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ABSTRACT
Therapeutic bioconjugates are emerging as an essential tool to combat human disease. Site-specific conjugation technologies are widely recognized as the optimal approach for producing homogeneous drug products. Non-natural amino acid (nnAA) incorporation allows the introduction of bioconjugation handles at genetically defined locations. Escherichia coli (E. coli) is a facile host for therapeutic nnAA protein synthesis because it can stably replicate plasmids encoding genes for product and nnAA incorporation. Here, we demonstrate that by engineering E. coli to incorporate high levels of nnAAs, it is feasible to produce nnAA-containing antibody fragments and full-length immunoglobulin Gs (IgGs) in the cytoplasm of E. coli. Using high-density fermentation, it was possible to produce both of these types of molecules with site-specifically incorporated nnAAs at titers > 1 g/L. We anticipate this strategy will help simplify the production and manufacture of promising antibody therapeutics.
Abbreviations
| AS tRNA | = | amber suppressor tRNA |
| ADC | = | antibody-drug conjugate |
| CFPS | = | Cell-free protein synthesis |
| CHO | = | Chinese hamster ovary |
| DAR | = | Drug-to-antibody ratio |
| DBCO | = | dibenzocyclooctyne |
| E. coli | = | Escherichia coli |
| Fab | = | fragment antigen-binding of an antibody |
| HC | = | heavy chain |
| IgG | = | immunoglobulin G |
| LC | = | light chain |
| LC-MS | = | Liquid chromatography-mass spectrometry |
| mAb | = | monoclonal antibody |
| NHS | = | N-hydroxysuccinimide |
| nnAA | = | non-natural amino acid |
| nnAA-IgG | = | nnAA-containing IgG |
| nnAA-LC | = | nnAA-containing light chain |
| pAMF | = | para-azidomethyl-L-phenylalanine |
| PBS | = | phosphate-buffered saline |
| PFLS | = | pre-fabricated light chain |
| RBS | = | Ribosome binding site |
| RS | = | aminoacyl-tRNA synthetase |
| SDS-PAGE | = | Sodium dodecyl sulfate polyacrylamide gel electrophoresis |
| SEC | = | Size exclusion chromatography |
| SPAAC | = | strain-promoted azide-alkyne cycloaddition |
| scFv | = | single chain variable fragment |
| T7 pr. | = | T7 promoter |
| T7 term. | = | T7 terminator |
| tRNA | = | transfer RNA |
Disclosure statement
E.C., J.L., and T.H. were employees and shareholders of Sutro Biopharma, Inc. during the work on this publication. All other authors are employees and shareholders of Sutro Biopharma, Inc.
Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2024.2316872