Floating droplets aid studies of challenging protein solutions

Researchers from University of Copenhagen, Technical University of Denmark and Lund University have recently published a paper showing that the acoustic levitation method can successfully enable studies on high concentrations of proteins in solution. The results have important implications for areas such as pharmaceutical, food and chemical industry.

Protein can precipitate under wrong storage conditions like the white solutions on the picture. This may be due to high protein concentration, high or low pH and high or low salt concentration.

For many applications, such as pharmaceuticals, it’s essential to keep a high concentration of proteins in solution. It’s not trivial though as the proteins tend to clump together, or aggregate, under such conditions, making the drug less effective. You have to add things like salts that will change the solution’s dynamics and make the protein molecules stay separate. So we need to learn more about how to do that optimally, which means we need to know more about highly concentrated protein solutions.

“The most important results of our study is that we have shown the possibility of characterising highly concentrated samples and that we can do it with small sample consumption,” says Pernille Sønderby Tuelung, first author of the study, University of Copenhagen. “It is a new type of sample environment and methodology that we now have at our disposal.”

With the novel methodology presented in the paper, different sample conditions in a wider range (for both proteins and excipients), can be studied with minimum sample quantities, in only one experiment.

Small Angle X-ray Scattering, SAXS, is used to study particles or molecules’ dynamics in solution. A conventionally used method is the flow cell where the solution flows through a capillary placed in the X-ray beam. The X-ray beam is scattered by the particles or protein molecules in the solution, and the researchers can make conclusions on things like shape or size of the dissolved protein. It doesn’t work so well for high concentrations though, but acoustic levitation, an already existing method, is an alternative. With this method, a droplet of the solution is made to float in the air, suspended by soundwaves, in the X-ray beam. The results are promising.

“We were surprised that we were able to do the data treatment so well. Even with larger noise due to the in-air sample environment, the data was of a quality and comparable to flow cell data,” says Tuelung.

Levitating droplet, suspended in the air by soundwaves.

As the droplet hangs there in the air, it will slowly evaporate, leading to salt and protein concentration changes that need to be accounted for.

“One of the challenges is to account for the evaporation of the buffer solution during the experiment. The droplet size also changes as the solution evaporates,” explains Tuelung. “It was important to be able to validate our results, so we compared with measurements done of the same protein at the same beamline, but in a different sample environment.”

The experiments were performed at MAX-lab, the predecessor of MAX IV, but are crucial preparatory work for implementing the method at the MAX IV CoSAXS beamline.

“Fast data acquisition of a large range of experimental conditions and concentrations is a big benefit of the method. Data analysis will be easier accessible for future academic researches but also, importantly, from the biopharmaceutical industry in areas of drug discovery, delivery and production,” concludes Tuelung. “With a decrease in divergence of the X-ray beam at MAX IV, we will get better data, and probe higher protein concentrations, pushing the limit of the method.”


Pernille Sønderby, Christopher Söderberg, Christian G. Frankær, Günther Peters, Jens T. Bukrinski, Ana Labrador, Tomás S. Plivelic, and Pernille Harris, Concentrated protein solutions investigated using acoustic levitation and small-angle X-ray scattering, J. Synchrotron Rad. 27, 396 (2020), DOI: 10.1107/S1600577519016977