Characterizing Nanoparticles in Liquids: Protein Aggregation Studies

Many patients’ lives have been improved with the use of monoclonal antibodies and recombinant proteins since their commercialization about 30 years ago. Work in the field of therapeutic proteins continues to be attractive for academia, pharma, and biotechnology, 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 up many challenges that are not generally encountered when working with traditional, small-molecule therapeutics. They have complex 3-D 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, thus requiring purification from complex media.

For a protein therapeutic to be effective, it needs to be available in its monomeric form, but another key feature of protein molecules is their tendency, to varying degrees, to bind to each other. This aggregation can progress more quickly or can even be initiated when the protein is subjected to stress such as exposure to interfaces (air–liquid or solid–liquid, for example), light, 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 In many cases, the mechanisms or pathways involved in aggregation are not well understood and are numerous, varying from protein to protein; further, multiple mechanisms can occur within the same sample4 (see Figure 1).

Figure 1 – Left: Possible types of particle found in therapeutic protein solutions. Right: Illustration of some example mechanisms responsible for protein aggregation (adapted from Ref. 4).

When prescribed to a patient, therapeutic proteins are dosed as liquid formulations, and are often produced prefilled, in syringes that 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 nucleation5 (see Figure 1).

As with all therapeutics, liquid formulations are regulated and are subject to the U.S. Pharmacopoeia <788> light obscuration test, which since 1995 has set limits on the allowable number of subvisible particles that are >25 μm and >10 μm as ≤600/container and ≤6000/container, respectively. These sizes are meant to control levels of process 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 the USP light obscuration test <788>.2 Subvisible protein particles (100 nm–10 μm) and those that are larger have the potential to impact the safety and efficacy of the therapeutic over its shelf life.2 In addition, small aggregates can grow into larger ones and eventually become a size and number that exceed the limits of USP <788>.

When working with proteins at any stage of research, development, and production, it is important to understand and control the profile of the smaller aggregates in order to identify the point of aggregation onset. The measurement of small aggregates has historically been investigated using size exclusion chromatography (SEC). However, this technique gives a readout of mass fraction and relies on having 100% sample recovery to be sure of the data profile; even 99% recovery means 1% of a particularly large aggregate may have been lost.3 Subvisible particles do not normally constitute a sufficient mass fraction to be quantified.2 In addition, SEC requires substantial dilution of the sample, which itself can change the aggregation profile. Techniques that can count and size individual species in undiluted therapeutic proteins may be more appropriate than mass fraction methods for studying protein aggregation.

Nanoparticle tracking analysis

Nanoparticle tracking analysis (NTA, NanoSight Ltd., Wiltshire, U.K.) is a method of visualizing and analyzing particles in liquids that relates the rate of Brownian motion to particle size. The rate of movement is related 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. A detailed description of the technology can be found in Ref. 6. Due to the low refractive index of protein, the limit of detection in NTA measurement is approximately 30 nm. This means that the protein monomer units, which are typically in the range of 3–10 nm, are not measured by NTA, but aggregates comprised of just tens of monomers to many thousands can be sized and counted. Since it is not necessary to dilute the sample to obtain the particle size distribution, the aggregation profile is not changed due to sample processing.

Dynamic light scattering

Dynamic light scattering (DLS), or photon correlation spectroscopy (PCS), is a commonly used sizing technique that also has its basis in Brownian motion but, 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 of the sample.7 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 a factor of r6, this allows the protein monomer to be sized, but a few larger particles in a population can greatly skew the data obtained. The technique struggles to resolve very polydispersed mixtures, which, as discussed above, are often observed when measuring protein solutions.

The NS500+DLS from NanoSight combines nanoparticle tracking analysis and dynamic light scattering in the same instrument, providing all the benefits of NTA in high-resolution sizing with DLS, extending the lower detection limit to 4 nm. This means that data from the same protein sample can be obtained to provide analysis of both monomer and aggregates. Figure 2 shows typical data expected from NTA data, DLS data, and the total sample size distribution of monomer and aggregates.

Figure 2 – Typical size distribution profile of a protein therapeutic, with the monomer being measurable with DLS (blue) and the high-resolution aggregate profile measured by NTA (red); the true distribution of the sample is shown in purple.

Using heat (50 oC), 1 mg/mL IgG was aggregated over time, with the particles scattering light increasing in number and intensity when observed in the NS500+DLS fitted with a 635-nm laser (Figure 3). At each time point, the NTA and DLS measurements were taken, and size data for both the monomer and the aggregates could be observed. After 20 min of thermally induced aggregation, the monomer peak described with DLS showed a sphere equivalent hydrodynamic radius of approx. 10 nm, with NTA measuring aggregate particles starting at approx. 30 nm, peaks observed at 50 and 85 nm, and the largest aggregates approx. 300 nm.

Figure 3 – Light scattering images of the aggregation of 1 mg/mL IgG at 50 oC over time.

Since NTA also gives an estimation of particle concentration, the increase in particle number during the time course of the thermal aggregation can also be tracked (Figure 4). These data suggest that for 30 min there are minimal aggregates above 30 nm in size. From 30 to 100 min, the number of aggregates at 30 nm or larger remains stable; after this time, the number of aggregates appears to increase in a more exponential manner. Use of NTA and DLS size data and NTA concentration data from the same sample gives an information-rich solution when studying the complex area of protein aggregation.

Figure 4 – Increase in aggregate number over time for thermally aggregated 1 mg/mL IgG.

Determining onset of protein aggregation

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 and DLS at different points in the process enables scientists to identify the point at which aggregation begins. This point/step can then be reviewed and potentially modified to prevent or slow the formation of protein aggregates. This measurement is now possible with the NS500+DLS conveniently combining NTA and DLS in one unit.


  1. Therapeutic Proteins Market to 2017;
  2. Carpenter, J.F.; Randolf, T.W. et al. Overlooking subvisible particles in therapeutic protein products: gaps that may compromise product quality. J. Pharm. Sci.  2009, 98(4), 1201–5.
  3. Philo J.S. A critical review of methods for size characterization of non-particulate protein aggregates. Curr. Pharm. Biotech.  2009, 10, 359–72.
  4. Philo, J.S.; Arakawa, T. Mechanisms of protein aggregation. Curr. Pharm. Biotech2009, 10, 348–51.
  5. Chi, E.Y.; Weickmann, J. et al. Heterogeneous nucleation-controlled particulate formation of recombinant platelet-activating factor acetylhydrolase in pharmaceutical formulation. J. Pharm. Sci.  2005, 94, 256–74.
  6. Carr, B.; Malloy, A.
  7. Engelsman, J.D.; Kebbel, F. et al. Laser light scattering-based techniques used for the characterization of protein therapeutics. In: Mahler, H.-C.; Jiskoot, W., Eds. Analysis of Aggregates and Particles in Protein Pharmaceuticals. John Wiley and Sons: Somerset, NJ, 2012; pp 43–8.

The authors are with NanoSight Ltd., Minton Park, London Rd., Amesbury, Wiltshire SL4 7RT, U.K.; tel.: +44 (0)1980 676060; e-mail:;