An amphipathic reagent to extract stabilize and purify proteins

Lead Research Organisation: University of Birmingham
Department Name: Sch of Biosciences

Abstract

The development of new therapeutic agents has undergone a revolution during the last two decades. For 80 years medicines have almost exclusively consisted of relatively small chemicals. However, biotherapeutics agents have recently emerged as the fastest growing type of new drugs being developed. They include antibodies that are part of our body's immune systems that fight off infections. Antibodies are a type of molecule called a protein; these proteins are highly complex and very fragile in contrast to the chemicals that were used in medicines in the past. Most biotherapeutic agents in clinical use are drugs manufactured in microorganisms or substances that are produced by living organisms. They include antibodies and enzymes. Most biotech pharmaceuticals are recombinant proteins produced by genetic engineering. Specific examples include cytokines like insulin and interferons, recombinant enzymes that combat cystic fibrosis (dornase alfa) and heart disease (Alteplase), hormones (Erythropoietin and human growth hormone), clotting factors, vaccines and monoclonal antibodies. Biotech pharmaceuticals have major limitations. Many are difficult to purify intact, and are unstable with limited shelf lives. Also, some are not absorbable in a medically useful form through the gastrointestinal tract, lungs or skin. Impurity is also a common problem, as they most be purified from complex biological sources. Protein impurities can cause allergic reactions or alter therapeutic effects. Hence, production of a stable protein particle that can easily be purified is very important. We have developed a new solution that increases both the yield and stability of the biotherapeutic. The chemical (called SPS) that provides this solution can act in two ways that helps the production of certain biotherapeutcics. Firstly it can be used to help release the product from the microorganisms in which it has been made. Once released the SPS can act like a stabilising bracelet, wrapping around proteins that are not generally stable in water preventing them from loosing activity. Both of these abilities will greatly increase the efficiency of biotherapeutic production which should result in cheaper and more available drugs. As an added advantage the reagent is safe for use in human beings and has been shown to facilitate the uptake of drugs into the body, increasing their effectiveness. This again has the potential to reduce drug costs. This project aims to demonstrate the effectiveness of SPS in biotherapeutic development while at the same time develop protocols so that it can be easily adopted by companies making biotherapeutics.

Technical Summary

Biopharmaceutical products represent the largest growth area in the pharmaceutical sector. These products offer a high degree of efficacy and selectivity compared to many conventional small molecule drugs making their development a high priority. However, although biopharmaceutics offer many health benefits, their production is a significant technical challenge. This is the result of a number of factors, but perhaps two of the most important being the difficult of specifically releasing the product from the feedstock and the low stability of the product. This project aims to continue our pioneering development of a reagent that addresses both of these issues. The reagent, a substituted polystyrene (SPS) is a highly amphipathic entity that has a number of remarkable properties that make it an exceptionally suited to downstream processing of biopharmaceutics. The reagent disrupts lipid bilayers to form a nano-disciodal structure in which the lipid bilayer is stabilised by a 'bracelet' of the SPS. The lipid-SPS assembly can also include membrane associated proteins allowing them to be stabilised in an entity that can be used in conventional chromatographic separations. The SPS can be easily removed from solution by a change is solution conditions leaving just the product solubilised in native membrane. In this project we will also demonstrate that SPS is multifunctional, being used to release products from the expression system and then acting as a stabilising agent for unstable products. The SPS has also been cleared by the FDA as a highly effective formulation and delivery system, allowing the potential to use a single reagent from cell disruption to administration. It therefore seems clear that, perhaps uniquely, SPSs can both improve product yield and purity of biopharmacuetic product as well as simplifying, and hence reducing the cost of the downstream process. This project aims to develop SPSs into a potent new entity for use in bioprocessing.

Publications

10 25 50
publication icon
Dafforn T.R. (2012) Detergent-free purification of membrane proteins in 243rd ACS National Meeting (Division of Biochemical Technology)

publication icon
Dörr JM (2014) Detergent-free isolation, characterization, and functional reconstitution of a tetrameric K+ channel: the power of native nanodiscs. in Proceedings of the National Academy of Sciences of the United States of America

publication icon
Jamshad M (2011) Detergent-free extraction of membrane proteins using a novel nano-encapsulation methodology in 8th European Congress on Chemical Engineering (ECCE 8) + 1st European Congress on Applied Biotechnology (ECAB 1)

publication icon
Kraemer J. (2013) Development of Novel Methods for Periplasmic Release of Biotherapeutic Products in 10th Annual Bioprocess UK Conference

publication icon
Thomas O.R.T. (2014) Styrene Maleic Acid (SMA) copolymers: One solution to two DSP problems? in Recovery of Biological Products Conference XVI

 
Description A novel way to purify proteins that are drug targets
Exploitation Route The methods are being adopted by:
1) other academics
2) commercial entities in the Pharma and Agrichem sectors
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description the findings have been used to enable other scientists to make membrane proteins in a form and allows further study
First Year Of Impact 2011
Sector Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology
 
Description Chair of Policy Committee - Biochemical Soceity
Geographic Reach National 
Policy Influence Type Membership of a guidance committee
Impact The policy committee at BS makes recommendations on a range of issues related to Biochemistry
 
Description BBSRC Responsive mode
Amount £376,277 (GBP)
Funding ID BB/I020349/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2012 
End 12/2014
 
Description BBSRC Responsive mode
Amount £423,879 (GBP)
Funding ID BB/J017310/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2013 
End 05/2016
 
Description BBSRC Responsive mode followon fund
Amount £151,570 (GBP)
Funding ID BB/J010812/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2013 
End 09/2014
 
Title Universal method for producing membrane proteins 
Description Universal method for producing membrane proteins 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact Has revolutionised the study of membrane protein structure and function 
 
Description Production of membrane proteins (Heptares) 
Organisation Heptares Therapeutics Ltd
Country United Kingdom 
Sector Private 
PI Contribution confidential
Collaborator Contribution confidential
Impact confidential
Start Year 2012
 
Title Extraction from cells 
Description A method for releasing the content of the periplasmic space of bacterial celis is provided, which comprises incubating the bacterial cells in a solution containing styrene maleic acid copolymer (SMA). Also provided is a method of preparing a substantially pure sample of recombinant polypeptide. The methods find application in the recovery of materials, such as proteins, from bacterial cells. 
IP Reference WO2012153089 
Protection Patent application published
Year Protection Granted 2012
Licensed No
Impact We have received strong interest from several biopharmaceutical companies (Biogen-Idec, Medimmune, Shire, BMS, Roche, UCB) on trialling the SMA mediated periplasmic processing technology disclosed in the PCT, and we are collaborating with UCB on the subject through a BRIC funded industrial PhD studentship (BB/K004441/1).
 
Title Extraction from cells 
Description New method for release of therapeutics from cells 
IP Reference WO2012153089 
Protection Patent application published
Year Protection Granted
Licensed No
Impact Reduces costs for therapeutics production
 
Title SOLUBILISATION OF MEMBRANE PROTEINS 
Description Development of a new reagent for extraction of membrane proteins in a stabilised form 
IP Reference EP2452199 
Protection Patent granted
Year Protection Granted 2012
Licensed Yes
Impact Establishment of a newco (Orbiscope BV) in the Netherlands based on selling materials detailed in the patent. Reagents currently being sold globally to academia and the pharmaceutical industry
 
Title SOLUBILISATION OF MEMBRANE PROTEINS 
Description New method for producing a membrane protein 
IP Reference US8754168 
Protection Patent granted
Year Protection Granted 2014
Licensed Yes
Impact 1) Formation of a new business spinout (Orbiscope) from Polyscope BV 2) Provision of new services to the pharmaceutical sector (Cube Biotech and Domainex Ltd)