The document describes HexaScreen® HexaBatch, a miniaturized bioreactor system for animal cell culture screening. It consists of a plate with 6 independent mini bioreactors and a workstation. Key features include: - Online monitoring of pH, dissolved oxygen, and cell density to obtain kinetic data - Automated control of temperature, agitation, and aeration - Reduced time and costs for screening compared to traditional methods like T-flasks or benchtop bioreactors due to parallel experiments and minimized manual labor - Applications include cell line selection, medium optimization, process development, and toxicity testing.

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HexaScreen®
Biotechnological Screening in Animal Cell Culture
Equipment to reduce required time &
costs for bioprocess development
EXPOQUIMIA 2011

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++
The Team
•Manufacturing
•Marketing & Sales
•Internationalization
•Cell Culture
•Bioreactors
• Measurement Systems
•Biocompatible materials
+

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Increasing importance of animal cell culture.

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The potential of animal cell culture arises from the capability of this type of
cells to carry out complex post-translational modifications providing proteins
with the required biological activity to be used for therapeutical and
diagnostic applications
CHO (Chineese Hamster Ovary)
NSO (mouse mieloma)
BHK (Baby Hamster Kidney)
HEK (Human Embryo Kidney)
PER.C6 (Human Retinal Cells)
MDCK (Madin Darby Canine Kidney)
Sf9 (insect cells, Spodoptera frugiperda)
Animal Cell Culture Bioprocesses

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Bioprocess Development: Animal Cell Culture
”The optimization of the bioprocess is specially important for animal cell culture”
– Less Cellular Concentration than Bacterium and Yeast:
• Bacterium and Yeast cultures: [X]f ≈ 109 cells/ml
• Animal cell cultures: [X]f ≈ 106 cells/ml
– More expensive & complex process:
• Culture media:
– Bacterium and Yeast cell culture: Simple undefined mediums
containing only salts (usually NaCl) and carbon & nitrogen sources
(usually within yeast extract and tryptone).
– Animal cell culture: Rich & complex mediums containing salts,
carbon & nitrogen sources, but also complements (AA, vitamins,
trace elements…) and serums (FCB, FBS…).
• Easier to be contaminated:
– Bacteria, yeast, mycoplasma or cross contamination.
– Specific challenges for animal cell culture: slow growth and specific
production rates, cell sensitivity (shear stress, nutrient limitation,
metabolite accumulation..).
If Ps=ct, we have 1000 times more product
with bacterium or yeast than with animal cells

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from F. Wurm. Nat. Biotechnol., vol. 22:1393-1398 (2004)
Bioprocess Development: Main Steps
Genetic Engineering Culture Conditions Operational Conditions
Best Clones:
Specific Productivity
Best Conditions:
[X]
Growth rate
Optimal Process:
[X]
Death times

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PROCESS
OPTIMIZATIONClonal
selection
Cell
line
Operation
strategy
Process
control
Purification
process
Scale-up
GMP
production
Culture
medium
Fisico-chemical
parameters
PROCESS
OPTIMIZATION
PROCESS
OPTIMIZATIONClonal
selection
Clonal
selection
Cell
line
Cell
line
Operation
strategy
Operation
strategy
Process
control
Process
control
Purification
process
Purification
process
Scale-up
GMP
production
GMP
production
Culture
medium
Culture
medium
Fisico-chemical
parameters
Fisico-chemical
parameters
Multiple aspects need to be optimized to establish optimal
and profitable production processes
High cell concentration, high specific productivity, maintain product quality

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Bioprocess Development: Phases
• PHASE I: Initial screening
1. Cell line selection (bacterium, yeast, animal cell…)
2. Cell modification (genetic engineering)
3. Clonal activity selection
Select which cells are producing the protein target
4. Protein target characterization:
Measurement of the protein activity
• PHASE II: Advanced screening
1. Clonal growth selection (select which cells grow faster…)
2. Culture medium composition: serum, glutamine, glucose…
3. Initial cell concentration.
4. Effects of stirring, [O2] and other culture conditions
5. Needs of the cell culture adaptation (ex: from adherent to suspension)
• PHASE III: Lab scale production
1. Scale-up the advanced screening optimum conditions
2. Purification process
3. Type of Bioreactor (Batch, Fed-Batch, Perfusion)
• PHASE IV: Pilot Plant and Industrial scale production
1. Scale-up the lab scale optimum production conditions
7/8
clones
1/2
clones
IMPORTANT!!!
Check the
activity of the
protein in all
phases

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– Reproducibility problems when scaling up from usual
screening phases into lab or pilot & production plant
scales due to:
• Homogeneity: Non agitated systems can produce cells or nutrient
accumulations giving not representative and impossible to repeat
experiments.
• [O2] limitations: Can produce a decrease of the cell culture growth
velocity, metabolic differences or stopping the cellular cycle leading to an
unreal productivity determination (higher or lower).
– Lack of probes: few knowledge of the main cellular
growth parameters : cell concentration, pH & pO2.
– Lack of automatization: human manipulation is highly
needed.
– Current screening agitated systems (spinner flasks)
characteristics include high volumes and post-
experiment treatments.
Screening Bioreactors: Differences
Cost and time
issues
Reproducibility
problems

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Vessel volume (ml)
0,1 1 10 100 1000+
T-flask
Spinner flask
Bioreactors
Multiwell plates
Advanced Screening
Usual scale-up procedure
New automated & controlled
screening platform
Initial Screening Lab ScaleInitial Screening
Screening : The HexaScreen Alternative

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Bioprocess Development: Phases
Bioprocess development for a biotechnology product
requires a number of steps
Scale-up Methodology: Laboratory to Pilot to
Industrial
Since Biotechnological Processes are not
completely known, the transition from bench to
final volume is done step by step.
Initial
Screening
Pilot Plant
Advanced
Screening
Production Plant
Lab scale
HEXABATCH

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– Simultaneous multiple experiment capability.
– Low minibioreactor volumes (10-15 mL), but not too low
to allow cell culture similarities with lab scale
bioreactors.
– Agitation requirement to allow system homogeneity, but
without upsetting cell viability.
– Parts in contact with cells must be manufactured with
single use biocompatible plastic (no contamination,…).
– Cell growth (optical density), pH and dissolved oxygen
monitoring via non invasive probes.
Screening: HexaBatch®Design Criteria

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HexaScreen®: HexaBatch Features
Initial/Advanced
Screening
HexaBatch
Pilot Plant
/ Industry
Equipment used &
environment
Stationary Culture
Systems: Heterogeneous
Stirred & O2 fed Culture
System to allow
homogeneity
Stirred Culture
Systems:
Homogeneous
Methodology Multiple experiments
Multiple experiments run
simultaneously to
decrease the time
required for the
screening phase
Unique cell
culture process
Vessel size Micro liters and milliliters
10-15 ml (bench top
scale)
From 2 to
thousands of
liters
Process control
Discontinuous and
manual
pH, pO2 and OD (cell
concentration) on-line
monitoring
Continuous
and monitored
Asepsis Difficult / uncontrolled
Disposable bioreactor
made of sterile
biocompatible plastic
material
Necessary

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– Single use Minibioreactors’ Plate. Previously sterilized
inside a plastic bag, includes 6 individual vessels equipped
with gas filters, one septum for inoculation, miniaturized
ports for probe’s allocation and a magnetic actuator for
stirring.
– Workstation. Contains the chamber where the
minibioreactors’s plate remains during its culture, while
maintaining optimal agitation & temperature (common),
sterility, providing individual aeration and acquiring pH, DO
and OD data. WorkStation is controlled via software.
HexaScreen®: HexaBatch Elements
HexaBatch version consists in two differentiated parts,
the 6-minibioreactors’ plate and the workstation with a
computer/software .

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HexaBatch: Minibioreactor plate characteristics
5
2
1
43
1-. Optical port for OD and pH measurements.
2-. Gas filters (inlet and outlet).
3-. Optical port for DO measurements via
fluorescence.
4-. Septum for cell inoculation.
5-. Low shear magnetic pendular
agitation (optimal for animal cell).
6-. Thermostated general bath.
7-. Vessel liquid volume: 10-15 ml.
8-. Biocompatible and disposable plastic,
sterile provided. Possible plasma treatment
to promote cell adherence / non-
adherence.
7 6
8

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HexaBatch: Minibioreactors Inoculation

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HexaBatch: Minibioreactors to Workstation

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HexaBatch: Variables, Controls & Monitoring
Variable Control Monitoring Information
Aeration Time Controlled No No
Agitation Set-point controlled No No
Vessel
Temperature
Set-point controlled Monitored System functionality
Gas & Filters
Temperature
Set-point controlled Monitored
Filters functionality –
Avoids evaporation
Optical Density Free evolution
Monitored and
correlated
Cell density information,
related to total cells
Dissolved
Oxygen
Free evolution
(constant aeration)
Monitored
Oxygen concentration,
related to alive cells
pH Free evolution Monitored
Cell activity information,
related to alive cells

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HexaBatch: Software Specifications
General specifications
• Automatic processes:
– Workstation’s configuration
– Thermal stabilization
– Oxygen calibration
– Optical calibration
– Data acquisition (minibioreactors’ behaviour monitoring graphs)
• Additional tasks:
– Create new or edit already existing experiment set-ups
– Create user defined graphs
– Create reports
– Export reports to EXCEL, PDF and HTML files
HexaScreen®’s control & acquisition program runs on a Windows
platform and will guide the user, as a wizard, through the processes of
workstation’s configuration, calibration and data acquisition.

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HexaBatch: Advantages & Benefits
Features Advantages Benefits
Specially designed for animal cell
culture.
- Stirring, gas exchange and oxygen supply are
specially designed for handling animal cell
culture.
- Focused.
- Suitable for both suspension and
adherent cultures.
Benchtop Scale:
Small Volumes from 10 to 15 ml”.
- Reducing working volume means less
development costs in medium, cell culture,
enzymes…
- Lower operation cost.
Parallel bioreactor system:
“6 multiple parallel experiments
for device”.
- Less time required to achieve final results
- Reproducible results: statistic data can be
obtained from replicated experiments.
- Faster time to market.
- Save time.
- Faster product development cycles.
Automated on-line
measurements.
- Real-time kinetic information:
cell concentration (OD), DO & pH.
- No off-line control processes required.
- No maintenance required during experiment.
- Precision & control over all
experimentation.
- Lower labour and time-consuming in
order to invest in other valuable
tasks.
Single use.
- No post-experiments treatments.
- Avoid possible cross-contaminations.
- Save money & time.
- Easier experiment validations.
Easy to use. - No expert personal required. - Less effort required.
Convenient. - Easier scale up to lab-size reactors. - Optimized culture parameters.
PC controlled.
- Save automatically the data acquired from all
the experiments done giving a comprehensive
documentation.
- Control on all acquisition
experiment data.
- Possibility to do final reports easier.

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HexaBatch: Applications & Cell Lines Cultured
Applications Examples
Cellular screening.
- Adherence and suspension lines.
- Clone selection.
Cellular characterization.
- Growth parameters measurements (cell
density and cell activity).
Cellular adaptation. - Adherent to suspension.
Medium definition and
optimization.
- Commercial medium comparison.
- Medium’s components definition.
Cellular tests.
- New drugs tests.
- Toxicity tests.
- Apoptosis tests.
Process optimization.
- Initial cellular concentration, culture
conditions…
Cell Lines Cultured
Suspension:
- Hybridoma.
- Genetically modified hybridoma.
- CHO cells (adapted).
- HEK (adapted)
Adherent:
- Vero cells.
- Ovine Mesenchymal Stem Cells.
- CHO.
- HEK.

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 Fast setup.
 No need for Cleaning.
 No risk of cross-contamination.
 Minimizes utility requirements.
 Minimizes validation.
 Minimizes space floor.
 Minimizes labor.
 Minimizes engineering design.
 Reduces COGS.
 Minimizes maintenance.
 Environmentally friendly:
 Use for generate electrical power by incineration.
 Estimated savings in WFI at over 80%.
 Estimated 72% saving in electricity compared to conventional
manufacturing facility.
Advantages of single-use technology

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Advantages of single-use technology

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• Systems to evaluate:
• HexaBatch
• 1L glass Bioreactor
• T-flasks for suspension cell cultures
• T-flasks for adherent cell cultures
Systems comparison for screening
On-line measurements
Off-line measurements

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• Case Study parameters:
• Number of conditions: 2
• Number of repetitions: 3
• Total number of cultures: 6
• Batch culture time: 3 days
• Cell line: CHO
• Culture media price (CDCHO): 103,52 €/L
• Qualified personnel costs: 30 €/hour
• It is assumed that there is only a one-liter bioreactor, so steps of the
experiment are performed sequentially.
• For off-line measurements (T-Flasks), only one sample is taken each
day, with a total of 3 cell concentration measurements during culture.
(no metabolites concentration measured)
HexaBatch Case Study: Conditions

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1
7
1 2
18
3
18
3
3
0
5
10
15
20
25
30
35
40
45
1 HexaBatch 1L Bioreactor T-flasks T-flasks (adherent cell)
Experimentaltime(days)
4 days
43 days
4 days
5 days
699 Total personnel cost (€)
Total bioreactor operation cost (€)
Total culture medium cost (€)
Fins a
5040
totals
Fin
19
tot
Inoculum Scale-up time
Bioreactor set-up time
(Set-up, sterilization, CIP, calibration, post-exp. cleaning)
Culture time
36 days
HexaBatch Case Study: Time Analysis

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HexaBatch Case Study: Costs Analysis
699
21
21
12
978
1800
0
500
1000
1500
2000
2500
1 HexaBatch 1L Bioreactor T-flasks T-flasks (adherent cell)
Costedeunexperimento(€)
699
151
12
22,5
150
219 74
Totalexperimentalcost(€)
Total personnel cost (€)
Total bioreactor operation cost (€)
Total culture medium cost (€)
Fins a
5040
totals
Fins a
1960
totals699
151
12
22,5
150
219 74
Totalexperimentalcost(€)
Total personnel cost (€)
Total bioreactor operation cost (€)
Total culture medium cost (€)
Fins a
5040
totals
Fins a
1960
totals
699 Total personnel cost (€)
Total bioreactor operation cost (€)
Total culture medium cost (€)
Fins a
5040
totals
Fi
1
to
Personnel costs
(staff hours x costs/hour)
Lab material costs
(Single-use plate costs + T-flask costs for scale-up)
Culture medium costs
183 €
2.520 €
161 €
507 €
699
151
12
22,5
150
219 74
Totalexperimentalcost(€)
Total personnel cost (€)
Total bioreactor operation cost (€)
Total culture medium cost (€)
Fins a
5040
totals
Fins a
1960
totals
699
151
12
22,5
150
219 74
Totalexperimentalcost(€)
Total personnel cost (€)
Total bioreactor operation cost (€)
Total culture medium cost (€)
Fins a
5040
totals
Fins a
1960
totals
Experimentcosts(€)

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HexaBatch Case Study: Manipulation Times
33 hours
45 min
60 hours
5 hours
0
10
20
30
40
50
60
1 HexaBatch 1L Bioreactor T-flasks T-flasks (adherent cell)
QualifiedPersonnelManipulationTime(hours)

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HexaBatch Results: On-line Optical Density
Total cells
, tdup
Final cell concentration
Off-line
Measurements
(T-FLASK)
- Hexabatch gives one on-line OD measurement every five minutes vs
just one/two per day in the case of T-flasks.

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HexaBatch Results: On-line pH Measurements
Gives
information
about cell
activity
Faster response than OD
(no death cells interaction)
, tdup (indirect)

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HexaBatch Results: On-line Dissolved Oxygen
Optimal Range

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HexaBatch System
=
1L bioreactor specifications
+
T-Flasks cheap and fast experimentation
• Technological advantages from a bioreactor
• System’s homogeneity: stirring and aeration
• On-line process monitoring (pH, DO, OD)
• Easy maintenance of asepsis
• T-flask experiment price, speed and flexibility
• Working volume at ml scale
• Multiple experiment capability
• Minimal needs on qualified personnel
Conclusion: HexaBatch system

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HexaScreen Applications: Index
1. Pharmacokinetic Tests.
2. Clone Comparison.
3. Media Comparison.
4. Inoculum Concentration.
5. Adaption to Suspension Cultures.

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Drug Functional Tests: Pharmacokinetics
Antibiotic effect in cell cultures
Functional Tests
Activity profiles
More advantages to perform functional tests
in cell cultures than in animals:
- Faster results
- Ethical issues

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Advanced Screening: Clone Comparison
Chose the best clone in terms of:
- Cell growth rates.
- Cell activity.
- Productivity at different sample times activity
activity
Growth
rates

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Advanced Screening: Media Comparison
• Best growth medium
• Time of action:
- Fed-Batch start point.
- Infection.
- Product recovery.
Medium
component
depletion

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Cell concentration profiles
obtained from pH profiles
(related to cell activity)
Advanced Screening: Inoculum Concentration

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Advanced Screening: Suspension Adaption
Control over adaption process
Cell concentration control along passages

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JAVIER AMAYRA: jamayra@hexascreen.com General Manager
GRACIAS POR SU ATENCIÓN:
HexaScreen Culture Technologies S.L.
Edifici Eureka, P1M1.2
Parc de Recerca de la Universidad Autónoma de Barcelona (UAB)
08193 Cerdanyola del Vallès (Barcelona)