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Once a necessary "magic ingredient" of media for mammalian cell culture, serum is rapidly being eliminated from media for biotherapeutic processes.
Cell culture productivity has increased dramatically over the last few decades. This productivity increase is due in large part to significant process development efforts in this area, efforts traditionally justified by the economic benefit of improving productivity. The economic pressure to increase productivity has been highlighted in recent years by the success of monoclonal antibodies, some of which have market requirements in the hundred-kilogram-per-year range. The demand for such large quantities of protein has, in turn, triggered concerns about a potential shortage of manufacturing capacity, intensifying the pressure to boost cell culture productivity.1
Development scientists have responded to this challenge admirably. Monoclonal antibody titers in cell culture have increased roughly tenfold, from a few hundred milligrams per liter a decade ago to grams per liter today, with reports of titers in excess of 5 grams per liter. If an industry-wide shortage of bioreactor capacity fails to materialize, this will be due not only to the recent burst in the construction of new manufacturing capacity, but also to the reduction of demand resulting from improvements in cell culture productivity.2
In addition to the strategic advantage of reducing the requirement for bioreactor capacity, increasing titer can have a significant impact on the cost of goods sold (COGS). Case studies show that a fourfold improvement in titer can cut COGS by one-half.3 Cell culture development has been so successful that downstream processing is beginning to surface in discussions as the next bottleneck to be addressed. Although the gain in COGS from increasing cell culture productivity may diminish due to the increasing impact of downstream steps as titers continue to improve, optimizing bioreactor output is still a primary concern for most companies.
Evolution of Mammalian Cell Culture Media
The steady increase in cell culture productivity is the result of intensive efforts to understand and optimize all aspects of the cell culture production process.Improvements have been made in molecular biology, cell line construction and selection, media and feed development, and bioreactor operating parameters. But arguably it is media and feed development, particularly feed development, that has been responsible for the largest share of this improvement.
While the strategic impact of decreasing capacity requirements and COGS makes boosting productivity a primary goal, it is but one of several important aspects to be considered when selecting or developing media. Others include safety (to assure patients are not adversely affected by therapeutics intended to improve quality of life), quality, robustness, economics, and operability.
Safety and the related requirements of product quality and regulatory compliance are achieved mainly through control of raw materials for media. Introduction of contaminants via raw materials may be possible at any step in a biomanufacturing process, but cell culture media pose a higher risk because the cell culture processes provide an opportunity for co-cultivation of microbes and replication of adventitious agents that may infect cells. This concern, which has been a driving force in the trend toward eliminating serum from media, has been deepened by an increasing awareness of transmissible spongiform encephalopathies.
Through good processing, controls, and documentation, manufacturers of serum may reduce the risk of a transmissible pathogen reaching patients. The impossibility of reducing the risk to zero, along with the increasing regulatory burden of assuring safety, has led to a strong effort to eliminate serum from cell culture media. This same concern has expanded to other animal-derived raw materials, and thus, many media are now "animal-free."
Comparability questions can arise from changing media in an established process. Changes in media may directly impact product characteristics such as glycosylation, or may alter cell culture conditions, subsequently impacting downstream steps. Introduction of new raw materials may also introduce new contaminants into the process stream. Even animal-free media may contain contaminants such as endotoxins, mycotoxins, or immunogenic compounds.4 Changes to media in an established process must be carefully assessed.
With current technology it is impossible to characterize all the variables in a biological process such as cell culture. Because of their complexity, media can be a significant source of variability. Mammalian cell culture media may have in the range of 60 individual components, some which may be uncharacterized or semi-characterized. Lot-to-lot inconsistency in these raw materials can translate into variability in the performance of cell culture processes. Preparation, including the method and conditions of sterilization, may also impact media quality and adversely affect cell culture operations. Minimizing the use of complex components, qualifying vendors, and screening new lots of media and media components are all strategies for reducing variability in media performance.
Media can impact process economics, operability, and logistics. The cost of media can vary greatly, depending upon the formulation. Small increases in productivity can typically justify significant increases in media costs, but expensive media or media supplements should be evaluated against their impact on productivity. Supply issues should also be considered. As cell culture media are critical process components, it is ideal to have multiple suppliers qualified, or at a minimum, to assure a high level of confidence in a single-source supplier. Logistic issues such as amenability to powder formulation, complexity of in-house preparation, and the number and difficulty of feed additions can all have an impact on economics and operability.
Media and feed development can be expected to increase volumetric productivity significantly, with up to fivefold improvements reported.
Increases in protein concentration are typically achieved through increases in cell density and duration of viable culture (viable cell-days), although increases in specific productivity have also been observed.
Media and feed development programs can have a major impact on process economics, but the quest for higher productivity must be tempered by the aforementioned considerations.
Once a necessary "magic ingredient" of media for mammalian cell culture, serum is rapidly being eliminated from media for biotherapeutic processes. Understanding the complex requirements of mammalian cells in culture has advanced to the point that it is possible to achieve good cell growth and productivity in chemically defined media. Two parallel trends — one toward eliminating animal-derived materials, the other toward eliminating complex and undefined components — have led to the development of a variety of media options. Basal, serum-free, protein-free, animal-derived-component-free, and chemically defined media are all now available.
Serum is usually added to supplement a simple basal medium, enhancing it with a rich mixture of functional proteins, growth factors, and trace elements; however, even richer complex media may benefit from the addition of serum. In addition to the safety concerns already discussed, serum is an expensive component due to its uncharacterized nature, with the potential to add lot-to-lot variability to the cell culture process.
Serum-free media often contain key serum proteins such as bovine serum albumin, transferrin, and insulin. They may also contain protein hydrolysates. While many of the drawbacks of serum are eliminated in such media, their high protein content may cause problems with purification, and many components of serum-free media may be derived from animal sources.
A number of commercial "animal-free" media have been introduced to address safety concerns associated with animal-derived raw materials. Although these media do not contain materials directly derived from animals, the exact definitions of "animal-free," including how far up the supply chain animal-derived components have been eliminated, may vary among vendors.8 The media may contain recombinant proteins and protein hydrolysates from non-animal sources.
Elimination of proteins takes media one step closer to being chemically defined. Protein-free media may both contain animal-derived components and protein hydrolysates.
In terms of reducing variability from raw materials, chemically defined media are the optimum choice because they contain no undefined components such as hydrolysates. Chemically defined media may still contain animal-derived components, although many chemically defined, animal-free media are available.
Although the gap is rapidly closing and exceptions exist, as a general rule media performance decreases as media move closer toward being chemically defined. Animal-free components may or may not provide performance equivalent to their animal-derived counterparts. In an effort to eliminate serum and other animal-derived components, hydrolysates have become a popular compromise. These are available from a number of non-animal sources, including yeast, soy, wheat, and other plant sources. They may contain an uncharacterized mixture of peptides and trace elements. In many ways, hydrolysates are today's "magic ingredient" that serum was several decades ago. Adding hydrolysates can significantly increase media performance, but at the cost of increasing variability due to their undefined nature. Some vendors offer hydrolysates that have been fractionated in an effort to reduce variability, and many biopharmaceutical companies have decided the advantages of hydrolysates outweigh their process-variability disadvantage. An effective raw-material screening program that can assess new lots of hydrolysates may go a long way toward reducing their inherent variability.
Before choosing or developing a medium or feed, the role it will play in the process and the criteria for a good formulation should be considered. These criteria will vary depending on the application. In a seed medium, for example, productivity is less important than a short doubling time and high viability. Production media formulated for perfusion cultures, where toxic by-products such as ammonia and lactate are constantly diluted and fresh nutrients are constantly supplied, will be different from media formulated for fed-batch cultures, where high osmolality and accumulation of toxic metabolites are often limiting factors. The best medium for un-fed-batch culture will not necessarily be the best starting medium for a fed-batch strategy. If medium is developed for an existing clinical or commercial process, comparability of product quality attributes is necessary criteria for a good formulation.
The scope of a medium-development program is apt to be driven largely by timelines and by the decision to either conduct the development work in-house or to outsource some or all of the project. Many media vendors, as well as some independent companies, offer media-development services. If choosing to outsource work, it is advisable to clearly establish parameters such as who will own the final medium formulation, what, if any, are the long-term obligations, and any other considerations to assure a high level of confidence in a second supplier or in a sole supplier.
Performing a sequence of screening, optimization, and confirmation is a logical approach to media and feed development. This sequence may involve a combination of methods such as automated model systems, component titration, media blending, and spent medium analysis, each of which has its own advantages and disadvantages.9 Although it is possible to design a medium entirely from scratch, it may be easiest to start by selecting a base medium from which to work. One approach is to screen vendor media and select one with good performance. Alternatively, if knowledge and control of the formulation is a requirement, one of the defined classical media (such as DMEM, F12, and RPMI), or a blend of classical media can provide a good starting formulation.
Once a starting medium is established, a panel of nutrient supplements can be tested. Traditionally, nutrients were adjusted individually, testing various concentrations in a series of shake flask experiments until optimal concentrations were identified. A more efficient approach is to split this effort into a screening step and an optimization step. If the cell type is commonly used in manufacturing, good starting ranges for many nutrients can be found in the literature. To determine the levels at which beneficial and toxic effects are seen, individual nutrients can be rapidly screened experimentally at various levels in parallel dilution series with the help of automation and high-throughput assays.
After establishing the nutrients and concentration ranges at which to begin testing, two approaches are available. The first approach, called spent medium analysis, involves monitoring a cell culture and measuring various media components and metabolites to determine what depleted nutrient or toxic metabolite may be limiting. Components depleted over the course of a culture can then be added at higher concentrations in the medium or feed in subsequent experiments. Toxic accumulation of metabolites may require decreases or changes in other nutrients. While statistical designs can be used for these experiments, the assay-intensive nature of the approach usually limits it to a relatively small number of flasks or bioreactors and produces results that must be interpreted by scientists with strong cell biology knowledge.
A recent alternative to spent medium analysis is a high-throughput approach. In this method, statistical design of experiments is coupled with automation and small-volume culture systems in order to investigate hundreds to thousands of medium or feed formulations in parallel. Due to the large number of samples, it is not practical to assay for and interpret the profiles of nutrient depletion and metabolite accumulation. Instead, high-throughput assays typically monitor only critical process criteria such as viable cell density and protein titer. Although these experiments can benefit from interpretation by cell biologists, they rely more on the statistical power of a large data set to uncover optimal conditions.
After optimizing media and feed compositions (as well as feed-delivery strategies) using either method, the most promising results should be confirmed in a model system such as a bench-scale bioreactor. Bioreactor parameters such as pH, dissolved oxygen, and pCO2 may have an additional impact on media and feed performance.
Media and feed development has helped to increase cell culture titers significantly in recent years. The introduction of new assay methods and high-throughput tools is expected to help continue this trend, and genomics tools also may prove beneficial for optimization of cell culture parameters including media.10,11 In the near future, media and feeds that are chemically defined and free of animal-derived components may be capable of achieving protein concentrations that exceed current benchmarks, while assuring the safety and reproducibility of cell culture processes.
Geoffrey Hodge is Vice President, Process Development and Technology, at Xcellerex, 170 Locke Drive, Marlborough MA 01752-7230, 508.480.9235 firstname.lastname@example.org.
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