from 147600 to 149126 Da.Among them,G0F/G1F glycoform(MW 148219 Da)had the highest content,while other glycoforms with relatively low content included G0F/G0F,G1F/G1F,etc.(2)a total of 10 glycoforms of adalimumab were identified,with the MW ranging from 147674 to 148858 Da.Among them,G0F/G0F glycoform(MW 148081 Da)content was the highest,and other glycoforms with relatively low content included GOF/GOF+Lys,G0F/G1F,G1F/G1F,etc.(3)a total of 10 glycoforms of bevacizumab were identified,with the MW ranging from 147752 to 149846 Da.Among them,G0F/G0F glycoform(MW 149198 Da)had the highest content,while other glycoforms with relatively low content included G0F/G1F,G1F/G1F,etc.Compared with the reported results,the native and denatured methods we established were able to identify more low abundance(<1%)glycoforms,and identify C-terminal lysine addition as well as other modifications.Native MS enables the protein to maintain its native structure and identify more glycoforms containing sialic acid,thus obtaining more accurate results of intact mass analysis of the mAbs.At the subunit level,a new middle-down analysis method was established after optimizing the LC gradient conditions and the CID and ETD fragmentation parameters.The established analysis method was able to achieve subunit baseline separation within 35 min for MW determination,amino acid sequence verification and N-glycosylation analysis.Trastuzumab,adalimumab and bevacizumab were analyzed at the subunit level.The average MWs of Lc and Fd subunits of trastuzumab were 23442.366 Da and 25382.794 Da,respectively.The average MW of Fc/2 subunit modified by GOF was 25236.626 Da.The average MWs of the adalimumab Lc and Fd subunits were 23411.667 Da and 25457.690 Da,respectively,and the average MW of the Fc/2 subunit modified by GOF was 25204.619 Da.The average MWs of the Lc and Fd subunits of bevacizumab were 23436.553 Da and 25929.795 Da,respectively.The average MW of the Fc/2 subunit modified by GOF was 25217.655 Da.The glycosites of the three mAbs was located at the Asn-61 of the Fc/2 subunit.CID and ETD data were integrated to obtain the complementary fragment information,yielding an amino acid sequence coverage comparable to that in the literature(up to 50%).At the peptide level,a new bottom-up analysis method was established by optimizing the sample preparation.The established analysis method was used to prepare peptides for amino acid sequence verification and N-glycosylation analysis within 3 h.Glycopeptides enriched of three mAbs were analyzed,and 20 glycoforms in trastuzumab,17 glycoforms in adalimumab and 15 glycoforms in bevacizumab were identified.After analyzing the intact peptides of three mAbs,the amino acid sequence coverage were 95.33%,86.62%,and 96.55%,respectively.The analysis of glycopeptides and intact peptides can accurately locate the glycosites of mAbs.Finally,the glycosites of the three mAb drugs are located on the asparagine residues at the Asn-300,Asn-301 and Asn-303 of the heavy chain,respectively.A method for the released N-glycan analysis was established by optimizing the sample preparation.The established method was able to rapidly label the N-glycan within 30 min,conduct N-glycosylation analysis of mAbs and investigate the difference between batches.Applying the established method to the N-glycosylation analysis of three mAbs,20 glycoforms of trastuzumab,18 glycoforms of adalimumab and 11 glycoforms of bevacizumab were identified,and the main glycoforms included G0F-GN,G0,GOF,etc.Compared with the literature,the established method was able to identify more low-abundance glycoforms(<0.5%,such as sialylated glycoforms)in the mAbs.The analysis results at the above four levels can be complementary and cross-verified.We established an integrated analysis strategy for the structural characterization of mAbs with fast speed,good repeatability and high sensitivity.Host cell proteins(HCPs),a common impurity in mAbs,are expressed by host cells that produce mAbs.The concentration levels of HCP impurities in mAbs are very different(even more than five orders of magnitude),and conventional mass spectrometers cannot effectively identify and quantitate HCP impurities with low abundance in mAb products.Therefore,HCP impurity analysis is one of the main challenges faced by the quality analysis of mAb drugs.In this study,based on the nanoLC-MS/MS technique,a highly sensitive method for HCP impurities with low abundance has been established through the optimization of MS parameters,LC gradient conditions and sample preparation methods.194 and 63 HCP impurities were detected in NISTmAb RM 8671 and adalimumab using the established nanoLC-MS/MS method.