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Challenges in Industrial Production of Peptides
Transcript of Challenges in Industrial Production of Peptides
Challenges in Industrial Production of Peptides
Dr. Daniel BourginDirector of Sales & BD LCM-TIDES, Lonza Ltd. Basel, Switzerland
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Agenda
Market TrendTechnology TrendChallengesLonza’s Technology portfolioStrategy of SynthesisEconomy
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Market Trend – Peptides
Total global market potential API level (in-house and CMO) = 1bn USDMarket growth = 10 – 15% p.a.Launched Peptides = 43250 in Clinical Phase>200 in Pre-ClinicalEmerging areas: Cancer, HIV, Cardiovascular, CNS and Metabolic disordersGeneric peptides
0
50
100
150
200
250
pre-
clin
ical
phas
e I
phas
e II
phas
e III
laun
ched
Worldwide Peptide Pipeline (synthetic)
Num
ber o
f can
dida
tes
Source: Market Research
2005 substantial investment (CHF 24 Mio) in Peptides
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Technology Trend
Market faces the challenge to produce peptides in kilograms and >100kg (industrial conditions)
Increasing importance of long peptides
3 Technologies available for production of PeptidesSolid-Phase SynthesisLiquid-Phase SynthesisRecombinant Technology
Proven scale ability of peptide chemistry up to 1000 l reactors
The supplier-customer relationship is becoming more complex requiring an intensive collaboration throughout the whole life cycle of the product
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Some characteristic PeptidesProduct Length Product characteristics Estimated API Demand
Bivalirudin 20 1 unnatural amino acid
Peptide amideCyclic peptide, S-S bridge2 unnatural building blocks
Long peptide, peptide amideN-Terminus modified
Calcitonins 32 All natural amino acids, chemicaland recombinant routes
>100kg
Teriparatide 34 Long peptide, hormone regulator(recombinant)
Zadaxin 28 Long peptide, chemical synthesis > 100 kg
Abarelix 10 6 unnatural amino acids,Peptide amideN-Terminus modified
10 kg
Symlin 39 All natural amino acids, recomb & chem. synthesis
>100kg
> 100 kg
Eptifibatide(Integrilin)
7 100 kg
Enfuvirtide(T-20), Fuzeon
36 >500 kg ( > 1 to ?)
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ChallengesRight strategy of synthesis applicable on all scales
Quality of the peptide, single impurity profile, scale-up issues
Regulation, ICH guidelines small molecules, status in thelifecycle
Economy, cost of goods
Down stream processing and isolation
Logistic
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Scale-up effect in Solid-Phase synthesis of a long peptide
Crude peptide, Qualification laboratory sample:
Crude peptide, Launch plant material:
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LONZA, Exclusive Synthesis
Production Concept
Recombinant Technology
Solid Phase Synthesis
Liquid Phase Synthesis
Solid Phase Peptide Synthesizer, Visp
Biotec pilot plant, Visp
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Strategy of Synthesis-Liquid Phase
ProMethod of choice for production of short peptidesBOC, CbZ strategy, less expensive raw materialsImpurity profile, high chance to be easyUnlimited capacity
ConLimitation in number of AA to be combinedConvergent SynthesisNumber of unit operationsLong cycle timeYield
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Strategy of Synthesis-Solid Phase
ProMerrifield, Fmoc Strategy, orthogonal protection groups opened the door to the rapid synthesis of long peptidesPhysical-chemical properties of the growing peptide can be controlledProcess is automatable and scaleableNo isolation of intermediatesShort production cycles
Con(Expensive raw materials)Might generate a complex impurity profile
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Convergent SPPS - Hybridal Technology
OHHH
H OH
Repeat n times
Final deprotection
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Solid Phase Synthesis of long peptides
Issues:Insertion, deletion, double-hits
With increasing peptide length, chain aggregation, leads to truncated peptides, purification, yield
Synthesis of peptides with 30-40 residuals for commercial production are possible (GLP-1 analogs)
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Solid Phase Synthesis
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DC
M
DCM
Selector
UV
Ok-Pool
Buffer BBuffer A
DC
M
0.45 mfiltration
HPLC 60 cm Column
Peptidecrude
dissolution
0.45 mfiltration
0.45 mfiltration
To recycle
HPLC 60 cm Column
Concentration
0.2 mFiltration
Lyophilization
Sidefractions
DSP: Purification
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Lyophilisation
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Logistical Aspects(Example:)
An average 9 mer peptide in conjunction with customers requirements (annual demand of peptide API):
Strategic (worldwide) sourcing activities, raw material supply in rail-tank-cars, tank-farms, no open handling, recycling systems, adequate warehousing.
Raw MaterialRequirements 20 kg 100 kg 200 kg 500 kg
Amino acid 0.4 to 2.0 to 4.0 to 10.0 toTCTU 0.3 to 1.3 to 2.6 to 6.5 toPiperidine 6.5 to 33 to 65 to 163 toNMP 100 to 500 to 1000 to 2500 toDCM 80 to 400 to 800 to 2000 toACN 35 to 175 to 350 to 875 toUSP water 300 to 1500 to 3000 to 7500 to
Peptide / API vs. raw materials
annual demand in metric tons
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Strategy of Synthesis: Recombinant Peptide Production
Production organism
Product localization as concatemer
Makes sense for Has limited sense for
Advantage
1. E. coli intracellular Hydrophilic peptides, no secondary or tertiary structure
Strongly hydrophobic peptides (Inclusion bodies)
High product concentrations 5 – 10 g/L fermentation br.
2. E. coli periplasmic Peptides/ proteins with sec./ tert. structure (S-S)
Peptides/ proteins without sec./ tert. structure (S-S)
Use of secretion to generate biological active conformation;
3. Pichiaangusta
fermentationmedium
Hydrophilic & slightly hydroph. peptide/proteins +/- sec / tert.str.
Strong hydrophobic peptides/proteins
Secretion of right confirmation, less unit operations
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KUP 15 KUP 15 –– FermentorFermentor 15m315m3
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Example of a tricky Peptide
Cationic peptide- 13 mer
H-AA1-AA2-Arg3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-Arg11-AA112-A13-NH2
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Cationic Peptide Synthesis
H-AA1-AA2-Arg3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-Arg11-AA12-AA13-OH
Expressed peptide
Protection with DiBOC
Chemical Amidation with NH3
H-AA1-AA2-Arg3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-Arg11-AA12-AA13-NH2
Yield 98%Purity 97%
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Cationic Peptide Synthesis
CTC
Cl
H-AA1-AA2-Arg3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-Arg(Pbf)
lenghtening1) Fmoc-AA-OH , HBTU, DIEA 2) Ac2O/pyridine/DMF if require3) 20% piperidine
2% TFA/DCM
95% TFA/H2O/anisole
cleavage
deprotection
H-AA1-AA2-Arg3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-Arg11-AA12-AA13-NH2
H-AA12(Boc)-AA13(Boc)-NH2 HBTU, DIEA
CTC
1) Fmoc-Arg(Pbf)-OH (1.5eq), DIEA 2) MeOH
CTCFmoc-Arg(Pbf)(0.4 mmol/g)
Purification yield 80%
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Results
Cationic PeptideSemi-synthetic route discontinuedSwitch to alternative solid phase route in Phase IIEconomy comparable with the bio-route at large scale
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Economical aspects-Production CostRaw materials
Production Determining factor
Solid Phase Route
30-40% of overall costs
60 % including purification& isolation
Raw material situation, waste stream, atom economy
Recombinant 5% 95 % including DSP-1, purification and isolation
Unit operations, high development cost including scale-up
Common bottleneck
HPLC-purification & Isolation
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Conclusion: Strategy of Synthesis: Technology Combination Chart
Entry Solid Phase Liquid Phase Recombinant
Solid Phase 10- 40 mer<20 mer fragmentsPeptides containing
modified/non-modified amino acidsScale: gram -MT
Fragment condensationPeptides containing
modified/non-modified amino acids
Scale: gram-MT
Liquid Phase Fragments <10 merFragment
condensationAll types of AAScale: gram-MT
Recombinant Fragment condensationScale: >100kg-MT
>10 mer-ProteinsComplexAll natural AAScale: >100kg-MT
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