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Bioreactor 1

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Atul Patel
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18 views16 pages

Bioreactor 1

Uploaded by

Atul Patel
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Mammalian Cell Cultivation

Systems
Let us begin
• Products from mammalian cells comprise viral vaccines, monoclonal
antibodies for diagnostic and therapy require higher production
• Due to the broad variety of cell types, purposes, scales etc. it is obvious
that no universal cultivation system or bioreactor exists
• The main challenges for techniques required for cultivation of mammalian
cells are slow growth rates
• Adherent cells cause severe problems in large scale production (large
surface area; microcarriers)
• Stirred tank bioreactors provide low shear environment by specially
designed agitation and aeration systems
• Cultivation systems for immobilized cells such as hollow-fiber, fluidized-
bed and fixed-bed bioreactors protect the cells from stressful conditions
Specific
requirements
• A well controlled environment with respect to pH, temperature,
dissolved oxygen, dissolved CO2-concentration etc.
• Homogeneous and low–shear mixing and aeration
• Effective mass and heat transfer
• A surface for cell attachment in case of adherent cells
• Measurability of process variables and key parameters
• Scale-up capability
• Long-term stability and sterility
• Ease of handling
• Maintenance
Types of Systems for production of
biopharmaceutical
Non-agitated system
Agitated system
Mechanical (external device, internal device)
Hydraulic (hollow fibre bioreactor, fixed bed and fluidized
bed bioreactors)
Pneumatic (bubble-column, air-lift)
Applied mode of cultivation
• Volumetric scale-up of lab-scale and pilot-scale-bioreactors at
similar process intensity
• Increase of process intensity (cells/volume), as cells can grow at
very high, tissue-like cell densities
Cell culture bioreactors

Static Systems Dynamic systems


(unenforced power Mechanically driven
input) Hydraulically Pneumatically
External device for Internal elements driven driven
Bioreactor movement
Rotating shaft Hollow fibre
Shaker With stirrer Bubble
Bioreactor column
Petri dish
Roller Unit Non rotating Airlift
Bed bioreactor bioreactor
T-flask shaft Fluidized
Spinner bottle Fixed bed
Culture bag

Rocker unit or
raising platform
Basic scheme of static cell culture bioreactors:
a) Petri dish, b) T-flask, c) Multi cell culture system, d)
Culture bag, e) Static membrane flask bioreactor, f)
Multi well plate
Dynamic cell culture bioreactors:
a)shake flask, b) Roller bottle, c) Rotating membrane flask, d) Spinner flask,
e) Rocking bag with induced motion, f)Hollow fibre bioreactor, g) Stirred
bioreactor, h) Bioreactor with eccentric motion stirrer, i) Bioreactor with
vibromixer, j) Bubble column, k) Airlift bioreactor, l) Fixed bed bioreactor, m)
Fluidized bed bioreactor
Bioreactor choice by scale ( volume) and cell density
requirements
Immobilization of Mammalian
•Cells
Cells are immobilized when they are restricted in their motility, while
their metabolic or catalytic activity is maintained
• Most techniques applied for immobilization relay on non-porous (solid) or
porous carriers
• Solid carriers provide a smooth surface for cell attachment
• A carrier is considered as macroporous, if the average pore size is
between 300 and 400 μm allowing for cell growth within the carriers
• Porosity of these carriers= the ratio between volume of the pores and the
total carrier volume (in percent) is normally between 60% and 99%
• Macroporous carriers are suitable for immobilizing adherent as well as
non-adherent cell lines
• Microcarriers are the most important technique for growth of adherent
cells in suspension type bioreactors, mainly stirred tanks
• The term “microcarrier” refers to small beads, either solid or
macroporous, having a diameter of approx. 100–300 μm with a density
slightly higher than the growth medium
• In microcarrier culture, cells grow as monolayers on the surface of solid
spheres or 3D within the pores of macroporous structures
Immobilization of Mammalian
Cells
• Cultivation of cells in fixed bed and fluidized bed bioreactors
requires carriers with high porosity, and large pore size among
others
• There are limited # of carriers for fixed bed and fluidized bed
cultures
• The earlier microporous carriers were mostly spherical, made of
glass and had usually a relatively low internal porosity
• Mass transfer limitations due to diffusive transport limit the cell
density within a carrier
• Smaller carriers could support a higher volume specific cell
density
• Therefore, for fluidized bed bioreactors a carrier size of 0.6–1 mm
is an appropriate choice
Bioreactor operation strategies
Discontinuous (batch, repeated-batch, fed batch)
Continuous (chemostat, perfusion)
Bioreactor operation strategies
Batch
• A batch culture is characterized by growth of cells without further
addition of substrate
• During duration of the culture, the cells pass through the typical
phases
• Reduction in cell growth and induction of cell death due to
substrate limitation or metabolite inhibition is mainly induced by
apoptosis
• During the cultivation additional compounds such as air or oxygen
for aeration, acid or base for pH-control or anti foam agents to
prevent foaming have to be added
• Batch processes are usually started with an initial cell density of
approx. 1–2 X 105 cells/mL and operate for 1–2 weeks
• After harvest the bioreactor has to be cleaned and refurbished
before the bioreactor is re-started.
• The productivity of batch cultures is limited by the initial
concentration of substrates
Repeated batch and Fed batch
• A first step to improve efficiency of a batch process can be seen in
a so called “repeated batch”
• Here the main part of the cultivation broth is harvested along with
the protein of interest at the end of exponential growth phase
• The remaining part is used as inoculum for the next run

• Cell and product yield can be significantly improved by applying a


fed-batch strategy, where nutrients are added after depletion
according to an appropriate feeding strategy
• Here the culture system is initially filled to 1/3 or 1/2 only and
started as batch
• As soon as substrates are consumed or have reached growth
limiting values, nutrients such as glucose, amino acids and
vitamins with trace elements are added mostly in concentrated
form to avoid substantial dilution
• The main advantage of a fed-batch compared to a batch can be
seen in a longer growth phase, higher cell concentrations (107–108
cells/mL) and higher product concentrations and a higher product
Continuous Culture
• A continuous bioreactor is a bioreactor that maintains a steady
state of culture growth to increase productivity
• In a continuous bioreactor, a limiting substrate is continuously
added to the bioreactor while culture broth is withdrawn at the
same rate
• Chemostat: The rate of addition of a single growth-limiting
substrate controls cell multiplication; cells are washed away with
the medium as it flows through the culture vessel
• The growth rate of cells can be controlled by varying the dilution
rate of the medium
• In continuous chemostat culture, fresh medium is continuously
pumped to the culture system and spend medium is removed
• Perfusion: This type of continuous bioprocessing mode is based on
either retaining the cells in the bioreactor or recycling the cells
back to the bioreactor; the cells are retained or recycled in the
culture vessel or in a device outside of it
• During steady state the values for cell concentration as well as
substrate and product (metabolite) concentration depends on the
flow rates for medium supply and harvest, expressed by the
dilution rate
Continuous Culture
• Perfusion culture can retain the cells in the culture system and
perfuse the system continuously with medium
• This can be done by connecting cell retention devices to a
suspension bioreactor (stirred tanks, bubble columns and air-lift
reactors) or by immobilizing the cells (e.g. fixed bed or fluidized
bed bioreactor, hollow fiber systems)
• Similar to a chemostat, here fresh medium is pumped
continuously to the culture system and spend medium (harvest
containing the desired product) is removed with the same flow
rate
• When dealing with adherent cells, perfusion reactor design
becomes slightly simpler (e.g. fixed bed and fluidized bed) due to
immobilization of cells within macroporous carriers
• The main advantage of perfusion cultures can be seen in the
reduced bioreactor size (approx. 1/10th of a suspension reactor
without cell retention)
Continuous Culture

• There are some difficulties in the perfusion concept


• Extra equipment such as the retention device itself, pumps for
feeding, harvest and medium circulation, storage tanks for feed
and harvest are required
• Harvest product titers are typically lower compared to fed-batch
cultures.
• The amount of media needed to complete a moderate to long term
run can be excessively large
• Process development and characterization can be more complex

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