Application of Vibrational Spectroscopy to the Structural Characterization of Monoclonal Antibody and its Aggregate

Abstract

Aggregation is often the major issue during formulation and manufacturing development of therapeutic proteins, in particular human monoclonal antibody. Currently, there is a lack of structural information of aggregates of such large protein as human antibodies, due to the large molecular sizes of the aggregates. In this article, we shall discuss the application of vibrational spectroscopies including FT-IR, Raman and Raman Optical Activity (ROA), to characterize the structures of various types of monoclonal antibody aggregates formed under different stresses. Two different classes of human monoclonal antibodies, namely IgG1 and IgG2, have been subjected to this structural investigation. The common stresses leading to antibody aggregation, mis-folding or unfolding during manufacturing and formulation include exposure to acidic pHs, heat and shear stress. The effect of different types of stresses on the structure and aggregate formation of human monoclonal antibodies has been investigated by employing vibrational spectroscopy. While data present only monoclonal antibody, the same technology can be used for any protein aggregates.

Particles in Therapeutic Protein Formulations, Part 1: Overview of Analytical Methods

“…As a main benefit, FTIR spectroscopy can be applied both on liquid samples (solutions and dispersions) and on solid samples (e.g., lyophilizates) 139. Also the analysis of highly aggregated protein formulations and particle containing protein formulations is possible.…”

Vibrational optical activity: From discovery and development to future challenges

“…While the principal application of VCD in the pharmaceutical industry is the determination of the AC, ROA shows considerable future promise as a sensitive diagnostic of higher‐order structure detection of biopharmaceuticals. The earliest such report is by Li and Li at Amgen in which they reported changes in ROA of pH stressed monoclonal antibodies human immunoglobins (IgG 1 and IgG2) without any perceived changes in the parent Raman scattering measured simultaneously . More recently, unique sensitivity of ROA was reported by researchers at Merck for the onset of spectral changes due to structural instability of monoclonal antibody formulations during heat stress.…”

Engaging with Raman Spectroscopy to Investigate Antibody Aggregation

“…A key cause of protein aggregation is the formation of non-native, partially unfolded intermediates with exposed regions capable of inter-molecular interactions [21,26,43]. The propensity of a protein to unfold, thereby exposing regions of residues, is not only influenced by changes in physiochemical conditions (e.g., temperature, pressure, pH and excipients) but can also be due to the intrinsic conformational properties of the protein (e.g., primary sequence, secondary and tertiary structure) [16,21,26].…”

“…As previously demonstrated in Figure 2, the Raman spectrum of a typical antibody has numerous features that can be easily assigned to both secondary and tertiary structure conformation. The Raman peaks observed at ~960, 1245 and 1674 cm −1 assigned to β-sheet indicate that this is the major secondary structure of the antibody, whilst features in the regions of ~500–550 cm −1 assigned to disulfide conformations, 800–860 cm −1 assigned to hydrogen bonding state of tyrosine residues and the peak at ~1550 cm −1 assigned to tryptophan are all markers of tertiary structure [17,24,43]. Raman spectroscopy is therefore an ideal tool for the characterisation of not only the native antibody but also the partially or fully-unfolded intermediates that lead to the formation of aggregates, including providing insight into the actual aggregation mechanisms [13,40,43].…”