Causes of industrial protein a column degradation, explored using Raman spectroscopy.
File(s)
Published version
OA Location
Author(s)
Type
Journal Article
Abstract
Monoclonal antibodies (mAbs) are used extensively as biotherapeutics for chronic and acute conditions. Production of mAbs is lengthy and expensive, with protein A affinity capture the most costly step, due both to the nature of the resin and its marked reduction in binding capacity with repeated use. Our previous studies using in situ ATR-FTIR spectroscopy indicated that loss in protein A binding capacity is not the result of leaching or degradation of protein A ligand, suggesting fouling is the principal cause. Here we explore binding behavior and resin capacity loss using Raman spectroscopy. Our data reveal a distinct Raman spectral fingerprint for mAb bound to the protein A ligand of MabSelect SuRe. The results show that the drop in static binding capacity (SBC) previously observed for used protein A resin is discernible by Raman spectroscopy in combination with partial least-squares regression. The SBC is lowest (35.76 mg mL-1) for used inlet resin compared to used outlet (40.17 mg mL-1) and unused resin samples (70.35 mg mL-1). Depth profiling by Raman spectroscopy indicates that at below saturating concentrations (∼18 mg mL-1), binding of mAb is not homogeneous through used resin beads with protein binding preferentially to the outer regions of the bead, in contrast to fully homogeneous distribution through unused control MabSelect SuRe resin beads. Analysis of the Raman spectra indicates that one foulant is irreversibly bound mAb. The presence of irreversibly bound mAb and host cell proteins was confirmed by mass spectrometric analysis of used resin beads.
Date Issued
2022-11-15
Date Acceptance
2022-10-19
Citation
Analytical Chemistry, 2022, 94 (45), pp.15703-15710
ISSN
0003-2700
Publisher
American Chemical Society
Start Page
15703
End Page
15710
Journal / Book Title
Analytical Chemistry
Volume
94
Issue
45
Copyright Statement
© 2022 The Authors. Published by American Chemical Society. This work is published under a CC BY licence.
License URL
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/36318727
Subjects
Staphylococcal Protein A
Spectrum Analysis, Raman
Ligands
Chromatography, Affinity
Antibodies, Monoclonal
Publication Status
Published
Coverage Spatial
United States
Date Publish Online
2022-11-01