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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_101_библиотеки_им_акад_М_И_Перельмана
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FIGURE 10.15 Bafed pivot bioreactors
Source: Niazi patent US 9238789
where Xm is the maximal attainable cell concentration, and X0 is the cell concentration at inoculation, tl is the lag time, and µ
10.12.2 fed- batch
In a fed- batch process, both the substrate (necessary medium components) and inoculum are
applied at the start, similar to a batch process. However, it also incorporates periodic feeding of
a concentrated solution of the growth- limiting supplement to the cells, allowing for controlled
1
t
c
is the exponential growth time.
max
ln
µmaxXmXo
t
l
Equation 10.2
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FIGURE 10.16 Exhaust- free bioreactor
Source: Niazi patent US 9469671
cell growth and possibly increased productivity. This process is commonly used in bio- industrial
applications requiring high cell density (Figure 10.17 and 10.18).
The feed added to the culture is typically a highly concentrated solution of critical nutrients
to extend the growth phase and enhance productivity while avoiding dilution of the fermentation
volume. Depending on the culture and components, the feed solution can be a concentrated glucose
solution with a single nutrient or a mixture of multiple components depending on the culture, the
nature of the components, such as precipitation, in which case it may be better to add various parallels
feeds to reduce the risk of precipitation. The feeding is generally intended to resolve any substrate
limitation via a sporadic introduction during fermentation. The feeding can either be a bolus feed
added intermittently to the culture or a continuous feed that is set up to be continuous, which may
be constant, linearly increasing with time, or cascade mode based on sensor measurements (e.g.,
glucose measurement in the culture). Implementing continuous feeds for large- scale processes can
be challenging and requires careful ne- tuning (Figure 10.12).
Fed- batch processes have been successfully employed for microbial (e.g., E. coli, Pichia pastoris)
and mammalian (CHO cells) cultures. In E. coli fermentation, cells are grown to a relatively high
density, and a bolus glucose feed is added to achieve a faster growth rate. However, the timing and
type of feed addition must be carefully balanced to prevent the accumulation of unwanted by- products
or metabolites(e.g., acetic acid in E. coli fermentation, ethanol for Saccharomyces cerevisiae, and
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FIGURE 10.17 Cell growth kinetics for a batch process
FIGURE 10.18 Fed- batch bioreactor prole
Source: https:// upl oad.wikime dia.org/ wikipe dia/ comm ons/ c/ cc/ Fed_ batc h_ pr inci ple.png
lactic acid in cell culture), which can inhibit growth or affect product impurity proles. For CHO cell
cultures, feeding may increase the medium and culture’s osmolality, impacting cellular functions.
In a fed- batch process, the maximal cell density and product production vary depending on
factors like the host cell, clone, culture medium, feeding schedule, and bioreactor control regime.
Microbial fed- batch operations are considerably faster, with E. coli taking 10– 48 hours and Pichia
pastoris taking 4– 7 days, depending on the product. CHO cell fed- batch methods typically take
10– 14 days but can extend to 20 days for increased output.
Fed- batch processes have become the preferred choice for bioproduction due to their operational
simplicity and ability to address some of the limitations of batch culture. With the application of
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single- use technology and advanced process control systems, fed- batch processes have become
more robust. However, the relatively long process times for mammalian cell culture or yeast cells,
required to achieve higher productivity, may be viewed as a disadvantage in terms of operational
efciency. Additionally, non- productive phases, including cleaning, verication, sterilization, and
batch preparation, are unavoidable for any type of bioreactor.
10.12.3 continuous ReactoR PRocess
A continuous reactor process includes a steady feeding of the substrate while simultaneously
removing an equal volume of culture out from the reactor. The concentration of the substrate can be
regulated to a xed value (Figure 10.19).
The primary form of a continuous process is the steady- state CSTR, also known as a chemostat.
In this system, the growth rate is determined by the availability of the limiting substrate or nutrition. The dilution rate, estimated by the chemostat’s ow rate, matches the net- specic growth rate,
giving users the ability to adjust the growth rate as needed. This control over cell growth allows for
precise control over product formation rates. By maintaining a constant dilution rate, specic growth
rate remains constant. The lag phase, commonly observed in a batch system, is not present in a continuous operation (Figure 10.21).
Productivity increases with the dilution rate until it reaches a maximum near the specic growth
rate (at a steady state). However, when the dilution rate becomes too high, cells can’t grow fast
enough to reach a steady state, resulting in a washout period without a steady state, leading to
decreased productivity (Figure 10.19).
Pr oductivity =
(
Biomass
reactorvolume time
)
(
=
xD
)
Equation 10.3
Using a CSTR for cell recycling can signicantly enhance volumetric productivity, especially
in processes with low- value products and large volumes, such as waste treatment and fuel- grade
ethanol production.
FIGURE 10.19 Schematic of a CSTR
Source: Brijesh Pratap Singh, under Creative Commons Attribution Share- Alike 4.0 International License
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FIGURE 10.20 Genetically modied Komagataella phafi aerobic culture showing the consumption of
glycerol (A), biomass (B) in different initial glycerol concentrations: 2% (blue circle), 6% (green diamond),
and 10% (orange triangle). Time prole for substrate and cells in a chemostat
Source: Microorganisms 2020, 8(5),781; https:// doi.org/ 10.3390/ microo rgan isms 8050 781; http:// crea tive
comm ons.org/ licen ses/ by/ 4.0/
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The industrial application of continuous fermentation processes has been limited. While
chemostats offer productivity advantages for primary products, concerns related to process exibility, genetic stability of cells, and contamination risks have restricted their usage (Figure 10.20).
10.12.4 PeRfusion
The perfusion procedure begins as a batch process, initially maintaining a constant volume in the
bioreactor. Fresh culture media is continuously supplied while spent medium is withdrawn at the
same rate when cells are in the exponential growth phase (typically 2– 4 days after inoculation).
A cell retention device, such as an ATF or TFF lter, retains reactor cells, allowing the removal
of low- molecular components along with spent media. This facilitates continuous harvesting and
product preparation for further purication. Although perfusion has been used in cell culture since
the 1980s, it has faced challenges related to equipment complexity and scaling up.
Perfusion processes typically run for extended periods, ranging from 20 to as long as 90 days.
This approach is less suitable for microbial fermentation due to their faster growth rates, which
result in shorter biomass production times. Mammalian cultures, such as CHO cells, are better
suited for perfusion processes, as their slower growth requires 15– 20 days in a fed- batch process to
achieve the desired product yield (Figure 10.21).
During perfusion, the continuous ow of culture medium ensures a steady supply of nutrients
to cells. Constant removal and recirculation of cells help maintain low levels of toxic by- products,
resulting in higher productivity compared to fed- batch processes. Cell densities ranging from 40 to
80 million cells/ mL are achievable with perfusion, in contrast to the 5– 20 million cells/ mL typically
observed in fed- batch processes. However, monitoring viabilities is crucial to prevent cell debris and
dead cell accumulation. Bleeding a small portion of the culture helps maintain high cell viability
and product quality.
Cell retention is achieved using membranes, screens, or selective cell removal centrifuges. The
most common methods are TFF and ATF systems with hollow ber lters. In TFF, medium is
pumped through a membrane lter, while a peristaltic pump recirculates cell culture supernatant
over the membrane surface. Smaller molecules pass through the membrane lter as permeate, while
larger molecules are retained as retentate. ATF, similar to TFF, uses a diaphragm pump to alternate ow directions over the membrane, minimizing fouling and cell shear. This maintains cells in
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FIGURE 10.21 Bioreactor set up operating in perfusion mode using an ATF system
Source: Copyright Cytiva LifeSciences
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constant equilibrium, promoting faster cell growth and higher productivity. Filters come in pore sizes
ranging from 10 kDa to 750 kDa with various surface area options, usually made of polysulfone or
polyethersulfone. ATF and TFF systems are available as reusable or single- use devices.
The recirculation ow rate is determined by the vessel volume exchanges per day (VVD) or
the cell- specic perfusion rate (CSPR) in pL/ cell/ day. The perfusion rate depends on both the cell
density as well as the culture medium. The perfusion rate is increased either manually or automatically, and this increase is decided either based on cell density or nutrient consumption, such as
glucose levels in the media. It’s advisable to keep the vessel volume exchanges per day to a minimum (typically 0.5– 2 VVD) to avoid diluting the desired product and manage medium usage and
cost. Higher perfusion rates (> 5 VVD) may require more complex liquid handling and can be more
demanding for subsequent processing. The ideal process condition maintains a steady state with
manageable high cell densities while controlling the bioreactor environment (foaming, osmolality,
pCO2, and O2) to sustain productivity.
The primary advantages of the perfusion process over the fed- batch process include improved
and controlled nutrient utilization, enabling steady- state conditions crucial for specic cell productivity. This increased cell concentration enhances productivity. When coupled with a microltration
device for harvesting, the ATF system eliminates the need for laborious clarication processes,
such as centrifuge or depth lter systems. Furthermore, perfusion processes contribute to efcient
facility utilization and extended production phases. The exibility to set up perfusion systems with
almost any type of bioreactor and scalability are key features preferred for bioproduction processes.
However, a drawback is the requirement for large media volumes, which may not fully utilize
nutrients compared to fed- batch processes. Media costs are generally prohibitive, especially for
chemically dened or custom- made media for specic cell types. Additionally, the preparation time,
labor, and media sterilization are challenging and require close supervision. The properties of the
media and the chosen cell line signicantly impact process efciency. The complex setups and validation of the systems and processes are areas that would require additional evaluation (Table 10.6).
10.13 KEY SYSTEM COMPONENTS OF A BIOREACTOR
The bioreactor plays a vital role in converting raw materials into the desired product. Running a
successful bioreactor operation requires an understanding of its key features, which enable control
of critical process parameters determining the bioreactor culture’s success. Factors determining the
bioreactor environment’s suitability for supporting cell growth and the production of interest (biomass or secreted protein) include agitation rate, oxygen transfer, temperature, and pH (as shown in
Figure 10.22 and Table 10.7).
10.13.1 agitation
The impeller or agitator within the bioreactor vessel maintains homogeneity of the bioreactor
contents, suspends solids in the liquid medium, facilitates efcient heat transfer, ensures uniform
mass and gas transfer between different phases, and disperses air for aerated culture conditions. Two
commonly used impeller types in conventional stirred- tank reactors are axial ow and radial ow
impellers, sometimes in combination. Other essential geometric considerations include impeller
size, vessel geometry, bafes, number of bafes, width, orientation, sparger form, and location. It’s
important to note that shear stress due to agitation can be highly damaging to sensitive and fragile
cells, so choosing the appropriate impeller type must be done carefully.
Axial ow impellers create an axial ow pattern where the liquid ows parallel to the axis around
the impeller shaft. Angled blades pump the liquid downward or upward, evenly mixing contents
from the top to the bottom of the bioreactor. The impeller’s orientation (clockwise or counterclockwise) determines the ow direction. Axial ow impellers are suitable for heat transfer and
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TABLE 10.6
215
Comparison of Batch, Fed Batch, and Continuous Modes of Operation
Operation
Mode Key Features Advantages Disadvantages
Batch Highest substrate concentration that
decreases with residence time,
suitable for small- scale production
Fed- batch A medium level of substrate control,
process conditions can help
prevent by- product accumulation,
the residence time of cells can be
controlled, distinctive batches,
most preferred for microbial and
mammalian cell culture processes
Continuous Cell growth kinetics is controlled
through substrate control, limited
by- product accumulation, consistent
quality
Easy operation, high
biomass accumulation,
assurance of quality/
sterility for each batch
High biomass yield,
limited by- product
accumulation, cost-
effective, economically
viable
High- throughput
downtime is
signicantly reduced
highest product
concentration
Extended downtime between runs,
high possibility of by- product
accumulation
Complex handling, ne- tuning
required for feed start times
to avoid substrate depletion;
demands tight process control
Highly complex, time- consuming,
increased burden on downstream
operations, steady- state is
challenging to achieve for
fast- growing organisms,
scale- up may be a concern,
requires a high level of process
control, challenging to change
operations
FIGURE 10.22 Schematic of a bioreactor and system components
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TABLE 10.7
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Parts and Their Function in a Bioreactor
Components Function
Impeller Mixing, air dispersion
Sparger Introduce air, oxygen into the liquid medium to support an aerobic culture
Bafes Prevent vortex and enable better mixing
Inlet air lter Filter air before introduction into the bioreactor
Outlet air lter/ exhaust To lter out the released gas
Sensor/ probes Measure and monitor temperature, pH, and DO in the medium
Cooling jacket Maintain temperature at its set point for the duration of fermentation
Ports (multiple) Enable sampling, the addition of inoculum, media, feed, and supplements, the addition
of acid/ base for pH control, the addition of antifoam to control foaming
FIGURE 10.23 Impeller types; radial left and axial right
Source: Daniele Pugliesi– Own work, CC BY- SA 3.0, https:// comm ons.wikime dia.org/ w/ index.php?curid=
4996 798
liquid- liquid mixing and are known for their low shear. Examples include marine blade impellers
and pitched blade impellers.
Radial ow impellers push liquid away from the impeller along its radius, creating four sections
or quadrants within the bioreactor. They are primarily used for gas- liquid and liquid- liquid mixing
and have a higher shear (Figure 10.23).
Pitch blade impellers are used for shear- sensitive cells like mammalian and plant cells. They have
at blades positioned at a 45- degree angle to the shaft, promoting both axial and radial ow. This
combination enhances mixing and oxygen transfer rates compared to traditional maritime blade
impellers.
Pitch blade impellers are used for shear- sensitive cells like mammalian and plant cells. They have
at blades positioned at a 45- degree angle to the shaft, promoting both axial and radial ow. This
combination enhances mixing and oxygen transfer rates compared to traditional maritime blade
impellers.
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TABLE 10.8
217
Impeller Types and Suitability for Cell Lines
Suitable Cell Line
Mammalian (Human
Impeller Type
Rushton impeller Escherichia coli,
Pitched blade
impeller
Marine blade
impeller
Spin- lter impeller SF9, Hi- 5, Sf21
Bacteria Yeast
Sacchromyces cerevisiae, Baker’s
Bacillus,
Streptomyces
Streptomyces Candida albicans HEK 293, HeLa, HL60,
yeast, Pichia pastoris, Candida
albicans
and Non- Human Cells) Insect
CHO, BHK
3T3, MC3T3, NS0
SF9, Hi- 5, Sf21
Flat- bladed or disk turbines, also known as Rrushton impellers, are commonly used in microbial
cultures where shear stress is not a major concern. They feature at blades set vertically along the
shaft, creating unidirectional radial ow (Table 10.8).
The impeller’s primary role is to disperse gas, while the sparger introduces air into the liquid
medium inside the fermenter. Three basic sparger types used in bioreactors are:
• Porous spargers are used in small- scale applications but limit gas throughput due to high
resistance. They are typically made from sintered metal, glass, or ceramics.
• Orice spargers, also known as perforated pipes, have been utilized in specic applications
and involve drilling small holes in piping, which is then shaped into a ring or cross and placed
at the reactor’s base.
• Nozzle spargers are employed in various bioreactors, providing a low- resistance gas ow and
minimal risk of blockage, making them superior to other designs.
Other sparger designs, such as hybrid sparger- agitator designs, have also been developed for
smaller fermenters, for example, gas and liquid are injected simultaneously through a nozzle to generate tiny bubbles, while air is delivered through a hollow stirrer- shaft.
The impeller needs to be powerful enough to distribute the gas bubbles in the vessel while also
extending the duration it spends there. When adding gas into a liquid media, an important issue is
to enhance the surface area to allow for faster gas absorption. Reducing the bubble size by using
microspargers to create tiny bubbles will increase the surface area of absorption and enable a higher
kLa. A consistent pore structure of the sparger device is key to generating even bubbles. The inuence of gas dispersion on cells, on the other hand, must be balanced. Eukaryotic cells can be damaged
by air bubbles. In microbial systems, where rapid dispersion and mass transfer across the vessel are
required to sustain cell growth and gas transfer rates, high shear impellers are commonly used. For
human cells, plant cells, and other cells, an oxygenation device capable of decreasing shear due to
bubbles is preferable. A gas basket and bubble- free aeration can be used to accomplish this. A ring
sparger, which creates bubbles, is used to introduce gases into the bioreactor tank. Between the
exterior surface of the inner tube and an outside membrane, these bubbles travel with the impeller.
The gas exchange occurs at the membrane- media interface, resulting in a bubble- free atmosphere
for the cells and negating shear induced by bubble breakage.
Bafes are used to increase mixing, prevent vortex formation, and improve gas dispersion and
aeration efciency. Typically, four bafes, accounting for 8– 10% of the vessel diameter, are attached
radially to the wall. Bafes also help reduce microbial growth on the bioreactor walls.
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