Particle Concentration

MALS is most often coupled downstream of a size-based separation method such as size-exclusion chromatography (SEC-MALS) or field-flow fractionation (FFF-MALS). Separation prior to measurement enables high-resolution particle size distributions across a size range of 13 nm to 650 nm in radius (equivalent to rms radius range of 10 nm to 500 nm), where the resolution is limited mainly by the separation method.

Because each size fraction is measured individually, the detector is not “blinded” to small particles by strong scattering from large particles so concentration values are generally quite accurate. If particles composed of different materials are separated, each peak may be assigned a corresponding n_particle value. SEC-MALS uses a standard HPLC system with a DAWN^™ MALS instrument; FFF-MALS adds an Eclipse^™ FFF controller and separation channel. ASTRA^™ software analyzes the particle size and concentration in each eluting fraction, and can integrate peaks to calculate the total number of injected particles in each size range. ASTRA supports 21 CFR Part 11 compliance, as does the VISION^™ FFF software.

In addition to size and concentration, a setup may be configured with UV, fluorescence and/or refractive index detectors in order to determine the molar mass, composition and payload of many types of nanoparticles.

Real-time MALS (RT-MALS) measurements of concentration are generally performed without separation in order to provide rapid feedback for process analytical (PAT) applications, reporting results 30 times per minute. The values may be integrated to calculate the final titer of, e.g., virions in the pooled fractions. At the same time, size is determined continuously for use as a biologics DSP pooling criterion or to monitor deviations of synthetic nanoparticles in the course of manufacturing.

The ultraDAWN^™ RT-MALS instrument is usually connected in-line with production or purification systems operating at lower flow rates. For static reactors or high-flow-rate processes, it is configured on-line, where a pump draws some of the product and runs it through the ultraDAWN. OBSERVER^™ software orchestrates the entire process; pre-defined trigger conditions may be set to alert the operator upon reaching an end point or deviating from acceptable product attributes.

RT-MALS only determines the average size and not a full distribution, so if the size distribution is wide, the particle concentration values may not be accurate. The reported concentration is most reliable for narrowly disperse nanoparticles.

DLS is usually performed without separation, in microcuvettes or microwell plates. This “batch” mode operation enables fast measurements of relatively low-resolution distributions, with less accuracy than SEC-MALS or FFF-MALS but potentially more than RT-MALS. High-throughput DLS in microwell plates is ideal for analyzing viral vector downstream bioprocess fractions or a nanoparticle formulation matrix.

The size range (hydrodynamic radius) over which concentration may be measured is 0.5 nm to 160 nm in the DynaPro^™ Plate Reader and 0.2 nm to 175 nm in the DynaPro^™ NanoStar^™, making it suitable for most viruses and nanomedicines as well as other types of nanoparticles. If more than one size population is evident in the distribution, DYNAMICS^™ software will provide the concentration of each.

Wyatt’s DLS instruments dramatically minimize sample requirements. Only 2 µL of sample are required in quartz cuvettes, and in the most common 384 well plates, 20 µL are needed for optimal measurements. Though these instruments cover several orders of magnitude in concentration, if particle concentrations are high and dilution is necessary, it is easy to do so directly in the measurement volume.

DLS is especially useful for quick and easy nanoparticle analysis with minimal instrument complexity or sample prep effort. Wyatt’s DLS instruments are supported by DYNAMICS^™ software, a comprehensive suite of instrument control, data acquisition and analysis that provides extensive automation, rich characterization capabilities and intuitive feedback on the quality and reliability of each and every measurement.

For use in quality control and at-line process analytics, DYNAMICS offer 21 CFR Part 11 compliance. In addition, it supports an application programming interface (API) that enables integration with third-party liquid handlers and high-level process or quality control software.