The Processes

Mar 10, 2006
By BioPharm International Editors

Fermentation is an industrial process, but it's probably easier to understand it as something more familiar: raising a living creature. Every fermentation process has to answer several basic questions: Where will the cells live and grow? What will they nourish themselves with? Will the cells live suspended in a mix of water and nutrients (the medium) or do they need to be contained or allowed to attach themselves to a solid base? How will food and air be distributed so all the individual cells get their share? Should all the nutrients the cells need be added at one time, or should the cells be fed additional food as fermentation progresses? How will the progress of fermentation be monitored to ensure the best possible results?

Fermentors and Bioreactors

The most basic piece of equipment for bioprocessing is the fermentor or bioreactor — the container where cells are grown in a liquid medium. The two terms are roughly synonymous (and will be used interchangeably in this discussion), but in the industry, fermentor refers to the vessel in which fermentation of single-celled organisms takes place. A bioreactor is the vessel for cell culture of animal cells.

Bioreactors come in many sizes, from lab-scale devices holding a liter or so to production tanks that can accommodate tens of thousands of gallons. Stainless steel is the material of choice, though bioreactors are also made of glass and plastic, especially if they are intended for use in the laboratory. The reactor is equipped with fittings and ports that allow water, air, and other ingredients to be added. Pumps move fluids about, and filters guard against impurities. Inlet gas is sterile filtered. Exhaust gas goes through condensers to remove water droplets and vapor, then through sterilizing filters. Valves direct fluid and gases in and out of the tanks.

Sterility is crucial in bioprocessing, so bioreactors are designed for easy cleaning. Smaller units can be sterilized in an autoclave, but most systems are cleaned in place (CIP) without disassembly, using chemicals or steam.

Cells require a very specific environment to grow. Technicians must control pH, dissolved oxygen levels, pressure, temperature, foaming, and concentrations of nutrients and waste products. Fermentors and bioreactors are equipped with sterile probe devices for process monitoring and control. Large-volume systems usually have back-up monitors in case of failure. Process sensors are calibrated regularly, and the medium is kept at optimum conditions for cell growth.

Growth is monitored by taking samples from the fermentor and counting cells on a hemocytometer, using chemical stains that distinguish between dead and living cells, or by measuring packed cell volumes or culture optical density. HPLC and ELISA are two of many methods for measuring product concentration in broth. One of the most straightforward ways of monitoring is photometry: A carefully controlled beam of light is passed through a sample of the liquid in the reactor. As the fermentation process proceeds, the light is dimmed more and more. Photometry does not provide detailed information about what is happening in the bioreactor, but it is quick and allows for rapid real-time adjustments.

Types of Bioreactors

The fermentation process begins with an inoculum culture, sometimes grown in small flasks or specially designed bottles called roller bottles. At a certain cell density, the contents of those bottles are aseptically transferred to a small bioreactor. When that volume has been filled, the inoculum is transferred to an even larger device. There are a number of different types of reactor to choose from.

Stirred-tank reactors are the workhorses of bioprocessing. Perhaps 90% or more of current biopharmaceutical production takes place in this sort of vessel. A stirred-tank reactor is just what the name suggests: a simple tank with a motor-driven impeller or agitator (which often looks a bit like a propeller) to stir the brew of cells and medium, ensuring that air and nutrients are evenly distributed. Air is added by sparging (spraying through a perforated plate in the bottom of the vessel) or surface aeration.

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