(M1230-06-32) Improve NTA Measurement Repeatability with Correct Diluent

Purpose: Nanoparticle tracking analysis is a new tool for evaluating particle concentration and size distribution. These measurements rest on repeatability of the measurement. Here, we examine repeatability of measured particle size and concentration for a commercial vaccine formulation. This system shows behavior to similar, less complex model systems. Careful attention to dilution is required to ensure repeatable results. A canine vaccine was chosen since it is readily available and represents a complete commercial vaccine formulation. Therefore, we encounter more issues compared to model systems such as ZrO2 or polystyrene latex.

Methods: Canine Spectra ® 6 was diluted in PBS and PBS with 0.2% and 0.02% amounts of Tween 20 (polysorbate 20) to ensure the concentration was within the linear range of the measurement. The as-received vaccine is otherwise too concentrated for correct analysis. Canine Spectra 6 is a canine vaccine from Durvet for Canine Distemper, Canine Adenovirus Type 2 (CAV-2), Canine Coronavirus, Canine Parainfluenza and Canine Parvovirus Type 2b. Particle size distribution was determined with the ViewSizer 3000 multi-laser nanoparticle tracking analyzer. Like many formulations, this vaccine has a reasonably broad particle size distribution and distribution had to be analyzed with three lasers with intensities optimized for each part of the size range and a color CCD detector. Otherwise, some particles could not be analyzed properly due to detector saturation or overly weak scattering. In order to ensure statistical robustness and no double counting, only particles present at the start of a video were tracked. That is particles that diffused away and returned to the video at a later point were not tracked. Each analysis experiment consisted of 25 short videos, with automatic mixing between frames. Diluted suspensions were analyzed six times in sequence. The variation in concentration with time is used to evaluation sample stability.

Results: Figure 1 shows measured size distribution of the vaccine diluted in PBS for repeated measurements. Note that the results are in terms of absolute concentration and show a pronounced decrease in total particle concentration (peak area) with time. This is an issue for routine measurement since that implies significant operator to operator variation as each introduces the sample at slightly different times. In contrast, vaccine diluted in 0.02% Tween 20 shows much less variation. Variation in measured total particle concentration with time are shown for each of the three diluents in is shown in Figure 2. Here, the ratio of the measured concentration to the initial concentration is plotted; values should remain at 1. It is apparent that the addition of Tween 20 significantly improves the repeatability of the result. A concentration of 0.02% Tween 20 allows about 15 minutes before significant concentration drift is observed while 0.2% suspension lasts longer. Unfortunately for this sample, particle size also changed with time, albeit on a much smaller scale. These changes are hard to discern in Figures 1 and 2. Measured values of Dn50 as a function of time are shown in Figure 3. Here, a decrease in measured size is most pronounced in the sample diluted with PBS and 0.2% Tween 20. The experimentalist is then left with a compromise choice of PBS and 0.02% Tween 20 to ensure stable size and concentration results.

Conclusion: For this particular vaccine, dilution with PBS and 0.02% Tween 20 gives the most stable results. This sequence of experiments illustrates the importance of careful choice of diluents and their effect on formulation stability. This case is particularly challenging due to the lack of details on the formulation components. Naturally, more details on formulation chemistry will help guide this method development. We recommend exploiting any such information.

Figure 1: Measured size distribution of canine vaccine diluted in PBS at various times. Note variation in concentration with time.



Figure 2: Variation in measured total particle concentration with time for various diluents (PBS only, PBS with 0.02% and 0.2% Tween 20). Note the improved stability obtained by using Tween 20.



Figure 3: Variation in measured total number median particle size with time for various diluents (PBS only, PBS with 0.02% and 0.2% Tween 20). Note that 0.02% gives good size stability as well as concentration stability (Figure 2).