Methodological Research And Application In Simultaneous Quantitative Analysis Of Trastuzumab And Pertuzumab In Cynomolgus Monkey Serum Based On LC-MS/MS

To establish a simple and robust method for simultaneous quantification of trastuzumab and pertuzumab antibodies in cynomolgus monkey serum based on LC-MS/MS.And in accordance with the "Guidelines for Validation of Quantitative Analysis Methods for Biological Samples" under the guidelines of the Chinese Pharmacopoeia(2015 edition,Part IV)and the laboratory’s standard operating procedures(SOPs)to validate the method,validation parameters include accuracy and precision,sensitivity,selectivity,matrix effect,stability,dilution linearity,carryover,maximum analysis batch,and autosampler reproducibility.The established method was applied to the pharmacokinetic studies of trastuzumab and pertuzumab in cynomolgus monkeys.Theoretical surrogate peptides were obtained by first determination the amino acid sequences of Trastuzumab and Pertuzumab through the drugbank database,then the silico trypsin digestion was executed by Expasy to predict the digested peptides and the corresponding molecular mass,and finally the online protein Basic Local Alignment Search Tool(BLAST)was used to predict the selectivity of each tryptic peptide in monkey serum by querying the protein databases.The final ion channels of surrogate peptides were obtained through the information dependent acquisition(IDA)data acquisition mode and after optimizing the source parameters by flow injection analysis(FIA)mode,the samples were analyzed by mass spectrometry.The quantification of trastuzumab and pertuzumab used infliximab as the internal standard,and the serum samples were denatured,reduced,alkylated and digested to obtain the surrogate peptides required for quantification.The analytical column was a Kinetex(?)C18 column(2.6 μm,2.1 x 50mm).The mobile phases were:Phase A:ultrapure water(containing 0.1%FA);Phase B:acetonitrile(containing 0.1%FA),using gradient elution and using a switching valve,the running time of a single sample was 6 minutes.ESI electrospray ion source and the multiple reaction monitoring mode(MRM)were used for detection.The ion pairs used for quantitative analysis were m/z 485.4→m/z 721.4(trastuzumab),m/z 419.2→m/z 589.0(pertuzumab),m/z 773.2→m/z 576.0(Internal standard,infliximab).The linear range of quantitative methods for trastuzumab and pertuzumab in cynomolgus monkey serum were both 2.0～400 μg/mL.The intra-day precision of trastuzumab was less than 13.5%,and the inter-day precision was less than 7.0%(except 15.1%for LLOQ),and the accuracy was between-15.0～10.3%.The intra-day precision of pertuzumab was less than 8.9%(except 18.4%for LLOQ),the inter-day precision was less than 14.6%,and the accuracy was between-13.0～14.0%.After intravenous injection the cocktail antibody of trastuzumab and pertuzumab in cynomolgus monkeys,the t1/2 of trastuzumab in serum was 58.70±53.16 hr,Tmax was 0.O33±0.00 hr,Cmax was 120.33±5.51 μg/mL,AUC(0-t)was 5629±806.01 hr*μg/mL;the t1/2 of pertuzumab in serum was 67.51 ±57.89 hr,Tmax was 0.033±0.00 hr,Cmax was 116.67±11.93 μg/mL,AUC(0-t)was 5677±890.76 hr*μg/mL。The present study successfully developed and validated a simple,fast and high-throughput LC-MS/MS method for the simultaneous quantitative of trastuzumab and pertuzumab concentrations in cynomolgus monkey serum,and the method was successfully applied to a pharmacokinetic study in cynomolgus monkeys.