Keeping tags on biomolecules

Radiolabelling using 14C is used for assessing the ADME profile of new peptide-based active pharmaceutical ingredients. The label is placed within the core of the drug, avoiding the risk of wash out or the need for a modified structure. The limitation of its modest maximum specific activity can be overcome through the use of Accelerated Mass Spectrometry (AMS)

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.

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 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. 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).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 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 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.

Figure 1. Synthesis of functionalised 14C peptides

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.

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 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 and 14C 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.



2. Salehpour M; Accelerator mass spectrometry offers new opportunities for microdosing of peptide and protein pharmaceuticals, Rapid Comm. Mass Spec., 24, pp1481-1489, 2010

3. Kitson SL; Accelerated Radiochemistry; PMPS Manufacturing, pp68-70, 2010