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: matthieu.giraud@lonza.comhttp://www.lonza.com