Neutron Reflection Study of Surface Adsorption of Fc, Fab, and the Whole mAb.

Characterizing the influence of fragment crystallization (Fc) and antigen-binding fragment (Fab) on monoclonal antibody (mAb) adsorption at the air/water interface is an important step to understanding liquid mAb drug product stability during manufacture, shipping, and storage. Here, neutron reflection is used to study the air/water adsorption of a mAb and its Fc and Fab fragments. By varying the isotopic contrast, the adsorbed amount, thickness, orientation, and immersion of the adsorbed layers could be determined unambiguously. While Fc adsorption reached saturation within the hour, its surface adsorbed amount showed little variation with bulk concentration. In contrast, Fab adsorption was slower and the adsorbed amount was concentration dependent. The much higher Fc adsorption, as compared to Fab, was linked to its lower surface charge. Time and concentration dependence of mAb adsorption was dominated by Fab behavior, although both Fab and Fc behaviors contributed to the amount of mAb adsorbed. Changing the pH from 5.5 to 8.8 did not much perturb the adsorbed amount of Fc, Fab, or mAb. However, a small decrease in adsorption was observed for the Fc over pH 8-8.8 and vice versa for the Fab and mAb, consistent with a dominant Fab behavior. As bulk concentration increased from 5 to 50 ppm, the thicknesses of the Fc layers were almost constant at 40 Å, while Fab and mAb layers increased from 45 to 50 Å. These results imply that the adsorbed mAb, Fc, and Fab all retained their globular structures and were oriented with their short axial lengths perpendicular to the interface.

Feasibility of Freeze-Drying Oil-in-Water Emulsion Adjuvants and Subunit Proteins to Enable Single-Vial Vaccine Drug Products.

To generate potent vaccine responses, subunit protein antigens typically require coformulation with an adjuvant. Oil-in-water emulsions are among the most widely investigated adjuvants, based on their demonstrated ability to elicit robust antibody and cellular immune responses in the clinic. However, most emulsions cannot be readily frozen or lyophilized, on account of the risk of phase separation, and may have a deleterious effect on protein antigen stability when stored long term as a liquid coformulation. To circumvent this, current emulsion-formulated vaccines generally require a complex multivial presentation with obvious drawbacks, making a single-vial presentation for such products highly desirable. We describe the development of a stable, lyophilized squalene emulsion adjuvant through innovative formulation and process development approaches. On reconstitution, freeze-dried emulsion preparations were found to have a minimal increase in particle size of ∼20 nm and conferred immunogenicity in BALB/c mice similar in potency to freshly prepared emulsion coformulations in liquid form.

Critical considerations for developing nucleic acid macromolecule based drug products.

Protein expression therapy using nucleic acid macromolecules (NAMs) as a new paradigm in medicine has recently gained immense therapeutic potential. With the advancement of nonviral delivery it has been possible to target NAMs against cancer, immunodeficiency and infectious diseases. Owing to the complex and fragile structure of NAMs, however, development of a suitable, stable formulation for a reasonable product shelf-life and efficacious delivery is indeed challenging to achieve. This review provides a synopsis of challenges in the formulation and stability of DNA/m-RNA based medicines and probable mitigation strategies including a brief summary of delivery options to the target cells. Nucleic acid based drugs at various stages of ongoing clinical trials are compiled.