(a) Trastuzumab scFv (lane 1: soluble lysate (reducing),lane 2: purified (reducing),lane 3: purified (non-reducing)

(a) Trastuzumab scFv (lane 1: soluble lysate (reducing),lane 2: purified (reducing),lane 3: purified (non-reducing). antibody fragments have emerged as a particularly exciting therapeutic modality due to their high specificity for a given target, which is usually thought to reduce the Ceftiofur hydrochloride adverse effects associated with treatment.1Because of this, the market for monoclonal antibody (mAb)-derived drug products has grown rapidly in the past few decades; the FDA recently approved the 100thmAb product for use in the United States, and nearly one-fifth of its yearly new drug approvals belong to this class.2mAbs and their derivatives have been especially impactful in the field of malignancy treatment, with dozens of products currently approved and many more in clinical development.3 It has long been recognized that this therapeutic properties of mAbs Ceftiofur hydrochloride can be enhanced through covalent fusion to small molecules. For example, the antitumor properties of these drugs can be significantly improved by tethering a cytotoxic small molecule to the protein to form a derivative called an antibodydrug conjugate (ADC).4,5Traditionally, ADCs were generated using reactive side groups on naturally occurring amino acids such as the thiol of cysteine or the -amine of lysine as the bioconjugation sites.6,7Cysteine conjugations have several drawbacks, however. Cysteine conjugation can result in heterogeneous ADC populations with varying drug-to-antibody ratio (DARs). This heterogeneity can affect the stability, pharmacokinetics, Ceftiofur hydrochloride and efficacy of the ADC. The heterogeneity also makes characterization and optimization more challenging. A more advanced technique referred to as THIOMAB utilizes designed cysteines that are introduced as unpaired cysteines on the surface of the protein for thiol conjugation.8Surface-exposed cysteines can lead to protein dimerization or be capped by glutathione, requiring reduction prior to conjugation. Also, cysteine conjugations typically require prior reduction, increasing the number of process actions.7In an alternative approach, conjugation is performed by reacting N-hydroxysuccinimide (NHS) esters with surface-exposed lysines.7Amine-based conjugation technology similarly suffers due to partial conjugation of surface-exposed lysines that results in heterogeneous mixtures. This approach has been used to improve the therapeutic properties compared to mAbs in their native form; however, traditional stochastic conjugation leads to heterogeneities in the number and location of modifications, which can affect the biophysical properties of the protein and result in undesirable outcomes such as compromised activity, stability and Mouse monoclonal to MDM4 pharmacokinetics.5,6,9 Site-specific protein bioconjugation approaches allow researchers Ceftiofur hydrochloride to build highly tunable protein constructs capable of improving the specificity and efficacy of targeted drug delivery while minimizing negative effects on therapeutic activity.6,10Producing an ADC this way has the potential to widen the therapeutic window due Ceftiofur hydrochloride to the selection of the single species that optimizes physicochemical properties, efficacy, and safety. Non-natural amino acid (nnAA) incorporation is usually a particularly exciting and well-studied bioconjugation method based on the site-specific genetic insertion of amino acids altered with bio-orthogonal functional groups. nnAAs are most commonly incorporated co-translationally using an designed, orthogonal transfer RNA (tRNA) that recognizes the amber stop codon, ochre stop codon, or other nonsense codons, as well as an aminoacyl-tRNA synthetase (RS) that catalyzes the aminoacylation of the orthogonal tRNA with the nnAA.11The charged tRNA then inserts the nnAA at a genetically specific location(s) during protein translation. However, nnAA incorporation has been difficult to implement in traditional mAb production hosts such as mammalian cells due to limited amber suppression efficiency, which adversely impacts production titers and product homogeneity.11,12 The production of nnAA-containing antibodies in stable Chinese hamster ovary (CHO) cell lines has recently been reported.12However, incorporation of nnAAs into antibodies produced in CHO is limited by low amber suppression efficiencies of ~ 40%, which results in low nnAA-containing IgG titers. In addition, truncated IgGs (resulting from translation termination at the TAG codon) were co-purified with the nnAA-IgGs, necessitating additional chromatography steps to remove truncated product. In an earlier report, the co-purification of truncated and nnAA-containing species was prevented by placing the nnAA incorporation site closer to the N-terminus.