Proteins
The Nicomp® DLS and the AccuSizer® SPOS systems are frequently used to measure the size, aggregation, and surface charge (zeta potential) of proteins.
The Nicomp DLS system has the sensitivity to measure monomers, dimers, and trimers at small sizes and at low concentration – including lysozyme at 0.1 mg/mL. The AccuSizer SIS system can be used to measure to measure protein aggregation per the new USP <787> test. The dual sensor AccuSizer FX Nano system is the only optical particle counter capable of measuring protein size and count down to 0.150 µm using only 150 µL of sample.
Featured Application Note
Featured Application Note
Featured Application Note
Protein Aggregation Size and Concentration Analyzer
The AccuSizer SPOS system is ideal to measure subvisble particles for therapeutic injections following the USP <787> test protocol.
The AccuSizer FX-Nano system is a single particle optical sizing system (SPOS) designed to work at the smaller particle sizes and at higher concentrations required to quantify protein aggregation. This is accomplished by using two sensors; the FX-Nano sensor to measure from 0.15-0.6 µm and the LE400 sensor to measure from 0.5-20 µm. The FX-Nano sensor uses a focused beam to reduce the total volume inspected, thus increasing the concentration limit of the sensor. This sensor is coupled with the SIS sampler, modified to allow for sample volume as low as 150 µL. Aggregated proteins are sized and counted one at a time using the two sensors to cover the entire dynamic range. This is an ideal system to measure subvisible particles for therapeutic injections following the USP <787> test protocol.
Example 1
Example 1: IgG protein (1%) was prepared and then allowed to aggregate. The sample was measured before (blue) and after (red) filtration (0.2 µm filter). After filtration, the concentration reduced from 9.7 to 3.1 x 108 particles/mL. The decrease in the tail of aggregated proteins is clearly visible and easily identified by the AccuSizer FX-Nano system.
Example 2: IgG protein was measured before and after incubation. The results show how the tail of aggregated protein changed with time.
Example 2
Using DLS to Characterize Protein Stability
The ability to characterize proteins will become increasingly important as the study of the human genome leads to the discovery of proteins with therapeutic properties or with actions involving specific diseases.
Bovine Serum Albumin (BSA) is a protein with a molecular weight of 69,000 Daltons that is commonly used in protein studies. Properly dispersing BSA is important to obtaining clean particle size distributions. Figure 1 contains the particle size distribution of 35% BSA in saline diluted in filtered distilled water. The sample was centrifuged for 2 minutes using special glass sample tubes to remove large particle contaminates from the diluent. The tubes can go from the centrifuge straight into the particle analyzer.
As can be seen in this intensity-weighted PSD, there are two peaks, one at 6 nm which is the primary BSA and another peak at 40 nm which are aggregates (dimers, trimers, etc.) with an intensity contribution of 40%. Figure 2 contains the particle size distribution of the same BSA but diluted in saline. The PSD is still a bimodal with a primary particle size at about 7 nm however the aggregate peak seems to be larger, with an almost 50% intensity contribution. Finally, Figure 3 contains the same BSA but in PBS. The particle size distribution has changed considerably. The size of the primary particle size grew to 10 nm.
Figure 1
Figure 2
These results indicate how important the chemical environment is to the dispersion of BSA.
Figure 3