The Invisible Threat: Unraveling the Role of Particle Size Analyzers in Drug Safety

Therapeutic protein products are an important weapon in the fight against disease. However, their effectiveness can be compromised if proteins become blocked or inactive due to antibody responses from the body’s immune system. ¹

Protein aggregation has been linked directly to these adverse reactions, making it an important risk factor to consider when assessing product quality. ¹

That’s where particle size analyzers come in. These instruments are key tools in drug development and manufacturing as they measure the size and distribution of particles in a solution. ¹

Dynamic Light Scattering (DLS) Particle Size Analyzers

These tools (e.g., Malvern Zetasizer and Horiba nanoPartica) are used to measure particles in the range of ~0.3 nm to ~10 μm. They work based on the principle of Brownian motion, where lighter particles move faster and this speed is directly proportional to particle size. The methodology involves using a laser to illuminate the particles and then analyzing the scattered light and fluctuations in intensity to determine the intensity-weighted size distribution within a sample. However, DLS may not always be the best method for characterization. ³ One of the limitations of DLS particle size analyzers is that they might not detect aggregates in the subvisible range and can be difficult to interpret when samples are polydisperse or if larger aggregates are present. Often the solution to improve data quality is to filter samples before running—which only removes the larger particles so they escape analysis entirely. ^4,5

Laser Diffraction Particle Size Analyzers

These instruments (e.g., Malvern Mastersizer 3000 and Horiba Partica LA-960V2) leverage the diffraction patterns produced by a laser beam when it passes through an object ranging in size from nanometers to millimeters. This technique quickly gauges a particle’s geometric dimensions, ranging approximately from 10 nm to 2,000 μm, and it functions independently of the volumetric flow rate, which measures the number of passing particles. However, there are limitations to this method. It requires dilution, which might alter the aggregate size, and it also necessitates a relatively large sample quantity. ^4,6

Size and Exclusion Chromatography (SEC) Particle Size Analyzers

This technique is used to evaluate the size distribution of molecules in a sample, typically ranging from 4 to 12 µm. SEC particle size analyzers (e.g., Agilent 1260 Infinity II Bio-SEC Multidetector System) separate molecules based on their size using a column packed with tiny porous beads. However, SEC may fail to detect aggregates that fall in the subvisible range. Other drawbacks include potential matrix interactions, dilution of formulation, and a limited dynamic range for sizes. ^4,7

Light Obscuration (LO) Particle Size Analyzers

This standard method can quantify particles ≥ 10 µm and ≥ 25 µm, as well as measure down to 2 µm. Complying with USP <1058> and 21 CFR Part 11 requirements, light obscuration particle analyzers (e.g., Beckman Coulter HIAC) pass a sample through a narrow opening in a device, measuring the light obstruction caused by individual particles to establish their size and concentration. Despite its effectiveness, this technique faces challenges with high viscosity formulations and particle morphological data, resulting in an undercounting of transparent, non-spherical particles. In addition, it requires a relatively large sample volume.^4,8

Flow Imaging Particle Size Analyzers

Commonly employed by Yokogawa FlowCam and Bio-Techne (formerly ProteinSimple) MFI, flow imaging microscopy captures particle images in a flow cell to calculate size, shape, count, and other attributes. These standard instruments typically cover a size range from about 1 to 400 µm. However, flow imaging does have drawbacks including possible clogging requiring low viscosity samples, the need for pre-and post-experiment cleaning, low throughput, and high sample volumes (>1 mL).^4,9

Light Microscopy

This traditional technique can directly count and characterize subvisible particles in the size range of 0.8–150 µm, as described in both USP 788 and Ph.Eur.2.9.19. Still, manual light microscopy is low-throughput and produces less accurate results than the light obscuration technique.^4,10