SCALE UP IN FERMENTATION PROCESSES/FERMENTERS

Importance of Scale Up

The detection or induction of a novel product, metabolite or protein is the first stage
in the development and study of the molecule of interest. Once an interesting
metabolite has been detected, it has to be produced in large volumes for
characterization, trials, structural elucidation and potentially for commercialization.
In order to obtain a product in increased quantities the culture system that was
originally used for detection/induction can be replicated multiple times. Or else, an
efficient process in a much larger vessel could be cultivated in larger volumes.

The intermediary steps that are involved in translating a successful bench product to
production scale are referred to as Scale up. This involves microtitre plates, shake
flasks, laboratory and pilot and production scale equipment. The production of a
metabolite under large scale is essentially a costly process and takes place under
GMP hence experimentation is impossible at large scale. It is therefore much
important to develop small scale venues where experimentation can take place

Variables to be Considered when Changing Fermentation Scale

1. Aeration and Agitation

Aeration provides two related functions in an aerobic fermentation process: (i) to

provide mixing, and (ii) to supply oxygen. Resistance is offered to the oxygen flow
in a process which increases proportionally as the scale of the process is increased

2. Sterilization of Fermentation Media and System

Batch thermal sterilization processes tend to be used for liquid and equipment in
fermentation systems, as these tend to be reliable and cost-effective options for both
small and large scale systems. Continuous sterilization of medium can be used for
fermentation systems, only under specific cases t, e.g. reduction in utilities or specific
nutrient characteristics. The sterilization methodology at different scales and in
different items of equipment may be a major source of variation due to chemical
degradation of medium components. In small scale processes medium is sterilized
by using autoclaves and for heat sensitive reactants, filters may be used. For pilot
and large scale processes, sterilization processes take place by steam injection.

3. Inoculum Development and Culture Expansion

Once inoculum development in small scale has been developed, there may be a need
to expand it – introducing another transfer step to a culture expansion or ‘seed’
vessel, with the aim of increasing biomass in the vegetative phase to sufficiently
larger volumes to use a sufficient volume of mature inoculum usefully to inoculate a
production vessel.
4. Raw Materials and Nutrient Availability

A medium that is useful for producing product in small vessels may not be viable
from a cost basis at the large scale, e.g. bacteriological grade yeast extract. However,
process becomes costly if used in large-scale fermentation operations, and cheaper
raw material options supplying a similar mixture of vitamins, minerals and cofactors
may require evaluation at pilot tank scale; e.g. testing corn steep liquor, dried yeast,
or mixed bulk protein sources as a replacement.

5. pH

pH tends to be a sensitive fermentation output variable and potentially a controlling

parameter, so it is therefore valuable to track pH at all stages of a scaling operation.
At shake fl ask scale stages, it may be relevant, to consider using buffers to ensure
pH does not drift to an extreme. pH changes can be the result of degradation or
uptake processes, e.g. proteolytic breakdown can result in release of ammonia,
causing pH increases. When carbon sources become limiting, the organic acids are
then consumed, altering pH. As nutrient availability tends to change with changing
scale, then pH changes can also alter and the changes may have positive or negative
impacts on the required outcome.

6. Shear

Shear tends to be low in shake flask cultivation systems, increasing dramatically

when a process is scaled to stirred tank reactors, as these are designed to be high
shear mixing systems for optimal gas transfer. Shear rates can also change during a
fermentation, if the organism produces a polymeric product such as, for example,
Xanthamonas campestris, producing xanthan gum, which shows pseudoplastic
tendencies.

7. Temperature Maintainence

7. Temperature Maintenance

At a small scale venue, biomass quantities are relatively low and heating can be
supplied relatively easily to an incubator or stirred tank reactor. However, as reactor
volume increases, so significant heat is generated during aeration and agitation.
Cooling capacity can be delivered to a large fermenter by using a jacketed vessel
supplied with cooling or chilled water, or the vessel may have internal cooling coils
or the vessels can be sited in the open air and be sprayed with water to cause cooling
by evaporative loss

8. Partial Pressures
In larger fermenters, back pressure tends to be used to help protect the sterile
envelope of the fermenter. In addition, in production scale venues of 100 000 litres
plus, hydrostatic pressure at the base of the fermenter can be significant.

9. Genetic Changes in the Population

Culture degeneration can be minimized by ensuring that a master culture stock is

used to generate a working cell bank and to ensure that each inoculum unit is taken
from the working cell bank and not from serial subculture. if unexplained results are
demonstrated during scaling, it can be valuable to check the culture microscopically
and by plating out to establish that the single colony phenotype has not altered
during the scaling operation

Approaches for deciding the number of stages in a scale up programme

(i) To have a scale-up plan with a relatively large number of scale changes, ideally
each scale change being 10 times larger than the former volume, to avoid large
increases of volume for each stage of scale up, and to minimize the risk of volume
increase providing novel variables during the scaling exercise. This approach focuses
on supply-side requirements. Production operations were relatively typically set up
with vessels for four or five scale transitions in the 1960s to 1980s.

(ii) To employ the minimum number of stages, so enabling maximum focus of

resource (manpower particularly) for investigating the biological responses at each
stage. This approach focuses more on demand-side requirements than does the first
approach. New production facilities are generally set up with one, or at most two,
scale transitions

Steps followed during planning of scale up

The recommended approach to a scaling exercise is to:

(i) Identify the most appropriate equipment to use based on preliminary analysis of
available information about process, potential impact of scaling on the process
variables and equipment availability.

(ii) Identify minimum success criteria for each scaling stage – criteria that have to be
met before it is worth transferring the process to the next scale stage. For example a
target for a microtitre stage may be relatively crude, such as the identification of a
condition that would generate a result 10% statistically different from control.
Criteria for a transferring a process to the next stage for a pilot result are likely to be
much more similar to the target criteria, such as achieving a product quality meeting
a minimum specified limit, and yield sufficient to enable generation of material in
quantities for clinical trials.
(iii) Transition to the next stage: start by trying to replicate the most productive
output of the previous stage, then explore a response surface to key variables and
focus on those results that shift the process closer to target.

(iv) Transfer to the next scale of operation once at least the minimum success criteria
have been achieved. If time and resource are available, continue to optimize at the
smaller volume stage until time runs out. Invariably it is more cost effective to
optimize in pilot scale vessels than in the production scale. It is also extremely
helpful to have licence submissions of at least partially optimized processes, with
practical ranges relating to product quality attributes.

Different Vessels used for scaling up

Microtitre/Minidish Scale Process Stage (10–1000 μL)

Microtitre dishes are first stage venues for liquid cultivation in high throughput
screening and are typically in arrays of 8 × 12 wells in both deep- and shallow-well
plate format (with working volume of 100–400 mL working volume). They can be
used in manual and automated modes and can be incubated in static and shaking
conditions

Shake Flask Scale Process Stage (50–500 mL) and Minifermenter

Shake fl asks are useful screening systems for starting to evaluate the output of a
high throughput screening programme or evaluate a range of clones, to explore
potential for expression of a specific product, evaluating raw material options, or
raw material variability. Evaluation of Temperature ranges, pH ranges , speed of
incubation, range of nutritional responses on a media using either inorganic, organic
or mixed sources of carbon and nitrogen and inoculum preparation

Stirred Tank Reactor Process Stage (1 litre – 500 + litre)

i) Laboratory fermenters Typically these are of the order of 500 ml–5 litre working
volume equipped with agitators, temperature control and are sparged with air

ii) Pilot-scale fermenters (in-situ sterilizable). Pilot-scale fermenters usually tend to have
working volumes of 20, 100, and 1000 litres

SCALE UP OF CELLS IN SUSPENSION CULTURES

Scale-up in suspension is the preferred method as it is simpler. Scale-up of

suspension culture primarily involves an increase in the volume of the culture. Small
scale generally means the culture capacity less than 2 litres volume (or sometimes 5
litres).
1. Stirred suspension cultures: It is usually necessary to maintain cell strains in
stirred suspension cultures, by agitation (or stirring) of the medium. The stirring of
the culture medium is achieved by a magnet encased in a glass pendulum or by a
large surface area paddle. The stirring is usually done at a speed of 30-100 rpm. This
is sufficient to prevent sedimentation of cells without creating shear forces that
would damage cells.

There are four common types of stirred suspension cultures

1. Stirrer Culture:

A diagrammatic representation of a stirrer flask is shown in Fig 37.2. The size of the
stirrer flask is in the range of 2-10 litres. It is fitted with a magnetized rotating
pendulum, and two side arms — one for the addition of cells and medium, and the
other for the supply of CO2.

The stirrer culture vessel is autoclaved (at 15 lb/ in2 for 15 minutes), and is then set
up as in Fig. 37.2. The flask is seeded with the culture. Then medium along with an
antifoam agent is added. The flask is connected to CO2 and stirred at a speed of 60
rpm. The flask is incubated for about 2 hours.

The contents of the small stirrer flask are transferred to a large flask and the entire
set up is restarted. Incubation at 37°C is carried out for 4-7 days. The growth of the
cells is monitored daily, and the cells are counted. There is a tendency of the cells to
enter apoptosis, if the concentration exceeds 1 × 106 cells/ml.

2. Continuous Flow Culture:

In a continuous flow culture, it is possible to keep the cells at a desired and set
concentration, and maintain. This is carried out by a bio-stat or chemo-stat (Fig 37.3).

Continuous flow culture consists of growing the cells at the mid-log phase, removal
of a measured volume of cells, and replacement by an equal volume of medium. The
equipment, specially designed for this purpose has the facility for removal of the
cells and addition of medium.

The flow rate of the medium addition can be determined from the growth rate of the
culture. The medium flow can be regulated by a peristaltic pump. By this technique,
it is possible to keep the culture conditions constant rather than to produce large
number of cells. The continuous flow cultures are useful for monitoring metabolic
changes in relation to cell density. However, these cultures are more susceptible to
contamination.

3.Air-Lift Fermenter Culture:

The major limitation of scale-up in suspension culture is inadequate mixing and gas
exchange. For small cultures, stirring of the medium is easy, but the problem is with
large cultures. The design of fermenter should be such that maximum movement of
liquid is achieved with minimum shear to damage the cells.

A diagrammatic representation of an air-lift fermenter is depicted in Fig. 37.4. A 5%

CO2 in air is pumped through the bottom of the fermenter. The bubbles formed
move up to agitate and aerate the culture. These bubbles carry a flow of liquid along
with them and release at the top which goes to the bottom for recycling.
It is possible to continuously supply O 2 to the culture in this technique. Air-lift
fermenter culture technique is suitable for fragile animal as well as plant cells. This
fermenter is extensively used in the biotechnology industry for culture capacities up
to 20,000 litres.

4. NASA bioreactor:

NASA (National Aeronautics Space Administration, USA) constructed a bioreactor

to grow the cells at zero gravity by slowly rotating the chamber (Fig. 37.5). The cells
remain stationary and form three-dimensional aggregates and this enhances the
product formation. In the NASA bioreactor, there is almost no shear force; hence the
cells are not damaged.
As the culture chamber stops its rotations, the cell aggregates sediment and the
medium can be replaced.

2. Static suspension cultures: Some cells can grow in suspension cultures, without
stirring or agitation of the medium, and form monolayer cells. However, static
suspension cultures are unsuitable for scale-up.

SCALE UP OF CELLS IN MONOLAYER CULTURE

1. Roller Bottle Culture:

A round bottle or tube is rolled around its axis (by rollers) as the medium along with
the cells runs around inside of the bottle (Fig. 37.6). As the cells are adhesive, they
attach to inner surface of the bottle and grow forming a monolayer.
Roller bottle culture has certain advantages.

i. The medium is gently and constantly agitated.

ii. The surface area is high for cell growth.

iii. Collection of the supernatant medium is easy.

There are limitations in roller culture.

i. Monitoring of cells is very difficult.

ii. Investment is rather high.

2. Multi-surface Culture:

The most commonly used multi-surface propagator of monolayer is Slunclon cell

factory (in short Nunc cell factory). It is composed of rectangular petri dish-like units
with huge surface area (1,000-25,000 cm2). The units are interconnected at two
adjacent corners by vertical tubes (Fig. 37.7). The medium can flow between the
compartments from one end.

The cell factory is almost like a conventional petridish or a flask with multiplayer
units. The main limitation of cell factory is that it is very difficult to monitor the
growth of cells. The major advantage however, is its simple operation to produce
large number of cells.

3. Multi-array Disks, Spirals and Tubes:

The surface area for growth of monolayer cultures can be increased by using disks,
spirals or tubes. They are however, not in common use as their commercial
importance is limited.

4. Micro-carrier Culture:

Monolayers can be grown on small spherical carriers or micro-beads (80-300 pm

diameter) referred to as micro-carriers. The micro-carriers are made up of any one
the following materials (trade names given in brackets).

i. Plastic (acrobeads, bioplas).

ii. Glass (bioglass, ventreglas).

iii. Gelatin (ventregel, cytodex-3).

iv. Collagen (biospex, biospheres)

v. Cellulose (DE-52/53).

vi. DEAE Dextran (cytodex I, dormacell).

The micro-beads provide maximum surface area for monolayer cultures. This
actually depends on the size and density of the beads. The cells can grow well on the
smooth surface at the solid-liquid interface. However, micro-carriers need efficient
stirring without grinding the beads. The main advantage with micro-carrier culture
is that it can be treated as a suspension culture for all practical purposes.

Micro-carriers can be cultured in stirrer flask (See Fig. 37.2) or in continuous

suspension (See Fig. 37.3). In fact, the suppliers of micro-carriers provide the
technical literature and other relevant information for setting up a micro-carrier
culture.

SCALE UP OF CELLS IN PERFUSED MONOLAYER CULTURE:

The growth surface areas of the monolayer cultures can be perfused to facilitate
medium replacement and improved product formation and recovery. The perfusion
can be carried out with pumps, oxygenator and other controllers. Perfusion of fixed
and fluid-bed reactor is briefly described.
 I.
Fixed-bed reactors:

The fixed-bed reactor has a bed of glass. The medium is perfused upwards through
the bed. The cells are grown on the surfaces of the beads. The products can be
collected from the top along with the spent medium. Instead of glass beads, porous
ceramic matrix with micro-channels can also be used in fixed-bed reactors.

II. Fluidized-bed reactors:

In a fluidized-bed reactor, the beads are suspended in a stream of medium. These

beads are porous in nature, and are made up of ceramics or a mixture of ceramics
mixed with natural products such as collagen. They are of low density and float in
the medium. The flow rate of the perfused medium is equal to the sedimentation
rate of the beads. The cells can grow as monolayers on the outer surfaces and inside
of the porous beads.

III. Other perfused monolayer cultures:

Membrane perfusion, hollow-fiber perfusion, matrix perfusion and

microencapsulation are among the other techniques for perfusion of monolayer
cultures.