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Carbon-14 Labelled Peptide APIsSolid Phase Peptide Synthesis, BIOTINylation & PEGylation

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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

OHF3C

OH

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

14C6

H314CO

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 C14

O

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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-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

CO2H

CH3*

* **

*

83-merRESIN

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: PEGylation

C14

N

O

O

N O

O

O

C14N

O

O

C14N

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

C14N

O

O

N

O

O

SC14

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)