Rapid deamidation of recombinant protective antigen when adsorbed on aluminum hydroxide gel correlates with reduced potency of vaccine.

Deamidation of the recombinant protective antigen (rPA) correlates with decreased effectiveness of the vaccine in protecting against infection by Bacillus anthracis. We present data demonstrating dramatic deamidation of amino acid positions 713 and 719 of rPA adsorbed onto aluminum hydroxide gel, an adjuvant, relative to rPA stored in solution without adjuvant. Although deamidation did not impact total levels of rPA-specific antibodies in a mouse model, it did correlate with a decrease in toxin-neutralizing antibodies. On the basis of these data, we hypothesize that interactions of rPA with aluminum hydroxide gel are destabilizing and are the direct cause of reduced vaccine efficacy.

Evaluation of the effect of syringe surfaces on protein formulations.

Packaging of drugs in prefillable syringes offers considerable advantages over conventional vials. Almost all major biotech molecules are available on the market today in prefilled syringes, and are safe and efficacious. Newer high-concentration liquid formulations, especially fusion proteins, however, can suffer from instability in prefilled syringes due to syringe components like silicone oil. To assess the effect of siliconized and modified syringe surfaces on protein formulations, the stability of the recombinant protective antigen (rPA) for anthrax, abatacept, a fusion protein formulation with known silicone oil sensitivity, and an antistaphylococcal enterotoxin B (anti-SEB) monoclonal antibody (mAb) was assessed in siliconized, uncoated, and BD-42-coated (a proprietary coating developed by BD Technologies) prefilled syringes under different conditions. Both the soluble protein content and the number of subvisible particles were followed over time. When filled in siliconized syringes, all three protein solutions showed increased number of subvisible particles relative to uncoated or BD-42-coated syringes; the abatacept formulation with known silicone sensitivity also developed visible particles. Although rPA and anti-SEB mAb formulations mainly showed individual droplets, presumably of silicone, the abatacept formulation also showed droplets entangled in a fibrous structure. Uncoated glass and BD-42-coated syringes considerably reduced the formation of both visible and subvisible particles after immediate contact and after agitation. The anti-SEB mAb also adhered as a thin layer to the siliconized surface after agitation, irrespective of storage temperature. The development of visible particles could not be correlated with the loss of soluble protein fraction at protein concentrations above 4 mg/mL. It appears that protein formulations interact differently with different surfaces. The BD-42 coating appears to be a promising solution for packaging silicone-sensitive proteins in prefillable syringes and needs to be investigated further. It is demonstrated that BD-42 provides an inert surface with adequate lubrication while limiting the formation of visible and subvisible particles. It is hypothesized that these particles are formed due to the release of silicone droplets in the solution and result in the formation of silicone-induced visible aggregates.

Biophysical characterization and formulation of F1-V, a recombinant plague antigen.

The recombinant plague antigen, F1-V, was studied for its structural characteristics using several biophysical techniques. A larger apparent molecular weight relative to its calculated molecular weight obtained from size exclusion chromatography, an unusually large R(g) obtained from MALS, and ANS dye binding studies which indicate that all hydrophobic regions of the protein are exposed to solvent demonstrated that F1-V exists like a disordered protein with a worm-like conformation. The pH-solubility profile of F1-V showed a solubility minimum at pH 5, close to its pI, consistent with the lack of repulsive forces that result in aggregation. Thus, in contrast to most globular proteins that exhibit a secondary and a tertiary structure, F1-V seems to lack tertiary structure and like an unfolded protein is more prone to aggregation via hydrophobic interactions. Despite this, when renatured gradually using descending guanidine hydrochloride concentration dialysis, in the presence of Mg+2, a surfactant and arginine hydrochloride at a pH of 7.5, F1-V appears to populate predominantly in its monomeric state.

Anthrax vaccine powder formulations for nasal mucosal delivery.

Anthrax remains a serious threat worldwide as a bioterror agent. A second-generation anthrax vaccine currently under clinical evaluation consists of a recombinant Protective Antigen (rPA) of Bacillus anthracis. We have previously demonstrated that complete protection against inhalational anthrax can be achieved in a rabbit model, by intranasal delivery of a powder rPA formulation. Here we describe the preformulation and formulation development of such powder formulations. The physical stability of rPA was studied in solution as a function of pH and temperature using circular dichroism (CD), and UV-visible absorption and fluorescence spectroscopies. Extensive aggregation of rPA was observed at physiological temperatures. An empirical phase diagram, constructed using a combination of CD and fluorescence data, suggests that rPA is most thermally stable within the pH range of 6-8. To identify potential stabilizers, a library of GRAS excipients was screened using an aggregation sensitive turbidity assay, CD, and fluorescence. Based on these stability profiles, spray freeze-dried (SFD) formulations were prepared at pH 7-8 using trehalose as stabilizer and a CpG-containing oligonucleotide adjuvant. SFD formulations displayed substantial improvement in storage stability over liquid formulations. In combination with noninvasive intranasal delivery, such powder formulations may offer an attractive approach for mass biodefense immunization.