The development and manufacture of emerging viral therapies is highly dependent upon accurate infective virus titers as well as total particle counts. But traditional quantification technologies have not kept pace with the needs of the viral vector market. Quantification methods based upon infectious particle counts can easily underestimate total particle counts. Indirect measurements such as enzyme-linked immunosorbent assays (ELISAs) and quantitative polymerase chain reaction (qPCR) are prone to overestimating the number of virus particles because they measure virus components to derive titer rather than enumerating full viruses and particles. Antje Schickert (virus analytics product manager at Sartorius Stedim North America, SSB) delivered an “Ask the Expert” webinar on 17 September 2019 to explain how her companyās Virus Counter 3100 platform enumerates virus particles more quickly, directly, and precisely than traditional methods.
Precise virus quantification is difficult because production process samples are highly heterogeneous, comprised of fully-formed and infectious virions as well as empty and defective particles, unassociated viral proteins, and nucleic acids that were not assembled into virus particles. Depending on the virus and manufacturing process, infective particles can make up as few as 0.0001% of total particle counts. The industry needs rapid full-particle enumeration tactics to meet cell and gene therapy manufacturing demands.
SSBās Virus Counter 3100 system addresses those needs. It is currently the only automated technology designed to quantify virus particles directly. With just the instrument, particle detection software, and quantification reagents, the platform executes a simple, no-wash enumeration in minutes instead of hours. Virus samples are labeled with a reagent and incubated for 30 minutes before being placed in the instrument. The device then guides samples through a laser that excites the fluors used to label the particles. The instrument directly measures the concentration of total viral particles with high specificity from the light emitted by the associated fluors. The ability to distinguish particles from unassociated viral components yields more precise results than other assays.
In a recent collaboration with Yposkesi (Genethon, Corbeil-Essonnes, France), the Virus Counter platform was used to enumerate total particle counts in two lentiviral-vector purification workflows: one for an adherent-cell process, the other for a suspension-cell process. The first compared results from the Virus Counter platform, an ELISA test, and an infectious qPCR assay; the latter compared the 3100 system, an ELISA, and a flow cytometry test.
Virus Counter measurements and ELISA assays tracked well, but the ELISA measured consistently higher than total particle titer determined with the Virus Counter platform. This is likely the result of the detection of free p24 capsid proteins above and beyond intact virus in the sample. Infectious titers for the two processes were analyzed with infectious qPCR and flow cytometry and measured 10 and 1000 fold below total particle concentration. The change in the infectious-to-total particle ratio could be related to the changes in manufacturing processes. Yet it should be noted that functional titer is evaluated by different methods for suspension and adherent processes.
The Virus Counter platform offers a rapid, highly specific method for vector enumeration ā and with significant logistical advantages over competing assays. SSBās system generates results in under an hour, whereas the ELISA takes eight hours to complete. SSB also offers a comprehensive line of ViroTag and Combo Dye reagents to tailor assays to desired viral vectors. With such advantages, researchers can gather crucial drug product information in near-real time and at any stage of manufacturing. Then they can make more informed bioprocessing decisions and be confident about the quality of their vector products.
Questions and Answers
What is the sensitivity of the instrument for virus particles per mL? Its lower quantification limit is 5 Ć 10āµ particles per milliliter of sample.
Can the instrument quantify human papillomavirus (HPV) particles, retroviruses, and other adventitious viruses? HPV detection is not possible currently. The instrument’s quantification limit might prevent adventitious virus detection if the expected titers are very low.
Does serum ever appear in the harvest samples? Serum is often present in sample harvests. We have not observed any interference with the performance of ViroTag reagents.
Can users confirm during an assay whether all viral particles in a sample have been labeled? Yes. Quantification reagents are optimized during product development to guarantee complete staining of viral samples.
The full presentation of this webcast can be found on theĀ BioProcess International website at the link below.