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.
Highlights
Therapeutic monoclonal antibodies (mAbs) (Monoclonal Antibodies) are major biopharmaceuticals, making up 22% of the Food and Drug Administration’s (FDA) newly approved drugs between 2016–2018.1 A total of 550 mAbs were in phase 1 or 2 clinical trials in 2019,2 increasing to 743 mAbs in 2020.3 In addition, a record 44 cancer and 44 non-cancer targeted mAbs were in FDA and European Medicines Agency (EMA) phase clinical studies as of November 2020.3 Currently, therapeutic mAbs available on the market are primarily of the subclass of Immunoglobulin type gamma (IgG).[4]
The greatest loss in static binding capacity compared to the unused resin is seen in the resin samples at the inlet of the column, where the cell culture supernatant (CCS) would be loaded for purification
Using a combination of in situ ATR-FTIR spectroscopy and SBC assays we show that the reduction in Protein A Affinity Chromatography (PrAc) static binding capacity is heterogeneous throughout a used pilot scale chromatography column, with the greatest loss of binding capacity occurring at the column inlet
Summary
Various steps are employed in downstream processing of recombinantly produced mAbs in order to reduce HCP and HCDNA to safe levels as well as remove high molecular weight species (HMWS) and culture media components.[5,10] Safe levels of HCP and HCDNA are recommended at below detectable limits by the FDA11 but are typically in the 1 ng mg−1 range.[5]. We used a combination of static binding capacity (SBC) analysis and Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy to explore the performance, ligand density and secondary structure of both Protein A ligand and mAb on MabSelect SuRe samples obtained from a used Protein A column. The exact position of peaks and shoulders within the amide bands are dependent on the protein secondary structure present,[26] the amide bands are extremely important when characterizing proteins.[24,25,27,28,29] Partial least squares (PLS) analysis of our spectroscopic data showcased ATR-FTIR spectroscopy as a simple and effective method of predicting performance of affinity resin for mAb capture and exhibiting additional molecular information of measured samples when compared to traditional OD280 nm based static binding capacity assays. The loss of binding capacity is not due to a reduction in the amount or conformation of the Protein A ligand bound to the resin in the column but is likely due to irreversible binding of mAbs or HCP fouling within the porous matrix of the Protein A resin
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
