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

Periodic Reporting for period 2 - AMECRYS (Revolutionising Downstream Processing of Monoclonal Antibodies by Continuous Template-Assisted Membrane Crystallization)

Reporting period: 2017-10-01 to 2019-03-31

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 expected impact is the decrease of both Capex and O&M costs in mAbs DSP, footprint reduction, and high-purity solid dosage formulation with preserved biological activity, that would lead to the generalized reduction of the manufacturing costs for anti-cancer mAbs.
Complete development and transfer of both mAb and dAb processes (USP, DSP, analytics) have finalized and baseline performance in terms of process assessment, including yield and critical to quality attributes, was collected.
Nanotemplates (NTs) have been synthesized to range of porosity and optimized to specific requirements for mAb crystallisation from several solution composition with improved results.
A sustainable method has been optimized to produce customized polymeric and blend membranes for membrane-assisted crystallization. The protocol for membrane preparation at laboratory scale has been successfully transferred to pilot industrial plants. A pristine membrane was manufactured in a continuous roll-to-roll pilot-industrial equipment.
Polymeric and blend membranes were successfully functionalized by several strategies. Screening and selection of suitable membranes for membrane crystallization of mAb was performed and protein was crystallized in several working conditions, starting from solutions of increasing complexity.
Three different multilevel microfluidic devices, each with specific features were developed and used to investigate a wide range of mAb crystallization conditions at the scale of a few nano-, micro- or milli-litres, also in high throughput screenings.
Phase diagram of mAb were defined and study on heterogeneous nucleation in microfluidic experiment commenced.
SAXS experiments have allowed to quantify the structural flexibility of mAb in solution, and to study the effect of several additives on structural flexibility and stability upon temperature changes.
The determination of protein conformational and colloidal stability and crystallization propensity was evaluated by CD, DLS and SLS.
Computational studies concerning the optimization of the NTs were accomplished through: i) MD simulations of water to calibrate ftDFT results; ii) ftDFT calculations concerning solvent-induced forces; iii) Construction of Monte Carlo code with Umbrella Sampling for determining nucleation free energy barriers; iv) Calculations of nucleation barriers for different pore geometries to determine optimal configurations.
A theoretical approach was developed to: i) estimating theoretical parameters as input for simulations by analysing most relevant characteristics of the membranes (pore size, roughness, contact angle); ii) studying aspects related to mAb crystallization kinetics in order to support the activity of prototype scale-up. A kinetic Monte Carlo (kMC) code with Forward Flux Sampling (FFS) to simulate crystallization was written and implemented.
Optimal crystallization conditions for dAb and mAb, aiming at improving X-ray diffraction, have been inferred. Structural characterization of crystals has been carried out by multi-campaign at synchrotron light source. Suitable additives have been found to have a positive effect on crystal size and crystal order. Multivariate methods to process optical images from crystallization trials are under development.
Process flow diagrams for 250 mL and 10 L feed volume plants, including crystals recovery stage, have been performed and technical scenarios defined in terms of residence time, flowrate, concentration polarization and fouling, mechanical stress, membranes geometry.
Baseline performance from traditional chromatography for mAb’s and dAb have been determined. A cost-of-goods analysis of the traditional chromatographic purification versus the developed method for both molecules of interest, were commenced.
Work has started to develop a technology roadmap, initially focussed on baselining conventional DSP processing and identification of key aspects that would need to be addressed by the proposed technology.
We have achieved the main objective of the project, i.e. proving the possibility of selectively crystallization of a full-length mAb by membrane –assisted technology and with the support of NTs, thank to a combination of experimental and modelling insights.
Customized functionalized membranes suitable for MCr process were developed and successfully used for the crystallization of monoclonal antibody from solutions at different levels of purity, thus proving the ultimate AMECRYS project main idea.
Structural characterization of mAb by SAXS data has highlighted a high conformational flexibility of Anti-CD20 in solution, which could be the reason for the poor diffraction properties of mAb crystals. Investigations about the effect of several additives on structural flexibility will be used to explore new crystallization conditions for obtaining more ordered mAb crystals.
In terms of computational work, the results obtained make a firm prediction for the optimal NTs properties and show the unexpected importance of trying to minimize grain boundaries forming. The secondary importance of the substrate lattice parameter on polymorph selection has been uncovered. Furthermore, model predictions concerning protein crystallization in nano-confined environment showed the potential to drive the manufacturing process towards membranes with better performance in terms of nucleation kinetics.