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

Nazer Rajoub, Charline J. J. Gerard, Elvira Pantuso, Enrica Fontananova, Rocco Caliandro, Benny D Belviso, Efrem Curcio, Fiore P Nicoletta, James Pullen, Wenqian Chen, Jerry Y Y Heng, Sean Ruane, John Liddell, Natalie Alvey, Joop H. ter Horst, Gianluca Di Profio

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)


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.

Original languageEnglish
Pages (from-to)2998-3049
Number of pages52
JournalNature Protocols
Issue number10
Early online date11 Sept 2023
Publication statusPublished - Oct 2023


  • membrane crystallization
  • monoclonal antibody purification


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