Micrometer-scale particle sizing by laser diffraction: critical impact of the imaginary component of refractive index.

PURPOSE: This study evaluated the effect of the imaginary component of the refractive index on laser diffraction particle size data for pharmaceutical samples. METHODS: Excipient particles 1-5 microm in diameter (irregular morphology) were measured by laser diffraction. Optical parameters were obtained and verified based on comparison of calculated vs. actual particle volume fraction. RESULTS: Inappropriate imaginary components of the refractive index can lead to inaccurate results, including false peaks in the size distribution. For laser diffraction measurements, obtaining appropriate or "effective" imaginary components of the refractive index was not always straightforward. When the recommended criteria such as the concentration match and the fit of the scattering data gave similar results for very different calculated size distributions, a supplemental technique, microscopy with image analysis, was used to decide between the alternatives. Use of effective optical parameters produced a good match between laser diffraction data and microscopy/image analysis data. CONCLUSIONS: The imaginary component of the refractive index can have a major impact on particle size results calculated from laser diffraction data. When performed properly, laser diffraction and microscopy with image analysis can yield comparable results.

Aggregation of granulocyte colony stimulating factor under physiological conditions: characterization and thermodynamic inhibition.

We have investigated the aggregation of recombinant human granulocyte colony stimulating factor (rhGCSF), a protein that rapidly aggregates and precipitates at pH 6.9 and 37 degrees C. We observed that native monomeric rhGCSF reversibly forms a dimer under physiological conditions and that this dimeric species does not participate in the irreversible aggregation process. Sucrose, a thermodynamic stabilizer, inhibits the aggregation of rhGCSF. We postulate that sucrose acts by reducing the concentration of structurally expanded species, consistent with the hypothesis that preferential exclusion favors most compact species in the native state ensemble. Thermodynamic stability data from unfolding curves and hydrogen-deuterium exchange experimental results support the above hypothesis. Thus, the strategy of stabilizing the native state of the protein under physiological conditions using thermodynamic stabilizers, especially ligands binding with high affinity to the native state, is expected to protect against protein aggregation occurring under such nonperturbing solution conditions.

A Redox-Sensitive Resin Linker for the Solid Phase Synthesis of C-Terminal Modified Peptides.

With the rapid development of combinatorial chemistry using solid phase synthesis, there is a great deal of interest in developing new solid phase linkers, which are stable during the solid phase synthesis process and yet readily cleavable under mild conditions. By taking advantage of a "trimethyl lock"-facilitated lactonization reaction, we have developed a redox-sensitive resin linker for the synthesis of C-terminal-modified peptides. The cleavage only requires mild reducing agents such as sodium hydrosulfite, which is not expected to cause any problem with the commonly seen organic functional groups. Using this new linker, three short peptides were synthesized with high isolated yields (70-90%). Such a linker could potentially be used for the synthesis of modified peptide libraries, which allows for the ready cleavage of the linker under mild conditions.