Hosts for Biosimilars Production: What is the future? Lloyd - - PDF document

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Hosts for Biosimilars Production: What is the future?

Lloyd Ruddock, Scientific Director, Paras Biopharmaceuticals Finland

Company executives meet and decide 10 year roadmap

2004

Paras Biopharmaceuticals Finland is conceptualized; details finalized

2006 2008

Biosimilars capabilities realized

2009

Paras Biopharmaceuticals Finland is established and executives look for interested investment groups

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Reproduced with permission from Helsinki University Collection

Paras research labs

Paras Business

Paras Biopharmaceuticals Finland works

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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

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Paras target areas

Diabetes Rheumatoid Arthritis Osteoporosis All high-growth, high-value areas with multiple products coming off patent over the next 6 years

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As well as utilizing existing local expertise, Paras has developed multiple novel technologies including:

• 1. Diabrid technology
• 2. Noble CleavR
• 3. BioMultifoldR
• 4. BioEnhancedR

Paras novel technologies

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“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.

Promoter Linker Terminator Paras propreity Therapeutic partner (PPP) Protein Cleavage

Diabrid technology

30 years ago FDA approved first recombinant biologic

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In the early years prokaryotic production dominated the market Sales of proteins produced in prokaryotes e.g. E.coli have continued to increase

Sales

• Low cost
• Rapid growth
• High biomass
• Easy cultivation and manipulation
• FDA friendly

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In the last decade market share of sales for biologics production in eukaryotes rapidly increasing

• Increased yields
• Decreased costs
• Eukaryotes can produce some

proteins, in an active state that prokaryotes cannot.

Market share

prokaryotes eukaryotes

Biologics production is predicted to continue to grow rapidly, but what will be the hosts in the future?

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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

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• No simple generic ”perfect” host
• Protein specific
• No simple generic ”perfect” host
• Protein specific
• Both eukaryotic and prokaryotic production systems have

their place

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• 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

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Prokaryotic production + ex vivo refolding

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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 C C N H H H PAM

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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

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ex vivo refolding to allow correct disulfide bond formation

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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

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This can be used to generate defined N-glycan structures by two distinct methods In vivo N-glycosylation + Ex vivo transglycosylation

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Engineered pathways based on PglB promiscuity

These are very successful for the production of some proteins

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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)

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Limitations of the periplasm:

• Low volume / low capacity => low yields (?)

Ignicoccus hospitalis: Karl Stetter (Regensburg) E.coli: Ronald Wetzel (SKB)

Limitations of the periplasm:

• Low volume / low capacity => low yields (?)
• Secretion machinery easily overloaded

E.coli: Ronald Wetzel (SKB) Ignicoccus hospitalis: Karl Stetter (Regensburg)

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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

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Cytoplasmic expression Naturally reducing environment => inclusion bodies

Pathways for disulfide bond reduction in E.coli cytoplasm

X X

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DtrxB Dgor 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

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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

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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)

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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

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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 The system is amenable to fermentation Cytoplasmic N-glycosylation in E.coli Uses N-glycosylation system from Actinobacillus pleuropneumoniae

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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

• f a prokaryote.

Why?

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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

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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

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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)

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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,

• ut-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

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Acknowledgements: Paras Biopharmacuticals Finland research team Biocenter Oulu Paras advisory board For further information: ikaur@parasbiopharma.com

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info@parasbiopharma.com Paras Biopharmacuticals Finland Kurkelantie 5 C-43 Oulu Finland