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To date, ocular antibody therapies for the treatment of retinal diseases rely on injection of the drug into the vitreous chamber of the eye. Given the burden for patients undergoing this procedure, less frequent dosing through the use of long-acting delivery (LAD) technologies is highly desirable. These technologies usually require a highly concentrated formulation and the antibody must be stable against extended exposure to physiological conditions. Here we have increased the potential of a therapeutic antibody antigen-binding fragment (Fab) for LAD by using protein engineering to enhance the chemical and physical stability of the molecule. Structure-guided amino acid substitutions in a negatively charged complementarity determining region (CDR-L1) of an anti-factor D (AFD) Fab resulted in increased chemical stability and solubility. A variant of AFD (AFD.v8), which combines light chain substitutions (VL-D28S:D30E:D31S) with a substitution (VH-D61E) to stabilize a heavy chain isomerization site, retained complement factor D binding and inhibition potency and has properties suitable for LAD. This variant was amenable to high protein concentration (>250 mg/mL), low ionic strength formulation suitable for intravitreal injection. AFD.v8 had acceptable pharmacokinetic (PK) properties upon intravitreal injection in rabbits, and improved stability under both formulation and physiological conditions. Simulations of expected human PK behavior indicated greater exposure with a 25-mg dose enabled by the increased solubility of AFD.v8.

Controlling ice nucleation during lyophilization of parenteral drug products increases the homogeneity of critical quality attributes, such as residual moisture, across drug product batches and shortens lyophilization cycle time. In the present study, we compare three mechanistically different techniques to control ice nucleation during the freezing step of lyophilization, which are referred to as “depressurization”, “partial vacuum”, and “ice fog” techniques. The techniques are compared with respect to their operational limitations and challenges. Installation considerations are also discussed. Using the aforementioned nucleation techniques, we investigated a monoclonal antibody formulation and an enzyme formulation at different protein concentrations using feasible nucleation temperatures and different vial formats and fill volumes. Samples were compared for solid state properties and other critical quality attributes on stability. When nucleated at the same temperature, the three techniques produced products with the same quality attributes and stability behavior. Under conditions resulting in micro-collapse, stability behavior can be different. We found that each technology had considerations for achieving robust nucleation. The present comparison may serve as guidance in selecting a nucleation method.

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