Therapeutic antibody technology heavily dominates the biologics market and continues to present as a significant industrial interest in developing novel and improved antibody treatment strategies. Many noteworthy advancements in the last decades have propelled the success of antibody development; however, there are still opportunities for improvement. In considering such interest to develop antibody therapies, this review summarizes the array of challenges and considerations faced in the design, manufacture, and formulation of therapeutic antibodies, such as stability, bioavailability and immunological engagement. We discuss the advancement of technologies that address these challenges, highlighting key antibody engineered formats that have been adapted. Furthermore, we examine the implication of novel formulation technologies such as nanocarrier delivery systems for the potential to formulate for pulmonary delivery. Finally, we comprehensively discuss developments in computational approaches for the strategic design of antibodies with modulated functions.

This study reports the synthesis, characterisation and antimicrobial activity of five novel silver N-heterocyclic carbene (Ag–NHC) complexes obtained by N-propylphthalimide and N-methyldioxane substituted benzimidazolium salts with silver oxide. The reactions were performed at room temperature for 24 h in the absence of light. The obtained complexes were identified and characterised by ¹H and ¹³C NMR, FT-IR and elemental analysis techniques. The minimum inhibitory concentration (MIC) of the complexes was determined for E. coli, P. aeruginosa, E. faecalis, S. aureus, C. tropicalis and C. albicans in vitro through agar and broth dilution. The results indicated that these complexes exhibit antimicrobial activity. In particular, complex 3 presented the significant broad spectrum antimicrobial activity.

Protein aggregation constitutes a recurring complication in the manufacture and clinical use of therapeutic monoclonal antibodies (mAb) and mAb derivatives. Antibody aggregates can reduce production yield, cause immunogenic reactions, decrease the shelf-life of the pharmaceutical product and impair the capacity of the antibody monomer to bind to its cognate antigen. A common strategy to tackle protein aggregation involves the identification of surface-exposed aggregation-prone regions (APR) for replacement through protein engineering. It was shown that the insertion of N-glycosylation sequons on amino acids proximal to an aggregation-prone region can increase the physical stability of the protein by shielding the APR, thus preventing self-association of antibody monomers. We recently implemented this approach in the Fab region of full-size adalimumab and demonstrated that the thermodynamic stability of the Fab domain increases upon N-glycosite addition. Previous experimental data reported for this technique have lacked appropriate confirmation of glycan occupancy and structural characterization of the ensuing glycan profile. Herein, we mutated previously identified candidate positions on the Fab domain of Trastuzumab and employed tandem mass spectrometry to confirm attachment and obtain a detailed N-glycosylation profile of the mutants. The Trastuzumab glycomutants displayed a glycan profile with significantly higher structural heterogeneity compared to the HEK Trastuzumab antibody, which contains a single N-glycosylation site per heavy chain located in the CH2 domain of the Fc region. These findings suggest that Fab N-glycosites have higher accessibility to enzymes responsible for glycan maturation. Further, we have studied effects on additional glycosylation on protein stability via accelerated studies by following protein folding and aggregation propensities and observed that additional glycosylation indeed enhances physical stability and prevent protein aggregation. Our findings shed light into mAb glycobiology and potential implications in the application of this technique for the development of “biobetter” antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include...

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.