Study of the resolution and detection of SEC-LS and DLS

A comparison of the resolution and detection sensitivity of SEC-LS and DLS

Introduction

This case study demonstrates the resolving and detection capabilities of size exclusion chromatography light scattering (SEC-LS) and dynamic light scattering (DLS). To mimic the appearance of small oligomers, mixtures of two different proteins were used. A 65kDa human serum albumin (Albucult, Novozymes Biopharma UK) sample was used for the monomer protein. This protein shows very good stability in its formulation buffer and is therefore a very useful DLS reference sample [1, 2]. Alcohol dehydrogenase (ADH) was used as it is similar in molecular weight to a dimer of Abucult, as ADH has a molecular weight of about 150kDa.

Experimental Methods

Stock solutions of both proteins were prepared in PBS buffer and filtered through a 0.02µm Whatman Anotop10 filter to ensure that no aggregates or dust particles were present in either sample. The Albucult sample was spiked with increasing amounts of ADH. The level of ADH is given in % of ADH (in terms of moles of ADH compared to total moles of protein in the sample). The samples were first measured by DLS on a Zetasizer Nano ZS and then duplicate measurements of 100µl of each sample were injected for analysis by SEC-LS on a Viscotek TDAmax.