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Viral Safety Are You Doing Everything

to Mitigate Your Risk?

CO N T E N TS
 dvances in Upstream  iral Safety in
PREVENT REMOVE
5 A 20 V
Technologies Reduce Monoclonal Antibody
Viral-Contamination Manufacturing
Risks
PREVENT Tutorial 25 Laying the Foundation
10 Upstream Virus Safety: Protect for Viral Safety
5 Your Bioreactor by Media Filtration
30 Infographic: Virus Safety
DETECT
20 33 Poster Gallery
13 Keeping Up with
34 Webinar Corner
Viral Safety Trends
in Bioprocessing 35 Product Showcase
Copyright © 2018 by GEN Publishing Inc., New Rochelle, NY. All rights reserved.
DETECT Tutorial
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Assessment of Biological Products Cover photo: Betelgejze / Getty Images
 Welcome
Dear GEN Readers,

Viral safety is a constant concern for all biopharmaceuticals as a contamination can

cause significant regulatory concern and business disruption.

A multilayered approach to virus safety across the full production landscape involves
treating raw materials to prevent virus from entering the upstream process, testing
intermediates to detect virus, and implementing purification and filtration technologies
to remove virus downstream.
John Sterling Darren Verlenden
Editor-in-Chief, Vice President,
GEN Bioprocessing, Recognizing there is no single solution that works for every process, experts from
MilliporeSigma
MilliporeSigma are hoping to stimulate discussion by highlighting different aspects of
viral safety for well-established platforms and newer virus-based therapies. We hope you
find these perspectives infomative and look forward to working with you to solve the
Listen Now Listen Now toughest viral safety challenges.
 Viral Safety PREVENT

Advances in
Upstream Technologies
Reduce Viral-
Contamination Risks
Multilayered Approach Includes Virus-Resistant CHO Cell Lines,
Advanced Filtration Technologies, and Careful Raw Material Selection

K. John Morrow, Jr. Ph.D.

Viral contamination in biopharmaceutical processes is
the enemy within—creating major problems for the
biomanufacturer and significant potential risk for
patient safety. Contamination constitutes a major
challenge to the biopharmaceutical industry, and is
now being vigorously attacked on multiple fronts.
On the upstream end, the CHO cell line that has been
the workhorse of the biologics industry for decades
is being reinvented to improve the line’s robustness
and performance. At the same time, improvements
in treatment and testing of media components have
dramatically lowered the risk of introducing virus
contamination to the downstream process. This multi-
Viral Risk Mitigation
layered approach has dramatically lowered the risk of Strategy
catastrophic failures.
Engineering Better Cell Lines
According to Joaquina X. Mascarenhas, Ph.D., team lead,
host cell-line engineering at MilliporeSigma, CHO cell
lines are the preferred host-expression system for many
therapeutic proteins, such as antibodies, hormones, Kevin Kayser, Ph.D.
and blood factors. The team's focus is on manipulating Head of Upstream R&D,
sublines of CHO cells to enable them to work faster and Listen Now MilliporeSigma
more effectively for biomanufacturing purposes.

5 | October 1, 2018
 Viral Safety PREVENT
Advances in Upstream Technologies Reduce Viral-Contamination Risks
“We are moving toward next-generation
expression systems for the manufacture of
recombinant therapeutic proteins and
vaccines with superior attributes, such as
improved product quality, higher productivity,
and shorter development timelines,”
asserted Dr. Mascarenhas.
Dr. Mascarenhas led a team of scientists that
developed a genetically engineered CHO
host cell line refractory to viral contamina-
tion. She and her colleagues have employed
genetic engineering to make the CHO line
resistant to the parvovirus, minute virus of
mice (MVM). This was accomplished by
modifying the major receptors used by
the virus to enter the cell. “Our goal was to
ensure that the cell lines were inherently
resistant to MVM contamination, as this is
the cell’s last line of defense,” she
explained.
In a peer-reviewed study, the group reported
that having recognized the role of sialic acid
in mediating virus entry into the cell, they
developed a cell line with modified sialic acid
glycan structures which lacked the ability to
bind MVM. The development of this cell line
has resulted in the first commercially avail-
able gene-editing approach for the creation
Upstream suite in a mAb manufacturing plant. A major component
of MVM-resistant CHO cells: Centinel
of a virus safety strategy is preventing viruses from entering the
Intelligent Virus Defense™ technology.
upstream process.
Dr. Mascarenhas explained that while a
majority of the known viral contaminants
in CHO lines were introduced through con-
taminated animal-derived components,
6 | October 1, 2018
MVM contamination continues to be a
threat, even in chemically defined processes.
Although it is impossible to completely
remove the risk of viral contamination, using
CHO cells that are resistant to one of the big-
gest threats to the industry should greatly
enhance even the most comprehensive
viral-mitigation programs in use.
Ensuring Product Quality
“Companies need to implement a multilay-
ered approach for their viral risk mitigation
strategy,” explained Kevin Kayser Ph.D., head
of upstream R&D at MilliporeSigma. “For ex-
ample, we are constantly evaluating our raw
materials used in cell culture-media manu-
facturing. We establish robust procedures for
the selection and approval of vendors and
raw materials to aid in this risk mitigation.”
According to Dr. Kayser, viral contaminants
ordinarily enter the process train from
various sources, such as cells (endogenous),
raw materials (serum), or manufacturing
processes.
“Companies cannot detect viral contamina-
tion at the level of one viral particle per liter,
yet it is known that this is sufficient to infect
a biologics manufacturing run, causing
contamination and loss of the product,” Dr.
Kayser explained. “The industry’s sampling
and detection methods aren’t capable of this
low-level detection. We take the risk very
seriously, and use a variety of strategies to
mitigate against viral contamination risk.”
 Viral Safety PREVENT
Advances in Upstream Technologies Reduce
Viral-Contamination Risks
Technologies such as high temperature
short time (HTST ) or virus filtration
designed specifically for upstream media
components provide alternatives for
upstream viral safety.
In considering the future direction of the
cell lines used in biologics production,
Dr. Kayser believes that modified and
engineered versions of the CHO line will
be the protein synthetic factory of choice
for the foreseeable future. “CHO cells
have proven to be a very successful tool
in protein drug production,” he stated.
“Yes, there are alternatives that people
are thinking about, but certification
of a new cell line is a costly and time-
consuming process and there would
have to be a powerful incentive in order
to embark upon this course of action.”
To date there have been no
instances of disease-causing
organisms transmitted through
biotechnologically designed
therapeutics.
7 | October 1, 2018
Virus-risk reduction may be one of those
powerful incentives.
Dealing with adventitious agents in
culture systems is a work in progress.
“We are always trying to find process
changes that will save costs and ulti-
mately lower the economics of drug
production,” Dr. Kayser asserted. “The
field is constantly evolving, and new
adventitious-agent threats are always on
the horizon. I foresee there will be ad-
vances introduced in the near term that
companies will employ to control viral
and other contaminating agents.”
Focus on Biosafety Issues
“In advising customers on production
issues, we employ what we refer to as
The mechanism of action for viral resistance in a Centinel
Intelligent Virus Defense™ modified CHO line.
 Viral Safety PREVENT
Advances in Upstream Technologies Reduce Viral-Contamination Risks
Survey by the CAACB
“Viral contamination events may cause
significant impact on a bioproduction
facility,” according to Paul W. Barone, Ph.D.,
director of the Consortium on Adventitious
Agent Contamination in Biomanufacturing
(CAACB) at MIT. “For this reason, our con-
sortium focuses on collective learning to
prevent these events in biopharmaceutical
manufacturing.”
The CAACB provides a forum for network-
ing, sharing experiences, collaborating on
projects and promoting new technology
to mitigate the risk of a contamination.
According to Dr. Barone, viral contamination
of mammalian cell lines is hardly an aca-
demic exercise. From a survey performed
by the consortium, the group is aware of
26 contamination events, the majority of
which occurred in the CHO cell line.
“Given its ubiquitous presence in biopro-
duction protocols, this is not surprising.
The consequences of these events affect all
aspects of a company’s production facility,
and can be catastrophic, costing millions of
dollars and causing shutdowns for months,”
he explained. “Moreover, contamination
events have been reported in all stages
of development, from preclinical through
commercial, with the cost of their mitiga-
tion rising astronomically in the late stages.”
8 | October 1, 2018
It is noteworthy that for the CHO cell
cultures, even though all virus contami-
nants were suspected to have come from
culture-media components, testing did
not eliminate the risk of contamination—
an observation highlighting the difficulty
of detecting very low levels of contamina-
tion.“ In at least one instance, a non-animal
raw material was directly identified as the
source of the virus contamination.
“As a way to reduce the risk from different
media components, the consortium has
evaluated the effectiveness of different
technologies to remove or inactivate
viruses in media,” Dr. Barone explained.
“UV-C irradiation, physical separation using
filtering devices, and heat treatment were
all, in general, found to be effective.”
On the other hand, for human and primate
cell culture, the source of virus contami-
nants was attributed to human sources.
Dr. Barone said he is proud of the
bioindustry’s long-term record.
“In 30-plus years of cell culture-based
biopharmaceutical manufacturing, no
recombinant DNA-derived product has
been shown to transmit a viral safety
problem,” he pointed out. n
the biosafety triangle: prevention, detection and removal,” explained
Darren Verlenden, Vice President of Bioprocessing at MilliporeSigma.
“Because we monitor the guidelines of regulatory bodies worldwide,
we can assist customers in the interpretation of directives established
by different countries.”
Verlenden expanded on the comments of the other interviewees. “We
have been developing this pathway over the last few years,” he noted.
“We recognize that newly emerging companies might not have the
level of resources of more established clients, so we have to tailor our
responses to match the client’s individual situation. The approach is
really holistic in which we assist the customer to understand and
quantify risk. We help evaluate different risk mitigation approaches
dependent on customers risk perception.”
To prevent virus entry into production processes, Verlenden stresses
that it is essential to carefully monitor extraneous input, as contami-
nants are invariably introduced through outside sources. “This means
we have to look very closely at suppliers, and be assured that they have
a long-standing pattern of mature quality control, and tight warehouse
A Tailored Approach
to Viral Safety
Darren Verlenden
Vice President
Bioprocessing,
Listen Now
MilliporeSigma
 Viral Safety PREVENT
Advances in Upstream Technologies Reduce Viral-Contamination Risks
management, which is especially critical.” Advances
in preventing virus entry into upstream purification
increase confidence for viral safety in your purified
product.
The Expanding Power of New Technologies
Increasing awareness of the risks associated with up-
stream vial contamination has fueled development of
products and technologies to meet the needs of today’s
biomanufacturing processes. New filtration technology,
capable of removing virus, mycoplasma, and bacteria,
enables efficient processing of cell culture media before
entry to the bioreactor. Importantly, these novel filters
don’t change the properties of the cell culture media
and provide an easy-to-implement solution that can
be integrated into upstream processes.
Colette Côté, Ph.D., expanded the discussion on recent
technologies describing next-generation sequencing
(NGS) or massively parallel sequencing (MPS) in detect-
ing viral contaminants. “It has proven to be a powerful
tool in the maintenance of sterility from early develop-
ment to the final product,” she explained.
NGS, and the power of computers to analyze and
search bioinformatics databases, is a robust new tool to
complement more traditional cell-based methods for
virus detection. It is not limited to virus detection, but
9 | October 1, 2018
will detect a broad range of adventitious agents such
as bacteria or mycoplasma without assumptions of the
nature of the agent.
“My colleague, Dr. Arifa Khan at the U.S. FDA, provided
an interesting example” pointed out Dr. Côté. “They used
NGS to identify a rhabdovirus contaminant in the sf9 cell
line from Spodoptera frugiperda, an important cell line
used in biotechnology in protein production protocols.”
Because sf9 is an insect cell line, it would be expected
to present fewer safety issues during protein produc-
tion than mammalian cells. Identification of this virus
contaminant by NGS was critical to risk mitigation in
the sf9 system: “We find that our bioinformatics
capability enables us to answer questions asked by
the entire industry in a much shorter time frame than
many of the traditional assays,” said Dr. Côté.
It is important to note that NGS or MPS is a so-called
“reactionary tool.” Dr. Côté described it in this fashion:
“Say your bioreactor just crashed and you want to know
why, without making assumptions as to the cause. You
can analyze your material in an unbiased fashion, quickly
and in an affordable manner.”
To facilitate sequence analysis, Dr. Côté described an
essential one-step identification tool, referred to by
the acronym BLAST. Developed by the National Center
for Biotechnology Information, the Basic Local
Alignment Search Tool is remotely accessible and
accepts sequence inputs, comparing them to the
local database of sequences on record. Widely used
in the virus-identification process, it has proven
invaluable in rapid identification protocols for viral
contaminants. The software also includes tools for
identification of mutational changes in target sequences
that may have been introduced during the PCR
amplification process.
Conclusions
While workers in the field recognize that total elimina-
tion of viral contaminants at the upstream end of the
process is not possible, it is clear that new technologies
have lowered the risk to manageable levels. Whereas in
the past there have been instances of virus transmission
to patients through contaminated plasma and blood
samples, subsequent improvements to screening
procedures and downstream purification operations
have reduced this risk.
To date, there have been no instances of disease-
causing organisms transmitted through recombinant
DNA-derived therapeutics; a strong endorsement of the
step-by-step improvements that we have seen over the
years within the industry, and a positive harbinger for
the future. n
 Viral Safety PREVENT Tutorial
Upstream Virus Safety:
Protect Your Bioreactor by Media Filtration
Christina Carbrello Ph.D., David Nhiem,
Mary Priest, Kimberly Mann, and
Trish Greenhalgh Ph.D.
MilliporeSigma
Biopharmaceutical manufacturing processes
involve a multilayered approach to microbial
and virus testing to assure that the drug
product is safe for human use. Screening raw
materials, testing in-process intermediates,
and demonstrating the virus removal
capabilities of the downstream process
are critical to biosafety assurance.
However, despite careful screening of raw
materials, there remains a risk of introducing
adventitious agents into bioreactors, which
could impact manufacturing operations,
cause significant business disruption, and
ultimately threaten drug supply to patients.
Various technologies may be used to mini-
mize this risk. One of these, filtration, is a
point-of-use operation that is easy to imple-
ment in the upstream process. This tutorial
summarizes the performance of a filter spe-
cifically developed for virus removal from
chemically defined cell culture media.
10 | October 1, 2018
The Viresolve® Barrier Filter removes high
levels of virus, mycoplasma, and bacteria
without impacting cell growth, antibody
titer, or protein quality. The filter has robust
performance over a broad range of condi-
tions and offers an effective, easy-to-imple-
ment solution for media treatment.
Risk reduction for viral contamination of
upstream processes has traditionally relied
on careful sourcing of raw materials, screening
cell banks for adventitious virus, and
control of facilities and workflow. Despite
these precautions, bioreactor contamina-
tions have occurred, resulting in significant
disruption and cost for the companies
involved. More recently, other options for
virus reduction in upstream applications
have been employed, but generally
Table.
require costly investment and are often
not suitable for all media components.
Filters specifically designed for
upstream processing offer an alternative
to capital-intensive methods, using
proven membrane technology to
assure robust, broadly effective,
size-based virus removal (Table).
 Viral Safety PREVENT Tutorial

Upstream Virus Safety: Protect Your Bioreactor by Media Filtration

The Viresolve® Barrier Filter can be integrated into Figure 1.

single use or stainless steel processes, and can be used
in place of a 0.1 µm or 0.2 µm sterilizing-grade filter;
high retention (above the detection limit) has been
demonstrated for large virus, mycoplasma, and
bacteria (Figure 1).

The graph (Figure 2) illustrates sustained high level of

retention for minute virus of mice (MVM), a relevant
small virus contaminant, during extended processing
times for two different membranes. Membrane made
near the limit of the manufacturing window shows
MVM retention of approximately four logs sustained
over 8 hours of processing. Typical nominal membrane Figure 2.
shows over five logs of MVM retention over the same
time period. ➜
Virus retention across the Viresolve® Barrier Filter was
evaluated at a range of processing conditions with
different representative cell culture media. The filter is
designed to retain a minimum of four logs Phi-X174,
which is used as a surrogate organism for MVM. Virus
retention remains high across a range of operating
pressures and pH levels, even after extended
processing times (Figure 3).

Virus filters designed for downstream applications are

inefficient for processing cell culture media. (Figure 4A). Figure 3.
The Viresolve® Barrier Filter leverages the proven
technology of the Viresolve® platform with asymmetric
polyethersulfone (PES) membrane technology and a
novel secondary chemistry formulated for optimal
processing of chemically defined media. This unique
filter provides good volumetric throughput across a
range of “off the shelf ” and proprietary media
(Figure 4B).

11 | October 1, 2018
 Viral Safety PREVENT Tutorial

Upstream Virus Safety: Protect Your Bioreactor by Media Filtration

Comprehensive analysis (mass spec or amino acid and

soluble vitamin HPLC, NMR, and ICP-OES) of two cell
culture media and their respective feeds before and
after filtration through Viresolve® Barrier Filter indi-
cated no changes in media composition that could be
attributed to filtration with the Viresolve® Barrier Filter.

Cell culture was performed using filtered media

in shake flasks (Cellvento™ CHO-200 medium
with MAb01) or Mobius® 3L Bioreactors (Ex-Cell®
Advanced™ CHO medium with MAb02). No significant
changes in viable cell densities (Figure 5A) or antibody
titers (Figure 5B) were observed, and analysis of
antibody charge heterogeneity, aggregate profile,
Figure 4. and glycan profile indicated no changes as a result
of the cell culture media filtration (not shown).

Summary
Risk-based analysis of bioprocess manufacturing pro-
cesses highlights weaknesses in design and, therefore,
offers opportunities for improving specific elements
that can impact virus safety. The Viresolve® Barrier
Filter is specifically designed to reduce risk early in
production by adding a final layer of protection before
the bioreactor, enhancing existing materials sourc-
ing, selection and facility control processes. The filter
is easy to use, does not impact cell culture processes,
and provides a high level of virus removal across a
range of conditions, increasing confidence that micro-
Figure 5. organisms will not be introduced to the bioreactor. n

Christina Carbrello, Ph.D., is a senior scientist; David Nhiem

is a development engineer; Mary Priest is a lab manager;
Kimberly Mann is a senior scientist; and Trish Greenhalgh,
Ph.D., is an R&D manager, MilliporeSigma.

12 | October 1, 2018
 Viral Safety DETECT

Keeping Up with
Viral Safety Trends
in Bioprocessing
Next-Generation Sequencing (NGS)
and Quality by Design (QbD)

Kristen Slawinski, Ph.D.

Viral safety is a key component to assuring the safety
of biopharmaceuticals for human use. Manufacturers
need to verify that their products are free of harmful
viruses, while regulatory agencies are tasked with
upholding standards for viral safety for all types of
biopharmaceutical products. As live cells are used to
express the biopharmaceutical product, and many
of the components used in cell growth are of animal
origin, the risks of virus contamination are high.
New technologies and methodologies are emerging
for both producing and testing biopharmaceuticals,
so the industry is entering a new era of viral safety
assurance.
Cell Culture Media
The High Cost and Likely Suspects
of Viral Contamination
Filtration
“If virus is detected anywhere in a biomanufactur-
ing process, the entire process must be shut down
and an intensive investigation embarked upon,” says
Bala Raghunath, Ph.D., director of global manufac- Martin Wisher, Ph.D.
turing sciences and technology at MilliporeSigma. A Global Head of
contamination incident in 2009 triggered a facility Regulatory Affairs
decontamination, extensive investigations of root Focusing on the
cause, and penalties from concerned health authori- BioReliance® Portfolio
Listen Now
ties, not to mention the significant loss in revenues as of MilliporeSigma
a result of the plant shutdown. “This impacted supply

13 | October 1, 2018
 Viral Safety DETECT
Keeping Up with Viral Safety Trends in Bioprocessing
of critical drugs which, in turn, affected
patient access,” says Dr. Raghunath.
Several reports detailed the incidence
of this contamination.
Raw materials have often been impli-
cated in viral contamination incidents
and there is a general acceptance of their
vulnerability to virus contamination.
Testing cell culture media for the pres-
ence of virus is inherently constrained
by assay sensitivity and an inability to
detect low levels of virus contaminants.
As a consequence, manufacturers are
considering adding steps to inactivate
or remove potential virus contaminants
from cell culture medium and other raw
materials used in upstream manufactur-
ing processes.
“Some companies have evaluated high
temperature short time (HTST) pas-
teurization methods to kill pathogens in
cell culture media,” says Dr. Raghunath.
“However, not all media components
are stable at high temperatures. Further,
HTST requires some significant initial in-
vestment and equipment, so only a few
companies have taken that approach.
More recently, companies have devel-
oped novel filters designed for cell cul-
ture-media treatment. Cell culture media
is at risk for introducing viral contamina-
tion into a bioreactor, so having that viral
filtration step is considered a good way
to enhance viral safety assurance in the
manufacturing process.”
14 | October 1, 2018
In addition to cell culture media, cell
banks used for manufacturing biothera-
peutics are also considered high risk for
introducing contamination and need
to be well characterized before starting
manufacturing.
“There is extensive characterization of
the master, working, and end of produc-
tion cell banks before bioprocessing,”
said Kathryn Remington, Ph.D., principal
scientist focusing on the BioReliance®
portfolio of MilliporeSigma. “Companies
need to verify that the cells are the type
that they think they are and also look for
purity of the cells. They’ll then need to
be screened for bacteria, fungi, myco-
plasma, and viruses.”
Martin Wisher Ph.D., global head of
regulatory affairs focusing on the Bio-
Reliance® portfolio of MilliporeSigma
added that “in vivo studies need to be
Scientists involved in performing cell line characterization testing.
completed in the master cell bank and
end of production cell line. You want to
be sure that your cell banks are free of
viral contamination. Testing the bulk har-
vest with in vitro assays gives you further
evidence that the cell banks were clear
and gives you assurance that there is no
other contamination coming in through
the process.”
Regulatory testing standards for cell
banks and bulk harvest are outlined in
the International Council for Harmoni-
zation of Technical Requirements for
 Viral Safety DETECT

Keeping Up with Viral Safety Trends in Bioprocessing

Lab scientist testing raw materials.
Pharmaceuticals for Human Use (ICH
Q5A) guidelines. The ICH brings together
the regulatory authorities from Europe,
Japan, and the U.S. as well as industry
experts with a purpose of providing
harmonized recommendations and
technical guidelines for product regis-
tration. Current guidelines recommend
extensive in vivo testing for viral safety
purposes in bioprocessing. But, with the
development of new technologies, the
industry is questioning the utility and
relevance of these more traditional tests.
In with NGS, out with
In Vivo Analyses
“One technique that the industry is hop-
ing to get rid of is the in vivo assay,” says
Dr. Wisher. “In the last few months, there
has been a move to rewrite specific ICH
guidelines to remove the requirement
for in vivo testing. The industry wants to
reduce the number of animals used in
pharmaceutical testing in general. The
old, classical in vivo techniques made
sense when there wasn’t an option to
use tissue culture, but they’re not as
sensitive as people thought they were.”
An emerging technique that could help
to replace at least some in vivo testing
is next-generation sequencing (NGS),
otherwise known as massively parallel
sequencing or “deep” sequencing. NGS
is a technique that allows unbiased
sequencing of all DNA or RNA in a given
sample, and has been commonly used in
a research setting for over a decade, but
is only more recently being implemented
for viral safety testing.
“Traditional testing will only test for a
panel of viruses that we know can impact
human health,” explained Colette Coté,
Ph.D., principal scientist focusing on the
BioReliance® portfolio of MilliporeSigma.
“But the reality is that there may be
emerging viruses that we haven’t identi-
fied that can also affect human health,
Zika being a perfect example. NGS en-
ables a deeper search to look for viruses
that we may not have known about.”
As promising as the technique is for
giving more insight into potential viral
contaminants, regulatory guidelines are
slow to adopt.
“The industry is trying to get their hands
and minds around the technology,” con-
tinued Dr. Coté. “Consistency, robustness,
reproducibility and sensitivity need to
be shown. They need answers to ques-
tions about what the technology can
do, what the limitations and advantages
are, and how it compares against current
technologies.” When these questions are
answered, regulatory expectations and
guidance will likely change.
The use of NGS for testing biologics
poses greater challenges as compared
to standard PCR or other molecular tests

15 | October 1, 2018
 Viral Safety DETECT
Keeping Up with Viral Safety Trends in Bioprocessing
due to its technical complexities and requirement for
Big Data bioinformatics. To address these concerns, the
Advanced Virus Detection Technologies Interest Group
(AVDTIG) was formed as a joint effort by regulatory and
industry scientists to share data and experiences using
advanced virus-detection technologies such as NGS.
“There are about 30 companies working in the AVDTIG
right now,” said Dr. Wisher. “This year, the group is
publishing papers that will discuss best practices
for NGS methods and data analysis. They’re also
developing a curated database of useful sequences
for viral safety studies as well as making a number of
standard purified virus preparations so that spiking
studies can be done to get a better idea of the
sensitivity of the technique.”
Although regulatory agencies will not yet accept NGS
as a replacement for standard virus detection tests, the
technology is already impacting industry expectations
and standards. “Last month at a viral safety meeting,
Sanofi announced during a presentation that they
will be using NGS for screening cell lines and vaccines
and submitting that data along with all of the
conventional testing that was done for all new
products,” said Dr. Wisher.
Viral Safety Testing in Quality by Design
Another trend in the industry, which has been
introduced and consistently encouraged by regulatory
authorities is the implementation of Quality by Design
(QbD) approach in process operations. The QbD
approach encourages an understanding of how raw
16 | October 1, 2018
materials and manufacturing processes impact the criti-
cal quality attributes of the product, i.e., attributes that
impact the safety, efficacy, and quality of a drug product.
Ultimately, a design space is defined, which represents
the operating range, within which the process meets
the critical quality attributes of the product. The
“absence of virus” can be considered a critical quality
attribute that impacts the safety of the drug product.
“The QbD approach enhances a manufacturer’s under-
standing of their process and unit operations as well
as the impact of operating parameters on quality attri-
butes,” explained Dr. Raghunath. “Following QbD meth-
odology, we have put together a knowledge base that
details the impact of various process conditions and
operating parameters on the viral clearance performance
of a downstream virus filter. The impact of parameters
that can typically change during the process, such as
pressure, conductivity, pH, protein concentration, and
membrane lots, on virus retention is reported. Under-
standing the effects of these parameters on quality
attributes like virus retention allows those parameters
to be controlled within a given range.”
Using the principles of QbD for viral safety testing
results in a more thorough understanding of the design
space for viral clearance unit operations and the
appropriate controls that are needed to maintain the op-
erating parameters. Key to the methodology is upfront
identification of risk factors and incorporation of testing
protocols to manage those risks, and NGS helps to
identify viral contamination risk. “When using novel
substrates in bioprocessing, extensive NGS character-
ization can be performed on several lots of the raw
materials to identify potential contaminants that may be
present,” said Dr. Remington. “This can help focus testing
so that manufacturers have a rational testing strategy
for specific contaminants, and appropriate assays
can be designed.”
Integrating sensitive new technologies for viral
detection into a QbD approach to biomanufacturing
provides confidence for both the manufacturer and
regulatory agency that the risks of virus entry to the
process are minimized, low levels of virus can be
detected, and the process parameters are controlled
to assure the expected levels of virus removal in the
downstream process, thereby assuring the integrity
and safety of the drug product. n
Traditional Testing
and Unknown Viruses
Colette Coté, Ph.D.
Principal Scientist,
Focusing on the
BioReliance® Portfolio
Listen Now
of MilliporeSigma
 Viral Safety DETECT Tutorial
Utility of Next-Generation
Sequencing (NGS) for Biosafety
Assessment of Biological Products
Colette Côté Ph.D., Lakshmi
Viswanathan, Sindy John,
and Audrey Chang, Ph.D.
The advent of next-generation sequencing
(NGS), also referred to as massively parallel or
deep sequencing, affords a radically different
approach to the challenge of identifying and
characterizing known and unknown agents
(sequences) with precision and sensitivity.
By delivering significantly more data than
traditional Sanger-based sequencing methods,
NGS opens a range of possibilities for the
analysis of diverse DNA and RNA populations.
NGS does not require any prior knowledge
of the sample sequence; the technology is
capable of detecting all sequences in a sample,
whether known or not. An NGS library is
constructed from sample nucleic acid and
then sequenced (Figure 1). Comparison of a
sequence to target sequences or to libraries
of known reference sequences using bioin-
formatics programs reveals identities. Identi-
fication of novel sequences is made possible
by virtue of homology to known elements/
sequences.
17 | October 1, 2018
By combining custom sample preparation
with tailored sequencing and bioinformatics,
NGS is ideal for the characterization of bio-
logical products (e.g., viral vaccines/products,
raw materials, cell lines used in biomanufac-
turing, and final drug products [Figure 2]) as
part of a Quality by Design (QbD) approach.
NGS is also particularly well suited for bio-
safety testing, including the identification
of unknown contaminants in biological
samples or systems (e.g., those that result in
Figure 1.
bioreactor/fermentor failures or unexpected
morphological changes/cell death during
cell culture). In such instances, a rapid inves-
tigation, combined with the ability to detect
contaminants without bias or prejudice, is
essential and NGS can be the critical first step
for contamination remediation.
NGS continues to prove itself, not only as a key
supplementary tool, but also a key alterna-
tive method to address testing requirements
specific for virus-based therapeutic products.
Virus-based therapeutic products (Figure 3)
are viruses that are converted into therapeutic Figure 2.
agents by reprogramming them to treat
 Viral Safety DETECT Tutorial

Utility of Next-Generation Sequencing (NGS) for Biosafety Assessment of Biological Products

disease. They can be grouped by application:
• Viral vaccines—viruses designed to prevent
replication and elicit an immune response
• Oncolytic virotherapy—viruses that selectively
target cancer and tumor cells and elicit an
antitumor immune response
• Viral gene therapy—viruses that deliver thera-
peutic genes to cells with genetic malfunctions
• Viral immunotherapy—viruses that introduce
specific antigens to a patient’s immune system
Plenty of regulatory guidance exists around the
manufacture of viral-based medicinal products.
FDA and EMEA guidance documents and
reflection papers outline testing strategies
and recommendations. While use of NGS is
not specifically defined, it may be inferred
under use of “state-of-art” technologies.
and the requirement for producing neutralizing
antisera, required for many traditional assays.
NGS offers opportunities for circumventing some
of these challenges by offering a consistent and Figure 3.
sensitive method compatible with a diverse
range of products to test for and identify
adventitious virus (Figure 5).
Specific NGS Applications for Virus
Product Safety and Characterization
One key application of NGS is identity testing
and variant detection, which is of great value
when large or difficult-to-sequence genomes
are evaluated, or when structural and rare
variants are involved (Figure 6).

Challenges for Traditional

Testing of Viral Products
Traditional tests for adventitious virus are lengthy
(e.g., sterility, 14 days; mycoplasma, 28 days;
[Figure 4] and RCR, 28 days). Although they may
detect a contaminant, they generally do not
directly identify it. Often, only small lots with
limited sample volumes of viral-based therapeutic
products are produced, resulting in limited
availability of starting material for process,
product, and test-method development.
Another testing challenge for these types of
products is the lack of reference standards Figure 4. Figure 5.

18 | October 1, 2018
 Viral Safety DETECT Tutorial

Utility of Next-Generation Sequencing (NGS) for Biosafety Assessment of Biological Products

Equally important is its application in contaminant
detection. Here, NGS allows both detection and identifi-
cation of viral, bacterial, or fungal sequence signatures,
of both known and unknown agents (Figure 7).

In summary, NGS has clearly emerged as an effective

molecular tool with a wide range of applications in
biosafety testing and biomanufacturing. NGS offers
not only a supplementary method, but also a novel
alternative testing strategy, enabling solutions
where traditional testing approaches struggle or fail.
Regulatory guidance driving the use of NGS for specific
applications is still a work-in-progress. However, the
technology can clearly be developed for, and applied
in, a regulatory setting, and meet, if not exceed, the
requirements and expectations. n

Colette Côté, Ph.D., is principal scientist, head of next-

generation sequencing and bioinformatics, development
services; Lakshmi Viswanathan, serves as senior scientist,
next-generation sequencing, development services; Sindhu
John is bioinformatics engineer supervisor, next-generation
sequencing, development services; and Audrey Chang,
Ph.D., serves as head of development services, at the
BioReliance® portfolio of MilliporeSigma.

Figure 6. Figure 7.

19 | October 1, 2018
 Viral Safety REMOVE
Viral Safety in
Monoclonal Antibody
Manufacturing
Various Technologies to Prevent,
Detect, and Remove Virus Contamination
Angelo DePalma, Ph.D.
The significance of viral safety is apparent through-
out the biopharmaceutical production process. The
ultimate goal is to protect patients from pathogenic
viruses, and biopharmaceutical manufacturers need
to demonstrate viral safety and validate viral
clearance capability of the manufacturing process
before market approval.
Viral safety is critically important in both upstream
and downstream processes. Factors to consider in
upstream processes include: choice of expression
system, degree and type of genetic manipulation of
those cells or organisms, and how the cell culture is
run. All approaches are selected to efficiently produce
20 | October 1, 2018
the biotherapeutic product while minimizing
the possibility of virus entry into the system. In
Uniqueness in
downstream purification, virus removal or Addressing Viral Safety
inactivation is accomplished by a combination
of orthogonal or complementary approaches that
include chromatography, chemical inactivation,
and filtration.
For this article, we turn to four experts from
MilliporeSigma, all with unique perspectives on the
various technologies that assure that biotherapeutic
David Beattie, Ph.D.
protein products not only comply with requirements Head of Bioprocessing
for cGMPs and expectations of regulators, but ultimately Listen Now
R&D, MilliporeSigma
provide the highest level of patient safety.
 Viral Safety REMOVE
Viral Safety in Monoclonal Antibody Manufacturing
Adventitious viruses can enter the production
processes from multiple different routes:
from cells, raw materials, personnel, or the
environment. In addition, mammalian
expression cells contain endogenous viruses.
“These viruses are known, quantifiable,
and represent fixed-input virus levels that
establish a baseline demand for removal or
inactivation,” explains David Beattie, Ph.D.,
Head of Bioprocessing R&D, MilliporeSigma.
Virus titers vary according to cell type, trans-
fection methods, and expressed protein. For
example, the NS0 cell line expresses higher
virus titers than the CHO cell line, as do
expression systems that produce cytokines.
Thus, the inherent variability and uniqueness
in addressing viral safety, according to Dr.
Beattie, is “having a variable level of input
A mAb downstream suite. The mAb downstream viral clearance virus that defines a baseline demand for
is accomplished by a combination of orthogonal or complementary clearance means your virus removal might
approaches including filtration, chromatography, and chemical be very effective in one situation, but
inactivation. inadequate in others.”
Using well-characterized cell lines, such as
Continuous Processing is a
CHO, the preferred expression system for
monoclonal antibodies (mAbs), is one way
huge change, a revolutionary
to reduce inherent virus loads, assuming the
expression system is appropriate for achiev-
development with profound
ing desired product yield and quality.
implications for viral safety.
The second significant source of virus con-
tamination are media components derived
from animal sources. This risk, along with the
fear of prion contamination, has been the
major impetus for adopting animal-free or
chemically defined raw materials. Even then,
rodent infestations in plants from which raw
21 | October 1, 2018
materials are sourced can result in
completely unanticipated viral contamination.
“Genentech and Merrimack both experienced
bioreactor contaminations by Minute Virus
of Mice (MVM), a virus associated with mice,”
notes Dr. Beattie. “Such adventitious con-
taminations can result in viruses entering
the downstream purification train.”
Standard mAb Purification Platform
The standard mAb purification platform
includes protein A capture, where significant
viral clearance occurs, followed by one or
more chromatography steps. Anion exchange
chromatography “polishes” process streams
of host cell protein and nucleic acid, and
can be an important step for viral clearance.
Cation-exchange chromatography, which
removes process-related impurities like
aggregates and charge variants, provides
one to three logs of virus removal, which
is modest but not insignificant. Viral
clearance numbers are logarithmic, so
three logs reduction is equivalent to a
thousand-fold reduction in virus levels.
However, “if you determine on the basis
of product yield or quality that you don’t
need that process step, you lose its
associated clearance,” Dr. Beattie points
out. “The desire to trim downstream
processing to simplify and enhance their
productivity carries the risk of eliminating
or modifying unit operations, thereby
reducing or losing their capacity to
remove viruses.”
 Viral Safety REMOVE
Viral Safety in Monoclonal Antibody Manufacturing
Critical steps in the downstream process
are those dedicated to viral clearance,
including low pH and/or detergent treat-
ment to reduce levels of enveloped
viruses, and virus filtration, which
removes both enveloped and non-
enveloped viruses. Most processes rely
on these dedicated steps to make major
contributions to overall viral clearance
targets. However, the impact of unit
operations on the properties of the
molecule can be quite complex. Low pH
hold is highly effective for inactivating
viruses but is hard on therapeutic
proteins and may affect yield, highlighting
the interplay of product-quality assess-
ments with requirements for viral
clearance throughout downstream
purification.
Similarly, upstream-processing con-
ditions will have a direct effect on
performance of the downstream unit
operations, and higher cell densities and
volumetric productivity will also likely
affect the amount of virus entering the
purification train. These changes can all
impact the efficiency of the purification
operations for both impurity removal
and virus reduction.
Clearance Strategy
Establishing and conducting viral clear-
ance testing for biopharmaceutical
customers is the focus of Kathryn
Remington, Ph.D., principal scientist
22 | October 1, 2018
focusing on the BioReliance® portfolio
of MilliporeSigma. At a previous job at
a large biopharmaceutical company, Dr.
Remington collaborated with in-house
process-development groups to build
viral safety into processes from an early
stage, and then evaluated the viral clear-
ance potential of the manufacturing
process. Today, services related to viral
clearance are largely outsourced, as the
time and cost of a dedicated viral safety
group and laboratories are beyond the
resources of most companies.
A process-centered view of viral safety
makes sense since downstream unit
operations serve as a “safety net” to clear
any adventitious virus that might escape
upstream testing. But Dr. Remington
cautions that “while some downstream
steps provide very good clearance, some
don’t provide any at all. The level of
clearance is process-specific, molecule-
specific, and even virus-specific.”
Some measures, like chemical inactiva-
A scientist developing a chromatography step at bench-top scale.
tion through detergents, inactivate
broad classes of viruses, as for example,
lipid-enveloped viruses. Low-pH inac-
tivation provides good inactivation of
enveloped viruses. But, as mentioned
earlier, not all products are stable under
acidic conditions. As the pH increases
above pH 3.5, inactivation of enveloped
viruses becomes less robust. Chromatog-
raphy clears viruses based on their inter-
action with the resin. “Each virus has its
 Viral Safety REMOVE
Viral Safety in Monoclonal Antibody Manufacturing
own isoelectric point and other physical
characteristics that make its interaction
with resins unique,” Dr. Remington says.
Removal of virus by filtration is based on
size, and high levels of both enveloped
and non-enveloped virus can generally
be expected to be removed.
Ideally, process developers build in suf-
ficient steps to remove or inactivate as
many potential virus threats as possible.
“The overall strategy should aim broadly
because we don’t know a priori what
viral contaminants we may encounter,”
says Dr. Remington.
The same step may not always provide
the same level of clearance from process
to process. “People believe that if a col-
umn works great at a certain pH for one
molecule that it will provide the same
level of clearance in other instances. But
that doesn’t always work out,” she contin-
ues. “Sometimes the conductivity or the
virus’ isoelectric point is not right.”
An operator setting up a virus filtration step. Virus filtration removes both
enveloped and non-enveloped viruses. Given appropriate resources, optimizing
purification, recovery, and viral clearance is
possible. However, development groups
typically focus on the first two objectives,
then work clearance in afterwards by
adding or enhancing certain steps.
“It helps to have sufficient resources to
conduct feasibility or even a design of
experiment study, to understand the
viral clearance potential within certain
23 | October 1, 2018
ranges of operating parameters,” Dr.
Remington adds. “This will provide
greater confidence in implementing
future process steps, especially if a
platform approach is involved.”
Process Intensification
Viral safety often depends on dedicated
inactivation and removal steps, and in
good part on downstream-chromatog-
raphy operations with inherent viral
clearing capabilities. As biomanufacturers
squeeze processes for even greater
productivity they must examine if those
process improvements affect virus
clearance of the individual unit
operations.
Michael Phillips, Ph.D., director of
next-generation processing R&D,
MilliporeSigma, notes that three levels
of process intensification could affect
viral safety. These are particularly salient
for CHO-based mAb manufacturing,
where many new ideas in bioprocessing
are first implemented.
The first level involves mitigating bottle-
necks in fixed facilities, leaving unit
operations in place but scheduling or
locating them more effectively. The flip
side of level one is adopting new tech-
nologies to enable faster processing.
“You’re not really eliminating any step
or operation, so the potential impact on
viral safety is minimal,” says Dr. Phillips.
 Viral Safety REMOVE
Viral Safety in Monoclonal Antibody Manufacturing
Compressing processes by connecting unit operations,
the second level, presents modest challenges regarding
viral safety. However, such strategies, he notes, “need
not be serious, provided one remains vigilant that virus
removal is not compromised.”
The third level of process intensification—continuous
processing—is where issues raised by connecting unit
operations appear “in spades,” according to Dr. Phillips.
“Continuous processing is a huge change, a revolutionary
development with profound implications for viral safety.”
As with some revolutions, however, this one will be
slow in coming. Although many companies and sup-
pliers are evaluating continuous processing, Dr. Phillips
cautions not to expect the coup to occur overnight.
“There are regulatory concerns and technical gaps in
the ability to implement continuous processing in the
near future,” he explains. For example, current-generation
sensors to ensure reliable operation of continuous
processes are lacking, as are control strategies. Dr.
Phillips believes that as the technology improves and
regulations coalesce around continuous processing,
viral safety within that environment will catch up.
“But don’t expect it for at least five to ten years.”
A more modest implementation of continuous
processing exists within the confines of individual
unit operations. Perfusion cell culture and variants of
simulated moving bed chromatography are two ex-
amples. Another is inline low pH virus inactivation. The
discovery that low-pH incubation could be significantly
shorter than the usual one-hour hold, coupled with the
inconvenience of standard two-tank virus inactivation,
led Dr. Phillips’ group to investigate continuous low-pH
treatment of process fluid as it emerged from a protein
A chromatography capture column, obviating the need
24 | October 1, 2018
for an intermediate incubation/hold step.
When viewed against the backdrop of an entire process,
these individual steps less represent continuous
processing than optimized or streamlined batch
operations in which feedstock enters, is processed,
and then awaits the next step. Under ideal continuous
operation, process fluids feed directly and continuously
into and through operations, and purified product con-
tinuously flows out. Continuous or “next-generation”
bioprocessing promises huge advances in productivity,
but process developers must be aware of how those
advances could affect viral safety. The improvements
as one progresses along various levels of process
intensification also bear a potential cost.
Herb Lutz, global principal consultant at Millipore-
Sigma, described recent results where a tangential
flow filtration-based protein concentration step was
performed before flow-through anion-exchange chro-
matography polishing. “We reduced the volume of the
protein solution by a factor of four to make the chroma-
tography column more efficient, but we were obliged
to test how this might affect viral clearance,” said Lutz.
Using this highly concentrated feed, they demonstrated
consistent 5 logs clearance of MVM and XMuLV at high
product loadings, confirming that viral clearance was
maintained in the smaller footprint process.
“We’re running processes in new ways,” he says. “The data
doesn’t yet exist to guide processors on the implications
of all aspects of how process changes impact viral clearance.
Conventional viral-clearance testing assumes that
purification steps behave uniformly throughout their
operation. Aliquots of feed are spiked with model virus,
subjected to normal filtration or chromatography, and
the concentration of virus before and after the operation
are measured and results are reported as a log reduc-
tion. Lutz, however, does not believe this accurately
represents conditions of continuous processing:
“Standard virus-spiking strategies are inadequate when the
feed solution changes because a protein peak is coming
through or some other event is occurring,” he maintains.
To better assess such operations, Lutz developed a
technique termed inline spiking, which enables moni-
toring of viral clearance under more representative
conditions, when the process feed changes due to
fluctuating concentrations of proteins and salts.
It turns out that in most cases (e.g., during cation-
exchange chromatography), virus retention is fairly
constant over a wide range of protein concentrations.
Nevertheless, the value of inline spiking is that for a
minor investment in time it provides a clear answer.
“Regulators like that,” Lutz adds. n
Inline Spiking
Herb Lutz
Global Principal
Consultant,
Listen Now
MilliporeSigma
 Viral Safety Feature
Laying
the Foundation for
Viral Safety
Mitigating the Risk of Viral Contamination
in Vaccines, Cell, and Gene Therapies
Meghaan M. Ferreira, Ph.D.
Viral contamination can result in lost product batches,
equipment sterilization costs, and facility shutdowns,
costing biotherapeutic manufacturers millions of dol-
lars. Even more importantly, undetected contamina-
tion can compromise patient safety and trust.
In 2009, the discovery of Vesivirus contamination
forced a biopharmaceutical manufacturer to shut
down a manufacturing facility, which caused a se-
vere shortage of expensive, single-supplier enzyme-
replacement therapies used to treat rare genetic
diseases. The incident highlighted the importance of
having a strong system in place to mitigate the risk of
25 | October 1, 2018
Raw Material Testing
viral contamination, as well as a contingency plan in
the unfortunate case that contamination does occur.
In contrast to chemically derived, small-molecule
drugs, biologics are generally produced in animal or
human cell lines, which carry the risk of virus contami-
nation. While cell culture media and supplements for
monoclonal antibody (mAb) production are often
chemically defined, the same is not true for vaccines Martha Rook, Ph.D.
and cell- and gene-therapy products, which gener- Head of Gene Editing,
ally rely on some animal-derived materials for their Novel Modalities,
Listen Now
production. While a trend toward animal-origin-free MilliporeSigma
materials has begun, it’s not always easy or possible
 Viral Safety
Laying the Foundation for Viral Safety
to implement, and the use of animal- or
human-derived materials significantly
increases the risk of adventitious viral
contamination.
To reduce this risk, manufacturers rely
on three fundamental pillars to support
their viral safety strategies: preventing
virus entry by careful source-material se-
lection, detection of viral contaminants
by multistage testing, and virus removal
by multiple operations in downstream
purification. This three-tiered strategy
provides a solid foundation for viral
safety in the production of recombinant
protein and mAb therapies, but the one-
size-fits-most manufacturing strategies
used for protein production often don’t
work well for non-protein biologics like
viral vaccines, cell therapies, and gene
therapies delivered via recombinant
viruses.
Alternate Approaches for
Non-mAb Therapies
The diverse characteristics of these novel
therapies make them incompatible with
the majority of technologies currently
used to inactivate and remove viral con-
taminants during downstream purifica-
tion, thus eliminating one of the three
pillars that form the foundation of viral
safety in mAb manufacturing. For exam-
ple, the large size of cell therapies makes
nanofiltration and other downstream,
size-based separation techniques used
26 | October 1, 2018
to remove virus from protein-based ther-
apies impractical. Gene therapies that
use recombinant viruses (also known as
viral vectors) to deliver DNA into cells
face a similar challenge, since the absence
of a sufficient size difference between
adventitious and therapeutic virus may
also prohibit the use of size-based filtra-
tion technologies. Downstream methods
traditionally used to inactivate viruses,
such as low-pH incubation and gamma-
or UV-irradiation treatment, also leave
manufacturers without many viable
options. Sensitive cell therapies cannot
withstand these harsh conditions, and
anything that would disrupt a contami-
nating virus would also likely inactivate
vaccines or gene therapies whose effec-
tiveness depends on virus activity.
Although the underlying cause of viral
Lab scientist testing raw materials to ensure their quality and purity.
contamination is not identified in the
clear majority of occurrences, a few
reports have suspected raw materials, such
as serum, medium, and trypsin. Since
While biopharmaceutical
manufacturers often cannot apply virus
inactivation and removal systems to the
downstream purification of therapies,
they are moving to implement treat- companies work toward establishing
ments for source materials before they
enter the manufacturing train. Treatment better methods to ensure viral
of media with high temperature short
time (HTST) or irradiation with gamma or safety, they are also ushering in a
new wave of novel therapies for
UV can help reduce virus levels in some
components, but not all supplements
diseases with an unmet need.
are compatible with these treatment
methods and the equipment can be
 Viral Safety
Laying the Foundation for Viral Safety
A HTST mid-scale unit operation
expensive to install and validate. Newer developments
include specially designed filters for quickly and easily
processing cell culture media and upstream supple-
ments as well as increased focus on single-use tech-
nologies to minimize the likelihood of virus entry from
operators or the environment.
Novel Filters and Chromatography Media
Many of the downstream virus, clearance technolo-
gies simply weren’t designed for upstream use, making
their performance in upstream applications less than
optimal. Viral clearance filters, for example, “Are mostly
designed for downstream purification of protein-based
therapeutics,” commented Priyabrata Pattnaik, Ph.D.,
27 | October 1, 2018
head of biologics operations, Asia Pacific, MilliporeSigma.
Dr. Pattnaik also mentioned that the R&D team at
MilliporeSigma has developed a novel filter specifically
designed to handle complex cell culture media.
“[The team] optimized the membrane chemistry and
design configuration to adapt the product to deal with
media filtration where it can offer high-throughput
volumes so that it’s economically feasible to imple-
ment,” he explained.
Merck KGaA, Darmstadt, Germany has also partnered
with the DiViNe consortium to develop an affinity
chromatography resin for downstream vaccine
purification. The DiViNe project, which received funding
from the European Union’s Horizon 2020 research and
innovation program, aims to improve yields, decrease
costs, and reduce the environmental impact of vaccine
production by employing an affinity-based strategy,
similar to the use of Protein A in mAb production. The
chromatography resin uses Nanofitin® ligands, with
tailored affinities, to capture and elute viral- vaccine
products. Affinity chromatography can provide an
option for selective removal of adventitious viral
agents from vaccine processes.
While current ion-exchange chromatography
methods typically work for viral-vaccine and viral-vector
purification, Dr. Pattnaik contended that they generally
exhibit low capacity and poor selectivity.
Tailoring virus-reduction technologies to better fit the
needs of both upstream and downstream processes of
vaccine, and cell- and gene-therapy production exem-
plifies some of the ways in which manufacturers have
adapted their processes to ensure viral safety in these
novel classes of biologics.
Viral Safety Pillars
While individual cell and viral therapeutics may be
amenable to some virus inactivation and removal
methods, “By and large you’ve lost one of your [viral
safety] pillars,” remarked Martha Rook, Ph.D., head of
gene editing, novel modalities, MilliporeSigma. “There’s
a much bigger focus on the selection of your source
material, on the testing of the product in various stages
of manufacturing, as well as, now, the processing
conditions themselves.”
In addition to placing a greater emphasis on qualifying
raw materials and their suppliers, the movement
to adopt closed, single-use systems throughout the
manufacturing train, from 2,000 L bioreactors to
prepacked chromatography columns, has gained
traction. Ensuring a closed, aseptic manufacturing
process that does not introduce adventitious viruses
is important, because “you’re not going to have an
opportunity to clear them later,” noted Dr. Rook.
Biopharmaceutical developers have also started
moving toward chemically defined cell culture media
for cell-therapy production. Chemically defined media
often contain recombinant proteins, cytokines, and/or
growth hormones in lieu of animal-derived components,
which can cause significant batch-to-batch variation
and increased risk of viral contamination.
While chemically defined media mitigates safety risks
and generates more robust, reliable processes, its
adoption may impact the clinical efficacy of a cell-
therapy product, where media and supplements can
significantly influence cell phenotype. Finding the
perfect recipe for chemically defined media that
maintains phenotype, potency, and efficacy of the
 Viral Safety

Laying the Foundation for Viral Safety

therapeutic product still relies heavily on scientific
discovery and, as a result, remains a long-term
strategy for most companies.
Screening the product for adventitious viruses through-
out the manufacturing process forms the third pillar
that safeguards against viral contamination. Cell thera-
pies in particular may require testing methods with
high enough sensitivity to detect viral contamination,
despite the smaller volume of material available for
testing compared to more traditional therapeutics.
“Classic viral tests are still [used]—they’re still
defined, they’re still approved, they’re still relevant,”
affirmed Alison Armstrong, Ph.D., senior director,
global head of field development services focusing
on the BioReliance® portfolio of MilliporeSigma. “It’s
really about where modifications may be applied…
to address whether a virus, and perhaps a novel
virus, is present.”
Virus Detection Assays
The amount of knowledge needed a priori to detect ad-
ventitious viruses depends on the different techniques
employed. PCR-based methods require a high degree
of homology (usually 95%) to the sequence selected
for testing and may not detect a novel virus, even if it
comes from the same family as the virus of interest.
Culture-based infectivity assays offer a broader range
of detection for nonspecific viral agents. These assays
expose indicator cell lines to a test sample, which then
undergoes an observation period to determine if an
infection event has occurred. However, infectivity as-
says are not completely agnostic as cell-line dependent
variations in susceptibility to a particular pathogen can
still bias detection.
Testing for adventitious virus with cell-based assays in
viral vaccine products can also be challenging because
the viral vaccine must be completely neutralized before

Novel Virus Detection

Alison Armstrong, Ph.D.

Senior Director,
Global Head of Field
Development Services
Focusing on the
BioReliance® Portfolio
Listen Now
of MilliporeSigma

Mobius® FlexReady solution with smart Flexware®

Operator installing a single-use bag in a assemblies for chromatography and prepacked
Mobius® 2000 L single-use bioreactor. Chromabolt® chromatography column.

28 | October 1, 2018
 Viral Safety

Laying the Foundation for Viral Safety

testing. “[It’s] a really hard assay to do,”
noted Julie Murrell, Ph.D., head of cell
therapy bioprocessing, MilliporeSigma.
“It takes a while to do it, [and] it takes
a skilled operator to make the call on
whether or not a cell is infected” with
adventitious virus.
In contrast, Next Generation Sequencing
(NGS) offers a nondirected, completely
agnostic approach that makes it a pow-
erful tool for virus detection. It’s taken
a while for the technology to make a
home for itself in the industry. Both
the high computational power and the
extensive bioinformatics required to
transform sequencing data into useful
information remain drawbacks of the
technology. But, unlike directed methods,
NGS can detect novel viruses without
a priori knowledge. “The universe of
viruses is large, and new viruses are
constantly being identified or discov-
ered,” said Dr. Murrell, “so what we’re
testing for today might be different
than what we’re testing for in
the future.”
to ensure viral safety, they are also
ushering in a new wave of novel
therapies for diseases with an unmet
need, like rare genetic conditions and
advanced cancers. Even as companies
work to address the viral-safety chal-
lenges these therapeutics bring with
them, “The risk-benefit ratio is still very
much in the balance of benefit to the
patient,” Dr. Rook reminded us.
As these new modalities advance and
expand to include a broader spectrum
of indications, Dr. Rook is confident that
manufacturers will mature and grow
with them to ensure a safe product:
“[Viral-safety strategies are] going to
evolve. We’re not going to stand still.
We’ll see these kinds of advances, and
they’ll be needed as the use of these
types of therapies expands.” n

The discovery and development of

Scientist involved in performing cell line characterization testing.
biologics offers hope to many patients,
but only with security of supply. The
shortage of medication caused by the
Vesivirus contamination in 2009 high-
lighted the criticality of viral safety to
the industry and patients alike. While
biopharmaceutical companies work
toward establishing better methods

29 | October 1, 2018
Viral Safety Assurance
Prevent, Detect,
for mAbs: Prevent, 3 What’s
What’snext
nextto
tominimize
minimize risks?
risks?

Remove
Detect, Remove
Current
Current and and
FutureFuture
Solutions Solutions
for Biopharmaceutical
Virus Safety
AArisk
risk mitigation
mitigation strategy
strategythat
thatincludes
includes the prevention,
the detection,
prevention, detection, and
and removal
removal of viral
of virus contamination
contamination willwill
helphelp ensurevirus
ensure process safety.
safety.

A viruscontamination
A viral contaminationcancan shut
shut down
down a biopharmaceutical
a biopharmaceutical plantplant for months
for months impacting
impacting manufacturing
manufacturing operations,
operations, causing
causing significant
significant businessand
business disruption disruption and
ultimately ultimately
threatening threatening
drug drug supply.
supply. Fortunately, Fortunately,
a range a range
of technologies ofavailable
are technologies
todayare available
to help today
prevent
to help
viral prevent virus
contamination andcontamination and and
assure an efficient assure
safean efficient and safe
biopharmaceutical biopharmaceutical
production process. production process.
Fig.3 Virussafety
Fig.3 Viral safetyassurance
assurance

1 Adventitious
AdventitiousViruses
Viruses
AAConstant
ConstantProcess
ProcessThreat
Threat
200-300
$1b
$

million Expression System

Expression System
Raw Materials
Raw Materials
Media Preparation
Media Preparation
Cell Culture
Cell Culture
Purification
Purification
Potential loss of
revenue due to a • Chemically defined
Potential cost of • Chemically defined
1993
1993 2004
2004
contamination
contamination • Genetic raw materials • Virus barrier filtration
Cache • Genetic raw materials • Virus barrier filtration
MVM
MVM Cache Valley
Valley 2010
2010 Prevent modification
1988
1988
2008
2008 PCV-1
Prevent modification •• High
High Temperature,
Temperature, Short
Short
Vesivirus PCV-1 ofhost
of hostcell
cell • Treatment
• Treatmentofof Time (HTST) Treatment
EHDV
EHDV 2000
2000 Vesivirus Time (HTST) Treatment
high-riskcomponents
components

0.04
high-risk

0.04%
Cache
Cache Valley
Valley
2006
2006 %
MVM
MVM
1994 ••PCR
PCRtesting
testing
1994 2009
2009 of
ofunprocessed
unprocessed bulkbulk drug
drug • Adventitious
• Adventitiousvirus
virustesting
testing
MVM substance
substance samples
samples tested
MVM MVM
MVM
tested ••In
Invitro
vitrotesting
testing
2003 Detect
Detect

1
positive
positive for
for viral
2003

1
viral • Next
• Next Generation
Generation
Vesivirus
Vesivirus contamination
contamination during
during
Cache
Cache Valley
Valley in Sequencing
Sequencing(NGS)
(NGS) ••Next
NextGeneration
Generation
invitro
vitro screening
screening
Vesivirus
Vesivirus assays
assays Sequencing (NGS)
Sequencing (NGS)
1999
1999
Reovirus Viral
Viralparticle
particleper
perliter
liter
Reovirus is issufficient
sufficienttotoinfect
biopharmaceutical
biopharmaceutical
infect
••Low
Low pH/chemical
pH/chemical
manufacturing
manufacturing
inactivation
inactivation
Remove
Remove ••Chromatography
Chromatography
Fig.1 Reported
Fig.1 Reportedmajor
majorviral
viral contamination
contamination events
events in biopharmaceutical
in biopharmaceutical manufacturing
manufacturing • Virus filtration
• Virus filtration
Fig.4 Various technologies help minimize viral contamination risks throughout the process
Fig.4 Various technologies help minimize virus contamination risks throughout the process

2 Traditional
TraditionalSolutions
Solutions A diverse
A diverse range
rangeofofnew
production, including
newtechnologies
technologiesfurther minimizes
further thethe
minimize riskrisk
of introducing
of introducing viral virus
contamination into biopharmaceutical
contamination into biopharmaceutical
production, includingvirus-resistant
virus-resistantengineered
engineeredCHO cell cell
CHO lines, novelnovel
lines, filtersfilters
designed specifically
designed for cell for
specifically culture
cell media,
cultureand
media,
Viral
Virussafety
safetysolutions that
solutions remove
that virus
remove fromfrom
virus monoclonal antibody
monoclonal and recombinant
antibody protein protein
and recombinant production
production innovative technologies for sensitive detection of unknown viruses. More traditional virus filtration technologies have been
are and innovative technologies for sensitive detection of unknown viruses. More traditional virus filtration technologies
arewell
wellunderstood.
understood. augmented by new prefiltration options enabling more efficient processing of a broader range of feed streams.
have been augmented by new prefiltration options enabling more efficient processing of a broader range of feed streams.

Prevent
Prevent Detect
Detect Remove
Remove

Affinity Cation Exchange Anion Exchange Virus

Affinity Low pH Cation Exchange Anion Exchange Virus
Chromatography Low pH Chromatography Chromatography Filtration
Chromatography Chromatography Chromatography Filtration
Parvovirus LRV 1-3 <1 ~1 2-5 >4
Parvovirus LRV ~2-3 n/a ~1 ~4-5 ~4-5
Retrovirus LRV 1-5 >5 ~2-3 ~4-5 >5 Gene Editing Technology
Gene Editing Technology NewNew barrier
barrier technologies
technologies likelike Next
Next Generation
Generation Prefiltersremove
Prefilters removeplugging
plugging
Retrovirus LRV ~4-5 4-6 ~2-3 ~4-5 ~5 created engineeredCHO
created engineered CHOcell
cell filtration
filtration products
products designed
designed Sequencing
Sequencing opens
opens doors
doors for for speciesfrom
species frommAbmAbprocesses
processes
lines that
lines that are
are resistant
resistanttotoMVM,
MVM, forforcell culture
cell culture media offer
media offer more
morerapid, sensitive
rapid, detection
sensitive detection significantly
significantlyimproving
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filtration
Fig.2 Expected viral clearance by manufacturing unit operation, log reduction value (LRV). one of the
one the main
mainviral
virus easy
easytotouse and
use andimplement
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virus contaminants of the filtration
capacity of thedevice
devicewhile
while
Fig.2 Expected
Miesegaes viral
G., Lute, S., clearance
Brorson by Analysis
K., (2010) manufacturing unit operation,
of Viral Clearance logforreduction
Unit Operations Monoclonalvalue (LRV).
Antibodies. Biotechnology and Bioengineering Vol 106, No 2, June 1 2010 p 238-246 contaminants in
contaminants inthe
theindustry.
industry. solutions
solutionsforfor
risk reduction.
risk reduction. maintaining
maintaininghigh highparvovirus
parvovirus
Miesegaes G., Lute, S., Brorson K., (2010) Analysis of Viral Clearance Unit Operations for Monoclonal Antibodies. Biotechnology and Bioengineering Vol 106, No 2, June 1 2010 p 238-246 removal
removaland andflux.
flux.

Learn more
Downstreamchromatographic
Downstream chromatographic purification
purification Downstream
Downstream virus
virus filtration
filtration www.EMDMillipore.com/virus-prevent-detect-remove

Downstream processing
Downstream processing separates
separates thethe
protein of interest
protein fromfrom
of interest cell cell Flexible
Robust prefiltration optionscan
viral clearance enable increased mass
be maintained loading
during virusonfiltration
virus filters to
culture harvest
culture harvestand
andresults in in
results a purified, concentrated
a purified, concentratedmolecule with with
molecule meet the demands
following plannedof today’s biomanufacturers.
or unplanned Robust viral assuring
process interruptions, clearance
The life science business of Merck KGaA, Darmstadt, Germany
low levels
low levelsof
ofimpurities.
impurities.Various
Various technologies
technologies withwith
a variety of base
a variety of base should be maintained
performance during virus filtration
and consistency following
of this critical planned
virus or unplanned
reduction operation.
media, ligands operates as MilliporeSigma in the U.S. and Canada.
media, ligandsand
andformats
formats offer multiple
offer multipleoptions for purification.
options for purification. process interruptions,
Flexible assuring
prefiltration optionsperformance and consistency
enable superior of thisacross
mass capacity critical
Although purification is the primary goal, reliable virus removal is also virus reduction operation.
Although purification is the primary goal, reliable virus removal is also
30 a broad range of molecules and conditions to meet the needs of MilliporeSigma and the vibrant M are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their
|
required to meetOctober
required to meet the viral safety 1, needs
the virus safety 2018 of the downstream process.
needs of the downstream process. today’s biomanufacturers.
respective owners. Detailed information on trademarks is available via publicly accessible resources.
© 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. Lit. No. MK_FL2621EN00 Ver. 1.0
 Viral Safety Advertorial

Prevent, Detect, Remove:

Prevent, Detect, Remove Virus Different Considerations for Upstream vs Downstream

Is your bioprocess protected from viral threats? Do you know how safe your raw
Master Cell Bank Working Cell Bank Seed Train
materials are? Do you have a holistic viral safety strategy to protect your process
from start to finish? If not, we’re here to help. Our comprehensive offering of viral
safety products and services will enable you to develop, implement and validate
your process to meet regulatory requirements.

Raw Materials

Viral Safety—Prevent Viral Safety—Remove Filtration Bioreactor Primary Clarification

Viresolve Barrier filter for cell culture media

®
Mobius MIX systems with Emprove products
® ®

for low pH and solvent/detergent treatments

HTST treated glucose for bioreactor feeds
Centinel Intelligent Virus Defense™ technology ProSep®, Fractogel® and Eshmuno® Secondary
Clarification
chromatography resins
(MVM-Resistant CHO cells) Column Column
Protection Protection
Viresolve® Pro Solution
BioReliance® viral clearance services or testing

Column Chromatography Chromatography

Protection Purification Viral Inactivation Protein A

Viral Safety—Detect Support

BioReliance® Biosafety Testing: cell line Full suite of Emprove® buffers and
characterization, virus bank characterization, process chemicals
Chromatographic
Next-Generation Sequencing, raw material Comprehensive support from initial process Polishing
testing, bulk lot and final product development through final implementation
release testing

Concentration Final Sterile

Virus Filtration and Formulation Filtration

Upstream Downstream
Prevent bioreactor contamination Demonstrate the process can remove virus
The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada. MilliporeSigma,
the Vibrant M, Centinel, Centinel Intelligent Virus Defense, Emprove, ProSep, Fractogel, Eshmuno, Mobius, and Viresolve are
trademarks of Merck KGaA, Darmstadt, Germany. BioReliance is a trademark of Sigma-Aldrich Co. LLC. or its affiliates. All other
trademarks are the property of their respective owners.

31 | October 1, 2018
 Viral Safety Advertorial

Centinel Intelligent Virus Defense™ Technology

A New Line of Defense Against MVM Contamination

Powered by MilliporeSigma’s advanced gene-editing platform, Centinel Intelligent Virus Defense™ technology represents the most significant advancement in viral
safety since the removal of serum from manufacturing processes.

The Risk Of MVM Contamination

One of the major threats for manufacturers using CHO cells is minute virus of mice (MVM) contamination.
As it is so small, tough and heat-resistant, MVM is hard to remove, and contamination often
goes undetected until a lack of productivity in CHO cell cultures is noticed in the bioreactor. MVM
is highly virulent and specifically targets rapidly dividing cells; just one virus particle per liter
can quickly take down a bioreactor full of CHO cells, costing millions of dollars. Regulators
now expect manufacturers to test every bulk harvest for MVM. To demonstrate the virus
safety of the manufacturing process, no virus should be detected. Now, there’s a new way
to guard cells against this pernicious viral threat.
Generate MVM-Resistant CHO Cells with Centinel™ Technology
from MilliporeSigma
Centinel™ technology makes it possible to develop MVM-resistant CHO cell lines, giving
manufacturers another layer of defense. Created in a partnership between BioReliance®
Services viral safety experts and MilliporeSigma’s cell-line gene editing team, Centinel™
technology represents an entirely new way to prevent viral contamination. Cell lines can be
engineered using MilliporeSigma’s gene editing technology, zinc finger nucleases (ZFNs), to
suppress expression of sialic acid – a key component for MVM cell binding and entry. With no
sialylated glycoproteins or glycolipids on the cell surface, MVM has no means of entering the cell,
and so cannot replicate.
Extensive testing by BioReliance® Services team, both with lab strains and viruses isolated from
biopharma producers, show that the cells are highly resistant to MVM, with no adverse effects on
protein production. While the risk of viral contamination can never be totally ruled out, using
cells that are MVM-resistant can benefit even the most thorough viral safety program.
Centinel™ Technology: A Collaborative and Innovative Idea
If the receptor that MVM uses to gain entry could be eliminated, the virus may be
unable to infect the cell.
“Viral safety is an issue that our customers – and the industry as a whole – take very
seriously, and several major biopharmaceutical companies have championed efforts
to improve viral safety in the industry,” says the Centinel™ development team. “We
developed Centinel™ technology in close collaboration with the industry and see it as
another layer of protection in a company’s overall viral mitigation program. After all, why
take a risk you don’t have to?”
Minute virus of Mice (MVM)

Centinel™ Technology in Brief

What is ZFN Technology?
A technology that provides security and sustainability by offering reagents, cell-line engineering
services and viral challenge assays to promote viral resistance. Zinc finger nucleases (ZFNs) are a class of
engineered DNA-binding proteins that
• No detectable MVM replication after viral challenge facilitate targeted editing of the genome
• No drop in protein productivity or quality by creating double-strand breaks in DNA
• Suitable for production of asialylated recombinant proteins at user-specified locations.
• Centinel™ technology can be applied to multiple cell types and gene targets A) DNA-binding domain B) DNA-cleaving domain

The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada. MilliporeSigma, the Vibrant M, Centinel, Centinel More information: www.Sial.com/centinel
Intelligent Virus Defense, Emprove, ProSep, Fractogel, Eshmuno, Mobius, and Viresolve are trademarks of Merck KGaA, Darmstadt, Germany. BioReliance is a trademark
of Sigma-Aldrich Co. LLC. or its affiliates. All other trademarks are the property of their respective owners.

32 | October 1, 2018
 Poster Gallery
Implementation of a Virus Barrier Media Efficient Virus Clearance Across
Filter into Fed-Batch Bioprocesses the MilliporeSigma Downstream
Kimberly Mann, Michael McGlothlen, Joe Orlando, Jonathan Broe, Patricia Kumpey, Kristina Cunningham, Yuanchun Zeng, David Nhiem,
Robert Smith, Christina Carbrello, Venkata Raman, Jeremy Perreault, Kevin Rautio
Purification Portfolio
Ushma Mehta, Chris Gillespie, Kevin Galipeau, Michael Doty, Trish Greenhalgh, and Michael Phillips
EMD Millipore Corporation, Bedford MA, USA EMD Millipore Corporation, Bedford MA, USA

No change in cell culture performance Introduction Protein A Affinity Chromatography

Introduction MAb #1 Cell Growth in Shake Flasks MAb #1 Viability in Shake Flasks The downstream purification process of any biologic has Eshmuno® A resin is a rigid, high capacity, acid and pool co-spiked with MVM at 1E+06 TCID50/mL (0.1%
Upstream bioprocesses are at particular risk of contamination from adventitious 25 100 several objectives: purity, yield, and safety for humans alkaline resistant Protein A affinity chromatography (v/v)) and XMuLV at 5E+05 TCID50/mL (~1% (v/v)).
agents. Typical 0.1 µm filters used at this step protect bioreactors from 90 or animals. A critical component of safety assurance is media for the purification of Fc-containing proteins Columns were washed in 100 mM citric acid pH 5.6,

Viable Cell Density (cells x 106/ml)

bacteria and mycoplasma, but offer no protection from viral contaminations.
20 VB- #1
VB- #2
80 VB- #1
VB- #2 reducing virus to levels that meet stringent regulatory such as monoclonal antibodies. Following preliminary then protein was eluted in 100 mM acetic acid pH 3.0.
A new polyethersulfone (PES) virus barrier filter, Viresolve® Barrier filter, has
VB- #3
VB- #4
70 VB- #3
VB- #4 requirements. Virus reduction can be achieved optimization of load, wash and elution conditions, Samples were assessed for titer by infectivity assays
through multiple complementary approaches and most Eshmuno® A resin delivered robust performance (MVM and XMuLV) or qPCR (XMuLV only). Protein A
15 VB+ #5 60

% viable
VB+ #5

demonstrated high levels of microorganism retention - full retention for bacteria VB+ #6 VB+ #6

processes rely on a combination of technologies that under a variety of conditions, consistently removed viral clearance results are summarized below and
VB+ #7
50 VB+ #7

and mycoplasma (>8.0 LRV - Log Reduction Value) and ~ 5 LRV for small viruses, 10
VB+ #8
40
VB+ #8

are designed primarily to achieve purification targets, high levels of HCP, and could be loaded to 40 g/L show moderate removal (~2.5 log) of both MVM and
such as parvoviruses. This filter is optimized for use with upstream components, 30
but may also offer opportunities for virus reduction. with protein recoveries of at least 95%. XMuLV, and approximately 0.5-1 log inactivation of
and therefore exhibits higher flow and capacity than downstream virus filters. 5 20

XMuLV, attributed to the low pH elution conditions.

In this study, we evaluate the effects of implementing a Viresolve® Barrier filter 10
The purpose of this project was to establish Duplicate columns were challenged with clarified harvest
capabilities for producing a monoclonal antibody
0
into upstream processing.
0
- 2 4 6 8 10 12 - 2 4 6 8 10 12
Eshmuno® A MVM Infectivity XMuLV Infectivity XMuLV qPCR
Days Days
feed enabling the development of new filtration and Column Sample Virus Load (log TCID50) LRV Virus Load (log TCID50) LRV Virus Load (log GC) LRV
Cell performance in shake flasks chromatography products. The purified antibody
should have industry-representative titer, yield and Hold 8.31 6.94 8.51
Two recombinant CHO cell lines were cultured in 125 mL shakers in fed batch culture. MAb #1 utilized Cellvento CHO-200 medium and feeds ®

and mAb #2 (data not shown) utilized Ex-Cell® CHO media and feeds. No change in cell growth was observed when media and feeds were purity, achieved using the Eshmuno® chromatography Eshmuno® A Flow-through 8.54 6.92 8.41
processed with (VB+) or without (VB-) Viresolve® Barrier filters. Cell viability was also unaffected. Osmolarity, pH, glucose, glutamate, lactate, 2.5 3.1 2.5
Direct implementation of Viresolve® Barrier filter into bioreactor processes or NH3 levels were within limits (as measured by BioProfile® FLEX biotech analyzer). Titer, as measured by POROS® Protein A HPLC, was also
resin platform in combination with Viresolve® resin: C1 Wash 5.7 4.02 4.3
consistent. Pro filtration devices as seen in Figure 1. Eluted 5.78 3.85 6.05
Viresolve® Barrier filters are sterilizable and may be used in place of a 0.1 μm filter.
Hold 8.31 6.94 8.51
3000 MAb #2 Cell Growth in Mobius® 3L bioreactors MAb #2 Titer
1800
Figure 1. Monoclonal Antibody Process Operations and Eshmuno® A Flow-through 8.29 6.72 8.31
1600 Model Stream A
16 3 VB-
resin: C2 2.5 3.5 2.5
Purification Media Wash 6.19 4.63 4.34
Liters filtered in 4 hours/m2

Liters filterin in 4 hours/m2

2500 VB-
Viable Cell Density (cells x 106/ml)

VB-
1400 Model Stream B 14 VB-
VB- 2.5
Eluted 5.85 3.47 6.05
VB+
2000 VB-
1200 12 VB+
VB+ VB+
1000 VB+ 2 Protein A Virus Virus
Clarification CEX AEX Concentration

Titer (g/L)
1500 10 Capture Inactivation Filtration
VB+
800
8 1.5
600 1000
Eshmuno® A Eshmuno® S Eshmuno® Q Viresolve® Pro
6

Low pH Virus Inactivation

400 1 resin resin resin filter device
500
200 4 Time of incubation Replicate # 1 Replicate # 2
0 0 0.5
Each unit operation was optimized to maximize step at pH 3.6 (min) XMuLV LRV XMuLV LRV
2

purity and yield. Following optimization, virus clearance This step is dedicated to the reduction of enveloped
MilliporeSigma
Thermo Fisher Scientific
Lonza
GE Healthcare Life Sciences
0
0 2 4 6 8 10 12 14 16
0

was assessed using two viruses: Minute Virus of virus and a short hold at low pH fits well in the 0 3.2 3.5
VB- VB- VB- VB+ VB+ VB+

Days Days Mice (MVM), and Xenotropic Murine Leukemia Virus process following elution at low pH from Protein A
5 ≥4.2 ≥4.2
(X-MuLV), Table 1. column. Duplicate aliquots of post Protein A elution
More than twice the volume of media can be filtered using Viresolve® Barrier filter shows high capacity for efficient filtration Cell performance in 3L bioreactors pool were adjusted to pH 3.6 with 100 mM 10 ≥4.7 ≥4.7
Viresolve® Barrier filter in 4 hours as compared to existing of most commercially available chemically defined media.
downstream virus filtration technologies.
Recombinant CHO cell mAb #2 was expanded for fed batch production in 3L bioreactors utilizing Ex-Cell® CHO media and feeds. As in the Table 1. Virus Properties Acetic Acid then challenged with XMuLV at 1E+06

View Larger View Larger

small-scale shake flasks, no change in cell growth was observed when media and feeds were processed with (VB+) or without (VB-) Viresolve®
Barrier filters. Titer, as measured by POROS® Protein A HPLC, was also consistent. Physico- TCID50/mL (~1% (v/v)), and incubated at room 30 ≥4.9 ≥4.9
Virus Family Virus Size (nm) Enveloped chemical temperature for 60 mins. Rapid virus inactivation
resistance 60 ≥4.9 ≥4.9
was observed and after 5 minutes, no infectious virus
Methods Parvoviridae MVM 18-26 No High was detected, resulting in LRVs of at least 4 log.
Given the small pore size of virus retentive filters, implementing virus filtration upstream Product quality of recombinant antibody was unaffected Retroviridae X-MuLV 80-110 Yes Low
of the bioreactor raises the question of whether critical cell culture media components
might be removed. Therefore, it is important to evaluate the cell culture performance

Genetic Engineering of CHO cells for

using filtered media to ensure that there has not been any negative impact to the Cation (CEX) and Anion (AEX) Exchange Chromatography
VB - VB + VB - VB +

process. Ex-Cell® CHO media, Cellvento® CHO-200 medium and corresponding feeds
VB - VB +
Methods
Prefiltration and Process Improvements:
VB - VB +
Absorbance at 230nm

VB - VB + VB - VB +
Absorbance at 230nm

were processed through Viresolve® Barrier filters, and media composition was compared Eshmuno® S resin is a strong cation exchange media

Viral Resistance to MVM

All aspects of clone development, selection, cell culture XMuLV
XMuLV removalusing
removal usingEshmuno
Eshmuno®® Q
Q with Post CEX
CEX pool
pool
to 0.2 µm Millipore Express® PLUS filtered controls. Fed batch cultures were performed with tentacle structure enabling fast, efficient purification.
media selection, scalable production in 3-200L Mobius® 6

Enhancing Virus Filter Performance with

in both shake flask and Mobius® 3L stirred tank bioreactors to verify that surfactants, Excellent purification across Eshmuno® S resin was
bioreactors, and clarification over Clarisolve® 20MS

XMuLV Log Reduction Value ( LRV)

such as poloxamer (which are essential for shear protection in stirred tank bioreactors achieved with binding at pH 5.5 and elution with Tris pH
depth filters were optimized as part of the benchmark 5
8.5, 10 mS/cm. These conditions avoided dilution of the
and can be difficult to filter), had not been removed during filtration. Cell culture
the Use of Adsorptive Depth or Surface
process development. feed before AEX polishing.
performance and protein quality were evaluated. 4
Table 2 summarizes the properties of the feeds used in Eshmuno® S resin did not contribute to virus
Joaquina X Mascarenhas 1, Nikolay Korokhov2, Ademola Kassim 1, Jason Tuter 1 Trissa Borgschulte 1, Henry George 1, Audrey Chang 2,
, each step. Media were selected with consideration to the 3

Modified Prefilters
Retention time (min)

safety targets as less than 1 log virus was removed

Retention time (min)

David Onions 2, Kevin Kayser 1

Media composition was not affected
1SAFC® MilliporeSigma, Saint Louis, Missouri, 63103, USA Charge heterogeneity Aggregate Profile
efficiency of purification with a view to minimizing dilution
or modification for downstream purification.
(data not shown). However, purification over CEX media 2
2Bioreliance®, MilliporeSigma, Rockville, Maryland, 20850, USA Weak cation-exchange chromatography was employed to analyze The size exclusion chromatography profile for mAb#2 showed very was critical to achieving acceptable viral clearance over
1.0
the Eshmuno® A purified antibody mAb#2 from bioreactor culture. high amounts of monomer for antibody purified from cultures, Eshmuno® Q AEX resin as shown in the graph. 1
No notable differences were seen in the amount of acidic, neutral which was unaffected by filtration with (VB+) or without (VB-)
Amanda Katz, Damon Asher, Patricia Greenhalgh, Janice Lonardo, Ben Cacace, and Jeff Hartnett
Concentration Ratio (Viresolve® Barrier Filtered/

Unfiltered
Millipore Express® PLUS filter
or basic peaks between antibody purified from either culture Viresolve® Barrier filters. Consistently, very small amounts of a
Tryptophan:
Clearance of MVM and XMuLV across Eshmuno® Q resin 0
EMD
TableMillipore Corporation, Bedford, MA
2. Feed Specifications
0.8
photo unstable;
system with (VB+) or without (VB-) Viresolve® Barrier filtration. high molecular weight species were noted and no fragments were
was evaluated at pH 7.5, 8.0 and 8.5. MVM removal was
non-Viresolve® Barrier filtered)

good indicator
Normalized Intesity

Viresolve® Barrier +
Millipore Express® PLUS filters
of media
stability
observed. 0.1 0.25 0.5
0.6

G0F
Step Protein Conductivity insensitive to loading pH, with LRVs of at least 5 log out Mass Loading ( kg/L)
Feed Conc pH @ 25°C to the 0.25 kg/L target loading (data not shown). XMuLV
LU
Media
Abstract
140 VB - VB +
0.4 G1F
evaluated (mg/mL) (mS/cm)
Vitamins: Low concentration

MVM Viral Resistance Testing

VB - VB +
removal was highly dependent on pH with effective
components are detectable
CHO Cells Sialic Acid Mutant Library Post CEX : pH 7.5 No CEX : D1: pH 7.5
Fluorescence Emission (420nm)

120 VB - VB +
0.2 Protein A Clarified reduction at the target 0.25 kg/L loading only achieved Post CEX : pH 8.0 No CEX : D2: pH 7.5
The introduction of animal origin free (AOF) media has significantly reduced
100
CHOZN WT Slc35A1- COSMC- Mgat1- Eshmuno® A Supernatant 1.3 7.2 12-15 at pH 8.0 and pH 8.5, the optimal conditions for HCP
resin pool Post CEX: pH 8.5
the incidences of adventitious virus contamination in biological production 0.0 80 120%
removal.
Abstract Viresolve® Pro Shield H
CHO Wild-Type SLC35A1 - COSMC- MGAT-/-

systems. Nevertheless, contamination by the parvovirus Minute Virus of Mice Genotypes CHOZN GS EX-CELL® CD CHO Fusion EX-CELL® Advanced™ CHO Fed Batch
-/-
EX-CELL® Advanced™ CHO Feed w/Glucose
60
G0
Man5
G2F
CEX
Figure 2 Viresolve® Prefilter Conditioned Feed Arrow denotes complete XMuLV retention
100% Post
(MVM) also known as Mouse Minute Virus (MMV) remains a continuing Eshmuno S ®
23.2 5.5 3-4 (15 g/L)
Viral genome copies % of wild type

Protein A
challenge
40
resin
Improvements in upstream process development often
Nuclearand on average
Magnetic Resonance we Fingerprinting
see one major – episode of MVM contamination
Inductively Coupled Plasma/Optical Structure Emission 80%
Surface modified membrane prefilter
Aromatic
every ~5 years.Region Although infrequent, infection of a Spectrometry fermenter can be N-Linked
(Asn)
20
60% generateAEX complex, high titer process streams, placing 500 0.9
catastrophic for ofa Ex-Cell
producer, resulting in the loss of product, temporary •downstream
Support membrane: 0.2 µm PES
considerable
Virus Filtration
NMR fingerprinting ®
Advanced ™
CHO Fed-batch medium The graph above shows the concentration ratio of metals in the 0 Eshmuno®demands
Q on downstream
Post CEX 11processing
8 steps. 6 of the filtration devices, even following
shows no change
withdrawal frombefore
the and after filtration.
market and NMR shows thatclean
extensive the downEx-Cell
®
costs,CHOwhich
media andcanfeed followingO-Linked
Viresolve® Barrier filtration 10 12 14 40% 16 18 20 resin 450 0.8
component levels have not changed and no new components have (Ser-Thr)
compared to Millipore Express® PLUS filtration as measured Retention time (min)23.5% Protein aggregates in these feeds influence hydraulic •product recovery,
PES membrane resulting
surface in reduction
modified of greater
via cross-linked than
mixed-
reach a total in the tens of millions of dollars. In addition to the loss of 20%
performance of
Nanofiltration virus filters resulting in over-sized platforms
400
0.7
been introduced. This same trend was observed in the aliphatic
associated
by ICP-OES. No significant differences were observed in metal In most biologic processes, virus filtration is the 5.9 log.chemistry
mode
region – of revenue, a the
contamination event can also haveconcentrations.
a potential impact Viresolve® Pro Post AEX 10 5 6
4.3%
particular note Pluronic® F68 peak was unchanged 0.6% 0.0% 350
Glycan Analysis and a significant impact on process economics. Virus filters dedicated step for reduction of both enveloped and non-

Relative Flux (J/J0)

Phenotype N-linked and Asialylated N-linked WT N-linked 0% 0.6
on drug supply,
(data not shown). patient safety and have regulatory implications. O-linked WT O-linked trunc Solution
T=0 T=21
300 • Surface chemistry binds to aggregates and other foulants

Flux (LMH)
Glycananalysis of the purified mAb#2 was performed using 2-AB fluorescent labeling and NP-UPLC. Consistent glycan patterns for both culture from a broad range of manufactures provide robust viral
trunc O-linked
WT HoursViresolve
systems were observed for the antibody produced from media and feeds with (VB+) or without (VB-) post- infection
Barrier filtration.
enveloped virus by size exclusion. Post AEX pool was 0.5
®
Device Sample description LRV
In this work, we evaluated engineering a CHO parental cell line to create a clearance but the impact of aggregates on flux is dependent on pH
250
adjusted then processed over a pre-filter Viresolve
Viresolve Pro Device
Viresolve Prefilter with 0.4
®

®
®
• Effective operational range: pH > 5.5 or conductivity
new host 1.0 cell line that would be resistant to MVM infection by eliminating the the filter. The impact of conditioning protein solutions using Pro
200
Shield, spiked with MVM atViresolve
2E+06 TCID50/mL
Pro Device ®
> 25 mS/cm (see Figure 7)Pool at 5.0 Kg/m2 ≥5.93
Purification Summary
0.3
Figure 1: Schematic of major glycosylation structures resulting from specific gene knockouts and the correlation of cell Viresolve Pro # 1
Concentration Ratio (Viresolve® Barrier Filtered/

®
major receptors used by the virus to enter cells. The goal was to engineer a prefiltration was assessed with several monoclonal antibody (~0.2%
150
(v/v), then processed over Viresolve® Pro filter
surface sialic acid and MVM binding and internalization. Pool+ wash ≥5.93
host cell0.8
line resistant to MVM infection, while maintaining desired feed streams. Adsorptive depth or surface modified membrane device
100 at constant pressure of 30 psi.
0.2
non-Viresolve® Barrier filtered)

productivity and product quality profiles. While the exact functional receptor Fluorescent microscopy panel shows the staining of fluorescent labeled Mal II Lectin. The Mal II lectin binds exclusively to sialic acid The purification
upstreamscheme metfilter
mAb yield and topurity Pool at 5.0 Kg/m ≥5.93
2

bound in an α-2,3 linkage conformation. CHO cell lines engineered to express lower α-2,3 surface sialic acid demonstrate increased prefilters of the virus were shown remove 50 0.1
Figure 5 Viresolve® Pro Shield H Test Summary
for MVM 0.6
binding to CHO cell surface is unknown, sialic acid on the cell
surface has been implicated. The CMP-sialic acid transporter, solute carrier resistance to MVM infection compared to the wild-type controls. Summary targets.from protein solutions, enhancing performance of the
foulants
At the 5.0 kg/m2 target loading, processing was 0.0
0
stopped, and product recovered. No virus
Viresolve® Pro # 2
Pool+ wash ≥5.93
HCPHCPLevels ininProcess Intermediates 500 was detected
family 35A1 (Slc35A1) is responsible for transporting sialic acid into the The risk of virus contamination of the bioreactor remains a concern for virus filter. Levels Process Intermediates 0 100 200 300 400 600
0.4 500 0.9
Throughput (L/m2)
Golgi. Knocking out function of this gene in a cell results in asialylated
glycan structures, thus eliminating the ability of MVM to bind to and enter
Slc35A1 KO in an recombinant biotherapeutic IgG ProducingasCell
manufacturers, thereLine
is no universal technology that provides a 10000000
100,0000 0.8
reliable, cost effective solution for virus removal of all components of cell culture 769,231 Viresolve® Pro Device

Why Do Virus Filters Plug?

1000000
0.2 Viresolve® Pro Shield H
the cell. While the wildtype cells displayed immediate loss of viability and 400

Viresolve Pro Shield

with Viresolve® Pro Device 0.7
HCP PPM (ng/mg)

media. This study evaluated the Viresolve® Barrier filter, which provides an efficient ®
inhibition in cell growth upon MVM infection, the absence of sialic acid on the Recombinant Thawed & Passaged 100000
Virus Clearance Summary confirmed our downstream purification technologies0.6can

Relative Flux (J/J0)

Working Cell
SLC35A1 knock-out cell line lead to complete
0.0
resistance to MVM infection.
IgG
producing
2x
Bank (10 and easy way to protect bioprocesses from adventitious virus contamination. • Pores of virus filters are sized to pass most proteins and
10000 be300run in a connected process to deliver viral clearance

Flux (LMH)
Media components: Amino acids and soluble vitamins vials)
This approach could be EX-CELL applied to different CHO host cell lines, as well as clone
7/14/15
exclude viruses Surface modified
The purification membrane
scheme achievedprefilter
virus clearance performance required by customers.
0.5
CD CHO Fusion EX-CELL Advanced™ CHO Fed Batch EX-CELL Advanced™ CHO Feed w/Glucose
1000
Study results demonstrated that media and feed compositions were unaffected by
® ® ®
Equivalency/Preliminary Studies
therapeutic protein producing clonal cell lines. Incorporation of viral 323 186 173

• Therapeutic protein feed streams may contain low levels of targets with a margin for safety. Importantly, the project 200
0.4
resistance to the MVM virus in the CHO host and subsequent production cell ZFN filtration One through
WCB Vial thawed the Viresolve
®
Barrier filter. Cultures, both in shake flasks and 100 • Support membrane: 0.2 µm PES
lines adds yet another Liquidlevel of “defense” to the current risk mitigation
Transfection on 12/7/15 and Figure 2: Case Study SLC35A1 Knockout in an Expressing aggregates
10
or other foulants that gradually plug the virus
0.3
High Performance Chromatography scaled prior uptoto stirred
passaged for a week
beginning of tank Production
bioreactors, showed no differences in cell growth or titer.
Line
10
• PES membrane surface modified via cross-linked polymeric
strategies
Amino acidused, adding by even greater assurance postof production of safely filter. Virus 0.2
1 filter fouling may become more pronounced as Unit Operation Target Loading MVM LRV XMuLV LRV
analysis conducted ion-exchange chromatography column Ninhydrin method and water-soluble vitamin analysis by
In addition, the secreted antibodies showed no differences in the glycosylation 100
growth & productivity
Single-Cell Cloning, Screening &
delivered cell derived
reversed-phase products.The graph above shows no significant difference in the media concentration ofSequencing
C-18 chromatography. amino acids and soluble
assay
A sulfonic acid cation exchange chemistry
vitamins following Viresolve® Barrier filtration compared to Millipore Express® PLUS filtration for Ex-Cell® CHO media and feed. pattern, amount of aggregates or charge
Schematic variants.
of work flow Preliminary
of studying work
the effect (not shown)
of Slc35A1 in in
knockout
the feed streamifi
ed titer increases.
ifi
ed o o
S
oa
d
o
Q ® ® ®
Protein A (Eshmuno® A resin) 40 mg/mL 2.5 2.5
0.1
r r un L
cla un un • Surface chemistry binds to aggregates and other foulants
Cla EX
WCB
Knockouts:1B1, 1C3, 1C8, 1G8, 1G11, 2A6 Wild-Type: 1A5, 1A10, 1A11, 1D8, 1E12, 2B3, 2B8, 2C1, 2C6

Mobius® 50 L bioreactorsa shows similar

recombinant results.
IgG expressing cell line. hm
• AdsorptionUnof monomer variants or hm
fragments may m
halso 0 0.0
Bank
A
Es Es Es Low pH hold at pH 3.6 NA 0 100
n/a 200 300
≥4.9 400

MVM: A Threat Even in ACF Free Processes contribute to membrane plugging • Effective operational range: pH < 6 and conductivity Throughput (L/m2)

MilliporeSigma, Viresolve, Millipore Express, Cellvento, Eshmuno, Mobius and the Vibrant M are trademarks of Merck KGaA, Darmstadt, Germany. and product quality characterization
Growth, productivity
Filtration of media and feeds with Viresolve® Barrier filter exhibits efficient < 30 mS/cm CEX(see FigureS7)
(Eshmuno ®
resin) 80 mg/mL 0 0.9
Cell
Virus Ex-Celland Advanced are trademarksYear Company Co. LLC. All other trademarks
of Sigma-Aldrich Reported By are the property of their respective owners. filtration performance, high virus retention and minimal cell culture impact, and The life science business of Merck KGaA, Darmstadt Germany AEX ( Eshmuno ® Q resin) 250 mg/mL ≥5.3 ≥4.9
EHDV CHO 1988 Bioferon GmbH Bioferon GmbH
MMV
Lit. No. PS6649EN00, Ver. 3.0 Copyright © 2017 EMD Millipore Corporation. All Rights Reserved.
CHO 1993 Genentech Genentech offers a viable option to improve the overall virus risk mitigation strategy for the operates as MilliporeSigma in the U.S. and Canada. In the absence of a prefilter, average mass capacity =
HPLC Titers Virus Filtration (Viresolve® Pro Solution) 5 kg/m2 ≥5.9 ≥5.9*
MMV CHO 1994 Genentech Genentech manufacture of biotherapeutics. 0.7 kg/m2.
The life science business of Merck KGaA, Darmstadt, Germany 10

View Larger View Larger

100
Reovirus Homo 1˚Kidney 1999 Abbott Labs FDA MilliporeSigma, Eshmuno, Viresolve, Clarisolve and Mobius are Cumulative mean LRV 13.7# 18.2#
virus protein aggregate
trademarks of Merck KGaA, Darmstadt, Germany. All other Using the Viresolve® Pro Shield H prefilter, average mass
operates as MilliporeSigma in the U.S. and Canada.
Reovirus CHO ? ? BioReliance 9

Cache Valley CHO 1999 Amgen / CMO Amgen 8 80

trademarks are the property of their respective owners. Lit. No. *Not tested, LRV from MVM used as surrogate for XMuLV #conservativecapacity
estimate when no virus
= 5 kg/m 2 detected downstream
Cache Valley CHO 2000 ? BioReliance
Slc35A1 KO Clone
PS1245EN00 Ver. 2.0 07/2017
Vesivirus 2117 CHO 2003 Boehringer-Ingelheim Boehringer-Ingelheim
7
©2017 EMD Millipore Corporation. All rights reserved.
Wild Type Clone
Titer (ug/ml)

Cache Valley CHO 2003 ? BioReliance

Viable Cell Density

6 60
CHO-K1 producing (WCB BANK)
Figure 6 Viresolve® Pro Shield H Conditioned Feed
% Viability

Cache Valley CHO 2004 ? BioReliance 5 0.2 µm Cross-linked polymeric

Hu Adenovirus HEK 293 ? Eli Lilly Eli Lilly
4 40
PES surface coating (13 g/L)
MMV CHO 2006 Amgen Amgen
Vesivirus 2117 CHO 2008 Genzyme, Belgium Genzyme 3
Vesivirus 2117 CHO 2008 Genzyme, USA Genzyme 14
2 20
Vesivirus 2117 CHO 2009 Genzyme, USA Genzyme nanoporous
MMV CHO 2009 Merrimack Merrimack 1
membrane Figure 3 Viresolve® Pro Shield Test Summary 12
Viresolve® Pro Device
Viresolve® Pro Shield H
PCV-1 Vero 2010 GlaxoSmithKline GlaxoSmithKline 0 0 protein with Viresolve® Pro Device
PCV-1/PCV-2 Vero 2010 Merck Merck 0 2 4 6 8 10
Days in culture
16 9

Capacity (kg/m2)
Table 1: Known incidences of viral contamination events in
biomanufacturing. Figure 3: SLC35A1 knockout in an expressing production line: Growth and Productivity 14 Viresolve Pro Device
®

Both wildtype and SLC35A1 knockout clones were isolated from the transfected population. Viability, viable cell density and Viresolve® Pro Shield 7

Viresolve® Prefilter
with Viresolve® Pro Device
12
productivity were measured. A range of titer and VCD were obtained in both knockout and wildtype clones. It was determined that the
genetic modification did not impact these parameters.
)

10 5
2
 Webinar Corner
PREVENT
Defend your bioreactor:
Using nanofiltration to prevent virus
contamination in cell culture processes
REMOVE
DETECT Parvovirus-retentive filter:
Emerging viral risks and mitigation Performance and removal mechanisms
strategies in biologics manufacturing during process interruptions

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PREVENT REMOVE
Viral risk mitigation strategies: DETECT Optimizing viral clearance filtration:
Key considerations in the prevention Rapid methodologies for biosafety testing Enhancing performance with the use of
and detection of viral contamination of biologic therapeutics adsorptive depth or surface modified prefilters

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34 | October 1, 2018
 Product Showcase
Viresolve®
BioReliance® Pro Virus Filtration
Biosafety services Solution
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Chromatography products for
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BioReliance® Biosafety Testing

EXPLORE
CONFIDENCE
For an unparalleled experience throughout the life cycle
of your therapy, BioReliance ® world-class biosafety
solutions offer a full range of GMP cell banking
services, cell line and virus bank characterization,
viral clearance and lot release testing. MilliporeSigma’s
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36 | October 1, 2018