Mimotope Peptide Modified Biomimetic Polymer For Purification And Quantification Of Trastuzumab

Therapeutic monoclonal antibodies(m Abs)play an important role in the treatment of tumor and immune system diseases.However,the clinic response of therapeutic antibody varies greatly among different patients,which affects the development of disease and treatment.Monitoring the concentration of therapeutic antibody in patients' serum not only provides data for pharmacokinetic studies,but also benefits individualized treatment.Currently,ELISA is the most commonly used method for measuring the concentration of m Abs.Taking the advantages of antigen-antibody recognition,ELISA has high specificity and sensitivity.However,it also has some disadvantages to be overcame,such as high cost of antigen,cross-reaction and system deviation.As an important supplementary technology to ELISA,LC-MS/MS has also been developed for the quantitative analysis of monoclonal antibody.While it often requires complex sample preparation,expensive equipment and time-consuming enzymatic hydrolysis.Mimotope peptides can specifically recognize antibody by simulating the homologous amino acid sequence or functional conformation of the antigen epitope.It is one of the best candidates for antigen because of its high specificity,small molecular weight,low cost,and better chemical stability.Polymeric materials are good solid support for ligand attachment thanks to their easy preparation and modification,good permeability and porous structure large surface area.The objective of this thesis is to combine highly specific mimotope peptide with polymer material to prepare a novel biomimetic material for the separation and analysis of therapeutic monoclonal antibodies in serum.The specific biomaterial based sample pretreatment was further coupled with a common protein quantification method to set up a simple,low-cost and specific bioanalytical platform.The proposed platform was also applied to serum samples of breast cancer patients or CHO cell culture fluids,to explore its clinical application and industrial production purification potential.The main content of the paper is as follows:In the first chapter,the background of HER2-positive breast cancer monoclonal antibody,including research progress,pharmacodynamic mechanism and clinical applications of trastuzumab,and quantitative detection methods for therapeutic monoclonal antibodies in serum were comprehensively introduced.The difficulties in separating and quantifying monoclonal antibody in serum were analyzed,and the advantages and disadvantages of the current quantification methods were summarized.Based on these,the research ideas,experimental design,and innovations of this thesis were proposed.In the second chapter,based on analyzing the binding sites between HER2 antigen and trastuzumab,HH24 mimotope peptide was designed.The affinity between the proposed peptide and trastuzumab was determined using microscale thermophoresis technique.Molecular dynamics simulation technology was used to analyze the binding sites between the HH24 peptide and trastuzumab.The results show that the novel mimotope peptide ligand HH24 could form multiple interactions(in particular electrostatic force)with the CDR in the Fab region,which can lead to moderate binding of trastuzumab.In the third chapter,a HH24 peptide functionalized biomimetic material was prepared,and a series of characterizations were performed to verify the successful modification of the HH24 peptide.The adsorption capacity of the material for trastuzumab was measured through adsorption isotherms and breakthrough curves.FITC-labeled BSA was used to investigate the anti-nonspecific adsorption capacity of the biomimetic material.MADIL-TOF MS and LC-MS/MS were used to analyze the elution of spiked serum.The results show that the HH24 peptide functionalized biomimetic material has a high adsorption capacity for trastuzumab,high specificity and excellent anti-non-specific adsorption capacity.Trastuzumab in the spiked serum was efficiently captured with recovery and purity of 90% and 98%,respectively.In the fourth chapter,the new bioanalytical platform based on HER2 mimotope peptide functionalized polymer was set up and applied for the quantitative analysis of trastuzumab in complex biological fluids,such as serum samples of breast cancer patients and trastuzumab biosimilar in a cell culture medium.This proposed platform integrated the high specificity of the HH24 functionalized polymer and high sensitivity of fluorescence spectrophotometry.The results show that the obtained quantitative results were similar to those obtained with a commercial ELISA kit,indicating that the novel bioanalytical platform has great potential in clinical monitoring of m Abs.In the fifth chapter,the main achievements and innovations of this research were summarized,and the shortcomings were also addressed.Finally,the prospect of the HH24 peptide functionalized biomimetic material and the proposed preparation strategy were discussed.