Bioreactors are the places where proteins for biotechnology are made during upstream processing. Today, bioreactors can be vessels made of plastic, glass or steel in which cells are cultured or, alternatively, whole plants or animals can be genetically engineered to produce a particular protein (transgenics). In this discussion we will focus on the types and characteristics of the glass, steel or plastic bioreactors used to house suspension or fixed cells for the purpose of making proteins during upstream processing.
Bioreactor vessels (also referred to as fermenters) can be almost any vessel capable of holding cells. In this course we will be using 50ml and/or 500ml glass Bellco Spinner Bottles with a plastic cap for holding a shaft with an impeller for mixing and a glass 5000ml (5liter) Bioflo 3000 bioreactor with a stainless steel headplate for holding a shaft with an impeller for mixing, various sensors, a sparging tube and a sampling port for culturing suspension cells for upstream processing of proteins. In addition, during the scale-up of bacterial and yeast cell cultures for upstream processing, we will also be using shake flasks and/or test tubes which also could be termed "bioreactors". Other simple bioreactors include t-flasks and roller bottles. The illustrated SOP's for assembling a Bellco Spinner Bottle and the Bioflo 3000 reactor vessel can be found by double clicking on the appropriate hyperlinked text of this paragraph. Once assembled, the bioreactor is sterilized either before or after the addition of media. The sterilization is either in place (SIP) or the bioreactor is placed in an autoclave and sterilized there. Following sterilization of the first bioreactor used in the scale-up process, one milliliter of cells to be cultured is added to the media in this bioreactor. Typically, the one milliliter of cells comes from a master cell bank laid down during process development and stored in liquid nitrogen or in a -86 degree Centigrade freezer. This one milliliter of cells from the master cell bank ordinarily contains about 1 million live cells. After these cells have grown and multiplied a number of generations so that the concentration of cells reaches 1 million cells/ml again, this batch of cells is added to a larger bioreactor vessel containing a larger volume of media. Scale-up continues until the cells reach the final bioreactor where the final stage of upstream processing occurs. The media or the cells are then harvested for the downstream processing of proteins produced during culture in the final bioreactor.
Animation of a Bellco Spinner bioreactor vessel
The final bioreactor used in industry for upstream processing of proteins is far larger than the final bioreactors used in this class. Typically, for the production of human therapeutic proteins, a stainless steel bioreactor constructed of the best quality (316) virgin steel with a working volume of 2000 to 5000 liters is used. However, stainless steel bioreactors with working volumes of up to 100,000 liters are also in use today. The most common shape for these stainless steel bioreactors is a vessel that is about as wide as it is tall containing a Rushton impeller which looks a bit like a boat's propeller. This is a stirred tank bioreactor. An alternative to this shape is the airlift bioreactor which is quite a lot longer than it is wide (a kind of bullet shape) that uses air sparged at its bottom as a mixing device.
There are many more alternatives to these typical final bioreactors. At one company, the final bioreactor for the manufacture of the protein, tissue plasminogen activator (tPA), is carried out in multiple 10 liter glass Bellco Spinner Bottles, with previous scale-up steps carried out in 1000ml and 100ml Bellco Spinner Bottles. (Bellco Spinner Bottles are available from 25ml to 36,000ml.)
For industrial upstream processing of proteins various types of plastic bioreactors are also in use including fixed cells in a fluidized-bed bioreactor. In this bioreactor the cells are fixed in a compartment and media is constantly pumped to the cells in the fixed compartment and constantly withdrawn and pooled for downstream processing. Another plastic bioreactor is the hollow fiber bioreactor in which cells grow inside hollow fibers or tubes and media is pumped either through the hollow fibers or over the surface of the hollow fibers and constantly withdrawn and pooled for downstream processing. Another sort of bioreactor is the perfusion bioreactor.
In industry, where quality control in the form of cGMP is a necessity, many parameters of bioreactors must be controlled and recorded. It is typical to use biosensors connected to computers to control and record these parameters. This is referred to as process control.
This allows for the monitoring of various parameters and provides negative feedback to insure that the bioreactor will operate within pre-defined limits. This is not un-like our bodies which have sensors and negative feedback mechanisms to keep us operating in homeostasis (or within pre-determined limits). The physical parameters that can be measured on-line during fermentation or cell culture include pH, temperature, dissolved oxygen (DO), dissolved CO2, impeller speed (or agiation rate), torque, power, foaming, volume of liquid, CO2 and O2 in exit gasses, substrate concentration, product concentration, and biomass concentration (or turbidity). Some of these parameters are still measured by fermentation or upstream processing operators or technicians, by "hand". For instance, it is common for the operator or technician to remove samples at periodic intervals and check the biomass concentration or turbidity by taking an OD reading on the spectrophotometer and also by staining the cells in the sample with vital dyes to determine the live cell number/ml of medium. Quality Control technicians often measure the concentration of the protein of interest in such samples. Many sensors are still under development inlcuding those that would measure the concentration of various enzymes, substrates, ions, ATP, DNA and RNA and others.
In our laboatory, the process controlled New Brunswick Bioflo 3000 bioreactor is connected to a computer loaded with New Brunswick AFS Software. In our yeast culture protocol for the making of human serum albumin (HSA) control of pH, DO, and the addition of media will take place automatically. A schematic representation of a pH control loop and a dissolved oxygen control loop taken from one of your texts is shown below.
REFERENCES:
BIOTOL 1992 Chapter 7: Control of environmental factors influencing growth. In In Vitro Cultivation of Micro-organisms, Butterworth-Heinemann, Stoneham, MA.
BIOTOL 1993 Chapter 8: Large scale animal cell cultures. In In Vitro Cultivation of Animal Cells, Butterworth-Heinemann, Stoneham, MA.
Hoare, Mike, Andrew Gill and Peter A. Lowe 1996 Optical biosensors to quantify product for fermentation monitoring. Genetic Engineering News, August 1996: 6.
Clapp, Ken and Lisa W. De Garrido 1996 Improving bioreactor performance by using mass flow controllers. Genetic Engineering News, August 1996: 13.
Merchuk, Jose C. 1990 Why use air-lift bioreactors? Trends in Biotechnology 8: 66-70.
New Brunswick BioFlo 3000 and other products made by New Brunswick
Bellco Glass Spinner Flasks and other products made by Bellco Glass
Sonia Wallman, NHCTC. 1997