Table 1.
Comparison between different bioconjugation technologies for posttranslational antibody modification. The technologies presented here are able to reconstitute antibody fragments in vitro on the protein level. Tethered variable CL as well as common LC bsAbs are generated on a molecular biology basis and not recombined on the protein level.
| Technology | Linkage | Component Number | Handle of Motif | Residual Amino Acid Imprint | Activation Conditions | Reaction | Cofactors | Downstream Purification | HTS Compatibility | In Vivo Ligation | Covered Formats |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Duobodies | None | 2 | F405 and Y407 substitution in CH3 | Seamless | Mixed mAb precursor fragments under mild reducing conditions | Controlled Fab-arm exchange | Reducing agent, 2-MEA | Protein A, SEC | Yes | No | Full length-IgG format, DuoHexaBodies |
| Transglutaminase | C-terminal, N-terminal, site specific | 2 | LLQGA | LLQGA | Addition of Transglutaminase in high concentrations | Acyl-transfer | None | Protein A, SEC | No | No | Antibody drug conjugates |
| Sortase A | C-terminal, N-terminal, site specific | 2 | LPXTG | LPXTGGG | Excess of one reconstitution partner required based on a reversible reaction, high concentrations of Sortase A | Transpeptidation | None | IMAC | yes | Yes | Fc fusions, scFv, VHH |
| SpyTag/SpyCatcher | C-terminal, N-terminal | 3 | Lys in SpyCatcher, Asp in SpyTag | SpyTag/SpyCatcher | Isopeptide bond formation after bringing precursor proteins in close proximity | Amidation | None | Protein A, SEC | Yes | Yes | Full length-IgG format, Fc fusions, scFv, VHH |
| Split inteins | C-terminal, N-terminal | 3 or more | Int N and Int C | Extein sequences | Isopeptide bond formation after bringing precursor proteins in close proximity under mild reducing conditions | Protein trans splicing | Reducing agent TCEP, DTT if using cys containing split inteins | IMAC | Yes | Yes | Full length-IgG format, Fc fusions, scFv, VHH |
| Tethered variable CL or common LC bsAbs | None | 2 | VL-HC fusion (VLfH) or none | Short (Gly4Ser)4-linker or none | Intact BsAb generation in a single cell line by fusing the VL domain residues (1-R108) genetically to the antibody HC via (G4S)4 linker and CL co expression | co expression | None | ProteinA | yes if supernatant compatible | Not needed | Full-length-IgG format |