DESIGN BIOREACTOR

FOR THE PRODUCTION OF

BIOPHARMACEUTICALS
FROM ANIMAL CELLS

2017/8/9
 CHE 654
BIOREACTOR ENGINEERING

EH242 (5A)
14 DECEMBER 2016
IR. PN NORMADYZAH BT

PRESENTED BY:

SHAZREEN IZZATI BT AHMAD AZMI (2014880054)

NURUL ALIA AMIRA BT JASMANI (2014671946)

NURUL IZZATY BT SIDDIK (2014607304)

ZURAIDAH BT DOLLAH (2014274422)

NUR AMIRA BT ABDUL RAHMAN (2014623042)

2017/8/9
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SUBSTRATE /
BIOMASS / PRODUCT
 ANIMA NUTRI
CELL BIOM
PROD ASS
L CELL ENTS UCT

SUBSTRATE, CO2
Collagen,(ossein) PRODUCTS,
S (Protex) Lactate
(Gelatin)

Animal cells, X is the cell that will be use to produce the
products.
Example: Collagen getting from bone of animal, ossein
Nutrients is the substrate that is being consume by the
cell to live.
Example: Protex
Cell products is the products that being harvest from the
reactor.
Example: Gelatin
Biomass is the left over substance from the reactions.
Example: Usually carbon dioxide and lactate

DESIGN
BIOREACTOR
Reactors use for suspension culture
The reactors use for large scale suspension cultures are of 3
main types:-

Stirred tank bioreactor (main use in industry)

Airlift bioreactor

Continuous Flow bioreactors

STIRRED-TANK BIOREACTOR
Simplicity and the ease of monitoring and controlling scale up
Single use
Able to provide efficient gas transfer to cells.
Homogenous systems when used at smaller scales.
Successfully used in the production of several bio the rapeutic products.
Bioreactor types range from small, <1-L benchtop units to 10,000-L systems
for large-scale industrial applications.
Can be batch type or continuous flow
Tight control over parameters such as temperature, moisture, pH, oxygen,
and stirring rate will produce the most satisfactory results.

Data Required for Bioreactor Selection and Design

Microorganism species
Final use product
Growth and oxygen requirements
Cleaning and sterilizing
Shear and rheology effects
Heating and cooling
Sterility
Measurement systems
Volumetric output
Materials construction
Impellers:
- The ratio of the impeller diameter to the diameter of the tank (di/dt) should be between 0.3 and 0.5.

Impeller Spacing:
- The spacing between impellers should be 1.0di to 2.0di , where di is the diameter of the impeller.
- Proper spacing between impellers should be Di < Hi < 2Di

Baffling:
- Stirred tank fermenters generally use baffles because of the need to disrupt the bulk fluid flow in the
tank.
- 4 baffles if the tank diameter less than 3m
- 6-8 baffles if the tank diameter larger than 3m
- Width of baffles, Dt/10 and Dt/12
- Diameter of vessels baffle 10< Dt/Db< 12

Tank Height:
- The height to diameter ratio of the tank is typically between 2.0 and 3.0; however, taller tanks (up to
HL/dt=4.0) have been used to reduce the power requirement of the impellers.
- Diameter ratio between 2:1 to 3:1
- If H=D, one agitator is enough
- If H=2D or more, additional set of agitator should be added.

Volume:
- 75% of the total vessel volume is filled with liquid
- 25% filled for gas space
- If foaming takes place, there is a chance of immediate contamination
Standard geometrical Dimension of STR
STIRRED-TANK BIOREACTOR
Advantages Disadvantages
Low operating cost Foaming is often a problem but it is
overcome by using proper antifoaming
agents
Low investment need Size limitation by motor size, shaft
length and weight
Continuous operation
Easy to clean
Simplicity of construction
Good temperature control
Easily adapts to two phase runs
AIRLIFT FERMENTORS
Air driven
External and internal loop system
Used in a number of large-scale processes

CONTINUOUS FLOW BIOREACTORS

Produce a cheaper better quality product at reduced energy and
environment cost
Optimum mixing ( efficient mass transfer )
Optimum temperature control ( efficient heat transfer)
The application of the scalling out approach (as opposed to the
traditional scale up approach)

PARAMETERS
INVOLVED IN DESIGN /
SCALE UP /
OPTIMIZATION
 REQUIREMENTS FOR A BIOREACTOR FOR
ANIMAL CELL CULTURE

Properties of animal cell that set constrains on design of animal cell
pharmaceutical bioreactor:
Cell are large (10-20m diameter)
Slow growing than most bacteria and fungi (td = 10 to 50h)
Very shear sensitive
More fragile
Toxic metabolites (eg. ammonium & lactate produced during growth)

General features of animal cell pharmaceutical bioreactor:

Reactor should be gently aerated and agitated
Agitation speeds = 20 rpm
Bubble column and airlift reactors operating at high aeration rates (cause shear
damage to cells)
Well-controlled homogenous environmental
Supply of CO2-enriched air
A large support material-surface-volume ratio (for anchorage-dependent cells)
Removal of toxic products of metabolism (lactic acid)
Require gentler culture condition and control systems
PARAMETERS FOR DESIGN/SCALE-UP



pH control
For small scale operation, can maintain pH by using gaseous CO2.

The acid is added by sparging carbon dioxide from the bottom of the reactor. As
the CO2 bubbles its way to the top, it forms carbonic acid decreasing the pH. The
base is added with carbonate as a fluid.
 For large scale cultures, can directly add acid or base

How?

- insert probe into culture to detect change of pH

acid or base pumped accordingly, under automatic control

lower the pH, add base (concentrated sodium bicarbonate)

increasing the pH, add acid (concentrated HCl)


Why control the pH?

Although cell culture media typically provides substantial buffering of pH,

mammalian cell metabolism routinely decreases the culture pH due to the
production of lactate and carbon dioxide, both of which are acidic in
nature.

Excessive hydrogen ion concentration may alter normal cell metabolism

and proliferation by impairing substrate uptake and product release.
Agitation
The mixing inside the bioreactor must be appropriately controlled, then the cell

will experience a very similar environment.

Eventually keeps the cells in the perfect homogenous condition for better
transport of nutrients and oxygen for adequate metabolism of cell to the desired
product(s).

Different impellers for suspension cells and cells grown on carriers were
investigated for their suitability to ensure homogeneous gentle mixing.

Agitation speeds = 20 rpm

Examples:

I. Large pitch blade impeller & novel 3-blade segment blade to ensure
homogenous gentle mixing.
Pressure Control
The stainless steel bioreactors are maintained under positive pressure to create an

environment that is more conducive to axenic operation.

Industrial bioreactors are designed to withstand a specific working pressure.

Pressure measurements are required as a factor of safety.

It is important to fit the equipment with devices that sense, indicate and control
pressure.

Pressure measuring sensors:

1. Bourdon tube pressure gauge

2. Diaphragm gauge

3. Piezoelectric gauge
Foam Formation
The appearance of foam is very undesirable phenomenon, since there is a risk to lose
an essential part of medium broth.

During foaming, it is not possible to perform high quality analysis and measurements.
How to eliminate foam formation?:
Addition metering of antifoam based on sensor
Probe is inserted through top of bioreactor, stainless steel rod set at a defined level above
broth surface.

When foam rises and touches the probe tip, pump is activated and antifoam is released into
bioreactor

Mechanical metering of foam

Mechanical antifoam devices: discs, propellers, brushes or hollow cones attached to agitator
shaft above broth surface.
Foam is broken down when it is thrown against the walls of bioreactor.
Temperature

Ensure actively growing and productive cell

Bioreactors temperature is regulated by adjusting the temperature of the jacket

surrounding the bioreactor and through the use of heat exchangers

Increase temperature cause increase in rate endothermic reaction, thus enhance

speed of conversion in cells

Excessive increase temperature lead to enzyme denaturation, reduce speed the

reaction they catalyse

It varies between 36 and 38C for mam-malian cells, while it lies between 25 and 30C

for insect cells

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Cell Density

Continuous culture reach higher cell densities than batch system

- because they provide more nutrients to cells by medium exchange

Approximately = 30-80million cells ml-1

This densities occupy about 10-30% of volume in bioreactor

The high cell density, must be in control of mixing, aeration and process

control
Dissolved oxygen & Dissolved carbon dioxide

DISSOLVED OXYGEN

Cell culture require oxygen for production of energy from organic carbon sources (eg, glucose)

The control of oxygen flow is needed to ensure it does not become a rate-limiting factor in process.

The hyperoxygenated air supply can give impact to culture performance.

High DO level can increase reactive oxygen species causing oxidative damage to cells and products.

At constant temperature, the DO concentration in culture media is proportional to amount

of oxygen in vapour phase within media.

DO probes is used to measure the reduction of oxygen.

Probes is allowed to polarize prior to the use.

Calibration is first performed.

DISSOLVED CARBON DIOXIDE

Dissolved and evolved carbon dioxide level can be indicative of cellular

metabolism

High carbon dioxide may inhibit the growth, metabolism and product

quality

PH and temperature may effect the CO2 control


BEST PRACTICES
IN LAB SCALE /
INDUSTRIAL SCALE
 1. Behaviours of cell culture process in
bioreactors.

During this process, factor that can reduced the
productivity is:
Shear stress
Oxygen supply
Gas compositions
 2. Physical characteristics

Mixing time
Shear
Mass oxygen transfer
Heat transfer
pH
 3. Process characteristics

Reactor geometry
Volumetric oxygen transfer coefficient
Maximum shear
Power input per unit volume of liquid
Volumetric gas flow rate per unit
Volume of liquid
Super ficial gas velocity
Mixing time
Impeller Reynold Number
Momentum factor
4. Aeration and agitation strategy

Need to be carefully design
-To prevent dissolved CO2 accumulation on a large
scale
How?
- Decrease in top impeller position will increase the
aeration rate, will help lower the dissolved oxygen level
and increase the final fiter by 100%
 THANKYOU :)

2017/8/9