Critical considerations in the formulation development of parenteral biologic drugs.

Biopharmaceuticals, unlike chemically synthesized small-molecule drugs, are marginally stable, with most of them requiring 3D structures to retain their activity and/or potency. This implies challenges to formulate these molecules for a shelf life >2 yrs and also to minimize the cost of goods for manufacturing. Patient compliance has become a key consideration in the design and development of suitable dosage forms in the modernized world. Thus, here we describe different classes of biological therapeutics, with an emphasis on molecular properties, formulation challenges, and development strategies. We also present statistics on the different classes of approved biologic drugs and dosage forms.

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.

Impact of formulation and particle size on stability and immunogenicity of oil-in-water emulsion adjuvants.

Oil-in-water emulsions have gained consideration as vaccine adjuvants in recent years due to their ability to elicit a differentiated immunogenic response compared to traditional aluminum salt adjuvants. Squalene, a cholesterol precursor, is a natural product with immunostimulatory properties, making it an ideal candidate for such oil-in-water emulsions. Particle size is a key parameter of these emulsions and its relationship to stability and adjuvanticity has not been extensively studied. This study evaluates the effect of particle size on the stability and immunogenicity of squalene emulsions. We investigated the effect of formulation parameters such as surfactant concentration on particle size, resulting in particles with average diameter of 80 nm, 100 nm, 150 nm, 200 nm, or 250 nm. Emulsions were exposed to shear and temperature stresses, and stability parameters such as pH, osmolarity, size, and in-depth visual appearance were monitored over time. In addition, adjuvanticity of different particle size was assessed in a mouse model using Respiratory Syncytial Virus Fusion protein (RSV-F) as a model antigen. Temperature dependent phase separation appeared to be the most common route of degradation occurring in the higher particle sizes emulsions. The emulsions below 150 nm size maintained stability at either 5 °C or 25 °C, and the 80 nm diameter ones showed no measurable changes in size even after one month at 40 °C. In vivo studies using the emulsions as an adjuvant with RSV F antigen revealed that superior immunogenicity could be achieved with the 80 nm particle size emulsion.

Comparative evaluation of disodium edetate and diethylenetriaminepentaacetic acid as iron chelators to prevent metal-catalyzed destabilization of a therapeutic monoclonal antibody.

Understanding the effect of metal chelators with respect to their ability to inhibit metal-catalyzed degradation in biologic products is a critical component for solution formulation development. Two metal chelators, disodium edetate (Na(2)EDTA) and diethylenetriaminepentaacetic acid (DTPA), were evaluated for their ability to stabilize IgG2 mAb in solution formulations spiked with various levels of iron. Real-time stability attributes such as oxidation, soluble aggregate formation, deamidation, and fragmentation demonstrated that DTPA was equivalent to Na(2)EDTA with respect to inhibiting iron-induced degradation over the range of iron concentrations studied. When sufficient chelator was present to stoichiometrically complex trace iron contamination, both Na(2)EDTA and DTPA exhibited the capacity to reduce protein degradation. However, substoichiometric ratios of both chelators were unable to inhibit the degradation induced by free iron ions, which were found to bind weakly to the mAb. This bound iron did not measurably alter the secondary or the tertiary structure of the mAb but appeared to decrease its intrinsic thermodynamic stability, probably by causing subtle perturbations in the tertiary structure. These destabilization effects were not observed when the chelators were present at stoichiometric ratios highlighting the feasibility of using DTPA as an alternate trace metal chelator to Na(2)EDTA in biologic protein formulations.

Rapid and refined separation of human IgG2 disulfide isomers using superficially porous particles.

A rapid reversed-phase HPLC separation of recombinant human immunoglobulin gamma 2 (IgG2) disulfide isomers using columns packed with superficially porous particles is reported. Under optimal conditions, a separation of monoclonal IgG2 disulfide isomers was achieved in 10 min using a Poroshell™ 300SB-C8 column via a combination of high column temperature (85°C), mobile phases with high eluotropic strength (e.g. isopropanol) and high flow rate (1.5 mL/min). Thermodynamic stability analyses of chromatographically enriched IgG2 disulfide isomers revealed differences in their individual denaturation temperatures, which correlate with the observed temperature-dependent refinement of peak profiles by reversed-phase HPLC. This reversed-phase HPLC method in conjunction with other orthogonal analytical techniques (e.g. capillary gel electrophoresis, peptide mapping, ion exchange chromatography, etc.) is being used to characterize disulfide isomers in the development of therapeutic IgG2 antibodies.

Quantitation of plasmid DNA deposited on gold particles for particle-mediated epidermal delivery using ICP-MS.

DNA-plasmid-based vaccines are a promising class of next generation therapeutics. Particle-mediated epidermal delivery is an attractive method for the administration of DNA plasmid vaccines. This technology utilizes minute quantities of DNA plasmid which have been deposited onto the surface of 2-3-microm gold particles, and so the development of this technology requires the use of analytical methods that can accurately quantitate the amount of the DNA on the particle. Spectroscopic methods are generally insufficient for this task due to interference from the gold particle. ICP-MS circumvents this issue while allowing for the sensitive, reproducible, and accurate determination of the quantity of DNA on the particle surface. This report will detail the development and application of such a method.