Protein stability: The key to casting your next stable protein superstar

Thermal stability and stability over time are important factors to consider when running a protein stability assay. Uncle, an innovative biologics stability screening platform, combines full-spectrum fluorescence, static light scattering (SLS), and dynamic light scattering (DLS) on just nine microlitres of sample. This automated and streamlined method allows efficient and effortless observation of protein candidate unfolding and aggregation. Once the most stable molecule has been identified, you can focus on enhancing its stability by modifying formulation conditions and excipients.

In this SelectScience^® webinar, now available on demand, Dr. Andre Mueller, product manager for Uncle at Unchained Labs, demonstrates how an antibody and a model antibody-drug conjugate (ADC) play out the details of protein stability over thermal ramps and isothermally. Plus, discover how these perform under Uncle’s spotlight in different formulation conditions.

Register now to watch the webinar on demand, and read on to explore highlights from the live Q&A session:

How is the sample introduced into Uncle?

AM: The sample introduction into Uncle uses a set of capillaries we refer to as Unis. A Uni has 16 nine microlitre capillaries which you load with your sample using a single-channel pipettor or a multi-channel pipettor. This Uni is then transferred into the Uni frame that has silicone seals at the ends of the capillary, and then that Uni seal and the Uni are put into Uncle.

The silicone seal ensures that evaporation is not an issue, and you can characterize your sample over longer periods of time basically without having to worry about loss and change of concentration.

Does Uncle use one optical system for fluorescence in SLS and DLS?

AM: Uncle uses two optical systems. One optical system is used for fluorescence in SLS using a UV laser of 266 nanometers and a blue laser of 473. Then a spectrometer quantifies the wavelengths for fluorescence and also the light at the excitation wavelengths.
DLS is measured in an independent system, and that uses a 660-nanometer red laser and independent detector.

How long does a thermal ramp experiment take?

AM: Thermal ramp experiments depend on the ramping rate that you do with the number of samples you will have. I think a good rule of thumb is about two hours for a thermal ramping experiment.

Does Uncle measure DLS during the thermal ramp?

AM: Uncle measures the DLS readout before starting a thermal ramp and after ending a thermal ramp. During the thermal ramp, we measure the SLS as a readout of aggregation, and we do not measure the DLS during the ramp. It is an orthogonal technique to give you a second readout of aggregation, but we find that SLS and the determination of that and using it for aggregation quantification is the more sensitive readout.

How does a stability test of a viral vector sample differ from that of an antibody?

AM: The viral vector stability measurement can be done the same way as any other protein can be done. We can assess the stability of the protein capsid by doing a thermal ramp and looking at the intrinsic fluorescence and the changes of the intrinsic fluorescence.

Another effect, or another phenomenon that we see for AAVs, for example, is the ejection of the genome that they're carrying at lower temperatures. Uncle allows you to measure that by adding an external dye. We use SYBR Gold, and that way, we can assess at what temperature the DNA is ejected. For an AAV, as it’s a more complex system, we have several parameters of stability. One is the genome ejection, which we can measure in Uncle. The other one is the capsid stability, which we can do just like any other protein in Uncle.

How does Uncle differ from a standalone differential scanning fluorimetry (DSF) instrument like the Prometheus?

AM: The big difference between the Prometheus and Uncle is that Uncle uses full-spectrum fluorescence. We're not measuring only two wavelengths and determining a ratio of those two wavelengths to assess the change of fluorescence in the temperature gradient, but we can monitor the entire spectrum, giving the experimenter more flexibility.

Uncle, because of the two excitation layers of wavelengths and full-spectral monitoring, gives you more flexibility to design the experiment based on the needs of your sample. For example, if your protein intrinsic fluorescence is low, Uncle doesn't have an issue with you using an external dye. If your buffer conditions quench the protein intrinsic fluorescence, you are also free to use an external dye. Another difference is the sample introduction in Uncle; the Unis are sealed. That allows you to do long-term experiments without having to worry about evaporation.

Can we obtain protein and viral particle concentration and the empty/full ratio like with the Stunner?

AM: Yes, we can. Stunner is a UV-vis detector and is, therefore, able to determine the empty/full ratio using that capability. But in Uncle, what we can do is measure the genome ejection in the presence of dye.

We can determine how much DNA is present at the beginning of the experiment before the ejection happens, and then how much DNA is present at the end of the experiment. Thereby, we can determine how much DNA was present in the sample. Uncle can determine these parameters and would be your instrument of choice when looking at the parameters of your vector and its stability.

If your interest is more in empty/full ratio through UV-vis, then Stunner would be your instrument of choice. Both can do this; they just use different techniques to get to that same result.

To learn more about protein stability screening, watch the full webinar here>>

SelectScience runs 10+ webinars a month, discover more of our upcoming webinars>>