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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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Upstream Processing
synthesis is required for optimizing the bioproduction stages. Given the underlying nature of
microorganisms and cells, the behavioral pattern is not always expected to make the upstream
process challenging since variations can lead to signicant process outcomes. However, a thorough understanding of these concepts and their relationship with each other can help minimize
these differences and design a process that can operate within an established range for a robust
and consistent process.
All organisms share a basic metabolic pathway involving both degradation and biosynthesis.
Cellular energy is generated through carbon consumption, driving overall metabolism.
A mass balance for aerobic culture can be represented by a generic equation:
Rate of accumulation = Rate of inow + Rate of outow + rate of generation
Understanding substrate utilization and cell growth is essential in determining optimal conditions
for product formation.
Cells + Substrate = Product Formation + Cell Growth
The exponential growth of cells can be represented by
r = μ X, Equation 10.1
where r is the cell growth rate (g/ Ls), X is the cell concentration, and r μ denotes the specic
growth rate.
10.4.1 cell gRoWth
Cell growth or reproduction, also known as proliferation, occurs through regulated events that vary
depending on the organisms. In a fast- growing E. coli culture, the doubling time is typically 15– 20
minutes, whereas eukaryotes like mammalian CHO cells can take 12– 24 hours to double. These
times are highly dependent on the available conditions required to maintain the cell in a dividing
state, including the right nutrient supply, oxygen source, and temperature, among others. A production cell line with a short doubling time, ease of scale- up, and the ability to mimic suitable
conditions across all stages while reducing batch time in the production bioreactor is always desirable. A fast- growing cell line shortens the time needed to produce seeds, allowing for a quicker
turnaround. The four stages of cell growth kinetics are the lag phase, exponential growth phase,
stationary phase, and death phase (Figure 10.2).
During the lag phase, cells adapt to the bioreactor vessel’s environment before proliferation
begins. Subsequently, when the cells have adjusted to the environment, they enter the exponential
growth phase. In this stage, cells reach the maximum cell division rate under favorable nutrient and
environmental conditions. The length of this phase can vary greatly depending on the cell type and
specic growth conditions. Following the exponential growth phase, cells enter a stationary phase,
where the cell population reaches its maximum, and the rate of cell growth becomes equal to the rate
of cell death. This ultimately leads to the death phase due to a decline in the growth rate caused by
nutrient depletion and the accumulation of toxic by- products.
The primary goal of any biomanufacturing process is to maintain cells in a state where productivity or product formation is maximized. This can involve increasing the number of cells (as in
the case of E. coli processes) or enhancing the secretion of proteins (e.g., CHO cells) for a growthassociated process. In non- growth- associated processes, productivity is increased by prolonging the
stationary phase.
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FIGURE 10.2 Phases of cell growth
10.4.2 cell death
While cell death is a natural part of the culture process cycle, it can lead to processing problems,
such as decreased overall product yield and increased impurities like DNA, HCP, and other cell
debris. Animal cell death can occur through necrosis or apoptosis. Necrosis results from unfavorable
conditions like shear stress and extreme pH levels, causing cells to swell and eventually burst due to
osmotic pressure, releasing their intracellular components. Apoptosis, on the other hand, is a genetically controlled process induced by certain environmental conditions such as nutrient depletion
and oxygen limitation. Therefore, delaying the onset of cell death and maintaining the culture in a
highly viable state is essential to minimize cell debris generation, which is critical for downstream
process purication.
10.4.3 Metabolic contRol
The upstream fermentation process depends on physicochemical parameters and culture media
composition that can impact cell metabolism. The metabolic demands differ between growing
cultures in a dividing state and those in a non- dividing state. Accumulation of by- products and
imbalances in nutrient uptake can obstruct cellular metabolism. For instance, in CHO cell culture processes, glucose consumption can result in lactate accumulation, affecting culture osmolality and pH and potentially causing apoptosis. Similarly, in E. coli cultures, the consumption of
carbon sources can lead to an acetic acid build- up, lowering the culture’s pH and impacting cell
growth (Table 10.5).
Oxygen supply is essential for supporting cell growth, especially in aerobic cultures. Oxygen
can be a limiting factor for microbial processes and animal cell cultures, particularly during scaleup. The oxygen transfer ratio should exceed the oxygen uptake ratio for a culture process. The
concentration of dissolved oxygen in the culture is expressed as a percentage and calculated as
follows:
=×
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TABLE 10.5
Upstream Processing
Operational Parameters of the Cell Culture Process have an
Impact on the Process Performance and Product Quality
Process Parameters
(Bioreactor Control) Control Strategy Impact
pH CO2 or base addition
DO Airow rate, oxygen ow rate,
agitation rate, sparger
Temperature RT D
Pressure Gauge
%*DO
C
L
100
C
L
Equation 10.2
where CL is the actual oxygen concentration in the medium, and C
Cell Growth,
Viability
Product concentration
Product quality
*
is when the medium is saturated
L
with air.
The difference between C
*
and CL drives oxygen transfer in the culture. Glucose consumption
L
can lead to lactate production in mammalian cells and acetic acid in microbial cultures. Tight control of pH is maintained by adding CO2 or base. The pH dead band and pH set point together form
the pH control strategy.
Microbial cultures have a higher oxygen demand that depletes rapidly. Oxygen is added through
headspace aeration and sparging using a microsparger. Variations in these parameters, as well as
other culture operating parameters such as temperature, agitation speed, carbon dioxide, pH, osmolality, and metabolite levels (including by- products such as lactate, acetic acid, and amino acids),
signicantly impact culture performance, yield, and product quality. pH can also have a signicant
impact on culture development and metabolism.
Process performance parameters are established, and the controls achieved in a small- scale process are used for nal scale- up. These variables are either volume- dependent (e.g., agitation, aeration, feed volume) or volume- independent (e.g., pH, temperature, DO set point).
10.5 CULTURE PRODUCTION PROCESS
This section presents the sequence of operations, timing, and precise control of process parameters
for fed- batch fermentation with E. coli and CHO mammalian cells. In a fed- batch process, cells progressively grow to high cell densities by increasing the quantity of ingredients in a time- dependent
manner to match exponential growth and minimize the accumulation of harmful by- products.
Typically, this growth is carbon- source limited to maintain low residual carbon concentration and
maximize productivity. Feeding can be both growth- dependent and time- dependent, with feeding
strategies often following a pre- set time prole using a pump integrated into the bioreactor’s control
system. It is common to time feeding with an expected DO spike in microbial cultures, primarily due
to carbon source depletion. However, during process development, careful evaluation and balance
are required to ensure that the depletion of the carbon energy source does not shift the cells’ growth
phase and metabolism, reducing overall biomass yield.
A sample growth curve and glucose consumption pattern are illustrated in Figure 10.3. Initiating
feeding before depletion can support exponential growth, although it is typical for the carbon source
to be entirely depleted as the culture reaches maximum cell concentration, leading to an accumulation of carbon source as cells enter the death phase.
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FIGURE 10.3 Cell growth and glucose consumption
FIGURE 10.4 Conventional culture process from vial to production bioreactor process
Source: Copyright Cetiva
10.5.1 seed tRain
A traditional fed- batch process involves multiple stages of seed expansion, starting from a
vial or vials, progressing to a large- scale production bioreactor. This is necessary to cultivate
a sufcient number of cells for inoculating the production bioreactor. The seed train begins
by thawing cells (one or multiple frozen vials) from a qualied cell bank, typically a WCB,
into a shake ask containing suitable growth culture media. Subsequently, the culture in the
shake ask is incrementally expanded in volume using bioreactors of increasing size, ultimately leading to the production bioreactor. A typical outline of this culture process is shown in
Figure 10.4.
In microbial cultures, seed train generation usually lasts for 24 to 48 hours until the production
bioreactor can be inoculated. However, in mammalian cell culture processes, the combined seed
train and inoculum train can take up to a month to generate a sufcient cell volume for inoculation
into the production vessel.
For mammalian cells, regular sub- culturing (also known as passage) is essential to keep the cells
in a dividing state and prevent them from entering the stationary phase before reaching the production stage. Subculturing can be based on a split ratio (volume) or a target starting (seeding) density
for the next stepwise expansion. It is crucial to minimize the number of passages (the number of
times the cells have been split) to prevent potential genetic drift or instability. The impact of passage
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FIGURE 10.5 Illustration of an intensied seed generation process
Source: Copyright: Cytiva
Upstream Processing
number on the overall production process must be carefully evaluated and validated. In contrast to
the conventional method, which takes around 30 days for seed generation, process intensication
can signicantly reduce the seed generation time by at least half (Figure 10.5).
The volume factor or scale- up factor indicates the increase in volume from one inoculum
preparation step to the next. It determines how much fresh culture media can be added to the
cells and whether the entire cell volume is required or just a portion. For example, a scale- up
factor of ten means that one seed reactor’s volume is ten times that of the previous seed reactor.
Unlike mammalian cell cultures, microbial cultures can sustain higher volume factors, which
may require a relatively lower volume factor. A signicant scale- up factor can greatly reduce
the seed train generation process. This scale- up factor is crucial in determining the number and
sizes of bioreactors needed to support a specic commercial- scale operation. Additionally, when
designing the seed train, one must consider the recommended or maximum working volume, as
it can impact the optimal performance and maintenance of process parameters supporting cell
growth.
Seed generation is a critical process in ensuring the overall success of a bioproduction process. The quality of the cell bank is paramount throughout the journey from vial to production
fermenter, ensuring that the upstream process remains robust and consistently productive and of
high quality.
Glass shake asks and stainless- steel bioreactors have been long- used for small volume and
expansion of seed culture stages, respectively. However, they have now been successfully replaced
with single- use disposable shake asks and single- use bioreactors available in various sizes for
the seed train stage. These are employed for small volume cultures (< 10 L). The increased use of
disposables at these stages enables a faster turnaround by eliminating cleaning steps and reducing
the risk of contamination. For cultures in the range of 10– 50 L and higher, a rocking bioreactor, such
as WAVE™, is more suitable than a stirred tank reactor. Shake ask cultures are maintained in an
incubator shaker under controlled humidity, carbon dioxide (for mammalian cells), temperature, and
agitation, which is adequate for microbial cultures.
10.5.1.1 Vial Thaw
The process of thawing the vial(s) should be swift and performed under controlled temperature
conditions using a water bath at approximately 37°C. This helps prevent damage to the cells from
ice crystal formation. Then, inside a laminar ow hood or biosafety cabinet, the cells are added to
fresh culture media in a shake ask and allowed to incubate until the next process step.
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10.5.1.2 Fermenter Setup
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The bioreactor vessel, whether for seed generation or production, is set up following the outlined
instructions. The vessel is lled with culture media (except for heat- sensitive ingredients) and then
sterilized. Any heat- sensitive components are added later through lter sterilization. In single- use
systems, culture media and other essential components are added to the bioreactor bag via a 0.2μm lter. It is standard practice to perform this operation at least 24– 48 hours before adding cells.
This allows the culture media to equilibrate to the process conditions, perform calibration checks,
and, most importantly, ensure sterility before inoculation. The composition of culture media and
process conditions for seed generation may differ from those used for the production stage. Various
procedures, including sterilization (SIP), sanitization, tube welding, tube sealing, and autoclaving,
are used to support sterile processing.
10.5.1.3 Inoculation of Production Vessel and Sampling
In the production bioreactor, cells are introduced (i.e., inoculated) under positive air pressure
to prevent contamination. Sterile air is used to pressurize the production bioreactor, and the
inoculum is delivered through sterile tubing equipped with a dedicated port. Establishing sterile
connections, for example, using pre- sterilized aseptic connectors such as AspetiQuik tubing
assemblies, simplies the process and eliminates the need for on- site sterilization of parts such as
piping and valves.
Production vessels are equipped with dedicated sampling ports to facilitate the collection of
culture samples according to process requirements. These samples are utilized for determining cell
density, either by using a spectrophotometer for E. coli cultures or a trypan blue- based method, or
automated cell measurement systems such as Vicell, in addition to other metabolite measurements.
In traditional stainless- steel fermenters, the sampling ports are positioned away from the vessel and
sterilized with steam before sample withdrawal. Bioreactors typically come equipped with sensors
or probes for continuous monitoring of pH and dissolved oxygen (DO), at the very least. They may
also feature more advanced capabilities, such as integrated cell density monitoring sensors and
metabolite measurement. The pH is initially calibrated using standardized buffers. After the culture media is added and equilibrated, a one- point standardization is performed using an ofine pH
meter. This adjustment is then incorporated into the control unit to enhance accuracy for subsequent
measurements. In some cases, processes may continue to use ofine pH measurements to intermittently adjust the pH drift.
DO calibration involves creating conditions of 100% saturation and 0% oxygen (achieved
by sparging nitrogen to remove air). Periodic ofine measurements are conducted to assess the
cellular environment conditions and determine appropriate feeding strategies. These ofine
measurements can employ various techniques, such as enzyme- linked membrane technology (e.g.,
NOVA Bioprole Flex), photometric methods (e.g., Opto Cell, Cedex, spectrophotometer), or ionselective approaches utilizing a reference electrode. For gas analysis and glucose measurement, online measurements can be integrated using advanced technologies like soft sensors and integrated
HPLC systems.
10.5.1.4 Fed- Batch Process Outline
10.5.1.4.1 Escherichia coli
1. Prepare culture media, feed, and other necessary supplementary solutions for the batch.
2. Sterilize liquid components using either ltration or autoclaving.
3. Assemble the bioreactor components, transfer lines, containers, pH and DO probes, sensors,
agitators, and gas lines required for the fermentation process.
4. Retrieve a vial from a −70°C freezer and thaw it in a water bath set to a temperature between
34°C and 37°C.
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Upstream Processing
5. Under a biosafety cabinet, transfer the cells into a ask(s) containing the required amount
of medium (e.g., LB culture media, TB culture media, etc.) with the appropriate antibiotic
(e.g., kanamycin) if the plasmid carries a resistance gene. Ensure that the ask is lled to
approximately 0.25– 0.3 of its capacity to ensure proper aeration of the culture.
6. Incubate the shake ask culture(s) in an incubator shaker set to a specic temperature and
agitation speed. Measure the optical density (OD), which assesses cell growth, using a
spectrophotometer at a wavelength of 600 nm. The incubation time can vary from 12 to 20
hours, depending on the target OD600 to be achieved.
7. Depending on the nal commercial scale, one or two seed generation stages may be
required. Use the shake ask culture as the inoculum for a 10 L fermenter vessel. Multiple
shake asks may be used to generate a sufcient volume of cells to inoculate larger or multiple fermenters simultaneously. This 10 L inoculum can, in turn, be used to inoculate a
higher volume seed bioreactor.
8. Calibrate pH and DO sensors, probes, pressure gauges, and pumps. Add an appropriate
amount of culture media to the production vessel and allow the system to equilibrate
to the process conditions (DO, temperature, pH, agitation) for at least 24 hours before
adding the inoculum. Conduct a sterility check to conrm that the culture media is free of
contamination.
9. Sparge air at the designated ow rate (e.g., 0.5– 1.0 vvm), which is expressed as volume
gas/ volume culture/ min, throughout the fermentation process.
10. Aseptically add the inoculum into the production vessel through a septum port for stainless
steel or a dedicated inoculation port for single- use systems. Perform a nal system
check to ensure that DO, pH, temperature, and agitation set points align with the process
requirements.
11. Periodically monitor the culture’s progress (e.g., every hour) by measuring OD600. If at
any point during the process, the DO or pH deviates from its set point, the sensors, through
a control strategy, work to correct it. For instance, the mass ow meter may open to aerate
the culture in case of low DO, or CO2 may be sparged to lower the pH or a base added to
increase the pH.
12. Implement an induction procedure for protein expression when the target cell concentration is reached by adding an inducing agent. Induction can be performed either earlier in
the production process or later at a higher cell density. Similarly, based on nutrient consumption, a bolus feed consisting of a concentrated solution of key nutrients (e.g., glucose
and other trace elements) is added to the production bioreactor, depending on fermentation
time or optical density.
• Induction for lac and tac promoters and T7: Add a specic concentration of IPTG
solution.
• Induction for pBAD promoter: a certain concentration of Arabinose solution.
13. Optionally, collect samples before and after induction for SDS- PAGE analysis to determine
yield and monitor protein expression progression during fermentation.
14. Terminate the fermentation process at a specied time once the target OD600 is achieved.
Proceed with harvesting the batch to recover the cell pellet for further downstream
processing.
10.5.1.4.2 CHO Cells
1. Prepare all the cultutre media, feed, and other necessary supplementary solutions for
the batch.
2. Sterilize the liquid components, either through ltration or autoclaving. Assemble the bioreactor components, transfer lines, containers, pH and DO probes, sensors, agitators, and
gas lines required for the fermentation process.
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Upstream Processing
3. Retrieve a vial from the liquid nitrogen cryo unit and thaw in a water bath set to the thawing
temperature (~37°C).
4. Under a biosafety cabinet, transfer the cells into a ask containing the required growth
medium supplemented with glutamine. Typically, use no more than 25– 30 mL of culture in
a 125 mL shake ask to ensure proper aeration.
5. Incubate the shake ask culture(s) in a humidied CO2 incubator (5% CO2) at a specic
temperature and agitation. Measure the viable cell density daily until the target cell density
is achieved.
6. Subculture the cells by gradually increasing the volume for subsequent passages until the
target inoculum volume is achieved. This may involve multiple shake ask steps (or spinner
asks) followed by small- scale/ pilot- scale bioreactors (rocking or stirred tank).
7. Calibrate pH and DO sensors/ probes, as well as pressure gauges and pumps. Add an appropriate amount of culture media into the production vessel and equilibrate it to the process
conditions (DO, temperature, pH, agitation) for at least 24 hours before adding the inoculum.
Perform a sterility check to conrm that the culture media is free of contamination.
8. Pump the initial amount of culture media into the production vessel and equilibrate the
system with air (headspace and sparger) and the process temperature setpoint.
9. Calculate the initial amount of culture media needed based on the target volume at harvest, the expected amount of inoculum to be added for a starting density, and any feed/
supplement solutions required during the fermentation process.
10. Establish the air sparging ow rate and DO setpoint before adding the inoculum. The feedback mechanism of the sensor and control unit allows the mass ow meters to respond
and sparge air and oxygen as needed. The agitation speed can either be kept constant or
adjusted to meet the process’s aeration requirements.
11. Add the inoculum to the production vessel, either using sterile connectors and pre- sterilized
transfer lines for single- use systems or following the traditional procedure for stainlesssteel systems.
12. Monitor the culture growth by measuring cell density daily, either once or multiple times
a day. Conduct ofine analysis of the culture sample for pH and metabolite measurements.
13. Start feeding or adding supplements as determined from process development studies. This
can be based on achieving the desired cell density or done periodically (e.g., adding 1 L of
feed per day from Day 2 to Day 10 of the fermentation process).
14. Temperature and pH set points as the process progresses.
15. Proceed to harvest and clarify the culture to recover the secreted protein.
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The upstream processes have advanced to the point where yields are no longer a limiting factor.
The integration of hardware and software components has enhanced our understanding of the key
parameters that can inuence the outcome of cell culture processes. Manufacturers are continually
seeking improved yields through an integrated approach, from cell line engineering to enhanced
process control, and more recently, through continuous manufacturing and process intensication. The adoption of Quality by Design (QbD) principles and the integration of Process Analytical
Technology (PAT) tools have become increasingly common to ensure compliance with every batch.
10.6 CELL SEPARATION AND HARVESTING
After completing the synthesis phase, the subsequent process stages include product isolation,
recovery, and purication. Product isolation, also known as separation, marks the beginning of
downstream operations. For the sake of continuity in concluding the production culture operations,
we include the separation process, i.e., isolating the product from the production system, in this
chapter on upstream processing.
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Upstream Processing
Once the desired product is produced, whether intracellularly in the cytoplasm, periplasm, or
secreted into the culture media by the respective production system (e.g., E. coli cells, mammalian cells such as CHO), it must be separated and recovered from the host system. Cell harvesting,
which involve,.es extracting or separating the host cells from the culture broth, is employed for this
purpose. In E. coli processes, the protein is held within the cells as inclusion bodies, requiring the
isolation and further processing of the cells for product recovery. In the case of mammalian cells,
the protein is secreted into the culture media. Therefore, removing the cells from the product stream
and clarifying it for further purication forms the general outline for mammalian- based production
processes.
Understanding the advantages, evaluating the success of these methods at the commercial scale,
and considering the limitations, including cost, are crucial for optimizing the process. It has become
common to follow a platform approach used for one product to apply for every product being
manufactured in the facility.
While this may be advantageous in some cases, it may not be suitable for all products. The selection of the primary clarication step can signicantly impact subsequent ltration and purication
operations. Variables such as the solid- to- liquid ratio, culture viability, cell debris, and other impurities can affect the outcome of the recovery.
The most widely used separation strategies for both E. coli and CHO systems are detailed below.
10.6.1 centRifugation
Centrifugation relies on differences in density between solid particles, such as cells and cell debris,
suspended in a feed stream, typically a culture broth. As a result, processing volumes in commercial-
scale operations can be quite high, making continuous centrifugation the preferred choice. While
the basic principles of centrifugation have remained largely unchanged for over 50 years, recent
advances in membrane technologies have improved process purity, control, efciency, speed, yield,
and cost (Figures 10.6 and 10.7).
FIGURE 10.6 Schematic of a continuous ow centrifuge
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FIGURE 10.7 Mechanism of continuous separation
197
A continuous ow centrifuge is capable of processing large volumes at high centrifugal forces.
The culture feed is introduced through the inlet at the bottom of the centrifuge bowl. The combination of centrifugal speed and feed ow rate causes solid particles, such as cells and debris, to
become trapped in the gradient as they move from the bottom to the top of the vessel. These solids
settle against the bowl’s walls under the centrifugal force, while the claried liquid, also known as
supernatant, moves up the channel and exits through the outlet. If the product of interest is present
in the supernatant, a collection tank is used for further processing. Alternatively, if the product is
within the solid particles, these settled solids can be continuously removed through nozzles or at
specied time intervals.
This process eliminates the need for lling and decanting centrifuge bottles or stopping and
starting the system multiple times, which is often required with conventional centrifuges or benchtop
models primarily suited for smaller volumes.
Continuous ow centrifugation offers several advantages, including high volumetric throughput,
strong centrifugal forces, and the ability to handle high solids feed streams, reducing the time spent
on sample handling. These attributes make it a preferred choice for large- scale operations involving
bacterial cultures, viruses, and as an initial step in mammalian cell clarication.
Factors that can affect the efciency of this process include
• Centrifugal force (g- force): Excessive centrifugal force can sometimes lead to shear that may
damage cells, releasing impurities such as DNA, host cell proteins, and additional debris. In
contrast, low centrifugal force may result in uneven settling of solids, leading to less claried
supernatant.
• Residence time: Extended residence time can reduce throughput and potentially harm product
quality (possible degradation).
• Flow rate: Solid particles’ sedimentation depends on both centrifugal force and ow rate.
Smaller particles require higher centrifugal force and more sedimentation time than larger
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