Analytical Techniques for the Characterization and Quantification of Monoclonal Antibodies

Abstract

Monoclonal antibodies (mAbs) are a fast-growing class of biopharmaceuticals. They are widely used in the identification and detection of cell makers, serum analytes, and pathogenic agents, and are remarkably used for the cure of autoimmune diseases, infectious diseases, or malignancies. The successful application of therapeutic mAbs is based on their ability to precisely interact with their appropriate target sites. The precision of mAbs rely on the isolation techniques delivering pure, consistent, stable, and safe lots that can be used for analytical, diagnostic, or therapeutic applications. During the creation of a biologic, the key quality features of a particular mAb, such as structure, post-translational modifications, and activities at the biomolecular and cellular levels, must be characterized and profiled in great detail. This implies the requirement of powerful state of the art analytical techniques for quality control and characterization of mAbs. Until now, various analytical techniques have been developed to characterize and quantify the mAbs according to the regulatory guidelines. The present review summarizes the major techniques used for the analyses of mAbs which include chromatographic, electrophoretic, spectroscopic, and electrochemical methods in addition to the modifications in these methods for improving the quality of mAbs. This compilation of major analytical techniques will help students and researchers to have an overview of the methodologies employed by the biopharmaceutical industry for structural characterization of mAbs for eventual release of therapeutics in the drug market.

Panda-UV Unlocks Deeper Protein Characterization with Internal Fragments in Ultraviolet Photodissociation Mass Spectrometry

“…Internal fragments increased the sequence coverage from 37 to 86% (Figure B) and accurately bracketed the glycosylation modification that cannot be localized by terminal fragments alone for Fc/2 (27+) (Figure C). For Lc (20+) and Fd (19+), internal fragments increased the sequence coverage from 56 to 86% and 46 to 79%, respectively (Figure E,I), in which six CDRs (complementary determining regions) , that are crucial for the characterization of mAbs were nearly completely sequenced (totally on 87%) (Figure F,J). Only terminal fragments with PCC scores greater than 0.6 and terminal fragments with PCC scores greater than 0.8 were retained to ensure confidence.…”

Multi‐dimensional technology – Recent advances and applications for biotherapeutic characterization

“…To enhance the potential success rates and improve the quality and safety of mAbs and related products as approved drugs, a comprehensive array of analytical techniques should be employed throughout the research and development (R&D) and quality control (QC) laboratories [11]. This approach facilitates thorough evaluation, supporting batch release.…”

Online Coupling of Size Exclusion Chromatography to Capillary Enhanced Raman Spectroscopy for the Analysis of Proteins and Biopharmaceutical Drug Products

“…25 fragmentation. 23,24,30 Besides, Raman spectroscopy (RS) is a powerful technique to investigate the secondary and tertiary structure of proteins, 25,26,28,29,31 but in pharmaceutical products its use for structural elucidation may be limited due to a low molar concentration of the active pharmaceutical ingredient (API) compared to the excipients. 32 In combination with size exclusion chromatography (SEC) proteins can be separated from matrix compounds, e.g., formulating excipients or protein aggregates, in an aqueous mobile phase, thus maintaining their native structure and preserving their pharmaceutical activity.…”

“…During biopharmaceutical product development, regulatory authorities such as EMA request a detailed characterization of drug candidates. The characterization includes the assessment of the higher-order structure, the molecular weight or size of the protein, and an impurity analysis regarding the formation of aggregates or fragments. , The last gained importance as recent studies indicate structural changes of aggregated protein species compared to the monomeric protein. − Size exclusion chromatography is suggested as an analytical technique for the investigation of molecular weight or size of the protein and the determination of protein aggregation and fragmentation. ,, Besides, Raman spectroscopy (RS) is a powerful technique to investigate the secondary and tertiary structure of proteins, ,,,, but in pharmaceutical products its use for structural elucidation may be limited due to a low molar concentration of the active pharmaceutical ingredient (API) compared to the excipients . In combination with size exclusion chromatography (SEC) proteins can be separated from matrix compounds, e.g., formulating excipients or protein aggregates, in an aqueous mobile phase, thus maintaining their native structure and preserving their pharmaceutical activity .…”