ADC & Bioconjugation
Bioconjugation refers to the process of joining two molecules together, where at least one of the molecules is a biomolecule, such as an antibody, protein, or oligonucleotide. These bioconjugates are crucial tools in biotechnology, medicine, and nanotechnology, particularly for purposes like detection, assay, and tracking of biomolecules. Common applications include attaching fluorescent probes to antibodies for use in flow cytometry and microscopy imaging, conjugating antibodies to beads for immunoprecipitation, linking antibodies to drugs for therapeutic purposes, and crosslinking proteins to investigate their biological interactions. One notable application of bioconjugation is in antibody-drug conjugation (ADC) technology, which uses monoclonal antibodies to selectively deliver highly potent pharmaceutical ingredients (HPAPIs) to targeted cells. In this conjugated form, the HPAPIs exhibit increased therapeutic activity specifically at the target site, reducing the effects on non-target cells and improving treatment outcomes. The choice of crosslinking reagents and reaction methods for bioconjugation depends on several factors, including the chemical and physical properties of the reagents, such as molecular size, water solubility, cleavability, and the specific functional groups targeted for coupling, all of which must be considered to achieve optimal conjugation efficiency and application success.
Overview
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.