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Confidential © Almac Group 2012
The manufacture of peptides for clinical trials
Steven McIntyreTeam Leader, Peptide Operations
2nd Irish Peptide Symposium14th-15th June 2012
• Background• The Almac approach• Case Studies• New technologies• Summary
Confidential © Almac Group 2012
• Contract Research / Manufacturing Organisation– Fully integrated service provider
– Pharmaceutical & Clinical Development Services
• Founded in 2003, privately owned
• Total employees ~3000
- 2000 UK, 1000 USA
- Headquarters in Craigavon, Northern Ireland
- R&D and Custom Synthesis Peptide site in Elvingston, Scotland
Confidential © Almac Group 2012
GMP Peptide GMP Peptide ManufactureManufacture
Drug ProductDrug ProductManufactureManufacture
GMP Small GMP Small moleculemolecule
ManufactureManufacture
Form.Form.Develop.Develop.
PackagingPackagingand Labellingand Labelling
RadioRadioLabellingLabelling
SS andSS andAnalysisAnalysis
StabilityStability
Peptide and Protein Technology Profile
• 35 Staff spread across research, development, manufacturing & analytical
• >150 man years Peptide manufacturing experience• Manufactured and supplied in excess of 9000 Peptides• Proven track record of high quality supply• Reputation for success with very challenging chemistries and
sequences• Routine synthesis of sequences up to 200 AA • Peptide and Protein ligation expertise (pegylation, labelling,
conjugation)• Site specific Protein engineering and chemical Protein synthesis
Non-GMP custom synthesis
• Rapid throughput, high quality manufacture• Manufacture and supply of >100 Peptides per month from mg to
gram scale• >75% of custom manufactures delivered within 20 days of order• Ways of working:
– Order accepted by phone, e-enquiry, fax– Technical owner assigned from order to dispatch – Highly experienced Peptide scientists available for direct contact – Multiple assets for parallel processing of multiple products– Range of analytical characterisation approaches available
Catalogue products
Human Chemokines and site-specifically labelled derivatives
Almac Methodology
• Extend scope of SPPS into long peptide manufacture• Methods offer high degree of control over synthesis• Flexibility to introduce unnatural building blocks• (custom or commercial)• Flexibility to introduce labels• Learning from each synthesis captured and applied
through “best-process” tool
• Conventional methodology enables step-wise synthesis of peptides up to 40-50 a.a.
• Novel methodology developed for synthesis of peptides and proteins(50-150 a.a.)
– Coupling reagents– Novel solubilising protecting
groups– Linkers and solid supports– Online monitoring– Tagging based purification– Folding conditions
Linker ResinHNFmoc
Linker ResinH2N
Linker ResinHNFmoc
PGSC
COOHHNFmoc
PGSC
Linker ResinH2N
PGSC
Linker Resin
PGSCPGSCPGSC
PGSCPGSC
H2N
COOHH2N
COOHHNFmoc
PGSC
Deprotection
Coupling
i) Couplingii) Deprotection
n
Cleavage and Purification
Refold
Deprotection
Almac Methodology
190 sub unit operations described
8 Families defined
Almac
Confid
entia
l
New process roadmap
• Almac Experience captured in a Process Best View summary• Best View updated based upon evolving experience• Peptides categorised into 8 families• Best View Process broken down into 190 sub unit operations for each family
• Best View Process establishes a high quality start point for any new synthesis
GMPManufacture
ProcessDefinition
Developmentlimited and only
if absolutelynecessary
ScaleUp
Proof ofProcess Make
Fit for purposeProcess
right first time
Done on accuratesmall scale modelsfor representative
data
New process roadmap
• Process developmentAlmac philosophy and approach to early phase makes:
Experience based>150 man years>8000 peptides
What is GMP?
• Manufacturing and testing practise that helps to ensure a quality product
• Manufacturing processes are clearly defined and details of each process are well recorded
• Any deviations are documented and investigated
In summary a system to ensure patient safety
The phases of clinical development (and typical purities of products)
• Pre-clinical: Determine toxicity of product in animal models (80-90%)
• Phase 1: Confirm safety and tolerance of compound in healthy volunteers (95%+ with no new impurity >0.1%)
• Phase 2: Confirm proof of concept in patients (97-99%, with no new impurity >0.1%)
• Phase 3: Larger studies (validated process, all impurities well understood and controlled)
• In summary a system to ensure patient safety
• Four independent manufacturing trains:
Able to simultaneous progress multiple manufactures
Akta
Varian80/150
Bench top
Virtis
CS Bio536
Wet Chemistry (cleave, PEGylation, conjugation)
full coverage 10 – 630 litre
PURIFICATION FREEZE DRYSYNTHESIS
100s of grams
Small scale modelling
Akta Bench topCS Bio536
Small scale modelling
Novasep50/80 VirtisCS Bio
536Up to 100 grams
Dev
GMP
CS Bio936
GMP manufacturing
GMP manufacturing
GMP manufacturing
• Manufacturing rate ca 15-20 batches per year• Scales up to 500g per batch• Multiple products >70mer• World’s first >100mer made to GMP by solid phase
synthesis• Current customer base UK / Europe / USA,
Small/Medium Biotech / Big Pharma
h-MDC required for clinical trial
Case study 1: h-MDC
Request: Vials of injectible h-MDC for clinical use
1. cGMP API Manufacture and Release• Define Manufacturing Route• Analytical Development for API Release
2. cGMP Drug Product Manufacture and Release
Case study 1: h-MDC
Human-Macrophage Derived ChemokineMember of the CCL22 family, binds to CCR4 receptor
GPYGANMEDS VCCRDYVRYR LPLRVVKHFY WTSDSCPRPG VVLLTFRDKE ICADPRVPWV KMILNKLSQ
• 69 Amino acids• 2 Disulfide bridges
Case study 1: h-MDC
Technical challenges• h-MDC is a small protein and technically challenging to synthesize• Effective characterisation of the product is required
Quality• A high purity product is required (typically >95% for clinical trial
using a suitably validated analytical method) • The manufacture is according to ICH standards
Commercial• The customer’s timelines and budget must be respected• Rapid development and delivery essential
Case study 1: h-MDC
For h-MDC there are several options for synthesis- Recombinant - Fragment synthesis- Linear SPPS
Preferred option: Linear
Offers rapid and cost effective entry to clinical programme
Appropriate to scale and customer timeline
Technology developed in house
Deep expertise in house
Case study 1: h-MDC
Synthesis of Linear 69 mer
Cleavage from Resin
Purification of Linear 69 mer
Folding of 69 mer
Isolation of h-MDC. Acetate
Technical Challenges
Finished Product. Acetate
Case study 1: h-MDC
Synthesis of Linear 69 mer
Cleavage from Resin
Purification of Linear 69 mer
Folding of 69 mer
Isolation of h-MDC. Acetate
Technical Challenges
Finished Product. Acetate
Achieve high coupling efficiency
Case study 1: h-MDC
Remove closely related impurities- Fold crude or isolate intermediate
Synthesis of Linear 69 mer
Cleavage from Resin
Purification of Linear 69 mer
Folding of 69 mer
Isolation of h-MDC. Acetate
Technical Challenges
Finished Product. Acetate
Case study 1: h-MDC
Identify critical folding parameters- Avoid dimers and misfolds- Drive reaction to completion- Achieve correct activity- Achieve yield and throughput
Synthesis of Linear 69 mer
Cleavage from Resin
Purification of Linear 69 mer
Folding of 69 mer
Isolation of h-MDC. Acetate
Technical Challenges
Finished Product. Acetate
Case study 1: h-MDC
Remove closely related impurities Achieve exchange to acetate
Synthesis of Linear 69 mer
Cleavage from Resin
Purification of Linear 69 mer
Folding of 69 mer
Isolation of h-MDC. Acetate
Technical Challenges
Finished Product. Acetate
Case study 1: h-MDC
High Purity DP requiredHigh Purity DS requiredAchieve correct activity of molecule
Synthesis of Linear 69 mer
Cleavage from Resin
Purification of Linear 69 mer
Folding of 69 mer
Isolation of h-MDC. Acetate
Quality Challenges
High level of control neededduring manufacture
Finished Product. Acetate
Case study 1: h-MDC
Synthesis of Linear 69 mer
Cleavage from Resin
Purification of Linear 69 mer
Folding of 69 mer
Isolation of h-MDC. Acetate
DevelopmentBest view process used as basisIncorporated multiple couplings at
appropriate points
Complex scavenger mixture requiredto minimise side reactions
Case study 1: h-MDC
Synthesis of Linear 69 mer
Cleavage from Resin
Purification of Linear 69 mer
Folding of 69 mer
Isolation of h-MDC. Acetate
DevelopmentComparison of two routes done• Direct fold of crude• Intermediate purification
Yield and quality similarIntermediate purification eliminates
variability
Case study 1: h-MDC
Purification developed to givecorrect selectivity and yieldTwo media compared
Synthesis of Linear 69 mer
Cleavage from Resin
Purification of Linear 69 mer
Folding of 69 mer
Isolation of h-MDC. Acetate
Development
Case study 1: h-MDC
Critical to peptide activityFocussed development on
achieving correct activityTwo sets of conditions tested
against ‘standard batch’Bioactivity tested and confirmed
Synthesis of Linear 69 mer
Cleavage from Resin
Purification of Linear 69 mer
Folding of 69 mer
Isolation of h-MDC. Acetate
Development
Case study 1: h-MDC
Best view applied directly to achieve isolated acetate salt
Synthesis of Linear 69 mer
Cleavage from Resin
Purification of Linear 69 mer
Folding of 69 mer
Isolation of h-MDC. Acetate
Development
Case study 1: h-MDC
Effective synthesis achieved- UV deprotection profile is used to monitor assembly- Average coupling efficiency > 99%
GMP Synthesis of Linear 69 mer
010
203040
506070
8090
x S L K N L I M M K V W P V R P D A C I E K D R F T L L V V V G G P R P C S D S T W Y F H K V V R L P L R Y R V Y D R C C V S D E M N A G Y P G
h-MDC
Case study 1: h-MDC
Purification of Linear 69 mer
Folding of 69 mer
Isolation of h-MDC
Crude peptide purified to remove major impurities before folding- Major impurity is methionine oxidation product (+16)- Separable by RP – HPLC - Deletions and truncates also removed
Case study 1: h-MDC
Folding monitored by HPLC Final purification used to isolate product at > 95.0 % purity
Purification of Linear 69 mer
Folding of 69 mer
Isolation of h-MDC
Case study 1: h-MDC
Proof of Identity Mass Spec Sequence by Mass Spec Sequence by AAA
CompositionPurity by HPLC (2 methods) Peptide Content Counterion Content Water Content Residual Solvents
Activity In vitro assay by client
Analytical Characterisation
Release - Sequence
Peptide digested and fragmentssequenced to build picture of peptide
GPYGANMEDSVCCR
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
GPYGANMEDSVCCR DYVR YR LPLR VVK
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
Phase appropriate validation during development programme
Selectivity/Specificity• Selectivity of h-MDC with Related Substances
Truncates Linear PeptideMisfolded peptide Oxidised product
• PrecisionRepeatability
• Linearityh-MDC Assay Range
• SensitivityLimit of Quantitation and Limit of Detection established
• Solution Stability
HPLC analysis
Method 2: 97.38%
Method 1: 97.73%
Orthogonal methods
HPLC analysis of h-MDC
Drug Product
• Drug Product formulation as lyophilisate in vials
• Sterile filtration of aqueous solution of drug substance through 0.2um filter
• Drug Product release testing• Minor amounts of oxidation (of methionine)
observed during formulation• Activity testing confirmed batch as suitable for
use in the clinic
Case study 2: Co-eluting impurity
• 49-mer peptide produced on solid phase• cGMP manufacture completed • Analysis of cGMP product indicated the presence of a
significant level (~5-10%) of an impurity -114 mass units from product as determined by mass spectrometry
• Product to be used in phase 2 studies – the -114 impurity was not present in any previous batch
• Principal of clinical development is that each phase of development should use material of higher purity
Case study 2: Co-eluting impurity – removal?
• Best purification conditions still failed to completely remove impurity
1716-143 TFA purification
0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
70.0%
80.0%
90.0%
100.0%
B8 B6 B4 B2 C1 C3 C5 C7 C9 C11
Fraction number
% p
urity HPLC purity
%impurity (TIC)
Case study 2: Co-eluting impurity – identification?
• Most likely impurity was thought to be capped truncate where UV profile had indicated coupling was not efficient
• Synthesis of this truncate had the correct mass but was not the impurity!!
• Reacted contaminated product with purification TAG (as activated ester) – both full length peptide and impurity reacted
• Trypsin digest confirmed that the impurity was a deletion product
• Still present in GMP batch however!
Case study 2: Co-eluting impurity – identification?
• Quantified level of deletion impurity by mass spec versus a standard of the impurity
• Initially reported level higher than actual level (actual level only ~1%)
• Deletion impurity tested in biological study – showed similar response to full-length peptide so material passed as fit for use in clinical study
New technology – tagging as a means of purification
• “Tagging” is a term we have coined for a process that facilitates more effective and efficient purification of crude peptides thanclassical methods.
• Analogous with common practices used in recombinant protein synthesis.
• It involves the temporary labelling of a peptide with a small molecule (the tag) that has been specially designed to aid purification.
• Due to the “capping” step commonly employed in peptide synthesis, the tag only attaches to the desired, full length peptide and not to truncated sequences.
The tag….
Cleavable spacer unit
Purification handle
Reactive group for attachment
to peptide
Tag-based purification – tag-peptide linkage
Tagged peptides on resin
Tag-based purification – resin cleavage
Crude tagged peptides in solution
Tag-based purification – binding
Immobilisation through Affinity Chromatography
Tag-based purification – cleavage
Elute recovered peptide
min5 7.5 10 12.5 15 17.5 20 22.5 25
mAU
0 100 200 300 400 500 600 700 800
min5 7.5 10 12.5 15 17.5 20 22.5 25
mAU
0 100 200 300 400 500 600
IMAC Purification of Tag2-12merCrude Tag-Peptide
Purified Peptide
Tag2-peptide
Peptide
Capped truncate
• Complete removal of co-eluting capped truncate through use of TAG
• Technology applied to range of peptides
Summary of services
• In-house services within PPT:• Process Development• Analytical Method Development• Phase-appropriate Method Validation• Manufacture (GMP & non-GMP) mg to kg• DS Release testing• DP Release testing• Stability Trials (DS and DP)• QA Support• Supply Chain Management
Summary of services
• Leveraged from wider Almac Group and Partners• Radiolabelling• Formulation development• Sterile formulation• Fill finish• Tox studies• QP release• CMC documentation / authorship of IMPD