Self-interaction chromatography: A tool for the study of protein-protein interactions in bioprocessing environments

Abstract

We describe a new protein characterization technique called self‐interaction chromatography (SIC), which exploits the specificity of protein–protein interactions that is common to protein aggregates and enables the rapid screening of protein formulation additives as physical stabilizers against aggregation. This technique also enables the identification of specific interaction sites and the determination of their relative importance for self‐association. Mannitol, glycine, and dextran 40 were tested for their stabilizing effect toward the model protein lysozyme. Dextran 40 exhibited a poor stabilizing effect. While mannitol stabilized both the native and acid‐denatured forms of lysozyme, glycine stabilized the native form with respect to the denatured species. These results are in good agreement with findings in the formulation literature. The SIC shows tremendous potential as a rapid formulation development tool. We also screened two putative interaction sites for involvement in the self‐association of lysozyme and estimated the associated binding energies using a binding isotherm model that we developed. The sites screened consisted of residues 41–48 and 125–128 and were selected based on their apparent importance in forming crystal contacts in several different crystal forms of lysozyme. Of the two sites, only residues 125–128 were found to influence self‐association under the conditions we employed. Because the success of this technique depends on the exploitation of self‐interactions between native species, several important applications are also suggested such as separating native from misfolded or variant species and probing site utilization in aggregation versus crystallization phenomena. © 1996 John Wiley & Sons, Inc.

Applications of the second virial coefficient: protein crystallization and solubility

“…These constraints led the Wilson, Henry and DeLucas laboratories to explore self-interaction chromatography (SIC), which had previously been demonstrated as an alternative experimental approach to measure B values (Patro & Przybycien, 1996). The technique is similar to affinity chromatography, with differences in the interpretation of protein solution times and the preparation of column media.…”

Lysozyme‐lysozyme self‐interactions as assessed by the osmotic second virial coefficient: Impact for physical protein stabilization

“…In 1996, Patro and Przybycien [28] introduced the application of self-interaction chromatography as a protein characterization tool for the rapid screening of protein formulation additives against aggregation or for protein purification [35]. However, in the presented study they did not derive from their results the osmotic second virial coefficient.…”

“…Various chromatography based techniques have emerged in the last years in order to characterize protein-protein interactions [17,[26][27][28][29][30][31]. For examples, Stevens [32] proposed the use of small-zone size exclusion chromatography for investigating protein-protein association and Wen et al [33] showed the benefit of the additional combination of various detection methods for analyzing protein-protein interaction (see also [17]).…”

High-Throughput Self-Interaction Chromatography: Applications in Protein Formulation Prediction

“…Each measurement consists of flowing a mobile microgram injection of the protein across the immobilized protein particles using an HPLC. The retention time of the mobile protein is directly related to its interaction with immobilized column protein (19), thereby providing a direct measurement of how two proteins (bound and injected) interact with one another. Formulas relating the chromatographic retention time to B 22 values can be found in the paper of Tessier et al (20).…”