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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