Biopharmaceuticals are large and complex molecules manufactured using biological sources, which can provide specific treatment to patients suffering from several pathologies. Amongst biopharmaceuticals, monoclonal antibodies (mAbs) represent today's most common class of biological therapies, with more than 1,200 mAbs-based drugs candidates currently in development for the treatment of several diseases including rheumatoid arthritis, multiple sclerosis, COVID-19 and many different types of cancer.
Industrial mAbs production presents the sizeable hurdles of purification costs and environmental sustainability that negatively affects market drivers. As mAbs have a common biological scaffold, they are produced in the upstream processing stage (USP) by Chinese hamster ovary (CHO) cells culture at scales of 10,000-15,000 L fermentation, and are then purified in the downstream processing (DSP) via a number of complex and expensive chromatographic steps, operated discontinuously, that relies heavily on protein A resin. The conventional purification of mAbs at appropriate yield and purity targets absorbs ca. 80% of the total production costs, with the most significant contribution given by the protein-A resin that can amount to more than ten thousand euro per liter. This translates into high market prices of mAbs: e.g., a single course of Anti CD20 - two 1000-mg intravenous infusions - costs about €2,000 per gram. Environmental aspects are also under consideration, especially with regard to the extensive use of toxic solvents and buffers for chromatography columns regeneration and operation that lead to an unsustainable environmental cost.
In this context, as main scientific and technological breakthroughs of the H2020 FET-OPEN AMECRYS project (https://cordis.europa.eu/project/id/712965/en) coordinated by Dr. Gianluca Di Profio (Cnr-Itm), an international consortium including researchers from Cnr-Itm (Enrica Fontananova, Gianluca Di Profio) and Cnr-Ic (Rocco Caliandro, Benny D. Belviso) has shown that conventional Protein A chromatography can be replaced with a single template-assisted membrane crystallization step to recover Anti-CD20 (Rituximab), a full-length mAb, at gram scale directly from clarified cell culture fluid. We have shown at laboratory scale (TRL4) that the membrane-based technology can completely replace the multi-step batch chromatography platform based on protein A traditionally used for the purification of the Anti-CD20 mAb, providing a cheaper and easily scalable alternative to chromatographic techniques, as a path to a potentially lower-cost and environmental sustainable mAb purification approach.
Crystallization is normally a cost-effective and easily scalable technique that is extensively used in the formulation of low molecular weight materials. Nevertheless, solution crystallization of large biomolecules, like mAbs, is challenging due to their high molecular weight, structural complexities, flexibility, and the presence of impurities that hinder the nucleation stage. This has prevented so far the wide use of crystallization in biopharmaceuticals purification at an industrial scale.
In the proposed approach, engineered templates (i.e. mesoporous silica) are embedded in a membrane to serve as heteronucleants for the target molecule that is normally reluctant to crystallize by conventional crystallisation methods. The paradigm shift from batch chromatography to continuous crystallization by means of modular and easily scalable membranes is turned into a viable manufacturing option for radical changes in efficiency and a drastic decrease of facilities size with lower capital and operating costs. Furthermore, compared to liquid formulations, solid-state normally affords higher product activity, enhanced stability and a sustained release capability (bioavailability).
Results of the AMECRYS project have been published in Nature Protocols. The objective of this workflow protocol is to establish a systematic and flexible approach for the design of a robust, economic and sustainable mAbs 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, inluding crystallisation conditions, choice of templates, choice of membrane, and includes advice regarding analytical and characterisation methods for both membrane and crystalline mAb product.
N. Rajoub, C.J.J. Gerard, E. Pantuso, E. Fontananova, R. Caliandro, B.D. Belviso, E. Curcio, F.P. Nicoletta, J. Pullen, W. Chen, J.Y.Y. Heng, S. Ruane, J. Liddell, N. Alvey, J.H. ter Horst, G. Di Profio, A workflow for the development of template-assisted membrane crystallization downstream processing for monoclonal antibody purification, Nature Protocols 2023, http://doi.org/10.1038/s41596-023-00869-w
Gianluca Di Profio
CNR - Istituto per la tecnologia delle membrane
Via P. Bucci Cubo 17/C, Rende (CS), Italy