Automated Hydrophobic Interaction Chromatography Screening Combined with In Silico 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.g., the nature of the salt, stationary phase chemistry, temperature, mobile phase additive, and ionic strength). Herein, we introduce a new HIC method development framework composed of a fully automated multicolumn and multieluent platform coupled with in silico multifactorial simulation and integrated fraction collection for streamlined method screening, optimization, and analytical-scale purification of biopharmaceutical targets. The power and versatility of this workflow are showcased by a wide range of applications including trivial proteins, monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), oxidation variants, and denatured proteins. We also illustrate convenient and rapid HIC method development outcomes from the effective combination of this screening setup with computer-assisted simulations. HIC retention models were built using readily available LC simulator software outlining less than a 5% difference between experimental and simulated retention times with a correlation coefficient of >0.99 for pharmaceutically relevant multicomponent mixtures. In addition, we demonstrate how this approach paves the path for a straightforward identification of first-dimension HIC conditions that are combined with mass spectrometry (MS)-friendly reversed-phase liquid chromatography (RPLC) detection in the second dimension (heart-cutting two-dimensional (2D)-HIC-RPLC-diode array detector (DAD)-MS), enabling the analysis and purification of biopharmaceutical targets.

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. Interestingly, of the additional 390 proteins characterized by the S-BPS, 300 proteins were low abundance (less than 4000 molecules/cell). Remarkably, the identification of proteins at very low abundance (less than 2000 molecule/cell) was improved by 106%. This suggests that the S-BPS is particularly advantageous for detecting low abundance proteins. Gene Ontology (GO) analysis was conducted to find fractionation pattern of proteins in our two-phase system, and in nearly every gene ontology category, the S-BPS provided greater coverage than the control experiment, i.e., coverage for integral membrane proteins and mitochondrial ribosome proteins are improved by 18% and 58%, respectively. The improvements in proteins coverage for low abundance and membrane proteins can be attributed to the strong solubilizing power of the amphiphile-rich phase of this S-BPS and its capability for concomitant extraction, fractionation, and enrichment of the complex proteomics samples. Each phase has selectivity towards specific yeast protein groups, this selectivity is generally based on pI and hydrophobicity of proteins. Therefore, more hydrophobic proteins and acidic proteins exhibit greater affinities for the amphiphile-rich phase due to the hydrophobic effect and electrostatic interactions.

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-(N,N-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. The coverage of the lower-abundance proteins (abundance below 2000 molecules/cell) improved by more than 100% using DMMAPS and DMMAPS + QUATS systems as compared to the conventional methods using urea or detergent solutions for protein solubilization. Additionally, these coacervate systems show increased coverage of integral membrane proteins and proteins with α-helices by up to 24 and 555%, respectively.

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. The incorporation of this innovative approach in the proteomics workflow would allow the fractionation of proteins in separate aqueous and coacervate phases and would also eliminate the need for using surfactants. Subsequently, proteins can be identified by the bottom-up proteomics approach where samples were digested in solution after phase separation. Yeast cell wall proteins, anchored membrane proteins, and proteins related to some regulatory activities were mostly found in the aqueous-rich phase. On the other hand, most integral membrane proteins, proteins involved in metabolic processes, and proteins responsible for ions or drug binding were identified in the coacervate phase. The detergent-free, facile, and rapid process of natural lipid coacervation increased the number of identified proteins by 8% (vs no-phase separation experiment). The identification of all IMPs and organelle IMPs was improved by 13% and 29%, respectively. In addition, 25% more low-abundance proteins (less than 20 ppm) were identified.

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 ClSO3H (85-95% w/w) and HOAc or H2SO4. The cation exchange capacity is controlled by the sulfonation time and the sulfonation solution composition and can be as high as 300 pequiv/mm2. Following sulfonation, the capillaries were coated with 65-nm-diameter anion exchanger (AEX) latex nanoparticles that attach electrostatically. The typical anion exchange capacities were ∼20 pequiv/mm2. The chromatographic behavior of the AEX latex-coated COP capillaries are greatly dependent on the degree of sulfonation. When the base is heavily sulfonated, neutrals elute after the anions. The position of the water dip varies with the degree of sulfonation; the elution order is normal (water dip appear before anions) only with lightly sulfonated columns. On silica (-SiOH) or poly(methyl methacrylate) (-COOH) surfaces, AEX latex attachment is not stable over long periods in significant concentrations of strong base (e.g., ≥10 mM NaOH). Latex attachment on sulfonated COP surfaces are much stronger; several types show sufficient binding to be used over long periods at practical eluent concentrations, paving the way for suppressed hydroxide eluent ion chromatography (IC), which is discussed in a companion paper. Another interesting feature of COP capillaries lies in their flexibility. If softened at modestly elevated temperatures (e.g., boiling water), they can be coiled down to <1 mm coil radii, revealing, for the first time, the beneficial effects, albeit small, of centrifugal force on mass transfer in open tubular columns.