The analysis, carried out following an increasing number of purification cycles, revealed marked HCP accumulation after 80 bind/elution steps.23 ATR-FTIR spectroscopy has been used to assess SPA ligand stability after repeated use27 and the effects of extended CIP exposure on protein A ligand20 as well as for quantifying mAb bound to the surface layer of resin beads in column and offline.26,27 ATR-FTIR spectroscopy is nondestructive and label-free, providing a chemical footprint which contains information around the structure of a protein sample. store (40.17 mg mLC1) and unused resin samples (70.35 mg mLC1). Depth profiling by Raman spectroscopy indicates that at below saturating concentrations (18 mg mLC1), binding of mAb is not homogeneous through used Ac-IEPD-AFC 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 Ly6a the Raman spectra indicates that one foulant is usually irreversibly bound mAb. The presence of irreversibly bound mAb and host cell proteins was confirmed by mass spectrometric analysis of used resin Ac-IEPD-AFC beads. Introduction In recent years, monoclonal antibodies (mAbs) have become the fastest growing class of biotherapeutics in the U.S. and EU, with 61 first approvals coming on the market between 2014 and 2020 compared to only 34 first approvals between 1997 and 2013.1 MAbs are extremely effective due to their high specificity2 and low uptake across the bloodCbrain barrier, resulting Ac-IEPD-AFC in limited off-target effects.3,4 Therapeutic mAbs are used to treat a range of chronic and acute conditions1 including triple-negative breast cancer,5 Ebola,6 COVID-19,7 and multiple sclerosis.8 Immunoglobulin type gamma (IgG) is the dominant subclass of commercially available therapeutic mAbs. Adalimumab, used to treat rheumatoid arthritis, was the bestselling drug of 2018, generating sales worth $19.9 billion.9 Although the global market in therapeutic mAbs is experiencing record sales, their very high cost (on average $100,000 per patient per Ac-IEPD-AFC year) limits patient access to these life-changing and life-saving drugs.10 Approximately 80% of the cost of therapeutic mAbs is attributable to downstream processing,11 essential to ensure the final product meets strict regulatory purity requirements.12,13 Typically mAbs are produced recombinantly in Chinese hamster ovary (CHO) cells2 and secreted into the growth media. The resultant cell culture fluid contains high levels of mAb as well as host cell proteins,14 media components, cellular DNA, and viruses12,13 which can cause highly undesirable immune responses in patients. 15 Effective purification of mAbs from cell culture fluid involves a number of different actions, with the key step exploiting protein A affinity chromatography to remove the vast bulk (98%) of contaminants.16 Protein A affinity chromatography utilizesprotein A (SPA) as a ligand to capture mAbs with high specificity. The reversible conversation between SPA binding domains and the constant heavy domains 2 and 3 (CH2-CH3) of the Fc region of IgG involves a combination of hydrophobic interactions, salt bridges, and hydrogen bonding.17 The mAb is bound to SPA immobilized onto chromatographic beads at neutral pH. Reduction of the pH to 3 results in protonation of histidine 137 of protein A and histidine 435 of IgG and subsequent release of the bound mAb due to electrostatic repulsion.18 To remove strongly bound contaminants in the column after repeated use, a cleaning in place (CIP) step is used, typically employing up to 0.5 M NaOH.19?21 Protein A affinity chromatographic resin costs over double that of other resins utilized in downstream processing and is thus responsible for most of the downstream processing costs.11,22 The high costs are exacerbated by lifetime degradation of protein A resins, discernible as a loss in mAb binding capacity over time.11,23 A range of analytical techniques have been used to better understand the cause of lifetime degradation of protein A resin including confocal laser scanning microscopy (CLSM),24,25 scanning electron microscopy (SEM),24 mass spectrometry,23 and, by our.