Chinese hamster ovary (CHO) cell substrates are widely used for manufacturing biologics, such as recombinant proteins and
monoclonal antibodies, used as human therapeutic and diagnostic agents. These cells are free of infectious endogenous viruses
and have proven to be relatively resistant to contamination by adventitious viruses derived from the environment, manufacturing
staff, and raw materials used in the processes. The few viral contaminants that have been detected with some frequency include the mouse parvovirus (mouse minute virus),
the respiratory/enteric virus reovirus, and the Bunyavirus Cache Valley virus (CVV). We describe the detection of CVV in biologics manufactured in CHO cells over a 10-year period.
BIOLOGICS TESTING EXPERIENCE
From 1996 through 2006, we performed viral safety testing of a variety of biologics manufactured in CHO cells in both bioreactor
and large-scale monolayer culture processes. A major component of such testing, the in vitro virus screen, is required by the US Food and Drug Administration for each manufactured lot of a biologic.5,6 The assay consists of inoculating the biologic bulk harvest samples (taken before purification) onto monolayer cultures
of three or more detector cell lines. Cache Valley virus causes a striking and characteristic response in this assay. It produces
an unusually rapid lytic infection of the detector cells, but does not produce hemadsorption or hemagglutination of red blood
cells. At high viral loads (>106 tissue culture infectious doses/mL), cytopathic effects are observed in the detector cells within 48–72 hours of inoculation.
Total cell lysis rapidly follows initial observation of cytopathic effects, typically within 24 hours.
Figure 1. Transmission electron micrograph of Vero cells infected with the year-2000 Cache Valley virus isolate (V, a particle
apparently budding from the plasma or vesicle membrane; S, external spikes on a viral particle). 99,800x magnification. Reproduced
from reference 14, with permission.
At least three separate encounters with CVV were documented at BioReliance. A biologic was found to be contaminated in the
year 2000. Cache Valley contamination of a cluster of five samples from one client was detected in the year 2003. In 2004,
a separate cluster of four samples from one client was contaminated. The viral infection in each case manifested itself as
the manufacturing process progressed (before collecting samples for viral safety testing). The manifestations included the
absence of cell sheets in the monolayer processes, and perturbations in parameters (oxygen and base consumption) indicative
of death of the cells in the bioreactor processes.
Table 1. Transmission electron microscopic findings for Cache Valley virus isolated from biologicals
Transmission electron microscopic evaluations of cells infected with the year-2000 isolate, the five year-2003 isolates, and
two of the year-2004 isolates were performed during the course of the contaminant identification process. Average viral particle
size, structure, and location in the infected cells were recorded. Viral particles were typically observed in vesicles in
the cell or were observed extracellularly. The electron micrograph of the year-2000 isolate displayed particles with characteristic
external spikes, including one particle apparently budding from a plasma or vesicle membrane (V in Figure 1). The particle
sizes for the year-2000 isolate were in the 80–100 nm range. Viral particle sizes for the isolates from the years 2003 and
2004 ranged from 70–90 nm (Table 1). A micrograph of one of the year-2003 isolates displays numerous 74–86 nm particles located
extracellularly (Figure 2).
Figure 2. Transmission electron micrograph of Vero cells infected with a year-2003 Cache Valley virus isolate (arrow points
to one of a cluster of viral particles). 90,700x magnification.
In each case, the contaminant was ultimately identified as CVV by means of reverse transcriptase–polymerase chain reaction
(RT–PCR) assays using bunyavirus-specific primer sets targeting the S- or M-segment G1 envelope glycoprotein genes, followed
by nucleotide sequencing of the resulting amplicons. Species level identity was obtained by comparing the sequences obtained
for the amplicons to published viral sequence data (GenBank) using basic local alignment search tool (BLAST) searches. For
the year-2000 isolate, 100% sequence identity was obtained for a 198 base-pair amplicon sequence and sequences posted for
CVV (GenBank accession numbers X73465.1, DQ315775.1). For one of the isolates obtained in 2003, a 98% sequence identity was
obtained for a 493 base-pair amplicon sequence and sequences posted for CVV (GenBank accession numbers AF186242, AF231119,
AF231113). A concensus region of 167 base pairs was obtained between the four isolates detected in 2004. There was 99% sequence
identity between the determined consensus sequence and sequences posted for CVV (GenBank accession numbers X73465, DQ315775).
For each BLAST search, the other close matches were all members of the Bunyamwera serotype.