Bioreactor Basics Mini-BIOMAN, July 2017

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

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

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

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

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)

Types of Bioreactors- Glass and Stainless Steel

Bioreactor and utilities and piping

Top of 20,000 liter final bioreactor

Stainless Steel Bioreactors

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

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

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

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

Disposable wave bioreactor and its mechanics

Single Use Bioreactors

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 ................37°C pH ...........................7.2 DO ...........................50% Agitation .....................75 rpm

Control Panel No foam parameter button on our model

Bioreactor Vessel

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

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

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

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

Bioanalyzer

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

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