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High concentration protein solutions are generally produced by spin column concentration (SCC) during early development and by tangential flow filtration (TFF) during later stages, when greater quantities of protein become available. This is based on the assumption that the protein generated by the SCC process would be fairly similar to the TFF process material. In this study, we report the case of high concentration solutions of an Fc fusion protein produced by the two processes using the same upstream drug substance (DS) with very different storage stability. The TFF and SCC batches were characterized for aggregation, viscosity, and hydrodynamic radius before and after storage at different temperatures (5°C, 25 °C, and 40 °C). Aggregation and viscosity of the solutions processed by TFF were higher than those processed by SCC upon storage at 25 °C and 40 °C for three months. Differential scanning fluorimetry (DSF) revealed differences in initial protein conformation. Upon exposure to shear stress, protein solutions showed conformational instability and increased aggregation upon storage at 35 °C. In addition, protein solution showed higher aggregation upon shearing under mixed (downstream purification process and final formulation) buffer conditions – which are more likely to be encountered during the TFF, but not SCC, process. These results were further confirmed in an independent experiment by Fourier transform-infrared (FT-IR) spectroscopy and aggregation analysis. Taken together, these data indicate that shearing the protein in intermediate, unstable buffer conditions can lead to conformational perturbation during TFF processing, which led to higher rate of aggregation and viscosity upon storage. This study highlights the importance of testing shear stress sensitivity in the transitional buffer states of the TFF process early in development to de-risk process related product instability.

Solution pH and ionic strength (I) have complex effects on protein stability. We developed an experimental approach based on exploitation of the zwitterionic characteristic of amino acid molecules to probe the relative contribution from each. A variety of types of amino acid solutions were adopted to investigate the effects of pH and I in a manner that allows independent evaluation of each factor. The same effect could not be achieved using conventional buffer solutions. Size-exclusion chromatography, capillary differential scanning calorimetry, and fluorescence spectroscopy were utilized to probe the protein aggregation and conformation. The results suggested that, in addition to pH, solution ionic strength as a function of ionization state of the amino acid molecules and the ions introduced by pH adjustment played an important role in the aggregation and conformation of the protein studied. This experimental approach offers a useful tool to aid fundamental understanding of the relative effects of solution pH and ionic strength on protein stability.

Amphiphilic block copolymers (ABCs) have been used extensively in pharmaceutical applications ranging from sustained-release technologies to gene delivery. The utility of ABCs for delivery of therapeutic agents results from their unique chemical composition, which is characterized by a hydrophilic block that is chemically tethered to a hydrophobic block. In aqueous solution, polymeric micelles are formed via the association of ABCs into nanoscopic core/shell structures at or above the critical micelle concentration. Upon micellization, the hydrophobic core regions serve as reservoirs for hydrophobic drugs, which may be loaded by chemical, physical, or electrostatic means, depending on the specific functionalities of the core-forming block and the solubilizate. Although the Pluronics, composed of poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide), are the most widely studied ABC system, copolymers containing poly(L-amino acid) and poly(ester) hydrophobic blocks have also shown great promise in delivery applications. Because each ABC has unique advantages with respect to drug delivery, it may be possible to choose appropriate block copolymers for specific purposes, such as prolonging circulation time, introduction of targeting moieties, and modification of the drug-release profile. ABCs have been used for numerous pharmaceutical applications including drug solubilization/stabilization, alteration of the pharmacokinetic profile of encapsulated substances, and suppression of multidrug resistance. The purpose of this minireview is to provide a concise, yet detailed, introduction to the use of ABCs and polymeric micelles as delivery agents as well as to highlight current and past work in this area.

We systematically altered the chemical structure of the core-forming poly(L-amino acid) block of an amphiphilic diblock copolymer series based on poly(ethylene oxide)-block-poly(N-hexyl-L-aspartamide), PEO-b-p(N-HA), acyl esters by varying the length of the attached acyl side chain. Drug-loaded micelles were prepared in good yield by a modified solvent evaporation procedure. In addition, the relative aggregation state and hemolytic activity of encapsulated amphotericin B (AmB) were analyzed by absorption spectroscopy. The length of the attached acyl side chain in PEO-b-p(N-HA) acyl ester micelles modulates the relative aggregation state of encapsulated AmB. Furthermore, acyl chain length appears to have a profound influence on the time-dependent hemolytic profile of encapsulated AmB toward bovine erythrocytes. For all acyl conjugate micelle-AmB formulations, the onset of hemolysis is delayed relative to free AmB. Particularly in the case of stearate ester micelles, the incomplete and gradual build-up of hemolysis might reflect the sustained release of drug over a period of 24 h. Based on the corresponding absorption spectrum, we speculate that encapsulated AmB may interact strongly with stearate side chains, resulting in sustained release. Via chemical manipulation of the core-forming region, it may be possible to fine-tune the release of encapsulated AmB from PEO-b-p(N-HA)-acyl ester micelles.

The original article to which this Erratum was published in Journal of Pharmaceutical Sciences 92(7) 2003, 1343–1355