体内单抗药物的精准定量分析是开展其PK/PD研究和保证安全有效的重要手段，但目前相关方法十分匮乏且面临严峻的技术挑战。如何高效富集生物基质中的单抗药物进而开发精密度和准确度高的精准定量分析方法是当前单抗药物研发中亟待解决的难题。前期研究发现基于亲和小肽配体的免抗体亲和整体柱可高效富集血浆中的IgG, 为解决该问题提供了一种新思路。本项目拟以Trastuzumab为模式药物，开发亲和小肽配体修饰的新型高效免抗体亲和富集整体柱，构建基于免抗体富集技术的离线、部分在线和全在线LC-MS/MS定量分析仪器系统，通过比对研究，最终建立体内单抗药快速高效的精准定量分析平台。该平台技术在克服传统免疫亲和富集存在的稳定性差、使用寿命短及价格昂贵等不足的同时，有望显著缩短分析时间、提高检测灵敏度，可推广至其他单抗药物，为其PK/PD研究提供强而有力的工具，对推动单抗Biosimilar行业的发展意义重大。

For successful drug development and quality control of therapeutic monoclonal antibodies (mAbs), reliable bioanalytical methods enabling the quantification of mAbs in biological fluids are highly required to generate toxicokinetic, pharmacokinetic and bioavailability data. However such reliable analytical techniques are very limited. Enzyme-linked immunosorbent assay, the current gold standards for mAbs quantification, still suffers from narrow linear dynamic range, crossreactivity of endogenous proteins, low accuracy and precision etc. LC-MS/MS considered as the most promising alternative technology still have to overcome some major challenges such as the low analyte concentrations and the interference of large amount of endogenous proteins. Previous studies found that synthetic peptide-based antibody-free affinity monolith can effectively enrich human IgG from plasma. In this study, a novel antibody-free affinity monolithic column will be carefully developed for the enrichment of Trastuzumab in biological matrices by using highly specific synthetic peptide as affinity ligand; three bioanalytical platforms based on the selectivity of antibody-free affinity monolith for sample purification, i.e. off-line, partial on-line and total on-line, will then be set up by combining with immobilized enzymatic reactor for proteolytic digestion and nano-LC–MS/MS for quantitative analysis. After a systematically comparative evaluation, an accurate, sensitive and rapid bioanalytical method will finally be developed for the quantitative determination of Trastuzumab in biological fluids. Antibody-free affinity enrichment based on small synthetic peptide ligands has many advantages over conventional immunoaffinity enrichment, such as low cost, less ligand leakage, better ligand stability and longer life time etc. The bioanalytical method based on the antibody-free affinity enrichment will be beneficial to significantly shorten the analysis time and improve the sensitivity. This research not only provides a powerful tool for the PK/PD studies of mAbs, but also shows great meaning in clarifying its mechanism of drug action and promoting the development of its biosimilar industry.