Glass delamination: a comparison of the inner surface performance of vials and pre-filled syringes.

The occurrence of glass delamination is a serious concern for parenteral drug products. Over the past several years, there has been a series of product recalls involving glass delamination in parenteral drugs stored in vials which has led to heightened industry and regulatory scrutiny. In this study, a two-pronged approach was employed to assess the inner surface durability of vials and pre-filled syringes. Non-siliconized syringes were used in order to directly compare glass to glass performance between vials and syringes. The vial and syringe performance was screened with pharmaceutically relevant formulation conditions. The influence of pH, buffer type, ionic strength, and glass type and source was evaluated. In addition, an aggressive but discriminating formulation condition (glutaric acid, pH 11) was used to ascertain the impact of syringe processing. Advanced analytical tools including inductively coupled plasma/mass spectrometry, scanning electron microscopy, atomic force microscopy, and dynamic secondary ion mass spectroscopy showed significant differences in glass performance between vials and syringes. Pre-filled syringes outperform vials for most tests and conditions. The manufacturing conditions for vials lead to glass defects, not found in pre-filled syringes, which result in a less chemically resistant surface. The screening methodology presented in this work can be applied to assess suitability of primary containers for specific drug applications.

Apolipoprotein A-I(Milano) anion exchange chromatography: Self association and adsorption equilibrium.

The self-associative properties of apolipoprotein A-I(Milano) (apoA-I(M)) were investigated in relationship to its anion exchange behavior on Q-Sepharose-HP with and without the addition of urea as a denaturant. Self-association was dependent on protein and urea concentration and both influenced interactions of the protein with the chromatographic surface. In the absence of urea, apoA-I(M) was highly associated and existed primarily as a mixture of homodimer, tetramer and hexamer forms. Under these conditions, since the binding strength was greater for the oligomer forms, broad, asymmetrical peaks were obtained in both isocratic and gradient elution. Adding urea depressed self-association and caused unfolding. This resulted in sharper peaks but also decreased the binding strength. Thus, under these conditions chromatographic elution occurred at lower salt concentrations. The adsorption isotherms obtained at high protein loadings were also influenced by self-association and by the varying binding strength of the differently associated and unfolded forms. The isotherms were thus dependent on protein, urea, and salt concentration. Maximum binding capacity was obtained in the absence of urea, where adsorption of oligomers was shown to be dominant. Adding urea reduced the apparent binding capacity and weakened the apparent binding strength. A steric mass action model accounting for competitive binding of the multiple associated forms was used to successfully describe the equilibrium binding behavior using parameters determined from isocratic elution and isotherm experiments.

Apolipoprotein A-I(Milano) anion exchange chromatography: mass transfer and adsorption kinetics.

The mass transfer and adsorption kinetics of self-associating apolipoprotein A-I(Milano) (apoA-I(M)) was investigated for the two anion exchangers Q-Sepharose-HP and Macro-Prep-HQ. At high salt where no protein binding occurs and without urea, mass transfer was controlled by hindered pore diffusion of multiple associated forms for both materials. Adding urea suppressed self-association, but resulted in higher viscosity and caused unfolding. As a consequence, the effective diffusivity decreased as urea was added and was greater for the larger pore Macro-Prep-HQ resin. At low salt, under strong binding conditions, the adsorption kinetics followed a more complex mechanism. In this case, the kinetics was very slow for both stationary phases up to 2 M urea. However, at higher urea concentrations, the adsorption kinetics for the smaller pore Q-Sepharose-HP matrix became much faster, suggesting a transition from pore- to surface-dominated diffusion. Microscopic observations confirmed that different transport mechanisms were in play below and above 2 M urea, which marked the approximate boundary above which self-association was suppressed and unfolding occurred. The net result was enhanced uptake kinetics at high urea concentrations (e.g., 4 M) where protein unfolding is thought to lead to a more flexible structure that can reptate along the pore surface. Although the observed enhancement was dependent on the pore size and, thus, the surface area of the resin, it was not limited to apoA-I(M). BSA showed a similar trend as a function of urea when its disulfide bonds were reduced.

pH Transients in hydroxyapatite chromatography columns-experimental evidence and phenomenological modeling.

Hydroxyapatite (HAP) columns, widely used for chromatographic separation of proteins and other biomolecules because of their unique selectivity and ability to resolve complex mixtures, exhibit limited stability at acidic conditions requiring careful control of pH. Even with buffered solutions, however, unintended pH transients can occur when the salt concentration varies. For example, the pH temporarily decreases below the feed value when the salt concentration increases and increases above the feed value when the salt concentration is decreased. The intensity and duration of these transients depend on the particular buffer used and the magnitude of the salt concentration step, but in extreme cases the pH can drop by as much as 1.5 pH units creating conditions where the HAP stability is potentially compromised. This work examines the mechanisms leading to pH transients in HAP columns generated by salt steps. The pH excursions are similar to those observed for weak cation exchange columns, but are accompanied by a transient evolution of phosphate which temporarily decreases below the feed value when the salt concentration is increased and increases sharply when the salt concentration is reduced before returning to the feed value. A phenomenological model is developed to describe this behavior by considering the reversible uptake of sodium ions by the P-sites and binding of phosphate ions by the C-sites. The interplay of these two adsorption mechanisms results in complex pH patterns that are consistent with those observed experimentally. In addition to helping understand the underlying mechanisms, the model also provides a useful tool to predict the effects of different buffers and salt concentration and develop corrective measures that can reduce the intensity and duration of the pH transients such as the addition of unretained co-buffers.

Theory and applications of refractive index-based optical microscopy to measure protein mass transfer in spherical adsorbent particles.

This work provides the theoretical foundation and a range of practical application examples of a recently developed method to measure protein mass transfer in adsorbent particles using refractive index-based optical microscopy. A ray-theoretic approach is first used to predict the behavior of light traveling through a particle during transient protein adsorption. When the protein concentration gradient in the particle is sharp, resulting in a steep refractive index gradient, the rays bend and intersect, thereby concentrating light in a sharp ring that marks the position of the adsorption front. This behavior is observed when mass transfer is dominated by pore diffusion and the adsorption isotherm is highly favorable. Applications to protein cation-exchange, hydrophobic interaction, and affinity adsorption are then considered using, as examples, the three commercial, agarose-based stationary phases SP-Sepharose-FF, Butyl Sepharose 4FF, and MabSelect. In all three cases, the method provides results that are consistent with measurements based on batch adsorption and previously published data confirming its utility for the determination of protein mass transfer kinetics under a broad range of practically relevant conditions.