基于免抗体亲和富集技术的体内单抗药精准定量分析方法研究

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.

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。

体内单抗药物的精准定量分析是开展其PK/PD研究的重要手段，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。本项目以Trastuzumab为模式药物，开发亲和小肽配体修饰的新型高效免抗体富集整体柱，进而构建基于免抗体富集技术的体内单抗药快速高效的精准定量分析平台，实现乳腺癌病人血样中Trastuzumab的定量分析及其互补性决定区（CDR区）关键多肽修饰的定性分析。所取得的成果如下所示：.1. 通过计算机辅助设计和微量热泳动等技术，设计筛选出一系列能特异性识别Trastuzumab的高亲和小肽，并结合整体色谱柱制备技术，研制出DAAG、HH24等免抗体SPE富集整体柱。特别是HH24肽整体柱能有效辨别复杂体系中的hIgG和Trastuzumab，进而实现了加标血清中目标单抗的精准捕获，建立了适用于体内Trastuzumab高效富集纯化的新方法。.2. 开发了不同种类的酶反应器和二维分离分析平台，为实现乳腺癌病人血样中Trastuzumab的定性定量分析奠定了基础。.3. 将上述富集纯化新方法成功应用于乳腺癌患者体内Trastuzumab的高效富集纯化；并结合荧光分析技术实现患者体内目标单抗血药浓度监测。最后结合LC-MS/MS技术应用于患者体内Trastuzumab-CDR区关键多肽序列的修饰分析，充分验证了本研究所开发的技术在临床上的实际应用潜力。.综上所述，本研究成功开发了能特异性识别复杂体系中Trastuzumab的高亲和小肽富集整体柱，并结合不同分析方法实现了乳腺癌患者体内Trastuzumab的血药浓度监测和目标单抗药CDR区关键多肽体内修饰的定性定量分析，从而为抗体药物研发和临床应用提供了重要技术支撑。