PSS to discuss protein aggregation
The technique of single particle optical sizing (SPOS) is known to produce particle size distribution (PSD) results of highest sensitivity and resolution over a wide size range, making it a uniquely powerful tool for assessing the quality and stability of a wide range of colloidal dispersions and suspensions.
In particular, SPOS based on the method of light extinction (LE) has recently been specified in USPto assess the quality and safety of lipid emulsions for injection by measuring the PSD “tail” of the largest fat droplets and thereby determining the percentage of total fat contained in fat globules larger than 5 microns (um). The SPOS technique for counting and sizing individual particles is obviously an attractive approach for assessing quantitatively the extent of aggregation of proteins and other macromolecules in suspension, provided it can be extended to sizes smaller than those accessible by the simple LE method. For example, we have combined the LE signal with the light scattering (LS) response from the same “view volume”. The resulting “LE+LS” hybrid sensor is able to count and size individual protein aggregates as small as 0.5 um, due to the more sensitive LS response, while still being able to measure those as large as 400 um, because of the wide dynamic range inherent in the LE method. By reaching further down in size than LE alone, this LE+LS approach is able to reveal in a detailed way a larger fraction of the overall “picture” of the protein aggregate distribution. However, it suffers from the need to dilute the starting sample, often extensively, in order to reduce particle coincidences and thereby avoid distortion of the measured PSD. Recently, SPOS technology has undergone a quantum leap in capability with respect to the lower size limit of single-particle detection and sizing. Based on an alternative LS approach that uses a focused laser beam to achieve a much smaller and more intensely-illuminated “view volume”, this new “FX-Nano” technology can count and size particles as small as 0.15 um. Because the intensity profile of the laser beam is highly non-uniform, novel and proprietary signal inversion algorithms are required to convert the detected pulse height distribution (PHD) into a final PSD that possesses very good resolution and absolute accuracy. Importantly, by design, the FX-Nano sensor functions at much higher particle concentrations (>100X) than those allowed by traditional SPOS sensors – i.e., above 1,000,000/mL. As a result, little or no dilution is required for most protein suspensions of interest. This is an important advantage for two reasons: 1) it avoids changes in the PSD if the aggregates are in dynamic equilibrium, sensitive to concentration; 2) it avoids the difficulty of diluting the starting sample to a major extent while keeping the level of background contaminant particles very low – a nearly impossible task in practice in the size range extending down to 0.15 um. Finally, the new SPOS-FX technology detects particles at a high count rate (2,500/sec), resulting in large total counts and much better counting statistics than provided by alternative technologies that have been used recently to characterize protein aggregation. Representative PSD results obtained from partially aggregated protein samples will be shown and discussed.