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Eppendorf – In touch with life®
Scalable Cell Culture Using Rigid-Wall, Single-Use Bioreactors Eppendorf BioBLU
®Single-Use Vessels
analytica, Dr. Christof Knocke, May 10, 2016
The Eppendorf Competence Areas
3Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
Sam
ple H
an
dlin
gC
ell H
an
dlin
gLiq
uid
H
an
dlin
g
Manual
pipettes
Electronic
pipettes
Pipette tips DispensersAutomated
pipetting systems
CO2
incubators Biological
shakers
Cell culture
and imaging
consumables
Photometers
and Spectrometers
Micromanipulators
and injectors
ElectroporatorsFermentors and
bioreactors
Centrifuges and
vacuum
concentrators
Ultra-low
temperature
freezers
Rotors and
accessoires
Mixers and
temperature
control systems
Thermal cyclers Test tubes
Single-use
bioreactors
CombitipsPipette tips Pipette
controllers
Multiwell plates
The Eppendorf Experience
History of Bioprocessing at Eppendorf
4
Premium partner in life sciences
Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
70 years of experience in the life science market
1946 2007 2012 20161991
Foundation of
Eppendorf-
Netheler-Hinz
GmbH
Foundation of
DASGIP® GmbH
1945
Foundation of
New
Brunswick™
Scientific Co
Eppendorf
Bioprocess
Acquisition of
New Brunswick
Scientific Co
Acquisition of
DASGIP GmbH
Our 3S Value Proposition
Scalable Systems
5Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
Scalable systems
Comprehensive
Eppendorf bioprocess
systems cover a wide range
of working volumes in
autoclavable, single-use and
SIP.
Single-use options
Ready-to-use
Ready-to-use single-use
bioreactors of different
volumes complement the
scale range.
Software solutions
Innovative
Comprehensive software
packages offer innovative
solutions for advanced
process control and
information management.
Scalable Systems
Scalable Systems
6Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
Bioprocessing needs scalable systems
> Process optimization (feeding, gassing,…) in the milliliter-scale
> Pilot/production scale in up to thousands of liters
> Scale-down model development to implement changes
The primary scope of this project was to investigate the scale-up
capabilities of Eppendorf BioBLU Single-Use Vessels for cell culture
Scale-Up Criteria
Bioprocess Scale-Up Criteria
Demands and objectives
8Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
Geometry
> Geometrical numbers
Performance
> Mixing
> Tip speed, shear stress
> Power input,
> Mixing times
> Gassing (kLa)
Eppendorf BioBLU® Single-Use Vessels
BioBLU® – Single-Use, but Rigid Wall: The Best of Both Worlds
> Inspired by industrial rigid-wall designs
> Typical stirred-tank (STR) dimensions
> Fast and efficient mixing
with magnetically coupled overhead drives
> USP Class VI certified and animal
component free material,
e.g. virgin monolayer polystyrene
Scalable and reproducible results
Single-use options
10Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
BioBLU® c – Concept
11Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
Effective exhaust
treatment, heat blanket
or liquid-free (Peltier)
Standard impeller sizes
and vessel dimensions
Overlay and sparge
gas options to best
suit your application
Magnetic drive assembly
Non-invasive sensors
BioBLU c Family
Weldable or easy to
connect tubing
Full featured highly
integrated headplate
BioBLU® c – The Widest Range of Rigid-Wall, Single-Use STRs
Single-use options
12Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
BioBLU 0.3c BioBLU 1c BioBLU 3c BioBLU 50c
Working volume 100 – 250 mL 320 mL – 1.25 L 1.25 – 3.75 L 18 – 40 L
Stirring 20 – 500 rpm 20 – 500 rpm 25 – 200 rpm 25 – 150 rpm
System/controller DASbox® BioFlo® 320, DASGIP®
BioFlo,DASGIP,
adaptor kits
BioFlo/CelliGen®,
adaptor kits
R&D and Scale DownProduct/Process
Development
Process
DevelopmentProductionPilot Scale
Geometry
Vessel name 0.3c 1c 3c 50c
Working vol : total vol 0.66 0.75 0.75 0.8
Impeller diameter :
Vessel ID 0.49 0.47 0.45 0.47
Vmax
height / vessel ID 1.22 1.51 1.5 1.27
*: Vmax
height = height from bottom of the vessel to liquid top surface at maximum vessel volume.
13Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
Scalable Systems
Mixing
> Tip speed range
> Numerical CFD analysis
Tip Speed
Scalable Systems
15Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
Tip
speed
(m/s)
0.3c
(rpm)1c (rpm) 3c (rpm) 50c (rpm)
0.3 174 115 87 36
0.4 231 153 116 48
0.5 290 191 145 60
0.6 347 229 174 72
0.7 405 267 203 84
> Tip speed as criteria for the
comparison of vessels
> Scale-up based on maintaining a
constant tip speed is common
approach for shear sensitive cells
> Range of available speeds by the
controllers limits the considered
tip speeds in the range of
0,3 – 0,7 m/s
Scientific Cooperation
Numerical investigation on the Eppendorf
BioBLU 0.3c, 1c, 3c, and 50c Single-Use Vessels
for cell culture
Scalable Systems
16Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
ZHAW Zurich University of Applied
Sciences
Department Life Sciences und Facility Management
Institute of Biotechnology, Biochemical Engineering
and Cell Cultivation Technique, Wädenswil
Scientists:
> Prof. Dr. Dieter Eibl
> Cedric Schirmer
> Valentin Jossen
CFD Modeling
Scalable Systems
BioBLU 50c mesh. Unstructured hexahedron volume mesh (A), and stirrer surface mesh (B) of the BioBLU 50c. The darker domain around the stirrer shows the multiple
reference frame zone (MRF) for the implementation of the stirrer rotation. For the investigation of the acting forces on the stirrer blades, a regular finely woven mesh
was used at the stirrer regions.
17Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
> Computational fluid dynamic
investigations to predict power
numbers and shear gradients
CFD Fluid Pattern
Simulation at a tip speed of 0,5m/s, maximum working volume
Scalable Systems
Steady state fluid flow pattern of the BioBLU 0.3c, 1c, 3c and 50c cell culture systems. Steady state fluid flow pattern of the BioBLU 0.3c , 1c, 3c and 50c at a stirrer
speed of 0.5 m/s at y-z-planes. The velocities are scaled to 90 % of the theoretical tip speeds.
18Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
0.3c 1c 3c 50c
Power Input
Scalable Systems
Power inputs of the BioBLU cell culture systems. Power inputs against tip speeds.
19Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
> Computational fluid dynamic
investigations to predict power
numbers and shear gradients
> Identified scalable range of power
inputs in function of tip speeds
Shear Gradients
Scalable Systems
20Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
> The mean, local shear gradients
as a function of impeller number,
agitation speed, and agitation
direction are below a critical
range
> Shear gradients of 500 s-1
to
5000 s-1
are considered to be
harmful for cell cultures (Yim and
Shamlou, 2000)
kLa Values
Scalable Systems
BioBLU Single-Use Vessel kLa-values determined with the static gassing out method
21Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
> kLa-based scale-up can be performed in the 2.0 – 6.0 h
-1range
Case Studies
> Case Study I: CHO cell culture 5L, 50L
> Case Study II: CHO cell culture and protein
production 250 mL, 3.75 L
Case Study I
Case Study I
23Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
Application note AA257; Nick Kohlstrom, Joseph Capone, and Ma Sha, Eppendorf Inc., Enfield, CT, U.S.A.
> Scale-up of CHO cell culture using
New Brunswick™ CelliGen BLU
stirred-tank bioreactors equipped
with 5 L and 50 L single-use
vessels.
Case Study II
Scale‐up of SEAP production with
CHO XM 111‐10 suspension cells
using the BioBLU 0.3c and BioBLU 5c
single‐use bioreactors
Case Study II
24Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
ZHAW Zurich University of Applied
Sciences
Department Life Sciences und Facility Management
Institute of Biotechnology, Biochemical Engineering
and Cell Cultivation Technique, Wädenswil
Scientists:
> Prof. Dr. Dieter Eibl
> Katharina Blaschczok
Case Study II – Setup
SEAP (secreted alkaline phosphatase)
expressing CHO suspension cell line
CHO XM
Scale-up criteria
> Equal mixing time
> Equal sufficient oxygen mass transfer
Case Study II
25Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
Schematic presentation of the BioBLU 0.3c bioreactor and feeding line setup.
Case Study II – Cell Growth and Protein Production
Case Study II
26Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
Production volume: 250 mL Production volume: 3.75 L
Case Study II – Glucose and Lactate Profiles
Case Study II
27Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
Production volume: 250 mL Production volume: 3.75 L
Summary
28Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016
> Eppendorf BioBLU c Single-Use Vessels of different sizes are
geometrically and proportionally similar
> Broad overlapping tip speed zone and kLa values for excellent
scalability
> Offer a low-shear environment
> Case studies demonstrated the suitability of Eppendorf BioBLU c
Single-Use Vessels for cell culture scale up
We Know BioprocessingContact: [email protected]
Disclaimer
Eppendorf®, the Eppendorf logo, In touch with life® and BioBLU® are registered trademarks of Eppendorf AG,
Germany. New Brunswick™ is a trademark of Eppendorf AG, Germany. BioFlo® and CelliGen® are registered
trademarks of Eppendorf, Inc., USA. DASGIP® and DASbox® are registered trademarks of DASGIP Information
and Process Technology GmbH, Germany. All rights reserved, including graphics and photos.
Copyright © 2016 by Eppendorf AG.
Eppendorf AG | Cell culture scale up in BioBLU Single-Use Vessels | Knocke, Christof | May 2016 30
