Multidimensional Chromatographic Coupling Strategies Implementing Capillary-Channeled Polymer Fiber Stationary Phases

Liquid chromatography (LC) is a widely employed technique in analytical separations due to the ease of implementing rapid analysis methods to isolate targeted analytes. While LC is commonplace, challenges often arise with complex macromolecules as it often effects isolation via only one attribute of the targeted analyte (i.e., physical, chemical). To cater to increasingly complex samples requiring multiple chromatographic workflows, two-dimensional liquid chromatography (2DLC) has emerged. Various applications, such as drug discovery, pharmaceutical manufacturing, and specialty chemical manufacturing, require highly pure final products for release to the market. Consequently, multiple chromatographic steps are necessary for isolation and purification. Within pharmaceuticals, therapeutic proteins and vectors, such as immunoglobulin G (IgG), and extracellular vesicles (EVs), specifically exosomes as vectors, are produced in highly complex matrices, meaning extensive purification is required before they can be used for patient treatment. IgG is often purified using Protein A (ProA) chromatography, and weak cation exchange chromatography (WCX) is used for the charge variant characterization, which is used to reveal information about the efficacy of the therapeutic end product. Another sought-after therapeutic for vectors is exosomes. Exosomes, or small EVs (sEVs), are excreted from cells, made up of a phospholipid bilayer containing biomarkers, and are defined by their 30 – 150 nm diameter. The role sEVs play in intracellular communication, along with their capacity for the targeted transport of their cargo, drives their interest as therapeutic vectors. The presented work will look at the isolation and characterization of IgG in terms of purification strategies and charge variants, along with the isolation and characterization of sEVs and IgG in a single platform. While therapeutics are commonly sought after, another field requiring highly pure products includes polymers, where they are used for everyday plastics to bioprosthetics. The physiochemical properties of polymers are tied to their function; therefore, their characterization is of utmost importance. Approaches detailing the molecular weight and chemistry characterizations using size exclusion chromatography (SEC) and reversed-phase (RP) chromatography will be presented. Additionally, the chromatographic approaches presented will incorporate capillary-channeled polymer (C-CP) fiber stationary phases for cost-effective, fast, and efficient isolations at high linear flow velocities and low backpressures (~ 70 mm sec-1 at \u3c1000 psi)