Protein charge ladders are an effective tool for measuring protein charge and studying electrostatic interactions. However, previous analyses have neglected the effects of charge regulation, the alteration in the extent of amino acid ionization associated with differences between the pH at the protein surface and in the bulk solution. Experimental data were obtained with charge ladders constructed from bovine carbonic anhydrase. The protein charge for each element in the ladder was calculated from the protein electrophoretic mobility as measured by capillary electrophoresis using the hindrance factor for a hard sphere with equivalent hydrodynamic radius. The protein charge was also evaluated theoretically from the amino acid sequence by assuming a Boltzmann distribution in the hydrogen ion concentration. The calculations were in excellent agreement with the data, demonstrating the importance of charge regulation on the net protein charge. These results have important implications for the use of charge ladders to evaluate effective protein charge in solution.

A new technique is described for the rapid and accurate measurement of electrophoretic mobilities of proteins in different solution environments using capillary electrophoresis. Data were obtained at different pH using surface-modified capillaries to reduce nonspecific protein adsorption and using hydrodynamic mobilization to improve reproducibility and overall accuracy. The net protein charge and extent of anion binding were evaluated from the mobility data obtained in different pH and ionic environments for bovine serum albumin. The results were in good agreement with titration data obtained using ion-selective electrodes and mobility data obtained using free solution electrophoresis. The method requires extremely small amounts of protein (picogram quantities and nanoliter volumes) and is easily automated, making it very suitable for protein characterization and for initial screening of possible separation techniques.

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Process modeling involves the use of a set of mathematical equations to represent key physical phenomena involved in the process. An appropriately validated model can be used to predict process behavior with limited experimental data, identify critical ranges for process variables, and guide further process development. Although process modeling is extensively used in the chemical process industries, it has not been widely used in purification unit operations in biotechnology. Recent FDA guidelines encourage the use of process modeling during process development, along with multivariate statistical methods, detailed risk assessment, and other quantifiers of uncertainty. This paper will review recent advances in the modeling of key downstream unit operations: chromatography, filtration, and centrifugation. The focus will be on the application of modeling for industrial applications. Relevant papers presented at a session on this topic at the recent American Chemical Society National Meeting in San Francisco will also be reviewed.