The band dispersion in channels with an orderly pillar structure with a pressure-driven liquid flow was determined. Several channels with different geometries were etched in a silicon wafer and enclosed by a glass wafer. The microchannels obtained had the same depth, pillar disposition, and overall porosity, but different pillar diameters and channel widths. The broadening of narrow bands of a fluorescent sample solution flowing through the channels was measured using a fluorescence microscope. It was shown that the peak dispersion occurring in the channels can be much lower than in conventional packed columns, as a consequence of the higher degree of order of the solid structure. Reduced plate heights of approximately 0.2 could be obtained for (nonretained) bands. No correlation was found between the aspect ratio (the ratio of the channel width and the pillar diameter) and band dispersion. The geometrical construction of the sidewall region was shown to play a critical role for channel performance. A good agreement was found with predictions for the optimal sidewall geometry obtained previously with simulation studies.
Charge variant analysis is a widely used analytical tool in characterization of monoclonal antibodies (mAbs). It depicts the heterogeneity of charge variant forms, some of which may differ by only minor modifications of a single amino acid. The analysis ensures product consistency with no unwanted changes to the protein. With increasing numbers of new mAb drug products emerging in the market, the need for a robust charge variant analysis has intensified. The charge variant profiles often display partially resolved peaks on shoulders of larger peaks. This puts considerably more pressure on the robustness of the method to maintain the suboptimum selectivity. New products and techniques have emerged to address these requirements, in addition to the pre-existing older methods that may not have been optimized correctly in the past. This has led to some confusion as to the best approach and strategies in optimization of charge variant analysis. We show studies from several different approaches using on-line pH monitoring to check the performance characteristics of the methods. This has led to new insights on the interactions between the protein, column, and buffer constituents. We dispel some inaccurate assumptions about the different ion-exchange elution mechanisms and suggest ways to develop high-throughput methods that remain robust and of high resolution. Streamlined automatable method development tools are presented that will result in more efficient method optimization. The mechanisms behind poor chromatography design have provided an alternative explanation behind some methods failing when in the QC laboratories.
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