Metal affinity protein precipitation: Effects of mixing, protein concentration, and modifiers on protein fractionation

Abstract

Previous work by us and others has shown that mixing impacts apparent protein solubility in single protein precipitations. In this work, we probe the effects of contacting conditions on fractional precipitation behavior at the bench scale. We have chosen metal affinity precipitation as our model system; the kinetics of this mode of precipitation are very rapid and largely irreversible and, consequently, mixing conditions govern the extent of fractionation and purity of the product in such a process. Our experimental strategy involved a three-pronged approach to control the effects contacting conditions on precipitate yield, purity, and particle size distribution. First, we studied the impact of process variables that control precipitant concentrations in the reactor including impeller speed and precipitant addition rate. Second, we controlled the rate of precipitation by changing the initial protein concentration to alter the protein-protein collision rate. Third, we examined the role of the molecular-level kinetics of affinity precipitation by using modifiers that compete with surface moieties to bind the metal ion, thereby reducing its availability. Our model process and protein system consisted of zinc precipitations of mixtures of bovine serum albumin and bovine gamma-globulins, carried out at a nominal 1-L scale; glycine was examined as a modifier. Faster impeller speeds and lower precipitant addition rates increased the desired protein yields, decreased purities, and reduced average precipitate particle size. Higher initial protein concentrations were found to produce precipitates with higher yields, lower purities and diminished particle size. Experiments with glycine indicated that modifiers in the precipitant solution serve to increase product purity, decrease yield, and increase the average particle size in bench-scale precipitations.

High‐performance liquid chromatographic determination of Levetiracetam in human plasma: comparison of different sample clean‐up procedures

“…Protein precipitation using zinc sulfate-1 N NaOH, however, was very effective, as the metal-ion precipitation yielded supernatants free of any particulate matter, and the precipitation with zinc sulphate gave satisfactory absolute recovery values even though such a low volume of precipitant was used. These results show the high efficacy of zinc sulphate as a precipitant agent and convalidate the data reported in the literature (Iyer and Przybycien, 1995;Polson et al, 2003). The chromatogram of a blank plasma sample spiked with 10 µg/mL of IS, and 10 µg/mL of Levetiracetam (subjected to protein precipitation with zinc sulphate) is shown in Fig.…”

A Model for Metal Affinity Protein Precipitation

“…All the binding sites that are not consumed by crossIn order to simplify the application of Eq. [5] for K(i, j) to the moment balance in Eq. [7], we can rearrange K(i, linking are available to bind and release metal ions and protons.…”

“…The first term in the braces on binding equilibria expressions that pertain are (21,22) the right hand side of Eq. [5] represents the number of ways in which the metal-bound sites of an i-mer can combine with the unbound sites of a j-mer, and the second term represents n…”

“…Therefore, groups on the surfaces of an i-mer and a j-mer, the collison 2(i 0 1) sites are removed from the solution during the kernel K(i, j) can be derived combinatorially as formation of an i-mer. If there are u sterically accessible histidines on a protein surface that are assumed to be identi- [5] cal in their ability to bind metal ions and protons, then the ion-binding equilibrium for such an i-mer in solution will where k is a function of the diffusion coefficients of an ibe with respect to the (iu 0 2(i 0 1)) sites that remain mer and a j-mer and any orientation effects accompanying exposed to the solution. In this scenario, the competitive such a collision (25-27).…”

“…While this has been shown theory (8,(10)(11)(12)(13)(14)(15)(16)(17). Using a classical thermodynamic apto be a promising alternative to organic solvent induced proach, Stockmayer (8) computed the most probable molecprecipitation of plasma proteins (5), the lack of fundamental ular size distributions for monomers with different functionwork in understanding metal ion induced precipitation hamalities by maximizing the entropy of the size distribution of pers efforts to predict solubilities, purities, and particle sizes the polymers formed. Later, Spouge (14) generalized the in such reactive aggregation processes.…”