How do I determine particle concentrations for multimodal samples in DYNAMICS?

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Introduction

With the release of the DYNAMICS® software version 7.10, multimodal samples measured with a DynaPro® Plate Reader III or NanoStar® can be analyzed to quantify particle concentration. This adds an important tool for high-throughput screening of complex samples where different sized populations are present. For example, monomeric capsid concentration can be determined while simultaneously screening Adeno-associated virus (AAV) samples for aggregation during formulation and process development.

Figure 1 shows an example of the regularization graph in DYNAMICS, highlighting the particle concentration of each component in a mixture of two polystyrene latex beads with nominal radii of 10 and 50 nm.

Prepare DYNAMICS

To enable particle concentration in the DYNAMICS software, under the Parameters/Sample, set “Particle Concentration Calculation” to “Yes” and select the Particle Material and Shape from the list of options. The particle concentration of each peak is calculated according to the procedure detailed in a previous Ask an Expert article, “How do I use the new particle concentration feature in DYNAMICS?“ The calculation uses the hydrodynamic radius measured by dynamics light scattering (DLS), total static light scattering (SLS) intensity at the instrument’s scattering angle, and the Lorenz-Mie shape model for a solid sphere.

For monomodal samples it is assumed that all the particles in the scattering volume are the same size and shape in the particle concentration analysis, so the hydrodynamic radius Rh obtained by the cumulants fit is used. For multimodal samples, regularization analysis results are used and each peak is considered to be monodisperse, while the peak width indicates the uncertainty.

After running your samples, the particle concentration of each peak range appears under the regularization graph as shown in Figure 1. You can also setup the datalog grid to show particle concentrations for each peak range as shown in Figure 2.

The peak ranges DYNAMICS uses for the regularization analysis can be customized for multimodal samples with different size ranges as shown in Figure 3.

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Figure 1. The regularization graph in DYNAMICS obtained for a mixture of two polystyrene latex bead sizes (represented by the inset) with nominal radii of 10 nm and 50 nm. Particle concentration for each peak is shown in the table below. To add the Particle Concentration column, right-click on an empty space in the column heading, select Table Settings, and choose the appropriate quantity.

Figures 2 & 3

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Figure 2. To display particle concentration in the datalog grid, right-click on the grid and select Table Settings. In the Table Settings menu (bottom), scroll down to Regularization and add the range corresponding to the peaks. In this specific case, Range 3 is not selected since there are no peaks in that size range.

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Figure 3. To set the peak ranges for your sample, right-click on the datalog grid, select Peak range Settings and enter the low and high limits for the radius. You can also check the box for a grid column to add it to the datalog grid. In this specific case the default ranges of 0.1 -10 and 10-100 nm were changed: 20 nm was entered as the upper limit of range 1 and the lower limit of range 2.

Limitations and important points:

  • Calibration: Because particle concentration calculations use SLS measurements, calibration is required. For NanoStar, calibration is performed with toluene.
    • When using a DynaPro Plate Reader III, the well plate must be calibrated. DYNAMICS 7.10 adds a new feature for plate calibration which allows auto-attenuation to be used with SLS measurements. Please refer to the DYNAMICS 7 User’s Guide for details.
  • The maximum allowable Rh for particle concentration analysis is 160 nm in a DynaPro Plate Reader III and 175 nm in a NanoStar. If the measured Rh is greater than this limit, DYNAMICS will not report the particle concentration.
  • The minimum concentration that can be measured depends on the radius of the sample. For a typical AAV the minimum concentration is ~6 x 1010 mL-1.
  • Multi-modal particle concentration analysis requires well-resolved regularization peaks. A size difference of at least 3-5x between the two modes is required to resolve them.
  • If the appropriate sample type is not listed under Particle Material, a priori knowledge of the refractive index of the particles is necessary. Select ‘Other’ and enter the real and imaginary components of the refractive index of the material comprising the particle.

Do you have a question? Contact our experts here in Customer Support. We’re happy to help! Call +1 (805) 681-9009 option 4 or email support@wyatt.com.