ADC & Bioconjugation

Bioconjugation Crosslinker Types

One of the most fundamental aspects of crosslinker design is whether the reagent is homobifunctional or heterobifunctional. The overwhelming majority of bioconjugate reagents are bifunctional, with two reactive groups usually located at the outer ends of an organic spacer. In a homobifunctional compound, the two reactive groups are identical, whereas in a heterobifunctional compound they are different. Heterobifunctional reagents have major advantages over homobifunctional ones when forming bioconjugates, since one reactive end group couples with only a specific functional group, while the other reactive end group reacts with a different functional group.

Bioconjugation Crosslinker Length

The dimensions or overall linear length of the target molecule before and after conjugation should be considered when choosing a crosslinker or modification reagent for the conjugation reaction. The spacer arm or cross-bridge of the reagent mainly determines the molecular length of the resulting compound. Crosslinkers of different sizes thus become molecular rulers for measuring the distances between functional groups in biomolecules.

Cleavable vs Non-cleavable Bioconjugation Crosslinkers

It is important for the crosslinker to be cleavable if the interacting biomolecules need to be isolated and analyzed, e.g. a crosslinker used to detect protein-protein interactions. Alternatively, a non-cleavable linker could be used where stability is required, e.g. an antibody attached to a resin for protein capture.

Hydrophobic vs Hydrophilic Bioconjugation Crosslinkers

In some applications, reagent hydrophobicity can be an advantage, especially when an application involves the penetration of cell membranes. Hydrophobic reagents without any strongly polar groups quickly pass through cell membranes to crosslink or label internal cell proteins. On the other hand, hydrophilic crosslinkers do not cause aggregation or precipitation of the interacting molecule and can lead to water solubility of antibodies and proteins modified by them. The use of hydrophilic bioconjugation reagents also results in greater biocompatibility.

Functional Groups Targeted in Bioconjugation

The most reactive functional groups in biomolecules are associated with the heteroatoms N, O, and S, which are nucleophilic due to an unshared pair of electrons and can spontaneously react with the compatible and electrophilic active groups on crosslinkers and modification reagents. In many cases, the nucleophilic functional groups in biomolecules are free and accessible. However, in some instances they are created to allow reactivity and coupling to take place. There are several specialized reagents available that facilitate the creation of an appropriate functional group for bioconjugation if the desired one is not available. The naturally occurring functional groups on biomolecules are amines, thiols, hydroxyls, carboxylates, aldehydes, organic phosphates, and reactive hydrogens on certain activated carbon atoms.

ADC Development and Manufacturing

The development journey of an antibody-drug conjugate (ADC) requires expertise in small and large molecule development, manufacturing, formulation and testing. Choosing an experienced partner, with these skills and the required containment facilities can help advance your ADC to market. ADCs are challenging molecules requiring advanced manufacturing suites and dedicated equipment to characterize the molecule and demonstrate its purity, homogeneity, and stability.