Bioconjugation for ADCs
Conjugation turns an antibody into a delivery vehicle. The chemistry decides how many payloads attach, where they attach, how uniform the product is, and whether the payload stays attached in circulation. Those four properties drive potency, tolerability and manufacturability more than the payload's own potency does.
The three routes
Lysine conjugation uses NHS esters on surface amines. An IgG has many accessible lysines, so the product is a distribution across both the number of payloads and their positions. It is simple and it produces heterogeneity that is hard to characterize and hard to reproduce lot to lot.
Interchain cysteine conjugation partially reduces the interchain disulfides with a mild reductant, then caps the freed thiols with maleimide-bearing payloads. The positions are limited to the interchain sites, so it is more controlled than lysine chemistry, but the product is still a distribution, commonly centered on an even number of payloads, and the antibody is left without those disulfides.
Site-specific conjugation fixes both the number and the position. Engineered cysteines at selected positions, unnatural amino acids, enzymatic attachment through transglutaminase or sortase, and glycan remodeling at the conserved Fc glycan are all in use. The product is homogeneous, characterization is straightforward, and the engineering cost is paid once.
DAR, and why the distribution matters
The drug to antibody ratio is usually reported as an average, and the average hides the distribution. A preparation with an average of four can be a tight distribution around four or a mixture of unconjugated antibody and heavily loaded species. Those behave differently: unconjugated antibody competes for the target without delivering anything, and heavily loaded species clear faster and are more aggregation-prone.
Measure the distribution, not only the mean. Hydrophobic interaction chromatography resolves species by load for cysteine conjugates, reversed-phase after reduction resolves light and heavy chain species, and intact or subunit mass spectrometry gives the composition directly. Report the distribution alongside the average.
Thiol chemistry in practice
Maleimide is fast and selective for thiols near neutral pH, and the succinimidyl thioether it forms is not permanent. It can undergo retro-Michael exchange, transferring payload to albumin and other thiol-bearing proteins in circulation, and it can hydrolyze to a ring-opened form that is stable to exchange. Designing for hydrolysis, by using self-hydrolyzing maleimides or by driving ring opening after conjugation, is a standard mitigation.
Free thiols are the other recurring issue. Partial reduction leaves unpaired cysteines, and unpaired cysteines promote disulfide scrambling and aggregation. Engineered cysteines usually arrive capped from expression, as mixed disulfides with cysteine or glutathione, so they need a controlled uncapping and reoxidation sequence before conjugation. Skipping that step is one of the most common causes of low conjugation efficiency.
Alternatives to maleimide exist for exactly these reasons, including bridging reagents that span the two thiols of a reduced disulfide and restore the covalent link while carrying the payload.
What to measure on the conjugate
- DAR and its distribution.
- Aggregation, before and after conjugation. Hydrophobic payloads promote it, and the increase scales with load.
- Free payload remaining after purification, since unconjugated payload gives cytotoxicity that is not target-mediated.
- Binding, re-measured. Conjugation near the paratope reduces it, and lysine chemistry can hit CDR lysines.
- Serum stability, as payload release over time in serum, which is where linker choices are actually tested.
How we run it
We treat the conjugate as a new molecule rather than a decorated antibody: DAR distribution, aggregation, retained binding and free payload all measured after conjugation, with the conjugation chemistry and the reduction and reoxidation steps recorded as part of the material's history.