10/23/2012
Hosts for Biosimilars Production:
What is the future?
Lloyd Ruddock,
Scientific Director, Paras Biopharmaceuticals Finland
2004
2008
Company executives
meet and decide 10
year roadmap
Biosimilars
capabilities
realized
Paras Biopharmaceuticals
Finland is conceptualized;
details finalized
2006
Paras Biopharmaceuticals
Finland is established and
executives look for interested
investment groups
2009
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10/23/2012
Paras research labs
Reproduced with permission from Helsinki University Collection
Paras Business
Paras Biopharmaceuticals Finland works on
development for biosimilars and their out-licensing
technology
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
Paras novel technologies
As well as utilizing existing local expertise, Paras has developed
multiple novel technologies including:
1. Diabrid technology
2. Noble CleavR
3. BioMultifoldR
4. BioEnhancedR
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Diabrid technology
Paras propreity
partner (PPP)
Promoter
Therapeutic
Protein
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
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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
• Rapid growth
Sales
• Low cost
• 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
prokaryotes
• Decreased costs
• Eukaryotes can produce some
proteins, in an active state that
prokaryotes cannot.
Market share
• Increased yields
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
or
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
C
C
H
H
N
C
H
H
COOH
PAM
R
O
C
C
H
N
H
H
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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
or
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 (?)
E.coli: Ronald Wetzel (SKB)
Ignicoccus hospitalis:
Karl Stetter (Regensburg)
Limitations of the periplasm:
• Low volume / low capacity => low yields (?)
E.coli: Ronald Wetzel (SKB)
Ignicoccus hospitalis:
Karl Stetter (Regensburg)
• Secretion machinery easily overloaded
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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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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
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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
of 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,
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- [email protected]
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Acknowledgements:
Paras Biopharmacuticals Finland research team
Biocenter Oulu
Paras advisory board
For further information:
[email protected]
or
[email protected]
Paras Biopharmacuticals Finland
Kurkelantie 5 C-43
Oulu
Finland
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