Elucidating aggregation mechanisms in antibody fragment-based therapeutics to improve their manufacturability
Lead Research Organisation:
University College London
Department Name: Biochemical Engineering
Abstract
Recent therapies for age-related diseases such as rheumatoid arthritis, macular degeneration, Crohn's disease, and some cancers are engineered forms of biological protein molecules called antibodies that form part of our own natural immune system. Such therapeutic proteins are being derived increasingly from simpler fragments of these antibodies with the hope that this will improve their behaviour in the body, reduce the frequency of injections required, allow them to target new regions of the body, and also allow them to be combined with other biological molecules without becoming too large or unstable. However the manufacturing of therapeutic proteins is extremely challenging due to their delicate and complex nature. Manufacturing processes aim to separate the protein molecules from the rest of the cellular components in which they were synthesised, to obtain extremely pure therapeutic material that is suitable for use in humans as a therapy. However, the processes available for large-scale manufacture place a great deal of stress on the protein due to changes in temperature or acidity, the addition of salts, the use of mechanical agitation, rapid changes in the rate of flow through machinery, and the interaction of proteins with air bubbles. This frequently causes the protein to deform slightly and to subsequently stick together to form tiny particles called aggregates. While these are often not visible to the naked eye, their presence in therapies can be hazardous to patients as they may cause severe inflammation and potentially more deadly immune responses. Therefore, one of the key challenges that the bioprocess development and therapeutic protein manufacturing industries would like to address is to be able to either predict the conditions that cause a protein to aggregate, or to increase their robustness so that they aggregate less frequently during their manufacture. We aim to carry out and demonstrate a suite of rapid experimental measurement techniques that allow a new therapeutic protein to be evaluated quickly for the conditions in which they have a greater tendency to form aggregates. The conditions to be tested will be same as those used throughout bioprocess manufacturing, and will therefore allow bioprocess engineers to rapidly identify the conditions in which their manufacturing processes will be best operated, or whether the protein is unlikely to be manufacturable. Having quickly determined the conditions at which the protein begins to form small and soluble aggregates, we will also carry out a detailed molecular analysis of the structure of proteins at these conditions and also those either side in which the protein remains in solution as a single molecule, and where it forms larger aggregates. This will allow us to see what changes in the protein structure occur before, during, and after the aggregation is initiated and therefore deduce which events are on the critical path to aggregate formation. Having achieved this we will then be able to target changes to the protein called mutations that will interfere with and suppress the aggregation process. Finally, by comparing a related set of therapeutic antibody fragment proteins, we will gain insight into those factors that are specific to each protein type, and those that occur more generally and hence become useful targets for the future engineering of therapeutic protein designs. It will also allow others to improve their mathematical modelling methods that aim to predict whether proteins will aggregate under certain conditions.
Technical Summary
Biopharmaceuticals are derived increasingly from Fab and ScFv antibody fragments. A major challenge for their manufacture is their tendency to aggregate during bioprocessing due to increasing protein concentrations, low pH, high salt, shear or surface effects in centrifugation, filtration, chromatography or viral inactivation steps. Aggregates are hazardous to patients and yet their necessary removal leads to longer and more costly bioprocess development times. Characterisation of the molecular events that occur as antibody-fragments aggregate under typical bioprocess conditions will provide a fundamental basis for designing more robust antibody fragment scaffolds and for improving current predictive tools. Several protein engineering efforts have selected antibody fragments resistant to pH or heat induced aggregation using libraries that vary only in the CDR loops, though these regions are also required for the selection of antigen binding affinity. Sequence algorithms have improved the prediction of sites with high aggregation propensity for small globular proteins by combining secondary-structure propensity (an indirect measure of local protein unfolding and beta-sheet forming potential) with amino-acid hydrophobicity and charge indices. However, these do not yet include the role of specific protein-protein interactions or the relative conformational positions of multiple domains in larger proteins which can both influence the initial formation of small soluble oligomers prior to aggregation. We aim to characterise the protein conformations, global and local unfolding, and specific interactions that occur within protein monomers and the small soluble aggregates formed as bioprocess conditions are changed. We will use a combination of microscale experiments, protein engineering, AUC, NMR and SAXS combined with constrained protein modelling. Identifying key protein sites and structural effects will fundamentally improve the design of robust therapeutic scaffolds.
Planned Impact
'WHO WILL BENEFIT FROM THE RESEARCH?' UK-based companies within the BRIC community will benefit from research which allows them to more effectively predict lead antibody-fragment candidates that are likely to aggregate during bioprocess development and manufacturing scale up, using high-throughput tools for a 'screen early - fail early' approach. A propensity to aggregate directly increases manufacturing process development costs due to time spent on finding bioprocesses that minimise it, and also due to absorbing the cost of failing to find a suitable bioprocess with earlier lead candidate molecules. Characterisation of aggregation under bioprocessing conditions for a variety of antibody-fragment based molecules will provide a broader and better mechanistic understanding of how aggregation is initiated, and ultimately how protein engineering can be used to intervene and produce scaffolds that are generally more robust to aggregation. This will in turn decrease the time, cost and risk of product development. Potential patients will benefit because the research will significantly aid reduction in development times of antibody-fragment based medicines, which is particularly crucial for those addressing previously unmet clinical needs. Benefits to the NHS relate to the possibility of constraining costs. Proteins are innately complex and labile so that bioprocess development times and hence costs tend to be high. The capacity to treat conditions such as rheumatoid arthritis much more effectively in ageing populations is vital but it still poses a problem with respect to stretched NHS budgets. 'HOW WILL THEY BENEFIT FROM THE RESEARCH?' The research will fundamentally characterise the early structural events that lead to antibody-fragment aggregation at a wide range of bioprocess conditions. This will reveal commonalities between several antibody-fragment types, as well as their idiosyncrasies. This improved understanding will better direct protein engineering and predictive tools in future for industry to minimise the aggregation propensity of new molecules with a significantly higher consistency. The research will also demonstrate simple microscale screening tools to rapidly identify the range of solution conditions in which bioprocesses can be operated safely without causing protein aggregation. These will be examined as indicators for molecular robustness at a broader range of in-process stresses including shear, surface interactions, air-liquid interfaces, freezing and freeze-drying. Together, the rapid evaluation tools and greater understanding of the structural mechanisms that initiate antibody-fragment aggregation will enable the UK biotechnology industry to design more efficient bioprocesses and robust protein scaffolds that minimise aggregation. This will continue to improve as academic beneficiaries in the UK will also be able to refine their predictive models for aggregation propensity by including the effects of solution conformation and specific protein-protein interactions elucidated here. Finally we will also compare different approaches to guide protein engineering for minimising aggregation, including existing predictive tools, previously identified mutations from other antibody models and guidance from the NMR spectra determined by us. The UK economy will benefit because academic research will complement the country's strength in bioscience discovery. Collaboration between bioprocess engineers and protein biophysicists on industrially relevant therapeutic proteins will ensure effective knowledge and skills transfer between the science and engineering base and UK industry. This will expand their position in the global healthcare market and attract further R&D investment from global business which recognises the UK as a good place to conduct these activities. Such retention of expertise, know-how and intellectual property will aid the capacity to remain internationally competitive.
Publications
Yu H
(2020)
A beginner's guide to molecular dynamics simulations and the identification of cross-correlation networks for enzyme engineering.
in Methods in enzymology
Pandya A
(2018)
An Evaluation of the Potential of NMR Spectroscopy and Computational Modelling Methods to Inform Biopharmaceutical Formulations.
in Pharmaceutics
Codina N
(2019)
An Expanded Conformation of an Antibody Fab Region by X-Ray Scattering, Molecular Dynamics, and smFRET Identifies an Aggregation Mechanism.
in Journal of molecular biology
Zhang C
(2021)
Comparison of the pH- and thermally-induced fluctuations of a therapeutic antibody Fab fragment by molecular dynamics simulation.
in Computational and structural biotechnology journal
Codina Nuria
(2018)
Elucidation of an Expanded Aggregation-Prone Conformation of Fab Using Saxs, Md Simulations and Smfret
in PROTEIN SCIENCE
Barata TS
(2016)
Identification of Protein-Excipient Interaction Hotspots Using Computational Approaches.
in International journal of molecular sciences
Chakroun N
(2016)
Mapping the Aggregation Kinetics of a Therapeutic Antibody Fragment.
in Molecular pharmaceutics
Robinson MJ
(2019)
Selective Stabilization and Destabilization of Protein Domains in Tissue-Type Plasminogen Activator Using Formulation Excipients.
in Molecular pharmaceutics
Spiteri VA
(2021)
Solution structure of deglycosylated human IgG1 shows the role of CH2 glycans in its conformation.
in Biophysical journal
Rayner Lucy E.
(2014)
The Fab conformations in the solution structure of human IgG4 restricts access to its Fc region: implications for low complement activity
in MOLECULAR IMMUNOLOGY
Rayner LE
(2014)
The Fab conformations in the solution structure of human immunoglobulin G4 (IgG4) restrict access to its Fc region: implications for functional activity.
in The Journal of biological chemistry
Rayner L
(2013)
The Solution Structure of Rabbit IgG Accounts for Its Interactions with the Fc Receptor and Complement C1q and Its Conformational Stability
in Journal of Molecular Biology
Rayner LE
(2015)
The solution structures of two human IgG1 antibodies show conformational stability and accommodate their C1q and Fc?R ligands.
in The Journal of biological chemistry
Robinson MJ
(2018)
Tm-Values and Unfolded Fraction Can Predict Aggregation Rates for Granulocyte Colony Stimulating Factor Variant Formulations but Not under Predominantly Native Conditions.
in Molecular pharmaceutics
Description | The overall aim was to characterise the aggregation behaviour of a Fab therapeutic protein, and then to delineate the mechanistic details using a range of biophysical methods. The aggregation phase map was determined in detail, as a function of temperature, pH and ionic strength, and monitoring by size exclusion chromatography. Thermal mid-points for denaturation were also determined across the same conditions using intrinsic fluorescence measurements. ThioflavinT was also used to determine the appearance of ordered fibrils. The overall kinetic pathway began with monomer loss and formation of intermediate disordered aggregates. Ordered aggregates formed on a slower timescale. AUC and SAXS were used to determine the monomeric state under the same range of conditions at time zero and also during incubations. The monomer size was found to vary in correlation with aggregation propensity, indicating some conformational or dynamic structure events. This was a novel finding. DLS based B22 measurements were obtained on the Fab and a range of charge variants. Electrostatic effects were found to be important at low ionic strength only. |
Exploitation Route | Aspects of the work are being taken forward in several current grant applications, and also two PhD projects. The findings have opened up new collaborations with both academia (Univ Manchester) and industry. |
Sectors | Healthcare Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
Description | Research findings have been reported to the BRIC community allowing industrial members to incorporate findings into their product development. Findings from this research, now published in the Journal of Molecular Biology, provided a platform of knowledge that underpinned further work on Fab antibody formulation and modelling, funded within the EPSRC Centre for Innovative Manufacturing grant. That work, published in Molecular Pharmaceutics characterised the aggregation kinetics of the Fab under a wide range of conditions, and paved the way to co-formulation studies using the same Fab in combination with IgG antibody therapies within the EPSRC Future Targeted Healthcare Manufacturing Hub. Thus the work started in this project have paved the way for deeper mechanistic understanding of protein aggregation, and also validated computational design methods for formulation design. |
Sector | Healthcare,Pharmaceuticals and Medical Biotechnology |
Impact Types | Economic |
Description | Membership of EPSRC Strategic Advisory Board for Manufacturing the Future |
Geographic Reach | National |
Policy Influence Type | Participation in a guidance/advisory committee |
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 | 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 | 09/2016 |
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 | Real World Handling of Protein Drugs - Exploration, Evaluation and Education |
Amount | € 3,139,983 (EUR) |
Funding ID | 101007939 - RealHOPE |
Organisation | European Commission |
Sector | Public |
Country | European Union (EU) |
Start | 06/2021 |
End | 07/2025 |
Description | Access to HDX MS at LGC |
Organisation | LGC Ltd |
Country | Global |
Sector | Private |
PI Contribution | UCL produces protein formulations to be further analysed at LGC. We have also helped LGC to develop the capability for HDX on solid state (freeze-dried) materials. |
Collaborator Contribution | LGC have provided access to their Mass spectrometry facility, particularly to carry out HDX and peptide mapping of proteins and their formulations. |
Impact | LGC have trained two PhD students - both funded by the EPSRC CDT, and also one PDRA from the EPSRC Hub. They have also provided access to their facilities to carry out a large body HDX peptide mapping for GCSF and IgG formulations. |
Start Year | 2016 |
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 |
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 |
Description | Neutron Characterisation in Fundamental and Applied Biotechnology, Abingdon, UK, September 2014. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Conference to bring together those interested in using neutron scattering for biophysical characterisation. Neutron Characterisation in Fundamental and Applied Biotechnology, Abingdon, UK, September 2014. |
Year(s) Of Engagement Activity | 2014 |
Description | News interview for Royal Society fo Chemistry (Chemistry World) on Nobel Prize Awards to Frances Arnold and Greg Winter in 2018 |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Media (as a channel to the public) |
Results and Impact | Interviewed to comment on the Nobel Prize Awards to Frances Arnold and Greg Winter in 2018. Quoted in an article online and also in Chemistry World magazine published by the Royal Society of Chemistry. https://www.chemistryworld.com/features/how-chemical-evolution-took-the-2018-chemistry-nobel-prize/3009626.article |
Year(s) Of Engagement Activity | 2018 |
Description | News interview for the Guardian on Nobel Prize Awards to Frances Arnold and Greg Winter in 2019 |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Media (as a channel to the public) |
Results and Impact | News interview for the Guardian on Nobel Prize Awards to Frances Arnold and Greg Winter in 2019. Quoted in article printed in the Guardian and online. |
Year(s) Of Engagement Activity | 2018 |