Use of protein charge ladders to study electrostatic interactions during protein ultrafiltration.

摘要

The overall objective of this thesis was to develop an improved fundamental understanding of the effects of electrostatic interactions on protein transport through charged ultrafiltration membranes using a novel experimental approach employing protein charge ladders. Charge ladders consist of a series of protein derivatives with essentially identical size and conformation that differ by an integral number of charge groups. The concentrations of the different species within the charge ladder were evaluated by capillary electrophoresis, allowing the transport properties of these proteins to be analyzed in a single experimental run.; Experimental data were obtained for the transport of myoglobin charge ladders through polyethersulfone and composite regenerated cellulose ultrafiltration membranes over a range of solution pH and ionic strength and in the presence of different buffer additives. These results provide the first published evidence for the presence of an attractive interaction between the protein and membrane in low ionic strength solutions. This unusual behavior was attributed to a shift in pH caused by preferential partitioning of hydrogen ions into the negatively charged membrane. A theoretical model was developed to evaluate the pH in the pores from measured values of the streaming potential. This required a combination of solutions to the Poisson-Boltzmann equation specifically formulated in the limits of high and low potentials. Model calculations incorporating the effect of this pH shift are in good agreement with experimental results.; The protein charge ladders also provide a method to study the use of membrane ultrafiltration for the separation of protein variants, which often arise during production of recombinant proteins. Greater than 7-fold selectivity was achieved for the separation of myoglobin from a variant differing by only one out of 153 amino acids by using a very low conductivity solution to exploit electrostatic interactions. A two-stage diafiltration process was designed which achieved over 9-fold purification and 90% yield for each protein variant, providing the first demonstration that ultrafiltration membranes can effect such high resolution separations. Theoretical simulations demonstrated that purification factors greater than 100-fold with 90% yield could potentially be achieved in a single-stage process using membranes with specially-designed pore size and surface charge density.