Most IVD tests requiring biologics run into cost and feasibility obstacles relating to their dependence on specialized or custom reagents. Such tests and devices often require enzymes and other proteins that are not commonly available from company catalogs. Nevertheless, these materials may represent the core of the diagnostic methodology.

One way such biologics can be produced is by means of fermentation. Essentially, fermentation is an enabling method that supports the growth of microorganism bioprocessors. Under the correct conditions, these bioprocessors produce large quantities of organic by-products useful as IVD raw material.

However, using fermentation technology to make the protein for one IVD product might require a multimillion-dollar facility. And there is no guarantee that a product will not falter in testing or when scaled up for evaluation in clinical trials. If a product does not receive regulatory approval for commercialization, a company's investment in raw-material manufacturing equipment can seriously affect product pipelines, especially for cash-strapped start-ups or grant-funded projects.

Contract manufacturers can often supply the necessary raw material quickly and cheaply while saving the IVD company from substantial capital investment. Some can even take the lead in developing the final product.

The process of fermentation is timeless but the modern technology involves complex components. The main fermentation vehicle for the manufacture of diagnostic raw materials is a closed cylindrical vessel called a fermenter. It is usually made of stainless steel to facilitate heat sterilization and withstand high pressure.

Fermenters range in size from benchtop models with a capacity of 1 L to tall several-thousand-liter vats. As fermenter size increases, some or all of its parts may be custom designed and constructed.

A benchtop unit with a volume of 1L can make enough material for up to hundreds of tests, depending on concentration. For commercial quantities, a larger fermenter is needed.

Most vessels are capable of variable-speed agitation via motorized internal impellers. Impellers are engineered so that their rotation elicits gentle aeration and dispersion of cells. Additional impellers at the top of the fermenter break the buildup of foam.

Probes are used to continuously monitor acidity levels, dissolved oxygen levels, temperature, and other conditions critical to the production of the biological material. Software monitoring systems are available that work with these probes to provide 24-hour surveillance of crucial measurements. In the event of a problem, these systems can even alert the operator via wireless communication.

Many other secondary pieces of equipment play a vital role in the fermentation process. At a minimum, a liquid nitrogen storage tank is required for storing cell banks. Peristaltic pumps and other pumping devices are required at various stages of the process to add nutrients to fermenters or to move product through filtration systems. Centrifuges and high-pressure filtration apparatus such as hollow-fiber or tangential-flow devices are essential for clarification and concentration. Glove bags and boxes facilitate manipulation of anaerobic organisms.

At the bare minimum, quality control equipment should consist of a spectrophotometer; however, electrophoresis and high-performance liquid chromatography equipment should also be available. Microscopes and other tools for basic microbiology must also be part of the arsenal for validating cells during the process as well as during QC functions such as testing for contamination.

Purification columns are kept on hand and, as a particular project develops, the resin used in the purification process is purchased. Large-quantity fermentation projects may require columns with volumes up to several liters.

Cell lines are processed in similar fashion, regardless of the species. The process involves the following steps.

Establishing and Validating a Master Cell Bank. The first steps in the fermentation process are to establish a master cell bank and validate its integrity. The purpose of these steps is to ensure viability, productivity, and lack of contamination. Validation is achieved through such microscopy methods as Gram's stains or wet mounts, product activity assays, or DNA analysis.

Culturing the Cell Bank. From the master cell bank, a working cell bank is cultured. In turn, that cell bank is cultured to a primary seed, which is propagated to suitable levels for inoculation of the fermenter of the desired size.

Characterizing the Primary Seed. The primary seed is tested to ensure that it conforms to the proper specifications. This process is known as characterization. Methods of characterization for cells include wet-mount observations, optical density tests, and plate counts.

Wet mounts entail cells being suspended on a slide and then observed under a microscope. Hybridomas are counted in this way. Bacteria can be distinguished by their shapes (rods, cocci, diplo, or chained, etc.)

In optical density tests, the turbidity of cultures is monitored. This process involves a sample being placed on a spectrophotometer and measured at a given wavelength.

In plate counts, cells are streaked onto solid media from a serial diluted sample.

Whatever the technique to be used, the procedure must be established prior to large-scale production. Failed campaigns resulting from insufficient or underdeveloped characterization techniques can contribute substantially to development costs.

Setting Up and Inoculating the Fermenter. The fermenter is filled with nutrient medium and heat sterilized. Upon cooling to the desired temperature, the inoculum is introduced.

The two primary types of fermentation methods are batch and fed-batch processing. In the batch method, the culture is established, and the product is harvested after cell growth reaches its peak. In fed-batch fermenting, nutrients such as additional carbon, amino acids, or other depleted media components can be added via ancillary pumping systems in an effort to increase cell numbers or product concentration.

Monitoring the Culture Kinetics. The cells grow and metabolize, depleting nutrients from the medium and producing the desired product, as well as by-products. The culture is sampled at appropriate intervals to verify data provided by monitoring probes and to determine cell or product concentration and integrity. Sample is taken from valves whose ports are sterilized with steam, also termed clean in place.

During the fermentation process, cells go through four phases called lag, log, stationary, and death (see Figure 1).

Figure 1. Density of cell growth during the phases of a typical fermentation process, from lag, to log, to stationary, to death. The majority of product is produced during the latter part of log phase and during stationary phase. In batch mode, this is the window for harvest, and therefore a limited amount of product is to be expected. Fed-batch fermentation enables the stationary phase to be extended by replacing depleted carbon source and nutrients, or delivering substrates that promote higher yields. Catabolic by-products and molecules related to cell lysis not only contribute to cell death but often break down the product of interest. In this particular run, the operator would harvest at 8 to 10 hours.

In lag phase, the number of cells does not increase. However, toward the end of this phase the cells will increase in size. Cells are synthesizing the enzymes and transport systems needed to grow in the new medium.

In log phase, the cells begin to multiply. Cells reproduce at a maximum rate for a given set of growth conditions.

In the stationary phase, the essential nutrients have been depleted from the medium, or there is buildup of a toxic metabolite that causes the growth rate to slow. When cell growth ceases, the culture has entered the stationary phase. Although they are not growing, the cells continue to metabolize, producing energy that maintains basic cell functions.

Once the energy of the cells has been exhausted, cells begin to die. Cell death follows the same first-order kinetics as log phase. In the production of IVD bioproducts, fermentation is halted before the onset of death.

All types of cells used in fermentation require off-line product characterization during the run. Assays for characterization include enzyme-linked immunosorbent assays (ELISA) for antibodies and substrate-specific spectrophotometric tests for enzymes. High-performance liquid chromatography and electrophoresis are also common tools used in this process.

Cell Harvesting. The fermenter is chilled immediately after run termination in order to slow metabolic processes and promote product stability prior to downstream processing.

Downstream processing consists of clarification and concentration and, ultimately, purification of the product. The finished product must be purified to homogeneity without affecting potency or quality. For some IVD products crude product is suitable; however, most platforms require a product that is isolated at the molecular level, leaving behind any contaminants that can inhibit the performance of the system.

Clarification and Concentration. The fermenter liquor must be separated from the cells and debris in processes known as clarification and concentration. During these steps, large volumes of dilute product are reduced to highly concentrated liquors or wet solids that facilitate downstream processing.

For mammalian cells clarification is accomplished by means of centrifugation or filtration, and purification of the final product is performed through ion exchange or by using affinity columns. Bacterial products are sometimes found in the supernatant and, in that case, should be handled as indicated for mammalian cultures. In bacterial fermentation, however, the product is more often part of the cell mass—that is, inside the cell wall or bound tightly to the surface. In that case, following centrifugation or filtration, a homogenizer or other device is used to lyse the cells. The protein can then be purified via columns or precipitation techniques.

High-speed centrifuges can process hundreds of liters of bioactive material per hour. On the other hand, filtration technologies have progressed to the point that large quantities of material can be intensely concentrated into very low volumes. Filtration thus offers a cost-effective alternative to centrifugation. A filter press is desirable when large amounts of biomass, such as that from yeast, must be processed.

Purification. In some instances filtration using the aforementioned techniques or gel filtration is the only step required for purification. However, it is more often the case that purification becomes a process within itself.

Purification is a costly process that, like fermentation, might most reasonably be outsourced to a contract manufacturer with the expertise and facilities to handle it easily.

Industrial-level purification is performed with columns ranging from low-volume types used during product development to production columns with the capacity to hold several liters of resin, which can cost several thousand dollars.

The concentrated filtrate material often contains extraneous proteins. When purifying this material with an affinity column, the resin selectively binds the protein of concern, allowing residuals to wash through. The product is eluted from the column using buf-
fers of varying acidity levels and ionic strength. Often the material will be passed through additional columns in an effort to achieve higher purity.

Many proteins can be precipitated using salts such as ammonium sulfate. Typically, proteins will precipitate at different salt concentrations, and thereby can be removed from the remaining liquid. To completely remove the product may require more than one salt cut followed by several buffer washings to eliminate any soluble (nonproduct) protein contaminants.

Quality Control. The level of commitment to quality by the contractor contributes substantially to the efficacy of the project. Fully developed QC departments will attest to the integrity of the process. The QC department should regulate raw materials as well as internally supplied materials such as reverse osmosis water. As mentioned previously, the QC department should also characterize the product and monitor the status of the culture throughout processing.

For example, some proteins such as antibodies may be tested for cross reactivity with other antigens. Electrophoresis, HPLC, and activity tests are common methods.

Packaging. Final purified material can be packaged in various ways. Frozen concentrate is very common. Manufacturers preferring to reconstitute the material when formulating their IVD test solutions need to be provided with lyophilized powder.

The results of the final product characterization will indictate the product's quality and whether it is ready to be sold. When the product is officially released, the product is shipped (including any banked strains) along with the batch record to the customer.

The bioprocessing microorganisms used in fermentation may be of mammalian origin, including hybridomas, or they may be bacteria or yeast. All can be grown in the same type of fermenter, with only minimal variation of the basic fermentation methods being necessary to accommodate the different organisms.

Mammalian Proteins. Even with the advent of perfusion hollow-fiber bioreactor technology—which enables gram quantities of antibody to be made one-tenth the volume of medium as required for a fermentation supernatant—fermentation is still an attractive alternative for such work. Many hybridoma cell lines, for example, have growth characteristics suitable for a batch or fed-batch culture.

In rare cases, mammalian cells are run in continuous culture where up to half of the fermenter volume is harvested and then replaced with fresh medium, allowing for lengthened production campaigns. This will only work as long as the culture remains asynchronous—meaning that the cells are in varying stages of life—and is therefore risky.

The principal disadvantage of using fermentation strategies for mammalian cell production is that the bulk product is usually somewhat dilute. For further purification, it must be diafilter concentrated severalfold in order to reduce the volume to a workable level.

Another drawback is that mammalian cultures often require serum proteins in the medium. Scale-up may include weaning cells to a serum-free medium; however, the results of using serum-free or protein-free media are usually lower product yields and slower culture growth.

Should mammalian proteins such as antibodies be produced by means of fermentation technology, handling becomes a critical concern. While many bacterial and yeast cultures use carbon sources that will not support the growth of contaminant organisms such as glycerol or alcohol, mammalian cultures use media rich with sugar. Such media are highly susceptible to contamination with environmental bacterial and mold sporulation. Furthermore, contaminating bacteria and mold will normally propagate faster than the mammalian cells, competing for media components.

All manipulations of mammalian cell lines should be performed either in a biosafety cabinet that is self-contained and circulates filtered air in a laminar- flow or, preferably, in a Class 100 cleanroom by means of sterile rather than aseptic techniques. Cultures for IVD use can be supplemented with low levels of antibiotics to enhance resistance to contamination.

Inocula are generally grown in seed flasks or roller bottles. Cell densities can excede 1 x 9 cells for every 50 L of fermentation volume. The inoculum seed concentration will dictate the lag time for growth. Agitation should be started at low settings and increased as the kinetics indicate that the culture is reaching confluence, thereby facilitating the penetration of dissolved oxygen and media components.

Fermentation of mammalian cells results in a low yield of product. About 0.1 mg/ml can be expected from an optimal run. Thus, very large or multiple runs will probably be necessary to achieve the desired quantity of material.

Bacteria. Fermentation is most often accomplished using bacteria ranging from various aerobic to anaerobic species as the host organism. Recent advances in genetics have enabled scientists to use recombinant DNA technology to produce a wide variety of proteins unrelated to the host organism. Escherichia coli, for example, is often chosen for this work because its genome is fully mapped and the organism is considered easy to work with. Challenges arise here because the desired protein may be part of the cell mass or secreted directly into the medium. Purification and recovery schemes require extensive development.

When using bacteria as the host organism, a media bottle incubated in a shaker is generally the vehicle of choice for inoculum development. A suitable inoculum volume dependent on the stability of the organism is grown. In the author's facility, when fermenters holding more than 1000 L are inoculated, the inoculum is propagated at volumes 2.5–10% of the fermentation volume in order to maximize cell concentration and thus ensure a successful run. This ratio is also applied when using a smaller fermenter on the order of 10–70 L.

Doubling times for bacteria are generally on the order of a few hours. Typically, bacteria are grown in batch mode; however, additional materials can be delivered in a fed-batch mode via ancillary vessels. Agitation can be increased to facilitate dissolved-oxygen penetration in aerobic fermentation or carbon dioxide penetration in anaerobic fermentation. Control of acidity level is achieved by adding acids or bases, depending on the buffer system used in the medium.

The key to a successful run is maintaining command of the culture kinetics. This is like having a detailed road map of the organism's metabolism with the destination being highest possible yields and suitable product integrity, while achieving the lowest possible levels of catabolic contaminants.

For example, the operator must pinpoint the time after log phase for harvest. The longer the culture runs past log phase, the greater the likelihood of cell lysis. Then, if it exists in the cell mass, the product will spill into the supernatant. If it exists in the supernatant, it will contaminate the supernatant with proteolytic molecules and other proteins. This contamination will place a greater demand on purification processes or else break down the desired product.

Bacterial clones, which are exact copies of the original bacterium, typically produce several times the quantity of product that native strains can, and may develop at faster rates. Where a typical bacterial run can take at least 24 hours, clone fermentation usually requires only 8–10 hours. Indeed, some recombinant E. coli systems run for 3 to 5 hours to effect induction, with fermentation taking another 3 to 5 hours before termination.

Yeast. Researchers have recently turned their attention to eukaryotic systems such as yeast, from which large quantities of stable protein are secreted into the medium. Like E. coli, yeasts are very suitable for recombinant systems. Enzymes and many mammalian proteins pertinent to IVD products can be produced using a yeast system. Because yeasts are eukaryotic, complex proteins maintain their integrity. An advantage of yeast is that there is no endotoxin contamination such as that associated with bacteria, or viral contamination such as that associated with mammalian cultures. Resourceful genetic research teams could theoretically clone most of the proteins derived from mammalian or bacterial cultures into yeast.

Yeast fermentation is similar to bacterial fermentation in that agitation increases dissolved-oxygen penetration and ancillary devices can be used to add nutrients in fed-batch mode. The difficulties associated with yeast production relate to the necessary storage of alcohol, which is the primary carbon source during fermentation, and the large amount of biomass involved in production.

Developing biotechnology companies with time and personnel constraints sometimes hire the services of research firms that not only offer product development and marketing experience but also have established connections for outsourcing raw material fermentation. These middlemen can, if necessary, be involved with a project from conception through market launch.

In establishing a contract manufacturing arrangement, raw materials technology transfer is clearly a central concern. The IVD company must divulge factual information and share its proprietary knowledge. The contract manufacturer integrates this input with its own operations to create a seamless system of industrial procedures. A confidentiality agreement is an essential part of any such contract manufacturing arrangement.

Some processes may need to be validated in accordance with specific requirements of the client or the contractor. The level of development of the process when it is handed over to the contractor has a significant effect on the cost of outsourced services and the time required for delivery of finished product.

Adaptation and scale-up of the process to industrial magnitude is likely to require modifications. The IVD manufacturer should be prepared to assist the contractor with any necessary process adjustments. Having developed the cell line, the contracting company has a feel for the growth characteristics of the organism and often sends a representative to the contractor's facility to be on hand for the first manufacturing run.

The manufacturing facility—in-house or outsourced—is a major determinant of product quality and integrity. Facility requirements include steam generation, deionized or reverse-osmosis water, plumbed high-pressure air, plumbed gas, and a substantial HVAC system. Successful facilities will have a washing area, incubators (shakers), media preparation space, and sufficient accommodation for refrigeration. This list of requirements is not exhaustive.

Diagnostic-grade materials need not be manufactured in facilities as strictly regulated as those that produce pharmaceutical products. Nevertheless, the IVD product will most likely fall under the good manufacturing practices (GMP) requirements of FDA's quality system regulation. If the facility is FDA registered, GMP compliance ensures that the product is from a federally regulated and inspected source. ISO 9000–series compliance is also desirable, as it indicates not only product quality but also that customer requirements will be met.

An unnecessarily elaborate facility will produce a costlier-than-necessary raw material. On the other hand, a regulated contract facility that can handle packaging and labeling and has a state-of-the-art quality control department might save an IVD manufacturer a lot of time and trouble. Such contract manufacturers can also be valuable resources for future research and development programs.

Some IVD companies choose to start out with materials produced at university facilities in small-scale quantities up to several liters. This can often be a cost-effective strategy. However, it adds another step in technology transfer when the process is moved to an industrial contractor because most universities are not regulated and are unable to provide GMP-compliant product.

Essentially, a facility being considered for outsourcing a process should possess all the equipment necessary for the process: fermenters of the right capacity, hollow-fiber separator-concentrators or tangential-flow devices that can rapidly handle production volumes of material, cell presses for large amounts of biomass, and ancillary equipment that should include analytical instruments for characterization. The critical criteria for equipment are validation and preventive maintenance.

Clients of contract manufacturers have a right to expect delivery of all materials generated throughout the process. They also need full documentation. All information pertaining to raw material manufacture should be incorporated into the batch record and provided to the IVD manufacturer at the completion of a contract.

Conclusion

The technology of fermentation has been around for a long time. And, as the IVD industry continues to expand, demand for fermentation equipment and services will increase. However, the pace of discovery and advances in genetics and biochemistry prohibits most IVD manufacturers from investing in production-scale raw material processing facilities of their own. For such IVD companies, outsourcing the fermentation process may be a suitable option.