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.
Publications
Mahendrarajah K
(2011)
A high-throughput fluorescence chemical denaturation assay as a general screen for protein-ligand binding.
in Analytical biochemistry
Sagar DM
(2013)
Optically induced thermal gradients for protein characterization in nanolitre-scale samples in microfluidic devices.
in Scientific reports
Hales JE
(2021)
Proof-of-concept analytical instrument for label-free optical deconvolution of protein species in a mixture.
in Journal of chromatography. A
Gaudet M
(2010)
Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale.
in Protein science : a publication of the Protein Society
Hales JE
(2019)
Virus lasers for biological detection.
in Nature communications
| 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 | TRACK TENSIONING SYSTEM |
| Description | A tensioning system for a tracked vehicle. The present invention provides for adjusting tension of a track by moving the sprocket relative to the rollers in a substantially vertical direction. The system comprises a sprocket assembly, a roller assembly, a track, and a tension adjustment assembly. The tension adjustment assembly includes a tension bolt threaded through a tension nut that is secured to the sprocket assembly. The bolt threads through the nut and contacts the roller assembly. As the bolt is threaded or unthreaded, the sprocket assembly is moved relative to the roller assembly in a substantially vertical direction. As the assemblies move relative to each other, tension in the track is resultantly adjusted. |
| 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. |