Enabling rapid liquid and freeze-dried formulation design for the manufacture and delivery of novel biopharmaceuticals

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

Abstract

Biopharmaceuticals have been approved to treat diseases including cancers, rheumatoid arthritis, multiple sclerosis, diabetes, leukemia and neutropenia. The next-generation of protein-based therapies, or biopharmaceuticals, are of increasingly complex engineered forms, with unpredictable solution properties. Proteins are formulated at high concentrations for clinical use, leading often to undesirable aggregate formation, high viscosity, opalescence, or phase separation, rendering them unsafe, or difficult to inject or manufacture. This is a major challenge to the biopharmaceuticals industry as approximately 50% of proteins in clinical trials have been freeze-dried as they were not readily liquid-formulated on timescales of months required during development. Formulation is an empirical process using combinatorial screens that aim to optimise stability, potency and ease of delivery to patients. Engagement with 36 industry leaders at a UCL EPSRC Centre for Innovative Manufacturing workshop identified the most significant protein formulation challenges such as the prediction of shelf stability over a two year period, at a time in development when not much material is available. Current surrogate techniques that accelerate protein degradation and minimize sample consumption provide poor indicators of 2-year shelf-life. Industry would benefit significantly from i) rapid analyses that more accurately determine long-term shelf-life, ii) low concentration analyses that indicate high-concentration solution behaviour, and iii) a better ability to use calculated protein and excipient properties to predict those formulations that are most likely to meet the required attributes.

State-of-the-art automated microplate and microfluidic analytics, purchased or established recently via EPSRC and BBSRC/BRIC awards at UCL and UoM provide a timely platform for generating large experimental datasets of aggregation kinetics spanning many different timescales, conformational and colloidal stabilities, rheological properties, phase-transition and glass transition temperatures, for liquid and freeze-dried formulations. A recent EPSRC funded £500k pilot-scale freeze-drying facility at UCL (EP/M028100/1), combined with DoE and 3D process simulations, will generate freeze-drying process models that elucidate the mechanisms linking critical process parameters to critical quality attributes for new formulations. Novel dipeptides emerging from recent UoM work will significantly expand the range of industry-accepted formulation excipients available. Novel microfluidic analytics will be tailored for formulation needs, bringing earlier, more sensitive, and lower-volume assessments of formulated protein heterogeneity and storage kinetics, ultimately in a high-throughput format using sealed microwells.

All data will populate a web-access database at UoM to provide modeling groups access to a much-needed experimental dataset. Informatics techniques initiated at UoM in a BioProNet PoC award will enable new proteins to be compared (via properties calculated from sequence and structure) to those in the database, and use their experimentally determined formulation behaviours in a predictive manner. Correlations between calculated protein properties and critical formulation attributes will identify the molecular basis of excipient behaviour.

Overall, this will benefit the biopharmaceutical formulation community with an ability to: a) identify better excipient combinations for input into formulation screens; b) predict those protein candidates most readily formulatable with current excipients and solution conditions; c) inform the rational design of novel peptide-based excipients through defined chemical modifications, d) predict long-term storage stability and concentrated solution behaviour from accessible experiments using minimal sample.

Planned Impact

"WHO WILL BENEFIT FROM THE RESEARCH?"

UK-based companies within the BioProNet/BRIC community will benefit from research which allows them to more effectively predict lead protein therapy candidates and formulations that are likely to be viscous, or lead to aggregation during storage, freeze-drying, bioprocess development and manufacturing, using high-throughput experimental and computational tools for a "screen early - fail early" approach. Industry will also have access to predictive tools and archived data that will inform the more effective design of their own product formulations and formulation screens. 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 formulation development times of protein therapies, which is particularly crucial for those addressing previously unmet clinical needs. Benefits to the NHS relate to the possibility of constraining costs that accumulate in industry due to late-stage failures of drug candidates. Proteins are innately complex and labile so that product 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 interactions between formulation excipients and proteins, as measured by their impact on aggregation kinetics, conformational and colloidal stability, and rheological properties, in a wide range of buffer conditions. This will reveal commonalities between several protein types, as well as their idiosyncrasies. This improved understanding will better direct protein formulation, engineering and candidate selection through the generation of predictive tools that identify formulation excipients most likely to stabilise each new therapeutic protein. The research will also demonstrate advanced microfluidic analytical techniques to rapidly identify the likelihood of a protein formulation to aggregate, and to detect aggregates with very high sensitivity. Together, the rapid analytical tools and the greater understanding of the structural mechanisms that lead to protein stabilisation by formulation excipients will enable the UK biotechnology industry to design more efficient formulation screening designs, novel excipients, and robust protein scaffolds that minimise aggregation, and high viscosity, and prolong product shelf-life. This will continue to improve as academic beneficiaries in the UK will also be able to refine the predictive models for formulation impact on aggregation and rheological properties, by adding their own data to the database, and though interactive use of the new algorithms.

The UK economy will benefit because academic research will complement the country's strength in bioscience discovery and development. Collaboration between formulation engineers, computational scientists 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

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Corbett D (2019) Measuring Nonspecific Protein-Protein Interactions by Dynamic Light Scattering. in Methods in molecular biology (Clifton, N.J.)

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Hales JE (2019) Virus lasers for biological detection. in Nature communications

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Kalayan J (2020) Model for Counterion Binding and Charge Reversal on Protein Surfaces. in Molecular pharmaceutics

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Yu H (2018) Coupled molecular dynamics mediate long- and short-range epistasis between mutations that affect stability and aggregation kinetics. in Proceedings of the National Academy of Sciences of the United States of America

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Yu H (2018) Exploiting correlated molecular-dynamics networks to counteract enzyme activity-stability trade-off. in Proceedings of the National Academy of Sciences of the United States of America

 
Description Our novel analytical device has been able to determine and identify complex interactions between protein molecules that are not detectable by standard methods. It is able to identify the relative amounts of two antibodies in the same solution based only on optical spectra. The device is also able to detect aggregate formation with much higher sensitivity than standard SEC detectors, enabling much earlier use of SEC in aggregation kinetics studies. This means that the device is very suitable for monitoring complex degradation pathways in protein formulations. It has attracted wide interest from industry, and is being developed further now in a partnership with Pall Europe.

We have revealed complex concentration dependent behaviour in aggregation kinetics of several proteins, in which aggregation slows at higher concentrations contrary to normally expected behaviour. We have been able to determine the mechanisms for this via rheology and other kinetic measurements.

A key factor controlling the critical quality attributes (CQAs) of biopharmaceuticals are hot spots or sticky regions on protein surfaces composed of hydrophobic groups. Through an informatics analysis of tyrosine groups in antibody complementarity regions, we have shown that empirical models for calculating the stickiness of hydrophobic surfaces can be improved by incorporating a shape-dependent term. (Hebditch et al (2019) J Pharm Sci 108:1434).

Large-scale measurement of biophysical properties for antibodies has opened the way to improved prediction models for CQAs. We have shown, using machine learning applied to sequence, that much of the variation in biophysical properties can be quantified in terms of sequence features such as charge and hydrophobicity. This for instance has allowed us to develop an improved model for predicting hydrophobic interaction chromatography (HIC), which indicates protein charge is a significant factor in controlling the retention time. The finding is directly relevant for formulation development as HIC is frequently used as a high-throughput readout for assessing biopharmaceutical developability (Hebditch M and Warwicker J (2019) PeerJ. DOI:10.7717peerj.8199).

We have discovered a novel class of promising excipients based on poly-phosphates for improving the CQAs of a broad range of therapeutics. We have shown their mechanistic action is different from other classes of excipients. The multivalent ionic excipients bind to and supercharge proteins leading to an increase in their colloidal stability (Bye J and Curtis R (2019) J Phys Chem 123: 593). In mixtures with other excipients, they exhibit synergistic effects, which provides much scope for developing formulations with even greater effectiveness at stabilizing proteins and biopharmaceuticals.
Exploitation Route The methods developed will be of very high value to the biopharmaceutical industry by improving both formulation design and also forced degradation for formulation screening.

The analytical methods we have developed are now being translated through partnership with Pall Europe, in an EPSRC EngD, to develop it for inline bioprocess monitoring purposes.

The formulation data and ML methods for predicting HIC properties from protein sequence will be of immense value to industry in the design of bioprocesses and protein formulations. We have developed our web-based tool (www.protein-sol.manchester.ac.uk), which is publicly available, for predicting protein solubility and aggregation propensity from protein structures (and models), which are key factors in controlling biopharmaceutical development. Key predictive indicators calculated by our server are charge and hydrophobic patches, and the pH-dependence of stability. Our server that includes these tools is currently supporting ~ 300 runs per month from external users (Hebditch and Warwicker (2019) Sci Rep 9:1969) and has already been cited in numerous publications concerned with predicting developability of biopharmaceuticals.

We have been awarded industrial acceleration impact funds from the University of Manchester in a collaboration with Albumedix for investigating the potential of using the multivalent ionic excipients in mixtures with recombinant human serum albumin (HSA) to improve upon Albumedix formulation technology. The preliminary experimental studies have demonstrated the effectiveness of the excipients for stabilizing liquid formulations. We are currently exploring patent opportunities for the technology. Along these lines, we are working with the Centre for Molecular Immunology (CIM) in Cuba to find optimal formulations with the excipients for stabilizing their IL-2 based biopharmaceuticals that are already in various stages of clinical trials.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Our early findings on the novel analytics have led us to form a collaboration between UCL and Pall Europe via an EPSRC EngD project. This is also aiming to lead to a commercial partnership in the near future to exploit the technology which has now been filed for a patent. The formulation prediction approaches are being exploited through a collaboration with Albumedix, supported by Impact acceleration impact funding in Manchester.
First Year Of Impact 2018
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Chair of the Royal Society of Chemistry Biotechnology Interest Group
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
Impact The RSC BTG committee responds to the community of Chemists working in the area of biotechnology (in its broadest definition). The committee runs symposia, conferences and training events that fit the needs of its community.
 
Description EPSRC Balancing Capability
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
 
Description Elected Management Board Member of BBSRC BioProNet
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
Impact BioProNet coordinates the entire UK academic and industrial community for Bioprocess Manufacturing, via network meetings, workshops and managed proof of concept funding. Research outputs feed into larger InnovateUK proposals.
 
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
 
Description Viral lasers for biological detection.
Amount £60,000 (GBP)
Organisation Royal Society of Edinburgh (RSE) 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2020 
End 01/2021
 
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 
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
 
Description Materials from Medimmune/AZ 
Organisation AstraZeneca
Department AZ Medimmune
Country United Kingdom 
Sector Private 
PI Contribution We have analysed the antibody samples using novel analytics
Collaborator Contribution Donation of five antibody and bispecific samples.
Impact Molecules have enabled an assessment of analytical techniques we have developed. Molecules have also been used to elucidate new mechanisms involved in protein aggregation in medical formulations. This will enable improved future formulations of antibody therapies.
Start Year 2018
 
Description Pall Europe collaboration 
Organisation PALL Europe
Country United Kingdom 
Sector Private 
PI Contribution Evaluating samples supplied by Pall in the new instrumentation.
Collaborator Contribution Supply of antibody samples of different grades for analysis.
Impact Led to a follow on EngD project in collaboration with Pall Europe
Start Year 2017
 
Description Formulation Specialist Working Group 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact The formulation Specialist Working Group meets every 6 months (twice in 2017, once in 2018 so far) to enable industry, regulatory agency, government laboratory and academic experts to discuss the arising challenges, and potential solutions on a 5-10 year horizon, in the area of formulation for stratified proteins, and cell and gene therapies. Over 30 different users have attended to date, and the meetings have refined the Hub research agenda, scoped out topics for review articles, and engaged the regulatory agencies which raises their awareness of future challenges that they will also need to address.
Year(s) Of Engagement Activity 2017
 
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