A new microfluidic tool for rapid analysis of protein stability and integrity in bioprocesses

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

Abstract

Analysis of protein stability is currently too slow and requires too much of an exceedingly valuable biopharmaceutical to be useful in guiding bioprocess development or control. Introducing the first microfluidic method for protein stability testing will reduce sample use and cost of analysis by up to 108-fold over microwell-based analysis. Combined expertise from biochemical engineering and the London Centre for Nanotechnology will enable this analysis with parallelism for up to 1000 samples per day. The new generation of protein-based medicines has rapidly become a $30billion-a-year industry addressing previously untreatable diseases. They have the potential for much further growth but a principal constraint is the high cost of the manufacturing methods required to preserve the structural integrity of proteins with limited stability. The ability to perform rapid and parallel protein stability characterisation experiments, at the microfluidic scale, is essential to enable: a) the rapid optimisation of therapeutic protein formulations; and b) the real-time monitoring of protein product quality in process-, microwell- and microfluidic scale bioprocess development experiments. Our preliminary research has demonstrated protein stability determination using fluorescence measurements at the microwell scale (Aucamp et al., 2005). The aims of this proposal are to a) explore the fundamentals that impact on measurement accuracy and sensitivity at the microfluidic scale, so as to significantly decrease the sample volumes required for protein stability measurement; b) establish a microfluidic denaturation technique; c) overcome the challenges that will enable broad application to bioprocessing and formulation of biopharmaceutical protein products.

Technical Summary

Analysis of protein stability is currently too slow and requires too much of an exceedingly valuable biopharmaceutical to be useful in guiding bioprocess development or control. Introducing the first microfluidic method for protein stability testing will reduce sample use and cost of analysis by up to 108-fold over microwell-based analysis. Combined expertise from biochemical engineering and the London Centre for Nanotechnology will enable this analysis with parallelism for up to 1000 samples per day. The new generation of protein-based medicines has rapidly become a $30billion-a-year industry addressing previously untreatable diseases. They have the potential for much further growth but a principal constraint is the high cost of the manufacturing methods required to preserve the structural integrity of proteins with limited stability. The ability to perform rapid and parallel protein stability characterisation experiments, at the microfluidic scale, is essential to enable: a) the rapid optimisation of therapeutic protein formulations; and b) the real-time monitoring of protein product quality in process-, microwell- and microfluidic scale bioprocess development experiments. Our preliminary research has demonstrated protein stability determination using fluorescence measurements at the microwell scale (Aucamp et al., 2005). The aims of this proposal are to a) explore the fundamentals that impact on measurement accuracy and sensitivity at the microfluidic scale, so as to significantly decrease the sample volumes required for protein stability measurement; b) establish a microfluidic denaturation technique; c) overcome the challenges that will enable broad application to bioprocessing and formulation of biopharmaceutical protein products.
 
Description An optical configurations for fluorescence detection of samples flowing in microcapillaries was established, characterised and optimised for sensitivity, signal-to-noise, accuracy and dynamic range. Improvements were obtained through exploration of both physical factors (laser type/power, lenses, gratings, filters, dichroic mirrors, collimators, PMT type, capillary type), and signal processing algorithms. A working optical bench and investigation of fundamentals on measurement sensitivity and accuracy was delivered on time using BSA and FKBP proteins.



A new confocal stage mounting for accurate alignment of an IR laser to induce heating of buffers within a microfluidic channel was created and a time-dependent 3D temperature profile determined by confocal microscopy using a temperature sensitive fluorescent dye. This provided accurate information on the temperature gradients, rate of thermal diffusion and the effective beam width of the IR laser in the channel. Various configurations of IR power and capillary flow rates have been investigated to control heating rate, create temperature gradients, and constant temperature profiles within the channel. The system was simulated in Comsol to improve fundamental understanding and to assist the design of improved microfluidic chips with integrated IR induced heating at single or multiple points along the channel. Temperature gradients with a 70°C range across 0.1-1 mm channel lengths were obtained, as well as constant temperatures of 20-98°C across 0.5mm, which can be established in ms timescales.

Throughput of data collection and processing was significantly improved with emissions for each laser pulse (at 1kHz) collected to a PC at 10E8 datapoints per second and processed in Labview. We developed signal processing algorithms to remove time-dependent variation of the laser pulse and to reduce the prompt scattered light signal. The detection limits for fluorescence intensity in the capillary (10E5 molecules) were compared to our microplate-based method (10E11 molecules). Equilibrium urea denaturation with the new technique was applied to BSA, FKBP-12 wt and an FL99 mutant to demonstrate application to different protein types and to an engineered variant with lower stability. The latter two were tested also in the presence of the drug rapamycin which binds to FKBP-12. These gave accurate unfolding dG and rapamycin Kd values consistent with literature measurements. This work has now been submitted for publication. This technique has great potential for both high-throughput drug screening in addition to biopharmaceutical formulation.
Exploitation Route The technique can be employed in standalone analytical instruments for rapid protein library screening of stability, or potentially for ligand affinity in microfluidic systems.
Sectors Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The device has been further improved via BBSRC Follow-on-funding, as well as a subsequent PhD project, to enable full feedback control on the IR-induced temperature gradient. The device is being further modified in BBSRC BRIC project BB/K011162/1 to include additional fluorescence modalities and enable it to be coupled to LC systems for detailed on-line monitoring of protein heterogeneity. This will be directly useable by the bioprocess industry. The previous system was evaluated by a UK company for assessing protein quality during crystallisation trials. More recently, the new techniques developed in this project have become the focus of an EPSRC EngD project in collaboration with Pall Europe.
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Steering Group Member of the BBSRC Bioprocess Research Industry Club (BRIC)
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
Impact The BRIC committee oversees research projects funded at the academic industry interface in bioprocessing, training events for PhD students and early careers researchers, and network events for the wider community.
 
Description BBSRC BRIC
Amount £430,000 (GBP)
Funding ID BB/K011162/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2013 
End 10/2016
 
Description BBSRC US Partnering
Amount £43,192 (GBP)
Funding ID BB/K021354/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2013 
End 12/2016
 
Description EPSRC Formulation
Amount £2,961,745 (GBP)
Funding ID EP/N025105/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 09/2021
 
Description Future Manufacturing Hubs
Amount £10,000,000 (GBP)
Funding ID EP/P006485/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 01/2017 
End 12/2024
 
Description High-throughput directed evolution to engineer thermostable therapeutic proteins
Amount € 195,454 (EUR)
Funding ID 795539 
Organisation Marie Sklodowska-Curie Actions 
Sector Charity/Non Profit
Country Global
Start 07/2018 
End 07/2020
 
Title Online monitoring tool for protein chromatography 
Description We have developed a new optical detection system capable of detecting the presence of, and quantifying the relative contributions of multiple protein species within a single sample, including the analysis of chromatographic peaks in real time. This will be a valuable research tool for protein purification and for protein formulation, and has the potential to become a useful online bioprocess analytical technique for PAT. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Impact is pending. Currently being patented and re-engineered into a demonstration unit to take to companies. 
 
Description Industry partnership for materials from UCB Pharma 
Organisation UCB Pharma
Country United Kingdom 
Sector Private 
PI Contribution We analysed the aggregation behaviour of a Fab protein obtained from UCB Pharma under a wide range of conditions. This has provided general information on the aggregation mechanisms, formulations and stabilising factors in Fab molecules, useful for therapeutic formulations and bioprocessing.
Collaborator Contribution UCB Pharma provided an E. coli strain that produces the A33 Fab fragment. They also provided advice for its expression and analysis.
Impact Scientific outputs on formulation of Fab and understanding of aggregation mechanisms. The access to this material has also enabled us to develop novel analytical techniques in other grants. The partnership has also led to three CASE-PhD collaborations with UCB in 2017.
Start Year 2011
 
Description Materials and facility access from NIBSC 
Organisation National Institute for Biological Standards and Control (NIBSC)
Country United Kingdom 
Sector Public 
PI Contribution We analysed the aggregation behaviour, and stability of a GCSF protein and mutants of this, obtained from NIBSC under a wide range of formulations. This has provided general information on the aggregation mechanisms, formulations and stabilising factors in GCSF molecules, useful for therapeutic formulations and bioprocessing.
Collaborator Contribution NIBSC provided an E. coli strain that produces the GCSF. They also provided advice for its expression and analysis. They also provided access to NMR, pilot-scale freeze dryers, Karl Fischer analysis, biological potency assays, and Mass spectrometry.
Impact This partnership has involved one EPSRC EngD, one BBSRC PhD, and two EPSRC CDT PhD students, formal partnership and strategic advice for the EPSRC Formulation project, Centre for Innovative Manufacturing and Future Targeted Healthcare Manufacturing Hub, as well as attendance by NIBSC at Hub events and workshops. The partnership is multi-disciplinary, bringing together protein biophysics (UCL), protein engineering (UCL), protein aggregation (UCL), freeze-drying (NIBSC), biological assays (NIBSC), NMR (NIBSC) and Mass spectrometry (NIBSC). Outputs therefore include, 3 graduated PhD/EngDs, 1 PhD currently running, 3 PDRAs receiving training and carrying out work in NIBSC facilities, 5 co-authored publications.
Start Year 2007
 
Title Microfluidic Method for Measuring Molecular Stability 
Description Patent filed and maintained covering a technique for measuring protein stability from intrinsic fluorescence in microfluidic samples. Useful in drug discovery, protein formulation applications 
IP Reference WO2010014995 
Protection Patent granted
Year Protection Granted 2010
Licensed No
Impact Holding patent has enabled engagement for further development of the work, currently with GSK.