Solutions for Vaccine Development
eBook
Last Updated: August 23, 2023
(+ more)
Published: August 18, 2023
From preventing deadly diseases to stopping the spread of illnesses, vaccines have made a profound impact on human health and have saved countless lives.
Traditional whole-pathogen vaccine platforms have achieved wide acceptance, but, the newer generation of vaccines, such as nucleic acid vaccines, can significantly accelerate development and manufacturing processes.
This eBook highlights ways that you can level up your vaccine research with selected bioreactor tools to fit your vaccine development workflow.
Download this eBook to explore:
- Cell-based vaccine production
- Transfection for vaccine applications
- Storage of viral samples
Solutions for Vaccine
Development
Level up your vaccine research with Eppendorf technologies
Eppendorf’s best-fit solutions with carefully selected tools for automation are tailored
to the individual needs of your vaccine-development workflow. Let us support you
to get your vaccine to market faster!
Vaccines have moved into the spotlight in recent
years and are now the subject of intense discussion
around the world. From preventing deadly diseases
to stopping the spread of illnesses, vaccines have
made a profound impact on human health and have
saved countless lives.
Each new vaccine development and the ongoing
study of vaccine effectiveness expand scientific
knowledge and contribute to research into these
life-saving treatments. Numerous vaccine technology
platforms have evolved over the past decades.
As a result of their proven track record in successfully combating many diseases, traditional whole
pathogen vaccine platforms have achieved wide
acceptance. While these vaccines still require
cultivation of the pathogen, the newer generation of
vaccines, such as recombinant-protein and nucleicacid vaccines only require the genetic sequence of
the pathogen.
These breakthrough platforms can significantly
accelerate development and manufacturing processes as well as unlock new potential to address
a broad range of indications at an unprecedented
speed. After all, to meet the growing need for new
vaccines, they must be developed quickly and
cost-effectively.
2 INTRODUCTION
Table of Content
INTRODUCTION
EDITORIAL
Development of Vaccines
WHITE PAPER
Vero Cell-based Vaccine Production
APPLICATION NOTE
Transfection for Vaccine Applications
APPLICATION BY MACHERY-NAGEL
NucleoSpin® 96 Plasmid Transfection-grade
INTERVIEW
Designing Experiments
SHORT PROTOCOL
Storage of Viral Samples
3 TABLE OF CONTENT
4 EDITORIAL
Increasing demands from governmental vaccination programs and pandemic events such as the COVID-19 outbreak
require scientists to work under pressure to shorten the timeto-market of developed vaccines. The current global vaccine
market valuation of approximately 50 Billion USD, with 80%
of the market on human vaccines. Altogether, a need for new
methods to increase speed and yield, and to produce new
vaccines in a cost-effective manner in order to remain
competitive is a constant concern for scientists. Although
the competition on the vaccine market is high, big vaccine
manufacturers are more and more forming and maintaining
collaborations with young companies and former competitors to speed up vaccine development. Additionally, we see a
trend towards collaboration with the biotechnology industry
in order to accelerate the research, development and largescale production of new vaccines.
Stirred-tank Bioreactors and How They
are Used in the Development of Vaccines
Bottlenecks for production arise from the use of twodimensional T-flasks and roller bottles. Therefore, a shift
to stirred-tank biological control systems is essential in
order to increase productivity. By enabling the parallel
control of several bioreactors at the same time, monitored
and controlled by powerful software solutions, vaccine
development processes can be optimized in small scales
and the parameters transferred in order to scale-up to
large production volumes.
Abstract
Parallel Processing - Learn from failures and optimize
the bioprocess
Process optimization consumes time when experiments are
running individually and sequentially. And these experiments are very costly. By utilizing scale-down strategies and
single-use bioreactors, the consumption of resources can be
reduced. Parallel bioprocess control systems are well suited
for scale-down approaches and offer the possibility to
change individual parameters in several bioreactors at the
same time, while monitoring and comparing the effect of
the changes in parallel (Fig 2).
Figure 1: Advanced technologies in upstream biotechnology.
Advanced technologies in upstream bioprocessing
enhance the efficiency of vaccine development
Parallel
processing
Single-use
technology
Scalable
systems
Process
intensivation
Efficient use
of data
Figure 2: DASbox® Mini Bioreactor
Parallel operation of up to 24 vessels. The optimal tool for PAT,
DoE and scale-down approaches.
EDITORIAL
5 EDITORIAL
Single-Use solutions – A step ahead of cross contaminations
Process optimization and development includes significant
manual interactions, increasing the risk of contaminations.
Traditional glass or stainless-steel bioreactors need to be
carefully cleaned and sterilized after each run before they
can be reused.
Especially nowadays, where time is crucial to find a cure
against COVID-19, the use of single-use bioreactors offer
the potential to speed up a bioprocess and prevent the loss
of a whole run due to contamination.
Scalable systems
Nearly five million people have been infected by the novel
corona virus so far. Due to its high infection rate, this
number is expected to increase tremendously before a
new vaccine will be available. In order to produce enough
doses of vaccines to help develop immunity at a global scale,
easy parameter and technology transfer if needed when
scaling up from bench- to pilot- and production is needed.
However, developing a scale-up strategy is time-consuming
and cost-intensive. High titer, robustness of the process,
constant product quality, fast turn-around times, and
scalability are some of the success factors that need to be
considered. It is important to work with bioreactors that are
comparable with bench- and pilot- and production sized
bioreactors. Keeping in mind critical scalability-related
engineering parameters like proportional vessel/impeller
geometry, oxygen transfer rate (OTR), impeller power
number (Np) and the impeller power consumption per
volume (P/V) helps to optimize a scale-up strategy.
Experience the power of data
One of the major benefits of working with advanced bioreactor control systems is the use of powerful SCADA software.
A powerful software suite, monitoring all critical parameters, automatically adapting feeding speed, gassing
conditions, and many more parameters, is the heart of each
process. With the help of software, limiting factors can be
detected and eliminated to efficiently optimize a process.
Thanks to the digitalization, the global lock-down did not
affect international collaboration of scientists and manufacturers. Like the scientists and manufacturers around the
globe are communicating with each other, it is also important that the software is able to understand all the information delivered by the various sensors connected to a bioreactor. This is especially true when they are manufactured
by different suppliers. Modern communication protocols
such as OPC UA enable the seamless communication among
devices, allowing the independent implementation into a
process while being safe and stable.
EDITORIAL
6 EDITORIAL
Conclusion
Stirred-tank bioreactors are one of the key technologies
needed on the journey of developing and producing a new
vaccine (Fig.3). They are optimal tools during each step in
upstream biology. Working with bioreactors enables for the
parallel control of several bioreactors resulting in a more
efficient and reproducible optimization of various process
parameters. The quality of the produced product greatly
benefits from the possibility to program automated responses such as feeding cycles or pH control. And finally, large
systems are available on the market that are suitable to
operate in cGMP environments to produce vaccines.
Figure 3. Scalable systems – Total upstream solutions.
Eppendorf bioprocess products cover the whole upstream bioprocess workflow
from early research to large scale production volume in development.
For more information about Bioprocessing
in Vaccine Development and Manufacturing
visit the Eppendorf website.
EDITORIAL
Small Scale Bench Scale Pilot/Production Scale
Fast selection of cell line
and cultivation medium
Monitoring and control of
process development
Industrial systems supporting
operation in cGMP enviroments
7 WHITE PAPER NO. 23
Vero Cell-based Vaccine
Production: Cell lines, Media
and Bioreactor Options
Executive Summary
The recent Covid-19 pandemic introduced new challenges
for the vaccine industry, it has also brought in new
innovations in vaccine development including DNA/RNA
based vaccines. The pandemic also increased demand for
well-established cell-based vaccine production
technologies.
We hereby review the strategies for optimizing Vero cell
based vaccine production using rabies and influenza as
examples. The Vero cell line is one of the most satisfactory
vaccine production hosts based on its infectability,
stability and well-documented performance in quality and
quantity of viral yield. It is one of the first cell lines that
received FDA approval for vaccine use and is used
throughout the world. Cell culture media technology has
advanced drastically in recent years, and a number of
serum free and protein free options are available through
commercial suppliers. Because serum tends to bind toxins
and contaminants, its elimination calls for adoption of the
highest quality reagents and careful monitoring of culture
conditions in order to achieve optimal performance.
Vero cell-based vaccine production often utilizes micro
carriers or similar types of cell attachment matrix. The
advancements of 3D cell culture matrix such as Fibra-Cel®
have been important additions to the range of possible
choices for optimizing in vitro production systems.
With a wide array of bioreactor options available, highdensity attachment cell culture will continue to be one
of the most productive methods for vaccine production.
In this era of daunting challenges from the Covid-19
pandemic, Eppendorf is constantly upgrading and expanding its cell cultivation technologies to meet the demands
of this unprecedented public health emergency.
WHITE PAPER No. 23
Ma Sha
Eppendorf Inc., Enfield, CT, USA
Contact: bioprocess-experts@eppendorf.com
Fig. 1. Fluorescent image of confluent Vero cells DAPI-stained
nuclei appear blue, and actin filaments stained with rhodamineconjugated phalloidin appear red (Eppendorf Inc.).
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Introduction
Long before Covid-19, various viral diseases, including
rabies and influenza, have been worldwide challenges to the
international biomedical community. WHO notes that in 1998
over 32,000 deaths due to rabies were reported, while
influenza has been responsible for millions of deaths worldwide over the course of the last century [1].
Rabies is often transmitted to humans from infected domestic animals. Dogs infected with rabies can become extremely
aggressive and attacks on humans are widespread, especially
in certain Asian countries where using unleashed dogs for
home security is common, and the dogs were often not
vaccinated. The virus is spread through their saliva and bites
by infected animals can be fatal. In China, the disease is
referred to as “Kuang Quan Bing”, i.e. “Mad Dog Disease”.
The annual number of deaths caused by rabies had grown to
about 59,000 worldwide according to the World Health
Organization (WHO) [2].
Since the 18th century, vaccination has proven to be the most
successful (and perhaps the only) route to the elimination of
viral diseases. The history of smallpox is well known, as is
the introduction of the use of cowpox virus from lesions in
infected animals by Jenner in 1796 [3]. Despite his work and
that of others, smallpox epidemics continued throughout the
19th century, due to improperly applied or non-existent
vaccination regimes. The work of Louis Pasteur and others
toward the end of the 19th century put vaccination on a sound
scientific footing [4].
Influenza has been a permanent worldwide scourge long
before Covid-19 virus. The CDC Influenza Division reported
an estimated range of deaths between 151,700 and 575,400
individuals resulting from the 2009 H1N1 virus infection
during the first year that the virus circulated [5]. Annual
deaths in the United States reached 61,000 in the 2018
season with over 800,000 hospitalizations according to CDC
statistics [6]. Anti-viral drugs are employed for acute treatment, but vaccination remains far and away the most
effective approach for combating viral illnesses.
There has been for years a constant, underlying concern
regarding the possibility of the emergence of a truly deadly
virus strain, on a level with the 1918 influenza outbreak, the
“Spanish Flu” that caused ~50 million deaths throughout
the world. This catastrophic possibility was realized this year
(2020) with the appearance and pandemic spread of the
Covid-19 virus, now responsible for over 50 million cases
and over 1.3 million deaths worldwide in its first year alone.
Currently numerous Covid-19 vaccine development programs
are underway throughout the world, many of them utilizing
the tried and true cell culture methods, including Vero, to
produce Covid-19 virus fragments and/or spike protein for
vaccine development purpose. The Vero cell line has been
used for years in various virus vaccine development, and is
recognized as a safe and efficient production tool. With the
current demands of pandemic vaccines development, we
believe the need for bioreactor-based Vero cell culture will
continue to grow.
Biological Systems for Viral Cultivation
Today the expanding demand for vaccine products has
necessitated the development of a range of techniques for
growing large quantities of antigenic proteins. Traditionally,
viruses have been grown in embryonated hen’s eggs, but
numerous shortcomings compromise their utility. These
include a bottleneck in the availability of high quality,
pathogen-free eggs, as well as low titers of emerging viruses
[7]. Another major concern is that when viruses are cultivated through extended passages in hens’ eggs, there is an
evolutionary process in the amnion and allantoic cavity of the
egg resulting in the selection of certain viral subpopulation,
antigenically and biochemically distinct from the original
inoculum. Because of these and other factors, well characterized permanent cell lines are coming to dominate the field.
As an alternative to egg-based vaccine production, the
advantages of mammalian cell culture systems have been
widely recognized. Cultured cells provide much shorter lead
times, a more controlled production process that takes
advantage of closed-system bioreactors, a reduced risk of
microbial contamination, and the opportunity to cultivate
viral stocks without significant egg passage-dependent
antigenic changes [8].
A WHO conference some years ago expressed concern
regarding the rapid emergence of pandemic viral strains.
It was concluded that insufficient time would be available
to generate the large quantity of high quality, fertile hens’
eggs that would be required to the demands of a worldwide
pandemic. In recent years, situation has only exacerbated.
Thus, the cell culture alternative provides a flexible and
scalable platform that can make use of the well-established
biopharmaceutical bioreactor cell culture infrastructure for
vaccine production.
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Cell Line Options
Over the years, a number of stable cell lines have been
approved by regulatory authorities for influenza virus
production, such as the Spodoptera frugiperda insect cell
line (Protein Sciences/Sanofi [9]), the Madin-Darby canine
kidney (MDCK) and the Vero cell line from African green
monkey, one of the most widely used. Another example is
the
PER.C6® cell line, which was recently announced for use in
a Covid-19 vaccine development program from J&J [10].
It is noteworthy that certain cell lines may provide an
environment favoring selection of viral subpopulations, and
these types may be inappropriate for vaccine production.
Anez et al attempted production of Dengue virus vaccine
candidates using FRhL-2 diploid fetal rhesus monkey lung
cells [11]. However, passage in this cell line resulted in the
accumulation of a mutational variant which was responsible
for reduced infectivity and immunity in Rhesus monkeys.
This phenomenon was not observed in viruses passaged in
the Vero cell line. Other lines of investigation support the
Vero cell line as the candidate of choice for viral vaccine
production, including: efficiency of primary virus isolation
and replication to high infectivity titers; genetic stability of
the hemagglutinin molecule, while maintaining the antigenic
properties of human-derived viruses; and similarities in the
pattern of protein synthesis and morphological changes
between virus-infected Vero and MDCK cells [12,13].
Given the regulatory acceptance as well as the abundance of
vaccines already successfully developed on Vero platform
(Table 1), The attachment culture of Vero cells remains to be
one of the most attractive options for cell based viral vaccine
production. The continued interest has also driven the
scientific community towards further development of Vero
cells into suspension cell lines, further expands the capability
of the Vero cells in the vaccine development and production
market [14].
Media Alternatives
There are a variety of different Vero isolates available from
commercial suppliers (Vero, Vero 76, Vero E6, Vero B4), but
all are quite similar, and their nutritional needs are comparable [17]. The search for the ideal mammalian cell culture
medium began in the 1950s, with the holy grail being an
economical, protein-free, serum-free medium that would provide strong growth support and have the property of scalability to large volumes, up to thousands of liters, while coming
in at an affordable price.
Table 1. Anti-viral vaccines using Vero cell culture production technologies. Modified from Barrett et al [15], and Kiesslich and Kamen [16].
Anti-viral Vero Cell-Based Vaccines
Study (year) Disease Vaccine Type Genus
Wang et al (2008) Chikungunya Fever Live attentuated Alphavirus
Howard et al (2008) Chikungunya Fever Inactivated Alphavirus
Blaney et al (2008) Dengue Fever Live attenuated or live
chimeric
Flavivirus
Tauber et al (2008) Japanese encephalitis Inactivated Flavivirus
Valneva Austria GmbH (Ixiaro, 2019) Japanese encephalitis Inactivated Flavivirus
Ruis-Palacios et al & Vesikari et al (2006) Rota gastroenteritis Live attenuated Rotavirus
GSK (RotaRix, 2008) Rota gastroenteritis Live attenuated Rotavirus
Montagnon (1989) Polio Live attenuated
Inactivated
Picornovirus
Aycardi E (2002) Rabies Inactivated Lyssavirus
Spruth et al (2006)
Qu et al (2005)
Qin et al (2006)
Severe acute respiratory
syndrome
Inactivated Cornovirus
Monath et al (2004) Smallpox Live attenuated Orthopoxvirus
Lim et al (2008) West Nile Encephalitis Inactivated Flavivirus
Monath et al (2006) West Nile Encephalitis Live attenuated Flavivirus
Baxter International Inc. (PREFLUCEL) Influenza Inactivated Orthomyxovirus
Chan and Tambyah (2012) Influenza Inactivated Orthomyxovirus
Merck & Co. (Ervebo 2019) Ebola Live-attenuated Zaire Ebolavirus
Wu et al., (2015) Hand-foot-and-mouth
disease
Inactivated Non-polio enterovirus
Pereira et al., (2015) Yellow fever Inactivated yellow fever virus
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Serum provides a protective function to cultured cells and
binds toxins and other contaminating materials. Thus
serum-free media must be extremely carefully formulated.
Albumin can be substituted for serum, but it may impede the
downstream steps of purification [18].
Chen et al. have tested five different serum free media,
combined with Cytodex 1 microcarriers [19]. The following
were evaluated: OptiPro SFM (Invitrogen®), VPSFM (Invitrogen), EX-CELL® Vero SFM (SAFC® Biosciences), Provero-1
(Lonza®) and HyQ SFM4MegaVir (HyClone®). The EX-CELL
Vero SFM gave one of the highest cell densities, demonstrating that the use of serum free media has become routine for
Vero cell cultivation. Comparable results were observed with
a commercial serum-free medium MDSS2N (manufactured
under the name AXCEVIR-VeroTM by Axcell Biotechnologies).
In this case, Vero cells were compared with MDCK cells
grown in T-flasks and microcarrier cultures.
Rabies Virus Cultivation Strategies
The Brazilian group led by Frazatti-Gallina has been active in
the field of Rabies vaccine production [20]. Using Vero cells
adhered to microcarriers and cultivated in a bioreactor with
serum-free medium, they generated an effective rabies
vaccine. With the aid of tangential filtration, they purified
the Rabies virus by chromatography and inactivated it using
beta-propiolactone.
Their protocol states that 350 cm2
T-flasks were harvested
and inoculated into a 3.7 liter New Brunswick™ CelliGen®
bioreactor, at a proportion of 16 cells per microcarrier
(Cytodex® 3-GE), yielding an initial seeding of 2.5 × 105
cell/
ml. The cells were grown in serum-free MDSS2 medium
(Axcell Biotechnologies). The serum-free VP-SFM medium,
according to the manufacturer, was developed for Vero,
BHK-21 and CHO cell growth. This medium drives the
adherence of the Vero cells to the microcarriers. After 4 days
of cultivation in VP-SFM medium, the cells were infected
with PV rabies virus (multiplicity of infection = 0.08). The
harvests of the culture supernatant were carried out 3 days
after the virus inoculation and four times thereafter at intervals
of 24 h. During this period, culture conditions were maintained at 60 rpm at a pH of 7.15 and 5% dissolved oxygen.
Only the temperature varied from 36.5 °C in the cellular
growth phase of the culture to 34°C after virus inoculation.
In the course of the program, seven batches of virus suspensions were produced in the bioreactor (16L per cycle) at a
mean viral titer of 104
. FFD50/0.05 mL.
The effectiveness of the preparation was demonstrated by
immunizing mice with three doses of the new vaccine (seven
batches), comparing it with the commercial Verorab and
HDCV (Rabies vaccine). Mean titers of neutralizing antibodies
of 10.3-34.6, 6.54 and 9.36 IU/mL were found, respectively.
The choice of the serum-free medium was fortunate. In this
case the amount of contaminating DNA was very low, and
tolerable, less than 22.8 pg per dose of vaccine. The authors
argue that this protocol is especially applicable in the developing world, where rabies is a constant hazard and a major
public health problem.
Fig. 2. New Brunswick Cell Lift Impeller (Eppendorf Inc).
Patented design consists of three discharge ports located
on the impeller shaft to provide uniform circulation without
traditional spinning blades for conducing microcarrier cultures
under ultralow-shear conditions. The flow is driven by centrifugal force, the rotation of the three ports creates a low-differential pressure at the base of the impeller shaft, lifting microcarriers
up through the hollow shaft and expelling them out through
its ports (The discharge ports must be submerged during
operation). Bubble-sheer is eliminated by the Cell Lift impeller,
which utilizes a ring sparger generating bubbles only within
the aeration cage, so that the oxygenation works without any
bubbles coming into contact with the cells.
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Yu et al. sought methods to increase yield in Vero cell culture
systems over that obtained using roller bottles [21]. In a
recent review, they summarized the production technology
developed over the course of the last seven years. They have
adopted the 30 L New Brunswick BioFlo® 4500 fermenter/
bioreactor.
The cells were cultivated in media containing 10% serum,
first grown as a monolayer, and when the cell density
reached 1.0–1.2 × 106
cells/mL, they were transferred to the
bioreactor containing 25 g/L of Cytodex-1 for perfusion
culture. The virus preparations, also cultured in roller bottles,
were infected with the PV2061 virus strain, harvested and
transferred to the bioreactors.
Wang et al have described a purified Vero cell rabies vaccine
that has been widely produced in China, which is responsible
for almost two-thirds of the total rabies vaccines used in Asia
[22]. This successful offering is a purified Vero cell vaccine,
referred to as ChengDa Vaccine (Liaoning ChengDa Biological Co., Ltd., Shengyang, China [23]). It is grown on a Vero
cell line utilizing the L. Pasteur 2061 strain of rabies virus,
inactivated with β-propiolactone, lyophilized, and reconstituted in 0.5 mL of physiological saline. It fulfills the WHO
recommendations for potency.
The process used at ChengDa was developed by Aycardi
[24]. A single Eppendorf bioreactor was capable of producing one million dose of rabies vaccine per year. The method
uses ultra-high density microcarrier cell cultures adapted to
a 30 L New Brunswick CelliGen bioreactor equipped with a
patented Cell Lift Impeller (Figure 2), specifically configured
for a perfusion system to feed the growth media into the
bioreactor. A specially designed decanting column (New
Brunswick Scientific) was used to prevent perfusion loss of
microcarrier and keep the cells in high concentration. The
system delivers high oxygen transfer, high nutrient level and
low shear stress, thus allowing cell growth up to 1.2 x 107
million cells/mL under continuous perfusion for up to
20 days.
ChengDa Vaccine was licensed by the Health Ministry of
China and the State Food and Drug Administration of China
(SFDA) in 2002 and has been marketed throughout the
country since that time.
Influenza Virus Cultivation Strategies
The application of Vero cells for the propagation of influenza
virus in animal-derived component free (ADCF) media was
extensively described by Wallace et al in their US patent
(no. 7,534,596 B2) [25]. The patent application includes the
steps of attaching ADCF-adapted cells to a microcarrier
(SoloHill® Engineering Inc.) and infecting the cells with
vaccine media, producing virus within the cells and harvesting of the virus. The influenza viruses produced by this
method achieved higher titer than that of the egg produced
vaccine (Table 2).
Table 2. Comparing egg-based influenza production with Vero-cell–
based production using Hillex II microcarriers (SoloHill Engineering).
Production System Panama H1N1 Titers (log10 TCID50/mL)
Egg 7.8
Vero: Serum-containing 7.9
Vero: Serum-free ADCF 8.0
60
0
10
20
30
40
50
x 104 Ni/cm2
Passage 1
(0.2 L spinner)
Passage 2
(2 L bioreactor)
Passage 3
(10 L bioreactor)
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 6 7
Days in Culture
Fig. 3. Vero-based expansion on microcarriers; seed train of
Vero cells cultured on Hillex II micocarrier beads (SoloHill
Engineering).
Top: Diagram detailing bioreactor based expansion scheme;
Bottom: Scale-up from Spinner flask to industrial bioreactors.
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A method for microcarrier-based expansion of cells from a
0.2 L spinner culture to a 2 L and 10 L bioreactor culture was
developed (Figure 3). A New Brunswick CelliGen 310
bioreactor with a 5 L vessel was used for the 2 L culture
stage. The vessel was equipped with a ring sparger, spin
filter, 3-segmented pitched blade impeller (up-pumping),
and 4-gas control at 100 mL/min (Air, N2
, CO2
, and O2
).
This expansion strategy couple with the demonstration of
viral productivity represents an ideal closed system platform
for vaccine production.
A similar method using Vero cell line for influenza vaccine
production was demonstrated by chen et al [26]. Using
Cytodex 1 microcarrier beads, these investigators were able
to achieve cell densities of 2.6 x 106
/mL in serum free,
protein free medium. These findings were obtained using a
250 ml Bellco microcarrier spinner flask equipped with a
paddle impeller, inoculated with 2.5 105
/mL Vero cells in 5%
CO2
atmosphere. In a subsequent expansion phase, starting
from an initial number of 5 x 105
/mL, the cells were expanded in a 3L bioreactor. After 24 hours the cells had adhered to
the microcarriers and the virus was added together with
fresh medium. Using these procedures, the authors were
able to obtain high virus titers up to 10 Log10 TCID50/mL.
They conclude that their approach could serve as a basis for
large scale commercial production of influenza virus.
In 2011, Baxter International Inc. announced the approval for
PREFLUCEL, the first Vero Cell based seasonal influenza
vaccine, available for 13 participating European Union
countries, including Germany, Spain, UK and the Scandinavian countries. Preflucel is comprised of purified, inactivated
split influenza virions, manufactured using Baxter’s adaptation of the Vero cell platform. Although not approved for sale
in the United States, data from a U.S. Phase III study with
over 7,200 healthy individuals has shown that Preflucel
provided 78.5% protective efficacy against subsequent
culture-confirmed influenza infection, and robust immune
responses against the three viral strains contained in the
vaccine.
Recent Examples of Vero Cell Cultivation in
Bioreactors
Although bioreactor based Vero cell culture has been widely
used in vaccine production, the cultivation methods were
typically guarded by vaccine manufactures. With increasing
demand from our customer base, Eppendorf bioprocess applications lab developed a number of bioreactor application
notes for attachment Vero cell culture.
In stirred-tank bioreactors, including both in R&D and in
actual vaccine production, Vero cells are often grown on
microcarriers or 3D support matrix such as Fibra-Cel. We
have evaluated our Fibra-Cel disks as an attachment matrix
because of their auspicious surface to-volume ratio. We find
that they provide an optimal three-dimensional environment,
protecting cells from damaging shear forces, allowing the
realization of much higher cell densities by enabling perfusion culture. In perfusion bioprocessing, it is possible to
constantly add nutrients and to remove byproducts, while
retaining the cells in the bioreactor. Therefore, higher cell
densities can be reached than in conventional batch or
fed-batch processes. We cultivated the cells in perfusion
mode, which ensures a consistent supply of nutrients and
the removal of toxic byproducts. We cultivated Vero cells in
BioBLU® 5p Single-Use vessels pre-packed with Fibra-Cel,
regulating the process with a BioFlo 320 bioprocess control
station. We achieved high cell densities, up to 4.3 x 107
cells
per mL (Figure 4) [27]. We believe that this provides strong
support for Vero-cell-based vaccine production using
Fibra-Cel packed-bed vessels (Table 3).
Fig. 4: High-density Vero cell culture in BioBLU 5p Single-Use
vessel pre-packed with Fibra-Cel.
Table 3. Comparison of growth surfaces of different cell culture vessels.
Vessel
Total growth
surface (cm2)
Growth surface equivalent
to (number of BioBLU 5p
vessels)
BioBLU 5p Single-Use
Vessel
180,000 1
T-25 flask 25 7200
T-175 172 1028
Roller bottle 850 212
10-layer stacked plate 6300 29
0
5
10
15
20
25
30
35
40
45
Calculated cell density (x 10
0 5 10 15 20
6 cells/mL)
Time (day)
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In another program to provide a method to inoculate larger
bioreactors packed with Fibra-Cel, we have evaluated spin
filter as the means to increase inoculation yields. The
spin-filer allows easy perfusion of microcarrier based cell
culture without the need for acquiring external perfusion
devices [28] We cultivated Vero cells on Cytodex 3 microcarriers at a density of 10 g/L in an Eppendorf 3 L glass vessel
using a microcarrier spin filter coupled with a pitched-blade
impeller. This device is a cylinder-shaped cage that spins
with the impeller shaft and is covered with a large 75-micron
screen designed to prevent microcarriers from being collected with the waste media (Figure 5). The process was
controlled with a BioFlo 320 bioprocess control station
without needing any additional perfusion devices. The
design of the spin filter permits cultivation of anchoragedependent Vero cells in perfusion mode on microcarriers
while ensuring a consistent supply of nutrients and the
removal of toxic byproducts.
At a modest microcarrier loading density of 10 g/L, we
achieved attachment Vero cell culture density of 8.0 × 106
cells/mL in 9 days [28], sufficient for inoculation of a 40 L
CelliGen 510 Fibra-Cel packed-bed bioreactor designed
for vaccine production. The CelliGen 510 has become the
leading Rabies vaccine production platform in China, and
the method, including CelliGen 510 itself, has been written
into Chinese FDA guidelines as part of their production
method recommendations. However, due to the very high
surface capacity, it typically requires several stacked-plate
culture devices to produce enough cells for inoculation. We
believe this simple spin-filter perfusion platform has great
potential to replace stacked plates for inoculation of larger
bioreactors in vaccine production.
Fig. 5: Microcarrier specific
spin filter with pitched-blade
impeller.
Acknowledgments
Our thanks to Ms. Alain Fairbank and Dr. Mark Szczypka from SoloHill Engineering, Inc. for their assistance and content
support. Special thanks go to K. John Morrow Jr. Ph.D, from Newport Biotech, Newport, KY, USA.
Conclusions
The combination of advances reviewed here provides strong
support for the use of cell culture systems for virus vaccine
production. The fact that Vero cells have been approved for
vaccine products represents an important step on the road to
technologies that do not rely upon hens’ eggs for generation
of adequate quantities of viruses. Advances in culture media
enable the elimination of serum, thus driving the rapid and
efficient purification of proteins. Whereas serum-containing
media may continue to occupy a default position, it is now
generally recognized that serum-free media are the optimal
choice. The use of microcarrier or Fibra-Cel add to the
efficiency of the culture technology, allowing greatly increased cell densities to be reached. Finally, improvements
in bioreactor design combined with these various technological advances result in a greatly improved and more functional production train.
The tumultuous events of the past year, dominated by the
Covid-19 pandemic, have brought us the overarching need
for a Covid-19 vaccine. The range of Eppendorf bioreactors
offers important opportunities for vaccine research, development as well as production. Our vaccine-enabling cell culture
technologies support on developing production strategies are
not limited to Vero cells, but can be used for other attachment
mammalian cell culture and vaccine production as well.
According to our existing vaccine customer base, the
Eppendorf packed-bed platform is the most effective attachment cell-based production platform on the market, achieving
well over 100 million cells/mL in mammalian cell culture.
With our expertise and specialized equipment, ranging from
microcarrier spin filter, cell-lift impeller, packed-bed bioreactors,
and single-use solutions, we offer a wide range of technologies
well suited for vaccine research and development.
WHITE PAPER
14 WHITE PAPER NO. 23
References
[1] World Health Organization. World survey of rabies number 34 (for the year 1998). WHO;
https://www.euro.who.int/en/health-topics/communicable-diseases/influenza/pandemic-influenza/past-pandemics
[2] https://www.who.int/rabies/epidemiology/en/
[3] Behbehani AM. 1983. The smallpox story: life and death of an old disease. Microbiol Rev. 47(4):455-509.
[4] Pasteur L, Chamberland, R. 1881. Summary report of the experiments conducted at Pouilly-le-Fort, near Melun,
on the
anthrax vaccination. (reprinted) Yale J Biol Med. 2002 Jan-Feb;75(1):59-62.
[5] https://www.cdc.gov/flu/spotlights/pandemic-global-estimates.htm
[6] https://www.cdc.gov/flu/about/burden/index.html
[7] Genzel Y, Reichl, U. 2009 Continuous cell lines as a production system for influenza vaccines.
Expert Review Vaccines 8(12):1681-92.
[8] Doroshenko A, Halperin SA. 2009. Trivalent MDCK cell culture-derived influenza vaccine Optaflu (Novartis Vaccines)
Expert Review of Vaccines. 8:679-688.
[9] https://www.genengnews.com/topics/drug-discovery/sanofi-to-buy-insect-cell-vaccines-company-protein-sciencesfor-up-to-750m/
[10] https://www.scienceboard.net/index.aspx?sec=sup&sub=Drug&pag=dis&ItemID=689
[11] Añez G, Men R, Eckels KH, Lai CJ. 2009. Passage of dengue virus type 4 vaccine candidates in fetal rhesus lung cells
selects heparin-sensitive variants that result in loss of infectivity and immunogenicity in rhesus macaques. J Virol.
83(20):10384-94.
[12] Govorkova, EA, Murti, G, Meignier, B, de Taisne, C and Webster, RG. 1996. African green monkey kidney (Vero) cells
provide an alternative host cell system for influenza A and B viruses. J Virol. 70(8): 5519–5524.
[13] Khadang B, Lapini G, Manini I, Mennitto E, Montomoli E and Piccirella S. 2012. Cell culture-derived influenza vaccines
from Vero cells: a new horizon for vaccine production. Expert Review of Vaccines 11(5):587.
[14] Lee DK , Park J, and Seo DW. 2020. Suspension culture of Vero cells for the production of adenovirus type 5. Clin Exp
Vaccine Res 9(1):48-55.
[15] Barrett et. al. 2009. Vero cell platform in vaccine production: moving towards cell culture-based viral vaccines Expert
Rev. Vaccines 8(5), 607–618.
[16] Kiesslich S and Kamen A. 2020. Vero cell upstream bioprocess development for the production of viral vectors and
vaccines. Biotechnology Advances44:107608
[17] Ammerman,NC, Beier-Sexton, M, and Azad, AF. 2008. Growth and Maintenance of Vero Cell Lines. Curr Protoc
Microbiol. November; APPENDIX: Appendix–4E.
[18] In Vitria website: https://invitria.com/application/vaccine-cell-culture-media/.
[19] Chen A, Poh SL, Dietzsch C, Roethl E, Yan ML, Ng SK. 2011. Serum-free microcarrier based production of replication
deficient influenza vaccine candidate virus lacking NS1 using Vero cells. BMC Biotechnol.11:81-97.
[20] Frazatti-Gallina NM, Mourão-Fuches RM, Paoli RL, Silva ML, Miyaki C, Valentini EJ, Raw I, Higashi HG. 2004. Vero-cell
rabies vaccine produced using serum-free medium. Vaccine. 23(4):511-7.
[21] Yu P, Huang Y, Zhang Y, Tang Q, Liang G. 2012. Production and evaluation of a chromatographically purified Vero cell
rabies vaccine (PVRV) in China using microcarrier technology. Hum Vaccin Immunother. Sep;8(9):1230-5
[22] Wang C, Zhang X, Song Q and Tang K. 2010. Promising Rabies Vaccine for Postexposure Prophylaxis in Developing
Countries, a Purified Vero Cell Vaccine Produced in China. Clinical and Vaccine Immunology. 17(4):688.
[23] https://adisinsight.springer.com/drugs/800041727
[24] Aycardi E. 2002. Producing human rabies vaccines at low cost. Genetic Engineering News (GEN). April 15, Vol. 22 #8.
[25] Wallace B and Hillegas W. 2009 Method and device for production. US patent 7,534,596 B2. May 19, 2009.
[26] Chen A, Poh SL, Dietzsch C, Roethl E, Yan ML, Ng SK. 2011. Serum-free microcarrier based production of replication
deficient influenza vaccine candidate virus lacking NS1 using Vero cells. BMC Biotechnol.11:81-97.
[27] Han X and Sha M. 2017. High density Vero cell perfusion culture in BioBLU 5p single use vessels. Eppendorf
Application Note #359.
[28] Han X and Sha M. 2020. Easy perfusion for anchorage-dependent cell culture using an Eppendorf vessel equipped with
microcarrier spin filter. Eppendorf Application Note #414.
WHITE PAPER
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16 APPLICATION NOTE 471
HEK293 Bioreactor Transfection for Vaccine
Applications Using the Eppendorf SciVario®
twin Bioprocess Controller: An Example with
COVID-19 Spike Protein Production
Jorge L. Escobar Ivirico and Ma Sha
Eppendorf, Inc., Enfield, CT, USA
Contact: bioprocess-experts@eppendorf.com
APPLICATION NOTE No. 471
Abstract
Bioreactor suspension cell culture platforms are essential
tools for vaccine production. They can support very high
cell densities, allowing for much higher production yields
and reduce the challenges associated with the scalability
process of adherent cell culture. Bioreactor transfection is
a method of deliberately introducing plasmids into large
numbers of cultured cells for protein production. With the
current global COVID-19 health crisis caused by the new
severe acute respiratory syndrome coronavirus 2 (SARSCoV-2), it is imperative to produce large quantities of
vaccine components such as spike proteins, the
predominant antigen of SARS-CoV-2 S vaccines.
To demonstrate feasibility, production of the SARS-CoV-2
S HexaPro spike protein was conducted using the
suspension-adapted HEK293 cell platform Expi293F™
transfected with spike-protein-encoding plasmid DNA.
To achieve high yield, the transfection was performed
using a BioBLU® 1c Single-Use Bioreactor controlled by
the SciVario® twin bioreactor control system. During the
cell culture run, parameters like cell density and viability,
as well as metabolite concentrations were monitored. In
the end, the transfection strategy reached a spike protein
titer of around 4 mg/L, which is in line with previous
reports [1].
Introduction
Vaccines are valuable tools to minimize the risk of infectious
diseases, such as the COVID-19 pandemic that started in
2020. Many vaccines subtypes exist, all with the goal to train
the immune system to fight certain infectious agents and
thus provide protection for future infections.
One such subtype is the protein subunit vaccine which
contains only components or antigens rather than the whole
pathogen. An example for an antigen used for subunit
vaccines is the COVID-19 spike protein (S-protein).
After delivering this antigen to the body, it is recognized
by the immune system and stimulates immune responses,
such as antibody production (Figure 1).
Adjuvanted spike protein vaccines are available on the
market, such as the Novavax COVID-19 vaccine NVXCoV2373 that recently received the emergency use
authorization from the FDA [2].
APPLICATION
17 APPLICATION NOTE 471
Furthermore, recent market analyses have concluded that
the global subunit vaccine market is anticipated to grow at a
significant Compound Annual Growth Rate of 10.9 % during
the forecast period of 2022-2028 [4].
In order to advance vaccine production and to enable
sufficient protection against prevalent and future pathogens,
scalable vaccine production strategies are needed. Bioreactors
offer a platform to develop such strategies.
In this application note, a bioreactor-based SARS-CoV-2 S
HexaPro spike protein production was developed using the
suspension cell culture platform Expi293F (Thermo Fisher
Scientific). In previous studies, this cell line demonstrated
efficient expansion to high densities [5] and was employed
for the production of Adeno-associated virus capsids [6], an
important component of vaccination but also in the emerging
field of gene therapy.
The bioprocess conditions were controlled and monitored by
the SciVario twin bioreactor control system in conjunction
with the BioBLU 1c Single-Use Bioreactor. Parameters like
cell growth, viability, and metabolic activity (glucose,
ammonia, and lactate levels within the medium) were
analyzed throughout the run. In addition, the SARS-CoV-2 S
HexaPro spike protein titer was determined at different time
points after transfection by ELISA, and its purity and
molecular weight were analyzed by SDS-PAGE.
Material and Methods
SciVario twin bioreactor control system and
BioBLU 1c Single-Use Bioreactors
The SciVario twin bioreactor control system was used to
perform two batch culture runs simultaneously using BioBLU
1c Single-Use Bioreactors equipped with a single pitchedblade impeller. Each bioreactor control system is equipped
with three universal port connectors for pH and dissolved
oxygen (DO) sensors, a temperature control block that
combines electrical heating and water cooling, agitation
control and a gas module that includes a Thermal Mass
Flow Controller (TMFC) with standard gas flow rates of 0.1 –
1,200 sL/h (resulting in an ultra-high turndown ratio of
1:12,000), as well as four solenoid valves (see Figure 2).
Figure 1: Schematic representation of SARS-CoV-2 S spike
protein production and potential immunization procedure.
Created with BioRender.com
Cell-derived SARSCoV-2 spike proteins
Spike protein
expression
SARS-CoV-2
(COVID-19 virus)
SARS-CoV-2
genome
Spike
protein
Spike gene
sequence
(mRNA)
Containing
Spike gene
(DNA)
Expression
plasmid
Plasmid
transfection
Spike protein is recognized by the
immune system (T cells) which
produces antibodies against the
COVID-19 virus (B cells)
Eective immune response to
the COVID-19 infection
to reduce disease severity
Transfected
mammalian cells
Vaccination
Protein subunit
formulated
APPLICATION
18 APPLICATION NOTE 471
Sensor calibration
Prior to the preparation of the BioBLU 1c Single-Use
Bioreactors, ISM® gel-filled pH sensors (Mettler Toledo®)
were connected to the SciVario twin bioreactor control
system where they were automatically detected by the
software. The calibration process was performed according
to the operations manual using buffer solutions of pH 7 and
pH 4 as “zero” and “span”, respectively. Hereafter, the pH
sensors were disconnected and sterilized in an autoclavable
pouch.
BioBLU 1c Single-Use Biorector preparation and process
parameters
Each BioBLU 1c Single-Use Bioreactor was equipped with
magnetic drives. The previously sterilized pH sensors were
then inserted in a spare PG 13.5 port under aseptic
conditions in the biosafety cabinet. In addition, the
polarographic DO sensors (Mettler Toledo) were fitted via a
non-invasive sensor sleeve into both bioreactors. DASGIP®
peltier exhaust condensers were connected to each
bioreactor and the sparge line (from the controller) was
connected to the submerged sparge filter on the bioreactor.
Three liquid addition ports were used on each bioreactor:
one for inoculation/medium addition, one for base addition
and another for the addition of 0.1% of Sigma Aldrich®
Antifoam C Emulsion (Merck). Then, the BioBLU 1c SingleUse Bioreactors were placed in their respective temperature
control block to keep the system at a constant temperature.
Finally, each bioreactor was filled with HEK ViP NX cell
culture medium (Sartorius) supplemented with GlutaMAX™
(Thermo Fisher Scientific) and conditioned for at least 24 hours
using the parameters and setpoints listed in Table 1.
SARS-CoV-2 S spike protein production workflow
An overview of the entire workflow schematic for SARSCoV-2 S spike protein production is shown in Figure 3.
The individual steps are detailed below.
SARS-CoV-2 HexaPro expression vector
Recombinant SARS-CoV-2 S HexaPro spike protein was
produced by transfecting the target cells with SARS-CoV-2 S
HexaPro expression vector obtained in-house from a
HexaPro variant plasmid in E. coli (Addgene, 154754).
Briefly, E. coli strain DH5 alpha cells were cultured in shake
flasks with LB medium (Merck) supplemented with the
appropriate selective antibiotic (ampicillin, Merck) for
18 hours at 37 °C and 200 rpm using an Innova® S44i shaker
incubator. After reaching the log phase, the cells were
pelleted by centrifugation at 11,000 rpm (16,639 × g) for
5 min at 4 °C. Plasmid DNA was purified from culture using
the PureLink™ HiPure Plasmid Filter Maxiprep Kit (Thermo
Fisher Scientific). Finally, purified plasmids were eluted
from the HiPure column by gravity flow after adding 15 ml
of elution buffer. The absorbance at 260 nm and 280 nm
was determined by using a BioSpectrometer® Kinetic D30
spectrophotometer. The ratio of A260/A280 estimates
sample purity.
Table 1: Process parameters and setpoints of the batch culture
experiments.
Working volume 1 L
Agitation 155 rpm (tip speed 0.4 m/s)
Temperature 37 °C
Inoculation density 0.4 × 106 cells/mL
Cell culture medium HEK ViP NX cell culture medium
DO setpoint 40 % (P = 0.1; I = 3.6/h)
pH setpoint 7.0 (deadband = 0.2), cascade to CO2
(acid)
cascade to 0.45 M sodium bicarbonate (base)
Gassing range 0.1 SLPH – 60 SLPH
Gassing cascade Set O2
% at 30 % controller output to 21%
and at 100 % controller output to 100%. Set
flow at 0 % controller output to 0.1 SLPH, and
at 100 % controller output to 60 SLPH
Figure 2: The SciVario twin bioreactor control system
allows the control two glass or single-use bioreactors, either
individually or in parallel, at the same time across a wide range
of vessel sizes from small- to bench-scale. It was developed for
both cell culture and microbial fermentation applications.
APPLICATION
19 APPLICATION NOTE 471
Ratios between 1.8 and 2 are commonly accepted as pure
DNA. Furthermore, an A260 value of 1 translates to ~50 ng/
µL of pure double-stranded DNA. Hence, the concentration
and purity of the plasmid were 2.58 mg/mL and 1.85,
respectively.
Suspension Cell Line (Expi293F)
In order to produce the SARS-CoV-2 S HexaPro spike
protein, the suspension adapted HEK293 cell line Expi293F
(Thermo Fisher Scientific) was used for plasmid transfection.
In previous transfection experiments for the production of
Adeno-associated virus capsids, this suspension cell line
demonstrated sufficient protein production and high cell
density [5,6].
Expi293F cell inoculum preparation for the BioBLU 1c
Single-Use Bioreactor
The cell’s expansion process was performed as shown in
Figure 4. Cells were cultured in HEK ViP NX Cell culture
medium supplemented with GlutaMAX in a New Brunswick
S41i CO2
incubator shaker at 37 °C, 8% CO2
and at an
agitation speed of 125 rpm. During the expansion process,
the inoculation density, percentage fill of the shake flasks
and other parameters were kept constant. Finally, 200 mL
inoculum containing 400 × 106
cells in HEK ViP NX cell
culture medium was added to each inoculation bottle.
Expi293F cell culture in BioBLU 1c Single-Use Bioreactors
Both BioBLU 1c Single-Use Bioreactors were inoculated with
the inoculum from the inoculation bottles (see section
“Expi293F cell inoculum preparation for the BioBLU 1c
Single-Use Bioreactor”) for a total working volume of 1 L
with a cell density of ~0.4 × 106
cells/mL and more than
95% cell viability. In both cases, the temperature was set
to 37 °C and the DO setpoint of 40 % was controlled by a
cascade (Table 1). To control foam formation, Antifoam C
Emulsion was added as needed. The pH setpoint was
controlled using a cascade of CO2
(acid) and 0.45 M sodium
bicarbonate (base) (Table 1).
Transfection procedure of Expi293F cells in
BioBLU 1c Single-Use Bioreactors
The transfection cell density of approximately
1 – 1.2 × 106 cells/mL was reached at two days after
inoculation in both bioreactors. Then, 1 µg of HexaPro
vector was diluted in 50 mL of Gibco™ Opti-MEM™ I
Reduced Serum Medium supplemented with GlutaMAX
(tube 1).
The transfection mix was prepared by adding 1 µl of
FectoVIR or FectoPRO per µg of plasmid DNA to 50 mL
of Opti-MEM medium (tube 2). The content of tube 1 was
filtered through a 0.22 µm syringe filter into tube 2 and
mixed by inversion. Then, the transfection mix solution was
incubated for 20 minutes at room temperature and finally
added onto the cells in the bioreactor.
Cell viability and metabolic activity
Samples were collected twice a day from the bioreactors
to determine the cell viability, cellular density, and the
concentration of metabolites (glucose, ammonia (NH3
) and
lactate), by connecting a sterile 5 mL syringe to the Luer
Lock sample port. Then, 3 mL of dead volume were
discarded and another 3 mL were collected again (using a
new 5 mL sterile syringe) as a viable sample for analysis.
Figure 3: The SARS-CoV-2 S HexaPro spike protein production workflow. Created with BioRender.com
Upstream Downstream
SARS-CoV-2
HexaPro
vector
Expansion
of Expi293F
cells
Plasmid
transfection
of the cells
Spike protein
expression by
the cells
SARS-CoV-2 HexaPro spike protein production workflow
Purification
Strep-Tactin
resin
Spike protein
quantification
RBD ELISA
Spike protein
size check by
SDS-PAGE
APPLICATION
20 APPLICATION NOTE 471
Cell density and viability were measured (via the trypan blue
exclusion method) using a Vi-CELL® XR Viability Analyzer
(Beckman Coulter®). pH values were monitored offline by
using an Orion Star™ 8211 pH-meter (Thermo Fisher
Scientific). Using the offline pH value, the pH calibration on
the controller was restandardized daily to prevent any
discrepancy between online and offline measurements.
Glucose, ammonia and lactate were measured using a
CEDEX® Bio Analyzer (Roche).
SARS-CoV-2 S HexaPro spike protein purification
and titration
Every day (until day 5) after transfection, 60 mL of sample
containing Expi293F cells and medium were collected using
a Labtainer™ BioProcess Container (Thermo Fisher
Scientific) with line sets. The cells were centrifuged at 300 ×
g in a centrifuge 5430R for 5 minutes to separate the cell
pellet from the supernatant. SARS-CoV-2 S spike proteins
from the supernatant were purified by affinity
chromatography. Briefly, 2 mL of Strep-Tactin® Superflow®
resin (as 50% suspension, IBA LifeSciences) were added to
Poly-Prep® Chromatography Columns (Bio-Rad). The resin
was equilibrated with 5 mL of Strep-Tactin wash buffer (IBA
LifeSciences) (five column volumes). The supernatant was
then added to the columns followed by another washing step
using Strep-Tactin wash buffer as above. Finally, 4 mL of
Strep-Tactin elution buffer (IBA LifeSciences) were added to
the column before the eluate was collected in a single tube
and concentrated using a 30 kDa cutoff spin concentrator
(Amicon® Ultra-15 Centrifugal Filter Units, Merck) at
4000 x g for 5 min (4 °C).
After the purification step, the pure SARS-CoV-2 S HexaPro
spike proteins from the supernatant were titrated through
the Invitrogen™ Human SARS-CoV-2 RBD ELISA kit
(Thermo Fisher Scientific). This ELISA antibody pair detects
the SARS-CoV-2 regional binding domain (RBD) of the S1
subunit of the spike protein. Then, the assay was conducted
according to the manufacturer’s protocol instructions.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE)
SDS-PAGE was performed with the Invitrogen™ NuPAGE™
4 to 12%, Bis-Tris, 1.0–1.5 mm, Mini Protein Gels (Thermo
Fisher Scientific) under non-reducing conditions.
SARS-CoV-2 S HexaPro spike protein samples were diluted
in Invitrogen™ NuPAGE™ LDS Sample Buffer (4x) (Thermo
Fisher Scientific) and 15 µl per sample were loaded onto the
gel. Gels were run in Invitrogen™ NuPAGE™ MES SDS
Running Buffer (20×) (Thermo Fisher Scientific) at 200 V
and the electrophoresis was completed in approximately
20 minutes. Then, gels were washed with DI water to remove
the SDS and buffer salts, stained with Invitrogen™
SimplyBlue™ SafeStain (Thermo Fisher Scientific) and
incubated for 1 hour at room temperature with gentle
shaking (30 rpm) using a New Brunswick S41i CO2
incubator
Shaker.
Figure 4: Expi293F cell culture scale-up from shaker to bioreactor for inoculum preparation.
Inoculum preparation in shake flasks for BioBLU 1c
Shake flask volume: 0.125 L
Initial cell density: 0.4 x 106
cells/mL
Performed in New Brunswick S41i CO2
incubator shaker
BioBLU 1c Single-Use Bioreactors
Final working volume: 1 L
Initial cell density: 0.4 x 106
cells/mL
Controlled by SciVario twin bioreactor control system
APPLICATION
21 APPLICATION NOTE 471
Finally, the gels were washed twice with DI water for 1 hour,
and photographs were taken with a clear background using
an Edvotek™ White Light Box (Thermo Fisher Scientific).
A pre-stained protein standard (Invitrogen™ Novex™ Sharp
Pre-stained Protein Standard, Thermo Fisher Scientific) was
used for accurate molecular weight estimation in a range of
3.5 to 260 kDa.
Results and Discussion
SARS-CoV-2 S HexaPro spike protein production in
BioBLU Single-Use Bioreactors
The expansion and transient transfection of Expi293F cells
were carried out in a 1 L BioBLU 1c Single-Use Bioreactor
culture. For transfection, approximately 1 µg of our in-houseproduced HexaPro expression vector was used for 106
cells.
Total volumetric DNA-to-transfection reagent (FectoVIR or
FectoPRO) ratio was 1:1.
Both bioreactors were inoculated with a cell density of
approximately 0.4 × 106
cell/mL. Transfection was carried out
at day 2 post inoculation with a cell density of approximately
1-1.2 × 106
cells/mL. As shown in Figure 5, two different
growth profiles were obtained. When using FectoVIR, a rapid
increase of cell growth was observed up to day 7 of culture,
reaching a peak in viable cell density at 9.5 × 106
cells/mL.
After transfection with FectoPRO, slower cell growth was
observed, and the culture reached 6.6 × 106
cells/mL at day 8.
It is worth pointing out that both bioreactors showed lower
cell growth after transfection compared to the Expi293F cells
growth in a 1 L bioreactor under the same conditions but
without transfection (data not shown, peak viable cells
density was approximately 13 × 106
cells/mL).
Figure 5:. Expi293F growth profile in BioBLU 1c Single-Use Bioreactors. Expi293F cell density and viability of two different
bioreactors containing different transfection mixes were shown.
0
20
40
60
80
100
0
1
2
3
4
5
6
7
8
9
10
11
12
0 1 2 3 4 5 6 7 8 9 10
Transfection
Cell Viability (%)
Cell Density/mL (x106
)
Time (days)
Viability: FectoVIR FectoPRO Cell density: FectoVIR FectoPRO
APPLICATION
22 APPLICATION NOTE 471
Furthermore, the concentration of glucose, lactate and
ammonia (NH3
) was analyzed on a daily basis. The lactate
concentration level were below 2 g/L in both bioreactors
throughout the run. NH3 concentrations were between
2 and 3 mmol/L until day 5 in both bioreactors and increased
drastically to toxic levels after day 6 towards the end of both
runs (Figure 6).
60 mL of sample was harvested daily post-transfection until
day 5 to determine the SARS-CoV-2 S HexaPro spike protein
titers in the supernatant by ELISA (Figure 7A). Spike protein
concentrations were reaching 3.5 mg/L for the FectoVIRtransfected and 4 mg/L for the FectoPRO-transfected cells.
Thus, the spike protein concentration in the supernatant
from cells transfected with FectoPRO as part of the
transfection mixture was noticeably higher (about 4 mg/L)
compared to the bioreactor in which FectoVIR was part of
the transfection mixture (3.5 mg/L) despite its lower growth
post transfection.
Finally, SDS-PAGE was used to analyze the SARS-CoV-2 S
HexaPro spike protein produced in both bioreactors. As
shown in Figure 7B, both samples showed high purity with
apparent molecular weight of ~ 190 kDa as expected [7].
Molecular weight standards in kDa are indicated on the left
side of Figure 7B.
Figure 6: Metabolic profile. A: Expi293F cells transfected using FectoVIR in the transfection mix. B: Expi293F cells transfected using
FectoPRO in the transfection mix.
0
1
2
3
4
5
6
7
8
9
10
11
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10
Lactate (g/L), N
H3 (m
mol/L)
Glucose (g/L)
Time (days)
Glucose Lactate NH3
0
1
2
3
4
5
6
7
8
9
10
11
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8 9 10
Lactate (g/L), N
H3 (m
mol/L)
Glucose (g/L)
Time (days)
Glucose Lactate NH3
A B
FectoVIR FectoPRO
APPLICATION
23 APPLICATION NOTE 471
Figure 7: Characterization of purified SARS-CoV-2 S HexaPro spike protein. A: Spike protein concentration obtained every day (until
day 5 post-transfection) in the cell culture supernatant of transfected cells. B: SDS-PAGE gel of SARS-CoV-2 S HexaPro spike protein
after elution from the Strep-Tactin column.
3.5
kDa
260
160
110
80
60
50
40
30
20
15
10
Fecto
VIR
Fecto
PRO
SARS-CoV-2
spike protein
Protein
ladder
B
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Time (days post-transfection)
FectoVIR-Supernatant FectoPRO-Supernatant
SARS-CoV-2 HexaPro Spike
concentration (mg/L cell culture)
A
0 1 2 3 4 5 6
Conclusion
This study successfully demonstrated the feasibility of
bioreactor-based plasmid transfection in BioBLU 1c
Single-Use Bioreactors under control of the SciVario twin
bioprocess controller.
COVID-19 spike proteins were produced from the plasmid
transfection, representing an important compound of protein
subunit vaccines. The suspension cell culture approach
allows a more straightforward scale-up into larger bioreactors
and offers much desired simplicity, as well as access to a
greater variety of production systems over attachment
culture-based methods.
The efficient setup of the SciVario twin enabled precise
control of the cell culture environment, resulting in robust
spike protein titers of 3.5 to 4 mg/L. To conclude, the
Expi293F cell line together with the advanced suspension
stirred-tank bioreactors and controllers offered by Eppendorf
provide an efficient platform for the development of
suspension cell-based protein production at various scales.
APPLICATION
24 APPLICATION NOTE 471
Literature
[1] Schaub JM, Chou CW, Kuo HC, Javanmardi K, Hsieh CL, Goldsmith J, DiVenere AM, Le KC, Wrapp D, Byrne PO,
Hjorth CK, Johnson N v., et al. Expression and characterization of SARS-CoV-2 spike proteins. Nat Protoc 2021;
16:5339–5356.
[2] Centers for Disease Control and Prevention (CDC). Grading of Recommendations, Assessment, Development,
and Evaluation (GRADE): Novavax COVID-19 Vaccine 2022.
https://www.cdc.gov/vaccines/acip/recs/grade/covid-19-novavax.html
[3] National Institutes of Health (NIH). Vaccine Types 2019.
https://www.niaid.nih.gov/research/vaccine-types
[4] Global Subunit Vaccine Market 2022-2028. Global: Orion Market Research Private Limited; 2022.
[5] Escobar Ivirico JL, Sha M. HEK293 Suspension Cell Culture Using the BioFlo® 320 Bioprocess Controller with BioBLU®
3c Single-Use Bioreactors. Eppendorf Application Note 447;
https://www.eppendorf.com/product-media/doc/en/1024474/Fermentors-Bioreactors_Application-Note_447_HEK293-
suspension-culture_HEK293-Suspension-Cell-Culture-BioFlo-320-Bioprocess-Controller-BioBLU-3c-Single-Bioreactors.pdf
[6] Escobar Ivirico JL, Sha M. Adeno-associated Virus Production in Suspension Cell Culture Using the SciVario® twin
Bioprocess Controller. Eppendorf Application Note 450;
https://www.eppendorf.com/product-media/doc/en/2079778/Fermentors-Bioreactors_Application-Note_450_SciVariotwin_Adeno-associated-Virus-Production-Suspension-Cell-Culture.pdf
[7] Hsieh C-L, Goldsmith JA, Schaub JM, Divenere AM, Kuo H-C, Javanmardi K, Le KC, Wrapp D, Lee AG, Liu Y, Chou C-W,
Byrne PO, et al. Structure-based design of prefusion-stabilized SARS-CoV-2 spikes.
Science (1979) 2022; 369:1501–1505.
APPLICATION
25 WHITE PAPER MACHEREY-NAGEL
MACHEREY-NAGEL
NucleoSpin® 96 Plasmid Transfection-grade
Purification of plasmid DNA with transfection-grade purity using the platform epMotion® 5075vt
Introduction
Transfection of cultured cells is one of the most common applications for isolated plasmids and requires highly pure DNA.
The main impurities in plasmid DNA preparations derive from
endotoxins. Endotoxins are lipopolysaccharides derived from
the bacterial cell wall that have cytotoxic effects and negatively
influence cell viability and transfection efficiency. Additionally,
endotoxins are known to influence gene expression in cell
cultures, leading to false results in gene expression analysis.
The efficient isolation of plasmid DNA from bacterial cultures is
essential for a variety of molecular applications utilized by many
research laboratories.
MACHEREY-NAGEL has developed a 96-well kit, NucleoSpin®
96 Plasmid Transfection-grade, for the isolation of endotoxin
reduced plasmid DNA based on silica membrane technology.
The kit combines a fast processing with novel endotoxin removal
wash buffers, enabling convenient and time saving isolation of
transfection-grade DNA (≤ 50 EU/μg DNA, endotoxin units per
μg DNA).
This application note describes the automated process on
the liquid handling workstation epMotion® 5075vt using
the NucleoSpin® 96 Plasmid Transfection-grade kit from
MACHEREY-NAGEL. The novel optimized protocol allows the
processing of a variable sample number in multiples of 8 (8–96).
The processing of 96 samples takes approximately 100 minutes
excluding cultivation and harvesting.
Product at a glance
NucleoSpin® 96 Plasmid Transfection-grade
Technology Silica membrane and endotoxin reduction technology
Sample material Up to 5 mL bacterial culture (E. coli, high-copy
plasmids)
Preparation time Approx. 100 min for 96 samples (excluding cultivation
and harvesting).
Format Variable sample number in multiples of 8 (8–96)
Typical yield 5–20 µg
Elution volume 100–200 µL
Binding capacity 20 μg
Material and methods
The optimized protocol is programmed to process up to 96
samples in parallel (Variable sample number in multiples of 8)
and developed for the epMotion® 5075vt platform. Cultivation
and harvesting of bacterial cells is recommended to perform
according to the NucleoSpin® 96 Plasmid Transfection-grade
user manual. Bacterial cell pellets from up to 5 mL cultures are
resuspended in Resuspension Buffer A1 and subsequently lysed
by addition of Lysis Buffer A2 for 5 min at room temperature.
Following lysis and neutralization by addition of Buffer A3, all
subsequent steps are performed on the epMotion® 5075vt. The
NucleoSpin® 96 Plasmid kit utilizes two different 96-well filter
plates in order to achieve a precise separation as well as high
yield and quality of plasmid DNA. Lysate clearance and Plasmid
DNA binding is performed by vacuum. Crude lysates are cleared
by the NucleoSpin® 96 Plasmid Filter Plate, removing cellular
debris as well as chromosomal DNA. Nucleic acids are subsequently bound to the silica membrane of the NucleoSpin® 96
Plasmid Binding Plate during the binding step. Contaminants,
such as salts or proteins, are then removed from the silica
membrane by three washing steps, and highly pure plasmid
DNA is finally eluted under low ionic strength conditions in a
slightly alkaline Elution Buffer AE.
IN PARTNERSHIP WITH
MACHEREY-NAGEL
26 WHITE PAPER MACHEREY-NAGEL www.mn-net.com Applikation Note NucleoSpin® 96 Plasmid Transfection-grade Eppendorf epMotion5075vt · 06/2019, Rev. 01
Application data
pGEM-T–359 bp pGEM-T–982 bp
14
10
6
0
Total yield [µg]
pGEM-T–1482 bp
4
2
8
12
Isolation of transfection-grade plasmid DNA from bacterial cultures
Plasmid DNA of three different bacterial strains, transformed with plasmids
containing either a 1482 bp, a 982 bp or a 359 bp insert, was isolated from
1.5 mL of bacterial cultures (E. coli DH 5α, high-copy plasmid pGEM®-T
Easy; n = 24) using the NucleoSpin® 96 Plasmid Transfection-grade kit on the
epMotion® 5075vt. Total yield was determined by UV spectrometry (dark blue
bars). All measured endotoxin contents showed significant less than 50 EU/μg
DNA (EU = endotoxin units)
A260 / A280
A260 / A230
pGEM-T–359 bp pGEM-T–982 bp pGEM-T–1482 bp
A260 / A280 A260 / A230
2.5
0
2.5
2
1.5
2
1.5
1
0.5
0
1
0.5
Purity of transfection-grade plasmid DNA from bacterial cultures
Plasmid DNA of three different bacterial strains, transformed with plasmids
containing either a 1482 bp, a 982 bp or a 359 bp insert, was isolated from
1.5 mL of bacterial cultures (E. coli DH 5α, high-copy plasmid pGEM®-T
Easy; n = 24) using the NucleoSpin® 96 Plasmid Transfection-grade kit on the
epMotion® 5075vt. Total purity was determined by UV spectrometry (A260/A280:
dark blue bars; A260/A230: orange squares). All measured endotoxin contents
showed significant less than 50 EU/μg DNA (EU = endotoxin units; data not
shown).
L pGEM 359 bp pGEM 982 bp pGEM 1482 bp L
Reproducible yields of plasmid DNA
Plasmid DNA of three different bacterial strains, transformed with plasmids
containing either a 1482 bp, a 982 bp or a 359 bp insert, was isolated from
1.5 mL of bacterial cultures (E. coli DH 5α, high-copy plasmid pGEM®-T Easy).
The reproducibility and integrity was analyzed by gel electrophoresis (10 μL
per eluate; 1 % TAE gel; Marker (L): GeneRuler™ 1 kb DNA Ladder – Thermo
Scientific).
Speed up and automate your transfection grade plasmid DNA extraction
MACHEREY-NAGEL and Eppendorf® deliver a fully automated solution for your high throughput plasmid DNA extraction in
transfection-grade purity. We adapted the NucleoSpin® 96 Plasmid Transfection-grade kit on the epMotion® 5075vt to speed up your
nucleic acid purification workflow.
n Endotoxin removal wash buffer and optimized filter plates for highly pure plasmid DNA with less than 50 endotoxin units per µg DNA.
n Flexible sample numbers (multiple of 8) and fast processing of 96 samples within 100 minutes (excluding cultivation and
harvesting).
n Reliable performance and excellent yields using NucleoSpin® 96 Plasmid Transfection-grade kit on the epMotion® 5075vt.
Ordering information
Product Specifications Preps REF
NucleoSpin® 96 Plasmid
Transfection-grade
Kit based on silica membrane technology for the isolation of
transfection-grade plasmid DNA from bacterial cultures in 96-well format
1 x 96 / 4 x 96 / 24 x 96 740491.1 / .4 / .24
epMotion® 5075vt Basic device incl. vacuum system, gripper, vac frame 2, vac frame
holder, Eppendorf ThermoMixer®, epBlue™ software, mouse, waste box,
100–240 V ±10 % / 50–60 Hz ±5 %, 0.2 µL–1 mL
1 5075000304
NucleoSpin® is a registered trademarks of MACHEREY-NAGEL; Eppendorf®, the Eppendorf Brand Design, Eppendorf ThermoMixer® and epMotion® are registered trademarks and epBlue™ is a trademark of
Eppendorf AG, Hamburg, Germany, GeneRuler™ and DH5α™ are trademarks of Thermo Scientific Inc; pGEM®-T Easy is a registered trademark of Promega Corporation in the U.S. and / or other countries
© Grebcha · pp77 – Fotolia.com – Fotolia
IN PARTNERSHIP WITH
MACHEREY-NAGEL
27 INTERVIEW / SEBASTIAN KLEEBANK
Sebastian Kleebank studied Bioengineering at the
University of Applied Sciences in Juelich, followed
by his Ph.D. at the RWTH Aachen, Institute for
Molecular Biotechnology.
In his actual position as a Product Life Cycle Manager
at Eppendorf SE, Bioprocess Unit, he is responsible
for the small-scale product portfolio.
During his 15 years in the bioprocess industry, he gained
a lot of experience in small scale upstream process
development, including several years working in the lab.
Contact: bioprocess-experts@eppendorf.com
ABSTRACT
In the Quality by Design (QbD) and Process Analytical
Technology (PAT) era, the generation and validation of a
large amount of data have become standard. Parallel
bioreactor control systems offer the possibility to test and
optimize different process parameters at the same time,
thus saving time and resources.
INTRODUCTION
Working with bioprocess control systems that help to develop and optimize processes in terms of time and cost efficiency is crucial. In the Quality by Design (QbD) and Process
Analytical Technology (PAT) era, the generation and validation of a large amount of data have become standard. This
data is not generated by random trial and error experiments.
Experiments are carefully designed with the information
gathered from previous experiments. This Design of Experiment (DoE) approach allows profound insights into the
process. Parallel bioreactor control systems offer the possibility to test and optimize different process parameters at the
same time, thus saving time and resources. This is especially
important in the fast-changing biotechnology industry.
We spoke with Dr. Sebastian Kleebank about the DASbox®
Mini Bioreactor System, a compact and flexible parallel
bioprocess controller for the control of up to 24 bioreactors.
What was one of the most significant experiences in your
laboratory life?
Sebastian:
Everybody who works in a laboratory running long, work-intensive, and expensive experiments knows the thrilling feeling,
and the question in mind: “How does my experiment look like
when I will be back In the lab after the weekend?”. One of my
most prominent incidents for me was, that I stepped into the
lab and found my culture everywhere on the floor as a consequence of a foam out.
This was the first time I realized that continuous monitoring of
the process is crucial and reliable process control is essential.
What are the major challenges in running a bioreactor
control system?
Sebastian:
The goal of each scientist is to gain as much process knowledge as possible in a short time. This is especially true for
scientists working in industrial laboratories, where the time-tomarket is important. To get to know your process, one needs to
perform many tests. To save resources, this is mainly done in
small working volumes of less than a liter. And here a precise
and reliable process control is key to gain high-quality results.
Regarding the foam out mentioned above it is a very comfortable feeling to know that the DASbox system can prevent this.
We developed a very sensitive level sensor that can identify
even the slightest foam formation and distinguishes precisely
between air bubbles and liquid. With a sensitivity in a range
between 1 – 20000 μS, foam can be reliably detected and destroyed, especially when you are investigating unknown, new
processes.
Knowledge Gain Through High Parallel Bioprocess Setups
The perfect tool for Design of Experiments
INTERVIEW
28 INTERVIEW / SEBASTIAN KLEEBANK
During fed-batch, perfusion, or continuous cultivations, it can
happen that the user underestimates the amount of substrate
that the organisms would need. This could lead to exceeding
the maximum working volume of the connected bioreactor.
With the combination of our very precise pumps in the DASbox
system and the powerful DASware software suite, we offer a
solution that mitigates the risk of such errors.
The software of our DASbox system sums up the added and
removed liquid volumes and automatically stops the feeding
if the maximum working volume is reached.
How can the DASbox help to optimize all the different
steps of a process?
Sebastian:
From the inoculation of the bioreactor to harvesting, the DASbox and its control software offer solutions to support you in
each individual step. After inoculation, the oxygen requirement
for aerobic cultures increases steadily.
With our oxygen cascade (flexible change of stirrer speed,
oxygen concentration of the input gas and/or gas flow rate) the
oxygen demand of the culture can effectively be covered.
The end of the batch phase can, for example, be reliably
identified by the so-called substrate consumption peak of the
dissolved oxygen concentration and the substrate pump can be
started automatically. When you add an exhaust gas
analyzer, the substrate can even be added in relation to the
metabolic activity of the cells. Our precise pumps ensure that
the substrate is added continuously even at very small feed
rates of down to 0.3 mL/h. In combination with submerged
liquid addition, this results in a very smooth DO (Dissolved
Oxygen) signal that enables a reliable process control.
And even the right time for harvesting can be defined for
example by reaching a certain number of cells (e.g. when
using a DASGIP OD sensor) or other measured variables and
the harvest pump can be started automatically.
With our scripting functionality, there are virtually no limits to
the flexibility of the control strategy. Controller inputs and outputs can be freely configured. Signals from balances, external
sensors e.g. for methanol or glucose, as well as internal
process values can be selected.
What are the benefits of highly parallel approaches?
Sebastian:
The obvious correlation is quite simple: the more experiments
are performed in parallel, the more results are collected at the
same time. The real advantage of highly parallel approaches is
that you can systematically investigate your process and thus
increase the knowledge gain. The knowledge about the process
is therefore the actual benefit here. The basis for this is that a
reliable and reproducible process control can be ensured and
that the results can be transferred to large scales. Both can be
achieved with the DASbox system. One way to systematically
investigate your process can be to consider the fact that many
processes are divided into two parts. The first part being the
growth phase and the second part being the production phase.
The tricky part is that the optimal growth conditions may not
be the optimal conditions for the production phase.
When using design of experiments (DoE) you can easily start
the growth phase in all setups under the same standard
conditions (resulting in the same amount of cells after the end
of the growth phase) and then switch the individual conditions
to analyze the impact of factors like temperature, pH, and
dissolved oxygen concentration on the product concentration
and product quality.
But highly parallel setups also own the risk of manual handling
errors. For example, a certain setpoint could be assigned to the
wrong bioreactor unit. To avoid such programming errors, our
DASware design software helps you to import complex
experimental designs and automatically assigns the correct
setpoints.
How do you keep an overview of all the many process
results?
Sebastian:
In high parallel experiments, it is crucial to identify which setup
worked as expected and which did not. Otherwise, the wrong
conclusions are made. DASware control gives a very good
overview on every single process using customizable online
charts. Thus, you can easily identify the setups which worked
as expected. For example, when using a pH setpoint of 7.0 you
can easily check if the process value was also 7.0 over the
relevant process time. The DASware control software is
capable of doing this for all relevant process parameters of
up to 24 vessels in parallel.
Additionally, the strength of the software is to compare
individual runs, also from past experiments. With DASware
control, you can start the inoculation time for each vessel
individually. This feature simplifies the comparison of relevant
process parameters like pH, DO, temp. profiles between
different units and even between different historical runs.
I don’t know how much time I spent manually synchronizing
the time axis of different runs to be able to compare them
when I was still actively working in the lab. Now the DASware
control software does this with one mouse click.
INTERVIEW
29 INTERVIEW / SEBASTIAN KLEEBANK
If I am experienced in bioprocessing, but not in programming, how can I benefit from the scripting possibility of
the DASware software?
Sebastian:
We have a collection of scripting-templates that can easily be
applied by beginners or being modified by expert users with
almost no limitation. Just ask our field service and benefit from
our long-term experience in the industry.
Where do you see the biggest advantage of the
DASbox system?
Sebastian:
The biggest advantage of the DASbox system is its compactness combined with the high precision of process control.
Together with the powerful DASware control software, the
DASbox system is the ideal tool for screening and process
development. Up to 24 vessels can be connected to one
process computer allowing for highly parallel setups requiring
only 1.80 meter/6 feet of bench space and the integrated
storage options for accessories ensure that everything is stored
where you need it.
Although the DASbox is a very compact system it uses industry-standard ports (PG13.5) that enable the use of standard
sensors with an outer diameter of 12 mm.
It offers the ability to use both single-use and glass vessels
side-by-side or go completely single-use or glass for certain
runs. This is a major advantage as the glass vessels allow users
to make impeller changes and accessory adjustments. With
the large variety of accessories and modular upgrades, you can
easily react to changing process requirements. For example,
if there is the need to add further features like exhaust gas
analysis, these devices can easily be added to the systems.
The installation is comparatively simple, as only a power
supply and the individual gas connections are required on site.
There is no cooling water needed for temperature control due
to our Peltier technology, which we also use for exhaust
gas condensation. With this, we keep the evaporation at a
minimum and effectively prevent clogging of the exhaust filters.
With easy workflow guidance on the one hand and options to
deeply adjust controller settings, the DASbox system is the
optimal tool for beginners and experts alike
INTERVIEW
For more information about DASbox®
please visit the Eppendorf website.
30 SHORT PROTOCOL
Large quantities of virus particles are often used in gene
therapy and vaccine production (i.e. SARS ..). For downstream purification of viruses and proteins various protocols
are used: filtration, ion-exchange chromatography and
gradient centrifugation.
All of these techniques rely on high quality plastic labware
for handling and storage of viral samples. During vaccine
production and purification viral samples are rather diluted
and viral particles are large. Therefore the unspecific adsorption to plastic labware often poses a major problem and leads
to sample loss. The solution to this problem may be the use
of high quality Protein LoBind tubes.
Dr. Rafal Grzeskowiak – Application Specialist
Brigitte Klose - Global Marketing Manager Consumables
Eppendorf Protein LoBind® Tubes – Your Excellent
Choice for Handling and Storage of Viral Samples
(in Vaccine Production Workflows)
Fig. 1: Virus loss in different containers (20mM HEPES,
150 mM NaCI. Ph 7.8: initial Ad5 concentration was
approximately 6 x10¹⁰ p/mL ≈ 0.02 g/L).
Eppendorf Protein LoBind Tubes and 8 competitor tubes.
Adapted from [1], page 7, Copyright 2007 by Elsevier B.V.
ScienceDirect® - “The leading platform of peer-reviewed
literature that helps you move your research forward” –
published the “Journal of Chromatography A”, Volume 1142,
Issue 1, 16 February 2007 with the detailed description of a
study on “Sorption processes in Ion-exchange chromatography of viruses” by E.I. Trilisky, A.M. Lenhoff, Department of
Chemical Engineering, University of Delaware, Newark, USA.
The authors of this article tested nine tubes from various
manufacturers in the ion-exchange purification protocols.
They clearly showed, that only by using Eppendorf Protein
LoBind Tubes the concentration of viral particles remained
stable during entire storage time of 120 hr. In all other tubes
tested the concentration of samples declined down to 60% –
18% of the initial one (Fig. 1).
0,8
0,4
0,2
0,6
0
1
0
Remaining fraction of virus
Time (hours)
24 48 72 96 120
Noticeably, it was also shown that concentration of viral
samples stored in Eppendorf Protein LoBind Tubes remained
stable under usage of various buffer systems (HEPES,
phosphate, Tris buffers) and broad range of ionic strength
conditions: NaCl concentration between 0 and 3 mol/L
(Fig. 2, adapted from [1]).
SHORT PROTOCOL
31 SHORT PROTOCOL
Fig. 2: Virus loss due to binding to the container as a function
of ionic strength (0.5 mL LoBind tubes, 20 mM HEPES, pH 7.8,
400 μL solution per tube with an initial concentration of
1.3 x 1010 p/mL ≈ 0.04 g/L, data were collected after 24 and
48 h with no significant differences between the two sets of
time points).
Adapted from [1], page 7, Copyright 2007 by Elsevier B.V.
Literature
[1] E I Trilisky, A M Lenhoff, Sorption processes in ion-exchange chromatography of viruses.
J Chromatogr A. 2007 Feb 16; 1142(1): 2-12
0,8
0,4
0,2
0,6
0
1
0
Fraction of virus remaining in solution
after 24 hrs
NaCl concentration (mol/L)
1 2 3 4
The authors conclude that of all containers tested, only one
type – Eppendorf Protein LoBind Tubes – did not bind viral
particles and is recommended for collection and storage of
viral samples.
Find more information on the above described article
https://pubmed.ncbi.nlm.nih.gov/17240385/
SHORT PROTOCOL
Learn more about LoBind Tubes
»How it works – Eppendorf LoBind®«
Watch video
Application Notes 382
»Comparative Analysis of Protein
Recovery Rates in Eppendorf LoBind®
and other ›Low Binding‹ Tubes«
Rafal Grzeskowiak¹, Sandrine Hamels², Eric Gancarek2
¹Eppendorf AG, Hamburg, Germany; ²Eppendorf Application Technologies SA, Namur, Belgium
APPLICATION NOTE No. 382 I October 2016
Abstract
Protein preparation and storage poses a critical step in a
wide range of laboratory applications. Unspecific adsorption of protein molecules or peptides to polymer surface
of lab consumables has been shown to be a substantial
factor contributing to sample loss during storage/handling
and to influence experimental results. Binding of protein
samples was investigated here by using a sensitive
fluorescence assay, and recovery rates were compared
between tubes of different manufacturers referred to as
“low binding”. The majority of tubes of different manufacturers tested showed very poor recovery rates (4% - 12%)
after 24 h storage time and do not protect sufficiently
against unspecific loss of protein samples. Eppendorf
LoBind Tubes provided highest recovery rates of proteins (95%) and thus ensure utmost protection of protein
samples.
Comparative Analysis of Protein Recovery
Rates in Eppendorf LoBind® and Other
“Low Binding” Tubes
Introduction
Protein preparation and storage pose critical steps in a wide
range of laboratory applications including various methods
in proteomics, molecular biology, forensics, diagnostics,
and bio-pharma. Protein sample purity and yield in these
methods have a strong effect on experimental results. They
are a function of biological material quality and availability,
of preparation and handling methods, but also of conditions
and consumables used during preparation and storage [1].
Unspecific adsorption of protein molecules and peptides to
polymer surface has been shown to be a substantial factor
contributing to sample loss during storage and handling in
lab consumables [2, 3, 4]. This process is largely conveyed
via unspecific binding of hydrophobic domains in peptides
and proteins to hydrophobic polymer surface, leading both
to structural denaturation and decrease in concentration over
relatively short time: up to 90% of protein sample may be
adsorbed within 24 h. [5] (fig. 1A). Unspecific sample and
activity loss may be a critical factor influencing experimental
results particularly when sensitive methods/assays or small
sample amounts are used in proteomic, forensic, and diagnostic protocols.
Download paper
Application Note 404
»Total Sample Recovery in
Eppendorf Protein LoBind
Conical Tubes«
Preparation and storage of protein samples are crucial steps
in various protocols in the fields of proteomics, molecular
biology, forensics and bio-pharma. Nonspecific adsorption
of protein and peptide molecules to polymer surfaces of
laboratory consumables has been shown to be a substantial
factor contributing to structural denaturation, diminishing
activity and decrease of sample concentration [1, 2, 3].
These effects are particularly prominent when
sensitive methods/assays or small sample amounts are
used in proteomic and forensic protocols.
In this Application Note, we investigated nonspecific binding
of low concentration protein samples by using a sensitive
fluorescence assay. Sample recovery was compared between
standard polypropylene conical tubes from various manufacturers and Eppendorf Protein LoBind Conical Tubes.
Introduction
Total Sample Recovery in Eppendorf
Protein LoBind Conical Tubes
Rafal Grzeskowiak¹, Sandrine Hamels², Blandine Vanbellinghen²
¹Eppendorf SE, Hamburg, Germany
²Eppendorf EAT, Namur, Belgium
Abstract
Protein preparation and storage are critical steps in a
wide range of laboratory applications including various
methods in the fields of proteomics, molecular biology,
forensics and bio-pharma. Nonspecific adsorption of
protein/peptide molecules to polymer surfaces of lab
consumables has been shown to be a substantial factor
contributing to sample loss and degradation. This may
adversely influence experimental results, particularly when
sensitive methods/assays or small sample amounts are
used. In this study, recovery rates of low concentration
protein samples were compared between conical tubes
from different manufacturers by using a sensitive
fluorescence assay. Among the conical tubes tested,
Eppendorf Protein LoBind Conical Tubes showed highest
protein recovery rates (mean of 100%) and thus ensured
the highest protection from sample loss.
APPLICATION NOTE No. 404
Download paper
Eppendorf® and the Eppendorf Brand Design are registered trademarks of Eppendorf SE, Hamburg, Germany. All rights reserved, including graphics and images. Copyright © 2022 by Eppendorf SE.
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32
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PER.C6® is a registered trademark of Janssen Vaccines & Prevention B.V., Netherlands. SoloHill® is a registered trademark of Sartorius Stedim North America, Inc., USA. Cytodex® is a registered
trademark of GE Healthcare Bio-Sciences AB, LLC, Sweden. HyClone® is a registered trademark of Hyclone Laboratories, Inc., USA. Lonza® is a registered trademark of Lonza AG, Switzerland.
Invitrogen® is a registered trademark of Life Technologies Corporation, USA. SAFC® Biosciences is a registered trademark of Merck KGaA, Germany. EX-CELL® is a registered trademark of
Sigma-Aldrich Co. LLC
Beckman Coulter® and Vi-CELL® are registered trademarks of Beckman Coulter, Inc., USA. Poly-Prep® is a registered trademark of Bio-Rad Laboratories, Inc., USA. Strep-Tactin® and Superflow®
are registered trademarks of IBA Lifesciences GmbH, Germany. ISM® and Mettler Toledo® are registered trademarks of Mettler-Toledo GmbH, Switzerland. Amicon® and Sigma-Aldrich® are
registered trademarks of Merck KGAA, Germany. Roche Diagnostics® and CEDEX® are registered trademarks of Roche Diagnostics GmbH, Germany. Thermo Fisher Scientific® is a registered
trademark of Thermo Fisher Scientific Inc., USA. GeneRulerTM and DH5αTM are trademarks of Thermo Scientific Inc; pGEM®-T Easy is a registered trademark of Promega Corporation in the U.S.
and / or other countries. ScienceDirect® is a registered trademark of Elsevier B.V., NucleoSpin® is a registered trademark of MACHEREY-NAGEL GmbH & Co KG.
Eppendorf®, the Eppendorf Brand Design, Eppendorf ThermoMixer®, epMotion®, epBlue®, LoBind®, CelliGen® Eppendorf SciVario®, SciVario®, BioFlo®, BioBLU®, Innova®, BioSpectrometer® and
New Brunswick are trademarks of Eppendorf SE, Hamburg, Germany. DASGIP® and DASbox® are registered trademarks of DASGIP Information and Process Technology GmbH, Germany.
All rights reserved, including graphics and images. Copyright © 2023 by Eppendorf SE.
Eppendorf SE reserves the right to modify its products and services at any time. This application note is subject to change without notice. Although prepared to ensure accuracy, Eppendorf SE
assumes no liability for errors, or for any damages resulting from the application or use of this information. Viewing the application notes alone cannot as such provide for or replace reading and
respecting the current versions of the operating manuals.
About Eppendorf
Since 1945, the Eppendorf brand has been synonymous with customer-oriented processes and innovative products,
such as laboratory devices and consumables for liquid handling, cell handling and sample handling. Today, Eppendorf
and its approximately 5,000 employees employees serve as experts and advisors, using their unique knowledge and
experience to support laboratories and research institutions around the world. The foundation of the company’s expertise
is its focus on its customers. Eppendorf’s exchange of ideas with its customers results in comprehensive solutions that
in turn become industry standards. Eppendorf will continue on this path in the future, true to the standard set by the
company’s founders: that of sustainably improving people’s living conditions.
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