Keeping tags onbiomolecules

36 manufacturing chemist April 2012 www.manufacturingchemist.com

Biomolecules are well recognised as a

significantly growing area within the

pharmaceutical and biotechnology

sectors.1 One subset of this is peptide

based APIs, many of which are being devel-

oped as potential new therapies for a range of

indications. A critical element of the develop-

ment of any drug is an assessment of its

ADME profile, most commonly performed

using 14C labelled versions of the parent drug.

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

Dr Sean Kitson of Almac explains how synergy between peptide and radiolabelling can speed up decision-making and problem-solving

in the drug development process

phase peptide synthesis (SPPS) within a peptide

synthesiser.

Stage 2 sees the introduction of the 14C amino

acid. In Figure 1 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. The most attractive amino acids

to target for introduction of the 14C label are

those with no sidechain (i.e. glycine) or aliphatic

sidechains (e.g. alanine or valine). These amino

acids can be introduced with specific activities

up to a maximum of 50-60mCi/mmol per 14C

label. The specific activity of the peptide can be

further increased by incorporating several 14C

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).2

The general approach to the synthesis of a 14C

labelled peptide is illustrated in Figure 1.

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

Figure 1. Synthesis of functionalised 14C peptides

peptide labelling

Stage 1

Stage 2 Stage 3

Stage 4resin

14C amino acid

further modificatione.g. biotin

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www.manufacturingchemist.com April 2012 manufacturing chemist 37

Two examples of syntheses at Almac that

have benefited from this synergy are as follows:

Example 1: Preparation of a biotinylated 14C

84-mer

In this target the unlabelled 83-mer resin bound

peptide was first synthesised using the SPPS

approach. The terminal Fmoc amino acid

protecting group was cleaved and the carbon-

14 label introduced via N-Boc-L-[U-14C]

isoleucine. Cleavage of the protecting group

followed by biotinylation then N-Boc cleavage

produced the 84-mer carbon-14 labelled

peptide. Resin cleavage released the [14C]

Peptide-Biotin, which was purified and

lyophilised, giving product with a

radiochemical purity (HPLC) >98 area%,

chemical purity (HPLC) >98 area% and

specific activity >300 mCi/mmol.

Example 2: Preparation of a PEGylated

5-mer

For the PEGylated target the unlabelled resin

bound peptide was synthesised by the SPPS

approach and the terminal Fmoc amino acid

was cleaved to enable the coupling of N-Boc-

[14C]glycine. The carbon-14 labelled peptide

was cleaved from the resin and purified,

followed by lyophilisation to give pure

[14C]Peptide. PEGylation of the peptide

followed by deprotection and purification gave

[14C]Peptide-PEG with a radiochemical purity

(HPLC) >98 area%, chemical purity (HPLC)

>98.0 area% and specific activity >20

amino acids. Coupling of the labelled amino acid

to the resin bound peptide chain is performed at

Almac in custom-made glassware that is

designed to maximise coupling efficiency

without damaging the resin support.

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.

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.

There are a number of companies that offer

excellence in peptide chemistry or in 14C

radiolabelling, but a very small subset that can

offer both. From Almac’s experience, there are a

number of important benefits that come from the

synergy between both peptide and radiolabelling

expertise.3

The primary benefit comes from the shared

pool of knowledge that enables well-informed

decision making and rapid problem solving

throughout the duration of each project. This

shared knowledge spans both the synthetic and

the analytical elements and is supported by the

appropriate equipment. A good example of this

was the early identification of a methionine

sulfoxide impurity by LC-MS during analysis of

a high specific activity 14C labelled peptide.

peptide labelling

mCi/mmol.

In summary, 14C labelling is attractive for

peptides, especially when analysis is performed

by AMS. The 14C peptide is typically made by

SPPS, using custom-made glassware for the key

coupling step(s). Further modification of the

purified 14C peptide can then be performed. A

company that offers both peptide synthesis and14C labelling can benefit from the synergies that

come from pooled knowledge and expertise and

shared analytical equipment. Almac has

successfully applied its deep experience with

peptides and radiolabelling in the synthesis of

some challenging targets. mc

References

1. http://www.peptidetherapeutics.org/PTF_report_summary_2010.pdf2. Salehpour M; Accelerator mass spectrometryoffers new opportunities for microdosing of pep-tide and protein pharmaceuticals, Rapid Comm.Mass Spec., 24, pp1481-1489, 20103. Kitson SL; Accelerated Radiochemistry; PMPSManufacturing, pp68-70, 2010

Contact

Sean Kitson, Investigator: Carbon-14 Ra-diolabelling at Almac Group● info@almacgroup.com● www.almacgroup.com/

Tel: +44 (0) 116 288 1588Fax: +44 (0) 116 281 3000

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