APR September/October 2020 - 108

MICROBIOLOGY

Rapid Methods for Adventitious Virus Detection
Acceleration of Clinical Timelines/"Speed to Patient"
Jürgen Müllberg
Principal Scientist / Viral Safety SME
Bristol Myers Squibb, Devens MA

Abstract
Contamination of a biological product with viruses, potentially harmful
for humans, is of concern for therapeutics derived from human and
animal sources, i.e. plasma, and for protein therapeutics and vaccines
produced in mammalian cells. More recent "Advanced Therapeutic
Medicinal Products (ATMPs)", such as gene and cell therapies, share
the same concern. Tests for viral contaminants are performed at
several stages of manufacturing. Current tests for detection of viruses
are lengthy; cell culture-based assays and animal experiments are
required. Alternative molecular tests, such as "next generation
sequencing (NGS)", provide faster results, and can eventually replace
in vivo animal studies. It is noteworthy that identification of a viral
contaminant that evaded detection in classical tests, by NGS, has been
demonstrated. Since NGS-based virus tests yield results significantly
faster than current assays, additional applications for in-process
testing and viral contamination control can be envisioned.

Viral Safety of Biologics
A potential viral product contamination is a concern for therapeutics and
vaccines either derived from human or animal source material, or manufactured in mammalian cells that are vulnerable to infection by viruses.
Requirements for manufacturing and safety of medicinal products, including viral safety, are outlined in the Code of Federal Regulations (CFR,
title 21), as well as in the US and European Pharmacopeia.
Three major pillars contribute to assurance of viral safety of products,
i.e. biologics (Figure 1). The first component is "Prevention" of
introduction of a viral contaminant into the manufacturing process.
Preventive measures include omission of raw materials of human or
animal origin from the process, audits of suppliers of consumables
and raw materials, design of the manufacturing facility, and
procedural controls for e.g. aseptic handling, gowning and material

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flows. "Testing" for viral contaminants at various stages, i.e. cell
banks and cell culture harvests, is another key component. The third
pillar is "Reduction", the process's capability to inactivate or remove
potentially present viral contaminants. Purification processes, aiming
to separate the product from impurities, have to include steps with
validated viral clearance capacity.

Current Methods for Viral Safety
Testing of Biologics
Acknowledging that products purified from human or animal material,
e.g. plasma, need to be tested for viral contaminants of concern using
appropriate assays, the focus of this article is virus detection methods
applicable to mammalian cell culture processes, i.e. Chinese Hamster
Ovary (CHO) cell processes.
Current test methods for evaluation of viral safety of biotechnology
products derived from cell lines of human or animal origin are outlined
in ICH Q5A, current version of guidance published 1999. A similar
guidance document for viral vaccines manufactured in cell culture was
published by the Center for Biologics Evaluation and Research (CBER)
in February 2010.
For the vast majority of biologics manufactured in mammalian cell
culture processes, the expression host is CHO cells. Table 1 lists methods
for detection of virus contaminants in CHO Master Cell Banks (MCBs).
A variety of tests is applied, including indicator cell-based infectivity
assays, in vivo animal studies, and molecular (Polymerase Chain Reaction
(PCR) based) tests. Several of the assays aim to detect adventitious
virus contaminants, possibly introduced during cell bank manufacture,
e.g. through the manufacturing environment, an operator, or a
contaminated raw material. Tests for specific animal viruses (e.g. porcine
and bovine viruses) can detect contaminants host cells have been
exposed to during their cell line history. At an earlier time, CHO cells
might have been cultured in media containing fetal calf serum, or when

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APR September/October 2020

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