Quantitative Bioanalysis By LC-MS/MS Methods And Pharmacokinetic Studies Of Two Monoclonal Antibodies

In recent years,monoclonal antibody drugs have developed rapidly and have become a hot spot in the development of new drugs all over the world.Monoclonal antibodies are mainly used in the treatment of tumors,autoimmune diseases and inflammatory diseases.The molecular weight,volume and polarity of monoclonal antibodies are much larger than those of small molecules,which makes there are great differences in pharmacokinetic characteristics between monoclonal antibody and small molecular drugs.Parenteral administration,slow tissue distribution and long elimination half-life are the most significant clinical pharmacokinetic characteristics of monoclonal antibodies.For a long time,the preferred method for bioanalysis of protein drugs is ligand binding assay?LBA?.However,due to the limitations of LBA,some researchers have proposed a supplementary/alternative analysis strategy of LC-MS/MS in recent years.In this study,LC-MS/MS methods were established to quantitatively detect two monoclonal antibodies?SHR-1603 and SHR-1222?in serum.The verified LC-MS/MS methods were applied to the preclinical study of these two drugs,and compared with the electrochemiluminescence method?MSD-ECL,based on LBA?of the two monoclonal antibodies.1.Determination of SHR-1603 in cynomolgus monkey and rat serum by electrochemiluminescence methodSHR-1603,a humanized monoclonal antibody developed by Jiangsu Hengrui Pharmaceutical Co.,Ltd.,its target is CD47,which is currently in clinical phase I and is intended to be used in tumor therapy.One MSD-ECL method for the detection of SHR-1603 in cynomolgus monkey and rat serum was established.The quantitative range of this method is 19.5 to 10000 ng/mL,and the serum sample is allowed to be diluted within 25600 times.It is proved that this method meets the requirements for the determination of biological samples and can be used to determine the concentration of SHR-1603 in cynomolgus monkey and rat serum.2.Determination of SHR-1603 in cynomolgus monkey and rat serum by two LC-MS/MS methodsTwo LC-MS/MS methods with different pretreatment methods,direct precipitation?straight LC-MS/MS?and affinity enrichment?IA-LC-MS/MS?,were established for the determination of SHR-1603 in cynomolgus monkey and rat serum.The basic process of LC-MS/MS quantitative protein drugs includes purification or enrichment of samples,enzymatic hydrolysis of target proteins,and finally detection of selected signature peptides?or surrogate peptides?to complete protein quantification.Straight LC-MS/MS means that all the proteins in the serum sample are precipitated directly,then the peptides are hydrolyzed by trypsin,and the mixture of peptides is detected by mass spectrometry after digestion.IA-LC-MS/MS refers to the affinity purification and enrichment of the sample before trypsin digestion,the SHR-1603 in the sample is captured by the antigen CD47,coated on the magnetic beads,and then the protein is digested,and the peptides mixture after digestion is detected by mass spectrometry.The development of LC-MS/MS method focuses on the selection of suitable surrogate peptides for SHR-1603,and the sensitivity,specificity and stability of peptides need to be investigated.This part of the work cooperated with Xiaotao Duan,a professor from the Academy of Military Medical Sciences,and the instrument system used is nano LC-LTQ/Orbitrap.The protein library and peptide attribution were searched by Mascot software,and the sequence information,charged state and daughter ions of all peptides were obtained.Through the pBLAST tool,we searched the Rattus,Macaca fascicularis and Homo sapiens Swiss Prot protein databases and screened out the unique peptides.The liquid conditions of the candidate peptides were optimized on Agilent 6495QQQ.According to the results of sensitivity,specificity and stability,the peptide LLIYSGSTLQSGVPSR on the light chain of SHR-1603 was selected for quantitative analysis.The digest conditions were optimized.For the consideration of throughput and simplicity of operation,the digest condition of DTT+IAA+60?incubation for 1.5 h was finally determined.In order to solve the surface adsorption of hydrophobic peptide LLIYSGSTLQSGVPSR,a variety of anti-adsorption methods were investigated.Finally,BSA digest was selected as the anti-adsorption reagent to effectively solve the problem of surface adsorption.Properly increasing the proportion of organic phase in the solvent?20%?is beneficial to improve the solubility of the peptide.Both LC-MS/MS methods have been verified.In cynomolgus monkey and rat serum,the linearity of straight LC-MS/MS was good in the concentration range of 250?500000 ng/mL,and the linearity of IA-LC-MS/MS was good in the concentration range of 100?100000 ng/mL.The precision and accuracy of the two LC-MS/MS methods meet the requirements.The two LC-MS/MS methods and MSD-ECL method were applied to the pharmacokinetic study of SHR-1603 injection in rats and cynomolgus monkeys,respectively,and the measured values were compared and cross-verified.The results showed that in the low-dose group,the results of the three methods were consistent,and there was no significant difference?P>0.05?.The ratio of the drug concentration obtained by the two LC-MS/MS methods to the corresponding concentration obtained by MSD-ECL method was between 1.05 to 1.11.In the middle-and high-dose group,the drug concentration measured by LC-MS/MS method was higher than that measured by MSD-ECL method,and there was significant difference among the three methods?P<0.05?.The drug exposure measured by Straight LC-MS/MS method was the highest in rat serum,while that measured by IA-LC-MS/MS was the highest in cynomolgus monkeys.The results of cross-validation also showed that the value measured by LC-MS/MS method was generally higher than that by MSD-ECL method,and there were differences between the two LC-MS/MS methods in different species.This may be due to the fact that free antibodies were detected by MSD-ECL method,while total antibodies?free and non-free?were detected by LC-MS/MS method.As for the species differences between rats and cynomolgus monkeys,it may be due to the huge differences in animal immune systems.Comparing the two analytical techniques,the dynamic quantitative range of LC-MS/MS method is better than that of MSD-ECL method.The quantitative range of the MSD-ECL method was 19.3?10000 ng/mL?500-fold?,100?100000 ng/mL of IA-LC-MS/MS?2000-fold?and 250?500000 ng/mL of straight LC-MS/MS?1000-fold?.The serum concentration of SHR-1603 in rats and cynomolgus monkeys can reach 1?2mg/mL,so the quantitative range of LC-MS/MS method is more advantageous.The sensitivity of LC-MS/MS method is lower than that of MSD-ECL method.This is mainly due to that the high abundance protein in the serum matrix improves the noise background and the peptide has multiple charges so that the MS signal is dispersed and the sensitivity decreases.Due to the purification and enrichment of serum samples,the sensitivity of IA-LC-MS/MS method is better than that of straight LC-MS/MS.Although the sensitivity of LC-MS/MS is lower than that of MSD-ECL,it can fully meet the detection requirements of pharmacokinetics study.LC-MS/MS method is less dependent on reagents and does not need specific antibodies,so it can provide a more rapid method for development.The standard curve was established by using monoclonal antibody SHR-1603?instead of surrogate peptides?and the isotope labeled peptide was used as internal standard so the detection accuracy of LC-MS/MS method was higher.The pretreatment of the LC-MS/MS method is simpler than that of the MSD-ECL method,and the straight LC-MS/MS method is the simplest,so the analysis throughput is higher,which can meet the detection needs of a large number of preclinical and clinical samples.Compared with MSD-ECL method,LC-MS/MS method is more cost-effective and efficient.3.Determination of SHR-1222 in cynomolgus monkey serum by LC-MS/MSSHR-1222,developed by Jiangsu Hengrui Pharmaceutical Co.,Ltd.,is a humanized monoclonal antibody targeting sclerostin.It is currently in clinical phase I and is intended to be used in the treatment of osteoporosis.One LC-MS/MS method was established for the determination of SHR-1222 in cynomolgus monkey serum to investigate the toxicokinetics of SHR-1222 injection.Compared with the MSD-ECL method established in previous studies,the reason why high concentration of anti-drug antibody?ADA?changed the concentration-time curve of SHR-1222 was explored.The development of LC-MS/MS method focuses on the selection of suitable surrogate peptides for SHR-1222,and the sensitivity,specificity and stability of peptides are investigated.This part of the work cooperates with Minjia Tan,a professor from Shanghai Institute of Meteria Medica,Chinese Academy of Sciences,and the instrument system used is nano LC-Orbitrap Fusion.The protein library was searched by Mascot software,and the homologous peptides were excluded by pBLAST tool.The liquid conditions of the candidate peptides were optimized on Agilent 6495QQQ.The Skyline software was introduced to optimize the mass spectrometry parameters,combined with the Agilent MassHunter data acquisition software to improve the efficiency.Finally,the peptide LLIYYTSNR on the light chain of SHR-1222 was selected for quantitative analysis.It was proved that the LC-MS/MS method in cynomolgus monkey serum was linear in the range of 2.00?500?g/mL,and the precision and accuracy of quality control samples met the requirements.The verified LC-MS/MS method was applied to the toxicokinetic study of SHR-1222?medium-dose group of 60 mg/kg?in cynomolgus monkeys.Compared with the MSD-ECL method established in the previous study,the concentration of SHR-1222measured by LC-MS/MS method was basically consistent with that measured by MSD-ECL method,and the ratio of C_max and AUC was between 0.89?0.95 and 0.80?1.00,respectively.The quantitative range of LC-MS/MS method is better than that of MSD-ECL method,and it can provide rapid method development and is more cost-effective and efficient.The standard curve was established by using monoclonal antibody SHR-1222?instead of surrogate peptides?,and the isotope labeled peptide was used as internal standard,which improved the detection accuracy.The pretreatment is simpler and the analytical throughput is higher than that of MSD-ECL method.The results of MSD-ECL method showed that in the toxicokinetic study of 60mg/kg SHR-1222,the detection values of individual serum samples of 2 cynomolgus monkeys were extremely low.It was found that in the three abnormal serum samples,the concentration of anti-SHR-1222 ADA was very high,and the titer was?16.In other serum samples,the titer of ADA was around 2.It is suggested that high concentration of ADA can change the concentration-time curve of antibodies.However,the accuracy of LBA detection is easily interfered by ADA.When a decrease in drug exposure was observed by LBA method,it may be difficult to distinguish whether ADA interferes with detection of SHR-1222 or ADA-mediated drug clearance in vivo.The LC-MS/MS method established in this study can overcome the interference of ADA and determine the total concentration of drugs in serum samples.The results of LC-MS/MS detection showed that the detection value of SHR-1222 in above three abnormal serum samples was also significantly lower than that of other animals in the same group.Therefore,it can be determined that high levels of ADA can accelerate the clearance of SHR-1222in cynomolgus monkeys,showing a decrease in drug exposure,rather than ADA interferes the detection.Sclerostin targeted by SHR-1222 is a soluble target.Antagonistic monoclonal antibodies targeting soluble targets generally do not produce long-term accumulation of biological activity due to the influence of ADA?and free targets?in serum.The quantitative detection results of SHR-1222 by LC-MS/MS and MSD-ECL were consistent,which is accorded with the characteristics of this type of drugs.4.ConclusionIn this study,we established two LC-MS/MS methods for quantitative detection of monoclonal antibody SHR-1603 in cynomolgus monkey and rat serum,and one LC-MS/MS method for quantitative detection of monoclonal antibody SHR-1222 in cynomolgus monkey serum,and compared them with the MSD-ECL methods of the two monoclonal antibodies respectively.Because the concentration of total antibody was detected by LC-MS/MS methods and free monoclonal antibody was detected by MSD-ECL methods,the detection value of LC-MS/MS was generally higher than that of MSD-ECL methods.LC-MS/MS method has advantages in rapid method development,quantitative range and accuracy,showing higher cost-effectiveness and efficiency.LC-MS/MS method provides a promising complementary or alternative analysis strategy for preclinical and clinical evaluation of monoclonal antibody drugs,and is a powerful tool to promote the research and development of monoclonal antibody drugs.