Microtools for downstream processing

Lead Research Organisation: University College London
Department Name: Biochemical Engineering

Abstract

Biocatalysis uses enzymes to enable greener production process. To fully harness the advantages of this technology, it is
necessary to understand not only the reaction steps, i.e. the steps where the desired product is produced, but also those
steps where the desired product is separated from by-products and residual (i.e. unreacted) substrate. This typically
requires several purification steps. And it is often the cost of these purification steps which determine the economic viability
of such a biocatalytic process. To rapidly understand the process and operating conditions of these purification steps -
which in turn determines the cost and viability of the whole process - it is necessary to rapidly analyse different purification
step 'candidates' and select the most efficient one(s). The more rapidly such an investigation can be conducted, the faster
critical decisions about the economic viability can be taken, and the faster one can bring a product to the market.
Here, we propose to use the advances in microfabrication to create miniaturised devices which can mimic the larger ones
typically used for purification, so that the different steps can be evaluated more rapidly. Miniaturisation will mean a more
cost-effective use of resources to undertake the investigations. Miniaturisation will also mean that particular effects can be
studied with greater detail. We envisage for example to study the effects of how product molecules 'transition' from one
solvent (e.g. aqueous one) to another solvent (e.g. an organic one), and under what conditions they do so best, in a
process that is called liquid-liquid extraction. We will then use this information to determine how larger systems (ie the
systems where the products will effectively be produced and purified) should be designed to improve their yield. Such an
improvement in yield should make biocatalysis an even greener process and make it also economically more viable.

Technical Summary

Biocatalysis can replace traditional chemical catalysis based procesess resulting in a greener production process. However, the reaction step of such a biocatalysis process needs to be integrated with one or several purification steps to recover products and/or remove inhibitory substances. The economical feasibility of a bio-based process is typically dependent to a large extent on the efficiency and the cost of the subsequent separation steps. Consequently, a series of separation process candidates have to be investigated with the purpose of achieving the most efficient downstream processing configuration. The more rapidly such an investigation can be conducted, the faster critical decisions about economical feasibility of a bio-based process can be taken, and the faster one can bring a product on the market. The main objective of this project is to establish a microscale downstream processing toolbox which can be used for rapid and high content screening or for process development. The following main results are expected to be obtained:
1) New and existing building blocks of a miniaturised downstream processing toolbox will be developed, standardized and evaluated. The toolbox will include separation processes based on extraction, pervaporation, adsorption, absorption and membrane technology.
2) The toolbox will be supplemented by advanced on-line measurements and rational experimentation protocols for rapid and accurate generation of data for downstream process characterization and development.
3) Scaling-up of the results obtained with the miniaturised downstream processing toolbox will be evaluated on the basis of lab-scale and pilot-scale experiments.
4) Separation process sequences will be developed for two challenging and industrially relevant case studies (transketolase, transaminase) in order to demonstrate the practical applicability of the miniaturised downstream process development toolbox.

Planned Impact

Production of chiral amines is important in the fine chemical industry, specifically for pharmaceutical applications.Transaminase-based processes, for example, are attractive for the pharmaceutical industry due to their capability of producing optically pure products. Time is money in the pharmaceutical industry, and any technological development that leads to shorter process development time - as is the case with the proposed miniaturised downstream processing toolbox - potentially has a major impact on the competitiveness of the traditional research-based pharmaceutical industry due to the
limited patent life time. Moreover, the fact that the microfluidic separation devices in the toolbox are all envisaged to be compatible with continuous process operation also fits nicely in the current trend in the pharmaceutical industry to replace relatively large batch reactors by continuous processes at micro-scale or meso-scale. Moving from batch towards continuous pharmaceutical manufacturing is one of the key strategies in the pharmaceutical industry for improving safety and product quality while decreasing waste generation and manufacturing costs. Development of continuous separation
methods at micro-scale is considered to be one of the current bottlenecks for the future development of such continuous micro-scale reaction sequences. Also here, the project results are expected to make a major impact. For process development, novel analytical methods are required. The proposed development of a bespoke UV absorbance based, dual-wavelength detection system (subcontracting to Paraytec) for the rapid detection and determination of product and substrate concentrations will provide valuable insight on how to optimise operation of liquid-liquid extraction systems. It will also directly offer assistance to an SME company in the UK who will receive funds to develop a new piece of equipment, and via the proposed research and the several collaborators in Europe, receive direct input on the performance of the device.

Publications

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Tan CK (2015) Electromagnetic stirring in a microbioreactor with non-conventional chamber morphology and implementation of multiplexed mixing. in Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)

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Raimes W (2017) Transfection in perfused microfluidic cell culture devices: A case study. in Process biochemistry (Barking, London, England)

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Marques MP (2017) Bioprocess microfluidics: applying microfluidic devices for bioprocessing. in Current opinion in chemical engineering

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Marques M (2019) Microfluidic devices towards personalized health and wellbeing in Journal of Chemical Technology & Biotechnology

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Marques M (2016) Stem Cell Manufacturing

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Lawrence J (2013) Microfluidic multi-input reactor for biocatalytic synthesis using transketolase. in Journal of molecular catalysis. B, Enzymatic

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Kirk TV (2016) Quantification of the oxygen uptake rate in a dissolved oxygen controlled oscillating jet-driven microbioreactor. in Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)

 
Description A device for liquid-liquid extraction, stable in a broad range of flow rates/dilution rates, has been characterised. We have developed in an associated PhD project a flocculation device. We have, also in an associated PhD project, developed a filtration system for liposome purification (submitted for publication).
We are exploring integration of dual-wavelength UV absorbance measurement with microfluidics.
We are awaiting delivery of an all quartz device of the liquid-liquid-extraction device to enable and test dual-wavelength UV absorbance measurements of substrate/product compounds. UPDATE: We did receive the all quartz device, but have had to wait for delivery of a cartridge to hold the device. We are expecting the company Denz Biomedical to provide us with a holder that is leak-tight and we will use doctorate or master students to finalise this part of the study.
Exploitation Route If established, the tool to measure in the UV will facilitate rapid real-time detection of small molecules from minute volumes.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The grant started one year ago. The company Paraytec benefits from this interaction with us by developing their analytical techniques further for integration with microfluidics. The company developed a new equipment, called D200, together with us in this grant
First Year Of Impact 2014
Sector Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
 
Title Assessing Downstream Processing Unit of Operations at Microfluidic Scale 
Description In order to assess microfluidic devices for downstream processing unit operations (DSP), we have investigated how to create trains of unit operations. This is still ongoing, but we understand the challenges better, such as matching of flow rates, necessity of integrating in-line or on-line analytics to make best use of microfluidic tools, avoiding undesired interactions between a unit operation placed downstream to the upstream unit operation so that assessment of the individual steps can occur, etc. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact We are looking into writing a review article once we have published the results from the individual unit operations realised at the microfluidic scale, notably the liquid-liquid extraction tool in continuous flow. 
 
Description 1-Day Teaching Seminar in Microfluidics 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Together with an industrial partner in microfabrication, I taught a one day seminar in microfluidics ahead of the Lab on Chip conference in Mumbai, India, 2018.
Year(s) Of Engagement Activity 2018
URL https://selectbiosciences.com/conferences/index.aspx?conf=MLOAC18
 
Description 17th European Biotechnology Congress (ECB2016) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Short talk on the development and integration of microfluidic devices for the production of chiral amines. Discussion afterwards and was invited to write a manuscript for the conference supported journal (New Biotechnology) and to give a lecture in Braunschweig University.
Year(s) Of Engagement Activity 2016
URL http://ecb2016.com/
 
Description 1st International Training Course - Application of microbioreactors (MBR) in bioprocess development. September 24-28, 2018, TU Braunschweig, Germany http://www.eurombr.nu/ 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact 5-day training course with lecture programme and laboratory hands-on
Topics:
- Microfabrication
- Microfluidics, Transport phenoma, Fluid flow - Sensors and Inline-Analytics
- Enzyme immobilisation
- Biocatalysis
- Cultivation
- Modeling and Design of microfluidic processes
Year(s) Of Engagement Activity 2018
URL http://www.eurombr.nu/
 
Description Biochemical Engineering Conference (ESBES2014) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presented ("Downstream Processing Toolbox: microscale Liquid-Liquid extraction") during the conference ESBES 2014. Level of engagement: low
Year(s) Of Engagement Activity 2014
URL http://esbes-ifibiop-lille2014.com/
 
Description Microfluidics Conference (IMTB2015) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Lecture given by team member on the implementation of microfluidics in bioprocess development. The engagement level was high
Year(s) Of Engagement Activity 2015
URL http://imtb2015.fkit.hr/
 
Description Protein and Antibody Engineering Conference (PEGSEurope) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Invited lecture by one of my team member in an International Conference. The topic was on the use of microfluidics for process development. The audience was mainly from Industry and the engagement level was high.
Year(s) Of Engagement Activity 2015