1. Insight into purification of monoclonal antibodies in industrial columns via studies of Protein A binding capacity by in situ ATR-FTIR spectroscopy†
- Author
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Monika Farys, Bernadette Byrne, Sergei G. Kazarian, James W. Beattie, Ruth C. Rowland-Jones, Richard Tran, and Biotechnology and Biological Sciences Research Council (BBSRC)
- Subjects
In situ ,medicine.drug_class ,Ataxia Telangiectasia Mutated Proteins ,Monoclonal antibody ,01 natural sciences ,Biochemistry ,Analytical Chemistry ,03 medical and health sciences ,Affinity chromatography ,stomatognathic system ,0399 Other Chemical Sciences ,Spectroscopy, Fourier Transform Infrared ,Electrochemistry ,medicine ,Environmental Chemistry ,Humans ,Denaturation (biochemistry) ,Least-Squares Analysis ,Staphylococcal Protein A ,Spectroscopy ,STAPHYLOCOCCUS-AUREUS ,030304 developmental biology ,FRAGMENT ,0303 health sciences ,Science & Technology ,Chromatography ,Downstream processing ,biology ,Chemistry ,Chemistry, Analytical ,010401 analytical chemistry ,technology, industry, and agriculture ,Antibodies, Monoclonal ,Ligand (biochemistry) ,0104 chemical sciences ,Physical Sciences ,biology.protein ,Leaching (metallurgy) ,Protein A ,0301 Analytical Chemistry - 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. Currently, industry favours Protein A affinity chromatography (PrAc) as the key step in downstream processing of mAbs. This step, although highly efficient, represents a significant mAb production cost. Fouling of the Protein A column and Protein A ligand leaching contribute to the cost of mAb production by shortening the life span of the resin. In this study, we assessed the performance of used PrAc resin recovered from the middle inlet, center and outlet as well as the side inlet of a pilot-scale industrial column. We used a combination of static binding capacity (SBC) analysis and Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy to explore the used resin samples. SBC analysis demonstrated that resin from the inlet of the column had lower binding capacity than resin from the column outlet. ATR-FTIR spectroscopy with PLS (partial least square) analysis confirmed the results obtained from SBC analysis. Importantly, in situ ATR-FTIR spectroscopy also allowed both measurement of the concentration and assessment of the conformational state of the bound Protein A. Our results reveal that PrAc resin degradation after use is dependent on column location and that neither Protein A ligand leaching nor denaturation are responsible for binding capacity loss., A combination of static binding capacity analysis and ATR-FTIR spectroscopy reveals that loss of binding capacity is not uniform through a used Protein A column and is not due to loss of Protein A ligand.
- Published
- 2021