Carbon-14 biologics are moving from niche curiosity to everyday tools in modern drug development. As more therapies are built around antibodies, fusion proteins, peptides and nucleic acids, there is growing pressure on radiochemists to deliver cleanly labelled, clinically acceptable materials that answer tough ADME and PK questions with single low-dose human studies.
In the last few years, the chemistry, modalities, and analytics have all shifted. Rather than simply “sticking a label on the surface”, the field is moving towards well-defined, metabolically robust ¹⁴C positions introduced late in the synthesis, often combined with accelerator mass spectrometry (AMS) to enable microtracer designs.
This article looks at where the real progress is happening: late-stage carbon-14 chemistry, radiolabelled antibodies and ADCs, oligonucleotides and newer strategies that integrate synthesis with AMS-enabled study design.
Late-stage ¹⁴C chemistry feeding biologics
For many years, ¹⁴C labelling meant rebuilding a molecule from a ¹⁴C building block near the start of the route. That approach is still common, but it is too slow and too expensive for many modern pipelines. Current work instead focuses on “late-stage” methods that introduce ¹⁴C at or near the final step of synthesis.
A wave of transition-metal and electrochemical methods now allows ¹⁴C to be dropped into carbonyls, carboxamides and related motifs using ¹⁴CO₂ or ¹⁴CO in a single step, often on fully elaborated drug scaffolds. Reviews by Batista and co-workers, and more recent work on electrocarboxylation, demonstrate how ¹⁴CO₂ generated from Ba¹⁴CO₃ can be used with palladium-catalysed or electrochemical methods to label complex molecules in a single operation.
From a biologics standpoint, these methods are important because they provide high-specific-activity ¹⁴C small molecules and linkers that can be bolted onto larger constructs. They are now feeding:
- ¹⁴C-labelled amino acids and non-natural residues for peptides and proteins.
- ¹⁴C-labelled linkers and payloads for antibody–drug conjugates (ADCs).
- ¹⁴C-labelled small-molecule heads in peptide or antibody conjugates.
The emphasis is on placing the label in metabolically stable positions so that the measured radioactivity reflects the fate of the parent drug or payload, rather than being quickly scrambled into one-carbon metabolites or CO₂.
Carbon-14 labelled antibodies: from concept to clinical reality
Monoclonal antibodies and Fc-fusion proteins dominate the biologics market, but radiolabelling them is technically challenging. Large size, multiple lysines, disulphide bridges and extensive glycosylation all work against tidy, well-defined ¹⁴C incorporation.
A significant development has been the demonstration of practical ¹⁴C labelling of full antibodies using ¹⁴C-formaldehyde. Kim and colleagues reported a method for reductive methylation of lysine residues on recombinant IgG and Fc-fusion proteins using ¹⁴C-formaldehyde, exploring the balance between specific activity, structural integrity and biological function.
By carefully tuning the equivalents of ¹⁴C-formaldehyde and the reducing agent, the team were able to introduce a controlled number of ¹⁴C-methyl groups per protein while maintaining monomeric purity and acceptable binding behaviour. They also evaluated storage stability and plasma stability, showing that label retention and aggregation profiles met expectations for clinical use.
The significance lies not in the novelty of reductive amination as a reaction, but in the optimisation and characterisation required to make it work under GMP-compatible conditions for human microdosing studies. When paired with an AMS readout, only a handful of ¹⁴C atoms per antibody are needed to support robust PK characterisation, thereby lowering both radiological burden and synthetic complexity.
At the same time, progress in site-specific antibody modification is opening new routes to more defined ¹⁴C placement. Enzymatic tools such as transglutaminase-mediated conjugation, glycan remodelling and various cysteine-based strategies already underpin many next-generation ADC platforms.
In many cases, the radiochemistry question now becomes: how do we make a ¹⁴C-labelled version of the linker or tag used in a well-established site-specific method? Late-stage carbonylation and related ¹⁴C techniques are a natural fit here, enabling convergent synthesis of radiolabelled linkers that can be coupled to antibodies with narrow drug-to-antibody ratios and clean conjugation profiles.
Radiolabelled ADCs and other protein–drug conjugates
ADCs are a particular focus because they raise complex distribution and catabolism questions: not only where the antibody goes, but where and how the small-molecule payload is released and cleared. ¹⁴C offers a convenient way to track payload and linker components through the body.
Industrial case studies now describe GMP manufacturing of ¹⁴C-labelled ADCs in which the label is attached to the linker connecting the antibody and the drug. This approach simplifies synthesis and ensures that any cleavage products carrying the linker can still be monitored by radiochromatography.
Meanwhile, academic work has shown the power of dual radiolabelling strategies, in which the protein and payload carry different isotopes, enabling separate in vivo tracking of the two components. Although much of this is done with gamma-emitting isotopes for imaging, the underlying conjugation chemistry can equally be adapted to ¹⁴C payloads for detailed ADME studies.
As conjugation technologies mature, the main synthetic bottlenecks are shifting towards:
- Reliable access to radiolabelled payloads and linkers using late-stage ¹⁴C chemistry.
- Scalable conjugation under GMP that preserves DAR, aggregation and potency.
Suppliers and service organisations are responding with specialised ADC radiolabelling offerings that integrate small-molecule ¹⁴C synthesis, conjugation, and AMS-compatible release testing into a single workflow.
Oligonucleotides and nucleic acid therapeutics
Oligonucleotide drugs – antisense oligonucleotides (ASOs), siRNA, splice-switching oligos and related formats – now form a key part of the modality mix. They pose different labelling questions to proteins: many atoms are chemically similar, solid-phase synthesis involves cycles of phosphoramidite coupling, and modern designs use heavily modified backbones and sugars.
Early work on ¹⁴C-labelled phosphorothioate oligonucleotides established protocols for manual synthesis, isolation, and purification of single-labelled 20-mers. More recent developments have explored multiple ¹⁴C inserts in 2′-modified RNA constructs, such as phosphorothioate 2′-methoxy RNAs containing two ¹⁴C atoms per strand, prepared using suitably labelled phosphoramidites.
Parallel progress in non-radioactive ASO design – including studies on 2′-O-methyl and 2′-O-methoxyethyl modifications and mixed phosphodiester–phosphorothioate backbones – helps guide where ¹⁴C should sit to avoid metabolic lability and maintain hybridisation.
For radiochemists, the practical message is that ¹⁴C must be introduced into positions that survive the known nuclease and biotransformation pathways for a given chemistry series. That often means:
- Labelling stable positions within the sugar or base, rather than the terminal phosphate.
- Avoiding positions prone to early β-elimination or oxidative cleavage.
- Using multiple labels judiciously when single-point labelling does not give sufficient sensitivity for AMS.
Recent reviews on radiolabelling of oligonucleotides, peptides and antibodies summarise this shift away from simply “adding a tag” and towards designing radiolabel positions alongside the underlying medicinal chemistry.
Metabolic labelling and complex biologics
For some classes of biologic – highly glycosylated proteins, virus-like particles, and cell-derived therapies – purely chemical ¹⁴C insertion is impractical. In these cases, metabolic labelling remains an essential tool.
Cells can be grown in media containing ¹⁴C-labelled amino acids, sugars or other precursors, leading to broad distribution of the isotope across the resulting biomolecule. When combined with high-resolution LC–MS and AMS, this type of “global” labelling can still give useful quantitative information on whole-molecule PK and clearance, even if the exact positions of ¹⁴C are diffuse.
Chemoselective ligation chemistry can then be used on top of metabolic labelling to introduce additional handles or to enrich label density at particular features. For example, metabolic incorporation of azido-sugars into glycans allows subsequent “click” attachment of ¹⁴C-bearing probes prepared via late-stage small-molecule chemistry.
This mix-and-match approach is still evolving, but it illustrates a significant trend: synthetic and biological labelling strategies are being combined more deliberately, rather than treated as separate toolkits.
AMS, microdosing and design for study
None of these synthetic advances exists in isolation from the analytics. The adoption of AMS for ¹⁴C quantification has changed what “good” looks like in radiolabelling.
Microtracer studies now use total radioactive doses in the microcurie range, often spiking ¹⁴C-API into a full therapeutic dose of cold drug. AMS can then detect attomole levels of ¹⁴C in plasma and excreta, enabling full human PK, mass balance, and metabolite profiles to be obtained from a single low-dose study.
For biologics, this is particularly attractive because phase 0 or early phase experiments can de-risk a candidate before large, expensive trials. Microdosing studies with ¹⁴C-labelled proteins have already shown that reliable human PK data can be generated in small cohorts, encouraging the wider adoption of similar designs for antibodies and complex biologics.
From a synthetic perspective, AMS relaxes some constraints and tightens others. Fewer ¹⁴C atoms are required to reach adequate counting statistics, but the purity, stability and characterisation of those labels become more critical. Radiochemists now need to demonstrate:
- Stability of the ¹⁴C–C bond and of conjugation linkages under physiological conditions.
- Clear mass balance between parent, metabolites and excreted radioactivity.
- Minimal impact of labelling on binding, potency and immunogenicity.
These requirements loop back to the choice of ¹⁴C position and method: late-stage, metabolically informed labelling gives the best chance of meeting them.
Where is the field heading?
Taken together, the latest developments in the synthesis of ¹⁴C biologics point to several clear themes.
First, small-molecule ¹⁴C chemistry is increasingly designed with biologics in mind. Electrocarboxylation, carbonylation, and isotope exchange methods are chosen not just for yield, but also for their ability to produce labelled linkers, amino acids, and tags that drop directly into established antibody, peptide, and oligonucleotide platforms.
Second, “one size fits all” protein labelling is giving way to modality-specific solutions. Reductive methylation of lysines may be entirely reasonable for an Fc-fusion protein intended for microdosing. In contrast, a site-specific glycan or cysteine conjugation route might be preferred for an ADC where DAR homogeneity is critical.
Third, oligonucleotide therapeutics are driving closer integration among medicinal chemistry, biotransformation knowledge, and radiochemistry. Understanding how a particular ASO series degrades in vivo now informs where ¹⁴C is placed in the sequence and whether single- or multiple-label approaches are appropriate.
Finally, the analytical power of AMS continues to reshape expectations. When a handful of ¹⁴C atoms in a large protein can support a full clinical PK and ADME package, the pay-off for careful, late-stage, metabolically robust labelling is clear.
For anyone working at the interface of synthetic chemistry, DMPK and biologics, the message is reassuring: ¹⁴C is no longer restricted to small molecules and labour-intensive resynthesis. With the current toolkit of late-stage methods, conjugation technologies and high-sensitivity detection, radiolabelled biologics are becoming a routine part of the development playbook rather than a rare, specialist exercise.
Disclaimer
This article is provided solely for general scientific and informational purposes. It does not constitute professional advice in radiochemistry, regulatory affairs, clinical development, or any related field. The discussion of carbon-14 labelling strategies, analytical techniques and study designs is intended to offer an overview of current practices and emerging approaches, but it should not be relied upon as a substitute for specialist guidance, validated protocols or formal regulatory interpretation.
Radiolabelling, GMP manufacturing, clinical study planning, and the handling of radioactive materials all require appropriately trained personnel, controlled facilities and compliance with applicable laws, safety standards and institutional approvals. Organisations must perform their own risk assessments, method validations and regulatory reviews before applying any of the concepts outlined here.
No guarantee is given regarding the accuracy, completeness or suitability of the information for any specific purpose. Any use of the content is at the reader’s own risk, and the author accepts no liability for actions taken or decisions made based on this material.
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