High-Throughput Biophysical Analysis and Data Visualization of Conformational Stability of an IgG1 Monoclonal Antibody After Deglycosylation

Abstract

The structural integrity and conformational stability of an IgG1 monoclonal antibody (mAb), after partial and complete enzymatic removal of the N-linked Fc glycan, was compared to the untreated mAb over a wide range of temperature (10° to 90°C) and solution pH (3 to 8) using circular dichroism, fluorescence spectroscopy, and static light scattering combined with data visualization employing empirical phase diagrams (EPDs). Subtle to larger stability differences between the different glycoforms were observed. Improved detection of physical stability differences was then demonstrated over narrower pH range (4.0-6.0) using smaller temperature increments, especially when combined with an alternative data visualization method (radar plots). Differential scanning calorimetry and differential scanning fluorimetry were then utilized and also showed an improved ability to detect differences in mAb glycoform physical stability. Based on these results, a two-step methodology was used in which mAb glycoform conformational stability is first screened with a wide variety of instruments and environmental stresses, followed by a second evaluation with optimally sensitive experimental conditions, analytical techniques and data visualization methods. With this approach, high-throughput biophysical analysis to assess relatively subtle conformational stability differences in protein glycoforms is demonstrated.

Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry

“…Dynamics-The conserved antibody glycosylation in the Fc domain is important for the structural integrity of antibodies, and partial or full deglycosylation has been shown to influence conformation, storage stability, and solubility (46,47). The impact of antibody glycosylations on HDX-MS was examined previously (40,48), and in our study on Ab wt and Ab degly we observed effects upon antibody deglycosylation similar to those seen in prior studies performed on another IgG 1 antibody.…”

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

“…30 It is possible that the increased flexibility of the peptide backbone disrupts the packing interactions between the peptide backbone and the glycan chains, leading to unfolding of this hydrophobic segment. This unfolded apolar segment, in turn, may act as a hotspot for subsequent structural alterations that could lead to irreversible aggregation in IgG1 mAbs, [55][56][57] for example, as observed by SEC analysis in this study. Limiting the flexibility of this particular segment may therefore serve as a good indicator for designing mAbs (and mAbrelated products such as IgG-Fc, fusion proteins, or antibody drug conjugates) with higher thermostability and resistance to aggregation.…”

Production, Characterization, and Biological Evaluation of Well-Defined IgG1 Fc Glycoforms as a Model System for Biosimilarity Analysis

“…A series of sequentially truncated glycoforms of IgG1 Fc (Figure 1), which differ only in the size of the N-linked glycan at N297 or a single conservative amino acid mutation (N297Q), were chosen as members for the model system. Previous studies have indicated that these glycoforms would display a range of biological activities and physical properties 1,32,45 that would be advantageous for our biosimilarity studies.…”