Microfabricated cantilever methods as nanoscale screens for early indicators of protein aggregation; a feasibility study

Lead Research Organisation: University of Nottingham
Department Name: Sch of Pharmacy

Abstract

It currently takes over $800 million and around 10-15 years for a new medicinal product to reach the market. Over one third of the products currently under development are biopharmaceuticals; medicines in which the active ingredients are large biological molecules, such as proteins or nucleic acids. Preparations containing monoclonal antibodies (e.g. for the treatment of immune disorders, cancer and infection) are currently the largest, and most important class of biopharmaceutical. The development of biopharmaceuticals is therefore both costly and time intensive. The approaches employed to design/discover new biopharmaceutical agents are very different to traditional synthetic drug-molecule based medicines, with large biological molecules bringing new challenges in terms of development processes during medicine design (formulation), manufacture and storage. At present this is often reflected in the high market price of biopharmaceutical therapeutics, and the pressure to provide cheaper products more quickly has resulted in a demand for new analytical methods/approaches to address issues which conventional drug approaches are struggling to meet. One such issue which directly impacts on protein-based medicines, and which can adversely affect any stage of the development process, is the unpredictable tendency of protein molecules to stick together, to form assemblies of molecules termed aggregates. During medicine manufacture, aggregation can result in highly viscous solutions, causing problems during processing and product packaging. It can also lead to decreased product stability, and hence difficulties in estimating product shelf-life. Within the final medicine, aggregation of the key therapeutic active ingredient can ultimately reduce the efficacy of the medicine and can in extreme cases produce severe unwanted side effects in the patient. By identifying as early as possible within the drug development process, which biomolecules have a tendency to aggregate (and also the conditions which encourage/discourage this aggregation) then the effort, costs and risks to patients associated with development of the therapeutic could therefore be significantly reduced. In this project we aim to explore the feasibility of addressing this difficult challenge, using microcantilever detection approaches (which employ ultrasensitive springs to measure interactions between biological molecules). Ultimately we aim to develop a novel approach to screen potential biological 'active' molecules for their tendency to aggregate through the use of devices capable of detecting the very early stages of aggregation or changes in molecular properties consistent with aggregation behaviour. Within this one year project, our aim is to test and explore the feasibility of a range of potential approaches to achieve this goal. Importantly, our experimental programme will use both model and therapeutically relevant proteins (including monoclonal antibodies). In close collaboration with the pharmaceutical industry the devices identified in these preliminary studies will also be assessed for viability and scale up for use in an industrial context. A sizable and increasing academic and industrial community have identified the occurence of protein aggregation as a critical issue in a number of fields, and have sought new methods to study this phenomenon. This project has the potential to provide an entirely new approach to detecting and investigating the origins of aggregation at its earliest stage. In the longer-term, the work, if built upon will impact upon significant areas of biotechnology and healthcare. Indeed, the methods developed could play an important role in bringing new generation medicines to the market in a cost-efficient and timely manner, and would thus have a very significant impact on public health and quality of life.

Technical Summary

Biopharmaceuticals are medicinal products where the active ingredients are large biological molecues (e.g. proteins or nucleic acids), and they constitute over a third of the medicines currently under development. Despite their increasing number, they continue to present many biomolecule specific challenges during product development - the considerable time and resource required to address these is ultimately reflected in products with high market prices. The vast majority of products have protein/peptide actives, with a large proportion of these based on monoclonal antibodies (mAbs). Aggregation of such molecules can be unpredictable and result in significant difficulties during formulation, manufacture and storage. Due to the severe immune responses that can be observed in patients if aggregates are administered, there is also a stringent regulatory environment associated with the levels of aggregate permissible in a final formulation. Identification of the tendency of a molecule to aggregate - and under what conditions - as early as possible within the development process could therefore help to significantly reduce the time, effort and costs associated with development, and also the potential risks to patients. The urgent need to address this issue is reflected by the support of MedImmune for this feasibility proposal. The proposed 12-month project will investigate the feasibility of using microfabricated cantilever approaches for the detection of protein aggregation propensity early within biopharmaceutical development. Importantly, through the 'development of new enabling tools' and obtaining biophysical data to extend our fundamental understanding of protein aggregation within biopharmaceuticals, the project aligns with the broad themes of BRIC2, and more specifically priority areas 1 (bioprocessing research challenges for protein products) and 4 (analytics for bioprocessing).

Planned Impact

Who will benefit from this research, and how? Statements on global market sizes are in general misleading at early stages in development, such as within this project, as the potential market share cannot easily be assessed. Nevertheless, it is useful to recognize that the biopharmaceutical market is immense (biopharmaceutical products accounted for 10% in 2006 of the total global market and is projected to account for about 15% or US$182.5 billion by 2015 (Biopharmaceuticals: A Global Market Overview, Industry Experts, April 2010)) and represents the one real long-term growth area in pharmaceuticals, a key UK market sector which has suffered major downsizing in recent years. The core project aim is to explore the feasibility of microcantilever detection methods for the early-stage detection of molecules susceptible to aggregation within biopharmaceutical development. The difficulties in predicting which molecules may or may not be prone to aggregation is currently a key barrier to bringing a new generation of biomacromolecular based treatments to the market. This hence represents a potential massive loss in benefits to public health, as well as a financial loss to the UK based biopharmaceutical sector. The urgent need to address this question is reflected by the support of MedImmune for this feasibility proposal, and within the broad themes of BRIC2 (and specifically priority areas 1 and 4). The long-term public benefits are wide ranging. Clearly the methodologies developed could play an important role in helping to bring viable bio-therapeutic formulations to the market in a cost-efficient and timely manner and would thus have a very significant impact on public health and quality of life. The knowledge gained would also have an impact in complementary areas such as sensors, diagnostics and food. What will be done to ensure that they have the opportunity to benefit? Within the proposed one-year project we aim to establish a clear proof-of-principle of the use of microfabricated levers as a viable and scalable approach to aggregation propensity screening. With the aid of our industrial supporters and the School of Pharmacy Business Development Officer, Dr Mark Gilbert (in-kind contribution) we will also carry out a preliminary study of the IP/patent landscape in the application area and collate market potential data. This will enable efforts to attract follow-on funding to be informed by a sound basis of the potential of the IP and the future needs of the healthcare and industrial communities. The ultimate commercial exploitation of the technology will benefit the industries to which it is licensed, or spun in to and hence the economy through the creation of value and new employment. The long-term benefits of this work will be achieved, by preference through follow-on support from the second round of BRIC2 funding, to achieve a level of outputs suitable to attract translation funding (such as from the Wellcome Foundation, TSB, an investment fund or direct industrial support, for example through a KTP). The applicants, supported by the Universities Research Innovation Services will also identify key application advances that have the potential to develop new/extend current IP and the development of an exploitation strategy. We will also aim to help maximise future technology transfer through engagement with regional and national networks (eg. East Midlands Development Agency, bioKneX, Medilink, East Midlands Innovation, iNets and KTNs), Nottingham University's wholly owned TSB funded healthcare-focused technology transfer company, Eminate Ltd and through extensive current industrial contacts. We will through these routes, or through direct contact engage industrial interest to support the project, and ultimately to form a potential partnership database. This process has already begun with support from MedImmune.

Publications


10 25 50
 
Description Based on atomic force microscopy (AFM) force measurement and microcantilever sensing techniques, this project established a method to detect molecular indicators of instability/aggregation tendency (e.g. unfolding), and explored complementary methods to directly detect aggregate formation. We obtained data for three types of model therapeutic molecules, namely albumin, insulin and IgG1 (monoclonal). In experiments investigating the effects of model stabilizing excipients (e.g. arginine) we have demonstrated that method developed in is able to detect its effects on the molecule. These measurements highlight the potential of this approach to be used as a screening method to quickly identify formulation conditions in which proteins are most stable. With the instrument manufacturer JPK we also developed and utilised their ForceRobot platform, with the long-term aim of developing automated AFM based screening protocols.
Exploitation Route The project has developed an approach for potential application as a new screening tool to predict protein stability/potential for aggregation within biopharmaceutical development. We are already exploring this potential through future projects (eg short projects feasibility projects to explore the translation/commericial potential of the approach). Similarly, the approach has also initiated new research using a very similar method for drug-discovery (for Fragment Screening). In addition the expertise developed in understanding and screening for protein aggregation within formulations has also initiated industrially funded projects utilizing other analytical screening tools and automated methods.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
 
Description Based on atomic force microscopy (AFM) force measurement and microcantilever sensing techniques, we established a method to detect molecular indicators of instability/aggregation tendency (e.g. unfolding), and explored complementary methods to directly detect aggregate formation. We obtained data for three types of model therapeutic molecules, namely albumin, insulin and IgG1 (monoclonal). In experiments investigating the effects of model stabilizing excipients (e.g. arginine) we have demonstrated that method developed in is able to detect its effects on the molecule. The measurements highlighted the potential of this approach to be used as a screening method to quickly identify formulation conditions in which proteins are most stable. With an instrument manufacturer JPK we also developed and utilised their ForceRobot platform, with the long-term aim of developing automated AFM based screening protocols. The project also generated intellectual property (IP) and supporting data that was assessed for its commercial potential by both the School of Pharmacy (via its Knowledge Transfer Committee) and the University of Nottingham (via its Commercialisation Panel). Nottingham approved the funding of a patent application in order to protect the IP, and a specialist patent agent was engaged to prepare the draft. On further discussion with the agent it was however decided to not pursue with the patent application. The expertise in protein aggregation screening (within formulations) developed through this project, has more recently led to industrial impact, leading to a two-stage project funded by industry (Janssen). The project aims to develop methods for the screening of aggregation with ocular proteins, with a specific focus on the development of new treatments for cataract treatment and/or prevention. This recent project thus extends the envisaged impact from the initial award to include improvements in our understanding of ageing processes and their treatment.
First Year Of Impact 2015
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic
 
Description BBSRC BRIC Doctorate Programme
Amount £100,000 (GBP)
Funding ID BB/J003840/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 12/2011 
End 11/2015
 
Description Janssen Protein Aggregation Project
Amount £450,000 (GBP)
Funding ID SH2617 (University of Nottingham Code) 
Organisation Janssen Research & Development 
Sector Private
Country Global
Start 01/2016 
End 11/2018
 
Description Secondment funding via University of Nottingham EPSRC Impact Accelerator Account
Amount £25,000 (GBP)
Funding ID EP/K503800/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 04/2015 
End 10/2015
 
Description BioProNET NIBB 
Organisation Networks in Industrial Biotechnology and Bioenergy (NIBB) - BioProNET
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Private 
PI Contribution The feasibility award funded through the BBSRC's Bioprocessing Research Industry Club, led to my involvement in writing the application to establish BioProNET. Once funded (2014), I was asked to become a member of its management board and was relected to this post in 2015.
Collaborator Contribution The BioProNET is one of the largest industry-academia funded BBSRC NIBB networks, and is led by Professor Mark Smales (Kent) and Professor Alan Dickson (Manchester).
Impact No direct outputs yet for myself, but the NIBB awards funds (e.g. business vouchers, proof of concept awards (up to £100k)) to others in the network. My involvement in the management board thus means that I regularly am asked to review and rank research proposals within the wider network/bioprocessing community.
Start Year 2013
 
Description Janssen Protein Aggregation Project 
Organisation Janssen Research & Development
Country Global 
Sector Private 
PI Contribution Due to our abilities to develop approaches for the screening of protein aggregation (as demonstrated through this project), Janssen approached our research group directly to work on a proof-of-concept study related to the set-up of a high throughput protein aggregation screen (further details of the protein and approach employed cannot yet be revealed due to confidentially).
Collaborator Contribution Our partners at Janssen, supply the protein of interest and also molecules of interest as potential modulators of protein aggregation.
Impact No outcomes yet.
Start Year 2016
 
Description Novozymes IAA 
Organisation Novozymes Biopharma UK ltd
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Private 
PI Contribution Following a presentation about the approach developed through this project Novozymes approached us, asking us to demonstrate proof-of-concept of our approach for the screening of their macromolecular excipients. This led to a research application to our University's EPSRC Impact Accelerator Account (EP/K503800/1) to fund this work.
Collaborator Contribution Supply of molecules and background knowledge on the macromolecular excipients of interest.
Impact No outputs yet.
Start Year 2015