Development Of Quantifying Method Of Protein And Peptide Drugs Based On LC-MS Technology And Its Application To Preclinical Pharmacokinetics

Protein/peptide drugs have attracted more and more attention due to their strong specificity,low immunogenicity,low toxic and side effects,and significant efficacy.However,the quantification of protein/peptide drugs are very difficult because of the large molecular weight,complex structure,low concentration in vivo and severe endogenous interference.The increasing industry interest and investment and the rising demand from the medical community for biotherapeutics come increased demand for an analytical platform that should be simple,robust and high-throughput.At present,the main methods for quantification of protein/peptide drugs are immunoassay,isotope labeling,imaging in vivo,and LC-MS/MS,among which the LC-MS/MS method is the best and most promising method.Although the indirect LC-MS/MS method is applicable for quantification of peptide/protein drugs,its highthroughput applications are severely limited by complex sample processing and high development costs.Therefore,we attempt to develop direct LC-MS/MS methods for quantification of peptide protein drugs.In this study,four different protein/peptide drugs with molecular weights ranging from 1000 Da to 20000 Da were selected,including APi1633,liraglutide,recombinant human insulin and EHL protein.We successfully developed and fully validated four LC-MS/MS methods for APi1633,liraglutide,recombinant human insulin and EHL protein,respectively,and these methods were successfully applied to preclinical pharmacokinetic studies.Firstly,a simple,sensitive and selective LC-MS/MS method was developed for the quantitative analysis of APi1633 and validated in rat plasma.Good linearity was observed in the concentration range 2.5~2500 ng/mL(r2>0.99).The accuracy ranged from 94.8~101%.The precision was within acceptable limit.The bioavailability of the APi1633 was 97.0% after administration in rats.Then,a simple,sensitive and selective LC-MS/MS method was developed for the quantitative analysis of liraglutide and validated in rat plasma.Good linearity was observed in the concentration range 0.5–250 ng/mL(r2>0.99).The accuracy and precision were within acceptable limits.The recovery ranged from 80~90%.The bioavailability of the liraglutide was 11.4% after administration in rats.A simple,sensitive and selective LC-MS/MS method for quantitative analysis of human insulin was developed and validated in dog plasma.Good linearity was observed in the concentration range of 1–1000 μIU/mL(r2>0.99),and the lower limit of quantification was 38.46 pg/mL.Direct comparisons of the LC–MS/MS and RIA methods demonstrated that our LC–MS/MS method exhibited superior accuracy,efficiency and costeffective for the PK assessment of human insulin.Finally,we successfully developed and validated an LC-MS/MS method for the direct quantification of the intact EHL protein in monkey plasma,and the direct quantification of intact macromolecules(MW>10 kD)in bio-sample came true.Good linearity was observed in the concentration range of 5~500 ng/mL(r2>0.99).The accuracy ranged from 90~101%.The precision was within acceptable limit.The recovery was about 25%.In this study,we successfully developed four direct LC-MS/MS methods for the quantification of APi1633,liraglutide,recombinant human insulin and EHL proteins,respectively.Based on the above studies,we summarized the methodologies and research strategies for direct LC-MS/MS quantification of intact peptides and protein drugs(molecular weight less than 20,000 Da).The research results promote the application of LC-MS/MS method in the quantification of protein/peptide drugs and provide a good analysis platform for the biological drugs.At the same time,this research is also a verification and breakthrough of the existing theories,which can provide reference and method support for the pharmacokinetic study of similar drugs.