Two-Trace Two-Dimensional Correlation Analysis for the Confirmation of Therapeutic Protein Identification and Evaluation of Extent of Glycosylation: A Rapid Method for Testing Critical Quality Attributes.

Abstract

Therapeutic proteins are an effective and highly selective, yet complex class of drugs for the treatment of diseases to improve the quality of life of patients. Their manufacturability is subject to evaluation to ensure product quality, safety, and efficacy. To this end, a need exists within the biopharmaceutical industry for a streamlined platform method that allows end-to-end therapeutic protein comparability assessment of multiple critical quality attributes. The implementation of a single platform method would de-risk development and accelerate speed to market. Herein, a novel method for therapeutic protein identification has been proposed that can be used to monitor regulated drug products throughout their lifecycle. The innovative streamlined method evaluates the intact protein in its formulation conditions. This breakthrough technology comprises a quantum cascade laser microscope with a heated accessory, an innovative slide cell array with a fixed path-length that allows for experimental flexibility and quantitative analysis, and dedicated software. Two-trace two-dimensional correlation spectroscopy (2T2D) was proven useful for the determination of spectral differences between two samples. The weighted absorbance differences, defined using the correlation between the cross-peaks, can be used to determine substantial molecular information, including secondary structure, the extent of glycosylation, amino acid content, and solvent accessibility, which is directly related to the therapeutic protein's critical quality attributes. Four different comparability assessments are discussed to demonstrate the usefulness of the platform method for the confirmation of protein identification and the comprehensive evaluation of the extent of glycosylation.