The Development Of LC-MS-based Proteomic Quantitative Method And Its Application In The Preclinical Pharmacokinetic Study Of Monoclonal Antibody

The The establishment of hybridoma platform provides large-amount ofmonoclonal antibody with highly specificity and homogeneity against specificantigens, and the technique innovation and improvement in antibody engineering helpthe investigation and research in chimeric antibody and full-human antibody, whichmake the therapeutic mAb one of hot spots in medical R&D. Compared with theconventional small molecular drugs, the mAbs can recognize and bind the targetsspecifically and effectively, with high efficacy, fewer side effects and longer half-life,which makes therapeutic mAbs widely applied in the diagnosis and treatment ofcancer and autoimmune diseases, relief and suppression of chronic inflammation andrejection reaction after organ transplant, and virus infection. In the R&D of noveldrugs, reliable, accurate and sensitive quantitative method is required. The currentgold standard, ELISA, features high sensitivity, simple operation procedure and highthroughput, but its application is limited by poor reproducibility, narrow quantitativelinear range, and potential endogenous interference. So the LC-MS based method hasbeen introduced into quantitative proteomics, such as absolute quantification of mAbs,as a promising alternative.Mass spectrometry, which can determine peptide sequence and proteinidentification, has become essential in proteomic research. The improvements ofsample preparation, and novel bio-informatical software, speeding up data-miningsignificantly, both promote the utilization of MS in quantitative proteomics. Howeverlimited by low efficacy of protein separation methods available, and detection rangeof mass spectrometer, it’s still a challenge to separate single target protein fromcomplicated biological matrix efficiently, and absolutely determine the proteinconcentration directly. So the shotgun strategy, a bottom-up method in which all theproteins in biological samples are be digested firstly, and the concentration of targetprotein is determined by quantifying the signature peptides, has been introduced andadvances quickly in quantitative proteomics. In the development history of LC-MSbased method, signature peptides, SP analogs, which are prolonged signature peptidesby several amino acids both upstream and downstream, and target proteins, areutilized in different trials. So are the corresponding internal standards. Obviously the quantitative results vary with different combination of calibrator and internalstandards, and no systematic comparison available now; meanwhile, for the key stepof shotgun strategy in quantitative proteomics, the selection and verification of SPs,there are no widely-accepted standards and procedures.In the thesis, against the deficiency of the shotgun strategy, a platform composedof a NanoLC system and hybridized LTQ/Orbitrap mass spectrometry was establishedfor peptide identification and SP selection. The digest of target protein is introducedinto hybridized LTQ/Orbitrap mass spectrometry after separation by NanoLC column,to get the chromatogram of LTQ analysis and corresponding spectra of Orbitrap MSanalysis. All the detected peptides can be listed by database searching, among whichcertain peptides can be assigned as SP candidates in specific biological matrix; apeptide quantitative platform made of a low-speed HPLC system and TSQ MS wasbuilded up for optimization of SRM parameters by performing online orthogonaltrials, and evaluation sensitivity and stability of SP candidates, to select the optimalSP and SRM for quantification. These two platforms were successfully used to chooseoptimal SP of anti-HCV mAb, and corresponding SRM parameters, andsystematically study the quantitative results of anti-HCV mAb in rat plasma undervarious combinations of calibrator and internal standards; the LC-MS basedquantitative method was established and validated for chimeric cT84.66andsuccessfully applied in pre-clinical PK study of cT84.66in mice.