Causes of Industrial Protein A Column Degradation, Explored Using Raman Spectroscopy

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.

Application of Raman Spectroscopy to Dynamic Binding Capacity Analysis

“…Overall column performance, assessed in terms of DBC, decreased as cycle number increased, likely due to irreversible binding of HCP contaminants. 25 An example of this column degradation is seen for MSP with BT B between cycle 6 ( Table S1, Supplemental Material ) and cycle 29 ( Table I ) where a 4% and 5% decrease in DBC is observed for the UV chromatogram and HPAC methods, respectively.…”

“…24 Previous research from our group utilized Raman spectroscopy to explore how column use affects mAb binding to protein A resin and provides insights into the causes of resin fouling. 25 Here, we have assessed the ability of Raman spectroscopy to establish both BT titer and DBC of two different, widely used protein A affinity resins, i.e., MabSelect SuRe (MSS) and MabSelect PrismA (MSP), Cytiva Life Sciences. Raman spectroscopy achieved results comparable to industry standard methods such as UV monitoring during chromatography and HPAC with the added advantage of producing multivariate data which has the potential to be further developed to quantify multiple protein characteristics crucial for BT quality and homogeneity such as host cell protein (HCP) titer, glycosylation status, and protein stability simultaneously.…”