Structure-Based Correlation of Light-Induced Histidine Reactivity in A Model Protein.

Light is known to induce covalently linked aggregates in proteins. These aggregates can be immunogenic and are of concern for drug product development in the biotechnology industry. Histidine (His) is proposed to be a key residue in cross-link generation ( Pattison , D. I. Photochem. Photobiol. Sci. 2012 , 11 , 38 - 53 ). However, the factors that influence the reactivity of His in proteins, especially the intrinsic factors are little known. Here, we used rhDNase, which only forms His-His covalent dimers after light treatment to determine the factors that influence the light-induced reactivity of His. This system allowed us to fully characterize the light-induced covalent dimer and rank the reactivities of the His residues in this protein. The reactivities of these His residues were correlated with solvent accessibility-related parameters both by crystal structure-based calculations of solvent-accessible surface area and by hydrogen-deuterium exchange (HDX) experiments. Through this correlation, we demonstrate that the photoreactivity of His is determined by both solvent accessibility and structural flexibility. This new insight can explain the highly complex chemistry of light-induced aggregation and help predict the aggregation propensity of protein under light treatment.

A Rapid High-Sensitivity Reversed-Phase Ultra High Performance Liquid Chromatography Mass Spectrometry Method for Assessing Polysorbate 20 Degradation in Protein Therapeutics.

Polysorbate 20 (PS20), a widely used surfactant in protein therapeutics, has been reported to undergo hydrolytic degradation during product storage, causing the release of free fatty acids. The accumulation of free fatty acids in protein therapeutics was found to result in the formation of particles due to their limited aqueous solubility at 2°C-8°C. Quantitation of free fatty acids originating from PS20 degradation is thus important during bioprocess optimization and stability testing in formulation development to ensure optimum PS20 stability as well as product and process consistency in final drug products. This work reports the development of a simple and robust, high-throughput, reversed-phase ultra high performance liquid chromatography mass spectrometry method for high-sensitivity quantitation of lauric acid and myristic acid by using isotope-labeled fatty acid internal standards. The high sensitivity (<100 ng/mL for lauric acid) and suitable precision (intermediate precision relative standard deviation of 11%) of this method enable accurate detection of lauric acid produced from the degradation of less than 1% of PS20 in a 0.2-mg/mL formulation. Using accelerated thermal stability testing, this method identifies processes that exhibit fast PS20 degradation within only days and consequently allows faster iterative optimization of the process.