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JUNE 1, 2014
www.GENengnews.com
TOOLS TECHNOLOGIES TECHNIQUES
Cell Culture Optimization Options
Angelo DePalma, Ph.D.
EMD Millipore
has developed
a completely
closed upstream
process that is
based on singleuse bioreactors.
The process
combines
disposables
with traditional
process steps.
Bioprocessors have adopted numerous approaches
to optimizing cell culture, from clone selection to
process conditions to feed and media strategies.
Finding high-producing cell lines has been a major
focus of development programs for protein
therapeutics.
At the recent BioProcess International European Summit in Prague, a number of presenters discussed various
methodologies and options for optimizing cell culture operations.
According to Tim Ward, strategic marketing director
Sticky Ends...
Life Limit Found
in Blood
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at TAP Biosystems, now part of Sartorius Stedim Biotech,
a clone’s specific productivity defines the size of the production facility and, ultimately, the drug’s profitability.
Given the importance of high-dose monoclonal antibody
therapies, bioprocessors have come to expect multigram-
Stem Cell
Breakthroughs
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Fears
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Expanding the
Transfection
Toolbox
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per-liter results from platform processes. “Achieving this
goal rapidly and efficiently is essential,” says Ward.
Because traditional cell-line optimization is often
costly and time-consuming, the
see page
industry has searched for tools to
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Corruption
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Microenvironment
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BIOPROCESSING
Cell Culture
Continued from page 1
improve screening and selection. Initially,
developers focused on automated clone
screening (such as Genetix’ ClonePix) and
microplate-based liquid-handling systems
(such as the TAP Biosystems’ Cello). More
recently, the focus has shifted to highthroughput microbioreactors.
The first such system, BioProcessors’ automated SimCell, used a custom-designed,
multiple-chamber cassette system that ran
hundreds of plates in parallel. Although several companies tested the system, SimCell’s
size and complexity led to limited adoption.
Smaller benchtop systems were also developed based on 24-well shaken plate designs.
Systems such as MicroReactor Technologies’ Micro24 (now sold through Pall) initially focused on microbial strain selection.
Later they were tested for cell cultures despite lacking automated pH control, a shortcoming that was overcome through liquid
base addition.
The first true microscale mimic of a benchtop bioreactor incorporating an impeller, gas
sparging, and full pH control using CO2 and
automated liquid base addition, was TAP
Biosystems’ ambr. This system combined individual bioreactor control for 24 or 48 15mL reactors in parallel, with the advantage
of fully automated liquid handling for media,
inoculate, feed, and sampling.
“Many reports note ambr’s scalability of
process parameters, which results from the
system’s ability to model all critical aspects
of a benchtop stirred tank reactor—from
one up to two hundred liters,” notes Ward.
Fully Closed Upstream System
Transferring cells from frozen vials to
shake flasks during cell expansion is one of
several open upstream steps that entail the
risk of contamination. Expansion may take
several days or weeks. A group at Merck
Millipore headed by Aurore Lahille, new
technology manager, has studied this problem and developed a completely closed upstream process, based on single-use bioreactors, that combines disposables with traditional process steps.
Lahille evaluated cell freezing and thawing
in disposable bags as the run-up to inoculation of a 1,250 L bioreactor. She demonstrated feasibility through a trial employing seven
different CHO cell lines using single-use bioreactors of the type and volume (3–200 L)
commonly used during process development
and as seeding or production bioreactors. Lahille also conducted several clinical-size runs
at 200 L and 1,250 L to ensure a meaningful
comparison, and compared glass and stainless-steel bioreactors ranging in size from 3.6
L to 1,250 L. “As a result, we have developed
a fully closed USP process by coupling cell
freezing in bags and disposable bioreactors
up to production scale,” Lahille says.
The new process saves time by protecting cells at every stage, and by virtue of its
larger working volumes compared with conventional amplification protocols. “We can
freeze 50 times more cells in bags than in vials,” Lahille explains, “and this cuts amplification time significantly.”
Another advantage of interest to contract
See Cell Culture on page 28
Because traditional cell-line optimization is often
costly and time-consuming, the industry has searched
A picture taken with a high-speed camera showing a 250 L drilled hole sparger (DHS) in operation.
The DHS is a film-based sparge disc with laser drilled pores that have a specific size and quantity
tailored for each single-use bioreactor volume. The larger air bubbles of the DHS support the microsparge with oxygen transfer and improve the removal of carbon dioxide.
Thermo Fisher Scientific
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JUNE 1, 2014 | GENengnews.com | Genetic Engineering & Biotechnology News
for tools to improve screening and selection.
BIOPROCESSING
Cell Culture
Continued from page 26
manufacturers or companies with robust
pipelines is greatly improved facility utilization through the realization of fully disposable multiproduct suites.
The upstream process, which Merck Millipore has validated, is based on existing single-use bags and connectors from the company’s catalog. Lahille has now turned her
attention toward a fully closed downstream
process, for which she says new single-use
technology may need to be invented.
Single-Use for
Microbial Fermentation
“We view single-use bioreactors in cell
culture as stable, almost mature,” says Ken
Clapp, senior global product manager for
Xcellerex bioreactors and fermenters at GE
Healthcare. Almost in parallel, CHO cultures
have become industrialized and highly opti-
mized for cell densities and productivity.
As single-use equipment evolved, the issue of a practical size limit has practically
disappeared. “No longer do customers ask
for volumes of 5,000 L or more,” Clapp remarks. “With 2,000 L being offered by us
and our main competitor, current production needs are addressed with one or more of
those reactors.”
What does Clapp see down the road for
mammalian culture? Mostly tools that facilitate efficiencies, such as perfusion culture,
single-use heat exchangers, and large-diameter sterile connectors.
The focus now turns to microbial fermentation, which as a process is much more
demanding than cell culture, especially with
respect to power, heat removal, and mass
transfer. Users of fermentation bioprocessing are now desirous, Clapp says, of the
benefits universally expected from singleuse, namely, lower capital costs, simpler
infrastructure requirements, decreased risk
of cross-contamination, lower water usage,
and greater versatility.
“Our data show that with the right design
and selection of materials, a scalable product line of single-use fermenters is possible,”
Clapp asserts. The typical performance and
scale-up metrics from stainless-steel stirred
tank reactors (such as kLa, oxygen transfer
rate, and power per unit volume) are readily used to make correlations in support of
wider industry adoption of disposables, particularly with respect to the challenges of microbial processes.
Materials of construction will play a significant role. Clapp notes that when various
grades of stainless steel failed to meet the
needs of animal cell culture, new ones were
invented or adapted: “In those cases, we
relied on a metallurgist to understand and
guide selection. Now, we have an alphabet
soup of polymer names, and polymer chemists are the new metallurgists.”
Super-Medium?
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JUNE 1, 2014 | GENengnews.com | Genetic Engineering & Biotechnology News
The Boehringer Ingelheim High Expression Technology (Bi-Hex®) is an established,
high-expression technology platform. Boehringer naturally has optimized media for
that platform, but what may not be generally known is that Bi-Hex media work quite
nicely with other cell lines, including GS
CHO, DG44 CHO, and CHOK1, according
to Benedikt Greulich, Ph.D., associated director of process science at the company. “We’ve
seen titer improvements of up to 3.5-fold,”
says Dr. Greulich.
The power of this medium arises from
how it was designed. Classically, media developers mix and match components, or
media, themselves. Then the developers test
for desirable characteristics in cells that have
been grown in the media.
Dr. Greulich and colleagues departed
from trial-and-error methodology, relying
instead on gene sequencing and expression
analysis, metabolomics, and DNA chips.
With these tools they discovered deregulated
genes and/or pathways, and identified media
ingredients that corrected the situation and
improved titers.
In one case, they achieved a 30% titer
increase by adding lipids. In another experiment that employed metabolomics, they discovered extracellular metabolites that transformed lactic acid producers to lactic acid
consumers.
Lactic-acid-consuming cells have a twofold greater citric acid flux as well as higher
energy production within the mitochondria.
BIOPROCESSING
By contrast, producers have an overall reduced carbon flux, disturbed energy production, and a reduction in pathways associated
with increased biomass.
“Several of these metabolites were suitable as media components, including three
that inhibited lactic acid production, and
thereby caused a 17% titer increase,” Dr.
Greulich asserts.
If the claims are correct, Bi-Hex and its
optimized medium represent a new level of
platforming. “It’s possible to obtain several
grams per liter of product simply by applying these process conditions, without complex handling or feed addition schemes, and
with a minimum of process development,”
Dr. Greulich explains.
ory equation. Here, kLa is a combined term
describing relative mass transfer efficiency
for a given set of operating conditions.
Brau measured kLa for both O2 delivery
and CO2 stripping. “The significance here
compared to most of industry is that we
look at and consider the tradeoff between
the two processes, as well as the limitations
that govern and drive the behavior in the
first place,” Brau says.
An excessively efficient sparge will achieve
the maximum possible oxygen delivery, but
is likely to generate excessive foam and ensure CO2 buildup. “That is why you have
to account for the ratio of performance a
sparge delivers under given operating conditions, specifically the ratio of O2 delivery and
CO2 stripping,” Brau explains. Going too
far toward CO2 stripping causes the overall
sparging efficiency to suffer.
“You have to strike a perfect balance
to achieve an O2 delivery and CO2 stripping kLa that is harmonious for your process conditions,” concludes Brau. “In other
words, [this is about] managing the quantity of O2 consumed and CO2 produced.
Striking this balance means you are maintaining your cell culture in ideal conditions
with [the smallest possible] gas flow rates,
relatively speaking.”
Biological Basis for
Media Development
Optimizing cell culture processes is a balancing act, where the more that is known
and controllable, the greater the opportunity
to create a more predictable process. Among
the critical variables are gas entrance kinetic
energy and vessel liquid height.
Gas entrance kinetic energy is determined
by the velocity of gas entering the bioreactor.
All things being equal, it would seem that
sparging at a higher gas volume per process
volume over time would result in higher gas
kinetic energy. Not so.
“Higher flow is only part of the issue,”
says Christopher Brau, associate engineer for
bioprocess production at Thermo Fisher Scientific. While kinetic energy may be directly
increased and decreased in existing systems
by raising or lowering the volume of gas delivered, in new systems one has the option of
changing the size and quantity of the pores
through which gas passes. Pore size, notes
Brau, directly affects not only the velocity at
which a given gas flows, but also how fast
the velocity will increase or decrease relative
to pore size.
Brau studied systems in which the velocity
or kinetic energy of the gas entering the vessel
was maintained below thresholds determined
to create more uniform behavior. “Excessive
kinetic energy in a sparge system risks damage to cells in addition to generating a wide
bell curve of bubble sizes which has its own
host of potential issues,” he explains.
Similarly, vessel liquid column height and
mixer flow pattern determine the total area
available for mass transfer between liquid
bulk and sparged gas. When bioprocessors
do not account for these factors and the
same sparge design is used despite increasing
liquid columns, the total area available for
mass transfer skews the behavior of a sparge
system, potentially quite unfavorably. “This
often manifests as a combination of CO2
buildup, a gas gradient in the column, excess
foam, high holdup volume, and higher cell
entrainment in the foam layer, while reducing the uniformity of scale up behavior between vessels,” Brau elaborates.
Brau’s work holds special significance for
kLa, the mass transfer coefficient (kL) multiplied by the area (a) available for mass transfer based on a simplified gas liquid film theGenetic Engineering & Biotechnology News
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