A novel microchip-based imaged CIEF-MS system for comprehensive characterization and identification of biopharmaceutical charge variants.

A microfluidic system has been designed that integrates both imaged capillary isoelectric focusing (iCIEF) separations and downstream MS detection into a single assay. Along with the construction of novel instrumentation and an innovative microfluidic chip, conversion to MS-compatible separation reagents has also been established. Incorporation of 280 nm absorbance iCIEF-MS analysis not only permits photometric quantitation of separated charge isoforms but also facilitates the direct monitoring of analyte focusing and mobilization in real-time. The outcome of this effort is a device with the unique ability to allow for both the characterization and identification of protein charge and mass isoforms in under 15 min. Acquisition, quantitation, and identification of highly resolved intact mAb charge isoforms along with their critical N-linked glycan pairs clearly demonstrate analytical utility of our innovative system. In total, 33 separate molecular features were characterized by the iCIEF-MS system representing a dramatic increase in the ability to monitor multiple intact mAb critical quality attributes in a single comprehensive assay. Unlike previously reported CIEF-MS results, relatively high ampholyte concentrations, of up to 4% v/v, were employed without impacting MS sensitivity, observed to be on the order of 1% composition.

Responses to familiar and unfamiliar objects by belugas (Delphinapterus leucas), bottlenose dolphins (Tursiops truncatus), and Pacific white-sided dolphins (Lagenorhynchus obliquidens).

Previous research with bottlenose dolphins (Tursiops truncatus) demonstrated their ability to discriminate between familiar and unfamiliar stimuli. Dolphins gazed longer at unfamiliar stimuli. The current study attempted to extend this original research by examining the responses of three species of cetaceans to objects that differed in familiarity. Eleven belugas from two facilities, five bottlenose dolphins and five Pacific white-sided dolphins housed at one facility were presented different objects in a free-swim scenario. The results indicated that the animals gazed the longest at unfamiliar objects, but these gaze durations did not significantly differ from gaze durations when viewing familiar objects. Rather, the animals gazed longer at unfamiliar objects when compared to the apparatus alone. Species differences emerged with longer gaze durations exhibited by belugas and bottlenose dolphins and significantly shorter gaze durations for Pacific white-sided dolphins. It is likely that the animals categorized objects into familiar and unfamiliar categories, but the free-swim paradigm in naturalistic social groupings did not elicit clear responses. Rather this procedure emphasized the importance of attention and individual preferences when investigating familiar and unfamiliar objects, which has implications for cognitive research and enrichment use.

Reductase-mediated metabolism of motexafin gadolinium (Xcytrin) in rat and human liver subcellular fractions and purified enzyme preparations.

The biotransformation of motexafin gadolinium (MGd, Xcytrin) was investigated in subcellular rat and human liver fractions. Microsomal MGd metabolism was dependent on NADPH in both species. Cytosolic metabolism in rat and human livers was dependent on NADPH or NADH. Under anaerobic conditions, MGd metabolism increased in liver microsomes and purified enzyme preparations. Cytochrome P450 (CYP450) inhibitors ketoconazole, proadifen, and carbon monoxide increased NADPH-dependent MGd metabolism in microsomes. Treatment of rats with beta-naphthoflavone increased cytosolic metabolism of MGd twofold, but had no effect on microsomal metabolism. Conversely, in liver preparations from phenobarbital treated rats microsomal metabolism of MGd was enhanced twofold, but not in cytosolic preparations. Purified CYP450 reductase from phenobarbital-treated rabbit or untreated human livers metabolized MGd suggesting involvement of CYP450 reductase. Dicumarol, a potent DT-diaphorase inhibitor, inhibited MGd metabolism in both rat and human liver cytosol. These data suggest MGd metabolism in rat liver involves CYP450 reductase and/or DT-diaphorase. In human liver preparations only CYP450 reductase is directly involved in MGd metabolism. A metabolite identified in microsomes and cytosol is a metal-free, reduced form of MGd, indicating that both enzymes generate metabolite 1, which appears to be PCI-0108, a synthetic precursor to MGd.