Automated Hydrophobic Interaction Chromatography Screening Combined with Optimization as a Framework for Nondenaturing Analysis and Purification of Biopharmaceuticals.

The mounting complexity of new modalities in the biopharmaceutical industry entails a commensurate level of analytical innovations to enable the rapid discovery and development of novel therapeutics and vaccines. Hydrophobic interaction chromatography (HIC) has become one of the widely preferred separation techniques for the analysis and purification of biopharmaceuticals under nondenaturing conditions. Inarguably, HIC method development remains very challenging and labor-intensive owing to the numerous factors that are typically optimized by a "hit-or-miss" strategy (e.

Improving identification of low abundance and hydrophobic proteins using fluoroalcohol mediated supramolecular biphasic systems with quaternary ammonium salts.

In this study, a newly discovered Supramolecular Biphasic System (S-BPS) was used in bottom-up proteomics of the Saccharomyces cerevisiae strain of yeast. We took advantage of S-BPS in bottom-up proteomics of this strain of yeast as the protein sample, while the results were compared to routinely used solubilizing reagents, such as urea, and sodium dodecyl sulfate (SDS). With the S-BPS, we identified 3043 proteins as compared to 2653 proteins that were identified in the control system.

Improved Protein Coverage in Bottom-Up Proteomes Analysis Using Fluoroalcohol-Mediated Supramolecular Biphasic Systems With Mixed Amphiphiles for Sample Extraction, Fractionation, and Enrichment.

A new class of supramolecular biphasic systems containing fluoroalcohol-induced coacervates (FAiC) provides concomitant fractionation of complex protein mixtures, high solubilizing power for extraction of various types of proteins, especially those with high hydrophobicity (such as membrane proteins), and enrichment of low-abundance proteins. Subsequently, the use of FAiC biphasic systems (BPS) in the bottom-up proteomics workflow resulted in significantly higher coverage for the whole proteome, various subproteomes, especially those embedded or associated with membranes, post-translationally modified proteins, and low-abundance proteins (LAPs) as compared to the conventional methodologies. In this work, we used a new type of FAiC-BPS composed of mixed amphiphiles, a zwitterionic surfactant 3-(,-dimethylmyristyl ammonia) propane sulfonate (DMMAPS), a quaternary ammonium salt (QUATS), and hexafluoroisopropanol (HFIP) as the coacervator for extraction, fractionation, and enrichment of yeast proteome in bottom-up proteomics.

Coacervation of Lipid Bilayer in Natural Cell Membranes for Extraction, Fractionation, and Enrichment of Proteins in Proteomics Studies.

This is the first report where hexafluoroisopropanol (HFIP) was used to induce the coacervation of lipid components in natural cell membranes that would concomitantly result in solubilization, extraction, and enrichment of hydrophobic proteins (e.g., integral membrane proteins, IMP) into the coacervate phase, and extraction of hydrophilic proteins in a separate aqueous phase.

Functionalized Cycloolefin Polymer Capillaries for Open Tubular Ion Chromatography.

We describe novel cycloolefin polymer (COP)-based open tubular capillary ion exchange columns. COP capillaries (inner diameter of 19-28 μm) were successfully sulfonated at room temperature using a cocktail of ClSOH (85-95% w/w) and HOAc or HSO. The cation exchange capacity is controlled by the sulfonation time and the sulfonation solution composition and can be as high as 300 pequiv/mm.