Taming Random Conjugation: A General Approach for Equimolar Linking of Proteins and Payloads
Sergii Kolodych, PhD, CSO, Syndivia SAS
Multiple conjugation sites are usually available on a biomolecule. Upon conjugation, they produce a mixture of species having different degrees of conjugation (DoC). We report a general conjugation approach for achieving a defined DoC, which is virtually applicable to any biological macromolecule and payload. Applied to native antibodies, the method yields highly homogenous antibody-drug conjugates, antibody-oligonucleotide conjugates as well as bispecific scaffolds.
Q: How is it possible to achieve a defined – and consistent – degree of conjugation (DoC)?
A: Everything started with our initial observation that conjugate homogeneity increases with low protein conversion rates. With very low conversion, only the unconjugated protein and the DoC 1 conjugate are detected in the mixture. We then went on to create a technology to efficiently separate DoC 1 conjugates and reintroduce the unconjugated protein into the process. This was achieved through a combination of automated affinity purification and recent developments in trans-tagging reactions, allowing the affinity tag to be replaced by any payload of interest.
Q: How can your ‘defined’ DoC be applied to any biological macromolecule and payload?
A: When developing this technology, we chose to exploit a very general mathematical feature of all statistical distributions of products obtained during conjugation reactions involving multisite macromolecules. Focusing on this general feature rather than on individual structural characteristics of distinct proteins, we were able to create a truly universal conjugation platform, which can be readily applied to any “off-the-shelf” protein and payload.
Q: How far along is this breakthrough technology, and how has it been applied to date?
A: Paradoxically, one of the major challenges was miniaturization of the conjugation setup. Whereas all components were available for a large biomanufacturing process, we had to create the components for small-scale conjugation from scratch. 3D-printing technology was very helpful in this task, since it enabled easy scaling and rapid production of prototypes. Our 3D-printed conjugation device has now been tested with a large number of proteins and payloads at different scales. Our next step is to make the technology compliant with GMP.
Q: What are you most looking forward to at the PEGS Europe Summit?
A: The PEGS Summit is a must for anyone working in the field of protein engineering. It is a superb opportunity to share novel approaches and gather valuable input from top industry professionals.