Assessment of IgG stability in a low pH elution buffer using ATR-FTIR spectroscopic imaging and microfluidics
File(s)
Published version
Author(s)
van Haaren, Celine
Byrne, Bernadette
Kazarian, Sergei G
Type
Journal Article
Abstract
Monoclonal antibodies (mAbs) represent the largest class of biopharmaceuticals, playing a vital role in the treatment of a wide range of diseases. Although the production of high quality mAbs has significantly improved over the last three decades, particularly in terms of scale and yield, the antibody's complex nature poses several challenges during bioprocessing. One of the main challenges in the production of mAbs is the formation of aggregates, which may cause harmful immunogenic responses in patients if not removed from the final drug product. Exposure to a low pH environment during protein A chromatography and viral inactivation is thought to be the major contributor to aggregate formation and has therefore been a topic of study for many years. Here, we investigate the stability of an IgG4 mAb in a low pH elution buffer (pH 3.5) under flow using ATR-FTIR spectroscopic imaging. This method, making use of a microfluidic set-up, enables non-destructive monitoring of mAb structural stability under bioprocessing-relevant conditions. Samples were (i) prepared through dialysis into the elution buffer and (ii) collected directly after elution from the protein A column, after which their stability was assessed under flow at two different temperatures (30 °C and 45 °C). Spectroscopic images and associated IR absorption spectra revealed that in both cases the protein in the low pH buffer underwent small, but measurable, structural changes at 30 °C. However, at 45 °C, the protein rapidly aggregated as indicated by a major shift in the Amide I peak position from 1637 cm−1 to 1625 cm−1, representing formation of inter-molecular beta sheets. These results confirm the destabilising effect of the low pH environment and demonstrate the applicability of ATR-FTIR spectroscopic imaging in combination with microfluidics as a powerful analytical tool for the analysis of protein structural stability under flow.
Date Issued
2025-09-21
Date Acceptance
2025-08-01
Citation
Analyst, 2025, 150 (18), pp.4201-4210
ISSN
0003-2654
Publisher
ROYAL SOC CHEMISTRY
Start Page
4201
End Page
4210
Journal / Book Title
Analyst
Volume
150
Issue
18
Copyright Statement
© The Royal Society of Chemistry 2025 Open Access Article This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.
License URL
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/40838564
Subjects
AGGREGATION KINETICS
Chemistry
Chemistry, Analytical
CONFORMATION
MECHANISM
MONOCLONAL-ANTIBODY
Physical Sciences
PROTEIN
PURIFICATION
Science & Technology
Publication Status
Published
Coverage Spatial
England
Date Publish Online
2025-08-05