Peptide at a Glance - Lonza: A Global Leader In Life Sciences
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Peptide at a Glance Dr. Matthieu Giraud / GSK, Stevenage, UK / 21-22- October 2009 Active Pharmaceuticals Ingredients (API) manufacturing
Transcript of Peptide at a Glance - Lonza: A Global Leader In Life Sciences
Peptide at a Glance
Dr. Matthieu Giraud / GSK, Stevenage, UK / 21-22- October 2009
Active Pharmaceuticals Ingredients (API) manufacturing
Insulin
slide 33-Nov-09
Increasing Molecular Weight (MW)
Chemical Molecule
Aspirin (180 Da)
Peptide
6mer (804 Da)
Antibody
Immunoglobulin G (150,000 Da)
Protein
Lysozyme(14,700 Da)
Complexity
Cell / Tissue
Surface of an Animal Cell
slide 43-Nov-09
Agenda
Agenda� Introduction to peptide Chemistry � Peptide chemistry at University � The market� Peptide chemistry in a CMO – case studies
Introduction to Peptide Chemistry
slide 63-Nov-09
Peptide – Backbone & Rules
NH
HN
OHH2N
O
O
OR
H R
H
H
OHO
n
Write from N- to C- terminalAlso Biosynthesis direction
Direction for the synthesis(C- to N-terminal)
slide 73-Nov-09
Options for Peptide Manufacture
Chemical Synthesis
� Solid-phase Peptide Synthesis (SPPS)
� Solution-phase Peptide Synthesis
� Hybrid of above-Convergent Peptide Synthesis
Biological Expression
� Bacterial
� Yeast
� Mammalian
� Transgenic
Fermentation
slide 9
Concatemer Approach
� Peptide multicopy gene � Peptide multicopy protein� Concatemer optimal design (i.e. for spacer and cutting principle)
Gene synthesis ���� Lonza Expression System ����High Cell Density Fermentation���� Concatemer Digest and Spacer Cleavage ���� Simple Precursor Purification
Cleavage
Peptide multicopy protein = concatemer
+ +
slide 10
Recombinant Peptides for The Medicines Company, Boston MA
slide 11
Pharmacology of Angiomax ™
slide 12
Generation of Bivalirudin18-mer Intermediate
Removal of medium ions via UF
Enzymatic digest of concatemer
Stop enzymatic digest with TFA
Remove enzymes, host cell impurit. via UF
concatemer
Trypsin
Leader-R-aa1---------aa1-R-aa1------aa1-R-aa1------ -aa1-R-aa1-----
Carboxy-Peptidase B
18-mer
slide 13
Peptide Impurity Profile rec. Bivalirudin-18mer: each peak characterised
18MER = RPGGGGNGDFEEIPEEYL
LEADER SEQ. = MSKTTKKFNSLPLDSMEGS 18MER
18MER -(YL)
ASP-18MER-(L)
18MER-(L)
VAL-18MER
ASP-18MER
18MER + 72 amu
CYCLIC 18MER
18MER + ARG
L R-18MER
KFNSLPLDSMEGS
KFNSLPLDSMEGSR
slide 14
Membrane purification Spray drier suiteHigh pressure homogenizer
Biotec Multi-Purpose Plant Down stream processing
� Launch plant: broad down stream processing capabilities
SPPS steps
slide 163-Nov-09
Solid Support: General Features
1.Stable to Variation in Temperatures2.Mobile, Well-Solvated, and Reagent Accessible Sites3.Acceptable Loadings4.Good Swelling in Broad Range of Solvents (if applicable)5.Acceptable Bead Sizes (if applicable)6.Stable in Acidic, Basic, Reducing, and Oxidizing Conditions7.Compatible with Radical, Carbene, Carbanion, and Carbenium8.Mechanically Robust: Batchwise and Flow-Continuous Modes
An optimal solid support should have the following characteristics:
slide 173-Nov-09
The Stress of the Bead
slide 183-Nov-09
Polymeric Support: Gel Type, Polystyrene
+
Styrene Divinylbenzene
Polymerization
Physical Characterization of beads• 100-200 ( 75-150 µm) or
200-400 (75- 38 µm) mesh• 1 or 2 % crosslinking• Mechanically robust• > 99% of the pendant functional
groups are located within the matrix
Solvent Compatibility• DCM• DMF• THF• Toluene• TFA
slide 193-Nov-09
Polymeric Support: Gel Type, Polystyrene
+
Styrene Divinylbenzene
Polymerization
CH2Cl
CH2Cl
CH2Cl
CH2Cl
CH2Cl
CH2Cl
Polymerization
slide 203-Nov-09
Polymeric Support: PEG-based resins
Aminomethyl-ChemMatrix®
• Highly crosslinked polymer based on PEG 2000.• Only primary ether bounds (stability towards
acidic conditions)• Loading: circa 0.95 mmol/g
NH2OH2NO
O( )n
OO
O( )n
OO
NH2H2N
H2N NH2
( )n
slide 213-Nov-09
Polymeric Support: Swelling
02468
1012141618
Aceto
nitrile
DCM
DMF
DMSO
Met
hano
lTFA
Water
Swel
ling
s (m
L/g
) Polystyrene
TentaGel
CLEAR
Hydroxymethyl-CM
Aminomethyl-CM HCl
slide 223-Nov-09
Solid-Phase Peptide Synthesis Strategy
NH
HN
OHH2N
O
O
OR
H R
H
H
OHO
n
C-TerminalCarboxyl
Side-ChainCarboxyl
N-TerminalAmine
BackboneAmide
Side-ChainFunctionality
slide 23
Stepwise Solid-Phase Peptide Synthesis
Fmoc cleavage
XFmoc cleavage
Repetitive reaction
XAA resin loading
XAA coupling
XAA coupling
OH+
OH+
OH+
X
H NH2Peptide cleavage
slide 24
Stepwise SPPS (2) – GD / DSP
H NH2 . TFA salt
Global Deprotection
Purification (HPLC)
Desalting
Lyophilization
H NH2. AcOH salt
H NH2
* Schema by Prof. F. Albericio, Institut de Recerca Biomèdica, Barcelona, IRB-PCB
slide 25
Convergent Solid-Phase Peptide Synthesis
OHHH
H OH
Repeat n times
Final deprotection
slide 26
Analysis Challenges
H OH
OHH
OHH
H OH
Main product
OHH
H OH
H OH
H OH
slide 27
Control of Reactions
� Colorimetric Methods:� ninhydrine� chloranil� Leclerk� bromophenol...
� Spectroscopic Methods: � FT-IR� Gel-phase� MAS-NMR
Peptide in Universities
slide 293-Nov-09
Tools Box Approach
slide 303-Nov-09
Some Typical Academic Focus
� Get the targets!� using routine elongation procedure (equipments, historical lab
know-how)� Routine purification (same stationary phase / buffer for any
peptides)� Isolation using lyophilization� Yield?� Productivity?� Counter ion?� impurities?� Analysis (Only ID & HPLC)
Market Analysis
slide 323-Nov-09
Global Pharmaceutical Market
slide 333-Nov-09
Peptide Pharmaceutical Market in 2008
Major Market Developments
� Growth: CAGR06-08 10% (revenues)
� 60% in-house vs. 40% CMO, to reach 50% by 2011
� Technologies: SPPS, HPPS (LPPS), recombinant technology
� Pipeline: Top 3 generics (Leuprorelin, Octreotide, Goserelin) = 50% of market revenues. Peptide products to be launched 2011-2015: 14-38
$1.3 bn(Peptide API Market
1400 kg )
$500 m(Peptide CMO Market)
LONZA
$9.5 bn(Peptide Pharmaceutical Market )
$9.5 bn and 1400 kg Peptide Market
slide 343-Nov-09
Top Peptide-based Drug Product
Top Ten Drug Product Drug Substance Amino acid
Copaxone Glatiramer (4)n
Lupron Leuprolide 9
Zoladex Goserelin 9
Sandostatin Octreotide 7
Byetta Exenatide 39
Decapeptyl Triptorelin 10
Integrelin Eptifibitide 6
Miacalcin Calcitonin 32
Fuzeon Enfuvirtide 36
Angiomax Bivalirudin 20
Sales
>USD 1
Billion
Sales
<USD 1
Billion
slide 353-Nov-09
Growing Market?
� After 2010: probably growth by 5 –10% per year
slide 363-Nov-09
Some Therapeutic Peptides & their Application
Desmopressin Mpa-Tyr-Phe-Gln-Asn-Cys-Pro-DArg-Gly-NH2
•bed wetting•diabetes insipidus•mild hemophilia
DPhe-Cys-Tyr-DTrp-Lys-Thr-Cys-Thr-olOctreotide(Sandostatin)
•digestive secreting tumors•acromegaly•pancreatic fistulae
LanreotideDNal-Cys-Tyr-DTrp-Lys-Val-Cys-Thr- NH2
•same as octreotide(but longer acting)
Leuprolide Pyr-His-Trp-Ser-Tyr-Dleu-Leu-Arg-Pro-NHEt
•prostate cancer•endometriosis•precocius puberty
Calcitonin(salmon) CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP-NH2
•osteoporosis•Paget’s disease
T20 (Fuzeon) Ac-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-NH2•HIV fusion inhibitor
slide 373-Nov-09
Fuzeon®
Fuzeon®
M.W. 4,492
O
NH
NH
ONH2
NH
NHO
NH
ONH
NH
O
OOH
NH
O
NH
O
O
OH
NH
O
NH2
NH
O
NH
NH
O
NH
O
OH
NH
O
NH O
NH
O
NH O
ONH2
NH
O
NH
OO
OH
NH
OO
OH
NH
ONH2 NH
O ONH2
NH
OO
NH2
NH
O
OH
NH
O
OH
NH
O
OH
NH
O
NH
O
NH
O
NHN
NH
O
OH
NH
ONHO
NH
O
O OH
NH
O
O OH
NH
O
OH
NH
O
O NH2
NH
O
O
NH2
NH
O NH2
O
OHO
O
NH2
O
Source: 2004_Trimeris Presentation NP2D event Zermatt
Model for HIV-cell fusion
ReceptorBinding
FusionIntermediate
Six-HelixBundle
Formation
HR2-Zipping
HR1
HR2
gp41
Target cell
membrane
Viral membrane
Fuzeon® inhibition of HIV fusion
Source: 2004_Trimeris Presentation NP2D event Zermatt
XXXXXXXXXXXXXXXXReceptorBinding
FusionIntermediate
Six-HelixBundle
Formation
HR1
HR2
HR2-Zipping
gp41
Target cell
membrane
Viral membrane
FuzeonFuzeon / T/ T--12491249 InhibitionInhibition
XXXXXXXX XXXXXXXXENF or TENF or T --12491249
Fuzeon® inhibition of HIV fusion
Source: 2004_Trimeris Presentation NP2D event Zermatt
slide 403-Nov-09
Fuzeon Route Development
Route 1:� Linear SPPS� Rink Amide MBHA Resin
Route 2:� SPPS of Peptide Fragments� Solution Fragment Condensation� 2CTC Resin
Common� Fmoc-Strategy� Side-chain protection (Boc, Trityl, and t-butyl)
slide 413-Nov-09
Fragment ApproachResinResin ResinResin Resin Resin
Fragment 1 Fragment 2 Fragment 3Ac
Purification
Side Chain Deprotection
API
H
Ac
NH2
NH2
NH2
Crude peptide
AA(13-26)AA(17-26)
AA(27-38)AA(27-35)
AA(1-12)AA(1-16)
OH
Fragment 3´
T-1249Fuzeon
H-Phe-NH2
slide 423-Nov-09
Purification & Process Summary
� Single pass RP HPLC purification � Isolation by precipitation near isoelectric point
� High purity� High recovery
THANKS to Roche / Trimeris (Fuzeon, T20): � Leverage a positive attitude from Pharma on Peptide
manufacture via SPPS� Positive effect of the supply chain (Fmoc amino acid +
CTC resin)
slide 433-Nov-09
Fuzeon Process Summary
� Conventional Plant Equipment� Minimal Isolations� No Lyophilization� Reagents and Starting Materials are Subject to Competitive Bidding� Overall yield, purity, and cycle time meet commercial requirements
slide 453-Nov-09 Sources: 1MMAS J Urol, 1994; 2Chew et al, Int J Impot Res. 2000
3.6 MM Men Seeking Treatment for ED 2
30 MM men with ED 1
618,000 new cases added each year
150 MM men with ED 1
18 MM Men Seeking Treatment for ED 2
Erectile Dysfunction Market
Worldwide USA
mill
ions
of p
eopl
e
Total
Severe
Moderate
Mild
slide 463-Nov-09
Visual & Tactile Stimulation
PDE-5
GMP
SelectiveNerve Signal
NO
cGMP
Vasodilation & Erection
ViagraLevitraCialis
X
cGMP
Bremelanotide
Bremelanotide – Mechanism of Action
slide 473-Nov-09
NH
NH
O
NH
NH3
+NH
NH
O
NH
NH
O
OHNH
O
O
NH
O
N
NH
NH
O
O
NH
O
CH3
O
O
Bremelanotide – PT141 - Structure
slide 483-Nov-09
� 14 synthesis steps
� Two main Fragments� Boc-Asp(OBzl)-His(Dnp)-(D)Phe-OH
� 2TFA.Arg-Trp(For)-Lys(Z)-OMe
NH
NH
O
NH
NH3
+NH
NH
O
NH
NH
O
OHNH
O
O
NH
O
N
NH
NH
O
O
NH
O
CH3
O
O
Liquid Phase Strategy & Challenges
slide 493-Nov-09
� Full length elongation� Cyclization� Selective side chain protection� Cost of Goods (Excess of reagents, Solvent,…)
O
O
NH
O
N N
NH
O
NH
NH
NH
NH
O
NH
O
N
NH
O
NH
O
O
S
O
O O
O
O
O
O
O
NH
Solid Phase Strategy & Challenges
slide 503-Nov-09
SPPS Route
NH
O
NH
O
O
O
O
O CTC
Cl CTC
O
O
NH
O
N N
NH
O
NH
NH
NH
NH
O
NH
O
N
NH
O
NH
O
O
S
O
O O
O
O
O
O
O
NH
CTC
peptide lenghtening:
1) 20% piperidine in NMP 2) Fmoc-AA-OH
1) Fmoc-Lys(Alloc)-OH, DIEA, DCM2) DIEA, MeOH
cycles
O
O
NH
O
N N
NH
O
NH
NH
NH
NH
O
NH
O
N
NH
O
NH
O
O
SO
O O
O
O
O
O
O
NH
CTC
OHO
O
NH
O
N N
NH
O
NH
NH
NH
NH
O
NH
O
N
NH
O
NH
O
O
S
O
O O
Ac-Nle-NH
1) Allyl/Alloc cleavage2) Cyclisartion3) Fmoc-Nle-OH4) 20% piperidine in NMP5) Acetylation6) cleavge
slide 513-Nov-09
Solution to selective Allyl / Aloc Cleavage
Catalyst� Pd(Ph3)4
� Pd(OAc)2 / P(oTol)3
� PdCl2(Ph3)4
Scavengers / proton donor� HCOOH/DIEA� Dimedone� Morpholine� AcOH/NMM� Phenylsilane� Me2NH.BH3 (DMAB)
Design of Experiments
slide 523-Nov-09
DoE Approach - Screening – Response Surface – Robustness Test
Factor Screening DoE
Optimization DoE
yield Contour79
80
80
81
81
82
82
83
83
83
84
85
86
87
88
88.5
666666
Design Space
slide 533-Nov-09
Solution to selective Allyl / Aloc Cleavage
Catalyst� Pd(Ph 3)4
� Pd(OAc)2 / P(oTol)3
� PdCl2(Ph3)4
Scavengers / proton donor� HCOOH/DIEA� Dimedone� Morpholine� AcOH/NMM� Phenylsilane� Me2NH.BH3 (DMAB)
slide 543-Nov-09
Individual Coupling Optimization
Purity 93A%NH O
N
NH O
NH
OH
O
O
O
O
N
N+
MW =615.76
O
ONH
O
N
NH O
NH
OH
O
O
O
O
MW =616.72
Prod. + 2
β−Ala impuritySM Prod.-Boc
Fmoc-Arg(Pbf)-Trp(Boc)-Lys(Aloc)-OH
slide 553-Nov-09
Final Global Deprotection
O
OF
F
F
NH
NH O
NH
NH3
+NH
NH O
NH
NH O
OHNH O
O
NH O
NNH
NH O
ONH
O
O
SNH
O O
O
ON
NH
NH O
NH
NH
NH O
N
NH O
OHNH O
O
NH O
N
NH O
ONH
O
1) reaction: TFAsolution + Scavengermixture
2) Precipitation inether
PI-001-PCN: 35169Molecular Weight =1619.96Exact Mass =1618Molecular Formula =C87H106N14O15S
PI-001-rohCN: 35193Molecular Weight =1139.22Exact Mass =1138Molecular Formula =C50H69N14O10 . C2F3O2
+ carbamate
slide 563-Nov-09
OptimizationReaction optimization (CAD):
� First set of experiments (screening)� cocktail composition
� Second set of experiments (optimization)� reaction conditions: Temp. / Time….
� residual solvent from SPPS…
Anisole
TIS
slide 573-Nov-09
Coefficient plot of the model
-4
-3
-2
-1
0
1
2
3
4
5
PH
L
H2O
DC
M
AN
I
TIS
PH
L*A
NI
PH
L*T
IS
H2O
*TIS
DC
M*A
NI
%
Investigation: PI-001-roh(sreening corr yield) (MLR)Scaled & Centered Coefficients for Yield-6
N=19 R2=0.946 R2 Adj.=0.893DF=9 Q2=0.888 RSD=1.9768 Conf. lev.=0.95
slide 583-Nov-09
Influence of TIS and Phenol
slide 593-Nov-09
Influence of TFA and Time
Yield
slide 603-Nov-09
Crude Material before DSP
slide 613-Nov-09
Manufacturing – Different Tools…
slide 623-Nov-09
Some typical Industrial Focus
� Get the target - on time and quality!� Using optimized elongation procedure (equipments, historical
know-how, DoE, QbD)� Best purification (best stationary phase / buffer)� Isolation using precipitation, lyophilization or spray drying� High yield & Productivity� Meet specification (Mass, Counter ion assay, peptide assay &
purity, enantiomeric analysis, residual solvent, heavy metal, bioorganisme…?
slide 633-Nov-09
Factors to Success - SPPS Approach
� Be innovative in the choice of protecting groups – Think out of the
box
� Strong sourcing department
� Back integration of key starting materials (Facility in China)
� State of the art cyclization is on resin
� Use DoE for screening & optimization
� Availability of tank farm for large volume & railway connection
� On site solvent recovery expert and assets
slide 643-Nov-09
Bright Peptide Future
� Exceptionally versatile molecules� Can address previously untreatable medical conditions.� Often very potent, offsetting (high) cost of manufacture.� Non-toxic: Physiological degradation to amino acids.� Deleterious side-effects are uncommon: Specificity honed by evolution. � Scale of manufacture increasing.� Cost of manufacture decreasing.� Drug delivery platforms becoming available
Any Questions:Dr. Matthieu GiraudDirectorResearch & Development Peptides phone: + 41 27 948 6631fax: + 41 27 947 6631 mobile: +41 79 645 27 46mailto: [email protected]://www.lonza.com
