The value and versatility of bioconjugation

The ability to combine the activity of a biomolecule with the activity of another entity through bioconjugation has been critical to basic research and discovery for decades.

Without it, we would not be able to detect specific targets of interest or create many of the proteomics and genomics discovery platforms that we use today.

More recently, bioconjugation has enabled new classes of therapeutic drugs and vaccines that further the promise of safer and more efficacious treatments.

Bioconjugation leverages chemical reactions to create covalent linkages between at least one biomolecule, such as a protein or oligonucleotide, and any other moiety to create a conjugate with distinct properties or functions.

As the field has gained a deeper understanding of biomolecule structure and function, researchers have expanded the breadth of applications of bioconjugate chemistry and the methods used to perform these reactions.

Continued innovation in bioconjugate chemistry will drive advancement in medicine and basic scientific research, bringing us closer to realising the full potential of this approach.

Established Applications of Bioconjugate Chemistry

The chemical modification of proteins was practically employed as early as the mid-nineteenth century, initially based on empirical observations but lacking a clear understanding of the processes involved at the molecular level.

The development of methods to couple antibodies or antigens to enzymes in the mid-1960s allowed for the creation of the enzyme-linked immunosorbent assay (ELISA), an alternative to radiometric immunoassays.

In the 1970s, landmark research demonstrated that the covalent attachment of polyethylene glycol (PEG) to a protein could drastically reduce its immunogenicity when injected into mice, paving the way for other protein-polymer conjugates.

Beyond these milestones, bioconjugation has become integral to the development of countless research tools, diagnostics, and therapeutics.

One of the most common applications of bioconjugation is the coupling of a small-molecule label, such as biotin or a fluorescent dye, to a protein.

The resulting bioconjugates allow for the detection, visualisation, quantification, and purification of biological targets, methods which are vital for probing basic biological functions and creating precise diagnostic tools.

Bioconjugate chemistry can also be used to label proteins within living cells, allowing researchers to visualize intracellular processes in real time.

Antibody-drug conjugates (ADCs) are another important class of bioconjugates that have demonstrated great promise in the treatment of cancers, with 14 different ADCs approved by the FDA as of late 2022 and more than 250 clinical trials currently underway.

ADCs are comprised of a monoclonal antibody coupled to a cytotoxic drug via a linker.

Because the associated antibody is specific to a tumour-associated target antigen, ADCs can reduce systemic exposure to the cytotoxic agent, thus reducing its toxicity.

Bioconjugation also plays a vital role in vaccine development by providing a method of linking antigens to carrier proteins, enhancing the immune response and improving vaccine efficacy.

Basic Tenets of Bioconjugation Strategy

While the concepts underlying bioconjugation are simple, its execution is more complex.

To create effective bioconjugates, researchers must understand the characteristics required of the final conjugate.

Beyond having the two critical entities linked together, researchers should consider how that linkage changes the biochemical properties of the final conjugate.

How should that conjugate behave once it reaches its target? Does one of the entities need to be released to perform its function?

Is the attachment point critical? How does it affect the functionality of the biomolecule?

For example, the resulting conjugate could be more hydrophobic than either of the original components, resulting in aggregation and stability problems, or the size of a drug conjugate may need to be increased to avoid renal clearance to improve half-life.

Site-specific attachment is another aspect to be considered.

It dictates the type of chemistry needed and the choice of linker may impact the final biochemical properties and functionality of the conjugate.

Some common targets for bioconjugation reactions include cysteine, lysine, and tyrosine residues.

However, because these residues are often present in large quantities or conjugation could have negative impacts on functionality, modification of amino acids, particularly at the N- and C-terminal position, is a common strategy for increasing the site specificity of a bioconjugation reaction.

Another strategy is the addition of a unique functional group onto a protein, followed by a second reaction to link this functional group with a second moiety.

Researchers can also employ site-specific mutagenesis to introduce specific amino acid residues at desired positions to promote selective conjugation to those sites.

More recent technologies like genetic code expansion can introduce non-canonical amino acids that provide specific chemistry for attachment.

Innovative approaches such as the SpyTag/SpyCatcher system can be used to join proteins through the recombinant introduction of a novel DNA sequence to create fusion proteins, which rapidly form a covalent bond when combined in a reaction.

Additionally, the linkers themselves can provide benefits beyond just being a tether.

They can be selected to adjust the distance between the two entities, to shield hydrophobic moieties, to increase size of a conjugate, or improve its solubility.

Leveraging Bioorthogonal Reactions and Click Chemistry to Overcome Challenges

Recent advances in the field of chemistry have enabled researchers to overcome longstanding challenges in bioconjugation chemistry.

Among these is “click chemistry,” which has gained increased attention since the 2022 Nobel Prize in Chemistry was awarded to three scientists for their work in developing the field.

Click chemistry is a powerful, versatile, and high-yielding chemical approach that emphasises the rapid and selective formation of covalent bonds between two molecular entities.

Its basic principles revolve around the use of small, modular, and highly reactive building blocks that can be easily combined under mild reaction conditions, typically in aqueous solutions and at physiological temperatures.

Bioorthogonal chemistry, a subset of click chemistry, plays a crucial role in this context by enabling the selective modification of biomolecules in complex biological environments.

In doing so, these methods avoid generating side reactions with endogenous functional groups or interfering with native cellular processes.

Bioorthogonal reactions thus must occur at physiologically relevant temperature and pH levels and must not be affected by the presence of water or endogenous oxidants, reductants, or other substances.

Additionally, the reactions must occur rapidly and form stable reaction products.

Its selectivity, efficiency, and compatibility with innate biological conditions make bioorthogonal chemistry an ideal tool for creating bioconjugates, overcoming many of the traditional challenges with bioconjugation to efficiently and effectively combine biomolecules and other moieties.

Driving Progress in Bioconjugation

In the evolving landscape of biomedical research and healthcare, the value of bioconjugation has become increasingly apparent.

This versatile chemical technique has enabled the development of research tools, diagnostics, and therapeutics.

Continued advances in the technology available to conduct effective and efficient bioconjugation chemistry will be vital in reaching the technique’s full potential across the biomedical sciences.

By enabling scientists to combine the native function of biomolecules with drugs, labels, other biomolecule classes, and more, bioconjugation adds to the molecular toolbox available for research and clinical applications.

As we explore new frontiers in science and medicine, bioconjugation will remain an indispensable tool, accelerating innovation from the bench to the clinic.

Pamela James, Ph.D., serves as Vice President, Product, for Vector Laboratories where she oversees research and development and leads quality assurance, product, and program management.

James has spent over a decade and a half with Vector Laboratories in multiple scientific and director roles.

James led the introduction of several impactful products from Vector Laboratories and is focused on Vector’s mission to bring glycobiology tools to the broader scientific community.

She earned a Ph.D. in Immunology from UMass Chan Medical School and a B.S. in Biochemistry from California Polytechnic State University-San Luis Obispo.