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How to obtain reliable information from light scattering

31 January 2013 No Comment

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My colleagues Kevin Mattison, Mark Pothecary and I gave a short course at Peptalk 2013 in Palm Springs, CA, last week. Attendees from Europe, Australia and the US proved to be a lively and participating audience in the Pueblo Meeting Room of the Renaissance Hotel.

Morning sky in Palm Springs, CA, with clouds scattering red light

Light Scattering in Palm Springs on the morning after the DLS short course

So what was the event about?

The first part introduced the fundamentals of dynamic light scattering (DLS, QELS, PCS), the technique to analyze fluctuations of the scattered light to find their corresponding hydrodynamic size.  While static light scattering (SLS) has an advantage for determination of absolute molecular weight and second virial coefficient (A2, B22), it can take considerable time and good preparation.  The ease and speed of DLS has in many publications become a method of choice for assessment of sample homogeneity. Details on data quality, intercept, fitting algorithms, ISO standards, mean size, mean polydispersity, intensity particles size distribution (PSD) and conversion to volume/mass size distributions, baseline resolution, and common errors rounded out the first hour. The take home message was: repeated runs are the best way to ensure reliable robust results.

Mark changed the focus to multi-detector size exclusion chromatography (SEC), with examples from the Viscotek LALS (=low angle light scattering) and RALS (=right angle light scattering) techniques, both contrasted to MALS (=multiple angle light scattering). He showed that specifically for proteins, the isotropic nature of their scattering profile eliminates the need for many angles. For example, a monoclonal antibody (mAb) has a hydrodynamic radius of about 5nm, and is thus significantly smaller than a tenth of the laser wavelength used in modern light scattering experiments. Even under the best of circumstances, determining an RG(= radius of gyration) from the angular dependence of the light scattering intensity becomes impossible to do for an antibody. However, one can use online intrinsic viscometry or dynamic light scattering to still obtain sizes down to 1nm. Using common protein examples (BSA, Trypsin Inhibitor, β-Lactoglobulin) Mark presented SEC-DLS (=combining single angle dynamic light scattering with chromatography). As with all good chromatography, stable baselines, robust columns, particle-free buffers, and optimum conditions are key requirements for obtaining reliable data.

Kevin led the final third of this workshop. Data interpretation tips are always bound to find an open ear, and there was no shortage of practical tips like a quick centrifuge spin, triplicate measurements, consideration for resolution expectations, etc.  Real-life examples followed on the popular empirical size/mass relationship, protein crystallization screening, and a thorough focus on formulation stability assessment and prediction. High concentration mAbs as part of late stage formulation optimizations were a popular topic discussed in this section. The session culminated in a group quiz: 12 real application examples challenged the participants to think about what they had learnt in the previous talks and discussions.

Thanks go to Kevin and Mark for their commitment as faculty, and thanks to the participants for the positive response on the feedback.