By Lavelay Kizekai, Stephen J. Shiner, and Matthew A. Lauber, Waters Corporation
One of the major limitations in protein size-exclusion chromatography (SEC) is the presence of undesired secondary interactions stemming from a lack of packing material and column hardware inertness. Due to their highly active surfaces, proteins (including mAbs, ADC’s, and other biotherapeutic molecules), have a propensity for interaction with metal oxide surfaces present in column hardware, both hydrophobic and electrostatically active sites present on silica and hybrid silica particles. These undesired secondary interactions create significant challenges for resolving protein aggregates, monomers, and fragments from one another, as well as for the accurate quantitation of these species. While there are several commercially available SEC columns that attempt to address these problems, either through column materials or protein pre-conditioning, most still require an appreciable amount of method development to achieve optimal results. Use of high ionic strength mobile phases and the addition of organic solvents are known to help suppress secondary interactions but are in many ways limiting and can make a chromatographer’s work more challenging. Improved column inertness would reduce the need for such measures and provide better method flexibility and robustness. To that end, Waters has developed a novel ethylene bridged-hybrid particle with a high coverage hydroxy-terminated polyethylene oxide (BEH-PEO) surface and coupled it with a first of its kind, hydrophilic high-performance surfaces (HPS) column hardware. A holistic solution to the problem of undesired secondary interactions in SEC is thereby achieved.