10/23/2012 Hosts for Biosimilars Production: What is the future? Lloyd Ruddock, Scientific Director, Paras Biopharmaceuticals Finland 2004 2008 Company executives Biosimilars meet and decide 10 capabilities year roadmap realized Paras Biopharmaceuticals Paras Biopharmaceuticals Finland is established and Finland is conceptualized; executives look for interested details finalized investment groups 2006 2009 1
10/23/2012 Paras research labs Reproduced with permission from Helsinki University Collection Paras Business Paras Biopharmaceuticals Finland works on technology development for biosimilars and their out-licensing Paras has multiple products already in pipeline The aim is to deliver at least one product API to market per year starting in 2013 2
10/23/2012 Paras target areas } Diabetes All high-growth, high-value areas with multiple products Rheumatoid Arthritis coming off patent over the next 6 years Osteoporosis Paras novel technologies As well as utilizing existing local expertise, Paras has developed multiple novel technologies including: 1. Diabrid technology 2. Noble Cleav R 3. BioMultifold R 4. BioEnhanced R 3
10/23/2012 Diabrid technology Paras propreity Therapeutic partner (PPP) Protein Promoter Linker Terminator Cleavage “Diabrid Technology” address two major problems in the biologics and therapeutic peptide industry: i) Higher cost of biologics production ii) Large scale production of long therapeutic peptides is very difficult, if not impossible by chemical synthesis. 30 years ago FDA approved first recombinant biologic 4
10/23/2012 In the early years prokaryotic production dominated the market Sales of proteins produced in prokaryotes e.g. E.coli have continued to increase • Low cost Sales • Rapid growth • High biomass • Easy cultivation and manipulation • FDA friendly 5
10/23/2012 In the last decade market share of sales for biologics production in eukaryotes rapidly increasing prokaryotes • Increased yields Market share • Decreased costs • Eukaryotes can produce some eukaryotes proteins, in an active state that prokaryotes cannot. Biologics production is predicted to continue to grow rapidly, but what will be the hosts in the future? 6
10/23/2012 Biologics production is predicted to continue to grow rapidly, but what will be the hosts in the future? Will the market share from eukaryotes systems continue to increase, while prokaryotic production decreases? • No simple generic ”perfect” host 7
10/23/2012 • No simple generic ”perfect” host • Protein specific • No simple generic ”perfect” host • Protein specific • Both eukaryotic and prokaryotic production systems have their place 8
10/23/2012 • No simple generic ”perfect” host • Protein specific • Both eukaryotic and prokaryotic production systems have their place • Things are not as bleak for prokaryotic production as is sometimes made out Current rule of thumb: Intracellular proteins Prokaryotic production Extracellular proteins Eukaryotic production or Prokaryotic production + ex vivo refolding 9
10/23/2012 In reality the issue is post translational modifications. PTM or not PTM, that is the question. Some PTMs can be added afterwards ex vivo e.g. amidation of the C-terminus R O H C C N C COOH H H H R O PAM C C N H H H 10
10/23/2012 Disulfide bond formation and N-glycosylation are not PTMs Both are co-translational modifications Both are difficult to add after the protein has been synthesised Some textbooks imply only eukaryotes can make disulfide bonds and N-glycosylate proteins Strict requirement for eukaryotic production system or ex vivo refolding to allow correct disulfide bond formation 11
10/23/2012 Periplasmic disulfide bond formation in E.coli Periplasmic N-glycosylation in E.coli Uses N-glycosylation systems introduced from other prokaryotes e.g. PglB from Campylobacter jejuni 12
10/23/2012 This can be used to generate defined N-glycan structures by two distinct methods In vivo N-glycosylation + Ex vivo transglycosylation 13
10/23/2012 Engineered pathways based on PglB promiscuity These are very successful for the production of some proteins 14
10/23/2012 These are very successful for the production of some proteins Can also be used as a route for other site specific modifications e.g. pegylation Limitations of the periplasm: • Low volume / low capacity => low yields (?) E.coli : Ronald Wetzel (SKB) 15
10/23/2012 Limitations of the periplasm: • Low volume / low capacity => low yields (?) Ignicoccus hospitalis: E.coli : Ronald Wetzel (SKB) Karl Stetter (Regensburg) Limitations of the periplasm: • Low volume / low capacity => low yields (?) Ignicoccus hospitalis: E.coli : Ronald Wetzel (SKB) Karl Stetter (Regensburg) • Secretion machinery easily overloaded 16
10/23/2012 Cytoplasmic expression in E.coli is ideal for protein production, but limited PTMs Periplasmic expression in E.coli can allow missing PTMs, but yields may be low. => An increased market share for eukaryotic systems in future? There are systems for disulfide bond formation and N-glycosylation in the cytoplasm 17
10/23/2012 Cytoplasmic expression Naturally reducing environment => inclusion bodies Pathways for disulfide bond reduction in E.coli cytoplasm X X 18
10/23/2012 D trxB D gor strains • Currently sold by Novagen (origami, rosetta-gami etc) and New England Biolabs (SHuffle). • Disulfide bond formation inefficient and dependent on external factors. • Yields of correctly folded protein often very low. • Strains can grow slowly on rich media and not at all on minimal media. These strains remove reducing pathways. They do not add a pathway for catalyzing de novo disulfide bond formation 19
10/23/2012 Either: • Take core catalysts from eukaryotes and express in the cytoplasm • Invert the natural systems present in prokaryotes so that they make disulfide bonds in the cytoplasm E.coli alkaline phosphatase 2 sequential disulfides, whose formation is essential for activity. Co-expression of Erv1p in a wild-type E.coli strain results in more active protein than rosetta-gami. Disulfide bonded 20
10/23/2012 E.coli phytase 4 disulfides, 1 non-sequential. Model protein that requires an isomerase. It folds correctly if Erv1p and an isomerase are co-expressed. Disulfide bonded Human tissue plasminogen activator (tPA ) kringle 2 + protease ≈ vtPA (9 disulfides, all non-sequential) 21
10/23/2012 Human tissue plasminogen activator (tPA ) kringle 2 + protease ≈ vtPA (9 disulfides, all non-sequential) This works in the cytoplasm of any E.coli strain Yields of up to 100mg/L of homogenously folded eukaryotic protein from shake flasks 22
10/23/2012 The system is amenable to fermentation Human interleukin 6: 1.0 g/L purified product Human growth hormone 1: 1.1 g/L purified product scFv: 0.6g/L purified product Cytoplasmic N-glycosylation in E.coli Uses N-glycosylation system from Actinobacillus pleuropneumoniae 23
10/23/2012 Several groups are now trying to combine cytoplasmic disulfide bond formation with cytoplasmic N-glycosylation, along with additional quality control systems. In effect they are mimicking the eukaryotic ER in the cytoplasm of a prokaryote. Why? 24
10/23/2012 Eukaryotic N-glycan heterogeneity Eukaryotic proteins are often heterogenous in the number and types of N-glycans added, even on a single protein This has major implications since N-glycans can modulate: • Biological activity • Stability • Clearance Eukaryotic N-glycan heterogeneity This can have some advantages, but is has a number of major disadvantages including: • Significant batch to batch variation • Biosimilars are not biosimilar 25
10/23/2012 Summary Efficient disulfide bond formation +/- N-glycosylation in the cytoplasm of E.coli offers up new, interesting, possibilities for the industrial production of homogenous proteins in high yields. It will not replace eukaryotic protein production, but offers an alternative solution that may create an interesting balance between eukaryotic and prokaryotic expression of biosimilars. Paras product developments Multiple product pipeline with delivery of at least one product API to market each each Products in our pipeline include: - Lantus (Glargine) - Teriparatide - A rheumatoid arthritis product 26
10/23/2012 Diabetes – Paras pipeline products validation Validation of production by: Mass Spectrometry SDS-PAGE Osteoporosis – Paras pipeline products validation Validation of production by: Mass Spectrometry SDS-PAGE Paras 7 (final product) 27
10/23/2012 Rheumatoid Arthritis – Paras pipeline product Paras 11 expresses product in high yields Purification of authentic product successful Purified yield equivalent to 0.7 kg from 500L Paras Pharmaceuticals Finland is looking for collaboration, out-licensing of technologies and partners. If interested please contact : Dr Inderjeet Kaur Director – Biosimilars & Biologics Paras Biopharmaceuticals Finland Kurkelantie 5 C-43 Oulu, Finland 90230 Email- ikaur@parasbiopharma.com 28
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