High-Throughput Thermal Stability Analysis of a MonoclonalAntibody by Attenuated Total Reflection FT-IR Spectroscopic Imaging

摘要

The use of biotherapeutics, such as monoclonal antibodies, has markedly increased in recent years. It is thus essential that biotherapeutic production pipelines are as efficient as possible. For the production process, one of the major concerns is the propensity of a biotherapeutic antibody to aggregate. In addition to reducing bioactive material recovery, protein aggregation can have major effects on drug potency and cause highly undesirable immunological effects. It is thus essential to identify processing conditions which maximize recovery while avoiding aggregation. Heat resistance is a proxy for long-term aggregation propensity. Thermal stability assays are routinely performed using various spectroscopic and scattering detection methods. Here, we evaluated the potential of macro attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopic imaging as a novel method for the high-throughput thermal stability assay of a monoclonal antibody. This chemically specific visualization method has the distinct advantage of being able to discriminate between monomeric and aggregated protein.Attenuated total reflection is particularly suitable for selectivelyprobing the bottom of vessels, where precipitated aggregates accumulate.With focal plane array detection, we tested 12 different buffer conditionssimultaneously to assess the effect of pH and ionic strength on proteinthermal stability. Applying the Finke model to our imaging kineticsallowed us to determine the rate constants of nucleation and autocatalyticgrowth. This analysis demonstrated the greater stability of our immunoglobulinat higher pH and moderate ionic strength, revealing the key role ofelectrostatic interactions. The high-throughput approach presentedhere has significant potential for analyzing the stability of biotherapeuticsas well as any other biological molecules prone to aggregation.