Almac's Steven McIntyre presented in June 2012 at the second Irish Peptide Symposium regarding the manufacture of peptides for clinical trials. See his presentation here.

1. The manufacture of peptides for clinical trials
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Steven McIntyre
Team Leader, Peptide Operations
2nd Irish Peptide Symposium
14th-15© Almac Group 2012
Confidential
th June 2012

2. • Background
• The Almac approach
• Case Studies
• New technologies
• Summary
Confidential © Almac Group 2012

3. • 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

4. Packaging
and Labelling
GMP Peptide
Manufacture
Form.
Develop.
SS and
Analysis
Radio
Labelling
GMP Small
Drug Product molecule
Manufacture Stability Manufacture
Confidential © Almac Group 2012

5. 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

6. 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

7. Catalogue products
Human Chemokines and site-specifically labelled derivatives

8. 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

9. Almac Methodology H
Fmoc N Linker Resin
Deprotection
• Conventional methodology PGSC
H2N Linker Resin
enables step-wise synthesis of H
Fmoc N COOH Coupling
peptides up to 40-50 a.a. H
PGSC
Fmoc N Linker Resin
Deprotection
• Novel methodology developed for PGSC
synthesis of peptides and proteins H2N Linker Resin
(50-150 a.a.) H
Fmoc N
PGSC
COOH i) Coupling
ii) Deprotection
– Coupling reagents PG SC
PG SC
PG SC
n
– Novel solubilising protecting H2N
PG SC
PGSC
Linker Resin
groups Cleavage and Purification
– Linkers and solid supports H2 N COOH
– Online monitoring Refold
– Tagging based purification
– Folding conditions

10. 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
8 Families defined
190 sub unit
operations
described
ac tial
lm en
A id
onf
C

11. New process roadmap
• Process development
Almac philosophy and approach to early phase makes:
Development
limited and only
if absolutely
necessary
Process Proof of Scale GMP
Definition Process Make Up Manufacture
Experience based Done on accurate Fit for purpose
>150 man years small scale models Process
>8000 peptides for representative right first time
data

12. 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

13. 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

14. GMP manufacturing
• Four independent manufacturing trains:
SYNTHESIS PURIFICATION FREEZE DRY
CS Bio Varian
936 80/150
Virtis 100s of grams
Wet Chemistry (cleave,
PEGylation, conjugation)
GMP full coverage 10 – 630 litre
CS Bio Novasep
536 50/80
Virtis Up to 100 grams
CS Bio
536
Akta Bench top Small scale modelling
Dev
CS Bio
536
Akta Bench top Small scale modelling
Able to simultaneous progress multiple manufactures

15. GMP manufacturing

16. 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

17. Case study 1: h-MDC
h-MDC required for clinical trial

18. 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

19. Case study 1: h-MDC
Human-Macrophage Derived Chemokine
Member of the CCL22 family, binds to CCR4 receptor
GPYGANMEDS VCCRDYVRYR LPLRVVKHFY
WTSDSCPRPG VVLLTFRDKE ICADPRVPWV KMILNKLSQ
• 69 Amino acids
• 2 Disulfide bridges

20. 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

21. 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

22. Case study 1: h-MDC
Synthesis of Linear 69 mer Technical Challenges
Cleavage from Resin
Purification of Linear 69 mer
Folding of 69 mer
Isolation of h-MDC. Acetate
Finished Product. Acetate

23. Case study 1: h-MDC
Synthesis of Linear 69 mer Technical Challenges
Achieve high coupling efficiency
Cleavage from Resin
Purification of Linear 69 mer
Folding of 69 mer
Isolation of h-MDC. Acetate
Finished Product. Acetate

24. Case study 1: h-MDC
Synthesis of Linear 69 mer Technical Challenges
Cleavage from Resin
Remove closely related impurities
Purification of Linear 69 mer - Fold crude or isolate intermediate
Folding of 69 mer
Isolation of h-MDC. Acetate
Finished Product. Acetate

25. Case study 1: h-MDC
Synthesis of Linear 69 mer Technical Challenges
Cleavage from Resin
Purification of Linear 69 mer
Identify critical folding parameters
Folding of 69 mer - Avoid dimers and misfolds
- Drive reaction to completion
- Achieve correct activity
Isolation of h-MDC. Acetate
- Achieve yield and throughput
Finished Product. Acetate

26. Case study 1: h-MDC
Synthesis of Linear 69 mer Technical Challenges
Cleavage from Resin
Purification of Linear 69 mer
Folding of 69 mer
Remove closely related impurities
Isolation of h-MDC. Acetate
Achieve exchange to acetate
Finished Product. Acetate

27. Case study 1: h-MDC
Synthesis of Linear 69 mer Quality Challenges
High Purity DP required
Cleavage from Resin High Purity DS required
Achieve correct activity of molecule
Purification of Linear 69 mer
High level of control needed
during manufacture
Folding of 69 mer
Isolation of h-MDC. Acetate
Finished Product. Acetate

28. Case study 1: h-MDC
Development
Synthesis of Linear 69 mer Best view process used as basis
Incorporated multiple couplings at
appropriate points
Cleavage from Resin
Complex scavenger mixture required
Purification of Linear 69 mer
to minimise side reactions
Folding of 69 mer
Isolation of h-MDC. Acetate

29. Case study 1: h-MDC
Development
Synthesis of Linear 69 mer Comparison of two routes done
• Direct fold of crude
• Intermediate purification
Cleavage from Resin
Yield and quality similar
Purification of Linear 69 mer Intermediate purification eliminates
variability
Folding of 69 mer
Isolation of h-MDC. Acetate

30. Case study 1: h-MDC
Development
Synthesis of Linear 69 mer
Cleavage from Resin
Purification of Linear 69 mer Purification developed to give
correct selectivity and yield
Folding of 69 mer Two media compared
Isolation of h-MDC. Acetate

31. Case study 1: h-MDC
Development
Synthesis of Linear 69 mer
Cleavage from Resin
Critical to peptide activity
Purification of Linear 69 mer Focussed development on
achieving correct activity
Two sets of conditions tested
Folding of 69 mer against ‘standard batch’
Bioactivity tested and confirmed
Isolation of h-MDC. Acetate

32. Case study 1: h-MDC
Development
Synthesis of Linear 69 mer
Cleavage from Resin
Purification of Linear 69 mer
Folding of 69 mer
Best view applied directly
Isolation of h-MDC. Acetate
to achieve isolated acetate salt

33. Case study 1: h-MDC
GMP Synthesis of Linear 69 mer
90
80
h-MDC
70
60
50
40
30
20
10
0
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
Effective synthesis achieved
- UV deprotection profile is used to monitor assembly
- Average coupling efficiency > 99%

34. 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

35. Case study 1: h-MDC
Purification of Linear 69 mer
Folding of 69 mer
Isolation of h-MDC
Folding monitored by HPLC
Final purification used to isolate product at > 95.0 % purity

36. Analytical Characterisation
Proof of Identity
Mass Spec
Sequence by Mass Spec Sequence by AAA
Composition
Purity by HPLC (2 methods) Peptide Content
Counterion Content Water Content
Residual Solvents
Activity
In vitro assay by client

37. Release - Sequence
Peptide digested and fragments
sequenced to build picture of peptide
GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ
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

38. HPLC analysis
Phase appropriate validation during development programme
Selectivity/Specificity
• Selectivity of h-MDC with Related Substances
Truncates Linear Peptide
Misfolded peptide Oxidised product
• Precision
Repeatability
• Linearity
h-MDC Assay Range
• Sensitivity
Limit of Quantitation and Limit of Detection established
• Solution Stability

39. HPLC analysis of h-MDC
Orthogonal methods
Method 1: 97.73%
Method 2: 97.38%

40. 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

41. 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

42. Case study 2: Co-eluting impurity – removal?
1716-143 TFA purification
100.0%
90.0%
80.0%
70.0%
60.0%
% purity
HPLC purity
50.0%
%impurity (TIC)
40.0%
30.0%
20.0%
10.0%
0.0%
B8 B6 B4 B2 C1 C3 C5 C7 C9 C11
Fraction number
• Best purification conditions still failed to completely remove impurity

43. 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!

44. 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

45. 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 than
classical 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.

46. The tag….
Purification Reactive group
handle for attachment
Cleavable to peptide
spacer unit

47. Tag-based purification – tag-peptide linkage
Tagged peptides on resin

48. Tag-based purification – resin cleavage
Crude tagged peptides in solution

49. Tag-based purification – binding
Immobilisation through Affinity Chromatography

50. Tag-based purification – cleavage
Elute recovered peptide

51. IMAC Purification of Tag2-12mer
mAU
800
Crude Tag-Peptide
700
Tag2-peptide
600
Capped truncate
500
400
300
200
100
0
5 7.5 10 12.5 15 17.5 20 22.5 25 min
mAU
600
Purified Peptide Peptide
500
400
300
200
100
0
5 7.5 10 12.5 15 17.5 20 22.5 25 min
• Complete removal of co-eluting capped truncate through use of TAG
• Technology applied to range of peptides

52. 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

53. 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