Improving biopharmaceutical production in microbial systems: Engineering GlycoPEGylation in E.coli
Lead Research Organisation:
University of Kent
Department Name: Sch of Biosciences
Abstract
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Technical Summary
The production of small therapeutic proteins or peptides in E.coli is advantageous in industry due to the relatively low costs of production. However, a specific limitation of the these therapeutic products is they often have suboptimal biophysiochemical features e.g. short elimination half life, instability and immunogenicity. An approach to improve these characteristics is post-production modification of the protein, which means the attachment of natural or synthetic polymers, and a very established method is PEGylation, the covalent attachment of PEG to a therapeutic protein.
We will develop a E.coli strain which can efficiently produce therapeutic proteins as well as add a site-specific glycan (GalNAc) residue. This glycan will act as the site for PEGylation in vitro using an sialyltransferase enzyme, also optimised in this project.
We have generated a prototype strain which adds the glycan effectively and on an in silico designed interferon alpha acceptance site.
We will use synthetic biology and an iterative metabolic engineering strategy to debottleneck production. At each stage, the exemplar therapeutic protein (IFNa-2b) will be tested in well established fermentation production platforms and tested for biophysical characteristics (i.e. solubility, stability etc.).
This proposal has great potential to generate an economically competitive therapeutic protein production system for the biopharmaceutical sector.
We will develop a E.coli strain which can efficiently produce therapeutic proteins as well as add a site-specific glycan (GalNAc) residue. This glycan will act as the site for PEGylation in vitro using an sialyltransferase enzyme, also optimised in this project.
We have generated a prototype strain which adds the glycan effectively and on an in silico designed interferon alpha acceptance site.
We will use synthetic biology and an iterative metabolic engineering strategy to debottleneck production. At each stage, the exemplar therapeutic protein (IFNa-2b) will be tested in well established fermentation production platforms and tested for biophysical characteristics (i.e. solubility, stability etc.).
This proposal has great potential to generate an economically competitive therapeutic protein production system for the biopharmaceutical sector.
Planned Impact
This is an interdisciplinary application at the interface of biological science, biological engineering, and chemistry with several potential impacts as follows.
Economic and societal impacts: Biopharmaceutical compounds in production internationally are valued at over £200 billion, with UK companies owning around ca. £20 billion (TSB, June 2012). Of this, 10% of licensed products sales are represented by biopharmaceuticals, with this figure rising to a third of the products in development (TSB). Industry has a choice of platforms for their manufacture, including animal, plant and microbial cells. Microbial production is industrially vital, and ca. 30% of the genuinely new biopharmaceuticals approved between 2006-10 employed the Escherichia coli platform (Walsh, 2010, Nat Biotechnol, 28, 917-24). We focus on E. coli here.
Academic Impact: Major beneficiaries include academics who study recombinant protein production, microbiology, peptide design and controlled biomolecular assembly, and most importantly industrial biopharmaceutical production. This would also be an exemplar project in the field of synthetic biology and metabolic engineering in the UK. The modular microbial glycosylation process, engineered high expression host strain and sialylation modules that are at the core of this proposal will also be of benefit to the synthetic-biology community more broadly as new parts/components will be generated that could aid in the assembly, production and/or secretion of other industrially important macromolecules. We believe this to be the case because many engineering changes we propose ought to be generic.
Research staff: The proposed development of improved glycoPEGylated proteins requires researchers who will work collaboratively with others from a number of different fields across biological engineering and micro/molecular biology, and the biologics production industries. The project provides opportunities for publishing in new scientific areas, presenting to diverse audiences and strengthening the direction of their careers. The research staff will benefit by meeting with BRIC member companies, publishing in high-impact journals and will be encouraged to be active in the preparation of subsequent funding applications. In addition, we seek, by leveraging the BBSRC-BRIC funding, to build critical mass of researchers in the area of synthetic biology with a focus on applications based research that will impact the area of microbial production of recombinant proteins - especially biopharmaceuticals (where there is a demonstrable need, and we have expertise). Thus any investment will have an impact on the face of interdisciplinary research in the UK.
Industry: Production of cost-effective antibody fragment drugs has great potential for translation into industry. The model we examine here (IFNalpha) appears to be a suitable model (discussed with BRIC member companies). This programme could place the UK at the forefront of using microbial systems to rapidly add site specific modifications to therapeutic antibody fragments and will be a significant advance for the UK to lead, with associated economic benefits.
Economic and societal impacts: Biopharmaceutical compounds in production internationally are valued at over £200 billion, with UK companies owning around ca. £20 billion (TSB, June 2012). Of this, 10% of licensed products sales are represented by biopharmaceuticals, with this figure rising to a third of the products in development (TSB). Industry has a choice of platforms for their manufacture, including animal, plant and microbial cells. Microbial production is industrially vital, and ca. 30% of the genuinely new biopharmaceuticals approved between 2006-10 employed the Escherichia coli platform (Walsh, 2010, Nat Biotechnol, 28, 917-24). We focus on E. coli here.
Academic Impact: Major beneficiaries include academics who study recombinant protein production, microbiology, peptide design and controlled biomolecular assembly, and most importantly industrial biopharmaceutical production. This would also be an exemplar project in the field of synthetic biology and metabolic engineering in the UK. The modular microbial glycosylation process, engineered high expression host strain and sialylation modules that are at the core of this proposal will also be of benefit to the synthetic-biology community more broadly as new parts/components will be generated that could aid in the assembly, production and/or secretion of other industrially important macromolecules. We believe this to be the case because many engineering changes we propose ought to be generic.
Research staff: The proposed development of improved glycoPEGylated proteins requires researchers who will work collaboratively with others from a number of different fields across biological engineering and micro/molecular biology, and the biologics production industries. The project provides opportunities for publishing in new scientific areas, presenting to diverse audiences and strengthening the direction of their careers. The research staff will benefit by meeting with BRIC member companies, publishing in high-impact journals and will be encouraged to be active in the preparation of subsequent funding applications. In addition, we seek, by leveraging the BBSRC-BRIC funding, to build critical mass of researchers in the area of synthetic biology with a focus on applications based research that will impact the area of microbial production of recombinant proteins - especially biopharmaceuticals (where there is a demonstrable need, and we have expertise). Thus any investment will have an impact on the face of interdisciplinary research in the UK.
Industry: Production of cost-effective antibody fragment drugs has great potential for translation into industry. The model we examine here (IFNalpha) appears to be a suitable model (discussed with BRIC member companies). This programme could place the UK at the forefront of using microbial systems to rapidly add site specific modifications to therapeutic antibody fragments and will be a significant advance for the UK to lead, with associated economic benefits.
People |
ORCID iD |
Colin Robinson (Principal Investigator) |
Publications
Alanen HI
(2015)
Efficient export of human growth hormone, interferon a2b and antibody fragments to the periplasm by the Escherichia coli Tat pathway in the absence of prior disulfide bond formation.
in Biochimica et biophysica acta
Matos CF
(2014)
Efficient export of prefolded, disulfide-bonded recombinant proteins to the periplasm by the Tat pathway in Escherichia coli CyDisCo strains.
in Biotechnology progress
Patel R
(2014)
Protein transport by the bacterial Tat pathway.
in Biochimica et biophysica acta
Description | The grant is led by Sheffield partner and is still ogoing. Our 6-month project is to develop constructs that can be exported to the periplasm and then modified by glycosylation. Preliminary results are very encourgaing. |
Exploitation Route | The aim is to develop a new means of producing glycosylated biopharmaceuticals that can be adopted by industrial collaborators |
Sectors | Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
Description | IB Catalyst grant |
Amount | £2,100,000 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 05/2015 |
End | 04/2019 |
Description | Marie Curie Initial Training Network 'ProteinFactory' |
Amount | £3,500,000 (GBP) |
Funding ID | 642836 |
Organisation | European Commission |
Sector | Public |
Country | European Union (EU) |
Start | 05/2015 |
End | 04/2019 |
Title | New E. coli strains |
Description | We have developed new strains of E. coli that export proteins to the periplasm by a novel pathway |
Type Of Material | Cell line |
Year Produced | 2010 |
Provided To Others? | Yes |
Impact | Several groups are testing these strains with a view to using them for production production on analytical or production scale |
Description | Open day presentations |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Schools |
Results and Impact | Hosted open day visits to my lab in majority of UCAS days - 2 groups of UCAS applicants + parents per open day, at least 10 open days per year Increased understanding of biotech projects at Kent |
Year(s) Of Engagement Activity | 2007,2009,2011,2012,2013,2014,2015,2016 |