Monoclonal antibody interactions with micro- and nanoparticles: adsorption, aggregation, and accelerated stress studies.

Therapeutic proteins are exposed to various wetted surfaces that could shed subvisible particles. In this work we measured the adsorption of a monoclonal antibody (mAb) to various microparticles, characterized the adsorbed mAb secondary structure, and determined the reversibility of adsorption. We also developed and used a front-face fluorescence quenching method to determine that the mAb tertiary structure was near-native when adsorbed to glass, cellulose, and silica. Initial adsorption to each of the materials tested was rapid. During incubation studies, exposure to the air-water interface was a significant cause of aggregation but acted independently of the effects of microparticles. Incubations with glass, cellulose, stainless steel, or Fe(2)O(3) microparticles gave very different results. Cellulose preferentially adsorbed aggregates from solution. Glass and Fe(2)O(3) adsorbed the mAb but did not cause aggregation. Adsorption to stainless steel microparticles was irreversible, and caused appearance of soluble aggregates upon incubation. The secondary structure of mAb adsorbed to glass and cellulose was near-native. We suggest that the protocol described in this work could be a useful preformulation stress screening tool to determine the sensitivity of a therapeutic protein to exposure to common surfaces encountered during processing and storage.

The effect of the glass transition temperature on the toughness of photopolymerizable (meth)acrylate networks under physiological conditions.

The purpose of this study is to evaluate how the toughness of photopolymerizable (meth)acrylate networks is influenced by physiological conditions. By utilizing two ternary (meth)acrylate networks, MA-co-MMA-co-PEGDMA and 2HEMA-co-BMA-co-PEGDMA, relationships between glass transition temperature (T(g)), water content and state, and toughness were studied by varying the weight ratio of the linear monomers (MA to MMA or 2HEMA to BMA). Differential scanning calorimetry and thermogravimetric analysis were performed to evaluate the thermal behavior and water content as a function of either MA or 2HEMA concentration while tensile strain-to-failure tests were performed at 37°C to determine network toughness. Both networks exhibited a maximum in toughness in PBS in the composition corresponding to a T(g) close to the testing temperature. This toughness maximum was achieved by adjusting the glass transition temperature and/or hydrophilicity through changes in chemistry. These relationships may be utilized to design tough photopolymerizable networks for use in mechanically rigorous biomedical applications.

Free-radical-mediated protein inactivation and recovery during protein photoencapsulation.

Photoencapsulation of protein therapeutics is very attractive for preparing biomolecule-loaded hydrogels for a variety of biomedical applications. However, detrimental effects of highly active radical species generated during photoencapsulation must be carefully evaluated to maintain efficient hydrogel cross-linking while preserving the structure and bioactivity of encapsulated biomolecules. Here, we examine the free-radical-mediated inactivation and incomplete release of proteins from photocurable hydrogels utilizing lysozyme as a conservative model system. Various protein photoencapsulation conditions were tested to determine the factors affecting lysozyme structural integrity and bioactivity. It was found that a portion of the lysozyme becomes conjugated to polymer chains at high photoinitiator concentrations and long polymerization times. We also found that the more hydrophilic photoinitiator Irgacure-2959 (I-2959, 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone) causes more damage to lysozyme compared to the hydrophobic photoinitiator Irgacure-651 (I-651, 2,2-dimethoxy-2-phenylacetophenone), even though I-2959 has been previously shown to be more cytocompatible. Furthermore, while nonacrylated PEG provides only limited protection from the denaturing free radicals that are present during hydrogel curing, acrylated PEG macromers effectively preserve lysozyme structural integrity and bioactivity in the presence of either photoinitiator. Overall, these findings indicate how photopolymerization conditions (e.g., photoinitiator type and concentration, UV exposure time, etc.) must be optimized to obtain a functional hydrogel device that can preserve protein bioactivity and provide maximal protein release.