Decoupling protein concentration and aggregate content using diffusion and water NMR Mark I. Grimes 1 , Matthew Cheeks 2 , Jennifer Smith 2 , Fabio Zurlo 2 , Mick D. Mantle 1 1 Magnetic Resonance Research Centre, University of Cambridge, UK 2 Cell Culture & Fermentation Sciences, AstraZeneca, UK Biopharmaceuticals, such as monoclonal antibodies (mAbs), accounted for 50% of the ten best selling drugs in 2020, and more than 800 are currently in clinical trials. 1,2 Water proton nuclear magnetic resonance ( w NMR) makes use of the transverse relaxation rate of the water signal [ R 2 ( 1 H 2 O)] to gain understanding about solutes. To date, it has been utilised to investigate the gene filling of adeno associated viral (AAV) capsids, 3 monitor vaccine adjuvant filling level 4 and sedimentation kinetics, 5 as well as to inspect biologic product vials. 6–8 It has also been used previously to obtain information about solution protein concentration and aggregate content, under both static 9,10 and flow conditions. 11 However, R 2 ( 1 H 2 O) is influenced by both characteristics, and is unable to differentiate between them. In this work, a method to decouple protein concentration and aggregate content has been developed using low-field NMR. R 2 ( 1 H 2 O) is demonstrated to show a combined linear effect with increasing protein concentration and reduced monomer percentage ( i.e. , increased aggregate content). By also recording the water diffusion coefficient [ D ( 1 H 2 O)], these two key characteristics can be separated. 12 This exploits the so-called “obstruction effect”, where solutes impede the diffusion pathways of water. 13 This method has been demonstrated on three different model protein systems – bovine serum albumin (BSA), a mAb, as well as a bispecific antibody (BisAb), in concentration ranges relevant for the maximum antibody titres found in fed-batch bioreactors. 14 This method allows for the rapid (<10 min) and facile determination of both protein concentration and aggregate content in a non-invasive manner, with minimal sample preparation. References 1. L. Urquhart, Nat. Rev. Drug Discov. , 2021, 20 , 253.
2. H. Kaplon and J. M. Reichert, mAbs , 2021, 13 , 1860476. 3. M. B. Taraban et al. , Anal. Chem. , 2021, 93 , 15816–15820. 4. M. B. Taraban et al. , Am. Pharm. Rev. , 2019, 22 , 70–73. 5. M. B. Taraban and Y. B. Yu, Magn. Reson. Chem. , 2021, 59 , 147–161. 6. K. T. Briggs et al. , AAPS PharmSciTech , 2019, 20 , 189. 7. M. B. Taraban et al. , J. Diabetes Sci. Technol. , 2022, 16 , 1410–1418. 8. K. T. Briggs et al. , Vaccine , 2020, 38 , 4853–4860. 9. M. B. Taraban et al. , Anal. Chem. , 2017, 89 , 5494–5502. 10. Y. Feng et al. , Chem. Commun. , 2015, 51 , 6804–6807. 11. M. B. Taraban et al. , Anal. Chem. , 2019, 91 , 13538–13546. 12. M. I. Grimes et al. , manuscript in preparation. 13. R. Kimmich et al. , Appl. Magn. Reson. , 1993, 4 , 425–440. 14. F. Li et al., mAbs , 2010, 2 , 466–479.
P10
© The Author(s), 2023
Made with FlippingBook Learn more on our blog