14C Labelled Peptide API\'s by Sean Kitson
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14C Labelled Peptide API\'s by Sean Kitson






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14C Labelled Peptide API\'s by Sean Kitson 14C Labelled Peptide API\'s by Sean Kitson Presentation Transcript

  • Carbon-14 Labelled Peptide APIsSolid Phase Peptide Synthesis, BIOTINylation & PEGylation 17-18 April 2012 Dr Sean Kitson sean.kitson@almacgroup.com 1© Almac 2012
  • 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
  • Introduction to 14C 3© Almac 2012
  • Discovery of 14CMartin Kamen & Sam Ruben (27-FEB-1940) T1/2 ~ 5730 Years 4© Almac 2012
  • 14C Starting Materials Ba(OH)2 5© Almac 2012
  • 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
  • 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
  • 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
  • 14C Amino acids 9© Almac 2012
  • Algae to [U-14C]-Amino Acids Ba14CO3 14CO 2 14 C O 14 14 C C OH NH2 10© Almac 2012
  • 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
  • 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
  • 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
  • 14C Peptide StrategyS L Kitson. ‘Keeping Tags on Biomolecules’, Manufacturing Chemist April 2012 14© Almac 2012
  • • 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
  • • 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
  • • 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
  • • 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
  • 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 sequence were coupled using a 433 peptide synthesiser by the Almac Peptide Group 20© Almac 2012
  • 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
  • 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
  • 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
  • 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
  • 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
  • Peptide Group: SPPS of Fmoc-Peptide RESIN ivDde Automated Peptide Synthesis Fmoc ivDde RESIN 83-mer 26© Almac 2012
  • Peptide Group: SPPS of Fmoc-Peptide Fmoc ivDde RESIN 83-mer Fmoc cleavage ivDde RESIN 83-mer 27© Almac 2012
  • Radiolabelling: [14C]-Peptide ivDde RESIN 83-mer * CH3 14C Boc * * H3C CO2H * * Boc * NHBoc 14C ivDde RESIN 84-mer 28© Almac 2012
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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© Almac 2012
  • Almac’s Radiochemistry Laboratory 41© Almac 2012
  • 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
  • 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