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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - AMECRYS (Revolutionising Downstream Processing of Monoclonal Antibodies by Continuous Template-Assisted Membrane Crystallization)

Teaser

Summary

The worldwide demand for therapeutic proteins is growing significantly driven by the increasing number and sales of recombinant monoclonal antibodies (mAbs), now a >$90 billion market, expected to grow up to $150 billion by 2020. To satisfy this request, industrial production of mAbs have been optimized for higher titres by significant improvements in cell culture media and recombinant technologies in the upstream processing (USP). However, this development created a bottleneck in the following downstream (DSP) stage, which currently relies on complex and expensive separations, primarily based on chromatography, usually operated in batch mode. With the therapeutic potential of mAbs well established for the treatment of several diseases, including cancer, the challenge now moves to rise access to such medicines through being able to isolate and purify them at target scale with reduced costs. On these premises, the overall objective of the AMECRYS Project is the development of an innovative, continuous, DSP for mAbs purification, based on Template-Assisted Membrane Crystallization as key-unit, leading to the complete replacement of the conventional multi-step batch chromatography-based platform. The central idea of the AMECRYS strategy is based on the combination of two innovative concepts: 1) membrane-assisted crystallization, a radically new separation technology based on the use of hydrophobic membranes, which allows the accurate control of supersaturation by solvent removal in vapour phase through the porous membrane, and 2) template-assisted nucleation by engineered 3D-nanotemplates (NTs), to enable the selective crystallization of structurally complex mAbs molecules from multicomponent solutions. In addition, paradigm shift from batch to continuous operation, to enhance process efficiency and product quality, is the further key tool in the proposed research approach. The expected impact is the decrease >60% for both Capex and O&M costs in mAbs DSP, 30-fold footprint reduction, and high-purity solid dosage formulation with preserved biological activity, that would lead to the generalized reduction of the costs for anti-cancer mAbs.

Work performed

As first step in the implementation of the AMECRYS Project, production processing for model full-length mAb and fragment dAb have been optimized for high final USP titre and conventional DSP purification. The optimized processes, other than generating materials to feed other work packages, will be used as reference to be compared with the developed membrane-based DSP.
An extensive experimental campaign has been implemented to assess the crystallization viability of available mAbs solutions, to identify general conditions for crystallization, and to understand molecular behaviour in several solution compositions.
NTs have been synthesized to range of porosity and surface chemistry introduced. These NTs have been tested in mAb crystallisation work with positive results.
A sustainable method has been developed and optimized to produce customized polymeric membranes for membrane-assisted crystallization processes, with tailored porosity, surface roughness and hydrophobicity. The protocol for membrane preparation at laboratory scale has been successfully transferred to pilot industrial plants.
A device integrating the best performing polymeric membranes have been successfully designed and fabricated by using soft lithography and mechanical clamping of microfluidic chips. The microfluidic system is to be used to investigate a wide range of mAbs crystallization conditions at the scale of a few nanoliters in high throughput screenings, implementing the â€œlab-on-a-chipâ€ concept.
Techniques for measuring template-assisted nucleation rates of proteins in solutions have been developed in view of screening tests by microfluidic membrane-assisted crystallization device, providing evidences on the effect of nanoparticles to enhance the nucleation rates for model proteins.
3D-Density Functional Theory code has been developed and applicate to study interaction between individual mAbs molecules and nanoscale pores, to support NTs optimization. Extraction of a coarse-grained description of the induced pore-molecular interaction due to water, for use in subsequent studies of mAbs crystallization in pores, has been performed. Molecular dynamics and DFT studies of the effect of substrate lattice mismatch on crystal polymorphism were accomplished.
3D-Monte Carlo simulation of the Ising model was implemented to investigate collective effects occurring in first-order liquid/solid phase transition, to enhance knowledge on the membrane-based crystallization process. A theoretical approach based on the extension of classical nucleation theory (CNT) was developed to study the influence of physical-chemical membrane properties on mAbs crystallization, to support membrane optimization work.
Structural analysis of protein shape and homogeneity by small angle X-ray scattering experiments and characterization of crystals by X-ray diffraction at synchrotron light source, confirmed the viability of the studied conditions to produce mAbs crystals.
Efforts were done by AMECRYS Consortium to define the data management and the communication, dissemination and exploitation plans and to translate efficiently them in practice.

Final results

For the template synthesis effort, a simplified one â€“pot strategy is currently being investigated.
Polymeric membranes specifically designed for membrane-assisted crystallization processes were produced using a combination of phase separation techniques and a green solvent. This outcome, which meet environmental and health regulations, could open new perspectives in the industrial membrane production field in the horizon of the transition from waste-intensive to green membrane production routes.
A device mimicking membrane-assisted crystallization at the microfluidic scale has been designed and fabricated for the first time, thus enabling the implementation of the â€œlab-on-a-chipâ€ concept for further optimizations and the upscale of crystallization conditions.
Suitable theoretical tool based on the probability distribution of induction times have been developed to evaluate heterogeneous nucleation/growth rate effects as a function of nanotemplates properties and impurity level.
Coarse-grained models for the interaction of mAbs and small pores were developed as the first step of a multiscale study of mAbs crystallization. These will be used to study crystallization accounting for the induced interactions with the substrate without having to explicitly model water molecules. The DFT code developed for these calculations is the only one known to be able to describe crystallization in arbitrary geometries. These tools enable to study crystallization in pores so as to provide guidance on the optimization of the template properties.
The Ising model implemented for a porous medium, in conjunction with the extended CNT, will drive the design and performance optimization of NTs and membranes for an enhanced and unprecedented control of crystallization kinetics of mAbs. The correlation between heterogeneous nucleation rate and induction time allows an appropriate tuning of simulation parameters on an experimental basis. Further studies will allow at estimating the energy barrier to nucleation as a function of roughness, solution velocity and impurity concentration, a critical issue in mAbs crystallization from multi-component solutions.