Purification of an Antigenic Vaccine Protein by Selective Displacement Chromatography

Abstract

A recent advance in the state of the art of displacement chromatography has been the development of selective displacement chromatography. In this process, the bioproduct of interest is selectively displaced while impurities with lower retention are eluted in the induced salt gradient and higher retained impurities are desorbed after the breakthrough of the displacer front. In this manuscript, selective displacement chromatography is employed to purify an antigenic vaccine protein (AVP) from an industrial process stream. Displacers were screened and an operating regime plot was employed to establish appropriate conditions for selective displacement. The selective displacement process was successful and resulted in AVP that was equivalent in purity to product obtained at commercial production scale after conventional step gradient chromatography. Methods used to characterize the purified protein include size-exclusion chromatography, SDS-PAGE, isoelectric focusing, N-terminal amino acid sequence analysis, and amino acid composition analysis. This is the first report of the purification of a commercially and pharmaceutically significant protein using selective displacement chromatography and thereby sets the stage for the implementation of selective displacement chromatography for the downstream processing of biologicals.

Purification of Recombinant Green Fluorescent Protein Using Chromatofocusing with a pH Gradient Composed of Multiple Stepwise Fronts

“…As a final consideration, the purity of 80% achieved in this study would appear to be somewhat less than the purities of 92.7% and 92.5% achieved in two other related studies where displacement chromatography using a traditional displacer component was employed to purify a target protein using cell lysate that had been subjected to an initial purification step as the starting material (36,37). This difference is likely due to corresponding differences for the various cases in the characteristics of the target protein and impurities and in the rationale for selecting the product fraction cut points.…”

Synthesis and Characterization of High-Affinity, Low Molecular Weight Displacers for Cation-Exchange Chromatography

“…6,9,[13][14][15][16][17] The SMA model involves three parameters: the characteristic charge (ν), which is the average number of sites that a molecule interacts with on a surface; the equilibrium constant (K); and the steric factor (σ), which is the average number of sites on the surface that are sterically shielded by the molecule. The equation for the SMA isotherm is:…”

“…[1][2][3][4][5] Recent reports have demonstrated the efficacy of displacement chromatography for the purification of proteins from industrial process streams. 6,7 Conventionally, large polyelectrolytes have been employed as displacers for ion-exchange systems. [8][9][10][11] Jen and Pinto 12 have used a 2 kD molecular weight poly-(vinyl sulfonic) acid as a displacer.…”

Unique selectivity windows using selective displacers/eluents and mobile phase modifiers on hydroxyapatite

“…Displacement chromatography has been successfully employed for the purification of proteins on multiple different stationary phases [2][3][4][5][6][7][8][9][10][11][12][13]. A wide variety of classes of displacers, such as polyelectrolytes [9], polysacchardies [14], low-molecular-mass dendrimers [11], amino acids [15], antibiotics [16] and aminoglycosidepolyamines [17] have been identified for protein displacement separations.…”