Carbon-14 Labelled Peptide APIsSolid Phase Peptide Synthesis, BIOTINylation & PEGylation                     17-18 April 2...
Objective   • This presentation will focus on a brief     introduction to carbon-14   • Leading onto synthetic strategies ...
Introduction to 14C                                3© Almac 2012
Discovery of 14CMartin Kamen & Sam Ruben (27-FEB-1940)      T1/2 ~ 5730 Years                                         4© A...
14C   Starting Materials                Ba(OH)2                            5© Almac 2012
Barium 14C carbonate staircase            OH                         MeO                                                  ...
14C    Drug Molecules 14C   Labelled drugs are used in human mass     balance (AME) or ADME studies to evaluate: •   Mass ...
14C        Labelling Strategy When designing a 14C labelled synthesis it is important to consider the following: • Identif...
14C   Amino acids                                   9© Almac 2012
Algae to [U-14C]-Amino Acids          Ba14CO3          14CO              2                          14                    ...
14C   Labelling   • The simplest approach to 14C labelling     involves acetylation of free amino groups in     the peptid...
14C       - Glycine Family       NH2             NH2                NH2                       *   CO2H           *        ...
A Synthesis of [1-14C]Glycine          O                                              O                           NaI / Ac...
14C        Peptide StrategyS L Kitson. ‘Keeping Tags on Biomolecules’, Manufacturing Chemist April 2012                   ...
• Stage 1 involves the synthesis of the peptide up to the   step prior to introduction of the 14C label • This is most typ...
• Stage 2 sees the introduction of the 14C amino acid  • This is shown ideally as the final amino acid in the    sequence ...
• Stage 3 involves cleavage of the crude labelled peptide   from the resin support and subsequent purification by   prepar...
• Stage 4 sees the (optional) further functionalisation of the   labelled peptide (e.g. by PEGylation, BIOTINylation or   ...
14C      Peptide API Case Studies     14C                                      19© Almac 2012
CASE STUDY 1:Synthesis of [1-14C]Valine 46-mer  • Manufactured by SPPS using the Fmoc    approach  • First 32 amino acids ...
14C   Radiolabelling  • Step 1 involved the synthesis of Fmoc-[1-14C]-L-    valine  • The 14-amino acid sequence containin...
H2N             32-mer                Resin                       1) Coupling of    14                            14      ...
H2N   13-mer      -V*                 32-mer        Resin                      TFA, Water                      Thioanisole...
Analysis• 0.22 mCi (8.7 MBq) of labelled [14C]-peptide  acetate salt• Radiochemical purity = 98%area• Specific activity = ...
Case Study 2:[14C]-BIOTINylated Peptide       14C                                           BIOTIN Customer Requirements: ...
Peptide Group: SPPS of Fmoc-Peptide   RESIN           ivDde               Automated                               Peptide ...
Peptide Group: SPPS of Fmoc-Peptide  Fmoc                                ivDde                                            ...
Radiolabelling: [14C]-Peptide                                                             ivDde                           ...
Radiolabelling: Boc-[14C]-Peptide-Biotin     Boc      14C                                          ivDde                  ...
Radiolabelling: [14C]-Peptide-BIOTIN     Boc     14C                                             BIOTIN                   ...
Project Strategy: Peptide & Radiolabelling Group  Peptide Group Core Tasks:  • Fmoc   protected 83-mer peptide on resin pr...
Project Strategy: Peptide & Radiolabelling Group Radiolabelling Core Tasks : •   Conversion of [U-14C]-L-isoleucine to Boc...
Summary • 4 mg of [14C]-BIOTINylated peptide delivered on schedule • HPLC Purity 98.9%area (RCP), 99.3%area (UV) • SA = 33...
Case 3: PEGylation & Bio-conjugation   • Stage 1: In corporation of [1-14C]glycine     into the peptide sequence   • Stage...
Stage 1: [14C]-Peptide                    H2N        AA-SEQUENCE         LINKER      Resin                                ...
Stage 2: PEGylation         14   Boc        C   AA-SEQUENCE              LINKER                                O          ...
Stage 3: Bio-conjugation                                         O   14     C      AA-SEQUENCE   LINKER   PEG   N         ...
Conclusion • Biomolecules are well recognised as a   significantly growing area within the   pharmaceutical and biotechnol...
Conclusion • For peptide labelling there are other options such   as tritium labelling or radio-iodination • One clear ben...
40© Almac 2012
Almac’s Radiochemistry Laboratory                                    41© Almac 2012
Northern Ireland HQ (32 acre site)www.almacgroup.com                                  IVRS          Clinical         Packa...
Thank youThe hexagonal shapes denote the famous Giant’s Causeway rock in Northern Ireland – these shapes also             ...
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14C Labelled Peptide API\'s by Sean Kitson

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14C Labelled Peptide API\'s by Sean Kitson

  1. 1. Carbon-14 Labelled Peptide APIsSolid Phase Peptide Synthesis, BIOTINylation & PEGylation 17-18 April 2012 Dr Sean Kitson sean.kitson@almacgroup.com 1© Almac 2012
  2. 2. Objective • This presentation will focus on a brief introduction to carbon-14 • Leading onto synthetic strategies towards labelling peptides with carbon-14 14C 2© Almac 2012
  3. 3. Introduction to 14C 3© Almac 2012
  4. 4. Discovery of 14CMartin Kamen & Sam Ruben (27-FEB-1940) T1/2 ~ 5730 Years 4© Almac 2012
  5. 5. 14C Starting Materials Ba(OH)2 5© Almac 2012
  6. 6. Barium 14C carbonate staircase OH MeO CH3 OH H H314CO N O 14C 6 OMe OH 14C 14 H3C 6 [ C]Combretastatin A-1 OMe N R T Brown et al. JLCR 2009, 52, 567-570 14CH H14C HO [14C]ZT-114[ C]Apomorphine H14CHO * MeO Cl 14CH I 3 14 N S L Kitson & L Leman et al. JLCR 2011, 54 760-770 C CH3 14CH OH H 3 HO [14C]XEN-D0401 Cu14CN H N O K14CN HO 14CO 14 2 F3C C OH S L Kitson. JLCR 2007, 50, 290-294 S L Kitson. JLCR 2006, 49, 517-531 OH Ba14CO3 Cl S L Kitson, S Jones. JLCR 2010, 53, 140-146 6© Almac 2012
  7. 7. 14C Drug Molecules 14C Labelled drugs are used in human mass balance (AME) or ADME studies to evaluate: • Mass balance and the routes of elimination • Identify circulatory and excretory metabolites • Determination of clearance mechanisms • To determine the exposure of parent compound and its metabolites • Used to validate animal species used for toxicological testing • To explore whether metabolites contribute to the pharmacological / toxicological effects of the drug - MIST C Prakash et al. Biopharm. Drug Dispos; 2009, 30, 185-203 7© Almac 2012
  8. 8. 14C Labelling Strategy When designing a 14C labelled synthesis it is important to consider the following: • Identify simple starting materials from the barium 14C carbonate ‘staircase’ which are commercially available or alternatively easily made • Plan, develop and execute the synthetic methodology to the final drug substance. This approach can often restrict the position of the label in the drug and will cause a change in the drug purity profile from the original laboratory synthesis route • Locate a biologically stable position for the 14C label S L Kitson ‘Accelerated Radiochemistry’,PMPS Manufacturing 2010, 68-70 8© Almac 2012
  9. 9. 14C Amino acids 9© Almac 2012
  10. 10. Algae to [U-14C]-Amino Acids Ba14CO3 14CO 2 14 C O 14 14 C C OH NH2 10© Almac 2012
  11. 11. 14C Labelling • The simplest approach to 14C labelling involves acetylation of free amino groups in the peptide with 14C-acetic acid via activation to provide peptides with a specific activity of up to 120 mCi/mmol O O O 14 14 C 14 C 14 OH C OH C OH 11© Almac 2012
  12. 12. 14C - Glycine Family NH2 NH2 NH2 * CO2H * CO2H CO2H * * • 14C-Glycine can be prepared with one or both carbon atoms labelled with carbon-14 leading to a maximum specific activity of 100-120 mCi/mmol • Incorporated during peptide assembly 12© Almac 2012
  13. 13. A Synthesis of [1-14C]Glycine O O NaI / Acetone K14CN N N Cl I Acetone O O O AcOH / HCl aq NH2 N CO2H * CN heat * O 13© Almac 2012
  14. 14. 14C Peptide StrategyS L Kitson. ‘Keeping Tags on Biomolecules’, Manufacturing Chemist April 2012 14© Almac 2012
  15. 15. • Stage 1 involves the synthesis of the peptide up to the step prior to introduction of the 14C label • This is most typically performed by incremental growth of the peptide chain by solid phase peptide synthesis (SPPS) within a peptide synthesiser 15© Almac 2012
  16. 16. • Stage 2 sees the introduction of the 14C amino acid • This is shown ideally as the final amino acid in the sequence although in practice further unlabelled amino acids may need to be added thereafter 16© Almac 2012
  17. 17. • Stage 3 involves cleavage of the crude labelled peptide from the resin support and subsequent purification by preparative HPLC • At this stage a full batch of analytical tests can be run to confirm identity, purity and, over time, stability 17© Almac 2012
  18. 18. • Stage 4 sees the (optional) further functionalisation of the labelled peptide (e.g. by PEGylation, BIOTINylation or conjugation to other high molecular weight biomolecules) • This additional chemistry is followed by further purification and analytical characterisation 18© Almac 2012
  19. 19. 14C Peptide API Case Studies 14C 19© Almac 2012
  20. 20. CASE STUDY 1:Synthesis of [1-14C]Valine 46-mer • Manufactured by SPPS using the Fmoc approach • First 32 amino acids sequence were coupled using a 433 peptide synthesiser by the Almac Peptide Group 20© Almac 2012
  21. 21. 14C Radiolabelling • Step 1 involved the synthesis of Fmoc-[1-14C]-L- valine • The 14-amino acid sequence containing the Fmoc-[1-14C]-L-valine residue were coupled manually • Cleavage of the labelled peptide from the resin and simultaneous deprotection using TFA • Purification by reverse phase HPLC • Conversion to acetate salt by preparative ion exchange HPLC 21© Almac 2012
  22. 22. H2N 32-mer Resin 1) Coupling of 14 14 CO2H CO2H Fmoc-OSu NHFmoc NH2 9% aq Na2CO3 2) Capping 3) Deblock V* H2N V* 32-mer Resin [1-14C]-L-VALINE 1) Coupling of the 13 AAs 2) Capping 3) Deblock H2N 13-mer -V* 32-mer Resin 22© Almac 2012
  23. 23. H2N 13-mer -V* 32-mer Resin TFA, Water Thioanisole TIS, EDT Phenol H2N 13-mer -V* 32-mer OH Purification by RP-HPLC (C18) in 0.1 % TFA Water / 0.1 % ACN H2N 13-mer -V* 32-mer OH TFA Salt Ion exchange HPLC H2N 13-mer -V* 32-mer OH Acetate Salt 23© Almac 2012
  24. 24. Analysis• 0.22 mCi (8.7 MBq) of labelled [14C]-peptide acetate salt• Radiochemical purity = 98%area• Specific activity = 54 mCi/mmol 24© Almac 2012
  25. 25. Case Study 2:[14C]-BIOTINylated Peptide 14C BIOTIN Customer Requirements: • 2 mg [14C]-BIOTINylated peptide (84-mer) • S.A. ≥ 300 mCi/mmol • Terminal amino acid radiolabelled with [U-14C]-L-isoleucine • Chemical and radiochemical purity ≥95%area • Stability Study at 2oC and –20oC for 4 weeks 25© Almac 2012
  26. 26. Peptide Group: SPPS of Fmoc-Peptide RESIN ivDde Automated Peptide Synthesis Fmoc ivDde RESIN 83-mer 26© Almac 2012
  27. 27. Peptide Group: SPPS of Fmoc-Peptide Fmoc ivDde RESIN 83-mer Fmoc cleavage ivDde RESIN 83-mer 27© Almac 2012
  28. 28. Radiolabelling: [14C]-Peptide ivDde RESIN 83-mer * CH3 14C Boc * * H3C CO2H * * Boc * NHBoc 14C ivDde RESIN 84-mer 28© Almac 2012
  29. 29. Radiolabelling: Boc-[14C]-Peptide-Biotin Boc 14C ivDde RESIN 84-mer 1. Cleavage of ivDde 2. Biotin Boc 14C BIOTIN RESIN Biotinylated 84-mer 29© Almac 2012
  30. 30. Radiolabelling: [14C]-Peptide-BIOTIN Boc 14C BIOTIN RESIN Biotinylated 84-mer 1. Boc cleavage 2. Resin cleavage 14C BIOTIN Biotinylated 84-mer [14C]-Peptide 30© Almac 2012
  31. 31. Project Strategy: Peptide & Radiolabelling Group Peptide Group Core Tasks: • Fmoc protected 83-mer peptide on resin preparation • Trials on final peptide coupling with reduced equivalents of radiolabelled amino acid in collaboration with radiochemistry • Trials on ivDde cleavage • Trials on BIOTINylation • Trials on resin cleavage (prevention of methionine oxidation) • Identification of suitable purification conditions 31© Almac 2012
  32. 32. Project Strategy: Peptide & Radiolabelling Group Radiolabelling Core Tasks : • Conversion of [U-14C]-L-isoleucine to Boc-[U-14C]-L- isoleucine • Trials on final peptide coupling with reduced equivalents of radiolabelled amino acid in collaboration with the Peptide Group • Radiolabelled [14C]-BIOTINylated peptide synthesis • Stability Study 32© Almac 2012
  33. 33. Summary • 4 mg of [14C]-BIOTINylated peptide delivered on schedule • HPLC Purity 98.9%area (RCP), 99.3%area (UV) • SA = 338 mCi/mmol Stability Study: • Material stable at –20oC over 4 weeks • 1% drop in RCP at 2oC over 4 weeks 33© Almac 2012
  34. 34. Case 3: PEGylation & Bio-conjugation • Stage 1: In corporation of [1-14C]glycine into the peptide sequence • Stage 2: PEGylation • Stage 3: Bio-conjugation to protein-SH 34© Almac 2012
  35. 35. Stage 1: [14C]-Peptide H2N AA-SEQUENCE LINKER Resin * 14 CO2H C Boc Coupling NHBoc 14 Boc C AA-SEQUENCE LINKER Resin Deprotection 14 Boc C AA-SEQUENCE LINKER SA Dilution 14 Boc C AA-SEQUENCE LINKER 35© Almac 2012
  36. 36. Stage 2: PEGylation 14 Boc C AA-SEQUENCE LINKER O O PE N O PEG N PEG O O O 14 Boc C AA-SEQUENCE LINKER PEG N O Boc Deprotection O 14 C AA-SEQUENCE LINKER PEG N O 36© Almac 2012
  37. 37. Stage 3: Bio-conjugation O 14 C AA-SEQUENCE LINKER PEG N O O 14 C AA-SEQUENCE LINKER PEG N S O 37© Almac 2012
  38. 38. Conclusion • Biomolecules are well recognised as a significantly growing area within the pharmaceutical and biotechnology sectors. Especially in the area of peptide APIs, many of which are being developed as potential new therapies for a range of indications • A critical element of the development of any drug is an assessment of its ADME profile, most commonly performed using 14C labelled versions of the parent drug 38© Almac 2012
  39. 39. Conclusion • For peptide labelling there are other options such as tritium labelling or radio-iodination • One clear benefit of using a 14C for the ADME programme is the fact that the label is placed within the core of the drug, without any risk of wash out or need to use a modified structure • One limitation of 14C is its rather modest maximum specific activity (62 mCi/mmol), a limitation that becomes ever more significant as the molecular weight of the molecule increases • This limitation can be overcome through the use of Accelerated Mass Spectrometry (AMS) 39© Almac 2012
  40. 40. 40© Almac 2012
  41. 41. Almac’s Radiochemistry Laboratory 41© Almac 2012
  42. 42. Northern Ireland HQ (32 acre site)www.almacgroup.com IVRS Clinical Packaging and Labelling Peptide & Protein Technology (PPT) Non GMP API Manufacture Form. Biomarkers Discovery Dev. & Research Diagnostics Solid State & Analytical Services Radio Labelling GMP API Drug Product Manufacture Manufacture Stability 42© Almac 2012 Confidential © Almac Group 2010
  43. 43. Thank youThe hexagonal shapes denote the famous Giant’s Causeway rock in Northern Ireland – these shapes also connect to the benzene ring used in science 43© Almac 2012

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