Advances in cell culture media technology have helped achieve safer biologics.
By Tom Fletcher, Holden Harris
Regulatory expectations for cell-culture-based biologics production processes changed dramatically once it was discovered in 1996 that a fatal disease (variant Creutzfeldt-Jakob disease or vCJD) appeared to be transmitted between species by something as small, simple, and difficult to eliminate as a prion. The threat of a previously unknown zoonosis (disease that can be transmitted from animals to humans) causing fatal harm was a call for biologics producers to implement immediate safeguards in their manufacturing processes and procedures to prevent the introduction of infectious agents into their products. These safeguards were intended not only to prevent infection from known pathogens, but also from unknown threats. The resulting push to eliminate animal-sourced materials drove the innovation of cell-culture media technology, which ultimately gave rise to the impressive chemically-defined, animal-component-free processes that have now become standard for producing recombinant antibodies, therapeutic proteins, and some vaccines. Although the same concerns for safety are ultimately expected to drive other cell-culture bioprocesses toward this goal, several challenges still remain, including for some of the most promising new opportunities to develop cellular therapy production processes for emerging immunotherapies and regenerative medicine.
Background
The development of serum-free mammalian cell-culture media technology has been impressive during the past few decades. The advances can perhaps best be appreciated when compared directly to a reference baseline--the native environment of mammalian cells. Today, healthy mammalian cell cultures are commonly grown in protein-free, chemically-defined media that contain only 60-70 pure components, none of which come from animals or are even exposed to animal-sourced materials during their manufacture. This is especially impressive when contrasted to the native environment of animal cells, which is generally a complex, protein-rich, milieu of thousands of 100% animal-sourced compounds continuously perfused by a pulsing stream of nutrient-rich blood serum. The efforts behind these achievements were driven not only by the opportunity to understand cell-culture science better, but by a variety of process-related concerns.
Many past efforts simply sought to improve process performance and/or consistency. Replacing complex bovine serum supplements with purified bovine proteins such as albumin, insulin, transferrin, and fetuin was seen as a major advantage in the early days of serum-free media technology (i.e., the first ‘serum-free’ media relied on these proteins in order to eliminate the serum) because it helped provide the technical process understanding required to implement desired process controls and reduce the risks of variation caused by serum. Other early efforts were driven by a desire for cost reduction and improving the reliability of raw-material supply. Some of these efforts resulted in the substitution of complex tissue digests or extracts for the more expensive and difficult-to-secure traditional serum supplements. It was common for these various features to be balanced against each other to find an ideal combination of advantages that best met a particular need, whether it was achieving a more defined formula by using more purified component proteins, or improving the availability of materials by resorting to complex ingredients instead. There was also a persistent interest in reducing the risks of high-molecular-weight proteins or other compounds that might co-purify with the desired product, especially when these proteins presented a potential risk for causing an immunogenic response in the patient.
Table I. History of viral contaminations in commercial cell culture manufacturing processes.
For the sake of safety, there were also aggressive efforts to reduce the potential risks of viral contamination (1-3). But because the chosen methods focused primarily on downstream virus removal or inactivation instead of strategic sourcing of lower-risk starting materials, the efforts had little effect on serum-free media technology. The good news is that, partly because of the efforts to remove or inactivate viruses, there have been no reported cases of disease transmission from a biopharmaceutical manufacturing process to a patient in more than 30 years. However, the reality is that the routine methods used for virus removal and inactivation have not always been sufficient to eliminate all of the upstream process risks presented by viruses. Viral contaminations of manufacturing processes have been reported to occur periodically in the past (4-16) (Table I), and until additional upstream process safeguards are implemented universally, periodic incidents are expected to continue. Examples of the viral clearance methods being implemented with increasing frequency fall into three categories:
• Inactivation—Includes methods such as high-temperature short-time (HTST), ultraviolet (UV), and gamma radiation
• Removal—Filtration with 20-50nm membranes
• Strategic sourcing—All materials from reduced risk sources.
Although avoiding risks right from the start is generally believed to be better than simply reducing or removing risks afterwards, none of the aforementioned approaches alone is expected to be the perfect solution. These methods are being developed and employed in various combinations in an effort to eliminate virtually all viral contaminations of commercial bioprocesses (17). But viruses were not the only adventitious agents presenting a serious risk of contamination.
As mentioned previously, in 1996, the appearance of vCJD forever changed our understanding of transmissible disease (18-20) and ultimately drove serum-free media technology to a new level of innovation. When the causal link was first discovered between bovine spongiform encephalopathy (BSE) and vCJD, it implied that diseases may be transmitted between species simply by exposure to an infectious protein, or “prion,” without involving any nucleic acids (21). Once this risk was known, it became urgent for the sake of patient safety to change the requirements for any materials used for manufacturing biopharmaceuticals to minimize the risks of transmitting disease through exposure. Patient safety immediately became a primary driver for developing innovative new cell-culture-based manufacturing processes, eventually leading the biopharmaceutical industry to achieve the impressive animal-component-free, chemically-defined processes that have become so commonplace today. The newly discovered risk became a reason to eliminate not only sources of known adventitious pathogens from biopharmaceuticals manufacturing processes, but also to eliminate potential sources of unknown pathogens. It was decided with support from various regulatory agencies to make every effort to avoid sources that were known as potential hosts for human pathogens, such as bovine sources, and also to require stringent testing and justification for any animal-sourced raw materials used in producing biopharmaceuticals, even for materials that are not associated with any proven threats (22, 23).
The new expectations challenged biologics producers to avoid all animal-sourced materials in any new processes without sacrificing the high yields and overall process performance that were commonly considered as the best measure of their success. It was not surprising that some resisted complying with the new expectation. Rational objections were expressed about what must have seemed to be an unreasonable new burden: “Don’t the policy makers realize that the cells at the core of these processes are animal derived?!”. There were also new, related challenges, such as an increased sensitivity of animal-component-free cultures to toxic contaminants, metabolic waste products, or leachables from polymers used in single-use equipment and flexible packaging films (24, 25). It has been difficult to identify functionally equivalent alternatives to some of the common animal-sourced media components. In some cases, satisfactory alternatives have still not been found. For example, many vaccine and cellular therapy production processes require a variety of animal-sourced attachment factors and cytokines for which viable alternatives have not yet been identified. Finding new animal-component-free materials was only one part of what was needed to successfully isolate animal-component-free processes from the risks of exposure to potentially infectious animal-sourced materials. Robust systems were required to prevent exposure.
Present situation
Several fundamental elements are mandatory for any systematic effort to successfully prevent exposure to animal-sourced materials. Coherent policies and procedures should describe precisely how the careful control of facilities, equipment, and materials will be used to prevent unacceptable exposure to animal-sourced materials. Using a safety-by-design approach that is risk-based can help focus and coordinate efforts to address the most important factors. Successfully avoiding exposure to animal-sourced materials requires that biologics manufacturers must go to great lengths to ensure that they understand the full risks of their material supply. Important materials for scrutiny certainly include cell-culture media and their components, but also other related equipment and reagents moving into and being used within the facility. Resins, columns, tubing, connectors, filters, gaskets, mixing blades, and other process contact materials or ingredients are included in this scope. The producers of these items also need to understand and control their own supply chains and the various components and equipment used in the related processes of their suppliers. Each additional level of control helps to reduce the risk of the final manufactured product being contaminated with infectious materials.
Figure 1: Potential exposure of media components made by fermentation. Diagram of a typical manufacturing process for recombinant insulin, a media component made by fermentation. This diagram helps illustrate a complex material manufacturing process that has multiple points of potential secondary or tertiary exposure to animal sourced materials. Exposure at any one of these points could cause concern and even disqualify the material for use in an animal-component-free cell culture process.
While determining whether a particular material is derived directly from an animal or not is elementary, understanding and controlling indirect (i.e., secondary, tertiary, etc.) exposure requires careful consideration of specific process details (Figure 1). Because this typically involves tracing materials and their manufacturing processes upstream multiple levels, the cooperation and transparency of each preceding supplier in the chain is essential to success. Effective control depends on close partnering between multiple levels of supplier/customer relationships. For example, cell-culture media suppliers routinely question their own suppliers on whether prospective raw materials are produced using any animal components. Determining the answer to a question like this may be more difficult than it might seem. For instance, a supplier may provide a recombinant protein produced from a non-animal source such as Escherichia coli.
However, upon further inspection, an animal component might be involved as part of the manufacturing process. The E. coli cultures may have been fed with culture media containing animal-sourced materials. The frozen cell bank may have been cryopreserved using animal-sourced materials. There may have been animal-sourced enzymes or other materials used in some of the particular process steps (Figure 1). These are examples of secondary exposure.
More subtle examples are common. Polymers and elastomers used in process equipment or plumbing may contain or may have been exposed to animal-sourced materials such as stearates, slip agents, etc. Recombinant enzymes from non-animal sources that are used as part of a process to produce an animal-component-free product may have been made in cultures containing animal-sourced materials. These would be examples of tertiary exposure. Suppliers that are several levels upstream do not always understand why this sort of information is important and may initially resist revealing their material sources or process details. They may object at first to questioning that seems intrusive and, for this reason, some suppliers may even opt out of the chance to participate. But many others will comply eagerly once the necessity is explained.
Once a prospective supplier is identified, they must be qualified. Paper and on-site auditing of suppliers can be used to gather relevant details about a supplier’s processes. A paper survey is commonly used to evaluate a supplier’s quality systems. Then an additional questionnaire can be used for collecting the important information regarding each individual material of interest. The material questionnaire typically requests details on all facets of how a material is produced, including descriptions of raw materials and equipment used, and the animal component status of these. Additionally, understanding the route the material might take from manufacturer to the final user can be important. For instance, the manufacturing site and materials may all be non-animal, but those assurances can be completely negated if the material is repacked by a distributor or subsidiary that compromises isolation from animal-sourced materials along the way. Each step where materials may be exposed after production should be questioned. A risk analysis can help determine which components and equipment may introduce the largest amount of risk. Steps with higher risks may also warrant an on-site audit to truly understand all facets of the production and points of potential exposure to risk.
Because cell-culture media contain a wide variety of materials obtained from various origins and have prolonged contact with the drug substance or final product, they typically rank higher than most other process materials on a scale of potential risks. Efforts to reduce these various risks have influenced cell-culture media technology in several ways. The earliest examples of successfully eliminating animal-sourced materials relied heavily on substituting complex, undefined alternatives such as vegetable protein hydrolysates or yeast extracts. These substitutes provided a way to eliminate animal-sourced materials for many processes, but carried risks of their own. Exploring the detailed heritage of almost any biologically derived material is likely to uncover reasons for concern, and these complex materials are no exception. Consider that the plant sources for these materials are grown in open fields and are subject to variation based on a wide range of factors such as geography, climate, season, etc. Even greater concerns are raised by the possibility that the materials may contain residual pesticides, naturally occurring bioactive compounds such as aflatoxins or alkaloids or, in contradiction to one of the main reasons for their use, they may be subject to exposure to animals and animal waste. These were some of the remaining concerns after the initial success with animal-component-free media that drove the technology even further--ultimately to achieve 100% chemically-defined solutions for some of the more mature applications such as those based on Chinese hamster ovary (CHO) cells. Eliminating hydrolysates helped to control variation and eliminate as much risk as possible from the media.
Remaining challenges
As successful as the efforts have been for these mature applications, sufficiently animal-component-free, chemically-defined media have not been developed for every cell-culture application. Some of the newer cell-culture applications, such as the production of cellular therapies, present significant challenges. Most of the media used in these methods contain serum-derived proteins and a variety of recombinant growth factors. Although the ultimate goal is to eliminate all animal-sourced materials from these methods, compromises have been reached by creating new categories of media, such as ‘xeno-free,’ which generally indicates all animal-sourced materials are human. Recombinant growth factors are often categorized as animal-component-free as long as their heritage can be verified to sufficiently avoid exposure to animal-sourced materials (Figure 1). By following the path of more established applications, such as methods for producing recombinant antibodies, those developing these newer applications can also build upon the success of others (Figure 2).
Figure 2: Reducing risks and complexity. Various concerns have driven cell culture media technology towards reducing risk of infection and reducing complexity (green arrow). Some newer cell culture applications, such as methods for producing cellular therapies, are following the path of more established applications, such as methods for producing recombinant antibodies.
Conclusion
The evolution of any technology can often be understood best by considering the various drivers at play in the process. Progress is driven by both desires and by needs, but the needs drive more forcefully. Cell-culture media technology is no exception to this. Recent innovations of the technology have been driven not only by desires for scientific or economic progress, but out of a more forceful, urgent need to meet required safety expectations. The demonstrated success of recently developed animal-component-free chemically-defined media for some processes suggests the possibility that the remaining challenges to develop safer media for producing cellular therapies and other newer, more difficult cell-culture applications may be met by following a similar safety-by-design approach.
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