Express Pharma

Sub-visible particles: Satisfying regulatory demands


Dr Michael Caves, India Business Development Manager, Malvern Instruments and Dr Namrata Jain, Product Technical Specialist — Nanometrics Bioscience, Malvern Aimil Instruments in this  describes how Malvern Instruments provides the technology and expertise required for characterisation and regulatory satisfaction

Dr Michael Caves

Most macromolecular drugs (proteins, peptides, DNA and RNA) are administered via intravenous (IV) injections and/ or through a drug delivery system (DDS) because of their poor oral bioavailability. Development of a viable oral drug delivery system for proteins and peptides requires a careful consideration of their physico-chemical properties (molecular weight, pH stability, hydrophobicity, molecular size, and ionization constant). Therapeutic proteins such as antibodies play a prominent role in the treatment of numerous diseases including cancer and rheumatoid arthritis. However, these proteins have a propensity to degrade and aggregate during manufacture and storage. In addition to affecting the efficacy of a drug, protein aggregation has been implicated in numerous cases of dangerous drug-induced immunological responses, and is the main cause of increasing regulatory concern over protein therapeutics. In recent years, the regulatory spotlight has been shone on sub-visible particles, due to their potential immunogenicity and the difficulties involved in their characterisation. In order to comprehensively characterise biopharmaceutical aggregation, we must take an orthogonal approach and look at the problem from a number of different angles.

The solution

Dr Namrata Jain

SEC is regularly used to assess the aggregation state of protein samples throughout the drug development pipeline. Static light scattering (often referred to as multi angle light scattering (MALS)) is used to measure protein molecular weight (MW). Malvern’s SEC-MALS 20 (Figure 1) is the most advanced multi-angle light scattering detector with 20 light scattering detectors arranged radially around a vertical flow cell. In addition to working in conjugation with Malvern’s Viscotek SEC systems, the MALS 20 detector is also compatible with any third party SEC system.

Figure 1: Viscotek SEC-MALS 20 is the most advanced multi-angle light scattering detector compatible with any GPC/SEC system

Figure 2: Malvern Zetasizer Nano is the market leading technology for measurements of hydrodynamic size and electrophoretic mobility, its reliability and ease-of-use being highly valued by QC departments the world over

Malvern Zetasizer range uses Dynamic Light Scattering (DLS) to detect early aggregates and also to monitor aggregate formation in response to stimuli such as time or raised incubation temperatures of biopharmaceutical formulations (Figure 2). Although this is a low resolution technique relative to SEC, the generation of accurate and consistent results in a short period of time makes DLS an essential biopharmaceutical characterisation tool. These two techniques used together provide a wealth of important information about the stability and aggregation profile of a sample. The Zetasizer Nano ZSP is the premium member of the Zetasizer Nano series designed to provide exceptional sensitivity for enhanced protein characterisation, while the Automated Plate Sampler (APS) system is capable of performing efficient QC and compatibility analysis on a large number of samples without user-intervention. Since the particle size is determined from fluctuations in scattered light intensity due to the Brownian movement of the particles, the fact that the intensity of the scattered light is proportional to the sixth power of the particle diameter makes this technique very sensitive to the presence of large particles. This can be an advantage where the purpose is to detect small amounts of large particles in relatively pure samples (during QC, for instance), though highly polydisperse samples are difficult to fully characterise using this technique. Moreover, immunogenicity is dependent not only on the particle size but also on level of sub-visible particles (0.1 to 10 µm) present in therapeutic protein products initially and over the course of shelf life.

Figure 3: NanoSight Nanoparticle Tracking Analysis (NTA) is a particle-by-particle technique that gives high resolution particle size distribution and concentration analysis, with visual validation giving extra confidence

NanoSight Nanoparticle tracking analysis (NTA) is an innovative system that combines laser light scattering microscopy with a high-sensitivity CMOS camera to track, visualise, count and size the particles individually but simultaneously using their light scattering signal (Figure 3). NTA uses the same scientific principles as DLS, but applies them to individual particles rather than light scattered from many particles. Measurement of the scattering intensity of each particle allows differentiation of particles of different composition in complex mixtures. NTA can also be used in fluorescence mode enabling specific nano particles to be tracked even in complex formulation. The ability to monitor individual particles provides a high resolution size distribution profile, whilst visualisation gives additional confidence for process optimisation.

Proof of concept

This article describes an IgG antibody formulation study using these Malvern technologies, IgG being the active ingredient in many commercially available biopharma. IgG has a very consistent molecular weight of about 150 kDa which was accurately measured using a SEC-MALS 20 detector (Figure 4). This sample also contains a secondary peak of 308 kDa identified as a dimer and finally a third peak of 674 kDa which represents some larger and more polydisperse aggregates. It can be seen that the molecular weight is stable across the monomer peak and also the dimer peak, with the polydispersity values, calculated using Malvern’s OMNISEC software, of 1.0 for each peak confirming their purity. The larger aggregate fraction formed a sloped molecular weight trace (in black) due to the higher polydispersity of this fraction. MALS data obtained from 20 detectors can also be displayed in a 3D plot as shown in Figure 4, showing the data collected for each of the 20 angles.

Figure 4: (Left) Chromatogram of polyclonal IgG measured by SEC-MALS 20 detector shows the 90 degree MALS signal in orange and RI signal in red and the molecular weight is overlaid in black. (Right) Typical MALS data of IgG showing light scattering signal at 20 measurement angles

IgG aggregates (obtained by heat stress) measured with NTA and DLS. The size distribution (middle panel) with the corresponding NTA video frame (left panels) and 3D graph (size vs. intensity vs. concentration; right panels) are shown

A heat-stressed IgG formulation was analysed using NanoSight NTA (Figure 5). Given that the lower detection limit of NTA for proteins is about 30 nm, protein monomers and aggregates smaller than this are not detected by this technique, hence the lack of NTA data for the unstressed sample (DLS reliably sizing the IgG monomer). Figure 5 demonstrates that NTA enabled sizing (and concentration calculation) of a subset of sub-visible particles between 10 and 100 nm in size that DLS could not reliably detect. Contrary to DLS, high peak resolution and suitability for polydisperse samples with the possibility of visualisation and particle count makes NTA a very useful technique for sub-visible particle quantification. NTA has proven to be a very suitable characterisation tool for research areas like extracellular vesicles including exosomes, drug delivery and virus nanoparticles, vaccines and protein aggregates with the possibility of live monitoring for heat-induced aggregation, providing information about aggregation kinetics.

An orthogonal approach that gives true comparability

The orthogonal approach described here gives confidence in sample purity and aggregate content, giving highly sensitive size and concentration characterisation of protein aggregates in biopharmaceutical formulations. Simple to implement, Malvern’s approach allows you to formulate and manufacture in a way that minimises the presence of aggregates in your final product and demonstrate your product purity to regulatory agencies.

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