Over the past two decades, we have seen continued advancement of virus characterization and quantification technologies come to market due to the progress made and demand from preclinical and clinical advancement of live viruses, vectored prophylactic vaccines, and gene therapy products to treat infectious diseases, cancer and various genetic disorders. The advancement of virus-derived therapies will be regarded as one of the great biotechnology outcomes of the 21st century. Even now, the number of products in clinical development and those in late-phase and obtaining marketing approval continues to increase annually. These products are also often more complex compared to chemical modalities of the past decades. The necessity to develop novel and robust tools for product characterization is evident, even amongst first-generation biologics comprising hormones, proteins, and monoclonal antibodies.
Challenges in selecting technology for virus particle analysis
Regulatory authorities insist upon clear definition, characterization, and quantification of product potency even in early clinical development. For live viruses, this is at minimum the infectious titer assay (plaque assay, TCID50, Immuno-Cytochemistry-based foci-assay, etc.) however, at least two titer assays (comprising genome copy and total physical particles titers) are generally also a part of the characterization and product release panel.
These analytical methods are highly relevant and comprise titre and a critical quality attribute (CQA) of a produced batch, enabling batch-to-batch quality comparison. It is important to characterize titer robustly with well-defined methods, as these assays define the percentage of virus particles within the vaccine or therapeutic having a therapeutic effect (infectious particles in a dose) and also develop an understanding of titer levels which may contribute to unwanted potential side effects (total particles in a dose).
While methods for genome copy titers are well established (amongst them optical density, HPLC, quantitative PCR, and digital droplet PCR), methods for total particle quantification are still needing improvements and opportunity exists for further establishment amongst analytical testing laboratories and drug development companies.
Assessing the options
For many years, the industry gold-standard for total particle analysis was Electron Microscopy and its derivatives; Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM), and Super-Resolution Fluorescence Microscopy (SRFM). However, for these technologies, investment costs are high, sample preparation is laborious and complicated, and a high level of training is required to use and maintain the equipment. Other methods for particle analysis also include ELISA, but this assay requires development and access to targeted antibody reagents, and is only available commercially for a limited number of viruses used in development.
Malvern’s MADLS Zetasizer Ultra Red
Malvern’s Multi-angle dynamic light scattering (MADLS) Zetasizer Ultra Red, was launched in 2018, and generates data for total particle concentration, particle size distribution, sample polydispersity index and zeta-potential (indicating particle net surface charge). The analyzer can be used to generate GMP batch release data (if good automated manufacturing practice [GAMP] compliance is in place) and for early process development, virus stability and aggregation studies – when carrying out formulation development projects for example.
Compared to many of the other technologies available, analysis of the most common gene therapy vector (26 nm diameter Adeno-Associated Virus), is straightforward. In fact, the instrument is suitable for size ranges of 0.3 nm to 10 µm, and for Zeta potential analysis of 3.8 nm to 100 µm, covering all (known) viruses. The equipment’s set-up requires minimal operator input which minimizes errors and eliminates operator subjectivity. The sample size can be as little as 3 µL and analysis is complete within a few minutes. Scattered light data from 173°, 13° and 90° are collected. The software is very intuitive and automatically generates summary reports containing all critical information and data. Because samples are loaded into closed single-use cuvettes, it is particularly suitable for live virus drug substance and drug product analytics comprising BSL-2/GMO products.
The MADLS technology analyses time-dependence in the intensity of the scattered light (auto correlation) to determine the diffusion speed (Brownian motion) of particles/molecules, and subsequently their hydrodynamic size – a calculation carried out based on the Stokes-Einstein equation. MALDS analysis should be accompanied by Refractory Index analysis and such values can be inserted into the Malvern instruments software settings to maximize the accuracy of particle concentration data. In the case of highly viscous samples, this must also be considered as it changes the particle’s mobility and therefore the size data.
Vibalogics has invested in the MALDS system and we are looking forward to offering this technology to our clients, who are focused on bringing life-saving products through the clinic to the patient. Stay tuned to find out more about further investments in viral analytical technologies and our full scope of services at Vibalogics’ Cuxhaven, Germany and Boxborough, Massachusetts GMP process development, manufacturing, testing, and fill & finish facilities.