on-column monitoring of protein purification using spectroscopic techniques.
Lead Research Organisation:
Imperial College London
Department Name: Life Sciences
Abstract
Monoclonal antibodies (Mabs) are of major interest to the pharmaceutical industry (1, 2), as they are highly specific and do not cross the blood-brain barrier. IgG4 type antibodies have been approved in the EU and US for treating Acute Lymphoblastic Leukaemia, Haemophilia A and Atopic Dermatitis(3). Immunogenic responses can occur when host cell proteins (HCPs) and DNA are present. It is recommended that <100pg DNA and <100ppm HCPs(4, 5) are present in final samples to prevent undesirable and potentially dangerous immune responses.
HCPs and DNA recommendations are achieved by downstream processing coupled with Protein A affinity chromatography (5-8). Protein A resins are expensive and additional costs are incurred by loss of binding capacity due to fouling, cleaning in place protocols (CIP) and a loss of protein A ligand associated with Mab purification(8-10).
Since continuous flow methodologies are optimal for large scale production (1, 11), it is becoming increasingly important to develop methods to monitor purification of Mabs on-column. Ways to monitor this process include micro flow columns using methods such as confocal laser scanning microscopy(12) and Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy (8, 10) although neither method is industrial scale as yet.
Aims:
To develop experimental methodologies for analysing purification on-column using ATR-FTIR and Confocal Raman Microscopy to observe changes in resin quality and performance in real time.
To apply these techniques to Protein A resin and other chromatography techniques used for isolation of mAbs.
To use these techniques in an industrial setting.
References:
1. Kelley B. Biotechnol Prog. 2007;23(5):995-1008.
2. Moorkens E, Meuwissen N, Huys I, Declerck P, Vulto AG, Simoens S. Front Pharmacol. 2017;8:314.
3. Kaplon H, Reichert JM. MAbs. 2018;10(2):183-203.
4. WHO Requirements for the Use of Animal Cells as in vitro Substrates for the Production of Biologicals (Requirements for Biological Susbstances No. 50). Biologicals. 1998;26(3):175-93.
5. Tarrant RD, Velez-Suberbie ML, Tait AS, Smales CM, Bracewell DG. Biotechnol Prog. 2012;28(4):1037-44.
6. Shukla AA, Jiang C, Ma J, Rubacha M, Flansburg L, Lee SS. 2008;24(3):615-22.
7. Goey CH, Alhuthali S, Kontoravdi C. Biotechnol Adv. 2018;36(4):1223-37.
8. Boulet-Audet M, Byrne B, Kazarian SG. Anal Bioanal Chem. 2015;407(23):7111-22.
9. Close EJ, Salm JR, Iskra T, Sorensen E, Bracewell DG. Biotechnol Bioeng. 2013;110(9):2425-35.
10. Boulet-Audet M, Kazarian SG, Byrne B. Sci Rep. 2016;6:30526.
11. Girard V, Hilbold NJ, Ng CK, Pegon L, Chahim W, Rousset F, et al. J Biotechnol. 2015;213:65-73.
12. Wang Y, Chen Q, Xian M, Nian R, Xu F. AMB Express. 2018;8(1):93.
HCPs and DNA recommendations are achieved by downstream processing coupled with Protein A affinity chromatography (5-8). Protein A resins are expensive and additional costs are incurred by loss of binding capacity due to fouling, cleaning in place protocols (CIP) and a loss of protein A ligand associated with Mab purification(8-10).
Since continuous flow methodologies are optimal for large scale production (1, 11), it is becoming increasingly important to develop methods to monitor purification of Mabs on-column. Ways to monitor this process include micro flow columns using methods such as confocal laser scanning microscopy(12) and Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy (8, 10) although neither method is industrial scale as yet.
Aims:
To develop experimental methodologies for analysing purification on-column using ATR-FTIR and Confocal Raman Microscopy to observe changes in resin quality and performance in real time.
To apply these techniques to Protein A resin and other chromatography techniques used for isolation of mAbs.
To use these techniques in an industrial setting.
References:
1. Kelley B. Biotechnol Prog. 2007;23(5):995-1008.
2. Moorkens E, Meuwissen N, Huys I, Declerck P, Vulto AG, Simoens S. Front Pharmacol. 2017;8:314.
3. Kaplon H, Reichert JM. MAbs. 2018;10(2):183-203.
4. WHO Requirements for the Use of Animal Cells as in vitro Substrates for the Production of Biologicals (Requirements for Biological Susbstances No. 50). Biologicals. 1998;26(3):175-93.
5. Tarrant RD, Velez-Suberbie ML, Tait AS, Smales CM, Bracewell DG. Biotechnol Prog. 2012;28(4):1037-44.
6. Shukla AA, Jiang C, Ma J, Rubacha M, Flansburg L, Lee SS. 2008;24(3):615-22.
7. Goey CH, Alhuthali S, Kontoravdi C. Biotechnol Adv. 2018;36(4):1223-37.
8. Boulet-Audet M, Byrne B, Kazarian SG. Anal Bioanal Chem. 2015;407(23):7111-22.
9. Close EJ, Salm JR, Iskra T, Sorensen E, Bracewell DG. Biotechnol Bioeng. 2013;110(9):2425-35.
10. Boulet-Audet M, Kazarian SG, Byrne B. Sci Rep. 2016;6:30526.
11. Girard V, Hilbold NJ, Ng CK, Pegon L, Chahim W, Rousset F, et al. J Biotechnol. 2015;213:65-73.
12. Wang Y, Chen Q, Xian M, Nian R, Xu F. AMB Express. 2018;8(1):93.
| Description | We have explored the causation for the reduction in Protien A resin lifespan. This step alone causes mAb based treatments to be extremely expensive. the projuct thus far has shown: Resin in a column used for the purification of monoclonal antibodies (mAb's) degrades at different rated depending on spatial location, This was shown using ATR-FTIR spectroscopy. A paper on this topic is currently awaiting legal approval from GSK before submission to a journal. ATR-FTIR can be used to monitor mAb purifcation in real time. MS-MS has shown indication to permanently binding proteins to the Protein A resin used in the purification of mAb's. Confocal Raman can be used to asses Protein A resin after use and with mAbs adsorbed. overall the project thus far has explored multiple spectroscopy methods to better understand the causation for protein A resin lifetime reduction. New methods are being developed to help improve monitoring methods of biopharamacuicals. Currently We are working to further use the aforementioned methods. |
| Exploitation Route | The research findings found by this project will be used by others to help improve current downstream processing of mAbs and other biopharmaceuticals. Findings on the causation will allow either the improved upstream processing or additional downstream processing steps to be utilised to ensure the longevity of Protein A columns. This will help reduce the cost of mAb treatments and improve the quality of the end product. |
| Sectors | Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Description | ICASE partner |
| Organisation | GlaxoSmithKline (GSK) |
| Department | Research and Development GSK |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | Carrying out and design of experiments, Reporting of findings and proposing new experiments aswell as implementing guidance to align experiments to project aims. |
| Collaborator Contribution | Provision of loaned items for experimental use. Supervision and guidance to align experiments to project aims. Provided opertunity to present my work to GSK internal Downstream Process Development meetings between UK and USA counterparts. |
| Impact | Collaboration is multi-disciplinary |
| Start Year | 2018 |