Bispecific Antibodies Produced via Chemical Site-Specific Conjugation Technology: AJICAP Second Generation

Bispecific antibodies are biotherapeutics that amalgamate the specificities of two distinct antibodies into one molecule. Bispecific antibodies can be utilized in a broad range of diagnostic and therapeutic applications; however, their engineering requires genetic modification and remains time-consuming. Therefore, in this study, we used AJICAP second-generation technology, which drives the production of site-specific antibody-drug conjugates in a practical and robust manner, without genetic modification requirements, to generate bispecific antibodies. Using haloketone chemistry as an alternative to maleimide chemistry, which carries reaction risks, we successfully produced site-specific antibody conjugates. Pharmacokinetic studies revealed that the haloketone-based antibody conjugate was stable in the rat plasma. The resultant bispecific antibodies were rigorously evaluated, and surface plasmon resonance measurements and flow cytometry analyses confirmed that antigen binding remained intact. Additionally, the affinity for the neonatal Fc receptor (FcRn) was retained after conjugation. Further cytotoxicity evaluation emphasized the pronounced activity of the generated bi-specific antibodies. These preliminary findings highlight the potential of AJICAP second-generation technology in BisAb production. This novel approach introduces a fully chemical, site-specific strategy capable of producing bispecific antibodies, heralding a new era in the field of biotherapeutics

Retardation Mechanism of Crystallization of Diacylglycerols Resulting from the Addition of Polyglycerol Fatty Acid Esters

Edible oils containing high concentrations (>80%) of diacylglycerols (DAG oil) have beneficial health effects on obesity and obesity-related diseases; however, at low temperature, undesired precipitation of high-melting fractions in DAG oil can occur. Thus, preventing the precipitation of high-melting saturated fatty acid moieties in DAG oil is crucial for its expanded use. In this study, we investigated the mechanism of retardation of crystallization of DAG oil through the addition of polyglycerol fatty acid esters (PGFEs). We observed the occurrence of birefringence under polarized crossed-Nicols conditions in the PGFE-added DAG oil. We also found that prior to the crystallization of high-melting DAG fractions, PGFE-added DAG oil showed shear-rate-dependent changes in viscosity, providing strong evidence for the existence of self-assembled structures that lead to the birefringence. Furthermore, small- and wide-angle X-ray diffraction patterns suggest the formation of a supramolecular assembly comprising DAGs and PGFEs, which is significantly different from the structure of DAG crystals. From these results, we conclude that the retardation of crystallization of DAG oil is caused by the formation of liquid-crystal-like supramolecular complex structures that contain high-melting fractions of DAGs and PGFEs. These complexes may disturb the formation of critical nuclei of high-melting DAG fractions during the prenucleation crystallization stage