As biologic based drugs become an increasingly important sector of the pharmaceutical industry, accurate and precision techniques for bioanalysis are required to support clinical trials and beyond. Ranibizumab, a fab therapeutic, is an FDA approved drug to treat wet age-related macular degeneration (AMD), as well as other eye related diseases. Ranibizumab’s mAb counterpart, bevacizumab, is often also used off-label to treat wet AMD. Ranibizumab and bevacizumab target circulating VEGF-A in the eye, reducing unwanted angiogenesis. Since these drugs are designed for local intravitreal administration, concentration levels in human plasma are expected to be significantly lower compared to vitreous fluid concentrations, presenting bioanalytical challenges. However, this is important for assessment of drug toxicity. In this manuscript, we describe the development, optimization, and validation of an LC-MS/MS method designed for quantitative bioanalysis of ranibizumab and bevacizumab in human plasma following intravitreal administration. In order to fully develop this method, evaluations were conducted to optimize the conditions, including selection of the surrogate peptide by in-silico experiments, optimizations of the immunocapture, denaturation, reduction, alkylation, and digestion extraction steps, as well as optimization of the LC-MS/MS conditions, and evaluation of a dissociation step to determine if there was interference from VEGF or ADAs. Once the method was fully optimized, it was then validated, following the 2018 FDA guidance on bioanalytical method validations. This method is now available for use during clinical trials and precision medicine, for the quantitative evaluation of systemic exposure of ranibizumab or bevacizumab in human plasma after intravitreal administration, with a linear calibration range of 0.300 to 100 ng/mL.

随着基于生物的药物成为制药行业中越来越重要的部门，需要精确而精密的生物分析技术来支持临床试验及其他。Ranibizumab是一种fab治疗药物，是FDA批准的药物，用于治疗湿性年龄相关性黄斑变性（AMD）以及其他与眼有关的疾病。雷尼单抗的mAb对应物贝伐单抗也经常用于治疗湿性AMD的标签外。雷尼单抗和贝伐单抗靶向眼睛中循环的VEGF-A，减少了不需要的血管生成。由于这些药物是为局部玻璃体内给药而设计的，因此与玻璃液中的浓度相比，人血浆中的浓度有望大大降低，这给生物分析带来了挑战。但是，这对于评估药物毒性很重要。在这份手稿中 我们描述了设计用于玻璃体内给药后人血浆中兰尼单抗和贝伐单抗定量生物分析的LC-MS / MS方法的开发，优化和验证。为了充分开发这种方法，进行了评估以优化条件，包括通过计算机模拟实验选择替代肽，优化免疫捕获，变性，还原，烷基化和消化提取步骤，以及优化条件。 LC-MS / MS条件，并进行解离步骤评估，以确定是否存在来自VEGF或ADA的干扰。方法完全优化后，将按照FDA 2018年生物分析方法验证指南进行验证。现在，该方法可用于临床试验和精密医学，