Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Abstract

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique. Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample. Video Link The video component of this article can be found at https://www.jove.com/video/59408/ 12 , resulting in flawed SEC-MALS analysis. Combining a high-specificity separation technique such as IEX with MALS can overcome the limitation

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“…Absorbance at 280 nm estimated a full capsid peak area of 85%. Light scattering estimated that the full capsid peak represented 78%, but that should be understood to be an overestimate due to the influence of higher mass in full capsids [ 1 , 54 , 55 ]. Intrinsic fluorescence gave the lowest estimate of full capsid peak area at 76%.…”

“…Measurement of light scattering intensity at a single angle is sufficient for capsid detection in crude and purified samples. Measurement of light scatter at multiple angles is necessary for accurate determination of particle size and mass in highly purified samples [ 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 ].…”

“…Larger particles produce more scatter than an equal mass of smaller particles. More recent publications show that light scattering detection combined with ion exchange chromatography produces useful information beyond the ability of SEC, despite the chemical environment changing across gradient elution profiles [ 52 , 54 , 55 ].…”

“…The size similarity of empty and full capsids makes it possible to estimate the relative proportions of dominantly-empty peaks and dominantly-full capsid peaks. However, full capsid peak area is inflated by the greater mass per full capsid [ 1 , 54 , 55 ]. Interference by proteins and other small host contaminants is negligible but chromatin contamination interferes with quantitative estimation in unpurified samples.…”

Fighting SARS-CoV-2 with green seaweed Ulva sp. extract: extraction protocol predetermines crude ulvan extract anti-SARS-CoV-2 inhibition properties in in vitro Vero-E6 cells assay

“…SEC analyses were performed at room temperature on a system (AKTA Pure, Cytiva), equipped with Superose 6 Increase 10/300 GL column (23 ml, Cytiva) and with an array of detectors, comprised of UV-vis detector (UV-900, Cytiva), a multi-angle light scattering (MALS; miniDAWN TREOS, Wyatt Technology) detector, a dynamic light scattering module (WyattQELS) and with a refractive index detector (Optilab T-rEX, Wyatt Technology). The elution was monitored at 280, 260, and 220 nm (UV–vis detector) and three angles (43.6, 90, and 136.4°) with a 658.9 nm laser beam (MALS detector) ( Amartely et al, 2019 ). The refractive index of the eluting solvent was determined to be 1.331, the viscosity was 0.8945 cP (typical for the PBS buffer) and the refractive index increment value (d n /d c ) for ulvan was defined to be 0.127 mL g −1 ( Paradossi, Cavalieri & Chiessi, 2002 ).…”