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European Biopharmaceutical Review

Characterisation Techniques

Many patients’ lives have been improved with the use of monoclonal antibodies and recombinant proteins since their commercialisation around 30 years ago. Work in the therapeutic protein field continues to be attractive for academia, pharma and biotechs, with the global therapeutic proteins market forecast to reach an estimated $141.5 billion in 2017. Monoclonal antibodies, insulins, interferons, growth hormones and blood factors are just some of the areas of interest (1).

Being large macromolecules, working with proteins brings an array of challenges that are not generally encountered when working with traditional, small molecule therapeutics. They have complex 3D structures that need to be maintained for binding efficacy, are generally more prone to degradation in the body, and their synthesis is typically achieved in a biological host, requiring purification from complex media.

Binding Together

For a protein therapeutic to be efficacious, it needs to be available in its monomeric form, but a further key feature of protein molecules is their tendency – to varying degrees – to bind to each other. This aggregation can progress more quickly, or even be initiated, when the protein is subjected to stress such as exposure to interfaces (air-liquid or solid-liquid) and light, or changes in temperature, ionic strength or pH (2).

Unfortunately these stresses are often inherent in the synthesis, purification, packaging, transport, storage and use of proteins. The formation of aggregates produces a wide spectrum of sizes, types and lifetimes (3).The mechanisms or pathways involved in aggregation, in many cases, are numerous and not well understood, varying from protein to protein, and multiple mechanisms can occur within the same sample (see Figure 1) (4).

When prescribed to a patient, therapeutic proteins are dosed as liquid formulations, often being produced prefilled in syringes, which are usually lubricated with silicon oil. The oil, along with other particulate debris that may be introduced with the processing of the protein, can also serve as sites for aggregate formation due to heterogeneous nucleation (5).

As with all therapeutics, liquid formulations are regulated and are subject to US Pharmacopoeia (USP) <788> light obscuration test which, since 1995, has set limits on the allowable number of sub-visible particles that are >25μm and >10μm as ≤600/container and <6000/container, respectively. These sizes are more to control process levels of debris that could potentially block blood vessels, while the risks associated with the administration of large aggregated protein particles were not considered in the establishment of USP light obscuration test <788> (2).

Sub-visible protein particles 100nm-10μm, as well as those that are larger, have the potential to impact the safety and efficacy of the therapeutic over its shelf-life. Furthermore, small aggregates can grow into larger ones, and eventually become the size and number needed to exceed the limits set out in the USP <788>.

Smaller Aggregates
When working with proteins at any R&D and production stage, it is important to understand and control the profile of the smaller aggregates in order to identify the aggregation onset point. The measurement of small aggregates has historically been investigated using size exclusion chromatography (SEC). However, this technique gives a read-out of mass fraction and relies on having 100 per cent sample recovery to be sure of the data profile; even 99 per cent recovery means one per cent of a particularly large aggregate may have been lost (3).

Sub-visible particles usually do not constitute a sufficient mass fraction to be quantified (2). In addition, the method of SEC requires substantial dilution of the sample, which can change the aggregation profile itself.

Techniques that can count and size individual species in undiluted therapeutic proteins may be more appropriate than mass fraction methods when studying protein aggregation.

Nanoparticle Tracking
Nanoparticle tracking analysis (NTA) is a method of visualising and analysing particles in liquids that relate to the rate of Brownian motion to particle size. The rate of movement is linked only to the viscosity of the liquid, the temperature and the size of the particles, and generates a high-resolution particle size distribution by sizing each particle individually, along with giving an estimation of the concentration of particles present in the sample.

NTA visualises, measures and characterises virtually all nanoparticles (10-2000nm). Particle size, concentration, zeta potential and aggregation can all be analysed, while a fluorescence mode provides speciation of suitably-labelled particles. This provides real-time monitoring of the subtle changes in the characteristics of particle populations, with all of these analyses uniquely confirmed by visual validation.

From loading the sample into the cell to obtaining the results can take as little as two to three minutes, with the ability to run batches of samples under the same conditions and directly compare results.

Individual Analysis
The Brownian motion of nanoparticles is analysed in real-time by a charge-coupled device or complementary metal-oxide semiconductor camera. Each particle is concurrently, but separately, visualised and tracked by a dedicated particletracking image analysis programme.

The NTA programme simultaneously identifies and tracks the centre of each particle on a frame-by-frame basis throughout the length of the video – typically 30 seconds. The average distance that each particle moves within the image is automatically calculated. From this value, the particle diffusion coefficient can be obtained and, by knowing the sample temperature and solvent viscosity, the particle hydrodynamic diameter can be identified.

Because each particle is visualised and analysed separately, the resulting particle size measurement and distribution does not suffer from the limitations of the intensity-weighted, z-average distribution found with dynamic light scattering (DLS). The ability of NTA to simultaneously measure particle size and particle scatter intensity allows heterogeneous particle mixtures to be resolved, and particle concentration can be directly measured. Because this is an absolute method, no user calibration is required.

The top of Figure 2 shows the particles present in liquid illuminated by the laser, while the middle shows the individual tracks of each particle. Finally, the bottom of Figure 2 shows the distribution of the particles under study.

Working with Proteins
Due to the low refractive index of protein, the limit of detection in NTA measurement is approximately 30nm. This means that the protein monomer units – which are typically in the range 3-10nm – are not measured by NTA, but aggregates comprised of just tens of monomers to many thousands can be sized and counted. As it is not necessary to dilute the sample to obtain the particle size distribution, the aggregation profile is not changed due to sample processing.

DLS, or photon correlation spectroscopy, is a commonly used sizing technique that also has its basis in Brownian motion. However, unlike NTA, the light scattered from all the particles in the sample is measured as a whole, thereby giving a single average size measurement for the sample, along with a guide to the level of polydispersity (6). As with NTA, the smaller particles scatter smaller amounts of light and larger particles scatter more light. The overall fluctuations in the scattered light over time are used to calculate the size of the particle population. Since the intensity of the scatter is of a factor r, this allows the protein monomer to be sized, but a few larger particles in a population can greatly skew the data obtained, and the technique struggles to resolve very poly-dispersed mixtures often observed when measuring protein solutions (7).

Analysing Findings
The experiment shown in Figure 3 demonstrates that when using heat (50ºC), 1mg/mL IgG aggregates over time, with the particles scattering light increasing in number and intensity. At each time point DLS and NTA measurements were taken, and the size data for both the monomer and the aggregates could be observed. After 20 minutes of thermally induced aggregation, the monomer peak described with DLS showed a sphere equivalent hydrodynamic radius of approximately 10nm, with NTA measuring aggregate particles starting at approximately 30nm, with peaks observed at 50 and 85nm, and the largest aggregates being approximately 300nm.

Since NTA also gives an estimation of particle concentration, the increase in particle number during the time course of the thermal aggregation could also be tracked (see Figure 4). These data suggest that for 30 minutes there are minimal aggregates above 30nm in size. From 30 to 100 minutes, the numbers of aggregates at 30nm or larger remains stable. After this time, the number of aggregates appears to increase in a more exponential manner.

To prevent the presence of large aggregates rendering a protein therapeutic unsuitable for patients, scientists need to have an understanding of where in the process of synthesis, purification, packaging, transport, storage and use the proteins monomer units begin to aggregate together. Taking size distribution measurements with NTA at different points in the process enables scientists to identify the point at which aggregation begins. This point can then be reviewed, and potentially modified, to prevent or slow down the formation of protein aggregates.

Leap Forward

The ability for NTA to be able to track aggregation processes in real-time is a huge leap forward for those working with proteins. Now that it is also recognised as current Good Manufacturing Practice, enforced by the US Food and Drug Administration, it is well-placed in the pharma industry for the research, manufacture and control of new and existing systems.

1. Therapeutic Proteins Market to 2017. Visit:
2. Carpenter JF, Randolf TW et al, Overlooking subvisible particles in therapeutic protein products: gaps that may compromise product quality, J Pharm Sci 98(4): pp1,201-1,205, 2009
3. Philo JS, A critical review of methods for size characterization of non-particulate protein aggregates, Curr Pharm Biotech (10): pp359-372, 2009
4. Philo JS and Arakawa T, Mechanisms of protein aggregation, Curr Pharm Biotech (10): pp348-351, 2009
5. Chi EY, Weickmann J et al, Heterogeneous nucleation-controlled particulate formation of recombinant platelet-activating factor acetylhydrolase in pharmaceutical formulation, J Pharm Sci (94): pp256-274, 2005
6. Engelsman JD, Kebbel F et al, Laser light scattering-based techniques used for the characterization of protein therapeutics. In: Mahler HC and Jiskoot W, Analysis of Aggregates and Particles in Protein Pharmaceuticals: pp43-48, 2012
7. Carr B and Malloy A. Visit:

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About the authors

Pauline Carnell is the Senior Application Scientist at NanoSight. She has over 20 years of experience in pharmaceutical research for Warner Lambert, UK and Pfizer Ltd, UK. Prior to joining NanoSight, she spent two years with a European Union biotech company working with proteinconjugated gold nanoparticles. Her current areas of interest are protein characterisation and fluorescent applications.

Sarah Newell is Marketing Communications Manager at NanoSight, a Malvern Instruments company. She has extensive experience in the life sciences industry, having spent 10 years working in Biochemical Research and Development at Merck, before moving into marketing at Sigma Aldrich and then onto Product Management at ThermoFisher.
Pauline Carnell
Sarah Newell
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