Peptide Manufacturing

Chemical Process Development Peptide Manufacturing Dr. Matthieu Giraud / London / 23rd & 24th Feb. 2012

Agenda Challenge life cycle, specification, scale up, solvent handling, bioavailability, sourcing Solution integrated process dev. suppliers formulation 24-Feb-12 slide 2

Challenges

Life Cycle Discovery Development Manufacture Distribution basic research disease discovery drug discovery drug development clinical trials production packaging marketing sales distribution Pre-clinical Toxicology Commercial Phase I Phase II Phase III non-gmp cgmp 24-Feb-12 (1-500g) Customer Process R&D (~100g - 5 kg) small scale Qualification / Validation ( ~0.1 kg - 10kg) Manufacturing ( >10 kg) slide 4

Purity: What are the Expectations? 95% 96-97% >98.0% Pre-clinical Toxicology Commercial Phase I Phase II Phase III European Pharmacopeia 6.5 24-Feb-12 slide 5

Solid-Phase Peptide Synthesis (SPPS) AA resin loading AA coupling AA coupling + Fmoc cleavage + Fmoc cleavage + X X X X X Repetitive reaction Peptide cleavage 2 slide 6

Stepwise GD / DSP 2 Global Deprotection (GD) 2. TFA salt Purification (PLC) Down Stream Process (DSP) Desalting Lyophilization 2. Ac salt * Schema by Prof. F. Albericio, Institut de Recerca Biomèdica, Barcelona, IRB-PCB slide 7

Cost Structure f(scale) 24-Feb-12 slide 8

Impurities! During the peptide assembly in in SPPS the the following following things willthings go wrong can go wrong Impurities in starting materials Impurities formed during peptide elongation Isomers Double hits Deletion products Side products from reactions with coupling reagents, deprotection reagents or solvents Side products from other reactions on side chain functionalities slide 9

QC Challenges Many impurities are co-synthesized: double hit AA, deletions, diastereomers Main product slide 10

Supply Chain: Example #1 ydrogenation workshop Centrifuge slide 11

Supply Chain: Example #2 Centrifuges Dryer slide 12

Too Fast? January 4th, 2011 slide 13

Manufacturing Challenges - Methods slide 14

Some Solutions

Integrated Process Design double hit AA, deletions, diastereomers Critical Impurities: Act on chromatography Act on chemistry on-critical Impurities: Act on chemistry! Main product slide 16

Inter-Department Synergy UPS DSP Product Quality Finnigan LCQDECA Applied Biosystems QStar Maldi Q-Tof Pre-clinical Toxicology Commercial Phase I Phase II Phase III slide 17

And the FDA? Stage 1 Process Design Stage 2 Process Qualification Stage 3 Continued Process Verification slide 18

Quality by Design (QbD): Conceptual Flow Chart Inputs Step utputs Target Route Selection Process Selection Qualification Plan Samples IS (GMP1) QFD Exp. Plan Parameter Screening Risk Analysis ptimization Design Space GMP1+3 Commer cial Robustness 24-Feb-12 slide 19

Audit Raw Material Supliers In the dryer building Finished product warehouse slide 20

Audit Raw Material Supliers Raw material warehouse liquids - solids slide 21

Audit Report Rating QC/QA Plant status SE Technology R&D verall rating fair to good fair to good good fair (focused on peptides) good (focused on peptides) fair to good slide 22

Sourcing Quality: strict specifications (purity, chiral purity, residual solvent, ) ow to accept new source QC analysis of 3 different lots Use-test in R&D BSA/TSE certificate Audit slide 23

Solid Support: General Features An optimal solid support should have the following characteristics: 1.Stable to Variation in Temperatures 2.Mobile, Well-Solvated, and Reagent Accessible Sites 3.Acceptable Loadings 4.Good Swelling in Broad Range of Solvents (if applicable) 5.Acceptable Bead Sizes (if applicable) 6.Stable in Acidic, Basic, Reducing, and xidizing Conditions 7.Compatible with Radical, Carbene, Carbanion, and Carbenium 8.Mechanically Robust: Batchwise and Flow-Continuous Modes 23-Feb-12 slide 24

The Stress of the Bead 23-Feb-12 slide 25

Polymeric Support: Gel Type, Polystyrene + Polymerization Styrene Divinylbenzene Polymerization C 2 Cl C 2 Cl C 2 Cl C 2 Cl C 2 Cl C 2 Cl 23-Feb-12 slide 26

Polymeric Support: Gel Type, Polystyrene + Polymerization Styrene Divinylbenzene Polymerization C 2 Cl C 2 Cl C 2 Cl C 2 Cl C 2 Cl C 2 Cl 23-Feb-12 slide 27

Polymeric Support: PEG-based resins Aminomethyl-ChemMatrix 2 ( ) n 2 2 ( ) n 2 2 ( ) n 2 ighly crosslinked polymer based on PEG 2000. nly primary ether bounds (stability towards acidic conditions) Loading: circa 0.95 mmol/g 23-Feb-12 slide 28

Polymeric Support: Swelling Swellings (ml/g) 18 16 14 12 10 8 6 4 2 0 Polystyrene TentaGel CLEAR ydroxymethyl-cm Aminomethyl-CM Cl Acetonitrile DCM DMF DMS Methanol TFA Water 23-Feb-12 slide 29

Polymeric Support: Productivity 10 kg peptide / Batch 3 kg peptide / Batch Resin + AC + peptide dry resin dry resin Resin + AC + peptide Resin + TFA + peptide Limiting steps (i.e ChemMatrix) Elongation: AC (6 L /kg) Clivage: TFA (16 L /kg!!!) o space for the clivage Limiting steps (i.e ChemMatrix) Elongation: AC (6 L /kg) Clivage: TFA (16 L /kg!!!) Productivity / 3 23-Feb-12 slide 30

Polymeric Support: Productivity dry resin Loading step Elongation Clivage Polystyrene: Loading step Loading: DMF (10 L /kg) Elongation: DMF (5 L /kg) PD: volume optimization in Loading step 23-Feb-12 slide 31

Integrated Process Design double hit AA, deletions, diastereomers Critical Impurities: Act on chromatography Act on chemistry on-critical Impurities: Act on chemistry! Main product slide 32

In Medicinal Chemistry vs Scale up 24-Feb-12 slide 33

In a CM (i.e. Lonza, Switzerland) Equipment for Solid-Phase Peptides Synthesis 280L SPPS reactor 800L SPPS reactor 1 600L SPPS reactor (150 kg resin per batch) 24-Feb-12 slide 34

Control of Reactions Colorimetric Methods: ninhydrine chloranil Leclerk bromophenol... Spectroscopic Methods: FT-IR MAS-MR slide 35

SPPS Scale Up time in the lab / time in production temperature effect local concentration holding point aging time 24-Feb-12 slide 36

ther considerations Reagent EDT (scavenger) Volatile Smell Bt Storage (explosive) Resins Availability on large scale Reliability from batch to batch Swelling (impact on productivity) 24-Feb-12 slide 37

DSP Scale Up Large-scale PLC Columns with 15, 30, 45 & 60 cm diameter Up to 100 bar USP standard water Amount up to 1-2kg per injection 24-Feb-12 slide 38

Example fractionation concept Lab chromatogram purity >80% Large-scale PLC Fraction size position to be defined First 5 runs: fraction IPC after each injection Process stable Fraction IPC every 6 injection IPC test K-pool before mixing fractions 1 2 3 4 5 6 7 8 9 24-Feb-12 slide 39

DSP Scale Up Lyophylization Lab scale (flask freeze-dryers) Industrial scale (12, 45, 200 kg ice capacity) Spray Drying Lab scale Industrial scale Amount up to 10-20kg per Lyo (5kg/d) Amount Continuous process (i.e 60kg/d) 24-Feb-12 slide 40

ow Does it Look Like? Lyophiliser, capacity 200kg ice/day Spray dyer, capacity 10 kg / h 24-Feb-12 slide 41

DSP - General Consideration Salt form (formulation impact, aggregation, drug delivery) TFA, Acetate, Cl, Pamoate, a Isolation (particle size effect: formulation impact) Lyophilized material Spray dried material 24-Feb-12 slide 42

Some umbers Price per g (i.e 500-1000 /g @ 10kg scale) 1 small batch in a Small Scall Plant (30 L SPPS) has a value of ca 0.5 Mio Typical campaign 1-3 Batches Success batch rate in UPS: 100% (a must). 1 kg of Peptide > 5 mt of Solvent Waste slide 43

Erectile Dysfunction Market Worldwide USA Total 150 MM men with ED 1 30 MM men with ED 1 millions of people Severe Moderate 18 MM Men Seeking Treatment for ED 2 3.6 MM Men Seeking Treatment for ED 2 Mild 618,000 new cases added each year 23-Feb-12 Sources: 1 MMAS J Urol, 1994; 2 Chew et al, Int J Impot Res. 2000 slide 45

Bremelanotide Mechanism of Action Bremelanotide Selective erve Signal Visual & Tactile Stimulation GMP X cgmp cgmp Viagra Levitra Cialis PDE-5 Vasodilation & Erection 23-Feb-12 slide 46

Bremelanotide PT141 - Structure C 3 3 + 23-Feb-12 slide 47

Liquid Phase Strategy & Challenges 14 synthesis steps Two main Fragments C 3 3 + Boc-Asp(Bzl)-is(Dnp)-(D)Phe- 2TFA.Arg-Trp(For)-Lys(Z)-Me 23-Feb-12 slide 48

Solid Phase Strategy & Challenges S Full length elongation Cyclization Selective side chain protection Cost of Goods (Excess of reagents, Solvent, ) 23-Feb-12 slide 49

SPPS Route Cl CTC 1) Fmoc-Lys(Alloc)-, DIEA, DCM 2) DIEA, Me CTC S CTC cycles peptide lenghtening: 1) 20% piperidine in MP 2) Fmoc-AA- 1) Allyl/Alloc cleavage 2) Cyclisartion 3) Fmoc-le- 4) 20% piperidine in MP 5) Acetylation 6) cleavge S S CTC Ac-le- 23-Feb-12 slide 50

Solution to selective Allyl / Aloc Cleavage Catalyst Pd(Ph 3 ) 4 Pd(Ac) 2 / P(oTol) 3 PdCl 2 (Ph 3 ) 4 Scavengers / proton donor C/DIEA Dimedone Morpholine Ac/MM Phenylsilane Me 2.B 3 (DMAB) Design of Experiments 23-Feb-12 slide 51

DoE Approach - Screening Response Surface Robustness Test Factor Screening DoE 88.5 88 yield Contour 79 80 81 82 83 87 6 86 Design Space 81 80 82 83 85 84 83 ptimization DoE 23-Feb-12 slide 52

Solution to selective Allyl / Aloc Cleavage Catalyst Pd(Ph 3 ) 4 Pd(Ac) 2 / P(oTol) 3 PdCl 2 (Ph 3 ) 4 Scavengers / proton donor C/DIEA Dimedone Morpholine Ac/MM Phenylsilane Me 2.B 3 (DMAB) 23-Feb-12 slide 53

SPPS Scale Up Filtration problem: Simplify the handling of Pd(Ph 3 ) 4 24-Feb-12 slide 54

Individual Coupling ptimization MW =615.76 MW =616.72 Purity 93A% + Prod. + 2 SM β Ala impurity Prod.-Boc Fmoc-Arg(Pbf)-Trp(Boc)-Lys(Aloc)- 23-Feb-12 slide 55

Final Global Deprotection S 1) reaction: TFA solution + Scavenger mixture 2) Precipitation in ether F F F 3 + + carbamate PI-001-P C: 35169 Molecular Weight =1619.96 Exact Mass =1618 Molecular Formula =C871061415S PI-001-roh C: 35193 Molecular Weight =1139.22 Exact Mass =1138 Molecular Formula =C50691410. C2F32 23-Feb-12 slide 56

ptimization Reaction optimization (CAD): First set of experiments (screening) cocktail composition Second set of experiments (optimization) reaction conditions: Temp. / Time. residual solvent from SPPS Anisole TIS 23-Feb-12 slide 57

Coefficient plot of the model Investigation: PI-001-roh(sreening corr yield) (MLR) Scaled & Centered Coefficients for Yield-6 5 4 3 2 1 0-1 -2-3 -4 PL 2 DCM AI TIS PL*AI PL*TIS 2*TIS DCM*AI % =19 R2=0.946 R2 Adj.=0.893 DF=9 Q2=0.888 RSD=1.9768 Conf. lev.=0.95 23-Feb-12 slide 58

Influence of TIS and Phenol 23-Feb-12 slide 59

Influence of TFA and Time Yield 23-Feb-12 slide 60

Crude Material before DSP 23-Feb-12 slide 61

Challenges on solvents & Waste Management

Regulatory: Example of Acetonitrile Specifications rigins of the waste of acetonitrile: in-house: Mainly Purification ur Waste only Dedicated tank and transport Use-Test / Equivalence needed 24-Feb-12 slide 63

Acetonitrile Shortage in 2008/2009 By-product of Acrylonitrile manufacture Decline of Acrylonitrile needs in Fibers / plastics / automotive Production shut down Chinese lympic Games urricane Ike in Texas 30 25 20 /L 15 10 5 0 Price evolution of C3C 2001 to 2008 Q1 2009 Q3 2009 24-Feb-12 Acetonitrile world shortage Chem. Eng. ews, ov 24th, 2008, p27. slide 64

Possible Solutions

All in the river? 24-Feb-12 slide 66

Solutions to Manage Solvent Waste Burn all? Sort Waste stream Basic Distillations Sold externally Re-use in Bulk Chemistry igh Performance Distillations Used in specific projects Used for all projects 24-Feb-12 slide 67

Acetonitrile Possibilities of Recycling n-line recycling Integrated Recycling Device Close Loop recycling (Analytic online) Efficient but limited ff-line recycling Pure AC or Azeotrope Dedicated Tank / transport Used in all Projects 24-Feb-12 slide 68

Patented Lonza Process for Acetonitrile W2005/044783 / US2008/0073201 P= 200 mbar 7 kg/h Light Purge 15% 20 P= 1 bara P= 1 bara r-ac 800 l 800 l 60m3 400 kg/h 10% 20 100 kg/h 60m3 Crude AC 20% AC 80% 20 24-Feb-12 B9170 10m3 300 kg/h 20 Very low AC needed for Waste water Disposal 50 ppm20 3 kg/h eavies Purge slide 69

Acetonitrile Results 90% waste recycled Fresh and recycled AC combined Ensure Security of Supply Lonza Self-independent Robust Process 24-Feb-12 slide 70

Alternative technology in DSP

Centrifugal Partition Chromatography rigin: Sequential extraction (Craig system for peptides 1944) Present: Kromaton 5L CPC CPC is a liquid liquid chromatography based on the partition coefficient of mixture components between two non miscible liquid phases (organic and/or aqueous). ne mobile phase and is continuously pumped through the equipment. The other stationary phase and is maintained in the rotor by mean of centrifuge force. 24-Feb-12 slide 72

CPC Advantages relative to PLC - Advantages relative to PLC: - o expensive solid support and no solid waste Reduced solvent consumption (10x) and disposal o acetonitrile igh recovery yields (> 90%) Reduced processing times Easier automation (only liquids) IPC sampling from both mobile phases possible. Depending on the mixture components, various mode of purification may be used: direct elution, dual mode, p zone refining, ion exchange, - more degrees of freedom. 24-Feb-12 slide 73

CPC disadvantages relative to PLC Drawbacks: Use of organic solvents under pressure Sample injection method must be well installed ften 2 runs or dual mode (reversal of mobile and stationary phases) will be necessary. Several exit streams to be managed More degrees of freedom require more intensive development and qualification 24-Feb-12 slide 74

API: ighly Potent Peptide APIs

API (ighly Potent API) Peptide ASK EL (ccupational Exposure Limit) ADI (Acceptable Daily Intake) 1 > 1mg/m 3 > 10mg/d 2 0.1-1 mg/m 3 1-10 mg/d 3 0.01-0.1 mg/m 3 0.1-1 mg/d 4 0.1-10 µg/m 3 1-100 µg/d 5 1-100 ng/m 3 0.1-1 µg/d 6 < 1 ng/m 3 < 10 ng/d ASK (Arbeitshygienische Stoffkategorie) (ccupational hygienic substance category) 24-Feb-12 slide 76

People and Experience Expertise established base on ighly Active Pharmaceutical Ingredients (API) track record 10 years Established safety team with regulatory experts from SE and QA Specialized Groups in Research & Development, Analytics and perations Training/Working habits API personnel in all areas Standardization work (clean and well organized labs and plants) know where the substance is! Additional training to work in dedicated API labs and plants Verification Control of Performance Personnel/Systems 24-Feb-12 slide 77

Labs Containment, Characteristics Containment adaptable to process, combination of Glove boxes a1-safetech hoods Laminar flow type work benches ccup. Exp. Lim. > 30 ng/m 3 IS 8 Air locks Peptides: Lyos/Prep PLC Small Molecules: <1 to 20 lt reactors 24-Feb-12 slide 78

Lab Suite for Clinical Supply IS 8 Clean Room EPA 14 filter on safety benches Combination Safetech/large reactor hood Qualified for cgmp API Lab 1080 Cleanroom API Lab 1080 large ood API Lab 1080 Safetech/Dryer 24-Feb-12 slide 79

Respirable Amount Volume of breath per work day 10 m 3 50 m 3 per week 2300 m 3 per year 70000 m 3 per professional life ASK EL Respirable Amount per Professional Life 1 > 1 mg/m 3 70 g 2 0.1-1 mg/m 3 7-70 g 3 0.01-0.1 mg/m 3 0.7-7 g 4 0.1-10 µg/m 3 7-700 mg 5 1-100 ng/m 3 0.07-7 mg 24-Feb-12 slide 80

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Conclusion UPS DSP Product Yield Quality slide 87

Any Questions: Dr. Matthieu Giraud Director Research & Development Peptides phone: + 41 27 948 6631 fax: + 41 27 947 6631 mobile: +41 79 645 27 46 mailto: matthieu.giraud@lonza.com http://www.lonza.com

Bio in Short Picture Dr. Matt Giraud (Switzerland / Group Leader R&D Peptides) Matt studied organic chemistry at the University of Sciences and Technology of Languedoc (Montpellier-France), obtained his PhD in Peptide, and joined Lonza in 2001. e has 15 years of peptide experience in all process development phases linked to API life cycle. Inventor of several patents (product specifics & general methodology). In his current position, in addition to his people management role, Matt is responsible for the IP peptide portfolio, and is leading the external collaborations with universities & with industrial partners in the peptide area to leverage new innovative ideas. More recently, Matt became a member of the business team acting toward strategic decisions (M&A, JV, partner collaborations, licensing new products, identification of new market opportunities), and is representing Lonza peptide in several tradeshow (oral presentations, booth, customer visits). slide 89