Uncle stays hot for biologics stability screening
Blog
How a protein handles heat is crucial for understanding its stability and effectiveness in the short- and long-term. Long-term stability testing is near impossible when time is tight and samples are limited. Uncle, the first all-in-one biologics stability screening platform, turns up the heat without eating up valuable time or sample with its isothermal protein stability assay. Uncle is perfect for spotting the most stable biologic or formulation and helping predict if it will fail long-term storage trials without investing months and years into the investigation.
Tm & Tagg and Isothermal Stability of a mAb
During a thermal ramp of a monoclonal antibody (mAb) in Uncle, two things typically happen in tandem: the protein unfolds, shifting its intrinsic fluorescence emission to higher wavelengths, and it aggregates, increasing static light scattering (SLS). Formulations that make mAbs unfold or aggregate at lower temperatures are less stable than ones that make it break down at higher temps.
Thermal ramps, melting curves, and aggregation curves are quick and powerful protein stability assays but holding samples for a longer time at steady temperatures can paint a more detailed stability picture. Quite often, incubating a mAb at a temp below its Tagg will, eventually, induce aggregation. For example, these three formulations of the same mAb had TaggS (dotted lines) above 65 °C. However, when they were held at 60 °C for six hours, SLS increased and the mAbs aggregated in the same order as during the thermal ramp. How quickly this happens, and at what rate, is another way of ranking the stability of formulations or protein variants.
Tm & Tagg and Isothermal Stability of RNase A with SYPROTM Orange
Reagent-free protein stability assays are preferable to using reporter dyes or tags since those can influence protein conformation or aggregation behavior. But intrinsic fluorescence measurements are not possible with all proteins. RNase A, for example, lacks any tryptophan residues and has exceptionally low intrinsic fluorescence. For difficult-to-characterize proteins, Uncle's full-spectrum fluorescence can use dyes like SYPRO Orange that are fully compatible with thermal ramps or isothermal holds.
Based on a SYPRO Orange protein stability assay, RNase A had nearly identical unfolding behavior in all three formulations and didn't aggregate in any condition. Isothermal experiments in which the proteins were held at 60 °C for 16 hours revealed something quite different - not only will the protein aggregate eventually, but high concentrations of NaCl will stabilize RNase A and prevent aggregation. Only an isothermal experiment made these further insights possible.
Out-of-the-box
Most longer-term protein stability studies rely on complex and time-consuming methods like HPLC, SEC, or mass spectrometry. Uncle has a better way. Simply stash your samples in an incubator and pop them into Uncle to grab the data with the Out-of-the-box apps. Whether it's for an hour, a day, or a week, Uncle has the flexibility to analyze any protein and point out which ones are acting up, whenever you want.
Once-a-day DLS reads of RNase A in three formulations, held at 60 °C in an incubator for three days, show no signs of an increase in the protein's hydrodynamic size in the high salt formulation. Just as in the isothermal experiment above, RNase A showed a significant increase of the z-average diameter in low salt and no salt after hours of storage. For RNase A, isothermal protein stability assays were necessary to determine the influence of the formulation condition on aggregation.
TL;DR
Uncle's high-throughput protein stability assays make identifying the best formulation to protect your protein from heat faster. Melting temps take as little as two hours with intrinsic or dye-based fluorescence. Thermal ramps show a protein's tendency to aggregate while isothermal holds complete testing by monitoring your analyte for hours or days. The Out-of-the-box app simplifies long-term data collection. Uncle helps you explore which proteins and formulations can take the heat in the most efficient way possible.
