Balancing Media Development with Patient Safety

ABSTRACT

Media compositions used for cultivating recombinant cell lines not only directly influence the cells' physiological phenotype and fermentation performance, and quality of the expressed product, but the development of novel media formulations also provide a mechanism of generating manufacturing intellectual property for a particular product. Biopharmaceutical companies and media suppliers are therefore recognizing the strategic importance of continued investment in the development of novel media formulations. Historically, raw materials involved in the production of biologics have included complex animal-derived components. Although a decade has passed since cases of bovine spongiform encephalopathy and the resulting transmissible spongiform encephalopathies were reported, their association with raw materials is still a major concern. Establishing a well-defined media formulation containing no animal-derived components is the ultimate aim for most biopharmaceutical companies. There are now well researched, chemically defined alternatives available that promise to meet these goals.

Eden Biodesign Ltd.

In recent years, a market surge in biopharmaceuticals has warranted extensive research and development in advanced cell culturing techniques and subsequent methods of optimizing bioprocesses. Media development is one of the most critical stages in biopharmaceutical manufacturing. Indeed media components have such a strong impact, they can account for up to 30% of the total production cost.¹ This area of development offers the potential to dramatically improve yield and quality of the expressed product.²

The ideal cell culture medium, whether for use in mammalian or microbial systems, is one that provides process consistency, robustness, and batch-to-batch reproducibility. Essentially, a raw material must meet most, if not all, criteria listed below.³

• It will produce maximum product yield or biomass per gram of substrate used.

• It will produce the maximum product concentration or biomass.

• It will permit the maximum rate of product formation.

• There will be a minimum yield of undesired products.

• It will be of a consistent quality and be readily available throughout the year.

• It will cause minimal problems during media preparation and sterilization.

• It will cause minimal problems during other steps of the production process, particularly fermentation, extraction, purification, and waste treatment.

For many years, economic constraints have dictated media composition. This has led to the widespread use of complex, readily available raw materials, making large-scale fermentations reliant on cheap sources of carbon and nitrogen, which are often by-products of other industries such as corn steep liquor from the corn-starch industry and beet molasses from the sugar industry. Combining such complex components with animal-derived hydrolysates has increased the scope of product manufacture, creating high yielding processes at minimal costs. The abundance of biosynthetic precursors and growth-promoting agents make complex media the composition of choice, primarily because they accelerate growth and enhance productivity. Complex raw materials usually are derived from animal-sourced processes, such as hydrolysed peptones and sera. They also may be non-animal–derived, such as the by-products beet molasses and corn steep liquor. They can contain numerous individual components, some of which are semi-characterized and many more are uncharacterized. In many cases, biomass yields are greater with complex and semi-defined media than with chemically defined media. However, data from physiological studies is more difficult to interpret and can be clouded or influenced by many intrinsic parameters.

Growth precursors found in such complex components may be channelled directly into anabolic pathways, thus saving metabolic energy.⁴ This offers an immediate advantage in terms of culture metabolism but ultimately makes process definition and development problematic.

Fermentations using complex media have worked successfully for many years, and strain selection is often based around current media and cultivation conditions. Media development, however, is largely empirical, with little research into defining such raw materials. For example, a modified strain of the yeast Saccharomyces cerevisiae, which had been labelled the ultimate solution to lignocellulose-derived xylose, was found to require yeast extract, additional hexose sugar, and oxygenation,⁵ thus making growth in a chemically defined media difficult without intensive investigation. Therefore, the final industrial environment must be considered to prevent unanticipated costs at later stages. Both S. cerevisiae and Escherichia coli have extensive biosynthetic capacity and can grow well in defined media. In contrast, the biosynthetic capacity of many lactic acid bacteria are limited and they require complex or extensively supplemented media for efficient growth.⁶

The quest for increased productivity combined with patient safety has encouraged biopharmaceutical companies to invest more time and money into their processes and for raw material suppliers to explore new chemically defined versions of media.

Complex Versus Chemically Defined

There are two areas that are generating the most interest these days. The first is the elimination of complex and undefined media components and their replacement with known chemical composition and the second is the replacement of animal-derived materials with non-animal–derived materials. With the exception of some processes, most non-animal–derived materials and chemically defined media equivalents do not meet the same performance criteria as their counterparts by not meeting the specific nutrient requirements of individual cell lines. Keeping this in mind, the most effective approach to the development of chemically defined media is to design the media formulation based on the specific nutritional needs of the cell line.⁷ In today's biopharmaceutical industry, the following criteria significantly dictate process development:

• primary raw materials will meet regulatory requirements

• they will consist of non-animal–derived components

• the process will be high yielding

• it will be scalable

• it will be reproducible

• it will meet economic criteria.

A shift toward chemically defined media increases process cost and economically this must be offset against improvement elsewhere in the process, whether this be increased titers or simpler purification requirements.

Chemical definition at the laboratory scale is relatively inexpensive and can be vital for the investigation of many factors including physiological and biochemical assessment of growth, but work at this scale must be centered on the eventual scaled-up process.

The economic implications imposed on a process following the switch to a chemically defined medium are somewhat offset by reductions in raw material variability and reduced controlled and uncontrolled process deviations. This in turn favors batch-to-batch consistency in products and permits simpler purification strategies. Process predictability has become important for biopharmaceutical manufacturers, and using components of a defined chemical composition can reduce process fluctuations, improve overall process control, and enhance operability.

Furthermore, it is far easier to optimize a process of known biochemical composition than to base development on empirical methods. Most mathematical Design of Experiments (DoE) based on raw material evaluation give more significant and meaningful results if used on chemically defined compositions and can be beneficial in the switch from complex to defined. The advantages and disadvantages of replacing complex media with chemically defined media are listed in Table 1.

Table 1. A comparison between the major advantages and disadvantages of complex and chemically defined media

Serum and the Impact on Patients

Media development and the need for chemically defined and non-animal–derived materials has become very significant in today's industry to eliminate potential risks to patients.

Animal-derived components have hit the headlines on several occasions. The 1970s brought cases of mycoplasma contamination, endotoxin concerns were raised in the 1980s, and bovine spongiform encephalitis (BSE) and the resulting transmissible spongiform encephalopathy (TSE) fears dominated the 1990s. The biopharmaceutical industry has since tightened controls over the sourcing and final composition of raw materials. In February 2002, the US Food and Drug Administration strengthened its regulations for BSE protection systems for the pharmaceutical sector to reduce theoretical risks associated with it.

Animal cell culture was first established for the production of viral vaccines in the 1950s, using monkey kidney cells to produce the polio vaccine. Successful growth of this adherent cell line depended on the addition of serum to the growth media.

Bovine serum contains numerous beneficial components, including macromolecular proteins, low-molecular weight nutrients, anti-oxidants, and carrier proteins for water-insoluble components such as hormones, lipids, amino acids, and globulins. In addition, serum contains a high level of albumin and transferrin. The former is believed to protect cells from shear forces and stress factors generated under bioreactor conditions, such as fluctuations in pH or nutrient concentrations, and the latter is an iron-carrying glycoprotein needed by CHO and NS0 cell lines for optimal growth.

Despite the positive properties of bovine serum, this undefined raw material can contain adventitious agents and detrimental by-products such as bacterial endotoxins or immunogenic contaminants. More commonly, it can contain viral contaminants, which often do not produce cytopathic effects or morphological changes in cell cultures, and once present, viral contaminants are almost impossible to remove. Other potential contaminants include fungi, prions, and bacteria/mycoplasma, thus raising concerns about patient safety.

It was not until the middle of the 1960s that a serum-free medium was first used for mammalian cell culture.⁸ To achieve the same growth-promoting effects as experienced with serum, it was necessary to replace the role of serum with other animal-derived components such as albumin, insulin, and transferrin, which for many years were obtained from animal-derived sources.

The adaptation of mammalian cells to serum-free conditions greatly increases the safety of the subsequent bioproduct and there are regulations to avoid animal-origin components for fermentation. Research into serum replacement gained momentum in the 1970s, when studies replaced the complex serum component with an equally complex animal-derived protein hydrolysate, thus still presenting a contamination risk to the bioproduct. This work was subsequently followed by their replacement with non-animal–derived protein hydrolysates, such as vegetable peptones. These are being used extensively in the industry to replace serum and now chemically defined serum replacements are widely available.

Unfortunately for a few cell lines, chemically defined media do not adequately meet the specific nutrient requirements, so media screening has become exhaustive and supplementation may still be necessary in many instances. Fortunately in these cases, many of the necessary supplements, including insulin, transferrin, and albumin are now readily available from non-bovine sources. This enhances their overall biological safety but side-steps the issue of eliminating media complexity and subsequently process complexity. Only further raw material development will make such changes easier.

Media Supplementation

Supplementing animal and human cell culture with sera of animal origin still remains a standard practice despite its reputation as an ill-defined, highly variable component. More and more companies are, however, choosing to develop biologics in serum-free conditions. For the manufacture of recombinant therapeutics using bacteria, casein- or meat-derived hydrolysates are frequently used in such fermentations. The trend in this area is to replace casein-derived hydrolysates because most of them are manufactured with animal-origin enzyme systems. Many media suppliers put a significant amount of work into developing hydrolysates produced using non-animal–derived enzyme systems such as vegetable hydrolysates. Quest International (Irvine, CA) was the first to market non-animal–derived protein hydrolysates for serum replacement, maintaining at least the same productivity as when serum was used. Since this breakthrough, a series of non-animal–derived protein hydrolysates have been developed such as the Hy-Pep series, including Hy-Soy, Hy-Rice, Hy-Cotton, Hy-Wheat, Hy-Pea, and Hy-Yest, all developed as serum replacement alternatives. Complementing these is a series of large-scale bulk fermentation hydrolysates. In July 2009, SAFC Biosciences (Lenexa, KS) introduced EX-CELL CD Hydrolysate Fusion, the first chemically defined cell culture supplement based on hydrolysate characterization that promises to match the capabilities of traditional undefined hydrolysate raw materials in CHO, NS0, and Sp2/0 bioprocesses. In November 2009, SAFC Biosciences further introduced EX-CELL ANTIFOAM, a non-animal–origin antifoam to increase consistency, efficiency, and stability of cell culture manufacturing.

Movement to less complex and animal-origin free culture media does not come without its share of problems. The term animal-origin free refers to any component not directly derived from eukaryotic animals (excluding higher plants, fungi, protozoa, and algae) and media suppliers are now being asked to provide certificates of origin of their components ensuring their source is non-animal–derived, and if it is, then it is from a country not affected by the BSE outbreak.

Suppliers of raw materials are under no obligation to provide raw materials free from contaminants, but audits performed by biopharmaceutical companies exercise stringent guidelines and specifications highlighted by the regulatory authorities. Raw materials must be fit for their intended use and show a high degree of consistency between batches. This generally presents no problem because most raw material suppliers adhere to current good manufacturing practices (cGMP) and are ISO 9001:2000 accredited, which is ensuring the relevant SOP documentation is readily available on request.

What Degree of Animal-Free?

The degree to which a product has come into contact with a raw material of animal origin is defined as a level. The primary level indicates that the finished product itself and its associated manufacturing process are free from animal-derived components.

Secondary level classification is the most common, and ensures that raw materials used to manufacture a primary raw material to be used in a manufacturing process are animal-origin free. For example a peptone derived from E. coli grown in a culture containing non-animal–derived components would be classified as secondary level.

The most challenging standard of a component is the tertiary level, requiring that starting raw materials, needed to produce secondary raw materials, are animal-origin free. A raw material can be classified as tertiary level if it contains animal-derived components that are classified as very low risk. This requires an extensive audit trail and because of difficulties in establishing, monitoring, controlling, and maintaining such stringent guidelines, many biopharmaceuticals manufacturers are avoiding tertiary level components and settling with secondary level materials. Even tertiary level components can cause a debate. Should material be classified as tertiary level if it has been at all associated with material of animal origin? Establishing animal-origin–free facilities requires that all of its suppliers provide animal-origin-free products, and that all of their suppliers do the same. Even if one company uses no animal-derived components, there remains a risk of contaminant carryover from the processing steps of its suppliers.⁹

The Future of Media Development

Biopharmaceutical companies are beginning to take a responsible approach to media formulation, and significant effort is being invested in the development of well defined, animal-origin free alternatives. It is important, however, to establish actual risk when deciding the level of animal-origin free components beyond the primary level. If a critical or expensive bioproduct such as insulin is to be manufactured, then the biopharmaceutical manufacturer may well be justified in setting raw material specifications. Many are seeking secondary level animal-origin–free insulin despite the injectable being a primary level product.¹⁰ This shows that before considering the animal-origin–free level of a raw material, it is important to review all aspects of the process and determine the overall potential risk of the product.

A shift toward the universal use of chemically defined media is a beneficial step forward in the long term. Companies must be prepared for a change in regulatory guidelines, which may include a ban of animal-derived components from biologics manufacturing or stipulate a complete chemically detailed profile of raw materials used in the process. Either way, a chemically defined process must be the ultimate goal of all biopharmaceutical companies, helping them meet their goal of balancing media development with patient safety.

MICHELLE LEA, PhD, is a senior fermentation development scientist at Eden Biodesign Ltd, Liverpool, UK, +44 151 728 1750, michelle.lea@edenbiodesign.com

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