1. Bioreactor Basics
Mini-BIOMAN, July 2017

2. What is a Bioreactor?
Vessels or containers designed to support the optimal growth and metabolic activity of cells producing a product of interest
Can be classified in several ways including:
Type of mixing
Stirred Tank
Airlift
Mode of operation
Batch culture
Fed batch
Perfusion
Type of vessel
Stainless steel/glass
Single use/ disposable

3. Ways to Prepare Bioreactors CIP/SIP Systems
CIP (Clean In Place)
automatic cleaning of processing equipment, vessels, piping and in-line devices
minimal manual setup or shutdown
little or no operator intervention during cleaning
strong base, rinse, strong acid, final rinse with WFI
conductivity tests to monitor content of cleaning solutions and rinse water
SIP (Steam In Place)
equipment and vessels sterilized with clean steam
After sterilization system must remain pressurized to maintain sterility
Vessels used during process and all associated piping/hoses must be free of any foreign substances prior to use
include cell debris, media, cleaning chemicals, and target protein from prior batch

4. Cell Culture Media for Bioreactors
Provides all the nutrition cells need within a narrow window of environmental conditions for optimal expression of the target protein
Major media components
Carbohydrate energy source – glucose
Nitrogen source such as amino acids
Lipids – often in the form of fatty acid
Cells also require
Trace minerals in the form of electrolytes (salts)
fetal bovine serum supplements – not so common due to risk of animal viruses
Chemically defined, serum free media commonly used – reduces threat of adventitious animal virus contaminants
Selective agents that cells require for optimal expression of the target protein
Cells with transfected gene will be able to thrive and make the target protein; cells without will die

5. Bioreactor- Mixing Types
Stirred Tank
Required gases (e.g. 02) nutrient media, cells are continuously stirred by agitator impellor (stirrer) at the bottom or top of the vessels
Baffles in the center of vessels ensures proper mixing and prevents formation of vortexes that might shear cells
Airlift
Gas is pumped from below through a sparge tube within the bioreactor creating bubbles which mixes the contents of the vessels
Contains baffle that guides gas up through bioreactor on one side of the baffle and then over and down the other side

6. Bioreactor Types – Stainless Steel
Made of durable material that can accommodate high volumes (up to 20,000L) of culture
Double walled, glycol jacketed with 4 layers that provide insulation/temperature control and sterile contact for cell cultures
Very large industrial sized reactors bioreactors have fixed vessel configurations with predefined port assemblies that can not be easily reconfigured
Expensive and time consuming cleaning procedures
high costs to produce purified water and steam for cleaning (CIP/SIP)

7. Types of Bioreactors- Glass and Stainless Steel

9. Bioreactor and utilities and piping

10. Top of 20,000 liter final bioreactor

11. Stainless Steel Bioreactors

12. Bioreactor Types – Single Use
Disposable bioreactors- intended for one time use
Components are typically made of plastic and are disposed of after use
Generally used for mammalian cultures
Cultivation chamber is inflated plastic bag
Single use technology in biomanufacturing is becoming widespread - has advantages and disadvantages

13. Advantages of Single Use Technology
Reduction or elimination of cleaning, sanitization, and sterilization steps. This reduces:
consumption of water – pure water is extraordinarily expensive to produce
energy used to produce purified water
consumption of cleaning and sanitizing chemicals (CIP)
eliminates the need to sterilize bioreactors (the vendor has done this)
eliminates the need to generate clean steam
need for cleaning and sterilization validation (and moves it to the vendor) – eases regulatory compliance

14. Single Use Advantages Continued
lower upfront capital costs – this becomes a major advantage for a small company or a new company wanting to start production quickly
faster cycle times and faster, less expensive changeover between campaigns – less fear of cross-contamination (by eliminating the need for cleaning)
lower risk – lower probability of cross-contamination with another product or microbial contamination

15. Single Use Disadvantages
Scale Limitations (only up to 2000 liter cell culture)
increasing product titers and cell densities are making this less important
Limited to mammalian cell culture - low oxygen transfer coefficient rates excludes use of bacteria
Increased reliance on outside vendors – potential supply chain problems
Concerns over leachables/extractables from the plastics
Additional consumables cost
Environmental impact – increase in solid waste that currently goes to landfill

16. Disposable wave bioreactor and its mechanics

17. Single Use Bioreactors

19. Applikon eZ Control BioreactorSystem
Jacketed Stirred Tank Reactor + Controller
Controller - Front view
eZ Controller
combines and supports actuators, pumps, and valves to optimize bench space
Controller for measurement and control of process variables –pH , temp, DO, agitation
Control Setpoints
Temperature °C pH
DO %
Agitation rpm

20. Control Panel
No foam parameter button on our model

21. Bioreactor Vessel

26. Cell Culture Parameters
Cell Density & Viability
Automated Cell Counter; Trypan Blue Assay
Temperature – 37°C
Monitored by temperature probe
Dissolved oxygen – cells need oxygen to live
Controlled by DO probe and computer system based upon the rate at which oxygen molecules diffuse a membrane covering a set of electrodes
Glucose and Lactate
Monitored by Biochemistry Analyzer or spectrophotometric method
Bioreactor filled with media; media equilibrated to meet setpoint ranges for temperature, DO and pH
Cells are inoculated into the bioreactor
Oxygen is a limiting nutrient - due to low solubility in nutrient media

27. Culture Parameters- pH
Critical process parameter
Media contains bicarbonate; when combined with CO2 from air, makes a buffering system to control pH
In bioreactor, pH is monitored by an in-line pH probe and controlled via bioreactor control system

28. Culture Parameters-Glucose
Glucose + O2
CO2 + H2O
cells
Main energy source for cells to grow, metabolize

29. Why measure Lactate? Lactate is a product of glucose break down
During cell proliferation glucose concentration decreases and lactate concentration increases

30. Bioanalyzer

31. YSI Analyzer 2900 User Interface
2700 SELECT
Reagent Bottle
Compartment
Buffer Bottle
Waste Bottle
Calibration Standard
Manual Sample
Printer
Keypad/Display
Test Tube Holder
BUFFER
WASTE

32. Inside the 2900 Sipper Assembly Buffer Pump Sample Chamber
WASTE
BUFFER
STD
Sipper Assembly
Buffer Pump
Sipper Pump
Cal Pump
Buffer Bottle
Waste Bottle
Cal Standard Bottle
Sample Chamber
w/ Electrodes