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Transcript
Challenges in Industrial Production of Peptides
Dr. Daniel Bourgin
Director of Sales & BD LCM-TIDES,
Lonza Ltd. Basel, Switzerland
Agenda
„
„
„
„
„
„
Market Trend
Technology Trend
Challenges
Lonza’s Technology portfolio
Strategy of Synthesis
Economy
slide 2
Market Trend – Peptides
„
2005 Â substantial investment
(CHF 24 Mio) in Peptides
150
100
50
0
Source: Market Research
launched
„
phase III
„
200
phase II
„
250
phase I
„
Worldwide Peptide Pipeline
(synthetic)
pre-clinical
„
Total global market potential API level
(in-house and CMO) = 1bn USD
Market growth = 10 – 15% p.a.
Launched Peptides = 43
250 in Clinical Phase
>200 in Pre-Clinical
Emerging areas: Cancer, HIV,
Cardiovascular, CNS and Metabolic
disorders
Generic peptides
Number of candidates
„
slide 3
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 Peptides
„ Solid-Phase Synthesis
„ Liquid-Phase Synthesis
„ Recombinant 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
slide 4
Some characteristic Peptides
Product
Length
Product characteristics
Estimated API Demand
Bivalirudin
20
1 unnatural amino acid
> 100 kg
Eptifibatide
(Integrilin)
7
Peptide amide
Cyclic peptide, S-S bridge
2 unnatural building blocks
100 kg
Enfuvirtide
(T-20), Fuzeon
36
Long peptide, peptide amide
N-Terminus modified
>500 kg
( > 1 to ?)
Calcitonins
32
All natural amino acids, chemical
and 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 amide
N-Terminus modified
10 kg
Symlin
39
All natural amino acids, recomb &
chem. synthesis
>100kg
slide 5
Challenges
„
Right strategy of synthesis applicable on all scales
„
Quality of the peptide, single impurity profile, scale-up issues
„
Regulation, ICH guidelines small molecules, status in the
lifecycle
„
Economy, cost of goods
„
Down stream processing and isolation
„
Logistic
slide 6
Scale-up effect in Solid-Phase synthesis of a
long peptide
„
Crude peptide,
Qualification laboratory sample:
„
Crude peptide,
Launch plant material:
slide 7
LONZA, Exclusive Synthesis
„
Production Concept
Recombinant Technology
Biotec pilot plant, Visp
Solid Phase Synthesis
Liquid Phase Synthesis
Solid Phase Peptide
Synthesizer, Visp
slide 8
Strategy of Synthesis-Liquid Phase
Pro
„ Method of choice for production of short peptides
„ BOC, CbZ strategy, less expensive raw materials
„ Impurity profile, high chance to be easy
„ Unlimited capacity
Con
„ Limitation in number of AA to be combined
„ Convergent Synthesis
„ Number of unit operations
„ Long cycle time
„ Yield
slide 9
Strategy of Synthesis-Solid Phase
Pro
„ Merrifield, Fmoc Strategy, orthogonal protection groups
opened the door to the rapid synthesis of long peptides
„ Physical-chemical properties of the growing peptide can be
controlled
„ Process is automatable and scaleable
„ No isolation of intermediates
„ Short production cycles
Con
„ (Expensive raw materials)
„ Might generate a complex impurity profile
slide 10
Convergent SPPS - Hybridal Technology
OH
H
H
Repeat n times
Final deprotection
H
OH
slide 11
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)
slide 12
Solid Phase Synthesis
slide 13
Buffer A
0.45 m
filtration
0.45 m
filtration
HPLC 60 cm Column
DCM
UV
Selector
Ok-Pool
Side
fractions
To recycle
HPLC 60 cm Column
Concentration
0.2 m
Filtration
Lyophilization
DCM
0.45 m
filtration
Peptide
crude
dissolution
DCM
DSP: Purification
Buffer B
slide 14
Lyophilisation
slide 15
Logistical Aspects
(Example:)
„
„
An average 9 mer peptide in conjunction with customers
requirements (annual demand of peptide API):
Raw Material
Requirements
Peptide / API vs. raw materials
20 kg
100 kg
200 kg
500 kg
Amino acid
TCTU
Piperidine
NMP
DCM
ACN
USP water
0.4 to
0.3 to
6.5 to
100 to
80 to
35 to
300 to
2.0 to
1.3 to
33 to
500 to
400 to
175 to
1500 to
4.0 to
2.6 to
65 to
1000 to
800 to
350 to
3000 to
10.0 to
6.5 to
163 to
2500 to
2000 to
875 to
7500 to
annual demand
in metric tons
Strategic (worldwide) sourcing activities, raw material supply in
rail-tank-cars, tank-farms, no open handling, recycling systems,
adequate warehousing.
slide 16
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 Peptides/ proteins Use of secretion
without sec./ tert. to generate
with sec./ tert.
biological active
structure (S-S)
structure (S-S)
conformation;
3. Pichia
angusta
fermentation
medium
Hydrophilic &
slightly hydroph.
peptide/proteins
+/- sec / tert.str.
Strong
hydrophobic
peptides/proteins
Secretion of right
confirmation, less
unit operations
slide 17
KUP 15 – Fermentor 15m3
slide 18
Example of a tricky Peptide
„
Cationic peptide- 13 mer
H-AA1-AA2-Arg3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-Arg11-AA112-A13-NH 2
slide 19
Cationic Peptide Synthesis
Expressed peptide
H-AA1-AA2-Arg3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-Arg11-AA12-AA13-OH
Protection with DiBOC
Yield 98%
Purity 97%
Chemical Amidation with NH3
H-AA1-AA2-Arg3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-Arg11-AA12-AA13-NH2
slide 20
Cationic Peptide Synthesis
Cl
CTC
1) Fmoc-Arg(Pbf)-OH (1.5eq), DIEA
2) MeOH
(0.4 mmol/g)
Fmoc-Arg(Pbf)
lenghtening
CTC
1) Fmoc-AA-OH , HBTU, DIEA
2) Ac2O/pyridine/DMF if require
3) 20% piperidine
H-AA1-AA2-Arg3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-Arg(Pbf)CTC
cleavage 2% TFA/DCM
H-AA12(Boc)-AA13(Boc)-NH2 HBTU, DIEA
deprotection
95% TFA/H2O/anisole
H-AA1-AA2-Arg3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-Arg11-AA12-AA13-NH2
Purification yield 80%
slide 21
Results
„
Cationic Peptide
„ Semi-synthetic route discontinued
„ Switch to alternative solid phase route in Phase II
„ Economy comparable with the bio-route at large scale
slide 22
Economical aspects-Production Cost
Solid Phase
Route
Raw
materials
Production
Determining factor
30-40%
of overall
costs
60 % including
purification&
isolation
Raw material situation,
waste stream, atom
economy
Recombinant 5%
Common
bottleneck
95 % including Unit operations, high
DSP-1,
development cost
purification and including scale-up
isolation
HPLC-purification &
Isolation
slide 23
Conclusion: Strategy of Synthesis: Technology
Combination Chart
Entry
Solid Phase
Liquid Phase
Solid Phase
10- 40 mer
„<20 mer fragments
„ Peptides containing
modified/nonmodified amino acids
„Scale: gram -MT
„Fragment
Liquid Phase
„
Recombinant
„
Fragments
Recombinant
condensation
„Peptides containing
modified/non-modified
amino acids
„ Scale: gram-MT
<10 mer
„Fragment
condensation
„All types of AA
„ Scale: gram-MT
„
„Fragment
condensation
„Scale: >100kg-MT
>10 mer-Proteins
„Complex
„All natural AA
„Scale: >100kg-MT
„
slide 24
THANK YOU FOR YOUR ATTENTION!
We invite you to our booth
9F12, Hall 9
slide 25