Microflow size exclusion chromatography to preserve micromolar affinity complexes and achieve subunit separations for...

For high throughput native mass spectrometry (MS) protein characterization, it is advantageous to desalt and separate proteins by size exclusion chromatography (SEC). Sensitivity, resolution, and speed in these methods remain limited by standard SEC columns. Moreover, the efficient packing of small bore columns is notoriously difficult. SEC sensitivity is inherently limited because solutes are not focused into concentrated bands and low affinity native complexes may dissociate on column. Recent work evaluated the suitability of crosslinked gel media in small bore formats for online desalting. Here, small bore format online SEC for native MS studies is again investigated but with alternative materials. We systematically studied the utility of diol and hydroxy terminated polyethylene oxide (PEO) bonded 1.7 µm organosilica particles as packed into 1 mm ID stainless steel (SS) hardware and hardware treated with hydrophilic hybrid surface technology (h-HST). For the equivalent diol-bonded particle and hardware, UV limits of detection (LODs) were reduced 32 to 89% with a microflow separation (15 µL/min) on a 1 × 50 mm column as compared to a 4.6 × 150 mm high-flow separation (300 µL/min) at the same linear velocity. Run times were also shortened by 45%. A switch from SS to h-HST hardware led to a significant reduction in secondary interactions and a corresponding improvement in detection limits for trastuzumab, myoglobin, IgG and albumin for both UV and MS. Coupling of the small bore columns to multichannel microflow emitters resulted in 10 to 100-fold gains in MS sensitivity, depending on the analyte. MS LOD values were significantly reduced into the low attomole ranges. Columns were then evaluated for their effects on the preservation of complexes, including concanavalin A, in its apo and ligand-bound states, and three therapeutically relevant noncovalent systems previously undetected on large column formats. The results suggest that the detection of large complexes by SEC is not just a function of sensitivity but is directly affected by chemical secondary interactions. The ability to detect 0.1 to 1 MDa complexes, with between 1 and 40 micromolar dissociation constants, represents a critical advancement for high-throughput native MS workflows as applied to the analysis of therapeutics.