Carbon-14 Labelled Peptide APIs Solid Phase Peptide Synthesis, BIOTINylation & PEGylation

Transcription

1 arbon- Labelled Peptide APIs Solid Phase Peptide Synthesis, BITINylation & PEGylation April 2012 Dr Sean Kitson 1

2 bjective This presentation will focus on a brief introduction to carbon- Leading onto synthetic strategies towards labelling peptides with carbon- 2

3 Introduction to 3

4 Discovery of Martin Kamen & Sam Ruben (27-FEB-1940) T 1/2 ~ 5730 Years 4

5 Starting Materials Ba(H) 2 5

6 Barium carbonate staircase H [ ]Apomorphine H H Me 6 H 3 6 N H3 H Me [ ]ombretastatin A-1 H H H H [ ]XEN-D0401 H N Me H H H 3 R T Brown et al. JLR 2009, 52, H 3 I H 3 H u N K N H Me H 3 N * H N [ ]ZT-1 S L Kitson & L Leman et al. JLR 2011, l S L Kitson. JLR 2007, 50, S L Kitson. JLR 2006, 49, F 3 H H 2 Ba 3 l S L Kitson, S Jones. JLR 2010, 53, 0-6 6

7 Drug Molecules 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 Prakash et al. Biopharm. Drug Dispos; 2009, 30,

8 Labelling Strategy When designing a labelled synthesis it is important to consider the following: Identify simple starting materials from the barium 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 label S L Kitson Accelerated Radiochemistry,PMPS Manufacturing 2010,

9 Amino acids 9

10 Algae to [U- ]-Amino Acids Ba 3 2 NH 2 H 10

11 Labelling The simplest approach to labelling involves acetylation of free amino groups in the peptide with -acetic acid via activation to provide peptides with a specific activity of up to 120 mi/mmol H H H 11

12 - Glycine Family NH 2 NH 2 NH 2 2 H * * 2 H * 2 H * -Glycine can be prepared with one or both carbon atoms labelled with carbon- leading to a maximum specific activity of mi/mmol Incorporated during peptide assembly 12

13 A Synthesis of [1- ]Glycine N NaI / Acetone N K N l I Acetone N AcH / Hl aq NH 2 N * heat 2 H * 13

14 Peptide Strategy S L Kitson. Keeping Tags on Biomolecules, Manufacturing hemist April 2012

15 Stage 1 involves the synthesis of the peptide up to the step prior to introduction of the label This is most typically performed by incremental growth of the peptide chain by solid phase peptide synthesis (SPPS) within a peptide synthesiser 15

16 Stage 2 sees the introduction of the 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

17 Stage 3 involves cleavage of the crude labelled peptide from the resin support and subsequent purification by preparative HPL At this stage a full batch of analytical tests can be run to confirm identity, purity and, over time, stability 17

18 Stage 4 sees the (optional) further functionalisation of the labelled peptide (e.g. by PEGylation, BITINylation or conjugation to other high molecular weight biomolecules) This additional chemistry is followed by further purification and analytical characterisation 18

19 Peptide API ase Studies 19

20 ASE STUDY 1: Synthesis of [1- ]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

21 Radiolabelling Step 1 involved the synthesis of Fmoc-[1- ]-Lvaline The -amino acid sequence containing the Fmoc-[1- ]-L-valine residue were coupled manually leavage of the labelled peptide from the resin and simultaneous deprotection using TFA Purification by reverse phase HPL onversion to acetate salt by preparative ion exchange HPL 21

22 H 2 N 32-mer Resin 1) oupling of 2 H 2 H Fmoc-Su 2) apping 3) Deblock NHFmoc 9% aq Na 2 3 V* NH 2 H 2 N V* 32-mer Resin [1- ]-L-VALINE 1) oupling of the 13 AAs 2) apping 3) Deblock H 2 N 13-mer -V* 32-mer Resin 22

23 H 2 N 13-mer -V* 32-mer Resin TFA, Water Thioanisole TIS, EDT Phenol N H 2 13-mer -V* 32-mer H Purification by RP-HPL (18) in 0.1 % TFA Water / 0.1 % AN N H 2 13-mer -V* 32-mer H TFA Salt Ion exchange HPL N H 2 13-mer -V* 32-mer H Acetate Salt 23

24 Analysis 0.22 mi (8.7 MBq) of labelled [ ]-peptide acetate salt Radiochemical purity = 98%area Specific activity = 54 mi/mmol 24

25 ase Study 2: [ ]-BITINylated Peptide BITIN ustomer Requirements: 2 mg [ ]-BITINylated peptide (84-mer) S.A. 300 mi/mmol Terminal amino acid radiolabelled with [U- ]-L-isoleucine hemical and radiochemical purity 95%area Stability Study at 2 o and 20 o for 4 weeks 25

26 Peptide Group: SPPS of Fmoc-Peptide RESIN ivdde Automated Peptide Synthesis Fmoc ivdde 83-mer RESIN 26

27 Peptide Group: SPPS of Fmoc-Peptide Fmoc ivdde 83-mer RESIN Fmoc cleavage ivdde 83-mer RESIN 27

28 Radiolabelling: [ ]-Peptide ivdde 83-mer RESIN Boc Boc * H 3 * * H 3 * * * 2 H NHBoc ivdde 84-mer RESIN 28

29 Radiolabelling: Boc-[ ]-Peptide-Biotin Boc ivdde 84-mer RESIN Boc 1. leavage of ivdde 2. Biotin BITIN RESIN Biotinylated 84-mer 29

30 Radiolabelling: [ ]-Peptide-BITIN Boc BITIN RESIN Biotinylated 84-mer 1. Boc cleavage 2. Resin cleavage BITIN Biotinylated 84-mer [ ]-Peptide 30

31 Project Strategy: Peptide & Radiolabelling Group Peptide Group ore 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 BITINylation Trials on resin cleavage (prevention of methionine oxidation) Identification of suitable purification conditions 31

32 Project Strategy: Peptide & Radiolabelling Group Radiolabelling ore Tasks : onversion of [U- ]-L-isoleucine to Boc-[U- ]-Lisoleucine Trials on final peptide coupling with reduced equivalents of radiolabelled amino acid in collaboration with the Peptide Group Radiolabelled [ ]-BITINylated peptide synthesis Stability Study 32

33 Summary 4 mg of [ ]-BITINylated peptide delivered on schedule HPL Purity 98.9%area (RP), 99.3%area (UV) SA = 338 mi/mmol Stability Study: Material stable at 20 o over 4 weeks 1% drop in RP at 2 o over 4 weeks 33

34 ase 3: PEGylation & Bio-conjugation Stage 1: In corporation of [1- ]glycine into the peptide sequence Stage 2: PEGylation Stage 3: Bio-conjugation to protein-sh 34

35 Stage 1: [ ]-Peptide H 2 N AA-SEQUENE LINKER Resin Boc * 2 H NHBoc oupling Boc AA-SEQUENE LINKER Resin Deprotection Boc AA-SEQUENE LINKER SA Dilution Boc AA-SEQUENE LINKER 35

36 Stage 2: PEGylation Boc AA-SEQUENE LINKER PE N PEG N PEG Boc AA-SEQUENE LINKER PEG N Boc Deprotection AA-SEQUENE LINKER PEG N 36

37 Stage 3: Bio-conjugation AA-SEQUENE LINKER PEG N AA-SEQUENE LINKER PEG N S 37

38 onclusion 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 labelled versions of the parent drug 38

39 onclusion For peptide labelling there are other options such as tritium labelling or radio-iodination ne clear benefit of using a 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 ne limitation of is its rather modest maximum specific activity (62 mi/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

40 40

41 Almac s Radiochemistry Laboratory 41

42 Northern Ireland HQ (32 acre site) IVRS linical Packaging and Labelling Peptide & Protein Technology (PPT) Non GMP API Manufacture Discovery Research Form. Dev. Biomarkers & Diagnostics Solid State & Analytical Services Drug Product Manufacture Stability Radio Labelling GMP API Manufacture 42 onfidential Almac Group 2010

43 Thank you The hexagonal shapes denote the famous Giant s auseway rock in Northern Ireland these shapes also connect to the benzene ring used in science 43