Insight into purification of monoclonal antibodies in industrial columns <i>via</i> studies of Protein A binding capacity by <i>in situ</i> ATR-FTIR spectroscopy

Beattie, James W.; Rowland‐Jones, Ruth C.; Farys, Monika; Tran, Richard T.; Kazarian, Sergei G.; Byrne, Bernadette

doi:10.1039/d1an00985k

Cited by 6 publications

(16 citation statements)

References 42 publications

(63 reference statements)

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“…This is similar in both the unused and used resin samples (Figure S4). This agrees with our earlier ATR-FTIR spectroscopic analysis, which revealed that changes in secondary structure of protein A are not responsible for the drop in SBC observed for the used resin samples …”

Section: Resultsmentioning

confidence: 99%

“…IgG4 (cB72.3) was prepared, isolated, and concentrated to 10 mg mL –1 as described previously. ,, MabSelect SuRe resin (Cytiva, UK) was analyzed in this study. Used MabSelect SuRe samples, harvested from the inlet and the outlet of an AXIChrom 981 mL column after 25 cycles of purification, were donated by GSK Biopharm Process Research as described previously .…”

Section: Methodsmentioning

confidence: 99%

“…This agrees with our earlier ATR-FTIR spectroscopic analysis, which revealed that changes in secondary structure of protein A are not responsible for the drop in SBC observed for the used resin samples. 27 In order to accurately perform band assignment of the measured spectra, a combination of literature values 32,36 and spectral comparison was used (Figure 2). MabSelect SuRe resin with/without mAbs bound and the nonfunctionalized Pierce agarose were used to identify and differentiate between protein bands in the measured spectra.…”

Section: Quantification Of Mabs Bound To Protein a Affinitymentioning

confidence: 99%

“…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 groups, attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. 20,26,27 CLSM showed an increase in both accumulation of foulant and bead pore blockages in protein A resin beads as a function of increased usage, a finding supported by SEM. 25 CLSM, although a powerful technique, requires fluorescent labeling which can change the absorption behavior of proteins.…”

Section: ■ Introductionmentioning

confidence: 99%

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

Beattie

Istrate

et al. 2022

Anal. Chem.

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Quantification Of Mabs Bound To Protein a Affinitymentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

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

Beattie

Istrate

et al. 2022

Anal. Chem.

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“…PVA + PA NF were electrospun from PVA solution with PA and crosslinker—1,1’-carbonyldiimidazole—similar to CA/PVA + PA NF. Comparing the spectra of PVA NF and PVA + PA NF, the peak of Amide I derived from the covalently conjugated PA was observed at 1630–1670 cm −1 [ 25 , 26 ] in PVA + PA NF, indicating the immobilization of PA on PVA. Likewise, the peak of Amide I was also observed in the CA/PVA + PANF by comparing the spectra of CA/PVA NF and CA/PVA + PA NF, indicating the presence of PA on the surface of the core-shell fiber; this is because ATR-FTIR of core-shell NFs emphasizes surface-localized chemical information [ 19 ].…”

Section: Resultsmentioning

confidence: 99%

One-Step Surface Immobilization of Protein A on Hydrogel Nanofibers by Core-Shell Electrospinning for Capturing Antibodies

Naganuma

Moriyama

Suye

et al. 2021

IJMS

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Nanofibers (NFs) are potential candidates as filter materials for affinity separation owing to their high liquid permeability based on their high porosity. Multiple and complex processes were conventionally performed to immobilize proteins for modifying NF surfaces. A simple method must be developed to immobilize proteins without impairing their biological activity. Herein, we succeeded in fabricating NFs with a core of cellulose acetate and a shell of hydrophilic polyvinyl alcohol immobilized with staphylococcal recombinant protein A by a one-step process based on core-shell electrospinning. A total of 12.9 mg/cm3 of antibody was captured in the fiber shell through high affinity with protein A immobilized in an aqueous environment of the hydrogel. The maximum adsorption site and dissociation constant evaluated by the Langmuir model were 87.8 µg and 1.37 µmol/L, respectively. The fiber sheet withstood triplicate use. Thus, our NF exhibited high potential as a material for membrane chromatography.

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Methods for addressing host cell protein impurities in biopharmaceutical product development

Tuameh

Harding

Darton

2022

Biotechnology Journal

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The high demand for monoclonal antibody (mAb) therapeutics in recent years has resulted in significant efforts to improve their costly manufacturing process. The high cost of manufacturing mAbs derives mainly from the purification process, which contributes to 50%–80% of the total manufacturing cost. One of the main challenges facing industry at the purification stage is the clearance of host cell proteins (HCPs) that are produced and often co‐purified with the desired mAb product. One of the issues HCPs can cause is the degradation of the final mAb protein product. In this review, techniques are considered that can be used at different stages (upstream and downstream) of mAb manufacture to improve HCP clearance. In addition to established techniques, many new approaches for HCP removal are discussed that have the potential to replace current methods for improving HCP reduction and thereby the quality and stability of the final mAb product.

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Insight into purification of monoclonal antibodies in industrial columns via studies of Protein A binding capacity by in situ ATR-FTIR spectroscopy

Abstract: Therapeutic monoclonal antibodies (mAbs) are effective treatments for a range of cancers and other serious diseases, however mAb treatments cost on average ~$100,000 per year per patient, limiting their use....

Cited by 6 publications

References 42 publications

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

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

One-Step Surface Immobilization of Protein A on Hydrogel Nanofibers by Core-Shell Electrospinning for Capturing Antibodies

Methods for addressing host cell protein impurities in biopharmaceutical product development

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