Such virus-detection assays are also used to evaluate specific contaminants, such

as retroviruses (endogenous to the cell line), bovine, and porcine viruses.

In the context of the 3Rs program (to Replace, Refine and Reduce [3Rs] the use

of animal in the safety evaluations of products) the sensitivity and scope of current

assays to detect adventitious agents have been reviewed [28] and suggests that in

vitro assays are more limited than in vivo assays. Several proposals were made to

ameliorate the current in vitro assays, including the potential inclusion of additional

cell lines and the integration of new molecular methods.

4.4.1.2

New Technologies

New assays for detecting adventitious agents are being developed to have higher

sensitivity or to detect new adventitious agents.

New technologies, based on nucleic-acid methods such as PCR, NGS, virus

microarrays, and broad-range PCR combined with mass spectrometry are powerful

tools for the detection and identification of viruses.

4.4.1.2.1

PCR-Based Methods

Today, the PCR-based assays are already widely used in the GMP environment, for

routine activities in the detection of specific adventitious agents, such as viruses

(PCV, MVM, Hep E) and bacteria (mycoplasma, mycobacterium).

In contrast to other nucleic-acid–based methods, such as quantitative PCR

(qPCR), or hybridization-based assays, NGS provides a sensitive and quick plat-

form assay for virus detection and identification in various types of biological

sample, without prior sequence knowledge of potential viral contaminants [29].

Several NGS platforms are currently available, and these platforms have the ca-

pacity to generate a huge amount of sequence data. Two main NGS platforms are

available today, primarily based on the read length; either short or long. Each

system shows strengths and limitations, related to the number of reads, read length,

error rate, run duration, and the complexity of the library construction. Compared

with short-read platforms, which show a limited capacity to detect new viruses due

to the length of the read, the long-read platforms are more able to identify new

adventitious agents. Bioinformatics tools including tools to manage the pipeline,

data quality, and data trimming/cleaning and data assembly, help establish the key

parameters for the breadth and accuracy of virus detection.

In contrast to conventional adventitious agent detection methods, nucleic-

acid–based methods report the genome copies/reaction or per volume present in the

test article. Because viral preparations contain noninfectious or defective viral

particles, in addition to infectious particles, the quantity of nucleic acid may not

match with the titer of infectious particles. The same considerations have been

largely discussed in the comparison of compendial tests for mycoplasma with PCR-

based assays.

4.4.1.2.2

Microarrays

Microarray technology uses oligonucleotide probes for the detection and identifi-

cation of known viral sequences. In contrast to NGS, the bioinformatic analysis is

performed during the probe-design phase, meaning that the analysis mainly consists

Cell lines for vaccine production

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