Mobile Affinity Sorbent Chromatography

Abstract

The objective in routine analyses is generally to determine a small number of analytes. With samples containing ~103 or more components there will be insufficient peak capacity to resolve analytes from nonanalytes. This issue was addressed herein through a new type of separation mechanism in which small groups of targeted analytes are bound with high affinity to a soluble analyte-sequestering transport phase (ASTP) composed of a ~25 nm Stokes radius hydrophilic polymer core (HPC). When introduced into a 30 nm pore diameter size-exclusion chromatography (SEC) column, ASTP/analyte complexes elute within minutes, together, unretained, and relatively pure in the first chromatographic peak. Nonanalytes, in contrast, enter pore matrices of the packing material, are retarded in elution velocity, and are eluted later, separated from analytes. Fabrication of ASTPs was achieved by covalently coupling an antibody or some other affinity selector to a high molecular weight HPC. Beyond sequestering analytes, the function of ASTPs is to act as a molecular weight shifting agent, conveying an effective molecular weight to analytes that is much larger than that of nonanalytes and causing them to elute in the SEC void volume. This mode of separation is referred to as mobile affinity sorbent chromatography (MASC). Subsequent to their purification, ASTP/analyte complexes were detected by fluorescence spectrometry.

Native detection of protein O-GlcNAcylation by gel electrophoresis

“…We next challenged SOPAGE with samples of near-stoichiometric O-GlcNAc modification. In such cases, if the affinity of the ligand (AI-OGA in this study) was too weak, or the ligand co-migrated with O-GlcNAc proteins due to unimmobilization, the theoretical plate number of SOPAGE gel will decline, 34 resulting in the failure to retard O-GlcNAc species. Besides, the applicability of SOPAGE to other types of proteins also needed to be evaluated.…”

Sample preparation and fractionation techniques for intact proteins for mass spectrometric analysis

“…Recently, Li et al. [194] published a new form of chromatography, referred to as mobile affinity sorbent chromatography (MASC), where the separation occurs in a three‐phase system. In MASC, the analytes of interest are immune‐specifically bound to the mobile sorbent transport phase and so these complexes are precluded from binding to the stationary phase and are coeluted from an SEC column in a single nonretained peak.…”

Mobile Affinity Selection Chromatography Analysis of Therapeutic Monoclonal Antibodies

“…To meet these requirements, we describe here a rapid mAb titer and aggregate analyses through an adaptation of mobile affinity selection chromatography (MASC). , MASC is a three-phase chromatographic process that differs from conventional two-phase chromatography in having a soluble third phase consisting of an affinity selector. The analyte of interest partitions with the mobile affinity selection phase (P as * ) and the stationary phase through different mechanisms. , In effect, two modes of chromatography, molecular sieving and affinity selection, are achieved simultaneously in MASC.…”