Viral Vaccines Application Notes
Viral vaccine preparations must be proven (validated) to be both stable and to contain known proportions of active elements. NanoSight allows an immediate and direct estimation of product purity and concentration. Similarly, the degree and rate of formation of aggregates in a virus preparation can be simply estimated using NanoSight allowing the manufacturer to develop improved product manufacturing processes and to optimise product shelf life.
As NanoSight allows all particles in the preparation to be visualised and sized, additional information about nanoparticle content is made available in a shorter time than would be available through conventional bio TCid50 or plaque assays. For instance, the presence of larger particles (which NanoSight can both size and count) could represent either non-viral cell debris from the cell culture process or aggregates of virus particles containing many individual virons.
In either case, such aggregates/contaminants represent a possible problem to the manufacturer, one which can be immediately identified with the NanoSight system.
Figure 2. 3-Dimensional plot of particle size vs relative particle insensity vs particle count.
Virus Clearance Studies
Virus clearance is assayed (validated) with high titre spikes of viruses (of various types depending on application). These are then used to challenge process steps which need to be qualified as a clearance step. The data must be obtained with pure, non-aggregated virus spike material which must first be prepared at high titer and stored before use as the spike. NanoSight is ideally placed to establish the status of aggregation both before and after storage.
The ability of NanoSight to rapidly establish the degree to which a virus preparation contains contaminants or aggregates and to be able to quantify the levels of such has proved invaluable to process developers interested in optimising purification protocols for virus preparation. For example, Figure 4 shows the difference between a partially purified virus preparation (white line) and the same sample having been successfully passed through an efficient purification protocol which effectively removed all contamination or aggregated material (red line). Note that the vertical axis represent particle concentration (virus particles/ml).
Bacteriophage-Based MRSA Protection – Phage Therapy
Virus particle detection and counting using NTA is providing essential information for researchers at the University of Strathclyde’s Institute of Pharmacy and Biomedical Sciences (IPBS). This team develops methods to employ naturally occurring bacteria to combat MRSA. MRSA (or Methicillin Resistant Staphylococcus Aureus) is a variation of the bacterium Staphylococcus Aureus which has developed resistance to most antibiotics making it difficult to treat and potentially deadly. Whilst the “Superbug” can be killed with detergents, detergent dilution and application is often inconsistent and ineffective, making the bacteriophage route an attractive alternative.
It is in characterising bacteriophage cultures that NanoSight is employed within a team lead by Dr Mike Mattey, honorary lecturer at IPBS. Prior to deploying bacteriophages as a dry-coat to protect high-risk bacterial invasion sites (sutures, instruments and wounds), the cultures need characterisation and their concentration needs assessing. NanoSight allows the team to view and size viral cultures rapidly in real-time and at low cost. “The characterisation of virus populations requires assessment of aggregation in the 20 nm – 1,000 nm range” says Dr Mattey. “NanoSight provides fast and easy quantitative sample characterisations not possible with other methods and at a much lower cost. Additionally, NanoSight’s technology provides a reassuring view of the particle population that supports the counting results.”
Figure 3: Overlaid particle size distribution plots of a virus preparation before (white) and after (red) a final purification step.
A wide range of virus types have been successfully analysed to date and include:
- Cytomegalovirus (MCMV)
- Porcine papillomavirus
- Lambda phage
- Avian flu stimulant (TMV)
- Murine Leukaemia virus (MULV)
- Japanese Encephalitis virus (JEV)
- Herpes Simplex
(Carr, B. (2009) Nanoparticle tracking analysis: direct visualisation for counting and characterising virus particles in liquids, Meeting the challenge of viral pandemics, 2009 - biotech-online.com)
The ability of NTA to visualize, size and count submicron nanoparticles on an individual basis underlie its potential in a wide range of application areas. In particular, the detection of aggregates and contaminants in proteinaceous biopharmaceutical therapeutic products is both an area of increasing importance and one in which NTA offers a solution to the limitations in current analytical techniques such as DLS. That NTA can both simultaneously and in real-time analyse such aggregate particles while generating high resolution particle size distribution information, is capable of analysing specifically fluorescent or fluorescently labelled nanoparticles serves to increase the information available to the user.
While NTA is limited in its lower limit of detection to approx 30-50 nm for viruses the addition of DLS data enables analysis down to 4 nm. Therefore the NS500+DLS instrument will give a far more realistic assessment of the true distribution of aggregating (or not) proteinaceous samples and is attracting increasing attention. See Protein Aggregation web page and application note for more information.