Hak-Kim Chan

Aggregation of rhDNase Occurred During the Compression of KBr Pellets Used for FTIR Spectroscopy

AbstractPurpose. To determine if a protein changes when it is compressed into a KBr pellet for FTIR spectroscopy measurement in the solid state, using recombinant human deoxyribonuclease I (rhDNase) as an example. Methods. Lyophilized rhDNase with KBr compressed at different pressures were analyzed by FTIR spectroscopy, size exclusion HPLC and enzymatic activity assay. Different protein/KBr weight ratios and residual water contents were studied for their possible effects on aggregation. Results. Depending on the pressure, a loss of enzymatic activity accompanied by an increase in soluble high molecular weight aggregates of the protein (up to ~15%) was demonstrated. Aggregation was reduced to less than 5% by a suitable dilution of the protein in KBr (1 in 1000). In contrast, water content variability (1–11 wt. %) did not affect aggregation. Conclusions. The findings emphasize the importance to examine for protein integrity when using the KBr method for FTIR sample preparation. Protein aggregation may be minimized by optimizing the sample preparation condition such as changing the protein/KBr weight ratio.

Application of the periodic bond chain (PBC) theory and attachment energy consideration to derive the crystal morphology of hexamethylmelamine.

The habit (external morphology) of a crystal is controlled by both the external (environmental) conditions of crystallization and the internal (structural) factors of the crystal. In order to separate the effects of the crystal structure and of the solvent and other external factors on the experimentally observed growth habit, the theoretical habit can be derived from the crystal structure using the periodic bond chain (PBC) theory and attachment energy considerations. According to the PBC theory the crystal habit is governed by a set of uninterrupted chains of strong bonds formed in the crystal lattice. In addition, the attachment energy (Eatt) is defined as the energy released per mole when a new layer is deposited on a crystal face. Since the habit of a crystal is determined by the relative growth rate (R) of the various faces, by taking R proportional to Eatt, the theoretical habit can thus be derived from Eatt. Using this approach, we obtained the theoretical crystal habit of an antitumor drug, hexamethylmelamine (HMM). The possible effect of solvents on the habit modification of HMM is discussed. This technique, based purely on the knowledge of the crystal structure, is directly applicable to other pharmaceuticals in deriving their theoretical crystal habit.