The manufacture of peptides for clinical trials               Please insert your title of                your presentation...
•     Background        •     The Almac approach        •     Case Studies        •     New technologies        •     Summ...
•   Contract Research / Manufacturing Organisation    – Fully integrated service provider    – Pharmaceutical & Clinical D...
Packagingand Labelling      GMP Peptide      Manufacture                         Form.                        Develop.    ...
Peptide and Protein Technology Profile•   35 Staff spread across research, development, manufacturing &    analytical•   >...
Non-GMP custom synthesis•   Rapid throughput, high quality manufacture•   Manufacture and supply of >100 Peptides per mont...
Catalogue productsHuman Chemokines and site-specifically labelled derivatives
Almac Methodology•   Extend scope of SPPS into long peptide manufacture•   Methods offer high degree of control over synth...
Almac Methodology                                                                  H                                      ...
New process roadmap•   Almac Experience captured in a Process Best View summary•   Best View updated based upon evolving e...
New process roadmap• Process development  Almac philosophy and approach to early phase makes:                         Deve...
What is GMP?• Manufacturing and testing practise that helps  to ensure a quality product• Manufacturing processes are clea...
The phases of clinical development (and typical                  purities of products)• Pre-clinical: Determine toxicity o...
GMP manufacturing•   Four independent manufacturing trains:       SYNTHESIS                PURIFICATION    FREEZE DRY     ...
GMP manufacturing
GMP manufacturing• Manufacturing rate ca 15-20 batches per year• Scales up to 500g per batch• Multiple products >70mer• Wo...
Case study 1: h-MDC     h-MDC required for clinical trial
Case study 1: h-MDCRequest: Vials of injectible h-MDC for clinical use1.    cGMP API Manufacture and Release      • Define...
Case study 1: h-MDC Human-Macrophage Derived Chemokine Member of the CCL22 family, binds to CCR4 receptor GPYGANMEDS VCCRD...
Case study 1: h-MDCTechnical challenges• h-MDC is a small protein and technically challenging to synthesize• Effective cha...
Case study 1: h-MDCFor h-MDC there are several options for synthesis  -   Recombinant  -   Fragment synthesis  -   Linear ...
Case study 1: h-MDC Synthesis of Linear 69 mer     Technical Challenges   Cleavage from ResinPurification of Linear 69 mer...
Case study 1: h-MDC   Synthesis of Linear 69 mer     Technical Challenges                                  Achieve high co...
Case study 1: h-MDC   Synthesis of Linear 69 mer     Technical Challenges     Cleavage from Resin                         ...
Case study 1: h-MDC  Synthesis of Linear 69 mer      Technical Challenges     Cleavage from Resin  Purification of Linear ...
Case study 1: h-MDC   Synthesis of Linear 69 mer      Technical Challenges     Cleavage from Resin  Purification of Linear...
Case study 1: h-MDC   Synthesis of Linear 69 mer     Quality Challenges                                  High Purity DP re...
Case study 1: h-MDC                                   Development   Synthesis of Linear 69 mer      Best view process used...
Case study 1: h-MDC                                  Development   Synthesis of Linear 69 mer     Comparison of two routes...
Case study 1: h-MDC                                  Development   Synthesis of Linear 69 mer     Cleavage from Resin  Pur...
Case study 1: h-MDC                                   Development   Synthesis of Linear 69 mer      Cleavage from Resin   ...
Case study 1: h-MDC                                  Development   Synthesis of Linear 69 mer     Cleavage from Resin  Pur...
Case study 1: h-MDC  GMP Synthesis of Linear 69 mer 90 80                                                                 ...
Case study 1: h-MDC  Purification of Linear 69 mer       Folding of 69 mer      Isolation of h-MDC Crude peptide purified ...
Case study 1: h-MDC  Purification of Linear 69 mer       Folding of 69 mer      Isolation of h-MDC Folding monitored by HP...
Analytical CharacterisationProof of Identity   Mass Spec   Sequence by Mass Spec       Sequence by AAAComposition  Purity ...
Release - Sequence        Peptide digested and fragments        sequenced to build picture of peptide        GPYGANMEDSVCC...
HPLC analysis  Phase appropriate validation during development programme  Selectivity/Specificity  • Selectivity of h-MDC ...
HPLC analysis of h-MDC                Orthogonal methods                Method 1: 97.73%                Method 2: 97.38%
Drug Product• Drug Product formulation as lyophilisate in  vials• Sterile filtration of aqueous solution of drug  substanc...
Case study 2: Co-eluting impurity• 49-mer peptide produced on solid phase• cGMP manufacture completed• Analysis of cGMP pr...
Case study 2: Co-eluting impurity – removal?                                          1716-143 TFA purification           ...
Case study 2: Co-eluting impurity – identification?• Most likely impurity was thought to be capped truncatewhere UV profil...
Case study 2: Co-eluting impurity – identification?• Quantified level of deletion impurity by mass spec versus astandard o...
New technology – tagging as a means of purification   •   “Tagging” is a term we have coined for a process that facilitate...
The tag….    Purification                 Reactive group      handle                     for attachment                   ...
Tag-based purification – tag-peptide linkage                        Tagged peptides on resin
Tag-based purification – resin cleavage                      Crude tagged peptides in solution
Tag-based purification – bindingImmobilisation through Affinity Chromatography
Tag-based purification – cleavage                     Elute recovered peptide
IMAC Purification of Tag2-12mer mAU  800               Crude Tag-Peptide  700                                             ...
Summary of services •   In-house services within PPT: •   Process Development •   Analytical Method Development •   Phase-...
Summary of services  •   Leveraged from wider Almac Group and Partners  •   Radiolabelling  •   Formulation development  •...
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Almac - The Manufacture of Peptides for Clinical Trials - 2nd Irish Peptide Symposium June 2012

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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.

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Almac - The Manufacture of Peptides for Clinical Trials - 2nd Irish Peptide Symposium June 2012

  1. 1. The manufacture of peptides for clinical trials Please insert your title of your presentation here Steven McIntyre Team Leader, Peptide Operations 2nd Irish Peptide Symposium 14th-15© Almac Group 2012 Confidential th June 2012
  2. 2. • Background • The Almac approach • Case Studies • New technologies • SummaryConfidential © Almac Group 2012
  3. 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. 4. Packagingand Labelling GMP Peptide Manufacture Form. Develop. SS and Analysis Radio Labelling GMP Small Drug Product molecule Manufacture Stability Manufacture Confidential © Almac Group 2012
  5. 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. 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. 7. Catalogue productsHuman Chemokines and site-specifically labelled derivatives
  8. 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. 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. 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. 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. 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 investigatedIn summary a system to ensure patient safety
  13. 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. 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 modellingDev CS Bio 536 Akta Bench top Small scale modellingAble to simultaneous progress multiple manufactures
  15. 15. GMP manufacturing
  16. 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. 17. Case study 1: h-MDC h-MDC required for clinical trial
  18. 18. Case study 1: h-MDCRequest: Vials of injectible h-MDC for clinical use1. cGMP API Manufacture and Release • Define Manufacturing Route • Analytical Development for API Release2. cGMP Drug Product Manufacture and Release
  19. 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. 20. Case study 1: h-MDCTechnical challenges• h-MDC is a small protein and technically challenging to synthesize• Effective characterisation of the product is requiredQuality• A high purity product is required (typically >95% for clinical trial using a suitably validated analytical method)• The manufacture is according to ICH standardsCommercial• The customer’s timelines and budget must be respected• Rapid development and delivery essential
  21. 21. Case study 1: h-MDCFor h-MDC there are several options for synthesis - Recombinant - Fragment synthesis - Linear SPPSPreferred option: LinearOffers rapid and cost effective entry to clinical programmeAppropriate to scale and customer timelineTechnology developed in houseDeep expertise in house
  22. 22. Case study 1: h-MDC Synthesis of Linear 69 mer Technical Challenges Cleavage from ResinPurification of Linear 69 mer Folding of 69 merIsolation of h-MDC. Acetate Finished Product. Acetate
  23. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 36. Analytical CharacterisationProof of Identity Mass Spec Sequence by Mass Spec Sequence by AAAComposition Purity by HPLC (2 methods) Peptide Content Counterion Content Water Content Residual SolventsActivity In vitro assay by client
  37. 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. 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. 39. HPLC analysis of h-MDC Orthogonal methods Method 1: 97.73% Method 2: 97.38%
  40. 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. 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. 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. 43. Case study 2: Co-eluting impurity – identification?• Most likely impurity was thought to be capped truncatewhere UV profile had indicated coupling was not efficient• Synthesis of this truncate had the correct mass but wasnot the impurity!!• Reacted contaminated product with purification TAG (asactivated ester) – both full length peptide and impurityreacted• Trypsin digest confirmed that the impurity was a deletionproduct• Still present in GMP batch however!
  44. 44. Case study 2: Co-eluting impurity – identification?• Quantified level of deletion impurity by mass spec versus astandard of the impurity• Initially reported level higher than actual level (actual levelonly ~1%)• Deletion impurity tested in biological study – showedsimilar response to full-length peptide so material passed asfit for use in clinical study
  45. 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. 46. The tag…. Purification Reactive group handle for attachment Cleavable to peptide spacer unit
  47. 47. Tag-based purification – tag-peptide linkage Tagged peptides on resin
  48. 48. Tag-based purification – resin cleavage Crude tagged peptides in solution
  49. 49. Tag-based purification – bindingImmobilisation through Affinity Chromatography
  50. 50. Tag-based purification – cleavage Elute recovered peptide
  51. 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. 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. 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

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