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© Almac 2012
Carbon-14 Labelled Peptide APIsSolid Phase Peptide Synthesis, BIOTINylation & PEGylation
17-18 April 2012Dr Sean Kitson
sean.kitson@almacgroup.com
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Objective• This presentation will focus on a brief
introduction to carbon-14 • Leading onto synthetic strategies towards
labelling peptides with carbon-14
14C
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Introduction to 14C
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Discovery of 14CMartin Kamen & Sam Ruben (27-FEB-1940)
T1/2 ~ 5730 Years
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14C Starting Materials
Ba(OH)2
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Barium 14C carbonate staircaseOH
C14
NH
O
OHF3COH
Cl
OH
OHOMe
MeO
OMe
NCH3
OH
OH
C14
H
NH
CH3
O
N
OH
ClMeO
CH3
*
Ba14CO3
14C6
14CO2
14CH3OH
14CH3I
H14CHO
Cu14CN
K14CN
14C6H3
14CO
14CHH14CR T Brown et al. JLCR 2009, 52, 567-570
S L Kitson, S Jones. JLCR 2010, 53, 140-146
S L Kitson. JLCR 2007, 50, 290-294S L Kitson. JLCR 2006, 49, 517-531
[14C]Apomorphine
[14C]Combretastatin A-1
[14C]XEN-D0401
[14C]ZT-1
S L Kitson & L Leman et al. JLCR 2011, 54 760-770
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14C Drug Molecules14C 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
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14C Labelling StrategyWhen 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 labelS L Kitson ‘Accelerated Radiochemistry’,PMPS Manufacturing 2010, 68-70
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14C Amino acids
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Algae to [U-14C]-Amino Acids
NH2
C14 C14O
OH
C14
Ba14CO3
14CO2
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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
C14
OH
O
C14 C14
OH
O
C14 OH
O
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14C - Glycine Family
• 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
NH2
CO2H
NH2
CO2H **
*
NH2
CO2H*
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A Synthesis of [1-14C]Glycine
NH2
CO2H*
N
O
O
Cl
N
O
O
I
N
O
O
CN
NaI / Acetone
Acetone
heat*
K14CN
AcOH / HCl aq
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14C Peptide Strategy
S L Kitson. ‘Keeping Tags on Biomolecules’, Manufacturing Chemist April 2012
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• 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
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• 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
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• 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
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• 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
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14C Peptide API Case Studies
14C
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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
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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
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[1-14C]-L-VALINE
NH2
CO2H
NHFmoc
CO2H
NH2
V*NH2
-V*NH2
1) Coupling of
2) Capping3) Deblock
Fmoc-OSu
9% aq Na2CO3
14 14
1) Coupling ofthe 13 AAs2) Capping3) Deblock
32-mer
32-mer
32-mer13-mer
V*
Resin
Resin
Resin
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© Almac 2012
-V*NH2
-V*NH2OH
-V*NH2OH
-V*NH2OH
TFA, WaterThioanisoleTIS, EDTPhenol
Purification by RP-HPLC (C18)in 0.1 % TFA Water / 0.1 % ACN
Ion exchange HPLC
32-mer13-mer
32-mer13-mer
Resin
32-mer13-mer
TFA Salt
32-mer13-mer
Acetate Salt
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Analysis
• 0.22 mCi (8.7 MBq) of labelled [14C]-peptide acetate salt
• Radiochemical purity = 98%area• Specific activity = 54 mCi/mmol
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Case Study 2:[14C]-BIOTINylated Peptide
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
14C BIOTIN
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Peptide Group: SPPS of Fmoc-Peptide
RESIN
ivDde AutomatedPeptideSynthesis
Fmoc
83-merRESIN
ivDde
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Peptide Group: SPPS of Fmoc-Peptide
Fmoc
83-merRESIN
ivDde
83-merRESIN
ivDde
Fmoc cleavage
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Radiolabelling: [14C]-Peptide
CH3
NHBoc
CO2HCH3
** *
*
*
83-mer RESIN
ivDde
84-merRESIN
ivDde
14C
14C
Boc
Boc*
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Radiolabelling: Boc-[14C]-Peptide-Biotin
84-merRESIN
14C
Boc
1. Cleavage of ivDde
ivDde
2. Biotin
RESIN
14C
Boc
BIOTIN
Biotinylated 84-mer
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Radiolabelling: [14C]-Peptide-BIOTIN
RESIN
14C
Boc
BIOTIN
Biotinylated 84-mer
14C BIOTIN
Biotinylated 84-mer [14C]-Peptide
1. Boc cleavage
2. Resin cleavage
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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
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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
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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
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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
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Stage 1: [14C]-Peptide
C14
NH2
C14
C14
C14
CO2H
NHBoc
*Boc
Boc
Coupling
Deprotection
Boc
Boc
SA Dilution
Resin
Resin
AA-SEQUENCE LINKER
AA-SEQUENCE LINKER
AA-SEQUENCE LINKER
AA-SEQUENCE LINKER
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Stage 2: PEGylationC14
N
O
O
N O
O
O
C14 N
O
O
C14 N
O
O
AA-SEQUENCE LINKERBoc
PEG
Boc PEG
Boc Deprotection
AA-SEQUENCE LINKER
PEGAA-SEQUENCE LINKER
PE
PEG
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Stage 3: Bio-conjugation
C14 N
O
O
N
O
OS
C14
AA-SEQUENCE LINKER PEG
AA-SEQUENCE LINKER PEG
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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
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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)
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© Almac 2012
Almac’s Radiochemistry Laboratory
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© Almac 2012
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Form.Form.Dev.Dev.
Clinical Clinical PackagingPackaging
and Labellingand Labelling
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Northern Ireland HQ (32 acre siteNorthern Ireland HQ (32 acre site))www.almacgroup.comwww.almacgroup.com
Confidential © Almac Group 2010
Peptide & Protein Peptide & Protein Technology (PPT)Technology (PPT)
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© Almac 2012
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
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