A case study of nondelamination glass dissolution resulting in visible particles: implications for neutral pH formulations.

Visible particles were unexpectedly observed in a neutral-pH placebo formulation stored in glass vials but were not observed in the same formulation composition that contained protein. The particles were identified as silica gel (SiO2 ) and polysorbate 20, suggesting dissolution of the glass vial. Time course studies were performed to assess the effect of variables such as pH, excipients, storage temperature, and duration on particle formation. Data suggest that glass dissolution occurred during the storage in the liquid state, as shown by increased Si levels in solution. Upon freezing, the samples underwent freeze concentration and likely became supersaturated, which resulted in the appearance of visible silica particles upon thawing. The glass degradation described here is unique and differs from the more commonly reported delamination, defined by the presence of reflective, shard-like glass flakes in solution that are often termed lamellae. This case study underscores the importance of an early assessment (during formulation development) of potential incompatibility of the formulation with the primary container.

Multivariate analysis of the sequence dependence of asparagine deamidation rates in peptides.

PURPOSE: To develop a quantitative scheme to describe and predict asparagine deamidation in polypeptides using chemometric models employing reduced physicochemical property scales of amino acids. METHODS: Deamidation rates for 306 pentapeptides, Gly-(n-1)-Asn-(n+1)-Gly, with the residues n-1 and n+1 varying over the naturally occurring amino acids, were obtained from literature. A multivariate regression technique, called projection to latent structures (PLS), was used to establish mathematical relationships between the physicochemical properties and the deamidation half-lives of the amino acid sequences. Three reduced physicochemical property scales, amide hydrogen exchange rates (to describe the relative acidity of the amide protons) and flexibility parameters for the sequences were evaluated for their predictive capacity. RESULTS: The most effective descriptors of the deamidation half-lives were reduced-property parameters for amino acids called zz-scores. The PLS models with the reduced property scales, combined with the hydrogen exchange rates and/or flexibility parameters, explained more than 95% of the sequence-dependent variation in the deamidation half-lives. The amide hydrogen exchange rate (i.e., amide proton acidity), hydrophilicity, polarizability, and size of amino acids in position n+1 were found to be the principal factors governing the rate of deamidation. The effect of amino acids in position n-1 was found to be negligible. CONCLUSIONS: Chemometric analysis employing reduced physicochemical parameters can provide an accurate prediction of chemical instability in peptides and proteins. The relative importance of these various factors could also be determined.

Self-buffering antibody formulations.

Monoclonal antibodies (mAbs) often require the development of high-concentration formulations. In such cases, and when it is desirable to formulate a mAb around pH 5.0, we explored a novel approach of controlling the formulation pH by harnessing the ability of mAbs to "self-buffer." Buffer capacities of four representative IgG(2) molecules (designated mAb1 through mAb4) were measured in the pH 4-6 range. The buffer capacity results indicated that the mAbs possessed a significant amount of buffer capacity, which increased linearly with concentration. By 60-80 mg/mL, the mAb buffer capacities surpassed that of 10 mM acetate, which is commonly employed in formulations for buffering in the pH 4-6 range. Accelerated high temperature stability studies (50 degrees C over 3 weeks) conducted with a representative antibody in a self-buffered formulation (50 mg/mL mAb1 in 5.25% sorbitol, pH 5.0) and with solutions formulated using conventional buffers (50 mg/mL mAb1 in 5.25% sorbitol, 25 or 50 mM acetate, glutamate or succinate, also at pH 5.0) indicated that mAb1 was most resistant to the formation of soluble aggregates in the self-buffered formulation. Increased soluble aggregate levels were observed in all the conventionally buffered (acetate, glutamate, and succinate) formulations, which further increased with increasing buffer strength. The long-term stability of the self-buffered liquid mAb1 formulation (60 mg/mL in 5% sorbitol, 0.01% polysorbate 20, pH 5.2) was comparable to the conventionally buffered (60 mg/mL in 10 mM acetate or glutamate, 5.25% sorbitol, 0.01% polysorbate 20, pH 5.2) formulations. No significant change in pH was observed after 12 months of storage at 37 and 4 degrees C for the self-buffered formulation. The 60 mg/mL self-buffered formulation of mAb1 was also observed to be stable to freeze-thaw cycling (five cycles, -20 degrees C --> room temperature). Self-buffered formulations may be a better alternative for the development of high-concentration antibody and protein dosage forms.