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Revolutionising Downstream Processing of Monoclonal Antibodies by Continuous Template-Assisted Membrane Crystallization

Deliverables

Communication events

Intermediate results of the project will be disseminated by organizing press releases and by realizing leaflets and a web video. All this material will be made available on the project website. Data coming from research activities, which will be decided to not protect by project governing bodies, will be uploaded on OpenAIRE 2020.

Website and project logo

Website and project logo

Technology roadmap

An assessment of the AMECRYS’s concept and results obtained during the project, including operational, financial and implementation considerations vs conventional downstream processing technology will be conducted. A technology roadmap describing the strategy to support the implementation of continuous crystallization-based purification in biopharmaceutical industry will be prepared.

Techno-economic comparison between conventional batch and innovative DSP

The comparison will take the form of a software-based comparative life-cycle and cost-of-goods analysis of the traditional chromatographic purification versus the developed crystallisation method for both molecules of interest (assuming full data sets available). As well as the economic and time implications, a full comparison of all the defined quality attributes of material produced from both purification methods will be reported.

Updated Data Management Plan

The data management plan will be adjusted for data management beyond the project. In principle, the data will be stored in the electronic archives of CNR for at least 5 years after the conclusion of the Project without additional cost. All the research data will be of the highest quality, have long-term validity and will be well documented in order other researchers to be able to get access and understand them after 5 years.

Report on prototype’s operation monitoring & compliance with QS/GMP/CQA regulations

Following the installation of the continuous nano-template-assisted membrane crystallizer at the CPI’s National Biologics Manufacturing Centre (UK), the crystallizer will be evaluated using the optimal conditions determined in the earlier work packages. This will include both the mAb and the HEL4 fragment, unless one of the molecules has already been ruled out by earlier results. The conditions selected will include biopharmaceutical product purity level, the product concentration, pH/buffer conditions, addition of precipitants or nanotemplates and transmembrane fluxes/input flow rates. During an experimental program, CPI will test the robustness of the new process and the ability to create product of a consistent quality and purity. A report will be prepared that describes how suitable the technology, in its current form, is for GMP operation as part of a biopharmaceutical manufacturing process. Recommendations will be made for further development that would be required for implementation of the technology in an industrial environment and compliance with relevant quality standards and regulations.

Report on structural/morphological/bioactivity properties of mAbs crystals produced by prototype operation crystallizers

The report will describe properties of crystals produced by the microfluidic system and by the crystallizer prototype, during its development and operation. They will be tested by carrying out X-ray diffraction analyses, mechanical tests, optical spectroscopy, electron and atomic-force microscopy and dynamic light scattering. The results will be used to optimize the prototype design and to assess its operational efficiency.

Report on preparation of nanotemplates for mAb crystallization

Report on preparation of nanotemplates of pore dimensions (3-20 nm) and characterization to determine pore dimensions and surface area. Nanotemplates will be derivitised to modify surface chemistry and characterized to confirm % of coverage.

Report on selective nucleation and growth kinetics of mAbs from screening tests

The measured heterogeneous nucleation kinetics using functionalized template surfaces and in microfluidic device will be translated into sound heterogeneous nucleation theories and help to apply the obtained nucleation knowledge on a larger scale. The report will provide specifications for nanotemplate synthesis, membrane optimization, process scale-up, and prototype design.

HEL4 domain fragment & Anti CD20 mAb process specification report

A process specification report will be prepared detailing the optimised upstream production and conventional downstream purification procedures for anti-CD20 mAb and HEL4 VH domain fragment. This will include details of the analytical methods developed to characterise the material at each of the three purity levels (post-harvest, post-capture column and final product) and the baseline quality attributes generated from these methods to allow for comparison with the crystallization process.

Dissemination and Exploitation Plan

A dissemination and exploitation plan will be delivered defining key dissemination opportunities within the network and to academia, industry and society outside the network. The plan will describe the dissemination of results to the Project Participants, Scientific Community, Academia, and Related Industry through internal meetings, deliverables publication in Project website, presentations at relevant conferences and symposia, and by publishing results in high-impact factor peer-reviewed journals. All scientific publications will be provided as free on-line open access (green/gold model). To disseminate our project outcomes we will organize an open conference or workshop at the end of the project for which key industrial and academic researchers will be invited. The plan will also define strategies and schedule for the exploitation of project’s foregrounds.

Robust microfabrication protocols to embed hydrophobic fluoropolymers membranes within microfluidic chips

Report on the development of fabrication protocols to embed the hydrophobic fluoropolymers membranes developed in WP3 within custom-made microfluidic devices obtained by standard PDMS soft lithography. These protocols may use mechanical clamping to seal the membranes between two micro-fabricated PDMS stamps or chemical linking with slight chemical modifications of the PDMS surface (preserving the requirements for mAbs crystallization).

Open-source MC-FFS computational simulation packages

The kinetic Monte Carlo codes with forward-flux sampling (and possibly other rare event techniques) used to simulate crystallization at membranes will be documented and archived on open-source repositories. The deliverable consists of the documented codes and an analysis of the results obtained for conditions corresponding to those of interest to the project.

Simulation code for thermodynamics of course-grained model of mAbs in confined geometry

This will consist of DFT/MC/Molecular dynamics codes (exact methods to be decided as the work progresses) for determining the phase diagram of a coarse-grained model for mAbs. The deliverable consists of the documented codes and the resulting phase diagrams.

Data Management Plan

A data management plan will be constructed for the generated data in the project. This will cover data in the form of chemical synthesis protocols, models/simulations/software, design documents including drawings and specifications, construction schedules, regulatory documents, quality control and assurance records. Generated data will be preserved on the intranet platform until the end of the Project.

Installation and validation of continuous flow template-assisted membrane crystallizer prototype

Fabrication and installation of the continuous flow template-assisted membrane crystallizer prototype: manufacturing the crystallization vessel and membrane modules, selecting and installing impellers/downstream filtration systems/nucleation probes, implementing PID controlers, testing and validation of the final product.

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Publications

On the aggregation and nucleation mechanism of the monoclonal antibody anti-CD20 near liquid-liquid phase separation (LLPS)

Author(s): Pantuso, Elvira; Mastropietro, Teresa F.; Briuglia, Maria L.; Gerard, Charline J. J.; Curcio, Efrem; ter Horst, Joop H.; Nicoletta, Fiore P.; Di Profio, Gianluca
Published in: Scientific Reports, Issue 10, 2020, Page(s) 8902, ISSN 2045-2322
DOI: 10.1038/s41598-020-65776-6

Solute particle near a nanopore: influence of size and surface properties on the solvent-mediated forces

Author(s): Julien Lam, James F. Lutsko
Published in: Nanoscale, Issue 9/43, 2017, Page(s) 17099-17108, ISSN 2040-3364
DOI: 10.1039/C7NR07218J

Lattice induced crystallization of nanodroplets: the role of finite-size effects and substrate properties in controlling polymorphism

Author(s): Julien Lam, James F. Lutsko
Published in: Nanoscale, Issue 10/10, 2018, Page(s) 4921-4926, ISSN 2040-3364
DOI: 10.1039/c7nr08705e

Tailoring PVDF Membranes Surface Topography and Hydrophobicity by a Sustainable Two-Steps Phase Separation Process

Author(s): Carmen Meringolo, Teresa F. Mastropietro, Teresa Poerio, Enrica Fontananova, Giovanni De Filpo, Efrem Curcio, Gianluca Di Profio
Published in: ACS Sustainable Chemistry & Engineering, Issue Number 6, Issue 8, 2018, Page(s) 10069–10077, ISSN 2168-0485
DOI: 10.1021/acssuschemeng.8b01407

Classical density functional theory, unconstrained crystallization, and polymorphic behavior

Author(s): James F. Lutsko, Julien Lam
Published in: Physical Review E, Issue 98/1, 2018, Page(s) 012604, ISSN 2470-0045
DOI: 10.1103/PhysRevE.98.012604

Solvent-mediated interactions between nanostructures: From water to Lennard-Jones liquid

Author(s): Julien Lam, James F. Lutsko
Published in: The Journal of Chemical Physics, Issue 149/13, 2018, Page(s) 134703, ISSN 0021-9606
DOI: 10.1063/1.5037571

Exploiting Fluoropolymers Immiscibility to Tune Surface Properties and Mass Transfer in Blend Membranes for Membrane Contactor Applications

Author(s): Carmen Meringolo, Teresa Poerio, Enrica Fontananova, Teresa F. Mastropietro, Fiore P. Nicoletta, Giovanni De Filpo, Efrem Curcio, Gianluca Di Profio
Published in: ACS Applied Polymer Materials, Issue 1/3, 2019, Page(s) 326-334, ISSN 2637-6105
DOI: 10.1021/acsapm.8b00105

Optimization of Vapor Diffusion Conditions for Anti-CD20 Crystallization and Scale-Up to Meso Batch

Author(s): Huaiyu Yang, Benny Danilo Belviso, Xiaoyu Li, Wenqian Chen, Teresa Fina Mastropietro, Gianluca Di Profio, Rocco Caliandro, Jerry Y. Y. Heng
Published in: Crystals, Issue 9/5, 2019, Page(s) 230, ISSN 2073-4352
DOI: 10.3390/cryst9050230