The Smarter Solution for Bioprocessing
Compendium
Published: January 28, 2025

Credit: Technology Networks
The journey to develop an upstream bioprocess is complex, involving process development, optimization and scale-up. Hence, it’s important to choose the right equipment to accelerate this journey while maintaining compliance.
Features like real-time parameter control, parallel processing, easy-to-use software interfaces are all crucial to enhance efficiency and mitigate risk.
This compendium highlights how a universal bioprocess control platform can support microbial and cell culture processes, adapt to batch or fed-batch workflows and seamlessly scale from lab to production, all within a compact design.
Download this compendium to discover:
- How universal control strategies optimize scalability and precision
- The role of advanced software in improving connectivity and risk mitigation
- Real-world applications enhancing productivity across various scales
BioFlo® 320 – Universal controller for cell culture and microbiology research
The Smarter Solution
2 BioFlo® 320
BioFlo® 320 3
Highly Evolved
The BioFlo 320 seamlessly combines form and function in one state of the art package regardless of whether your
process includes cell culture or fermentation, autoclavable or single-use bioreactors. A robust industrial design,
intelligent sensors, Ethernet connectivity, and enhanced software capabilities are only a few of the features that set it
apart from the competition. Combined with a sincere commitment to quality, the BioFlo 320 truly is the premium choice
in bench-scale bioprocess control systems.
Ease of Use
> Autoclavable vessels and our comprehensive portfolio of BioBLU® Single-Use Bioreactors provide
process customization
> Eppendorf exclusive packed-bed and cell-lift impeller designs for continuous and perfusion processes
> Thermal mass flow controllers for sparge and overlay gas can be upgraded in the field
> Universal gas control strategy for both microbial and
cell culture applications removes process limitations
> Industry standard Ethernet communication for
multi-unit control of up to eight systems
Application Driven
> Industrial design featuring stainless steel front,
back, and utility panels
> Left- and right-handed orientations to maximize lab
space efficiency
> Hemispherical vessel nest design for minimum
footprint
> Highly configurable gas flow control - Up to 5
configurable Mass Flow Controllers (TMFC) for
Overlay and Sparge
Scalability
> Extensive working volume range of 400 mL – 40 L
on a single control platform
> High-powered direct and magnetic drive motor assemblies
> Up to six integrated pumps capable of operating in
variable speed mode
> The combination of the BioFlo 320 with the
BioFlo 720 provides a perfect connection between
R&D and pilot/production suites
Risk mitigation
> Monitor and control the process directly at the large
touch-screen or with our Eppendorf SCADA software
> Alarm functions automatically stop all running loops
if needed
> Automatic recognition of the connected digital sensors and installed TMFCs
> Robust protection of sensitive electronic components (IP68-rated connections on utility panel and
IP22-rated connection for power entry)
> Universal connections for analog or digital Mettler
Toledo® ISM® sensors reduce sensor complexity
Small footprint... big impact
From R&D laboratories to pilot-scale production facilities, space is an important factor when selecting the right
equipment. The BioFlo 320 offers flexibility, better control, and maximum functionality while occupying a fraction of the
valuable lab space of similar systems. This means greater efficiency and productivity at a lower operating cost for your
lab.
The next generation of our BioFlo 320 bioprocess control system combines the benefits of a classical industrial design
with the power of our improved BioFlo control software.
Developed to be used in cGMP regulated environments, Emerson® and Eppendorf have partnered to develop seamless
communication between the DeltaVTM distributed control system and the BioFlo 320 bioprocess control system. Our open
communication protocol exposes controller information allowing it to be integrated into broader features and functions
of the DeltaV platform, simplifying tech transfer, scale-up, and recipe sharing in bioprocess research and process
development.
Application Driven
Designed for cGMP environments
4 BioFlo® 320
Single-use bioreactors:
0.40 L – 40 L
Vessel material:
Glass or
Single-Use
Process control:
Cell culture or
Microbiology
Autoclavable
bioreactors:
0.6 L – 10.5 L
Control up to eight
units from a single
user interface High-powered direct
and magnetic drive
motor assemblies;
capable of clockwise
and counterclockwise
rotation
Ultra-compact design
with vessel nest for
minimum footprint;
left- and right-handed
orientations to
maximize lab space
Unique variable/
fixed speed frontmounted pumps
capable of clockwise
and counterclockwise
rotation
Industrial, brushed
stainless steel panels
provide enhanced
corrosion resistance
Sixteen
interchangeable
autoclavable
vessels and the
comprehensive line
of BioBLU Single-Use
Bioreactors
Four universal connections
for analog or digital Mettler
Toledo ISM sensors
Interchangeable
TMFC drawers for
sparge and overlay
gas flexibility
BioBLU® Single-Use Bioreactors
BioFlo® 320 5
Scalability
The perfect connection from R&D to production
Seamless transition of your process from 400 mL to 40 L and beyond. In combination with the BioFlo 720 bioprocess
control system, we designed a bioreactor control platform that supports one of the widest range of single-use solutions,
allowing the scale-up of your process to up to 2,000 L.
The updated and improved BioFlo software ensures a consistent user experience across all BioFlo control systems and
comes with new features to improve your process.
> Compatible with 400 mL – 40 L BioBLU Single-Use
Bioreactors, including the BioBLU 5p, the first single-use
bioreactor to utilize the exclusive packed-bed impeller
> Built-in optical pH sensor technology
> Vessel and exhaust heat blanket connections provide precise temperature control and exhaust gas treatment
> Bench-scale single-use bioreactors specifically designed
for microbial fermentation
> Rigid-walled, stirred-tank design provides many advantages over single-use bag design
> Eliminates potential for tears, pits, and folds during installation
> Single-layer polymer removes both uncertainty for leachable and extractable data and the need for unnecessary
preventative actions, like pre-process media wash
Application flexibility
> Suitable for the use in all labs, from academia through
pilot-scale production
> Batch, fed-batch, perfusion, and continuous processes
> Universal control for mammalian, stem cell, insect,
microbial, plant, and algae cultures
> Secreted product, vaccine, and monoclonal antibody
production
> Biofuels research and manufacturing
> Scale-up and scale-down modeling
> Suspension or adherent cultures
> Micro-aerobic, anaerobic, exothermic
fermentation processes
> Specialized impellers for low-shear
and zero-shear process needs
> Food and beverage
> Fine chemical processes
> Integrated Mettler Toledo Intelligent Sensor Management (ISM) platform
> Universal connections for up to four analog or digital (ISM) sensors
> pH: analog or digital (ISM)
> DO: analog or digital (ISM)
> Optical DO: digital (ISM)
> Redox: analog or digital (ISM)
> CO2
: digital (ISM)
> Real-time sensor diagnostics anticipate sensor failure
Full integration of bioprocess software and data into
Emerson’s DeltaVTM system
Eppendorf and Emerson have collaborated to integrate Emerson’s DeltaV™ distributed control system
(DCS) with the BioFlo 320 bioprocess control system.
> Full integration of bioprocess software and data into Emerson's DeltaV system
> Simplified tech-transfer, scale-up, and recipe sharing in bioprocess research
> Manage data and use the same automation systems throughout the product
development process, from bioprocess research to manufacturing
Intelligent Sensors
6 BioFlo® 320
Monitoring the controller and the process is key to successful
process development. The BioFlo 320 software features
screens, designed to provide you with the status of both.
> Alarm functions automatically inform about the status of the
process, with automatic loop shutdown if needed
> Receive alarm notifications via email or text message directly to your mobile device
> The diagnostic screen provides information on the hardware
status and upcoming maintenance
Risk Mitigation
Parallel processing is key to efficiently optimizing a process or to producing reproducible results. Eight units of the
BioFlo 320 bioprocess control system can be connected to each other and controlled by a single unit. The control
software gives valuable insights into all processes, such as current process parameters, and the lifetime of connected
digital sensors.
Monitoring and control of critical system parameters
BioFlo® 320 7
Ease of Use
Advanced software making life easier
Auto Calibrate
> Automated calibration for all attached DO sensors
at once
> DO sensors can be run through an automatic
calibration sequence reducing touch time and
ensuring consistency
> User specifies calibration scheme, process conditions, and zero-point method (electronic or N2
)
> System completes calibration when slope stabilizes
The new release of the BioFlo software ensures a consistent user experience across all BioFlo control systems and
comes with new features to improve your process. Develop your process with the BioFlo 320 and seamlessly scale-up
from 50 L – 2,000 L with the BioFlo 720 bioprocess controller. New software features such as the Auto Calibrate and
Scale Up Assist function simplify your process, mitigate risk, and help to save time.
Parallel Control
> Control eight units from a single user interface
> Automatic gas mixing algorithms for simplified
control (4-gas, 3-gas, O2
enrichment, N2
enrichment)
> Ten-point cascade feature for sophisticated control strategies
> Built-in elapsed fermentation timer for batch
management
> Trend display with up to twelve process values
within a single view
Scale Up Assist
> Scale Up modeling software for the calculation of
important process parameters
> The integrated Scale Up Assist of the BioFlo
software calculates all parameters based on either
constant P/V or constant tip speed
> Up to 3 different vessel sizes can be selected from
> The software contains vessel specific data from the
Eppendorf vessel portfolio and the
ThermoTM Scientific HyPerformaTM 5:1 Single-Use
Bioreactors (SUBs).
Mettler Toledo® and ISM® are registered trademarks of Mettler Toledo AG, Switzerland. Watson-Marlow® is a registered trademark of Watson-Marlow Limited, UK. Emerson® is
a registered trademark of Emerson Electric Co., USA. DeltaV™ is a trademark of Emerson Electric Co., USA. Thermo ScientificTM and HyPerformaTM are trademarks of Thermo
Fisher Scientific Inc, USA. Eppendorf®, the Eppendorf Brand Design, and BioBLU® are registered trademarks of Eppendorf SE, Germany. DASware® is a registered trademark
of DASGIP Information and Process Technology GmbH, Germany. BioFlo® and BioCommand® are registered trademarks of Eppendorf, Inc., USA. All rights reserved, including
graphics and images. Copyright © 2022 by Eppendorf SE.
Order No.: AN00111020/GB5/0.5T/0824/EBC/STEFF · Carbon neutrally printed in Germany
www.eppendorf.com/BioFlo-320
Your local distributor: www.eppendorf.com/contact
Eppendorf SE · Barkhausenweg 1 · 22339 Hamburg · Germany
eppendorf@eppendorf.com · www.eppendorf.com
Technical Data
BioFlo 320 Specifications
Control Station
Dimensions (W x D x H) 40.6 x 40.6 x 66.0 cm (16 in x 16 x 26 in)
Net weight 32 kg (70 lb)
Touchscreen 38.1 cm (15 in) projected capacitive touchscreen
Communication 2 x USB (software updates, serial communication)
Ethernet (SCADA, IP Network)
3 x Analog Input/Output (defined as 4 – 20 mA or 0 – 5 V or 0 – 10 V)
Utility Connection Requirement
Electrical IEC (with regional plug types) 100 – 120/208 – 240 VAC, 50/60 Hz, 2270 VA, Single Phase
Water Stainless steel quick-connect 10 psig (0.69 barg)
Gas supply (Air, O2
, N2
, CO2
) Push-connect Autoclavable Single-use
10 psig (0.69 barg) 6 psig (0.44 barg)
Exhaust 0.5 psig (0.035 barg)
Operating conditions 10 – 30 °C, up to 80 % RH, non-condensing
Agitation
Direct drive 1 L, 3 L: 25 – 1500 rpm
5 L, 10 L: 25 – 1200 rpm
Magnetic drive (autoclavable vessels) 1 L, 3 L, or 5 L: 10 – 500 rpm
10 L: 10 – 150 rpm
Magnetic drive (single-use vessels) BioBLU 1f: 10 – 1200 rpm; BioBLU 3f: 25 – 1200
BioBLU 1c: 10 – 500 rpm
BioBLU 3c, 5c, 5p, 10c & 14c: 10 – 200 rpm
BioBLU 50c: 10 – 150 rpm
Temperature
Water-jacketed 5 °C above coolant to 55 °C above ambient (80 °C max)
Stainless steel dish-bottom 5 °C above coolant to 65 °C above ambient (90 °C max; 85 °C max for 10 L)
Single-use 5 °C above ambient to 40 °C (60 °C max for BioBLU 1)
Sensor type PT100
Gas supply
Sparge 1, 3, or 4 TMFC; ring or micro-sparger
Overlay 1 TMFC; headspace addition
Sensors Communication Control range
pH Analog or digital Mettler Toledo ISM 2 – 12
Optical pH Digital (Presens) 6 – 8
DO Analog or digital Mettler Toledo ISM 0 – 200 %
Optical DO Digital Mettler Toledo ISM 0 – 200 %
Redox Analog or digital Mettler Toledo ISM (-)2000 mV – (+)2000 mV
CO2 Digital Mettler Toledo ISM 0 – 100 %
Pumps Pump Head Variable Speed Fixed Speed
Pumps 1, 2, & 3 Watson-Marlow 114DV 5 – 25 rpm 25 rpm (0 – 100 % Duty Cycle)
Pump 4 Watson-Marlow 314D 20 – 100 rpm 100 rpm (0 – 100 % Duty Cycle)
External pumps 1 & 2 Watson-Marlow 120U/DV 0.1 – 200 rpm N/A
Specifications subject to change.
Seamless Integration of Glucose Control using Raman
Spectroscopy in CHO Cell Culture
Célia Sanchez, Laure Pétillot, Fabrice Thomas, Charlotte Javalet
Merck KGaA, Darmstadt, Germany
13 chemin du Vieux Chêne, 38240 Meylan, France
Contact: bioprocess-experts@eppendorf.com
APPLICATION NOTE No. 415
Abstract
In the context of Process Analytical Technologies (PAT)
implementation in the biopharmaceutical industry,
Quality by Design (QbD) is being developed and widely
implemented and used. In upstream processes, one
compound of great interest to monitor is glucose, and
specifically, being able to control its concentration during
the process. Such a monitoring leads to process quality
improvement, including glycosylation of the product
of interest. In this study, a Raman analyzer has been
successfully used to implement a feedback control loop in
a CHO cell culture based on glucose concentration. The
feedback control loop implied a direct OPC UA
connectivity between the analyzer and the bioreactor
control system The culture was fed with a complex feed
containing glucose. As a result, glucose concentration
was maintained steady for three days. The process
performance remained similar to the ones of regular
fed batch cultures and a noteworthy decrease in lactate
production was observed. The process was completely
automated for glucose concentration management and
did not require any human intervention throughout the
process.
Introduction
The Process Analytical Technology (PAT) and Quality by Design (QbD) guidelines, promoted by the US Food and Drug
Administration (FDA) and the European Medicines Agency
(EMA) aims to support the idea that quality cannot be tested
only into a product but must instead be deployed throughout
design.
Seamless integration of monitoring and control of analytical data into a bioprocess is crucial to understand a process
and to overcome manufacturing challenges.
One of the biggest challenges is the monitoring of quality
attributes such as glycosylation. Important characteristics
like stability and immunogenicity are affected by glycosylation. In order to receive regulatory approval, glycosylation
is a Critical Quality Attribute (CQA) ensuring the safety and
potency of biopharmaceutical products.
Maintaining the glucose concentration steady is key for
the control and optimization of processes’ yields and quality [1, 2]. Manual bioreactor sampling and feeding can be
a costly endeavor, both in terms of labor costs as well as
increased risk for contamination each time the sterile boundary is penetrated.
In this application note, researchers from Merck KGaA,
Darmstadt, Germany integrated the ProCellics™ Raman
Analyzer via OPC UA (Open Platform Communications
United Architecture) connectivity into DASware® control
software to optimize their bioprocess, controlled by a
BioFlo® 320 bioreactor control system. DASware control
allows the easy integration of third-party devices. OPC
UA allows the independent implementation into a process
while being safer, more stable, and more flexible than older
OPC versions (such as OPC DA – Data Access) [3]. The
programming of complex feedback loops as functions of
different parameters, and the accurate measurements of the
Raman analyzer, resulted in stable glucose concentrations
without the need of human interaction.
Merck KGaA, Darmstadt, Germany develops and commercializes a Raman analyzer for the biopharmaceutical industry,
dedicated to in-situ monitoring of bioprocesses.
APPLICATION NOTE I No. 415 I Page 2
Material and Methods
Media
We used FreeStyle™ CHO-S (Gibco®) cells cultivated in
CD-CHO medium (Gibco) with 8 mM glutamine, 1‰ of
Anti-Clumping Agent (Gibco) and 0.5 % of Penicillin/Streptomycin.
Bioreactor control system and process parameters
We performed the CHO cultivation, and process monitoring and control with a BioFlo 320 bioprocess controller with
a water jacketed 3 L glass bioreactor. The bioreactor was
equipped with a ring sparger and a pitched-blade impeller.
The DASware control 5.4.1 software was used to control the
experiment. The bioreactor was inoculated with cells at a
density of 0.4 x 106
cells/mL, with a starting volume of 2 L.
Bioreactor settings to control the process are listed in Table
1.
The bioreactor was shielded against external light to make
sure that the Raman measurements were not affected by
external light.
Feeding strategy
The culture was fed with 15% v/v EfficientFeed™ B (Gibco)
on day zero. Glutamine was added when the concentration
dropped below 4 mM. On day three, we started with constant
glutamine feeding. For glucose feeding, a control loop
was programmed based on the glucose concentration: the
pump rate of feed B (containing glucose) was controlled
by a normal law (on DASware control 5) based on the
glucose concentration read by the ProCellics Raman
Analyzer to maintain a glucose concentration of 5 g/L.
The communication was integrated via OPC UA. The used
function was:
Model building for Raman monitoring
To perform monitoring with Bio4C® PAT Raman Software,
a step of model building is needed to correlate the
reference values obtained by the BioProfile® FLEX2™ (Nova
Biomedical®) and ProCellics Raman Analyzer (Merck KGaA,
Darmstadt, Germany). The spectra were preprocessed on
the Bio4C PAT Raman Software (SNV on the water region,
Savitzky Golay derivative with 3 points (15 cm-1, polynomial
order 2nd and 1st derivative) and spectral selection (350-
1775cm-1 + 2800-300 cm-1) to create a dataset. The reference
values were automatically linked to their corresponding
spectra. The chemometric models for the monitoring are
based on four standard fed-batch cultures (total of 103 pt).
A PLS model was computed for each monitored parameter
using SIMCA® Software (SARTORIUS STEDIM BIOTECH®).
Models for Viable Cell Density (VCD), Total Cell Density
(TCD), glucose, glutamic acid, ammonium and lactate were
performed.
Raman monitoring
The ProCellics Raman Analzyer acquired and preprocessed
Raman spectra, and calculated the process parameters
Table 1: Process parameters and cultivation conditions.
Parameter Setpoint Control
Temperature 37°C Water jacket
pH 7.0 (deadband 0.1) Sparging CO2 or 0.5N NaOH
pO2 40% Mix of air and O2 sparging
(flow rate max 0.1 vvm)
Stirring 80 rpm
Direct OPC UA
communication
> Read the spectra
> Calculate glucose concentration
> Send the glucose value to
DASware® control
> Read the glucose concentration
sent by ProCellics®
> Adjust the pump rate for feed
(containing glucose)
Fig. 2: Experimental setup: The analyzer communicates directly with the biocontroller to send the glucose concentration
read in the bioreactor.
Fig. 1: Illustration of the complete system setup with the
ProCellics probe inside the bioreactor controlled by DASware
control 5.
Pump rate = 2000e (
-[Glucose]² ) 5
APPLICATION NOTE I No. 415 I Page 3
0.0
2.0
4.0
6.0
8.0
10.0
12.0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
0 24 48 72 96 120 144 168 192 216
VCD (cells/mL)
Glucose & lactate concentration (g/L)
Culture time (h)
A
Glucose - Raman
monitoring
Glucose - O line
values
Lactate - Raman
monitoring
Lactate - O line
values
VCD - Raman
monitoring
VCD - O line
values
0
1
2
3
4
5
6
7
8
9
10
11
12
13
Glucose & lactate concentration (g/L)
0 24 48 72 96 120 144 168 192 216
Culture time (h)
0.0
2.0
4.0
6.0
8.0
10.0
12.0
VCD (cells/mL)
Glucose - O line
values
Lactate - O line
values
VCD - O line
values
B
Fig. 3: Cell culture parameters evolution.
A: Glucose feedback control loop based on Raman measurement (full lines) in comparison with offline measurements for
reference (cross)
B: Control: Classical fedbatch culture (offline measurements
only)
including glucose concentration. Based on the scheduled
frequency, measurements were carried out every 30 minutes.
Following the previously described loop settings, the pump
rate was adjusted according to the measurements every 30
minutes.
Third-party sensor integration
Connectivity between the ProCellics Raman Analyzer and the
DASware control software was implemented with OPC UA.
Results and Discussion
Until day 4 glucose was consumed by the cells until the minimum set value of 5 g/L was reached. The glucose concentration was precisely maintained at 5 g/L for 3 days by the
programmed feedback loop (Figure 3A). In parallel, glucose
concentrations have been measured offline with FLEX2 in
order to assess Raman monitoring. These measurements
confirmed that the Raman analyzer accurately measured the
glucose concentration.
Feeding was stopped when the maximum vessel volume was
reached. The process was stopped, when the glucose concentration in the vessel dropped below 1 g/L.
As a control, a classical fed-batch run with manual glucose
addition was performed (Figure 3B). The cell growth kinetics and the maximum cell density in the feedback-controlled
run was comparable with the classical fed-batch control
run. However, the lactate concentration in the feedbackcontrolled run was lower (2 g/L) in comparison to the control
run (3 g/L). This is a noteworthy result, since to high lactate
concentrations can be toxic for cells.
Conclusion
DASware control 5 enabled the efficient and easy integration
of the ProCellics Raman Analyzer via OPC UA protocol. OPC
UA is more secure compared to OPC DA. With this setup, we
were able to prove that ProCellics Raman Analyzer is fully
ready for process automation. Once set up, the automation of
the feedback control loop was complete and reliable.
A great level of confidence for the extremely stable glucose
concentration, and accurately measured with the Raman
analyzer was achieved. This allows to reduce the number
of human interactions needed, thus reducing the risk of
contamination due to repeated sampling. Less sampling
is needed due to the automation, resulting in minimized
manual work and reducing the risk of contaminations.
Additionally, the risk of batch failures due to a lack of
glucose during the night or at weekends is reduced.
The feeding is stopped at maximum volume
TCD VCD Glucose (g/L) Lactate (g/L)
The feedback control loop set the
concentration at 5 g/L
Fig. 4: Cell culture parameters evolution over the cultures,
displayed on Bio4C PAT Raman Software.
APPLICATION NOTE I No. 415 I Page 4
Ordering information
Description Order no.
BioFlo® 320, overlay gas option, 1 TMFC (0.05 – 5 SLPM) 1379502111
Software License, DASware® control 5, for one culture vessel 78600166
Vessel Bundle, for BioFlo® 320, water jacket, magnetic drive, 3 L vessel M1379-0311
Pitched-Blade Impeller Kit, magnetic drive, 3 L M1379-5069
Harvest tube M1287-9483
www.eppendorf.com/bioprocess
Bio4C® is a registered trademark and ProCellicsTM is a trademark of Merck KGaA, Darmstadt, Germany. BioProfile® is a registered trademark and FLEX2™ is a trademark of NOVA BIOMEDICAL CORPORATION, USA. Gibco® is a registered trademark and FreestyleTM and Efficient FeedTM are trademarks of Life Technologies Corporation, USA. SIMCA® and SARTORIUS STEDIM BIOTECH® are registered trademarks of Sartorius Stedim Data Analytics AB, SE. Eppendorf®
and the Eppendorf Brand Design are registered trademarks of Eppendorf SE, Germany. BioFlo® is a registered trademark of Eppendorf, Inc., USA. DASware® is a registered trademark of the 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 note alone cannot as such provide for or replace reading and respecting the current version of the operating manual. AA415-020-03-062020
Your local distributor: www.eppendorf.com/contact
Eppendorf SE · Barkhausenweg 1 ∙ 22339 Hamburg · Germany
eppendorf@eppendorf.com · www.eppendorf.com
Literature
[1] Brandon N. Berry et al., “Quick Generation of Raman Spectroscopy Based In-Process Glucose Control to Influence
Biopharmaceutical Protein Product Quality during Mammalian Cell Culture,” Biotechnology Progress 32, no. 1 (2016):
224–34, https://doi.org/10.1002/btpr.2205
[2] Inn H. Yuk et al., “Controlling Glycation of Recombinant Antibody in Fed-Batch Cell Cultures,” Biotechnology and Bioengineering 108, no. 11 (2011): 2600–2610, https://doi.org/10.1002/bit.23218
[3] Jürgen Lange, Frank Iwanitz, and Thomas Burke, OPC From Data Access to Unified Architecture, 4th rev. Ed., OPC Foundation - Softing (VDE Verlag GMBH, 2010)
How can PAT help improving upstream
bioprocessing?
Find out, which parameters can process analytical
technology (PAT) be used to monitor and which sensor
types are used. Discover various application examples
in which the use of PAT improved the bioprocess!
Visit our website
www.eppendorf.link/bioprocess-pat
APPLICATION NOTE No. 438
Abstract
The yeast Pichia pastoris has become an important and
convenient workhorse for genetically engineered protein
production in the biotechnology industry. Currently, most
P. pastoris promoters used for efficient expression of heterologous proteins are derived from genes in the methanol
metabolism pathway. For example, PAOX1, the most widely
used promotor, requires the switch to methanol feeding to
activate protein production, referred to as methanol induction. Due to the flammable nature of methanol, this causes
safety concerns in both academia and industry, especially
when dealing with large volumes.
We explored the feasibility of a safer protein production
alternative using a new P. pastoris strain carrying a strong
methanol-independent promoter called pUPP. The pUPP
promoter requires glycerol feeding for protein production, thus eliminating methanol induction and its affiliated
safety risks. We carried out fed-batch P. pastoris fermentation runs for lipase production in the absence of methanol.
This workflow was initiated in an Innova® S44i Biological
Shaker for inoculum preparation, and followed with
BioFlo® 120 and BioFlo 320 bioprocess control systems
for bench scale fermentation.
We used 3 L BioBLU® 3f Single-Use Vessels as fermenters. At 27 h post inoculation, the BioFlo 120 controlled
run reached a maximum OD600 of 183.8. The BioFlo 320
controlled run reached a final maximum OD600 of 229.8 at
46 h post inoculation. Maximum lipase activity was detected simultaneously with peak optical density, reflecting
the positive correlation between yeast growth and protein
production.
We conclude that this methanol-free P. pastoris fermentation workflow can be most advantageous for Eppendorf
customers seeking a safer alternative to the traditional
methanol induced P. pastoris protein production.
From Shaker to Fermenter: Methanol-free Pichia pastoris
Protein Production Workflow
Ying Yang, Ma Sha
Bioprocess Applications Lab, Eppendorf, Inc., Enfield, CT, USA
Contact: bioprocess-experts@eppendorf.com
APPLICATION NOTE | No. 438 | Page 2
Introduction
The yeast Pichia pastoris has been developed as a highly
competitive expression system for heterologous protein production. Compared to mammalian cell culture-based protein
production systems such as CHO (Chinese Hamster Ovary)
cells, the advantages of eukaryotic P. pastoris expression
hosts include:
> Expedited growth, resulting in drastically shorter fermentation cycle,
> robustness,
> low cost of medium,
> absence of need for viral clearance,
> production of large quantities of the desired biomolecules
at much higher concentrations [1, 2].
Currently most P. pastoris promoters used for efficient
expression of heterologous proteins are derived from genes
that code for enzymes in the methanol metabolism pathway. The most widely used promoter is PAOX1 [2, 3], which is
strongly repressed in the presence of common carbon sources like glucose and glycerol. Upon depletion of the common
carbon source, the promoter is de-repressed and capable
of eliciting its full activity when induced by another carbon
source, most typically methanol. However, due to its volatility
and flammability, the addition of methanol to the bioprocess
system brings significant safety concerns. Therefore, alternative promoters which do not require methanol induction to
achieve high protein yield are much in demand.
Here we used a P. pastoris strain carrying a strong and
constitutive promoter pUPP which can rely on glycerol as
the only carbon source, meaning that both biomass growth
and protein expression are supported by glycerol consumption, whereas no methanol is needed. The objectives of this
study are: (1) to carry out fed-batch P. pastoris fermentation with the novel methanol-free induction technology for
protein production, here in this case, lipase expression and
secretion; and (2) to demonstrate the feasibility of using the
Innova S44i Biological Shaker, the two benchtop bioprocess
control systems BioFlo 120 and BioFlo 320, and the
BioBLU 3f Single-Use Vessel, for high-density P. pastoris
fermentation and protein production workflow.
Material and Methods
Yeast strain
The yeast strain Pichia pastoris Bg10-pJAG-Lip1 used in this
study was constructed by BioGrammatics, Inc. This strain
contains a lipase expression construct and a constitutive
pUPP promoter (also known as PGCW14) which is free of methanol regulation in the production of heterologous protein [4].
Upon receipt, we streaked the P. pastoris cells on a YPD agar
plate, incubated at 28 °C for 48 h, and picked a healthy single
colony to inoculate a 250 mL shake flask containing 75 mL
fresh YPD medium. We transferred the shake flask into the
Innova S44i Biological Shaker at 28 °C with 200 rpm agitation. After 48 h, we prepared the glycerol stock in multiple
cryogenic vials by adding 600 µL of the actively growing P.
pastoris suspension from the shake flask to each of the cryogenic vial containing 400 µL 75 % (v/v) sterile glycerol. Cells
were well mixed before being transferred into the freezer at
-80 °C (New Brunswick Innova U360, Eppendorf) for future
use.
Media preparation
We used two types of growth media in this study, YPD (Yeast
extract-Peptone-Dextrose) and BMGY (Buffered Glycerolcomplex Medium).
For YPD medium, both agar and liquid media were prepared. The agar YPD medium was made by dissolving 10 g
yeast extract (Fisher BioReagents™, BP1422-500), 20 g peptone (BD Gibco™ Bacto™ Proteose Peptone No. 3, 211693),
20 g glucose (Sigma-Aldrich®, G7021), and 15 g agar (BD
Difco™ Plate Count Agar, 247940) per 1 L DI water through
boiling, and autoclaved at 121 °C. We poured agar plates to
streak the P. pastoris cells after receipt as described above.
The liquid YPD medium was prepared by dissolving all the
compounds except agar at the same amount per 1 L DI water
and autoclaved at 121 °C. The liquid medium was used when
preparing the glycerol stock.
BMGY broth was prepared for bioreactor fermentation.
One litre of BMGY broth was made as follows:
> 10 g yeast extract, 20 g peptone and 7.5 mL glycerol
(Fisher BioReagents™, BP229-1) were disslved in 800 mL
DI water and autoclaving at 121 °C.
> Next we prepared 100 mL 1 M potassium phosphate buffer
(pH 6.0) by dissolving 2.405 g K2
HPO4
(Fisher Chemical™,
P228-500) and 11.73 g KH2
PO4
(Sigma Aldrich, P5655)
in 100 mL DI water, and sterilized the broth by passing it
through a 0.2 µm membrane.
We also prepared 10x YNB (Yeast Nitrogen Base, with ammonium sulfate) solution by dissolving 13.4 g of the YNB
powder (Invitrogen™, Q300009) in 100 mL DI water and
sterilizing through 0.2 µm membrane filtration into the
cooled broth to make a total volume of 1 L.
APPLICATION NOTE | No. 438 | Page 3
The fed-batch fermentation feeding medium was a 50 %
(v/v) glycerol solution, sterilized by autoclaving in the feeding bottle and aseptically connected to the liquid addition
port on the head plate of the bioreactor before the run.
Inoculum preparation
For each procedure, we removed one P. pastoris cryogenic
vial from the -80 °C freezer and thawed it under ambient
temperature. We prepared two 500 mL Erlenmeyer flasks,
each containing 150 mL fresh BMGY medium, and inoculated 500 µL of the P. pastoris glycerol stock from the vial
into each of the two flasks. A third control Erlenmeyer flask
contained 150 mL fresh BMGY medium without inoculation.
We transferred the three flasks into the Innova S44i Biological Shaker for the inoculum to grow at 28 °C with 200 rpm
agitation. After 48 h, the broth in the two Erlenmeyer flasks
which had been inoculated turned turbid, indicating active
growth of P. pastoris. The medium in the control flask should
stay clear, demonstrating sterility of the medium and aseptic
manipulation.
After 48 h shaking culture, we picked one of the two flasks
containing 150 mL actively growing P. pastoris suspension
and measured its optical density at 600 nm with the fresh
BMGY medium as blank using an Eppendorf
BioSpectrometer®. The OD600 of this shake flask culture was
30.9. We then carefully poured the 150 mL suspension into
two 500 mL sterile bottles. The 75 mL inoculum was pumped
at maximum speed into each vessel. The schematic flowchart
for glycerol stock preparation and bioreactor fermentation is
illustrated in Fig. 1.
BioBLU® 3f Single-Use Bioreactor and vessel setup
In this study, we performed all fed-batch fermentations in
BioBLU 3f Single-Use Bioreactors. The vessel has rigid walls
with a working volume range of 1.25 – 3.75 L and allows
a maximum agitation of 1,200 rpm. It is equipped with a
macrosparger and three Rushton-type impellers, specifically
designed for robust microbial applications.
All fed-batch fermentations started with an early-stage
batch run at 1.5 L working volume in BMGY broth. Towards
the end of the fed-batch, the final working volume was around
3 L. We added 0.9 mL Antifoam 204 (Sigma-Aldrich, USA)
to the BMGY broth when filling the BioBLU 3f vessel before
autoclaving to reach a final antifoam concentration of 0.03 %
(v/v).
We used a pH/Redox ISM® sensor for pH monitoring and
an analog polarographic dissolved oxygen (DO) sensor for
DO measurements (Mettler Toledo®, Switzerland). Both sensors are 12 mm in diameter with 225 mm insertion depth.
The two sensors were installed on the head plate of the
BioBLU 3f vessel through the two Pg 13.5 ports. A stainlesssteel cooling finger was installed through a compression
fitting taking another Pg 13.5 port. We extended three liquid
addition ports appropriately for connection with the external
bottles.
Fig. 2 illustrates the BioBLU 3f Single-Use Vessel as it appears before autoclave sterilization. We manually applied the
Ni-plated open jaw Hoffman tubing clamp (Eppendorf order
No. P0160-4830) to each of the head plate tubing which
connects to a submerged port into the medium. These ports
include gas sparger, submerged liquid addition line, harvest
port, and sampling port. We tightened these metal clamps
to prevent the medium from refluxing into the tubing, filters,
and possibly ejecting during high-temperature high-pressure
autoclaving. Additionally, to ensure effective venting during
autoclaving, the overlay gas port on the BioBLU 3f head plate
should be left open, as marked by a “Do not clamp” sign preattached to this specific port.
We autoclaved the medium-filled vessel and let it cool
to 28 °C. We then added sterile 1 M potassium phosphate
buffer and 10x YNB solution to the vessel to complete the
BMGY broth preparation as described earlier. Upon completion of DO sensor calibration, the inoculation bottle, the feeding bottle, and the base bottle were aseptically connected
before inoculation to the liquid addition ports on the vessel
head plate through a SCD®-II Sterile Tubing Welder (Terumo
BCT, USA). The detailed setup of the vessel can be found in a
previously published application note [5]. Fig. 1: Schematic flowchart of the Pichia pastoris fermentation
in this study.
A. Glycerol stock preparation. B. Bioreactor fermentation. Created with biorender.com
APPLICATION NOTE | No. 438 | Page 4
Sensor calibration
We followed the same sensor calibration protocols for both
BioFlo 120 and BioFlo 320 bioreactors, calibrating the pH
sensor outside of the vessel before sterilization. We used the
2-point calibration method by setting ZERO using buffer at
pH = 7 and setting SPAN using buffer at pH = 4.
We calibrated the pre-polarized DO sensor after autoclaving with the sterile BMGY broth in the vessel. We recommend calibration of the DO sensor under the same condition
as during the actual fermentation procedure. Therefore, we
checked to make sure the pH was at 6.0, set the agitation
at maximum 1,200 rpm, and set the temperature at 28 °C.
A 2-point calibration method was also applied. We sparged
pure nitrogen at 1 VVM, here in this case 1.5 SLPM, until the
DO value stabilized to set ZERO at 0 %; then switched the
gas supply to air under the same flow rate, waited till the DO
value stabilized again, to set SPAN at 100 %.
Pump calibration
Prior to the run, the pumps were calibrated on both control
systems. The same tubing applied to the peristaltic pump
head for liquid addition during fermentation should be used
for the pump calibration. It is recommended to add a section of PharMed® tubing (Saint-Gobain®, France) between
silicone tubing connections and fit it to the peristaltic pumps
for better chemical compatibility especially for base addition.
Pump calibration was performed by pumping DI water into
a fully filled section of tubing for a set period of time (3 min
for both control systems) and tracking the water volume collected in a graduated cylinder at the end of tubing. Then the
maximum pump speed specific to the tubing used can be recorded in the system. Pump calibration is critical here for the
addition of feeding medium and base during fermentation.
Process parameter setup from shaker to fermenters
The bench scale P. pastoris fermentations were maintained at
28 °C, pH 6.0, and 30 % DO. Most of the process parameters
retained for the two bioprocess control systems (see Table
1). To show the entire workflow, Table 1 also includes the
parameter setup of the early inoculum preparation. The key
equipment used in this workflow is illustrated in Fig. 3.
For the BioFlo 120, heating was controlled by an external
heat blanket. The DO cascade was designed to maintain the
DO at 30 % by first accelerating the agitation from 300 to
1,200 rpm, then increasing the air sparging rate from 0.8 to
Fig. 2: Preparation of the BioBLU 3f Single-Use Bioreactor
before autoclave sterilization.
A and B, side and top view of the vessel and its head plate; C,
D, and E, manually tightened tubing with Ni-plated open jaw
Hoffman tubing clamps for gas sparger, submerged liquid addition line, harvest port, and sampling port.
a b
c d e
Table 1. Key process parameters applied to Pichia pastoris fermentation and protein production workflow.
Parameter Configuration
Inoculum preparation
Shaker Innova S44i Biological Shaker
Temperature 28 °C
Agitation 200 rpm
Flask 500 mL Erlenmeyer flasks
Volume 150 mL in each Erlenmeyer flask
Duration 48 h
Bench scale fermentation
Controller BioFlo 120 and BioFlo 320
Vessel BioBLU 3f
Inoculation density 5 % (v/v), 75 mL inoculum to an initial
1.5 L working volume
Dissolved oxygen (DO) 30 %, maintained by DO cascade
Agitation Magnetic drive, maximum 1,200 rpm,
controlled by DO cascade
Gassing Automatic gas flow and mix, controlled
by DO cascade
Temperature 28 °C, cooling controlled by a single
stainless-steel cooling finger
pH 6.0 ± 0.1, controlled by the addition of
30 % (v/v) sterile ammonium hydroxide
solution
Impeller Three Rushton impellers
Sparger Macrosparger
Feeding Manually triggered by the DO spike, then
at a constant feeding rate of 0.4 mL/min
of 50 % (v/v) glycerol solution for the rest
of fed-batch fermentation
APPLICATION NOTE | No. 438 | Page 5
3.0 SLPM, and finally enriching oxygen in the sparged gas
stream from 0 to 100 %. These three steps corresponded to
the DO output of 0-50 %, 50-65 %, and 65-100 %, respectively, which are fixed in the BioFlo 120. Based on our
preliminary studies, to better support yeast growth, we found
the importance of keeping a minimum air sparging rate at
0.8 SLPM throughout the run rather than starting from zero
air flow. The detailed DO cascade setup in the BioFlo 120 is
shown in Fig. 4.
For the BioFlo 320, both cooling and heating were controlled by running water through the stainless-steel cooling
finger. We set the DO output between 0 % and 100 %, and
built a customized DO cascade the same as it was for BioFlo
120 (Fig. 5).
For a more direct comparison, Table 2 lists some configuration differences between BioFlo 120 and BioFlo 320 in this
P. pastoris fermentation. Besides the aforementioned heating
and DO cascades, the most distinctive differences are the gas
sparging and pumps. For BioFlo 120, the sparge gas module has 1 TMFC (thermal mass flow controller) which has 4
solenoid valves but only allows one gas to flow through at a
given time point without pre-mixing. Therefore, towards the
later stage of aerobic P. pastoris fermentation when oxygen
enrichment takes place, both air and oxygen are called, but
only one gas is sparged to the bioreactor at a given time
point depending on the real-time DO output. The BioFlo 320
is equipped with 4 TMFCs. Different kind of gases, air and
oxygen in this case, can be called simultaneously at different flow rates and mixed together, thus allowing a more
comprehensive and precise control of gas sparging during
the process. Furthermore, compared to the three fixed speed
pumps on BioFlo 120, BioFlo 320 has three variable speed
pumps and a fourth one with much more powerful performance, greatly contributing to the flexibility and efficiency of
the bioprocess.
An obvious DO spike indicated the depletion of the carbon
source, glycerol in this case, in the batch stage of P. pastoris fermentation. When glycerol depletion occurred, the
Fig. 3: Key equipment used in this Pichia pastoris fermentation and protein production workflow.
Top: Innova S44i Biological Shaker; bottom left: BioFlo 120
bioprocess control station; bottom right: BioFlo 320 bioprocess control station).
Fig. 4. DO cascade setup in BioFlo 120.
Fig. 5. DO cascade setup in BioFlo 320.
APPLICATION NOTE | No. 438 | Page 6
metabolic rate of P. pastoris cells significantly slowed down
including the consumption of oxygen. As a result, a DO spike
took place. Here the DO spike triggered control was achieved
by manual observation and handling with experience from
previous runs based on EFT (elapsed fermentation time). We
manually turned on the feeding pump at a constant pump
rate of 0.4 mL/min right after we detected the DO spike on
the trend page collected by both control systems.
Biomass formation – optical density measurement
Upon completion of DO calibration and right before inoculation, we took a sample of 30 mL fresh BMGY broth from one
vessel, and used 1 mL of this medium to set the blank for
optical density measurement at 600 nm using an Eppendorf
BioSpectrometer. The remaining volume was saved as the
diluent for the yeast suspension during the run. We took
suspension sample at 11 time points, 0, 3, 6, 21, 24, 27, 29,
46, 48, 51, and 54 h after inoculation, for optical density
measurement.
Protein production – lipase activity assay
The Lipase Activity Assay Kit (colorimetric) used in this study
was purchased from Abcam® (ab102524). Since lipase was
secreted from the P. pastoris cells, we took one extra mL
suspension at the last 7 sampling time points for OD measurements, pelleted the yeast cells down by centrifugation
in a MiniSpin® plus microcentrifuge at 14,000 rpm for 90 s,
and collected the supernatants for lipase activity colorimetric
assay. The basis of this assay is lipase hydrolyzation of a triglyceride substrate to form glycerol which can be quantified
enzymatically by monitoring a linked colorimetric change in
the OxiRed probe absorbance at 570 nm. We followed the
assay protocol to first load a series of standards, samples,
background control samples, and positive controls into a
96-well plate, then adding the reaction mix and background
reaction mix respectively to the designated wells. The OD570
of the plate was measured by EpochTM Microplate Spectrophotometer (BioTek Instruments) in kinetic mode for 60 min.
The standard curve was drawn and the lipase activity of each
supernatant sample was calculated accordingly. The detailed
protocol is available when purchasing the assay kit or accessed on the Abcam website [6].
Results
We ran the Pichia pastoris fed-batch fermentation two times
at 28 °C, pH 6.0, and a DO level of 30 % in BioBLU 3f bioreactors, one controlled by a BioFlo 120, and the other by a
BioFlo 320 bioprocess controller. We maintained DO by applying a customized DO cascade and initiated feeding right
after observing the DO spike. Throughout the fermentation,
we took intermittent samples for optical density measurements and lipase activity assays to evaluate the yeast growth
and secreted protein production.
Bioprocess trends in the BioFlo 120 and BioFlo 320 bioprocess control systems
As described previously, a DO spike during fermentation
indicates the depletion of the carbon source in the broth and
is the signal for initiating feeding. We observed a significant
DO spike at 9.25 h in both runs, indicating a synchronized
growth and metabolism pattern of P. pastoris in the two
vessels. Relative to a DO set point at 30 %, the peak of the
DO spike was 35 % and 50 % in BioFlo 120 and the BioFlo
320, respectively. Right before the appearance of DO spike,
agitation was ca. 800 rpm and still ramping up. The DO spike
was accompanied by a sharp drop of agitation, indicating
the largely reduced demand of oxygen since the metabolism
of P. pastoris slowed down. Immediately after feeding was
initiated, yeast growth quickly resumed, which caused the
DO concentration to decrease first and soon recover with agitation continuing its upward trend.
For both control systems, the agitation reached its maximum at 1,200 rpm at t = 11.5 h and maintained at this speed
for the rest of the fermentation. The air sparging rate ramped
Table 2. Differences in bioprocess control between BioFlo 120 and
BioFlo 320 in this study.
Parameter BioFlo 120 BioFlo 320
Heating An external heating
blanket
Heated water circulating
through the stainlesssteel cooling finger
Gas sparging 1 TMFC (0.04-20 SLPM) 4 TMFCs (0.04-20 SLPM)
DO cascade Fixed DO output range
for a series of defined
parameters: agitation, air,
and oxygen
Can be completely customized, the DO output
ranges, and the associated parameters are all
self-defined
Pumps Three front-mounted
pumps (type 114DV)
which run at fixed speed
with 0-100 % duty cycle
Four pumps which can
run at variable speeds,
the top three are type
114DV pumps, and the
fourth has a larger capacity (type 314D) and runs
up to 4x faster
Inoculation Through one of the three
same pumps
Through the fourth pump
which leads to a very fast
inoculation process
APPLICATION NOTE | No. 438 | Page 7
up from 0.8 to 3.0 SLPM in the next 3 hours before oxygen
enrichment took place at t = 14.5 h. From then on, the trends
collected in BioFlo 120 and 320 started to show some difference. In the BioFlo 120 controlled fermenter, oxygen enrichment gradually increased from 0 to 10 % in the sparging
gas for the next 7 hours till t = 21.5 h and then started to
decline back to 0 % at t = 36 h. Therefore, oxygen enrichment lasted for a total of 21.5 h. After that, the air flow rate
started to decrease from 3 to 0.9 SLPM from t = 36 h towards
the end of the run at t = 54 h. In the BioFlo 320 controlled
run however, oxygen input was only called intermittently at a
flow rate of 0.1 SLPM corresponding to 3.3 % in the 3 SLPM
sparging gas stream for 1.5 h until t = 16 h, then no oxygen
was needed for the rest of the run. The air flow rate started
to drop from 3 to 1.6 SLPM during the next 2.5 h till t = 18.5
h and then slowly recovered to 2.4 SLPM at t = 29 h. Beyond
that, the air flow rate declined again and was at 1.8 SLPM
when we ended the fermentation at t = 54 h.
Yeast growth and lipase activity
Based on the optical density measurements, growth curves
are drawn for both fermentations (Fig. 6). According to the
growth curve, P. pastoris grew faster during the exponential growth phase under BioFlo 120 with maximum OD600 of
183.8 at t = 27 h. After that, the stationary phase and death
phase were observed in the BioFlo 120 culture and OD600
started to decline till the end of fermentation. However,
the growth pattern of the BioFlo 320 culture was different.
Although its specific growth rate was lower than the BioFlo
120 counterpart during the early exponential growth, yeast
biomass continued to accumulate till t = 46 h with the maximum OD600 at 229.8 in the BioFlo 320 culture before entering
the stationary phase.
The growth curve correlates well with the culture’s realtime oxygen demand as described earlier. In the first half
of the fermentation, after air flow reached its maximum of
3 SLPM at t = 14.5 h, up to 10 % oxygen enrichment was observed for the BioFlo 120 culture and the enrichment lasted
for 21.5 h. The early high demand of oxygen supported
robust exponential P. pastoris growth controlled by
BioFlo 120. However, for BioFlo 320 culture, oxygen enrichment was minimal, but air sparging rate maintained at a
relatively high level despite some fluctuations throughout
the entire fermentation, which resulted in a long and steady
growth of P. pastoris till t = 46 h.
For lipase activity, we drew developed a linear standard
curve based on the assay protocol to correlate the concentration of glycerol loaded in each well with the OD570 reading associated with the OxiRed probe at the end of 60 min incubation [6]. The equation of this linear standard curve is OD570 =
0.1514 x glycerol concentration (nmol) with a R-square of
0.9937. Therefore, the lipase activity we can be calculated
the lipase activity accordingly from the colorimetric readings
collected from each well, indicating the glycerol released
from triglyceride hydrolysis.
We found the maximum lipase activity was 11.5 nmol/
(min·mL) at t = 27 h for BioFlo 120 and 22.5 nmol/(min·mL)
at t = 46 h for BioFlo 320. We notice that for both control systems, maximum lipase activity was detected simultaneously
with the peak of P. pastoris optical density in each vessel.
Since the pUPP is a constitutive promoter, heterologous
protein expression and secretion take place at the same time
with P. pastoris growth. Therefore, it is not surprising to see
Fig. 6. Growth curves of Pichia pastoris and the activities of
the secreted lipase in BioBLU 3f Single-Use Vessel controlled by BioFlo 120 (A) and BioFlo 320 (B) bioprocess control
systems.
0
5
10
15
20
25
30
0
50
100
150
200
250
0 10 20 30 40 50 60
Lipase activity (nmol/(min·mL))
OD600
Time (h)
OD600 Lipase activity
0
5
10
15
20
25
30
0
50
100
150
200
250
0 10 20 30 40 50 60
Lipase activity (nmol/(min·mL))
OD600
Time (h)
OD600 Lipase activity
a
b
APPLICATION NOTE | No. 438 | Page 8
that lipase activity and yeast biomass are generally positively
correlated, which provides us an easier approach to high
protein yield through simply boosting yeast growth. This is
very different from growing P. pastoris which has the widely
used adopted methanol-induced promoter like such as the
alcohol oxidase I promoter PAOX1 for protein production. As
a host with such a methanol regulated promoter, P. pastoris
needs to go through separate stages for biomass accumulation first and then heterologous protein production by methanol induction, a strategy which poses more challenges in
experimental design.
Conclusion
This study successfully demonstrates a workflow of methanol-free induction for heterologous protein production in P.
pastoris from shake flask culture to bioreactor fermentation.
The Innova S44i Biological Shaker, the BioFlo 120 and the
BioFlo 320 control systems, together with the BioBLU 3f
Single-Use Vessels, are all capable of supporting such an innovative and convenient fermentation bioprocess.
With a strong and constitutive promoter independent of
methanol regulation, the experimental design for such fedbatch P. pastoris fermentation is safer, simpler, and shorter
than the traditional PAOX1 induced protein production. P.
pastoris reached maximum OD600 of 183.8 at t = 27 h under
BioFlo 120, and maximum OD600 at 229.8 at t = 46 h under
BioFlo 320. Maximum lipase activity was detected simultaneously with peak optical density, indicating a positive correlation between yeast growth and protein production.
This detailed example of methanol-free induction in P.
pastoris fermentation can serve as a useful reference for customers seeking a safer alternative to the traditional methanol
induced bioprocess. We assert that the methanol-free P.
pastoris strain with robust and safe expression system can
grow into a key competitor to traditional protein production
technologies dominated by the use of methanol induced
strains.
Literature
[1] Pichia pastoris Revisited. Genetic Engineering and Biotechnology News, Vol 34(11). 2014
[2] Ahmad M, Hirz M, Pichler H, Schwab H, Protein expression in Pichia pastoris: recent achievements and perspectives for
heterologous protein production. Applied Microbiology and Biotechnology 98(12), 5301-5317. 2014
[3] Gonçalves AM, Pedro AQ, Maia C, Sousa F, Queiroz JA, Passarinha LA. Pichia pastoris: A recombinant microfactory for
antibodies and human membrane proteins. Journal of Microbiology and Biotechnology 23(5), 587-601. 2013
[4] Liang S, Zou C, Lin Y, Zhang X, Ye Y. Identification and characterization of PGCW14: a novel strong constitutive promoter of
Pichia pastoris. Biotechnology Letters (35), 1865-1871. 2013
[5] Yang Y, Sha M. A beginner’s guide to bioprocess modes – batch, fed-batch, and continuous fermentation. Eppendorf Application Note 408. 2019
[6] ab102524 Lipase Activity Assay Kit (Colorimetric) protocol booklet.
Available online at https://www.abcam.com/lipase-assay-kit-colorimetric-ab102524.html
APPLICATION NOTE | No. 438 | Page 9
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Ordering information
Description Order no.
Innova® S44i, incubated, 120 V, orbit diameter 2.5 cm (1 in) S44I200005
Innova® U360, 360 L, ULT freezer, 115 V/60 Hz U9425-0000
New Brunswick™ Excella® E24, orbit diameter 1.9 cm (3/4 in), 120 V/60 Hz M1352-0000
Eppendorf BioSpectrometer® kinetic, 230 V/50-60 Hz 6136 000.002
BioFlo® 320, all configured units include the same base control station
Base control station 1379963011
BioFlo® 120
Advanced control station bundle, TMFC 20 SLPM B120ACS000
BioBLU® 3f Single-Use Bioreactor, fermentation, macrosparger, 3 Rushton-type impellers, autoclavable 1386000900
Bioprocess system Accessories
Agitation motor for BioBLU® 3f 1386080000
Water manifold kit M1386-9909
Cooling finger for BioBLU® 3f, stainless steel M1386-0555
Exhaust condenser kit M1386-9905
Exhaust condenser bracket, BioBLU® 3f 1386930400
Heat blanket for BioBLU® 3c/5c/5p/3f M1379-8116
Heat blanket adaptor for BioBLU® (to be used with BioFlo® 120) 1386811900
pH/Redox sensor, Mettler Toledo®, InPro 3253i, ISM®, 225 mm P0720-6657
DO sensor, Mettler Toledo®, InPro 6830, angled T-82 connector, 220 mm P0720-6282
Ni-plated open jaw Hoffman tubing clamp P0160-4830
Centrifuge MiniSpin® plus, non-refrigerated, with Rotor F-45-12-11 5453 000 015
APPLICATION NOTE No. 447
Abstract
Mammalian cells cultivated in vitro represent one of the
most important manufacturing platforms for vaccine and
gene therapy developers. Especially, human embryonic
kidney 293 (HEK293) cells are an attractive and reliable
host for numerous biotherapeutic platforms. HEK293 cells
have a wide variety of advantages including low-maintenance, rapid proliferation, and convenient application to
both, transient and stable expression. Furthermore, they
are easy to transfect and can produce large amounts of
recombinant proteins and virus particles.
However, a major limitation of the cell line is its tendency
to clump when converted to suspension format and therefore has been limited to adherent cell culture. To achieve
large scale protein production, a new suspension-adapted
HEK293 cell line, Expi293F™, was developed by Thermo
Fisher. The new HEK293 cell line appears to be a robust
suspension cell line capable of achieving greater per cell
productivity in high density culture without clumping.
In this study, we evaluated the cell line using bioreactor
batch culture. We used a BioFlo® 320 bioprocess control
system and a BioBLU® 3c Single-Use Bioreactor to carry
out Expi293F batch culture in 2 L scale. In addition we
monitored and analyzed the metabolites as well as cell
density and viability during 11 days of culture.
HEK293 Suspension Cell Culture Using the BioFlo® 320
Bioprocess Controller with BioBLU® 3c Single-Use
Bioreactors
Jorge L. Escobar Ivirico and Ma Sha
Eppendorf, Inc., Enfield, CT, USA
Contact: bioprocess-experts@eppendorf.com
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APPLICATION NOTE | No. 447 | Page 2
Introduction
Developing innovative preventive solutions through new
vaccine technologies or gene therapy platforms that respond
to new diseases is an important challenge in present day biotechnology. The gene therapy market is projected to reach
more than $ 3 billion by 2023 and the global vaccine market
is expected to reach $46 billion by 2022, pushing them to
the apex of the biotechnology food chain [1-3]. To produce
high-quality biotherapeutics, process development involves
several demanding components including cost, cell line
development, small scale exploration, effective scaling, and
optimization of upstream/downstream processes.
The selection of the host cell is a key factor based on its
capabilities and properties, including its ability to grow in
suspension or adhere to a substrate. HEK293 is one of the
most versatile mammalian cell lines with a wide range of
applications including expression of recombinant proteins,
antibodies and viruses. The HEK293 cell line was immortalized in 1973 by the integration of a ~4.3 kbp adenoviral 5
(Ad5) genome fragment containing the E1A and E1B genes,
located on chromosome 19 [4,5]. E1A and E1B are essential
helper factors for adeno-associated virus (AAV) manufacture,
making these cells a popular host platform for AAV particles
production [6]. Our goal in this project was to evaluate the
suspension culture of Expi293F cell line using Eppendorf
bioprocess equipment and assess its suitability in virus production.
HEK293 in gene therapy
Gene therapy involves the transfer of functional genes into
cells to replace absent genes or correct defective ones. In a
typical protocol, the cells are extracted from the donor and
genetically modified by introducing a new or modified gene
to inactivate or to replace a disease-causing gene. These
modified cells are then reimplanted in the subject (ex vivo
strategy). In addition, a well-established cell culture platform,
such as HEK293, can be used to produce viral or non-viral
delivery vehicles to introduce the gene of interest (GOI).
A well-established method (transient transfection) is frequently employed to produce different vectors using adherent human HEK293 cells cultivated in T-flasks or bioreactors.
Prominent examples include lentivirus [7, 8], adenovirus
[9,10], non-viral vectors [11], and AAV [12,13]. AAV represents one of the leading platforms for gene therapy due to its
ability to provide in vivo long-term gene expression. Nearly
200 AAV clinical trials and biotherapeutic protocols are in
different stages of FDA review, in which the transient transfection of adherent HEK293 cells has been the predominant
platform [14,15]. We believe that the Expi293F cell line is a
significant improvement over traditional HEK293 cell lines
due to its robust growth under suspension culture conditions
as well as its property of stable expression in addition to
transient expression.
HEK293 in vaccine production
During the last century, vaccines saved billions of lives
throughout the world. Vaccine technology has distinguished
itself as the most important development in the history of
medicine. This unparalleled success has driven researchers to explore new and more efficient platforms to meet the
constantly expanding demands of the industry.
Conventional vaccines usually contain whole weakened or
inactivated viruses or protein subunits made by the pathogen
to trigger an immune response. The cell-based vaccine platform is a well-established technology [16], offering several
notable advantages:
> cell lines are well characterized and may be easily stored
for future applications,
> their use avoids dependence on embryonated chicken eggs
(ECE), whose quality is highly and unpredictably variable,
despite their being the most common method used to
develop vaccines,
> some viruses grow better in cells reducing the time to
achieve high growth profiles [17],
> compared to ECE, viruses propagated in mammalian cells
have shown an antigenic profile similar or identical to that
of the field virus [18], and
> scalability is superior to the ECE production platform [19].
Although numerous mammalian cell lines have been
evaluated for vaccine production [20-24], and performance
in gene therapy protocols, the HEK293 cell line is one of the
most widely used cell platforms for these demands. Specifically, high yield adenoviral vectors (~4 x 1015 viral particles)
have been obtained in stirred-tank bioreactor systems using
microcarriers [25,26]. Adherent HEK293 cells are easy to
cultivate at laboratory scale, and require less expert bioengineering know-how, but when biotherapeutics production
increases, suspension cell lines offer advantages in terms of
scalability and robustness, using established stirred-tank bioreactor platforms. Eppendorf bioprocess systems, including
BioFlo 320, are not medical devices and cannot be directly
used for Gene Therapy and Vaccine production without special approval process. However, it can be used for effective
HEK293 suspension cell culture and virus
production research including research in the Gene Therapy
and Vaccine production field at R&D level. To this end,
APPLICATION NOTE | No. 447 | Page 3
Expi293F cells (a suspension adapted HEK293 cell line) can
accelerate the therapeutics development by enabling rapid,
high yield and scalable production of proteins, viral antigens
and AAV particles.
In this study, we used a BioBLU 3c Single-Use Bioreactor
for Expi293F cell expansion and a BioFlo 320 as the bioprocess control system (Figure 1). We analyzed the cell growth,
the viability as well as the metabolic activity (levels of glucose, ammonia and lactate in the medium).
Material and Methods
Cell line and medium
We cultured the suspension Expi293F cell line (Thermo Fisher Scientific, USA) in Expi293 Expression Medium (Thermo
Fisher Scientific, USA) formulated with GlutaMAX-I reagent.
Expi293 Expression Medium is a chemically defined, serum
and protein-free medium, ready to use without need for additional supplements.
Inoculum preparation
We rapidly thawed the cryovial containing 1 mL of Expi293F
cells (Thermo Fisher Scientific, A14527) at 1 x 107
cells/mL,
from a previously prepared cell bank, using a ThawSTAR®
CFT2 instrument (MedCision®, USA). Just before the cells
were completely thawed we decontaminated the vial wiping
it with 70% alcohol before opening it in a laminar flow hood.
We transferred the entire content of the cryovial into a 125
mL disposable, sterile and vented shaker flask containing
30 mL (24 % of the total volume) of pre-warmed Expi293
Expression Medium (Thermo Fisher Scientific, A1435101).
We cultured the cells in a New BrunswickTM S41i CO2
incubator Shaker (Eppendorf, Germany) at 37 °C, 8 % CO2
and at
agitation speed of 125 rpm. We cultured the cells for 4 days
after thawing and then determined the cell viability and total
viable cells using a Vi-CELL XR cell viability analyzer (Beckman Coulter). We then performed the subsequent passages
when the viable cell density reached around 3 x 106
cells/
mL (typically 4 days after shaker flask inoculation) reaching
more than 900 x 106
cells in the third passage. During the
expansion process we kept the inoculation density, percentage fill of the shake flasks and other parameters constant.
Finally, we prepared the inoculum containing 800 x 106
cells
in 200 mL of Expi293F Expression Medium. The cell expansion workflow is shown in Figure 2.
Fig. 2: Inoculum preparation.
Created with: BioRender.com.
Cryovial of cells ( 1 × 107 cells)
125 mL shake flask
Cell density ( 0.4 × 106 cells)
3x 125 mL shake flask
Cell density ( 0.4 × 106 cells)
6x 250 mL shake flask
Cell density ( 0.4 × 106 cells)
BioBLU 3c (2 L working volume)
Inoculation density ( 0.4 × 106 cells/mL)
Fig. 1: BioBLU 3c Single-Use Bioreactor (left) and BioFlo 320
bioprocess control system (right).
Technical Features
www.eppendorf.group/bioflo320
In this study, pH, DO, and temperature were controlled
online. Get to know more about the possibilities for
bioprocess control with the BioFlo 320.
APPLICATION NOTE | No. 447 | Page 4
Bioreactor control
We used a BioFlo 320 bioprocess control station to perform
two batch cultures using BioBLU 3c Single-Use Bioreactors
equipped with a single pitched-blade impeller. The bioreactor unit is equipped with two universal port connectors for
pH (port 1) and DO (port 2) sensors, an heat blanket connection providing precise temperature control, agitation control
and a gas module that includes 3 TMFC, high-flow sparge
drawer with a gas flow range of 0.04 – 20 SLPM.
Sensor calibration
Prior to the preparation of the BioBLU 3c Single-Use Bioreactors, we connected the gel-filled pH sensor to the
BioFlo 320 bioprocess controller. The software automatically
detects the connected sensors to support an efficient workflow. We performed the calibration process according to the
operation’s manual using buffer solutions of pH 7 and pH 4
as “zero” and “span” respectively. Then, we disconnected
the pH sensor and sterilized it in an autoclavable pouch.
BioBLU 3c Single-Use Bioreactor preparation and process
parameters
We equipped the BioBLU 3c with a magnetic drive, the previously sterilized pH sensor, inserted in a spare PG 13.5 port
under aseptic conditions in the Biosafety Cabinet, a polarographic DO sensor (Mettler Toledo®), an exhaust condenser,
a 3-gas mixing line connected to the gas sparge port, and 3
liquid addition ports (one for inoculation/glucose addition,
one for base addition and another for the addition of 0.1 %
of antifoam (Pluronic®-F68 surfactant, Life Technologies®,
24040-032). Then, we controlled the temperature using a
heat blanket. Finally, we introduced the 1.8 L of Expi293F
Expression Medium into each bioreactor and conditioned for
at least 24 hours under the parameters and setpoints listed in
Table 1.
Expi293FTM cells culture on BioBLU 3c Single-Use Bioreactor
We inoculated the BioBLU 3c Single-Use Bioreactors with
the inoculum described above (see section “Inoculum preparation”) reaching 2 L as working volume with a cell density
around 0.4 x 106
cell/mL and more than 95 % of cell viability. We monitored the temperature at 37 °C and controlled the
dissolved oxygen (DO) at 40 % using the 3-Gas Auto mode.
In addition, we limited the oxygen flow to 0 – 1 SLPM and
the air flow to 0.04 – 1 SLPM in the controller setup screen
to avoid high gas flow that can cause DO fluctuation and
excessive foaming in the beginning stage of the cells culture.
In addition to the gas flow limit, we added Pluronic-F68 surfactant as needed. We used a gel-filled pH sensor to control
the pH during the cell culture run at 7.0 (deadband = 0.2),
using a cascade to CO2
(acid) and 0.45 M sodium bicarbonate (base). We took a sample from the bioreactor daily and
measured the pH, the cell viability and density as well as the
concentration of various metabolites offline.
Cell viability and metabolic activity
We collected samples on a daily basis from the BioBLU 3c
Single-Use 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, we discarded
5 mL of dead volume and collected again 3 mL (using a new
5 mL sterile syringe) as a viable sample for analysis. We used
1 mL to measure the metabolite levels employing a Cedex®
Bio Analyzer (Roche, USA), 1 mL to measure the cell viability
and density using a Vi-Cell® XR Viability Analyzer (Beckman
Coulter®, USA) and 1 mL to check the pH offline using an
Orion Star A211 pH meter (Thermo Fisher Scientific, USA),
which we calibrate daily using standard pH buffers.
Results and Discussion
To evaluate the Expi293F suspension culture robustness,
we performed two bioreactor batch culture runs using
BioBLU 3c Single-Use Bioreactors controlled by BioFlo 320
bioprocess controller. We used Expi293 Expression Medium with additional glucose supplementation to extend
the growth phase and increase the peak cell density. The
inoculum was ready after the cell expansion in the New
Brunswick S41i CO2
incubator Shaker at 37 °C and 8 % CO2
and agitation speed of 125 rpm. We then inoculated the
BioBLU 3c Single-Use Bioreactor with an initial cell density
of 0.4 x 106
cells/mL under a controlled environment (see
Table 1).
Table 1: Process parameters and setpoints of the first and second
experiments.
Parameters Setpoints
Starting volume 1.8 L
Ending volume 2 L
Initial agitation 120 rpm (0.4 m/s tip speed)
Temperature 37 °C
Inoculation density 0.4 x 106
cell/mL
Cell culture medium Expi293™ Expression Medium
DO Setpoint 40% (P=0.1; I=0.001)
pH Setpoint 7.0 (deadband = 0.2), cascade to CO2
(acid)
and cascade to 0.45 M sodium bicarbonate
(base)
Gassing range Air flow: 0.04 SLPM -1 SLPM
O2
flow: 0 SLPM -1 SLPM
APPLICATION NOTE | No. 447 | Page 5
In addition, we added Pluronic-F68 surfactant (0.1 %) to the
medium in the bioreactor to decrease foaming produced by
the gas introduced through the sparger.
As shown in Figure 3A, we observed a rapid increase of
cell growth between days 1 and 9 of culture, reaching a peak
in viable cells density at 13 x 106
cells/mL, followed by a
decrease in cell density and viability as anticipated. Furthermore, we determined the consumption of glucose and
production of lactate and NH3
while at the same time maintaining the concentration of lactate and NH3
below 2 g/L and
2 mmol/L respectively during the whole run (See Figure 3B).
We performed bolus glucose supplementation (to maintain
target concentration > 2 g/L in both runs) at days 3 and 5 to
extend the growth phase. The ammonia concentration gradually increased every day up to 2.2 mmol/L on day 11. We
believe the depletion of glucose and other nutrients
contributed to the decrease of the cell density, starting from
day 9. Overall, cell growth increased around 32-fold.
Conclusions
Using the BioFlo 320 bioprocess control system and
BioBLU 3c Single-Use Bioreactors, we demonstrated the
feasibility of applying glucose-enhanced batch culture
technique to expand Expi293F cells rapidly up to 13 million
cells/mL within 9 days. The efficient and straightforward
configuration of the BioFlo 320 allows precise control of the
cell culture environment, leading to reliable cell expansion.
Although the experiments were conducted as feasibility
studies and no optimization of conditions was attempted, we
observed vigorous growth of Expi293F in suspension culture
at a pace and simplicity close to Chinese hamster ovary
(CHO) cells, the accepted industry standard. We believe that
the Expi293F cell line has great potential in both vaccine and
gene therapy method development when used in conjunction
with Eppendorf’s advanced stirred-tank bioreactors.
Fig. 3 : Expi293F growth profile in BioBLU 3c Single-Use Bioreactor in Expi293 Expression Medium.
A: Expi293F cell density and viability. B: Metabolic profile.
0
20
40
60
80
100
0
4
8
12
16
20
Cell viability (%)
Cell Density/mL (x106)
Time (days) Cell density
Batch 1 Batch 1
Batch 2 Batch 2
Cell viability
1
1
1
987 121110
7
6
6
5
5
4
4
4
3
3
3
2
2
2
0
a
0 0
0 1 98765432 121110
Time (days)
Glucose (g/L)
Lactate (g/L), NH3 (mmol/L)
Glucose Lactate NH3
b
APPLICATION NOTE | No. 447 | Page 6
Literature
[1] Grand View Research, "Gene Therapy Market Size, Share & Trends Analysis," 2019.
[2] Markets and Markets, "Gene Therapy Market by Vectors," 2019.
[3] Vaccines Market by Indication, Type, Route of Administration & Geography / Global Vaccines Market Forecasts 2018-2025 /
ResearchandMarkets.com/CoherentMarketInsights, 2018.
[4] Graham FL, Smileyt J, Russell WC, Nairn R. Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5. J. gen. Virol 1977; 36:59–7.
[5] Louis N, Evelegh C, Graham FL, 1997. Cloning and Sequencing of the Cellular– Viral Junctions from the Human Adenovirus Type 5 Transformed 293 Cell Line. Virology 1997; 233:423–429.
[6] Clément N, Grieger JC. Manufacturing of recombinant adeno-associated viral vectors for clinical trials. Mol. Ther. Methods
Clin. Dev. 2016; 3:16002.
[7] McCarron A, Donnelley M, McIntyre C, Parsons D. Transient Lentiviral Vector Production in HEK 293T Cells Using the
BioFlo® 320 Control Station with a BioBLU® 5p Single-Use Packed-Bed Vessel. Application Note 2019; App-411.
[8] Ferreira CB, Sumner RP, Rodriguez-Plata MT, Rasaiyaah J, Milne RS, Thrasher AJ, Qasim W, and Towers GJ. Lentiviral Vector Production Titer Is Not Limited in HEK293T by Induced Intracellular Innate Immunity. Molecular Therapy: Methods &
Clinical Development. 2020; 17:209-219.
[9] Feng L, Wang Q, Shan C, Yang C, et al. An adenovirus-vectored COVID-19 vaccine confers protection from SARS-COV-2
challenge in rhesus macaques. Nat Commun. 2020; 11:4207.
[10] Fedosyuk S, Merritt T, Peralta-Alvarez MP, Morris SJ, et al. Simian adenovirus vector production for early-phase clinical
trials: A simple method applicable to multiple serotypes and using entirely disposable product-contact components. Vaccine. 2019; 37:6951–6961.
[11] Patil S, Gao YG, Lin X, et al. The Development of Functional Non-Viral Vectors for Gene Delivery. Int J Mol Sci. 2019;
20:5491.
[12] Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov.
2019; 18:358–378.
[13] Li C, Samulski RJ. Engineering adeno-associated virus vectors for gene therapy. Nat Rev Genet. 2020; 21:255–272.
[14] Rumachik NG, Malaker SA, Poweleit N, et. Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors. Molecular Therapy: Methods & Clinical Development. 2020; 18:98-118.
[15] Grimm D, Kern A, Rittner K, Kleinschmidt JA. Novel tools for production and purification of recombinant adeno-associated virus vectors. Hum. Gene Ther.1998; 9:2745–2760.
[16] Le Ru A, Jacob D, Transfiguracion J, Ansorge S, Henry O, Kamen AA. Scalable production of influenza virus in HEK-293
cells for efficient vaccine manufacturing. Vaccine. 2010; 28:3661–3671.
[17] Jung EJ, Lee KH, Seong BL. Reverse genetic platform for inactivated and live-attenuated influenza vaccine. Exp Mol Med.
2010; 42:116-21.
[18] Donis RO. The Influenza Cell Culture Working Group. Performance characteristics of qualified cell lines for isolation and
propagation of influenza viruses for vaccine manufacturing. Vaccine. 2014; 32:6583-90.
[19] Hegde NR. Cell culture-based influenza vaccines: A necessary and indispensable investment for the future. Hum Vaccin
Immunother. 2015; 11:1223–1234.
[20] Rimmelzwaan GF, Baars M, Claas ECJ, Osterhaus ADME. Comparison of RNA hybridization, hemagglutination assay, titration of infectious virus and immunofluorescence as methods for monitoring influenza virus replication in vitro. Journal of
Virological Methods. 1998; 74:57–66.
[21] Voeten JTM, Brands R, Palache AM, et al. Characterization of high-growth reassortant influenza A viruses generated in
MDCK cells cultured in serum-free medium. Vaccine. 1999; 17:1942–1950.
[22] Pau MG, Ophorst C, Koldijk MH, Schouten G, Mehtali M, Uytdehaag F. The human cell line PER.C6 provides a new manufacturing system for the production of influenza vaccines. Vaccine. 2001; 19:2716–2721.
[23] Kistner O, Barrett PN, Mundt W, Reiter M, Schober-Bendixen S, Dorner F. Development of a mammalian cell (Vero) derived candidate influenza virus vaccine. Vaccine. 1998; 16:960–968.
[24] Youil R, Su Q, Toner TJ, et al. Comparative study of influenza virus replication in Vero and MDCK cell lines. Journal of
Virological Methods. 2004; 120:23–31.
[25] Vemula SV, Mittal SK. Production of adenovirus vectors and their use as a delivery system for influenza vaccines. Expert
Opinion on Biological Therapy. 2010; 10:1469–1487.
[26] Fuenmayor Garcés J, Gutiérrez Granados S, Gòdia Casablancas F, Cervera Gracia L. Cell line and plasmid influence on
the efficiency of HEK293 cells transient transfection for the production of VLP-based vaccines. New Biotechnology. 2016;
33:S32-S33.
APPLICATION NOTE | No. 447 | Page 7
Your local distributor: www.eppendorf.com/contact
Eppendorf SE · Barkhausenweg 1 · 22339 · Hamburg · Germany
eppendorf@eppendorf.com · www.eppendorf.com
www.eppendorf.com/bioprocess
FlowStar® and MedCision® are registered trademarks of MedCision Inc., USA. Mettler Toledo® is a registerd trademark of Mettler-Toledo AG, Switzerland. Pluronic® and Life Technologies® are registered trademarks of
Life Technologies Corp. USA. Cedex® is a registered trademark of Roche Diagnostics GmbH, Germany. VI-Cell® and Beckman Coulter® are registered trademarks of Beckman Coulter, Inc., USA. BioBLU®, Eppendorf®
and the Eppendorf Brand Design are registered trademarks of Eppendorf SE, Germany. BioFlo® is a registered trademark of Eppendorf Inc., USA. Expi293F™ and Thermo Scientific™ are trademarks of Thermo Fisher
Scientific Inc., USA. All rights reserved, including graphics and images. Copyright © 2022 by Eppendorf SE.
Ordering information
Description Order no.
BioFlo® 320, base control station, no water connection 1379962911
BioFlo® 320, left-handed orientation/four front-mounted peristaltic pumps (3 @ 5 – 25 rpm/1 @ 20 – 100 rpm) 1379963211
BioFlo® 320, sparge gas option, 3 TMFC (0.04 – 20 SLPM) 1379501311
Accessories
Touchscreen Monitor Bundle, includes desk mount and 1 meter cable set, for BioFlo® 320 M1379-9906
Bioreactor
Single-Use Vessel Bundle, for BioFlo® 320, for BioBLU® 3c/5c M1379-0322
BioBLU® 3c, 1.25 L - 3.75 L, Microsparger, 1x pitched-blade 1386000100
BioBLU® 3c ,1.25 L - 3.75 L, Macrosparger, 1x pitched-blade 1386000300
BioBLU® 3c, 1.25 L - 3.75 L, Microsparger, 2x pitched-blade 1386120000
BioBLU® 3c, 1.25 L - 3.75 L, Macrosparger, 2x pitched-blade 1386121000
Executive Summary
Plasmids serve as important tool in genetic engineering,
gene therapy research or genetic vaccination. Recombinant plasmid DNA (pDNA) is produced in bacterial
cultures, mostly in Escherichia coli (E. coli). Successful
pDNA production, reaching high yield and quality, relies
on multiple factors, including the type of vector and
host strain, the insert, and the methodology chosen for
cultivation and downstream purification. Here, we review
the distinct steps in the plasmid production workflow and
give tips on optimization: from vector and strain selection
to cultivation, from shake flasks to fermenters up to downstream processing steps of harvesting and purification.
From Strain Selection to Purification – Key
Factors for Successful Plasmid Production
Introduction
Plasmids serve as a tool in genetic engineering, either to
clone and amplify DNA fragments, such as genes, express
recombinant proteins or to serve as templates e.g. for in
vitro mRNA production or transient CRISPR expression in
gene therapy research and genetic vaccination [1,2]. Plasmid DNA (pDNA) can be easily genetically manipulated and
produced in large amounts in bacteria, typically in E. coli.
Furthermore, a variety of ready-to-use solutions allows easy
subsequent downstream purification.
WHITE PAPER No. 89
Stefan Schlößer¹, Eric Vincent3, Ruchika Sharma3, Samuel Ellis4, Ines Hartmann2
1
Eppendorf SE, Jülich, Germany
2
Eppendorf SE, Hamburg, Germany
3Promega, Madison, WI USA
4Thomson Instrument Company, Oceanside, CA USA
Vector selection
& design
Host selection +
transformation
Shake culture Fermentation Harvesting Purification
Depending on the application, pDNA production ranks from
research laboratory scale (microgram range) to large industrial scale (milligram to gram range). The plasmid production includes multiple steps (Figure 1). In the following, we
review the factors affecting plasmid production and give tips
on optimization.
The production of pDNA can be separated in five parts:
1) Plasmid vector selection, page 2–3
2) Host strain selection and transformation, page 3
3) Production in shake flasks and fermenters, page 3–12
4) Culture harvesting, page 12
5) Purification, page 12–13
Figure 1: Plasmid production workflow
WHITE PAPER I No. 89 I Page 2
1) Plasmid vector selection
Since the availability of the first commercial cloning plasmid vectors in the 1970s, the number of available products
has increased substantially. Plasmids are small circular
double stranded DNA molecules. The typical size ranks from
thousand up to a few thousand base pairs. Minimal required
components of plasmids used in the lab include the origin of
replication (ori), which enables the independent replication
from the host’s chromosome, the multiple cloning site (MCS)
or polylinker region with the restriction digestion sites, that
allows to insert the gene of interest, and a selection marker
to ensure that only bacteria containing the plasmid survive
in culture. In expression plasmids, an additional promoter is
inserted upstream to the multiple cloning site, which drives
the transcription of the gene (Figure 2).
Figure 2: Minimal components of a plasmid vector. Important components
include the origin of replication (ori), the multiple cloning site with the restriction
sites (here shown with the inserted gene of interest) and the selection marker (here
an antibiotic resistance gene). In expression plasmids an additional promoter is
inserted upstream the multiple cloning site.
The following parameters are helpful for choosing the right
plasmid for your needs.
Replicon and copy number
One important factor affecting the yield of plasmids from
a given system is the copy number, meaning the expected
number of plasmids per host cell. A plasmid’s copy number
is determined primarily by the ori and the surrounding regulatory DNA sequences. This area, known as the replicon,
controls the replication of plasmid DNA.
Plasmids can be grouped into low and high-copy plasmids
(Figure 3). For example, plasmids derived from the plasmid
pBR322 contain the ColE1 origin of replication from pMB1.
This origin of replication is tightly controlled, resulting in
only ~25 copies of the plasmid per bacterial cell (low copy
number). On the other hand, pUC-derived plasmids contain
a mutated version of the ColE1 origin of replication, which
results in reduced replication control and ~200–700 plasmid
copies per cell (high copy number).
Figure 3: High and low copy plasmids. High copy plasmids (left) result in increased plasmid copies per cell, compared to low copy plasmid (right).
In most cases, a high-copy plasmid produces greater yields
of plasmid DNA and is the preferred choice for gene cloning. Another advantage of the high copy number is higher
plasmid stability when random segregation occurs during
cell division [3]. However, high-copy plasmids pose a high
metabolic burden on the host cell. That may lead to poor
culture growth and can favor plasmid instability. Especially
for ambitious cloning tasks, like amplifying plasmids with
large inserts >8 kb or adenine and thymine rich (‘AT-rich’)
sequences, a low copy plasmid may be considered to increase stability [4].
Excursus I: Considerations for protein expression plasmids
For most protein expression applications, high-copy
plasmids are well suited to produce high amounts of
protein. However, when high-level expression results
in protein aggregation, a slowed-down synthesis rate
can have a positive impact on yields and a low copy
plasmid may be considered [5]. Other measures, such as
introducing additional backbone features into a proteinexpression plasmid (for example protein-solubility
increasing fusion tags), using weaker promotors,
or co-expressing multiple protein components with
a compatible dual expression plasmid system, are
strategies to further improve solubility [6].
Bacteria cell
Plasmids
WHITE PAPER I No. 89 I Page 3
Insert size
Most commonly available plasmids like pUC and pBlueScript
can take inserts of up to around 15 kb [6]. However, larger
fragments can lead to decreased plasmid copy numbers or
problems in plasmid DNA replication and stability [7]. There
are special types of high-capacity vectors such as PACs, BACs,
cosmids, which are suitable for cloning large DNA fragments
[8].
Selection marker
Almost all plasmids have a selection marker, usually an antibiotic resistance gene, that ensures that only the cells that have
incorporated the plasmid vector can survive in the presence
of the specific antibiotic. Depending on the selection marker,
the referring antibiotic must be added to the solid or liquid
medium to keep the selection pressure and plasmid stability.
To avoid degradation, antibiotics should be added to the medium after autoclaving at temperatures lower than 60 °C and
always freshly prior use. For some therapeutic plasmid-based
products, the presence of antibiotics is restricted or not recommended by regulatory authorities. Kanamycin is the most
widely used and preferred selection agent for DNA vaccines,
as it does not present a significant allergic risk [9]. Engineering more stable vectors and alternative selection strategies to
antibiotics have been investigated [10].
2) Host strain selection and transformation
The choice of the E. coli host strain can have a significant
impact on the quantity and quality of the produced pDNA.
E. coli K-12 strains such as DH5α, JM109 and the slower
growing XL1-Blue are common host strains for gene cloning
applications generating high plasmid yields. They contain
several gene mutations, including one in the endonuclease A
(endA) gene to prevent plasmid degradation and one in the
recombinase A (recA) gene to avoid homologous recombination. Constant genetic engineering efforts, such as metabolic
engineering strategies targeting individual bacterial enzymes
relevant to the cell’s physiology, have been explored with the
aim of increasing plasmid production [11,12].
Excursus II: Host strain selection for protein expression
Amongst protein expression strains E. coli B-strains,
such as BL21 (DE3), are often chosen for high-level
production of recombinant proteins. They are deficient
in genes encoding certain proteases that can degrade
proteins during purification. Several strain modifications
for difficult protein expression have been engineered, such
as BL21(DE3)-RIL for expression of rare codon-containing
genes and BL21-AI for toxic proteins.
Two procedures are primarily used to transfer plasmids
into bacterial cells: chemical transformation and electroporation. Chemical transformation or heat shock is cost-effective
and well established for E. coli but is a more time-consuming
and labor-intensive method compared to electroporation.
Electroporation requires investment in a specific electroporation device and cuvettes but has been shown to increase
transformation efficiency [13] and to be beneficial for larger
insert sizes [14]. The transformation is followed by clonal
selection on agar plates containing selection markers, such
as antibiotics. Transformation efficiency can be calculated as
the number of colony-forming units (CFU) per microgram of
plasmid DNA used.
3) Production in shake flasks and fermenters
Cultivation technique – choosing a shake flask or fermenter
The growth environment plays a crucial role in pDNA
production. Productivity is usually proportional to the final
cell density and the specific productivity (amount of pDNA
per unit cell mass). The pDNA yield is commonly indicated
in milligrams of pDNA per liter of culture (mg/L). Typically,
0.5–1 g pDNA per kg of wet weight biomass is obtained from
using high copy plasmids [15].
Besides existing lab resources, the application and yield
demands determine the cultivation method of choice. Tube
cultures and small shake flasks are suitable for gene cloning
and laboratory scale protein production, where working volumes between 1 mL to 1 L per vessel and yields in the microgram range are usually sufficient. If higher plasmid yields are
required, for example for transfection purposes, large-scale
recombinant protein production, or if the plasmid is the final
product itself in genetic vaccination or gene therapy applications, a controlled fermenter is usually the device of choice,
being able to handle higher working volumes.
Knowing the bacteria´s growth behavior
Growth curve measurements based on optical density
(OD600) provide a simple tool to analyze the bacterial growth
behavior, for example to compare different strains, analyze
the influence of media composition, or determine the optimal point of harvesting. In a typical growth curve, one can
differentiate four distinct phases of growth: the lag, exponential (log), stationary, and death phase (Figure 4).
WHITE PAPER I No. 89 I Page 4
0.1 for shake flasks (often higher for fermenters). However,
the optimal inoculation density is dependent on the growth
characteristics of the microorganism and the given growth
environment and can be evaluated by performing initial
growth curve comparisons. Unlike mammalian cell cultures,
long, cost-intensive seed trains are not required, and, in most
cases, a one-stage inoculum train is sufficient also in industrial settings [20].
Stationary phase
B
b
t
[days]
Figure 4: Typical bacteria growth curve (batch culture). In the initial lag phase,
the cells are in a metabolically active state, but there is no or only little cell division.
The cells accommodate to the new conditions. The subsequent logarithmic (log)
phase is the phase of optimal growth with cell numbers increasing in a logarithmic
fashion. In the stationary phase the cell growth rate slows upon nutrients depletion
or toxic product accumulation and the rate of cell division and death is roughly
equal. In the death phase the cell number decreases. In red: The generation or
doubling time G of the cells is calculated during the exponential phase of growth.
B is the number of bacteria present at the start of the observation, b is the number
present after the time t.
From the growth curve, one can calculate the generation
(=doubling) time (G) in minutes or hours using the following
formula:
G = t
n = t
3.3 log b / B
In the formula, B is the number of bacteria present at the
start of the observation, b is the number present after the
time t, and n is the number of generations. The generation time is calculated during the exponential growth phase
(linear growth on a log-scale plot) (Figure 4).
Inoculation strategies
The most frequently recommended method to inoculate a
small liquid culture from a frozen stock, is to start from a
single colony grown on a freshly streaked selective agar
plate [16,17,18]. This ensures that the culture derives from
one clone. However, a recent study comparing direct inoculation from a glycerol stock versus inoculation from a single
colony did not identify significant differences between the
two methods in terms of growth behavior and final plasmid
yields [19].
Shake cultures and fermenter cultures are often inoculated
from a liquid pre-culture to reduce the lag-phase (Figure 5).
The pre-culture should be harvested within the logarithmic
phase. Inoculation with a pre-culture does not only shorten
the lag phase, but also allows a better synchronization of
cultures by adjusting to a defined start OD600. This is helpful
when comparing strains. A typical starting OD600 is around
0
1
2
3
1 2 3 4 5 6
OD600
Time [h]
Agarcolony
Liquid pre-culture
Figure 5: Influence of inoculation method on bacteria growth. Inoculation
of 250 mL Erlenmeyer flasks filled with 20 % LB medium with either a single
colony from an agar plate or from a liquid pre-culture, starting OD600 ~0.04. (E.
coli DH5ɑ, incubation at 37 °C and 250 rpm)
The optimal harvesting timepoint
The harvesting time point affects both the yield and quality of plasmid DNA. Bacterial cultures grown to insufficient
density will yield reduced amounts of pDNA. Overgrown
cultures may result in suboptimal yields and excessive chromosomal DNA contamination due to autolysis of bacterial
cells after they have reached the stationary phase. Harvesting for plasmid preparation is usually recommended in the
late logarithmic or early stationary phase [21]. By this time,
the culture possesses high biomass, hence plasmid yield,
but not too many dead cells. Growth times for cultures with
high copy plasmids under standard conditions are typically between 12-16 hours, and for cultures with low copy
plasmids ≥ 20 hours [18]. Due to the higher metabolic
burden posed on the bacterial cell by high copy plasmids,
the achievable maximum cell density of these cultures is
usually lower compared to low copy plasmids, resulting in a
shorter incubation period. The optimal cultivation time may
vary for several reasons, such as the utilized strain, plasmid,
temperature, agitation speed, flask type, media composition, and cultivation mode and should be evaluated based on
growth curve analysis and sampling at various time points
with subsequent plasmid preparations.
WHITE PAPER I No. 89 I Page 5
Optimizing tube culture
For small-scale approaches culturing is typically carried out
in 15 mL conical tubes. However, due to their small diameter,
handling can be difficult. They are also not optimal for efficient
aeration due to the small surface-to-volume ratio. A 25 mL
conical tube such as the one provided by Eppendorf has the
diameter of a 50 mL tube offering the advantage of a larger
surface for oxygen transfer but is half the size of a standard
50 mL tube, thus better accessible with a pipette. A comparison between the 25 mL Eppendorf conical tube and a standard 15 mL tube, using the same inoculum amounts of E. coli,
identified enhanced cell numbers and plasmid DNA yields in
the 25 mL tube [22] (Figure 6). This indicates improved mixing
and aeration due to the different shapes. As a rule of thumb, fill
volumes in tubes should not exceed 20 % to facilitate adequate
mixing. In addition, an increased liquid surface for oxygen supply can be achieved by cultivating the tube at an angle such as
30-45 °, especially when working at standard shaking speeds
of around 200 rpm [23].
800
600
400
200
15 mL culture
25 mL tube
7.5 mL culture
25 mL tube
7.5 mL culture
15 mL tube
0
Plasmid DNA Yield [µg]
Figure 6: Influence of tube design and size on plasmid yields. Total yield of
low copy plasmid DNA (PBR322) purified from bacterial cultures E. coli DH5α
incubated in Eppendorf Conical Tubes 25 mL with snap cap and standard
15 mL conical tubes. Inoculated from one seed stock and grown under similar
conditions.
Optimizing shake flask culture
Shake flasks are the most used cultivation vessels for production of plasmids on laboratory scale. Also, early process
development, such as initial strain and media selection,
starts typically in shake flasks prior to further process
development and expansion in fermenters. Shake flasks are
inexpensive, easy to parallelize and simple to use. There are
different parameters one can adjust in cultivation to increase
biomass and product yields in a shake flask. Although E. coli
is a facultative anaerobic microorganism, it grows best in
the presence of oxygen [24]. In a shake flask, oxygen supply
only occurs passively via the air-liquid interface. Oxygen
limitation is one of the most frequent problems in shake
flask culture.
Different shake flask designs are available to increase the
oxygen transfer to the culture (Figure 7).
Figure 7: DIfferent shake flasks
A) Classic non-baffled and baffled Erlenmeyer flask designs. Baffled flasks have
defined cavities in the bottom area B) Ultra Yield® flasks. Flasks with steep vertical
walls and six baffles at the flask bottom in 2.5 L size (Thomson).
Baffles for example, disrupt the regular swirling liquid
flow by the implemented cavities in the bottom area, thus
improving the aeration of the culture [25]. Comparisons of
different flask designs have shown that baffles can improve
cell growth and maximum cell density drastically compared
to unbaffled flasks (Figure 8).
0
5
10
15
20
25
30
2 4 6 8 24
OD600
Time post inoculation [h]
Ultra Yield®
Baed
Non-baed
Figure 8: Influence of flask design on bacterial growth. Ultra Yield® flasks
with the specialized baffled design resulted in the fastest growth and maximum
cell density, followed by the standard baffled design and finally the non-baffled
design (E. coli DH5α with pUC19 plasmid cultivated in modified TB media in
500 mL flasks with 25 % fill volume, 37 °C and 200 rpm).
The disadvantage of this design is a higher risk of foam
formation that may lead to cross contamination by wetting
the flask closure. However, this problem can be solved by
using an antifoam agent. Unreproducible results caused by
variances in baffle geometry and size have been described
for glass flasks [26]. However, this has not been seen in
disposable flasks [27], assuming these effects may be dependent on the manufacturing process in general.
a b
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Typical agitation speeds for flask cultures are 200-
250 rpm. An increase in agitation speed supports the oxygen
transfer to the liquid and increases both biomass and
plasmid yields independently of flask size [28]. An increase
from 250 to 350 or 400 rpm resulted in up to almost 2-fold
higher biomass [data shown in original app note 449] and a
~30-90 % increase in pDNA yields in 500 mL and 2.5 L Ultra
Yield® flasks, respectively [Figure 9A and B]. However, care
must be taken, especially when using high agitation speeds
with baffled flasks, to prevent cap wetting.
34.8 45.2
0
50
100
pDNA yield [mg/L]
250 rpm 400 rpm
A) DH5α transformed with pUC19
64.0
120.3
200 rpm 350 rpm
B) JM109 transformed with pGEM-3Z
Figure 9: Influence of agitation speed on pDNA yields. An incubation at higher
agitation speed resulted in higher pDNA yields for both E. coli strains.
A: Cultivation of E. coli DH5α transfected with pUC19 plasmid in 2.5 L Ultra Yield®
flasks (20 % fill, 37 °C, modified TB media, harvest 8 h post inoculation).
B: E. coli JM109 with pGEM®-3Z plasmid in 500 mL Ultra Yield® flasks (25 % fill,
37 °C, modified TB media, harvest 8 h post inoculation).
As a rule of thumb, fill volumes in a shake flask should not
exceed 20 % for microbial applications. In case of problems
with oxygen supply, one can further reduce the fill volume
to 10 or even 5 %. Some baffled designs, such as Thomson
Ultra Yield®, may allow also higher fill volumes under certain
conditions [28]. Again, when using higher fill volumes in
combination with agitation and/or baffles, care must be taken
to avoid cap wetting.
Flask size can also influence pDNA yields. A smaller flask
will give higher maximum oxygen transfer rates compared to
larger flasks (when similar conditions, same fill and agitation
speed are applied) due to the relative volumetric surface area
increase. Oxygen transfer rate (OTR) measurements with
2.5 L and 250 mL Ultra Yield flasks have been described [27].
Hence, when scaling up from small to large flasks, conditions
may need optimization, such as agitation speed or fill volume
to get similar yields per L.
Influence of temperature
The standard cultivation temperature for E. coli is 37 °C.
However, for some plasmids the copy number can be
increased by a temperature up-shift to 42 °C. Temperature
sensitivity has been described for high copy number pUC
plasmids and explained with the existence of certain mutations [29]. This strategy of induced amplification is used
primarily in fermenters. Typically, fermentation starts with a
lower temperature at 30°C, at which the plasmid is maintained stably at low levels while biomass is accumulating.
When cell density has reached a certain level, the plasmid
amplification is induced by shifting the temperature to 42°C
and by continued exponential nutrient feeding for a defined
time [29]. It has also been reported that plasmid yield and
quality can be improved by holding fermentation cells at a
reduced temperature post the 42°C induction phase [30].
Excursus III: Influence of temperature on protein expression
A cultivation at reduced temperatures may reduce the
problem of protein misfolding and aggregation and can
be one factor to optimize when working with difficult to
express proteins.
Influence of media composition
The media should support cell growth as well as plasmid
amplification and stability while minimizing other cell
activities. Other than in mammalian culture, the media
costs are relatively low due to the mostly simple formulations. One distinguishes minimal (defined) media and
complex (semi-defined) media. In minimal media the exact
chemical composition is known, whereas complex media
contains components of unknown chemical composition
and varying proportions, such as yeast extract or peptones
for example. Minimal media allows for highly reproducible
processes [31]. Complex media allows for high cell densities but may give more varying results and might interfere
in downstream processing in industrial settings [32]. Formulations based on Lysogeny Broth (LB) or Terrific Broth
(TB) are frequently used complex media.
Bacteria need as minimum set of nutrients to grow: water, a carbon source, a nitrogen source, and some mineral
salts. However, typical bacterial complex components are
protein hydrolysates like peptone or tryptone, yeast extract,
carbohydrates, nitrogen, trace metals, and minerals.
Glucose is the most common carbon source added, as it
is inexpensive and can be efficiently metabolized by E. coli,
leading to good biomass and specific plasmid yields [33].
However, it might favor the undesirable accumulation of
acidic by-products, such as acetate, due to metabolic overflows. Acetate accumulation is a major concern in aerobic
fermentation known to inhibit cell growth and protein
expression [34]. However, the effect of acetate on plasmid
DNA production has not been investigated much. One study
states that acetate formation can improve pDNA yields
when present at low concentrations (3 g/L) [35]. Reduced
growth rates have been linked to elevated copy numbers
before [36]. Glycerol can be chosen as an alternative or
complementary carbon source. It increases specific plasmid
WHITE PAPER I No. 89 I Page 7
productivity but may lower cell growth compared to Glucose
[36]. The carbon sources of LB media are metabolizable
amino acids in the form of tryptone and yeast extract with no
additional carbon source added. Beside a higher amount of
yeast extract and glycerol, TB media formulations contain an
additional carbon source, which may increase pDNA yields
compared to classic LB formulations (Figure 10) [27]. LB
medium is excellent for routine molecular biology applications, but bacteria growth is limited due to the presence of
the small amounts of utilizable carbon sources [37].
7.1
34.8
0
10
20
30
40
50
LB media Modified TB media
pDNA yield [mg/L]
Figure 10: Influence of media on pDNA yields. The TB media formulation
resulted in higher pDNA yields compared to LB media. Cultivation of E. coli
DH5α with pUC19 plasmid in 2.5 L Ultra Yield® flasks in either LB or modified
TB media (20 % fill, at 37 °C and 250 rpm).
Nitrogen is important to produce proteins and nucleic acids
and can be either supplemented from organic sources, such
as yeast extract or hydrolysates like tryptone/peptone or from
inorganic supplemented nitrate.
Beside the availability of nutrients, the pH of the medium
affects the culture. The optimal pH range for E. coli is between
6.5 and 7.5, depending on temperature [38]. During growth, the
pH is drifting as consequence of production of substrates and
metabolic compounds. A decrease of pH occurs for example by
acetate accumulation due to metabolic overflow when cultivating with glucose as a carbon source. However, after glucose
depletion consumption of acetate by the bacterial cells causes
the pH to increase again [39]. Hence, pH control is important,
especially when the medium contains additional carbon sources
like glucose. Phosphates are commonly used buffer systems.
Alternative buffer solutions are zwitterionic buffers like MOPS
[40]. An overview of commonly used media with a short description of their properties can be found in online sources of
suppliers [41]
There are also commercially available ready-to-use media
optimized for plasmid production that support high-density
growth and high plasmid yields (Table 2) [42].
Table 2: Plasmid+® Media yields higher plasmid concentrations over longer
time periods compared to other complex media. Shown are the plasmid yields of
Plasmid+ versus commonly used TB medium cultures in 2.5 L Ultra Yield flasks.
Plasmid concentrations are given in ng/µL. Yield differences between Plasmid+
and TB medium cultures are given in percent. Experiments were done at 310 rpm
and 37 °C (Source: Thomson)
Yield [ng/µL] % increase vs TB
Incubation time [h] 20 hr 22 hr 24 hr 20 hr 22 hr 24 hr
Plasmid+,
at 310 rpm 94.9 103.3 142.6 190.2 274.3 354.1
Terrific Broth,
at 310 rpm 32.7 27.6 31.4 – – –
Traceability and source documentation for media components, such as batch certificates of analysis, are especially
important in regulated areas. The trend in the industry towards eliminating the use of any animal-derived components
or even completely chemically defined medium to alleviate
potential regulatory concerns has been described [43].
Optimizing fermenter culture
A bioreactor or fermenter in microbial settings is a vessel that provides a biological growth environment for the
(micro)organism of choice and is usually used together with
a bioprocess control station and software to monitor, adjust
and maintain this environment (Figure 11).
Figure 11: Composition of a bioreactor. Components of a stirred tank‐bioreactor
(left) and a connected control station (right).
a: bioprocess control software
b: motor
c: pump
d: head plate
e: sensor
f: bioreactor
g: bioprocess control station
h: impeller
a
b
c d
e
f g
h
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The biggest advantage of bioreactors is a stable and adjustable growth environment due to precise control of various incubation parameters, such as temperature, agitation speed, gas
supply and pH. Furthermore, it offers the possibility to scale up
the bioprocess which enables reaching increased cell numbers
and plasmid yields [44].
However, also bioreactors offer certain challenges that
can influence process efficiency. This section aims towards
overcoming them and giving insights into the most common
considerations for the use of bioreactors. For this, we will look
at the example of the stirred-tank bioreactor type which utilizes
a motorized impeller to swirl the bacterial cell suspension to
support gas exchange, nutrient distribution, and culture homogeneity (Figure 12).
The topics of this section include the method or mode of
fermentation, considerations concerning culture conditions and
equipment, as well as bioprocess scale-up.
Fermentation mode
When starting fermentation in a bioreactor, one has the choice
between three commonly used fermentation modes, namely
batch, fed-batch and continuous fermentation.
In batch fermentation, inoculation is carried out in a fixed
volume without the addition, removal, or exchange of medium
or feeding solution. In the process of nutrient consumption,
the culture will undergo the typical cellular growth phases:
first a lag phase in which the microorganisms adapt to the
new environment, then an exponential phase marked by rapid
cell division and cell density increase, followed by a stationary
phase where growth is halted due to nutrient limitations and
lastly a death phase in which the cells start to die [45]. Batch
fermentation is easy to set up and bears a low contamination
risk. However, the microorganisms experience a constantly
changing environment due to the ongoing nutrient depletion
and accumulation of potentially toxic by-products within the
medium [45]. This might affect the growth of the culture resulting in lower cell density and ultimately decrease plasmid yields.
Therefore, fed-batch fermentation is an advancement
of the batch mode to overcome its disadvantages. Here,
nutrients are replenished during the incubation creating a
more stable environment for the cells. Usually, the feeding
is initiated during the mid-exponential growth phase (OD600
of 8-12) to prevent nutrient deprivation [46]. Thus, fed-batch
not only enables higher cell density and biomass production
than batch fermentation, but it also allows for longer cultivation periods and limits by-product accumulation [45, 47].
Consequently, higher plasmid yields were observed for fedbatch over batch fermentation [48, 49]. However, the more
complex structure of the fed-batch process also increases
the risk of contamination [45]. Thus, care needs to be taken
when preparing and adding the required feeding solutions.
The last fermentation method we look at, continuous
fermentation, is the most complex operation mode. However,
once it is ideally adjusted long cultivation of days, weeks and
even months are possible. This main advantage is achieved
by continuous removal of used medium and cells while fresh
medium and nutrients are added simultaneously [45]. The
resulting steady state ideally keeps the volume and nutrient
concentrations constant while simultaneously removing toxic
by-products, thus creating the most stable cell environment
yet. Furthermore, the long run times reduce process costs
and downtimes compared to batch and fed-batch, for example, due to fewer cleaning procedures [45, 50]. However, the
greatest strength of continuous fermentation also causes a
lot of its weaknesses. Long-term incubation makes maintaining sterile and stable growth conditions more challenging. As
a result, product yields are usually lower than for batch and
fed-batch fermentation which increases downstream costs
[50].
Therefore, if long-term production of plasmids in a bioreactor is not your main concern, fed-batch fermentation is
recommended as the best synthesis in terms of productivity,
downtime, and contamination risk. Table 3 summarizes the
advantages and disadvantages of each fermentation mode.
Batch Fed-Batch Continuous
Figure 12: Bioreactor operation modes. Batch cultivation carried out in a fixed volume, fed‐batch with medium and nutrient addition after the initial lag‐phase until the
end of the process, and continuous cultivation with constant medium/nutrient replenishment to ensure a stable growth environment.
WHITE PAPER I No. 89 I Page 9
MODE PROS CONS
Batch > Ease of use
> Low contamination risk
> Constantly changing environment
> By-product accumulation
> Low cell density
> High downtime for cleaning due to short run-time
Fed-batch > Longer cultivation time compared to fermentation and thus less
downtime
> More stable environment than batch fermentation due to feeding
> Higher cell density possible compared to batch fermentation
> Consequently, higher plasmid yield than batch fermentation
> By-product accumulation limited
> More complex structure than batch fermentation
> Higher contamination risk than batch fermentation due to
feeding
Continuous > Longest possible cultivation period (weeks to months)
> Ideally a constant environment
> Growth rate constant
> Possibility to remove by-products and prevent accumulation in
the culture
> Strongly reduced downtime
> Most complex process structure
> Highest contamination risk of all operation modes
> Compared to fed-batch lower productivity (cell density, plasmid yield)
Table 3: Pros and cons of common fermentation modes
Culture conditions and equipment
Once the fermentation mode is selected, the culture needs
to be maintained. Many parameters impact the efficiency of
cultivation and most of them can be precisely controlled in a
bioreactor.
The first and foremost is the optimal growing temperature
which is 37°C for most bacteria. As opposed to shake flasks
that need to be transferred to an incubator, the temperature
of a bioreactor can be set, controlled, and automatically adjusted in place [51]. This is achieved by temperature probes,
as well as heating blankets, thermowells, water jackets, and
cooling fingers which can provide heat to or remove heat
from the medium.
Another factor important for bacterial growth and plasmid
yield is the pH value. Of course, the optimal value varies
from microorganism to microorganism. However, in most
cases, Escherichia coli is used for plasmid production. Here,
we will look exemplarily at E. coli DH5α, one of the most
commonly used strains for plasmid production. Studies in
tubes and shake flasks identified its optimal pH range for
plasmid production to be between 6.7 and 7.5, depending
on the plasmid and the medium type [52, 53]. However, it is
worth mentioning that the pH changes over the course of nutrient consumption and growth. Studies showed that the pH
usually drops over the course of incubation [53,54]. As pH
shifts can impact the resulting plasmid yields, it is desirable
to monitor and adjust the pH accordingly, something that is
more difficult to achieve in shake flask cultures. Bioreactors
on the other hand offer precise and continuous pH measurements through sensors with the option of automatic adjustments by adding either acid or base solutions to the medium
[51].
Ensuring efficient mixing and aeration of the culture is
also critical for the bioprocess. Therefore, agitation speed
and the right impeller type need to be selected for optimal culture growth. Both parameters are usually a tradeoff
between sufficient aeration, mixing, and prevention of shear
stress on the cells. However, as most bacteria have a relatively low shear sensitivity, a commonly used Rushton or
Rushton-like impeller (Figure 13) with high agitation speed
can be recommended for bacterial fermentation [55].
Figure 13: Rushton-type impeller used for bacterial bioreactor cultures with
low shear-sensitivity.
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If the impeller is not sufficient to aerate the culture
efficiently, additional gassing can help. Oxygenation is
reached by addition of O2
, while anaerobic conditions can be
achieved by gassing with nitrogen or other anaerobic gasses
[51]. Gas supply can be provided by surface gassing as well
as submerged dip or sparging tubes which directly transfer
the gas into the medium, thereby increasing the surface for
gas exchange by introducing bubbles [51].
However, introducing bubbles combined with agitation
and the protein composition of the medium can facilitate
foam formation [56]. Amongst others, foam formation in
bioreactors is associated with reduced working volumes,
loss of medium and cells, as well as increased contamination
risk [57]. Thus, it is important to detect and control foam.
Detection of foam is usually achieved by using a probe
located in the bioreactor’s top part. It measures the electric
resistance, that is changing once the foam is reaching the
probe [56]. It can also be used to release surface-active
antifoam substances. However, these newly introduced
components within the medium might change the microbe’s
metabolism, reduce oxygen transfer, or impact downstream
processes and upscaling [56]. Therefore, testing the antifoam
with the target cell and medium of choice beforehand or
using mechanical antifoam solutions is recommendable.
Furthermore, to reduce foam formation in the first place,
agitation and gassing should be balanced with the culture’s
aeration needs.
Once the culture conditions are set, further steps
to support and monitor optimal culture growth can be
undertaken. One of them is shortening the lag-phase after
inoculating the main culture. This is achieved by (i) the right
growth phase indicated by the optical density at 600 nm
(OD600) within the pre-culture, (ii) the correct initial OD600
in the main culture after inoculation with the pre-culture
and (iii) the right volumetric amount of bacterial pre-culture
within the main culture. According to the literature, E. coli
inoculation pre-cultures from shake flasks are grown to
OD600 of 6-15 [58, 59]. As mentioned earlier, these values
are within the range of exponential growth and ensure
actively expanding bacteria [46]. When inoculating the main
culture within the bioreactor, the pre-culture gets diluted to
initial OD600 between 0.04 and 0.4, as described for E. coli
in various settings [58, 59, 60, 61, 62, 63]. At this point, it
is also important to keep the inoculation volume relatively
high. 0.1-10 % of the total working volume is described in
the literature but the ideal ratio should be determined by
testing [45, 46, 58, 64]. After preparing the main culture
this way, regular OD600 measurements should be carried
out during cultivation, for example, to identify the right
feeding or termination time points. Sample-taking for OD600
measurement can be performed by special sampling valves
within the bioreactor and even non-invasive options exist to
reduce the contamination risk even more. These tips should
enable an efficient start for bioreactor runs for small-scale
and scale-up processes.
Scale-up
Scale-up is the process of increasing the fermentation size
from a small scale consisting of several milliliters to several
liters to an industrial production scale with thousands or
even millions of liters [65]. In principle, the same parameters
important for small-scale approaches also apply to largescale, such as consistent temperature, pressure, dissolved
oxygen set point, or feeding solution sterilization [64].
However, scaling up is not a one-step process of simply
enlarging bioreactors and volumes. Many parameters are
influenced simultaneously during scale-up which can result
in inhomogeneous culture conditions.
Larger bioreactors with higher volumes lead to longer
mixing times with the risk of impaired oxygen and substrate
distribution. Additionally, the stronger hydraulic pressure
can influence the oxygen transfer rate, a critical parameter
for air-to-liquid oxygen delivery. Such O2
and nutrient
gradients can induce metabolic shifts within the cells,
resulting in the accumulation of unwanted by-products, such
as lactate or succinate which in turn affect the medium’s pH
value [66]. Furthermore, high-density microbial cultures can
produce significant amounts of heat which combined with
inefficient mixing can lead to zonal overheating and further
stress on the cells [64,66].
Thus, instead of moving immediately from small volumes
to the endpoint, intermediate steps are introduced before
the production scale is reached (Figure 14). Small and
bench scale with volumes up to about 10 L are translated
into so-called pilot scales with 1-10% of the full production
scale volume [65]. Once sufficient yields are achieved in the
pilot scale, translation into the manufacturing scale can be
initiated.
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Figure 14: Illustration of a scale-up process in bioreactors. In the process of translating small (left) into production scale (right), intermediate steps, such as bench scale
and pilot scale approaches, are introduced to ensure consistent bioprocess performance between the different bioreactor dimensions.
During this process it is crucial to ensure reproducibility
between the different scales. To this end, different scale-up
parameters were defined over time, all with the goal of overall
consistent culture conditions for the different scales. Such parameters include constant agitation power input per volume,
oxygen transfer rate and oxygen transfer coefficient (kLa),
heat transfer rate per volume, mixing time, impeller tip speed,
gas volumetric flow rate, and gas superficial velocity, to name
a few [64, 67].
However, due to the bioreactor’s physical properties it is
not possible to keep all the parameters constant at the same
time, as they can influence one another. Therefore, a selection of these parameters should be tested over all scale sizes.
Out of the above, constant agitation power input per volume,
constant oxygen transfer rate and constant heat transfer rate
per volume are among the commonly used ones in large-scale
microbial fermentation [64].
The agitation power consumption of an impeller is influenced by the agitation speed (rpm), the medium’s viscosity
(kg/m3
), the impeller’s outer diameter (m), and a dimensionless power number [64]. The power or Newton number is
experimentally determined for a given impeller by measuring
its torque but can also be obtained from standard impeller
references or vendors [64,67,68]. Once all these variables are
obtained, the final power consumption per volume given in
W/m3
can be calculated by a specific mathematical formula
[64, 68].
The oxygen transfer rate (OTR), given in mmol O2
/(L x h),
is a measure of the gas-to-liquid transfer efficiency of oxygen
during gassing. It is influenced by the agitation speed, airflow
rate, pressure, and pure oxygen supply [64]. Just as the power
consumption per volume, the OTR requires experimental
determination as well. Different methods exist, for example
the sulfite depletion method. Here, the oxygen-driven reaction
of Na2
SO3
to Na2
SO4
is used to determine how fast oxygen is
replenished after it was absorbed by sulfite within the medium
[69]. With these results the oxygen transfer rate for a specific
bioreactor can be calculated. Furthermore, oxygen transfer
rates can be obtained from equipment manufacturers. However, as these values often represent the possible maximum
capability of a bioreactor it should be considered to perform
own experiments for a given setup [69].
As mentioned already, high-density cell cultures can produce large amounts of heat which make the heat transfer rate
an important factor for keeping temperatures controllable. It is
influenced by the area covered by the cooling device, as well
as the culture’s and cooling agent’s temperature [64].
Besides parameters directly linked to the bioreactor, also
other things need to be considered when planning a scale-up
process. Deep freeze cell banks for storing backups of bacterial strains as well as the necessity of seed trains or bioreactor
plant stress tests need to be considered [65, 64].
As shown by these examples, scale-up processes are complex and lengthy. Transitioning from lab to production scale
WHITE PAPER I No. 89 I Page 12
can take several years [65]. Nevertheless, it is important not
to rush it. The scale-up should be planned out with the final
goal in mind and all your actions should be positioned towards
this goal. A detailed road map should be created beforehand,
containing considerations like timelines, costs, unit operation design, energy-balance, risk assessment and mitigation
plans [65]. Especially during this early stage of planning, input
from experts with scale-up experience and inclusion of such
expertise within your team are valuable [65, 64]. Once the
foundation is laid, the initial ideas should be regularly adapted
according to small, bench and pilot scale experimental results.
Furthermore, the smaller scale approaches should always be
designed in a way to represent the larger scales as closely as
possible to make the larger scales as reproducible as possible
[65].
In summary, there is no one-fits-all solution for upscaling.
Many parameters need to be evaluated for your individual
approach. However, this extra work will pay by receiving consistent yields from benchtop to production scale.
4) Culture harvesting
Small scale cultures from shake flasks or small fermenters
are typically harvested by centrifugation. A benchtop multipurpose centrifuge with swing-bucket rotor is typically used
for harvesting from conical tubes or conical bioreactors. A
typical E. coli pelleting run is performed at around 5,000 g for
~10–20 min at room temperature or 4 °C. If higher g-forces
are required to reduce the harvesting time or for harvesting
larger volumes, fixed-angle rotors are the appropriate choice.
The available purification system protocol usually gives a
good guideline on the centrifugation settings. If the recommended centrifugation time or speed is exceeded, the cell
pellet may be more difficult to resuspend. On the other hand,
insufficient centrifugation time or speed may result in incomplete pelleting of cells and loss of starting material. After pelleting the cells and removal of the supernatant, the cells can
be stored at -20°C for subsequent plasmid isolation.
Larger industrial-scale fermenter cultures are typically harvested by either centrifugation or by tangential flow filtration
(TFF) [70]. Besides conventional centrifuges, other types such
as continuous-feed centrifuges can be used for the harvesting
step [71].
5) Purification
The process of DNA extraction and purification consists of
five basic steps: 1) cell lysis, the disruption of the cellular
structure to create a lysate 2) separation of the soluble DNA
from cell debris and other insoluble material, 3) binding of
desired the DNA to a purification matrix, 4) washing step to
remove cellular contaminants from the matrix, 5) elution of
the DNA.
The primary consideration for plasmid purification is
separation of plasmid DNA from the host bacterium’s chromosomal DNA and cellular RNA. Besides these components,
other contaminants such as proteins, salts, and endotoxins
are removed as well. Furthermore, the process aims towards
eliminating undesired plasmid isoforms in the final product,
as plasmids may exist in supercoiled, open circular and linear
configuration, resulting from conformational changes within
the bacterial host cell, or during the purification process [72].
However, the supercoiled structure is considered the native
and active plasmid form and mostly the desired conformation.
To generate a cleared lysate with free plasmid DNA,
several methods have been developed, including SDSalkaline denaturation [73,74], salt-SDS precipitation [75],
or rapid boiling [76]. SDS-alkaline denaturation is a popular
procedure for purifying plasmid DNA because of its overall
versatility and consistency. This technique exploits the difference in denaturation and renaturation characteristics of
covalently closed circular plasmid DNA and chromosomal
DNA fragments. Under alkaline conditions (pH 11.5-12.5)
[77], both plasmid and chromosomal DNA are efficiently
denatured. Rapid neutralization with a high-salt buffer, such
as potassium acetate, in the presence of SDS has two effects
that contribute to the overall effectiveness of the method.
First, the rapid neutralization causes the chromosomal DNA
to base-pair in an intrastrand manner, forming an insoluble
aggregate that precipitates out of solution. The covalently
closed nature of the circular plasmid DNA promotes interstrand rehybridization, allowing the plasmid to remain in
solution. Second, the potassium salt of SDS is insoluble, so
the proteins and detergent precipitate and aggregate, which
assists in the entrapment of the high-molecular-weight chromosomal DNA.
Accurate execution of SDS-Alkaline lysis is critical for sufficient plasmid yields. First, the lysis pH of ~11.5-12.5 is close
to the denaturation pH of pDNA. Thus, small pH deviations
can favor the formation of undesired circular, single stranded
plasmid DNA isoforms. Lysis time should be long enough for
complete cell lysis but short enough to not denature pDNA.
Second, aggressive mixing may lead to degrading of pDNA
due to excessive shear forces, while insufficient mixing may
lead to incomplete cell lysis. Also, ensuring complete neutralization of the solution during the neutralization step is
critical. For research scale kit-based purification, adhere to
the given times in the supplier’s protocol.
WHITE PAPER I No. 89 I Page 13
The separation of soluble and insoluble material is accomplished by a clearing method (e.g. filtration, magnetic clearing, or centrifugation). The soluble plasmid DNA is ready to
be further purified.
There are several methods available to purify plasmid DNA
from the cleared lysate, including:
> binding plasmid to silica in the presence of high concentrations of chaotropic salts [78,79,80]
> differential precipitation of plasmid DNA from aqueous
chaotropic salt/ethanol solutions [81,82,83]
> ion exchange chromatography over DEAE-modified cellulose membranes [84]
> precipitation with polyethylene glycol (85,86) organic
extraction using phenol [87]
These methods can be adapted to various scales, from
centrifugation-based minipreps to large-scale automated and
semi-automated methods.
The volume of the bacterial culture should match the
distinct isolation system. For research scales, spin column
based mini, midi or maxi preps provide fast and easy purification usually together usually with a fixed-angle microcentrifuge. Typical volume ranges and yields from Promega [88]
plasmid preparation kits are listed (Table 4). Some suppliers
provide Mega and Giga preps, that allow purification from
even larger volumes up to 5 L with yields up to 10 mg [16].
Table 4: Typical volume ranges and yields with Promega plasmid purification
kits for laboratory scale purifications (Source: Promega). Please note that kits
from other suppliers can vary.
Culture size Sample
volume
Isolation
system
Average pDNA yield
high copy plasmid
Small 0.6-3 mL Miniprep 1.5–15.0 μg
Medium 50–100 mL Midiprep 100–200 µg
Large 250 mL Maxiprep Up to 1 mg
For low volume, high-throughput processing, isolation
systems based on a 96-well format are available based on
vacuum or magnetic beads.
Methods used at small scale are not readily scalable for
large scale. For large-scale pDNA production, the steps after
alkaline lysis are followed typically by different filtration
techniques or centrifugation with a final chromatography to
separate pDNA from residual impurities [72].
The biomass should be kept in a range that is acceptable
for the plasmid isolation system used. Most research scale
spin column based purification systems are optimized for
use with LB media [88,89]. Using enriched media like TB
that leads to higher biomasses per liter, that may cause an
overloading of the purification system and insufficient lysis
[16,88]. The culture volume per column should be reduced
in these cases to match the recommended biomass. That
also applies to extremely high copy plasmids and host
strains that show high growth rates. Furthermore, the
potential higher viscosity of the lysate will require more
vigorous mixing, which may result in shearing of bacterial
genomic DNA and contamination of the plasmid DNA [16].
For very high-sized plasmids (> 20,000 bps) a reduced efficiency in elution from the column is noted, the limitation in size
should be checked in the manual of the used supplier [16,88].
pDNA concentration in a laboratory scale is typically determined by measuring the absorbance at 260 nm (A260) in
a spectrophotometer, for example using a quartz cuvette.
Standard spectrometers allow measurement of the A260/230
and the A260/280 ratios to provide valuable information on the
plasmid DNA’s purity.
1) Determination of the purity ratios A260/A280 reveal nucleic
acid sample quality and possible contamination with protein,
phenols or other aromatic compounds. A ratio of 1.8-2.0
indicates sufficient pDNA quality [89]
2) The A260/A230 ratio reveals nucleic acid sample quality
and possible contamination with organic compounds. This
ratio should be > 2.0 [89].
To check the quality and presence of plasmid isoforms in the
eluate, agarose gel electrophoresis can be performed to separate the plasmids according to their conformation.
Appendix
Methods are intended for research applications. They are not
intended, verified or validated for use in the diagnosis of disease or other human health conditions.
WHITE PAPER I No. 89 I Page 14 I No. 8x I Page 2
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