NanoSight

Aggregation Studies

Understanding the degree and kinetics of particle aggregation is a ubiquitous requirement across a diverse range of applications. Particle aggregation can influence a range of parameters such as the efficacy of a therapeutic product, the degree to which it disperses in the body and the biological response to the product etc. Understanding the kinetics of particle aggregation is implicit in understanding the stability of a product with respect to time and perhaps understanding the rate and degree of particle aggregation has implications when applied to the field of biosensors.

The NanoSight technique is a single particle measurement technique, which provides high resolution number versus size distributions, in real time, making it an ideal analytical tool for measuring particle aggregation. Aggregation can be followed in three ways; qualitatively as the sample aggregates – it is clear to the user that the sample has aggregated by viewing the unique images generated by the NTA instrument, by following the change in the particle size with respect to time the kinetics of reaction can be followed, finally by measuring the concentration of particles the concentration of monomer can be measured with respect to aggregates and followed with respect to time. The NTA software can be automated such that measurements can be user defined to track the aggregation process over any time period (minimum discrete measurement time of 10 seconds).

Examples:

Nanoparticle Aggregation in Biosensing

Figures 1 and 2.

Figures 1 and 2 shows a suspension of a mixture of 60nm 3’- and 5’-oligonucleotide-functionalised Au nanoparticles before (Fig.1) and after (Fig.2) addition of a DNA sample which bound to the 20-mer oligonucleotides immobilised on the Au nanoparticles. Mean size was seen to increase from 61nm to 81nm following dimerisation. The quantities of DNA ligand added to induce this detectable level of aggregation were extremely low and potentially providing an extremely sensitive biosensor. These videos were captured following a time course experiment captured in the NTA software. The videos show the start and end point of a 15 minute experiment with videos taken every minute.

Nanoparticle Toxicology

Videos of the nanoparticles were recorded (Figure 2), tracked and analysed for size when diluted in citrate buffer and in human plasma, and corrected for track length (Figure 3).

Figure 3¹: NanoSight results using NTA.

Understanding the degree of nanoparticle aggregation is of paramount importance when trying to understand the biological response to engineered nanoparticles. Understanding how nanoparticles aggregate when introduced into a biologically relevant fluid is essential as this is the state in which the body will be exposed to the nanoparticles. Therefore NTA provides unique understanding in the field, its high resolution particle size distributions offers a unique insight into a complex, aggregating system. Also through fluorescent labelling, target particles can be tracked specifically in the background of a complex biological fluid.

Protein Aggregation

Characterising the state of aggregation in proteins is of paramount importance when trying to understand biopharmaceutical product stability and efficacy. Product quality, both in terms of biological activity and immunogenicity can be highly influenced by the state of protein aggregation.

A wide variety of aggregates are encountered in biopharmaceutical samples ranging in size and characteristics (e.g., soluble or insoluble, covalent or noncovalent, reversible or irreversible). Protein aggregates span a broad size range, from small oligomers (nanometers) to insoluble micron-sized aggregates that can contain millions of monomer units. Protein aggregation can occur at all steps in the manufacturing process (cell culture, purification and formulation), storage, distribution and handling of products. It results from various kinds of stress such as agitation and exposure to extremes of pH, temperature, ionic strength, or various interfaces (e.g., air–liquid interface). High protein concentrations (as in the case of some monoclonal antibody formulations) can further increase the likelihood of aggregation. Therefore, aggregation needs to be carefully characterised and controlled during development, manufacture, and subsequent storage of a drug substance and formulated product. Similarly, by monitoring the state of aggregation, modification or optimisation of the production process can be achieved.

NanoSight offer a new laser-based Nanoparticle Tracking Analysis (NTA) system which allows nanoscale particles, such as protein aggregates, to be directly and individually visualised and counted in liquid in real-time, from which high-resolution particle size distribution profiles can be obtained.

he NanoSight technique allows protein aggregates within the size range of 30 - 1000nm to be individually imaged and sized by tracking their Brownian motion on a particle-by-particle basis. Particle-by-particle analysis allows high-resolution number distributions to be generated. This region is often poorly served by DLS with high concentration of protein monomer and low number of large, bright aggregates often dominating the signal.

Whilst fractionation can be performed such as with FFF to aid DLS analysis, the dilution that is often required for FFF can make this route undesirable due to the potential for further aggregation. Furthermore, dilution of these ‘mid-sized’ aggregates often takes them below the concentration sensitivity limit for DLS. The NanoSight technique frequently requires no dilution as the 30 - 1000nm protein aggregates often fall within the optimum concentration range for this technique.

The cut off limit of the NanoSight technique (approx. 30 nm for protein aggregates) means that it is well suited to complement SEC/DLS or SEC/UV above the exclusion limit of SEC. The upper limit of the NanoSight technique represents the point at which conventional single particle imaging/obscuration techniques become applicable. With no prior separation of aggregates, DLS would typically produce a bimodal result for the aggregated sample shown in Figure 4.

Figure 4

The primary peak would be formed from the large number of monomeric particles, while the secondary peak would be formed by very large aggregates which scatter significant intensities of light. A poorly resolved DLS analysis would show no particles between these points despite their existence as the primary monomeric particles and the few larger aggregates would dominate the signal.

⁽¹⁾Montes-Burgos, Dorota Walczyk, Patrick Hole, Jonathan Smith, Iseult Lynch and Kenneth Dawson, (2010) Characterisation of Nanoparticle Size and State Prior to Nanotoxicological Studies, Journal of Nanoparticle Research, Volume 12, Number 1 / January, 2010 DOI: 10.1007/s11051-009-9774-z