eBook: Analytical Method and QC Testing Strategies for Biologics Production - 22

White Paper: Validation of a
Duplex qPCR Assay to Detect
Residual Sf9 Host Cell DNA and
Baculovirus DNA
Dr. Kara Norman
Introduction
In the bioproduction workflow, residual DNA
quantitation occurs downstream, right before or
after purification. It allows developers to determine
if their manufacturing process minimizes DNA
contamination in the final product.1
The US Food
and Drug Administration (FDA) recently released a
guidance, specifically for gene therapy (Chemistry,
Manufacturing, and Control Information for Human
Gene Therapy Investigational New Drug Applications),
which recommends limiting the amount of residual
DNA for non-tumorigenic cell lines to less than 10
ng per dose and the size of the DNA to be less than
200 base pairs. Other regulatory agencies such
as the World Health Organization (WHO) and the
European Medicines Agency (EMA) also have similar
requirements to ensure the safety and purity of
analytes in biological drugs.
Among the methods of detecting residual DNA, realtime
quantitative polymerase chain reaction (qPCR)
is the most widely used for residual DNA quantitation
due to its sensitivity, accuracy, and precision. It is also
the preferred method noted in the US Pharmacopeia
chapter 509 produced a guidance for host-cell DNA
quantitation in biopharma manufacturing.
This whitepaper discusses the validation of a new,
integrated qPCR solution for the detection and
quantitation of residual Sf9 and Baculovirus DNA to
help meet regulatory requirements.
Sf9 host cell and baculovirus
expression system
The Sf9 insect cell line is a clonal isolate derived
from the parental Spodoptera frugiperda (Fall
armyworm) cell line IPLB-Sf-21-AE, and it is now
being increasingly used as the workhorse of the
baculovirus expression system in the production of
recombinant proteins and vaccines.
In the gene therapy space, there are two main cell
lines used for therapy development: 1) the HEK293
human cell line, which does not require a helper virus
for expression of proteins, and 2) Sf9, which uses the
baculovirus expression system. According to Lisa Yan,
Senior Product Manager, Pharmaceutical Analytics at
Thermo Fisher Scientific, HEK293 cells are currently
the gold standard in the industry, but they come with
some limitations, particularly around scalability.
22

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