GENERAL BACKGROUND

• Virus Counting (Total Particle Count)
  
  
• Virus Sizing
  
  
• Virus Sample Characterisation (Aggregation / Impurity Profiling)
  
  
• Polydisperse and Monodisperse Preparations (Vaccines / Proteins / Plasmids)
  
  
• Protein Aggregate Profiling
  
  
• Adjuvant Characterisation

 ...in a couple of minutes...

Total virus count, aggregation profile, sample characterisation...accurate and quick with Nanoparticle Tracking Analysis, and most importantly, with typical polydisperse samples!

Nanoparticle Tracking Analysis (NTA) is a NEW and powerful technology, developed to offer both particle sizing and profiling as well as counting of viruses and aggregates in suspension. Protein aggregates, which frequently can involve complexes with other materials, eg DNA etc, can be visualised above about 30nm or so. Liposomes and other nanoparticles can also be seen.

NTA enables the user to quickly characterise either polydisperse or monodisperse samples within a couple of minutes, with little or no sample preparation.

How does NTA do this?

Samples of semipurified or purified virus or vaccine preparations are simply injected into an optical unit and approximately 2nl is examined for laser light scattered from individual nanoparticles between about 30nm and 1000nm within this field of view.

All particles are measured for diffusion speed due to Brownian Motion which provides a direct measurement of particle size, while individual particle identification permits a direct count of every particle. This data is then plotted as a particle size distribution curve.

How does NTA differ from traditional Dynamic Light Scattering (DLS)?

NTA identifies and evaluates individual particles, thus providing both accurate and fast sample profiling, together with a direct count. DLS (also known as Photon Correlation Spectroscopy or PCS), uses data from all particles to provide an average value of Brownian diffusion. Larger particles scatter more light than smaller particles (intensity varies with ratio of radius to log6) which weights data dramatically in favour of larger particles within polydisperse samples. Individual virions are not measured correctly as a result, even when present in massive excess.

DLS provides acceptable data for monodisperse samples, but only NTA can offer accurate profiling of polydisperse or lower purity preparations.

Why is NTA the most complete particle characterisation method for virus and vaccine preparations?

Biological nanoparticle preparations such as vaccines and other virus preparations are almost impossible to purify to monodispersity. Residual nanoparticulate host cell debris and a variable tendency towards aggregation typically result in degrees of polydispersity that demand characterisation and control during the manufacturing process. Electron Microscopy is known to present artefacts resulting from drying and other sample preparation procedures and cannot distinguish between such artefacts and naturally present aggregation. Density Gradient Centrifugation demands large volumes of sample, takes significant time and involves costly  equipment. DLS (PCS) is inherently unsuited to polydisperse samples and does not provide a count.  Apart from EM, measurement is essentially indirect.

Only NTA offers the speed, direct measurement (both size and count) and simplicity for characterisation of such polydisperse preparations, combined with low sample consumption (less than 0.5ml).

Current technologies used for vaccine and virus preparation include centrifugation, filtration and chromatography. It is possible to follow the step operations to final product characterisation with Nanoparticle Tracking Analysis, facilitating process optimisation or monitoring, and also product formulation and storage.

The range between 30 and 1000nm is ideally suited to characterisation of ~99% of all virus, VLP and even larger protein aggregates, and the limits of detection between 10⁷/ml and 10¹⁰/ml are such that most samples can be evaluated directly with no sample preparation or with simple dilution.