Monitoring Adenovirus Infections with On-Line and Off-Line Methods

Abstract

Several known process monitoring methods were tested for their efficacy in the detection of adenovirus infections. The methods that we explored include several indirect indications of viral infections, including metabolic rate analysis, secondary gauges of respiration, cell size measurement, cell number and cell viability determination, and changes in capacitance. Direct indications of the adenovirus infection were also applied, including total viral particle and infectious particle measurements, as well as a flow cytometry method for detecting infected cells. All of the methods tested in the study provide some positive indication of an adenovirus infection. Many of the methods require repeated sampling, which may limit their utility in a manufacturing process. All of the indirect measures of viral infection may be limited by the fact that they do not uniquely identify an infection. The simplest monitoring methods appear to be detection of changes in respiration or the capacitance of the culture, both of which seem to provide a clear indication of an infection. Further work will be required to demonstrate that these indications are characteristic of only a successful and productive adenovirus infection.

Monitoring of the Adenovirus Production Process by Flow Cytometry

“…Monitoring of virus productivity with FC (FS parameter) will prove to be of immense practicability when no marker or reporter proteins are available, and as indicated before, such proteins are not desirable in vectors destined for human therapeutics. Also, the results shown here and other reports of cell size increase of Ad infected cells indicate that the utility of FC may have a wider remit with regard to monitoring production processes, including adherent and suspension cultures (15, 16).…”

“…Alternatively, the FS parameter measured by FC is not dependent on cell density and therefore would provide a more conclusive measurement of cell size increase attributable to virus infection and propagation. Monica et al (16) did show an increase in capacitance post‐infection corresponding with no increase in cell density, which demonstrates the utility of the capacitance probe to monitor infection; however, this work was done with relatively high MOIs of 10−50, which still leaves a question of its utility at low MOI infections. Figure 6b shows the relative mean FS increase with infection at a low MOI of 1.…”

“…To our knowledge there is no reported use of granularity of Ad packaging cells as a parameter to monitor Ad infection. There also seem to be only a couple of reports regarding change in cell size upon Ad infection (15, 16). Henry et al (15) wrote that during the viral protein synthesis phase, the size of 293 cells can increase from 14 to 16 μm, leading to a 50% augmentation of the cell volume, and showed an increase in cellular volume of infected 293 cells using an on‐line measurement of capacitance.…”

“…Recently, several methods have been proposed to assess the progress of Ad infection based on GFP reporter, oxygen demand and cell membrane capacitance (15)(16)(17). However, some of these parameters are not easily measured other than in bioreactors with specialized monitors, for example, fluorescence and biomass monitors that can be very expensive.…”

Production of Adenoviral Vectors in 293 Cells: A Case Study of the Adaptation of Attached Cells to Grow in Suspension

“…The production of adenoviral vectors for gene therapy applications has also been reported using *Address correspondence to this author at the National Institutes of Health, National Heart, Lung, and Blood Institute, 9000 Rockville Pike Bldg 10, Rm 7D04, Bethesda, MD 20892, USA; Tel: +1-301-496-6104; Fax: +1-301-496-9985; E-mail: negretea@nhlbi.nih.gov attached or clumped 293 cells in serum contained medium (SCM) [12,14] and serum-free medium (SFM) in 293 cells grown in suspension [15,16]. According to the information available in current literature the production of adenoviral vectors in 293 cells grown in suspension in SCM in a 20 L bioreactor [17] and in a customized SFM in a 10 L bioreactor [18] has been reported. However, with the customized SFM a maximum yield of 10 3 infective particles was obtained at 48 hours post infection (hpi).…”