Uncle raises the benchmark for protein characterization methods
Blog
With the meteoric rise of biologics as therapeutic medicines, establishing effective protein characterization methods has become more critical than ever. However, all these methods suffer from similar drawbacks. High-resolution techniques, like size-exclusion chromatography (SEC) or differential scanning calorimetry (DSC), take too much time and require specialized expertise. High-throughput techniques, like dye-based differential scanning fluorimetry (DSF), usually require the addition of labels or other reagents that may skew results one way or another. These methods also all have the same problem - they only provide 1 kind of data output. SEC only tells you about aggregation, DSC and DSF only about unfolding. Efficient protein characterization methods should give a lot of data with only a little time and sample.
Uncle uses label-free detection methods to characterize microliters of up to 48 samples at a time in a few hours. Protein unfolding is examined with full-spectrum fluorescence while static light scattering (SLS), and dynamic light scattering (DLS) look at aggregation. These detection methods run together on the same samples in the same experiment to give deep insights.
Simultaneous detection of unfolding and aggregation
One useful standard for evaluating protein characterization methods is the National Institute of Standards and Technology (NIST) monoclonal antibody (NISTmAb) reference material, RM8671. This mAb is extensively characterized, highly controlled, and readily available. Uncle can easily determine the thermal stability of NISTmAb, but its real versatility comes from comparing the antibody in different conditions, for example, in different formulations.
Uncle identified 3 melting points (Tms) at 67, 80, and 90 °C of NISTmAb in 25 mM histidine, pH 6. When reformulated into 30 mM sodium phosphate, 200 mM NaCl, pH 8.5 it had 2 Tms at 68 and 76 °C. These values come from the BCM curves (solid, left y-axis). We can compare melting to the onset of aggregation, or Tagg, using the concomitant change in SLS intensity (dashed, right y-axis). This showed more aggregation in the phosphate buffer with a Tagg of ~77 °C. While aggregation was first visible by changes in SLS intensity, it also impacted the BCM curve in phosphate, causing a sharp drop at about 79 °C. Based on this data, NISTmAb is more stable in histidine than the phosphate buffer. Uncle’s software automatically assigned the Tms and Tagg, making it an easy-to-use protein characterization method.
Dive deeper into aggregates with DLS
SLS is effective for detecting the onset of aggregation of a protein, but sometimes orthogonal methods are needed. If a protein is already aggregated at the start of a thermal ramp, SLS alone won’t detect it. However, DLS will. DLS is also sensitive enough that you can use it to detect even small changes in protein size. Uncle checks the size and size distribution of proteins with DLS at the beginning and end of a thermal ramp with no extra sample or set-up time.
NISTmAb had a hydrodynamic diameter of about 11 nm and PDI <0.1 at 15 °C in both the histidine and phosphate buffers. These values are typical of well-behaved, monodispersed antibodies. After being heated to 95 °C, the size and PDI of NISTmAb increased in both formulations. The changes were small in histidine, indicating aggregate growth may have been arrested. NISTmAb’s particle size and PDI increased to over 1000 nm and 0.37, respectively, after being heated in the phosphate buffer indicating the formation of large aggregates.
| Histidine | Z-average diameter (nm) | PDI |
|---|---|---|
| 15 °C | 10.3 | 0.09 |
| 95 °C | 12.6 | 0.16 |
| Phosphate | Z-average diameter (nm) | PDI |
|---|---|---|
| 15 °C | 10.4 | 0.06 |
| 95 °C | >1000 | 0.37 |
TL; DR
With more data outputs and less hands-on time, Uncle shows just how simple protein characterization methods can be. Full-spectrum fluorescence detected NISTmAb unfolding while SLS spotted aggregates as they formed. DLS completed the picture with quantifiable sizes so we could see that NISTmAb formed larger aggregates in phosphate than in histidine. The versatility of Uncle helps researchers reduce their risks when developing biotherapeutic molecules by providing robust methods for characterizing proteins from only 9 µL of sample.
Uncle
Uncle is the all-in-one biologics stability platform designed to crank out data on stability and formulation in just hours with way less protein. Uncle combines dynamic light scattering, fluorescence and static light scattering with temperature control to deliver 12 powerful applications for characterizing biologics. Only 9 µL of sample gets you size info from DLS analysis, and info on protein unfolding and aggregation from a thermal ramp experiment.
