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
Dafforn T.R.
(2012)
Detergent-free purification of membrane proteins
in 243rd ACS National Meeting (Division of Biochemical Technology)
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
Gulati S
(2014)
Detergent-free purification of ABC (ATP-binding-cassette) transporters.
in The Biochemical journal
Jamshad M
(2015)
Structural analysis of a nanoparticle containing a lipid bilayer used for detergent-free extraction of membrane proteins.
in Nano research
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)
Jamshad M
(2011)
Surfactant-free purification of membrane proteins with intact native membrane environment.
in Biochemical Society transactions
Kraemer J.
(2013)
Development of Novel Methods for Periplasmic Release of Biotherapeutic Products
in 10th Annual Bioprocess UK Conference
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 | 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 |
Description | Unshackling Membrane Protein Research : New Amphiphilic Copolymers for Extraction of Stable, Active Membrane Proteins |
Amount | £492,954 (GBP) |
Funding ID | 223728/Z/21/Z |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2022 |
End | 02/2027 |
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 | SOLUBILISATION OF MEMBRANE PROTEINS |
Description | A method is provided for solubilising a membrane protein. The method is applied to cellular material comprising the membrane protein and an associated membrane lipid. A copolymer of styrene and maleic acid, wherein the styrene:maleic acid ratio is between 1:2 and 10:1, is mixed with the cellular material to cause the copolymer, lipid and protein to form soluble macromolecular assemblies. |
IP Reference | US2012142861 |
Protection | Patent granted |
Year Protection Granted | 2012 |
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) |
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 |