Optimization and method validation for the quantitative analysis of a monoclonal antibody and its related fab...

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 0.300-100 ng/mL.