PhD student Polly came into the lab on Monday morning to find that her purified protein had started to aggregate after a weekend in the fridge. The profile she got from her Dynamic Light Scattering (DLS) measurement clearly showed two peaks, with the second in the micron range. Her supervisor suggested she tried Nanoparticle Tracking Analysis (NTA) to get more information on the different sized aggregate species in her sample. This showed that there were aggregates of many different sizes present. The question was, what should she do next?

combine DLS and NTA for proteins

Monday 3pm

Polly: I’m going to purify another protein batch and then store it in buffer of different recipes. I’ll keep checking that I have my nice DLS monomer peak.

Is there more I can do to understand what’s already happened to my protein?

resonant mass measurement & size exclusion chromatography

Prof: So we have a number of other techniques available to us. As well as using DLS and NTA to obtain quick and easy profiles, we can measure relative amounts of monomer, dimer, trimer etc. by SEC (size exclusion chromatography), or use the Archimedes, to investigate larger aggregates by Resonant Mass Measurement (RMM).

This diagram shows how the range of techniques come together to help you see the whole picture:

Protein aggregate characterization toolkit

I suggest that when analyzing your protein, you use a flow chart like this one to decide which technique will give you the information you need (click on the image below to view the full size flow chart):

protein aggregate analysis

Polly: What else I could do to characterize the effectiveness of the new storage conditions?

Prof: As well as obtaining size data, we can also measure zeta potential of the protein in each buffer to see if one is potentially more stabilizing than another. Zeta potential can be measured on most Zetasizer instruments and on the NanoSight NS500.  Another option is to carry out a thermal stress study, which you can do on these instruments as well. This could predict long term storage stability.

Make sure you read this material, to get a good understanding of each approach to analyzing protein aggregation:

Direct visualization, sizing and counting of protein aggregates using NTA

Protein storage and stability analysis using the Zetasizer Nano ZSP

Differentiation and characterization of subvisible particulates in therapeutic protein products

Measuring protein aggregation with the Viscotek SEC-MALS 20

Formulation stability evaluation using light scattering techniques

Using Light Scattering to Study the Thermal Stability of Recombumin

diary of a protein scientist


Recap Part 1

Recap Part 2

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