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A workflow for the development of template-assisted membrane crystallization downstream processing for monoclonal antibody purification

Nature Protocols volume 18, pages 2998–3049 (2023)Cite this article

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Abstract

Monoclonal antibodies (mAbs) are commonly used biologic drugs for the treatment of diseases such as rheumatoid arthritis, multiple sclerosis, COVID-19 and various cancers. They are produced in Chinese hamster ovary cell lines and are purified via a number of complex and expensive chromatography-based steps, operated in batch mode, that rely heavily on protein A resin. The major drawback of conventional procedures is the high cost of the adsorption media and the extensive use of chemicals for the regeneration of the chromatographic columns, with an environmental cost. We have shown that conventional protein A chromatography can be replaced with a single crystallization step and gram-scale production can be achieved in continuous flow using the template-assisted membrane crystallization process. The templates are embedded in a membrane (e.g., porous polyvinylidene fluoride with a layer of polymerized polyvinyl alcohol) and serve as nucleants for crystallization. mAbs are flexible proteins that are difficult to crystallize, so it can be challenging to determine the optimal conditions for crystallization. The objective of this protocol is to establish a systematic and flexible approach for the design of a robust, economic and sustainable mAb purification platform to replace at least the protein A affinity stage in traditional chromatography-based purification platforms. The procedure provides details on how to establish the optimal parameters for separation (crystallization conditions, choice of templates, choice of membrane) and advice on analytical and characterization methods.

Key points

  • Monoclonal antibodies used in clinical applications need to be produced and purified at a gram scale. Purification by crystallization is less expensive and more environmentally sustainable than the standard chromatography approach. However, finding appropriate crystallization conditions is challenging.

  • In template-assisted crystallization, the template is a heteronucleant specifically designed to induce crystallization. In this approach, the template is attached to a membrane that is used, at scale, for osmotic membrane-assisted crystallization.

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Fig. 1: A model showing the macromolecular Y-shaped structure of a mAb molecule formed of two Fab (fragment antigen binding) regions and one Fc (fragment crystallizable) region.
Fig. 2: TMC versus conventional chromatography-based DSP purification of mAbs.
Fig. 3: Workflow for TMC of mAbs in DSP.
Fig. 4: Nucleation rate of mAb crystals versus supersaturation.
Fig. 5: Phase diagram of anti-CD20 in 1% (vol/vol) PEG 400 and 0.1 M Na2SO4 solutions as crystallization agent, at pH 7.4 and 20 °C after 48 h under stirring conditions without templates.
Fig. 6: Examples of X-ray diffraction patterns obtained from anti-CD20 crystals.
Fig. 7: MIP results for template pore size determination.
Fig. 8: Images of a macroporous template particle.
Fig. 9: SEM images of polymer membranes at two different magnifications.
Fig. 10: Setup and results for the I04 beamline.
Fig. 11: Average size of crystallite domains, as determined by fitting radial diffraction profiles with a Gaussian function for peak at q = 0.11 Å−1.
Fig. 12: Crystal size distribution of anti-CD20 crystals obtained by TMC.
Fig. 13: Process variables measured in real time during a batch process.

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Data availability

Data supporting this publication are reported in ZENODO under reference codes https://doi.org/10.5281/zenodo.3856655, https://doi.org/10.5281/zenodo.3874571, https://doi.org/10.5281/zenodo.3245362, https://doi.org/10.5281/zenodo.2605199 and https://doi.org/10.5281/zenodo.1322662, and in the database SASBDB at the entry SASDMX3.

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Acknowledgements

This work has received financial support from the European Union’s Horizon 2020 FET-OPEN research and innovation program within the AMECRYS project (http://www.amecrys-project.eu/) under grant agreement no. 712965.

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Authors and Affiliations

  1. CMAC Future Manufacturing Research Hub, c/o Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Technology and Innovation Centre, Glasgow, UK

    Nazer Rajoub, Charline J. J. Gerard & Joop H. ter Horst

  2. Consiglio Nazionale delle Ricerche (CNR), Istituto per la Tecnologia delle Membrane (ITM), Rende, Italy

    Elvira Pantuso, Enrica Fontananova & Gianluca Di Profio

  3. Consiglio Nazionale delle Ricerche (CNR), Istituto di Cristallografia (IC), Bari, Italy

    Rocco Caliandro & Benny D. Belviso

  4. Department of Environmental Engineering, University of Calabria, Rende, Italy

    Efrem Curcio

  5. Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Edificio Polifunzionale, Rende, Italy

    Fiore P. Nicoletta

  6. FUJIFILM Diosynth Biotechnologies, Billingham, UK

    James Pullen

  7. Department of Chemical Engineering, Imperial College London, London, UK

    Wenqian Chen & Jerry Y. Y. Heng

  8. Center for Process Innovation (CPI), Darlington, UK

    Sean Ruane, John Liddell & Natalie Alvey

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  1. Nazer Rajoub
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Contributions

All authors contributed to developing this protocol and writing, refining and revising this paper. G.D.P. coordinated the AMECRYS project.

Corresponding author

Correspondence to Gianluca Di Profio.

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Nature Protocols thanks Dorota Antos and Gaohong He for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Meringolo, C. et al. ACS Sustain. Chem. Eng. 6, 10069–10077 (2018): https://doi.org/10.1021/acssuschemeng.8b01407

Meringolo, C. et al. ACS Appl. Polym. Mater. 1, 326–334 (2019): https://doi.org/10.1021/acsapm.8b00105

Yang, H. et al. Crystals 9, 230 (2019): https://doi.org/10.3390/cryst9050230

Pantuso, E. et al. Sci. Rep. 10, 8902 (2020): https://doi.org/10.1038/s41598-020-65776-6

Chen, W. et al. Cryst. Growth Des. 20, 866–873 (2020): https://doi.org/10.1021/acs.cgd.9b01252

Gerard, C. J. J. et al. Cryst. Growth Des. 22, 3637–3645 (2022): https://doi.org/10.1021/acs.cgd.1c01324

Belviso, B. D. et al. Front. Mol. Biosci. 9, 823174 (2022): https://doi.org/10.3389/fmolb.2022.823174

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Rajoub, N., Gerard, C.J.J., Pantuso, E. et al. A workflow for the development of template-assisted membrane crystallization downstream processing for monoclonal antibody purification. Nat Protoc 18, 2998–3049 (2023). https://doi.org/10.1038/s41596-023-00869-w

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