The Processes

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.

Stirred-tank reactors are simple and familiar, and they scale-up easily from laboratory scale to full production. The problems they present have mostly to do with the damage that can be done to cells by the stirring and aeration process. Collision with the impeller or with the walls of the tank can break down cells, especially delicate animal cells. Gas bubbles released by the sparger subject the cells to surface tension forces; and to fluid mechanical forces from motion, disengagement, and bubbles bursting. Foaming can break cell walls or membranes, killing the cells and releasing lytic enzymes that denature proteins. Designers of bioreactors attempt to control these problems by modifying the design of the impeller and by adding baffles and other elements.

Airlift fermentors are designed to provide a gentler stirring action. Instead of using an impeller, these reactors create circulation pneumatically. Gas is sparged through the base of the vessel, rising in a cylindrical draft tube (the riser) to the surface, creating circulation of the contents of the reactor. They offer good oxygen transfer and easy scale-up, good mass transfer characteristics, and easy control. However, airlift fermentors do expose cells to considerable sparging and thus possible damage. Large-scale systems can require considerable space and large quantities of gases.

Packed bed reactors grow cells on a bed of glass or plastic beads, stainless steel bars, or a number of other materials — fibers, composite fabrics, even bits of seashell. A separate unit oxygenates medium that is circulated through the bed, and an aeration reservoir is used as an airlift pump. In two-chamber reactors, biomass growth occurs in a lower chamber, and cells are lifted to the upper chamber by the moving substrate and settle back down.

Some newer reactor designs attempt to create conditions closer to those found in the body to increase the productivity of animal cells.

Hollow-fiber reactors provide cells with a way to circulate oxygen and nutrients that is a little like the human circulatory system. Animal cells are grown attached to the outside of porous hollow fibers — the same type used in kidney dialysis machines. Fresh medium is circulated through the fibers, allowing nutrients to diffuse through the porous fiber walls to the cells, while toxic metabolites produced by the cells diffuse into the stream flowing away.

Hollow-fiber reactors can achieve great cell densities. In the human body, there are about 10⁸ cells/mL. Stirred tank bioreactors can normally support only about 10⁶ cells/mL. Using hollow-fiber units, biotechnologists have been able to support concentrations of about 10⁷ cells/mL.

High cell density means that more protein can be produced in a relatively compact bioreactor. Complex hollow-fiber reactors are not easy to control. Dead cells can secrete proteases and contaminate the protein products, complicating the process of isolating and purifying the desired protein. Fiber breakage can pose a sterility risk, and the fibers, which come bundled in single-use disposable cartridges, can break, causing a sterility risk.

There are other systems for allowing animal cells to grow in dense concentrations that approximate mammalian tissue. Ceramic matrix systems provide cells with a large surface area to attach to, and pack it into a relatively small volume.

One of the latest developments in bioreactors is the appearance of disposable manufacturing systems. In these systems, cells are grown in plastic bags or in tanks equipped with disposable plastic liners. Disposable systems offer many advantages in terms of scaleup, and they are a cost-effective way to eliminate much of the risk of cross contamination. At the moment, most disposable systems have a capacity of hundreds of liters, but larger systems are already in use for some applications.

Fermentation Processes

Process developers must consider a complex set of conditions that affect cell propagation, product yield, and concentration of nutrients, waste, and products. The performance of a fermentor or bioreactor is governed by thermodynamics (such as the solubility of oxygen in the medium), microkinetics (such as cell growth and product formation), and transport of materials (moving nutrients into the cells, removing waste products, and so on). Optimal mixing ensures effective oxygen transfer, heat transfer, and dispersal of materials. Minor deficiencies in circulation of the medium can have major effects on growth and protein production. System designers therefore have to consider the fluid viscosity and momentum and the sizes of the cells involved.

A key decision in designing a process has to do with the timing of how nutrients and other ingredients are added to the reactor. Batch and fed-batch processes are commonly used for microbial fermentations.

In batch fermentation, medium and an inoculum of cells are added to the fermentor at the beginning of fermentation, the system is closed, and nothing but sterile air is added for the rest of the process.

Batch processes are simpler to scale up than other processes. They offer the flexibility often required, especially in multiproduct facilities, where several different cell lines are grown to manufacture different products. Because each batch is processed separately, there are periods of downtime, which are useful for cleaning and sterilizing the reactor. In a batch process, the environment changes as nutrients are depleted and product and metabolites accumulate. In a fed-batch process, fresh nutrients are added periodically. When nutrients approach depletion, the hungry cells are "fed." Frequent addition of fresh culture medium replenishes the nutrient supply. Various methods are used for controlling the rate at which the feed medium is delivered.

In some cases, the feed is delivered at a fixed rate, which is matched to the growth rate of the culture. Tighter feed control is often accomplished by linking feed rate to the culture's demand for nutrients as measured by the pH, or the amount of dissolved oxygen being consumed by the culture. High cell densities can be achieved in fed-batch fermentations, which, in turn, result in high levels of product formation.

Continuous culture is often used for assessing scale-up issues. A continuous feed of fresh medium is supplied, and fluid containing cells and cell products is removed at the same rate. Continuous culture offers several advantages over batch processes. Fermentor use is more efficient, with less downtime. High cell density and product output can be maintained for longer periods. Labor costs may be reduced because of less frequent cleaning. This method is appropriate for proteins that are continuously produced in large quantities.

In perfusion propagation, animal cells are held at high concentrations inside a growth chamber, and fresh medium is circulated around them. That provides continuous addition of nutrients and removal of waste products. Perfusion systems are commonly used in antibody production.

Suspension vs. Anchorage

Another key distinction is between suspended and anchored cells.

In suspension cell culture, the cells being grown float freely in the culture medium. Suspension cell culture is simple and easy to manage, and it works well for bacteria and yeasts, types of organisms in which each cell is a separate, independent entity.

Animal cells are a different matter. Most animal cells evolved to live not independently but as parts of tissues or organs. They are not naturally well adapted to growing in suspension.

Scientists have taken two general approaches to working with animal cells. On the one hand, cells can be engineered to make them adapt better to growing in suspension. Today, most of the mammalian cells commonly used in bioprocessing can be grown in suspension, if desired.

A suspension cell culture system must be delicately balanced. The mix of cells in suspension needs a lot of oxygen — but too much will kill them.

Mechanisms such as agitation and air sparging are used, but both can cause hydrodynamic shear stress leading to cell damage or death. Decreased oxygen concentrations slow or stop cell growth, but keeping dissolved oxygen at a high level causes formation of precipitates after growth has leveled off. Detergents can be used to lower surface tension and increase viscosity, thus protecting against shear and foaming.

The alternative approach is to engineer the fermentation process so that cells can attach themselves to the surface of the culture vessel or some other support.

Anchorage-dependent cell culture, as it is called, takes a number of forms. In the simplest forms, cells are attached to beads or enclosed in microcarriers that are allowed to float free in the medium. In other cases, cells might be attached to the wall of the vessel, to a matrix, gel, ceramic cartridge, or to some other structure (such as the tubes of a hollow-fiber system).

Confining cells to a support protects them from mechanical stresses. Sometimes other sorts of cells can be added to the support to provide catalytic activity. The chemical composition of solid supports can create favorable microenvironments. Animal cells can achieve higher densities in attachment culture than in suspension.

Cells can be immobilized in several ways. Collagen-based beads or polymer agents protect against shearing. Gels or solid matrices can simplify downstream processing, and in stirred or aerated systems, they can offer some protection from air sparging.

At the laboratory scale, major concerns are cell viability, mass transport, and the size of membrane pores. At the industrial scale, issues such as price, complexity, and reliability come up. Problems can include leakage and polymer toxicity.

Perfusion culture is made simpler when cells are retained in place using a mesh screen. Some perfusion cell culture systems are based on ceramic matrices that immobilize cells. The technique can immobilize nonadherent cells, protecting them from shear.

Microencapsulation. Cell-containing beads are a useful immobilization method for both anchorage-dependent and independent cells because they provide sparge protection. Encapsulated cells can hold and concentrate product, but beads may not retain their integrity for long-term culture. Although capsule membranes allow small molecules such as nutrients and oxygen to diffuse through, limitations in mass transfer can lower cell viability and contaminate or degrade products. High-molecular-weight products are kept within the capsules, and low-molecular-weight products diffuse out. The cost of encapsulation can be a disadvantage, as can oxygen transfer limitations at large scales. And encapsulated cells may not receive optimal nutrients.

Microcarriers are commonly used in attached cell systems. Compared to microcapsules, microporous beads (to which cells attach themselves) are easier to use and scale up. Adsorption is the simplest system for attaching cells to a support. Cells are mixed with beads (for example) and attach themselves to their surfaces. But adsorbed cells are not protected from shear forces.

There are several ways to attach cells to a support. Covalent attachment involves a chemical bond between the cells and their support. Leakage is minimized, but chemicals can affect cell viability. Again, no shear protection is provided. With ionic to covalent crosslinking, a cell suspension is treated with polymers that form bridges between the cells, making them aggregate loosely. The resulting cloudy flocs are not particularly stable, and cell leakage is still a problem, but additives can improve the situation. Entrapment offers a gentle solution to many attachment problems. Cells are mixed with polymers or monomers to form a gel that encases them. Leakage is reduced, and many cells can be loaded.

A Medium for Growth

Cells deteriorate, die, and disintegrate (lyse) when they get too few nutrients. Nutrients are provided to cultivated cells in the form of a medium.

Different kinds of cells require different media, and vendors offer preformulated media designed for all of the cells widely used in bioprocessing. In addition to the nutritive elements, media sometimes contain additives designed to improve the fermentation process. Pluronic F68, for instance, is used to make cell membranes more resistant to shear forces. Polyethylene glycol, polypropylene glycol, or silicon-based surfactants may be used to reduce foaming.

Bovine serum has long been preferred as a culture supplement for mammalian cells because of its low concentrations of immunoglobulins, which can complicate downstream processing. It also contains mitogens and growth factors that promote cell growth. But adding bovine sera can increase costs and impede scale-up. For one thing, lots can vary in composition and quality so that cell lines can be sensitive to some lots and not to others. Availability is sometimes a problem, because of the products' traditional ties to the cattle industry. Another source of concern since the late 1990s has been the risk of "mad cow disease" or bovine spongiform encephalopathy (BSE). The prion believed to cause both that disease in cattle and Creutzfeld-Jacob disease in humans can be present in bovine serum, and it is virtually impossible to detect and remove.

To ease downstream processing of recombinant products, many companies have begun to use serum-free and protein-free media to reduce costs and increase safety in animal cell culture. The main focus in media formulation is consistency in production and performance of large lots, which can be more carefully monitored with serum-free media. But serum-free media may have some serum-sourced ingredients. By contrast, protein-free media are chemically synthesized and produced and thus well defined in composition. Both serum-free and protein-free media are manufactured with certain cell lines in mind, each providing just the nutrients a given cell line needs. Obviously, such media are more expensive. But they may make up for that cost by facilitating downstream purification. Selection of TSE-free culture medium components is also important in microbial fermentations.

Scale-Up

Some fermentation processes work well in the laboratory, only to fail at the industrial scale. Such failure can be attributed to many causes. For one thing, scaleup involves increasing bioreactor size as well as production capacity, but equipment might not function at the same efficiency on a larger scale. Scaleup affects other factors as well. Optimizing output means maximizing cell mass production and product formation. Most often, the parameters of oxygen-in and waste-out will change at higher scales. Perhaps the required raw materials are too expensive. Perhaps cells or proteins aggregate in large numbers.

Biotechnologists face many challenges when taking their processes from the laboratory to the manufacturing scale. Large-scale fermentation often gives lower yields than laboratory experiments would suggest. The goal of any commercial fermentation process is to achieve high productivities, product yields, and product concentrations. Large bioreactors are expensive, so bioprocess scientists must estimate their ultimate scale (and future needs) early. Here are some questions involved in scaling up a fermentation process.

Regarding the product. What amount will be required, and what size vessel will produce that amount? How big and how stable is the protein molecule? How will temperature and cell proteases affect it?

Regarding the cell line. What are its characteristics (such as genetic stability and anchorage dependence)? How fragile are the cells? What are their kinetics of growth and product formation? Does the product accumulate inside the cells or is it excreted to the culture medium? What will the product yield be? What levels of containment are required, and what safety issues are involved?

Regarding the fermentation vessel. From what materials (glass, plastic, or stainless steel) will it be constructed? How will media be mixed without damaging fragile cells? Will control, operation, and sterility maintenance of the reactor be difficult? What are the fixed capital costs (reactor and instrumentation) and the running costs (media, disposables, labor, maintenance, downtime, and so on) for different types of equipment? Will the process be batch, fed batch, perfusion, or continuous culture?

Cost. Can the process produce an acceptable amount of purified recombinant protein to justify all the money put into it? Or will it be so expensive that no patient will be able to afford the drug?

Viral contamination. Mammalian cell lines are prone to contamination because of the complexity and duration of their culture (one to two weeks per batch, many months for continuous culture). They host many of the same adventitious agents that humans do.

Regulatory Guidelines

Fermentation and cell culture practices in the biopharmaceutical industry are regulated by the Code of Federal Regulations (CFR), Title 21, Parts 210–211, describing current good manufacturing practices (cGMPs). Also important are 21 CFR 600–680, which talk about biologics specifically. Diagnostics are governed under Part 800, which covers medical devices. In addition to those laws, the FDA has released (through its Center for Biologics Evaluation and Research) many "Points to Consider" documents that detail further guidelines. Although such documents are not regulations that carry the force of law, FDA inspectors certainly expect to see their recommendations followed. When published in the Federal Register, however, guidelines from the International Conference on Harmonization (ICH) do carry the same weight as FDA regulations. Another source of guidance is the United States Pharmacopeia and National Formulary.

Regulations emphasize that biologic products must be produced in properly validated facilities. Validation of processes involves many different protocols. Equipment must be qualified for use. Installation qualification helps technicians determine whether equipment is installed according to requirements. Operational qualification documents the methodologies used to operate and validate each item and record measured information for analysis. Performance qualification ensures that the equipment will do the job it is intended to do. Process validation studies and conformance lots are used to demonstrate process reproducibility. Control qualification checks automation and controls. Standard operating procedures specify how work is done in the fermentation facility, and employees are trained to follow the letter of the law in all matters of research, bioprocessing, documentation, and cleanliness. In addition, regulatory agencies require characterization of all cell lines and cell banks used in producing pharmaceuticals.

Technicians working in bioprocessing plants must wear special clothing and have special training to work there. Such facilities require sophisticated process plants, with all fluid handling in enclosed systems. Process vessels and associated pipework systems must be pressure and integrity tested to ensure that there are no leaks. Stainless steel pressure vessels and pipework are sterilized using clean steam at high pressures and temperatures. All interior surfaces are designed crevice-free with drainage that prevents condensate buildup that can generate possible unsterilized cold spots. Pipework is specially welded and incorporates sanitary valves and pumps. Product contamination (by accumulation of deposits, for example) is prevented by thorough periodic cleaning and sterilizing.