Periodic Reporting for period 3 - AMECRYS (Revolutionising Downstream Processing of Monoclonal Antibodies by Continuous Template-Assisted Membrane Crystallization)
Período documentado: 2019-04-01 hasta 2021-03-31
Biopharmaceuticals are large, complex medicinal drugs manufactured using biological sources, which can provide targeted treatment with fewer side effects compared to traditional pharmaceuticals, to give health benefit to patients suffering from conditions ranging from diabetes, cancer, cardiovascular disease, infections, and immune diseases. The scope of biopharmaceutical products includes therapeutic proteins, monoclonal antibodies (mAbs), vaccines, and cell & gene therapies.
The worldwide demand for biologic drugs is growing significantly driven by the increasing number and sales of recombinant mAbs, which is expected to reach globally the market of $179.56 billion in 2025, at a CAGR of 11.9%. To satisfy the requests for mAbs-based therapies, 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 protein A chromatography, usually operated in batch mode. There is an ongoing need to reduce the costs and the risks associated with mAbs development and manufacture in order to address new treatments and extend the availability of these medicines, against a background of tightening and restricted health care budgets. Therefore, the challenge now moves to rise access to such medicines through being able to isolate and purify them at target scale with reduced costs and environmental impact.
On these premises, the main objective of the AMECRYS project is the development of an innovative, continuous, downstream processing for mAbs purification, based on template-assisted membrane crystallization (TMC) as key-unit, leading to the complete replacement of the conventional multi-step batch protein A chromatography-based platform. The expected impact is the decrease of both Capex and O&M costs in mAbs purification, footprint reduction, increased flexibility and high-purity solid dosage formulation with preserved biological activity, which would lead to the generalized reduction of the manufacturing costs and the wider use for mAbs and other biologic drugs, with beneficial effects also in the development of personalized medicine and the treatment of rare diseases with orphan indications.
Following the successful completion of the AMECRYS project, the final objective of the project has been matched, with the construction of a demonstration prototype and its fully operability demonstrated, proving the capability of TMC to produce significant quantities of crystals containing mAb, that can be easily scaled further with comparable results and continuously operated.
The worldwide demand for biologic drugs is growing significantly driven by the increasing number and sales of recombinant mAbs, which is expected to reach globally the market of $179.56 billion in 2025, at a CAGR of 11.9%. To satisfy the requests for mAbs-based therapies, 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 protein A chromatography, usually operated in batch mode. There is an ongoing need to reduce the costs and the risks associated with mAbs development and manufacture in order to address new treatments and extend the availability of these medicines, against a background of tightening and restricted health care budgets. Therefore, the challenge now moves to rise access to such medicines through being able to isolate and purify them at target scale with reduced costs and environmental impact.
On these premises, the main objective of the AMECRYS project is the development of an innovative, continuous, downstream processing for mAbs purification, based on template-assisted membrane crystallization (TMC) as key-unit, leading to the complete replacement of the conventional multi-step batch protein A chromatography-based platform. The expected impact is the decrease of both Capex and O&M costs in mAbs purification, footprint reduction, increased flexibility and high-purity solid dosage formulation with preserved biological activity, which would lead to the generalized reduction of the manufacturing costs and the wider use for mAbs and other biologic drugs, with beneficial effects also in the development of personalized medicine and the treatment of rare diseases with orphan indications.
Following the successful completion of the AMECRYS project, the final objective of the project has been matched, with the construction of a demonstration prototype and its fully operability demonstrated, proving the capability of TMC to produce significant quantities of crystals containing mAb, that can be easily scaled further with comparable results and continuously operated.
As first step in the implementation of the AMECRYS project, production processing for model molecules have been optimized for high final USP titre and conventional DSP purification. The cell lines, process and methods have been fully completed, which enabled to generate batches of feedstock at different purification levels, to supply other works, and the baseline performances in terms of traditional chromatography process assessment, including yield and critical to quality attributes, that were used to assess the relative performance of the AMECRYS concept in subsequent work.
Extensive studies based on a combination of experimental and modelling insights based on calculations, allowed to identify: (i) the general conditions for Anti-CD20 crystallization, (ii) the phase diagram and crystallization kinetics in specific solution conditions, and (iii) the production of suitable nanotemplates (NTs) and membranes for the crystallisation at laboratory scale from cell culture solution with a suitable productivity and operation stability, thus achieving the ultimate AMECRYS targeted objective.
The determination of Anti-CD20 conformational and colloidal stability, structural flexibility, and crystallization propensity were investigated, aiming to postulate a nucleation mechanism. Anti-CD20 crystallization trials performed in microgravity on board of the International Space Station were performed to gain additional insights on the crystallization kinetics.
Multi-campaign data collections at synchrotron light sources confirmed the viability of the studied conditions to produce mAbs crystals, since it allowed to prove the presence of the mAb in the crystal, confirming the crystallization protocols. Diffraction data obtained for Anti-CD20 was not sufficient to solve the crystal structure but it led to gain evidence of the packing and symmetry of molecule in the crystal form. Besides, crystals characterization by a multi-technique approach allowed evaluating morphological and biological properties. In addition, quantitative assessment of crystal properties and crystal yield have been related with operational parameters of the runs performed by the prototype crystallizer through a regression model. Once validated, the model allowed to extrapolate the ranges of operational parameters that guarantee maximum yields and optimal crystal properties.
On the bases of experimental results carried out at laboratory scale, the design of the TMC crystallizer prototype with capacity to treat initial mAb solution volume of several litres were completed. Several runs, demonstrating the ability to produce crystals of Anti-CD20 from cell culture feed material at large-scale, were performed. Process monitoring techniques were implemented and developed throughout the course of the project, proving useful for observing run progression. The TMC process was found to be a viable technique for producing significant quantities of crystallised mAb. The direct scalability of the technique with additional membrane modules was demonstrated.
A techno-economic analysis comparing the conventional approach to monoclonal antibody purification with the alternative approach realised by the AMECRYS project through application of TMC technology has been provided. The techno-economic analysis was developed to act as a guide to understand the potential advantages that achieving and implementing TMC would bring to mAbs production and where technology development is still required. The AMECRYS roadmap identified enablers for TMC supply for biopharmaceutical production.
Actions aimed at implementing efficient communication and dissemination activities, tailoring the information on the AMECRYS project to the needs of multiple audiences, were undertaken pursuant of G.A. articles 28, 29 and 38. A patent application was submitted and a spin-off company was activated (www.seligenda.com). Simulations codes and related published papers are provided as open sources in ZENODO (https://zenodo.org/search?page=1&size=20&q=AMECRYS).
Extensive studies based on a combination of experimental and modelling insights based on calculations, allowed to identify: (i) the general conditions for Anti-CD20 crystallization, (ii) the phase diagram and crystallization kinetics in specific solution conditions, and (iii) the production of suitable nanotemplates (NTs) and membranes for the crystallisation at laboratory scale from cell culture solution with a suitable productivity and operation stability, thus achieving the ultimate AMECRYS targeted objective.
The determination of Anti-CD20 conformational and colloidal stability, structural flexibility, and crystallization propensity were investigated, aiming to postulate a nucleation mechanism. Anti-CD20 crystallization trials performed in microgravity on board of the International Space Station were performed to gain additional insights on the crystallization kinetics.
Multi-campaign data collections at synchrotron light sources confirmed the viability of the studied conditions to produce mAbs crystals, since it allowed to prove the presence of the mAb in the crystal, confirming the crystallization protocols. Diffraction data obtained for Anti-CD20 was not sufficient to solve the crystal structure but it led to gain evidence of the packing and symmetry of molecule in the crystal form. Besides, crystals characterization by a multi-technique approach allowed evaluating morphological and biological properties. In addition, quantitative assessment of crystal properties and crystal yield have been related with operational parameters of the runs performed by the prototype crystallizer through a regression model. Once validated, the model allowed to extrapolate the ranges of operational parameters that guarantee maximum yields and optimal crystal properties.
On the bases of experimental results carried out at laboratory scale, the design of the TMC crystallizer prototype with capacity to treat initial mAb solution volume of several litres were completed. Several runs, demonstrating the ability to produce crystals of Anti-CD20 from cell culture feed material at large-scale, were performed. Process monitoring techniques were implemented and developed throughout the course of the project, proving useful for observing run progression. The TMC process was found to be a viable technique for producing significant quantities of crystallised mAb. The direct scalability of the technique with additional membrane modules was demonstrated.
A techno-economic analysis comparing the conventional approach to monoclonal antibody purification with the alternative approach realised by the AMECRYS project through application of TMC technology has been provided. The techno-economic analysis was developed to act as a guide to understand the potential advantages that achieving and implementing TMC would bring to mAbs production and where technology development is still required. The AMECRYS roadmap identified enablers for TMC supply for biopharmaceutical production.
Actions aimed at implementing efficient communication and dissemination activities, tailoring the information on the AMECRYS project to the needs of multiple audiences, were undertaken pursuant of G.A. articles 28, 29 and 38. A patent application was submitted and a spin-off company was activated (www.seligenda.com). Simulations codes and related published papers are provided as open sources in ZENODO (https://zenodo.org/search?page=1&size=20&q=AMECRYS).
AMECRYS project demonstrated the viability of the TMC process to recover in solid state and at gram scales Anti-CD20 - a full-length mAb - from various levels of solution purities; therefore, TMC is demonstrated as cheaper and easily scalable alternative to chromatographic techniques. This is expected to drive further interest, research and investment into this process and similar processes, providing a path to a potentially lower-cost, sustainable and disruptive alternative to current chromatography-based mAb manufacturing techniques, which will help in a broader use of biologic drugs with the consequent benefits on the health systems and society.