Bioreactor Basics MiniBIOMAN July 2017 What is a

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 inline 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

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

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, 000 L) 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 10

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 crosscontamination (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 e. Z Control Bioreactor. System Jacketed Stirred Tank Reactor + Controller - Front view e. Z Controller • combines and supports actuators, pumps, and valves to optimize bench space • Controller for measurement and control of process variables –p. H , temp, DO, agitation • Control Setpoints Temperature. . . . 37°C p. H. . . . 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

Culture Parameters- p. H § Critical process parameter § Media contains bicarbonate; when combined with CO 2 from air, makes a buffering system to control p. H

Culture Parameters-Glucose Main energy source for cells to grow, metabolize Glucose + O 2 cells CO 2 + H 2 O

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 Printer Keypad/Display 2700 SELE CT Manual Sample WASTE Test Tube Holder BUFFER Reagent Bottle Compartment Buffer Bottle Waste Bottle Calibration Standard

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