Animal serum and other complex biological materials have been employed in the cultivation of mammalian cells for approximately 100 years. Several factors led to the wide adoption of bovine serum as a standard tissue culture supplement. In comparison to serum from other animal species (horse, goat), bovine serum is easily sourced, and thereby more affordable. Many investigators choose to use fetal serum in their experimental systems because of concerns associated with antibodies present in newborn and adult serum that could cross-react with cells in culture and cause cell lysis through complement-mediated pathways. To eliminate that concern, heat was introduced to inactivate complement that was potentially present in the serum. Studies of FBS undertaken in the 1950s on the cultivation of low-density human cells to elucidate mechanisms of cell growth found that (1) the albumin component may serve as a carrier of essential small molecules; (2) fetuin, a glycoprotein present at high levels in the alpha globulin fraction, facilitates cell attachment and stretching; and (3) fetuin markedly inhibits trypsin, and this antiproteolytic activity may play a role in the ability of fetuin to stimulate cell growth.

In the 1960s and 1970s, serum supplementation of tissue culture media became the norm, thus facilitating biomedical research as well as the first large-scale vaccine manufacturing processes. Serum supplementation reduced the requirement for optimizing medium formulations for different cell types. FBS was shown to provide a variety of polypeptide growth factors. Albumin promoted cell growth presumably because of its abilities to function as a carrier protein for small molecules or lipids, to bind metal ions, to serve as a pH buffer, and to protect cells against shear. Similar functions were found for other serum components such as transferrin, hormones, and other serum-derived attachment factors such as fibronectin, vitronectin, and laminin.

Serum is a complex mixture of macromolecules that is required for cell growth and virus production, and its use as a raw material presents a number of challenges. These include its batch-to-batch composition and the risk of contamination by adventitious agents. The development of serum-free media has replaced serum in some new biotechnology manufacturing applications, but many cell lines used in manufacturing have not been adapted to these serum-free media. Regulatory constraints and scientific challenges generally make it impractical to alter existing manufacturing processes in which serum is used as a raw material.

FBS sometimes is required in cell and tissue bioprocessing, which often involves the cultivation of cells from tissue explants and biopsies. Some bioprocesses may also require the maintenance of specific cellular characteristics during cultivation. FBS often appears to facilitate such procedures and may affect the biological behavior of fastidious cell types. FBS has been shown to affect the transcription of developmentally important genes, apoptosis, and apoptosis-related gene expression, and to provide neuroprotective and antioxidative factors, all of which may be beneficial to the survival and development of cells in culture. Therefore, FBS will continue to play an important role as a cell culture supplement for production of cell- and tissue-based therapies.

In most viral vaccine manufacturing processes the media used for cell culture expansion and virus infection/production are supplemented with different types of serum at different concentrations. In these processes, bovine serum helps generate a mass of cells in an optimal physiological state for efficient viral replication.

For all types of bovine sera, blood should be collected in government-inspected and -registered premises (slaughterhouses, abattoirs, and donor farms). Blood should be collected by trained operators following the written procedures approved by the serum manufacturer and using either single-use disposable collection devices or reusable collection equipment for which cleaning procedures have been validated.

Trained personnel should perform serum separation (harvesting) and further processing activities following written and approved procedures. Serum is first separated and pooled, followed by filtration and filling into clean and disinfected containers. If the serum is subjected to one or more virus inactivation treatments in the production process, serum manufacturers should validate the virus inactivation processes against a range of relevant viruses. It is recommended that bovine viral diarrhea virus (BVDV) be included in any virus validation study because it is ubiquitous.

Finished product labels must contain the following information: product description, lot number, storage conditions, name and address of manufacturer, and a statement indicating the intended use. Materials intended for research purposes are exempt from labeling regulations (21 CFR 801). Typically, serum manufacturers supply a lot-specific Certificate of Analysis (COA) that is classified as part of the product’s labeling. See COA requirements in the following section.

TSEs are transmissible animal and human diseases that are characterized by degeneration of the brain, associated with severe neurological signs and symptoms. Since the outbreaks of TSE in cattle, termed BSE, which were transmitted to other species, public health officials have been concerned about the risk of TSE infection, including the possibility of TSE transmission by the use of therapeutic products manufactured using bovine serum. In cattle infected with BSE, lower titers have been found in the cerebrospinal fluid, lung, lymph tissue, spleen, kidney, liver, and ileum. Studies have shown that transfusion of blood from sheep infected with either BSE or scrapie but without evident disease can infect naive sheep. Although the risk of cross-contamination is always present, to date no studies have shown that blood can transmit disease from cattle with BSE. Embryos from BSE-affected cattle have not transmitted diseases to mice. Calves born of dams that received embryos from BSE-affected cattle have survived for up to 7 years, and examination of the brains of both the unaffected dams and their offspring revealed no spongiform encephalopathy.

No currently available procedures have been validated as being sufficiently sensitive for routine antemortem screening of asymptomatic animals, although analytical methods are under development for detection and quantitation from low-infectivity materials such as blood. The classic diagnostic test for TSEs is postmortem histological examination of brain tissue to confirm characteristic vacuolar degeneration. Other testing options include immunohistochemical tests that can confirm the presence of PrPSc, the abnormal disease-specific conformation of prion-related protein (PrP), in the vacuolated regions of the brain; and immunochemical tests such as Western blots and enzyme-linked immunosorbent assays that can detect PrPSc in tissues with high or moderately high titers. These tests typically take less time to perform than histological examination (6–8 hours vs. weeks, respectively) and can be partially or fully automated. Although most of these are postmortem tests, studies have demonstrated the feasibility of antemortem testing of lymphoid tissue samples from the tonsils or from the third eyelid of infected animals. Immunochemical tests require extensive sample collection and preparation and can be cost prohibitive for routine testing and monitoring the disease state of large herds. Diagnostic strategies must consider the sensitivity of testing in certain tissues as well as the test’s ability to detect infectivity in animals before the development of clinical signs of disease. Negative results do not ensure the absence of infectivity. Detection of infectivity is possible if suspect tissue is inoculated into experimental animals intracranially where the causative agent can amplify. This approach for detection of low infectivity can take months to years to yield a positive result.

Serum manufacturers should employ risk reduction strategies to eliminate the danger of cross-contamination of fetal blood with other tissues, including appropriate sourcing of animal-derived articles and using practices that have been shown to eliminate or minimize the risk of transmitting TSE, via either foods or health care products. Serum end users should perform a risk assessment of their sourcing strategy that takes into account the amount of bovine serum used in their application and should conduct supplier audits to ensure traceability of sourcing, handling, and appropriate quality control systems.

Rigorous testing procedures, strict processing and production guidelines, and appropriate risk assessments help ensure the safety of the different types of bovine serum. This section discusses specific tests that can detect and control adventitious agents.

The adventitious agents testing required for the evaluation of master seeds, master cells, and bulk and final products is described in 9 CFR 113.53 and by directives from the European Agency for the Evaluation of Medicinal Products (EMEA) (EMEA/CVMP/743/00 and EMEA/CPMP/BWP/1793/02). The testing methods outlined in these documents can detect a wide range of bovine microbial agents in serum products. These testing methods meet the requirements for most of the world’s regulatory agencies. Serum manufacturers should test a representative sample of each batch of serum to determine the presence of adventitious agents. Testing is performed after filtration but before any further processing that is intended to inactivate or remove viruses.

Filtration with 100-nm (0.1-µm) pore size filters is an accepted method for removing mycoplasmas and gamma irradiation (> 25 kGy while frozen), and chemical treatments (e.g., with betapropiolactone) are accepted methods of inactivating viruses and mycoplasmas; serum manufacturers routinely use these tools in both production and testing facilities. These treatments do not remove antibodies that may interfere with some applications. Additionally, the treatments do not ensure complete viral removal or inactivation, but can significantly reduce the risk of viral activity. The testing series to screen bovine serum for the absence of adventitious agents typically includes the following:

The procedures described in Sterility Tests 71 confirm the absence of bacterial and fungal infection. For viruses, only cultivation using suitable substrate cells can indicate viral infectivity and replication. Those who use serum for research or production should test the serum for the absence of adventitious agents in a manner that is consistent with the product's intended application, bearing in mind that testing indicates only presence or absence of adventitious agents within the limits of the test procedures used.

Mycoplasma contamination in tissue culture can arise from many animal origin sources, including serum, but more commonly it results from cross-contamination of infected cultures. Mycoplasmas are particularly insidious contaminants in cell culture because they

Classical mycoplasma detection is described in Mycoplasma Tests 63.

In addition to these methods, more recent detection procedures include luminescent and polymerase chain reaction (PCR) assay procedures. Nucleic Acid-Based Techniques—Amplification 1127 describes the general principles of PCR assays. The sensitive 20-minute luminescent assay measures a specific enzyme activity of mollicutes that converts adenosine diphosphate to adenosine triphosphate via a luciferase/luciferin reaction. Results are unequivocal and semiquantitative. PCR methods are quick and sensitive and display with good reliability, but occasional false positive results are a source of concern with commercial testing service labs. PCR may detect mycoplasmal DNA fragments that are non-infectious.

The virus testing procedures for serum products are outlined in 9 CFR 113.52 and 9 CFR 113.53. In addition, there are other documents that may include equivalent or relevant testing such as EMEA/CVMP/743/00-Rev.2 from the Committee for Veterinary Medicinal Products (CVMP) Revised Guideline on Requirements and Controls Applied to Bovine Serum Used in the Production of Immunological Veterinary Medicinal Products and EMEA/CPMP/BWP/1793/02 from the Committee for Proprietary Medicinal Products (CPMP) Note for Guidance on the Use of Bovine Serum in the Manufacture of Human Biological Medicinal Products. Serum manufacturers should perform virus testing in compliance with this regulation, using at least two different and sensitive detector cell lines, one of which should be of bovine origin. The tests include cultivation of detector cells in cell culture media supplemented with 15% test serum for at least 21 days. Cells are subcultured at least twice during this period, usually 7 and 14 days post inoculation. At the conclusion of the last subculture (after a total of at least 21 days of incubation), cells are examined for general signs of virus amplification. The following end points are used for general virus detection: microscopic cell examination for cytopathogenic agents such as infectious bovine rhinotracheitis virus, cell staining and microscopic examination for inclusion bodies, and hemadsorption test to detect hemadsorbing agents such as PI-3. In addition to this series of testing and at the conclusion of the last subculture (after a total of at least 21 days of incubation), cells are stained with specific fluorescent antibodies against the following specific viral agents:

In addition to the viruses listed above, other viruses can be causative agents of disease and may require testing in various bovine serum applications. The serum end user is responsible for determining whether full 9 CFR testing is sufficient, and if other specific viral agents should be tested for. Examples of specific viruses not covered by the current virus testing guide may include akabane, bovine herpesvirus 1 (BHV-1), Parainfluenza-3 virus (PI-3), bovine leukemia, bovine rotavirus, bovine circovirus, bovine polyomavirus, coronavirus, torovirus, bovine enterovirus, bovine astrovirus, foot-and-mouth disease virus (FMDV), and rinderpest. Appendix 2 provides a general description of some of these viruses as well as the ones for which testing is required. A serum end user’s thorough risk analysis should determine the scope of testing and serum treatment options.

Serum manufacturers and serum end users should carry out a comprehensive, science-based risk assessment (e.g. Failure Modes and Effects Analysis) in order to better understand the safety profile of the serum product. The following risk assessment elements can be taken into consideration, but other elements can be included as appropriate: country of origin, region of the country, animal disease status of the country/region of origin, animal age, blood collection process, animal stunning method and exsanguination method, serum manufacturing process, type of production quality system, production in-process controls, final product testing, virus inactivation, equipment segregation, equipment cleaning procedure, personnel training, serum use/application, pharmaceutical product type, and intended use.

The species barrier provides a degree of protection against infection by some animal etiologic agents. This barrier is not an alternative to proactively ensuring that pharmaceutical products are manufactured only from raw materials of animal origin that have undetectable levels of adventitious agents. Inoculation of viable organisms into a nonhost species carries a risk that the organisms could cross the species barrier. An appropriate test regimen of serum material should therefore include examination for potential contaminants associated with the species of origin and the species of intent. Serum treatments to inactivate viral agents are a factor in establishing the appropriate test regimen for a particular material. Lowest risk of contamination is associated with biological materials that are terminally sterilized.

Zero risk is neither possible nor reasonable. The serum manufacturers should fully describe specific testing regimens in the product specifications, and these will vary depending on the type and source of the serum. Therefore, the guidelines for screening described in this chapter are examples only, and screening for all viruses listed may not be required for a particular material. Some manufacturers may perform certain tests on the finished product or on in-process materials rather than on individual component(s). Manufacturers must also evaluate the dilution effect in relation to the limit of detection of the test procedure. Interference with growth or neutralization of viral activity by serum may be an indication of a specific antibody or certain nonspecific factors in serum masking the viral agent. It is recommended that serum manufacturers consider this possibility when determining an adequate level of treatment in their viral inactivation studies or in virus testing applications.

Serum manufacturers should confirm that the species of origin is bovine to ensure that no other nonbovine agents may be present. Manufacturers should perform extraneous virus testing in appropriate cell cultures (see Virology Test Methods 1237 for appropriate cell line choices dependent on assay and targeted agent). If necessary, seroconversion studies should be conducted in susceptible animal species using a host species immune antibody response as the method of detection. Studies should use this procedure following an inactivation step to detect whether the virus was present before the virus inactivation process.

Serum manufacturing processes should be conducted in a consistent manner, following the established manufacturing procedures, with adequate quality systems built into the production process. Furthermore, equipment segregation (by species of origin), equipment and facility cleaning procedures, and personnel training are important elements in the risk assessment of the process.

End users of donor bovine serum may require serum that does not have detectable antibodies against BVDV or other specific agents so that the users can propagate cell cultures used in vaccine production, diagnostic testing, and test kit preparation, especially for the maintenance of master seed and master cell stocks. More than 40 cell types are available for the production of veterinary biologicals, but fewer than 10 media types are available for their propagation. Some researchers have proposed serum-free media as an alternative in propagating certain cells and viruses; but this means adapting culture procedures, which may alter the cells and change production results. If new or different sera are imported into the U.S., serum end users will require confirmation of source, species, and documentation of the origin of the sera in countries that are free of FMD and rinderpest.

In the absence of end product-specific requirements, each lot of FBS should be tested to confirm that the serum meets the requirements of the proposed general chapter Fetal Bovine Serum—Quality Attributes and Functionality Tests 90. For all other types of bovine sera, this section describes several key procedures for characterization. These procedures are not mandatory but are guidelines that manufacturers may consider for their individual applications. The table in the Hemoglobin section shows samples of specifications for the different types of bovine sera.

Both inter- and intraspecies identification assays should be performed on bovine sera to confirm species identity and the integrity of the serum products, and to ensure that nonbovine agents are not present. The most commonly used assay for the identification of bovine species identity is based on the electrophoretic profile of specific serum proteins. With electrophoresis, the serum proteins usually separate into as many as six fractions: albumin, alpha 1, alpha 2, beta 1, beta 2, and gamma globulins.

Other procedures used for bovine speciation include radial immunodiffusion (RID) and the double diffusion Ouchterlony method. These procedures allow either qualitative or quantitative measurements of the immunoglobulin G levels in serum. The RID method is based on the diffusion of an antigen from a circular well into a homogeneous gel that contains specific antiserum for each particular antigen. A circle of precipitated antigen and antibody forms and continues to grow until it reaches equilibrium. The diameters of the rings are a function of antigen concentration. The Ouchterlony method is a double gel diffusion test wherein antigen and antibody diffuse toward each other in a semisolid medium to a point in the medium where optimum concentration of each is reached, forming a precipitate. The Ouchterlony plates contain cylindrical wells—a central 8-mm diameter antigen well, surrounded by six 3-mm antisera wells—which make possible the simultaneous monitoring of multiple antigen–antibody systems and the identification of particular antigens in a preparation. The proposed general chapter Fetal Bovine Serum—Quality Attributes and Functionality Tests 90 describes the accepted procedure.

Hemoglobin is a multi-subunit protein that forms an unstable reversible bond with oxygen in the red blood cells. The oxygen-loaded form is called oxyhemoglobin and is bright red. The oxygen-unloaded form is called deoxyhemoglobin and is purple-blue. Oxyhemoglobin is the predominant form in red blood cells.

Low hemoglobin content in sera is widely accepted as a good general indication of rapid and careful processing of blood that will be used for serum. Red blood cells are fragile and rupture easily, releasing hemoglobin into the serum. Rough handling of the harvested blood, poor temperature control, or delayed processing elevates hemoglobin content in serum. Acceptable levels of hemoglobin may vary with intended application. The hemoglobin levels are measured using spectrophotometric procedures (see Spectrophotometry and Light-Scattering 851) as described in the proposed general chapter Fetal Bovine Serum—Quality Attributes and Functionality Tests 90.

The testing of components such as cholesterol, alpha globulin, beta globulin, gamma globulin, albumin, creatinine, bilirubin, glucose, alanine aminotransferase, aspartate aminotransferase, phosphorus, potassium, calcium, and sodium usually is not considered a criterion for bovine serum lot release. Some manufacturers do not perform the tests on a routine basis but only as auxiliary tests. In some instances hospital clinical laboratories may run the tests. The levels of these chemicals in serum are important to end users and may also be used to assess lot-to-lot variability.

Although high endotoxin levels are not suitable for applications involving injectables, acceptable levels in bovine sera vary depending on the intended application. Some manufacturers may overlook the importance of low endotoxin levels in bovine sera used in cell culture applications. Endotoxin influences more than 30 biological activities. Some of these are macrophage activation, mitogenic stimulation, and induction of interferon and colony-stimulating factor (for some applications, these may be positive activities). Endotoxin can also lead to cytotoxicity by initiating complement activation. The most commonly used methods for endotoxin detection are the semiquantitative gel clot Limulus amebocyte lysate procedure and the quantitative kinetic chromogenic method described in Bacterial Endotoxins Test 85. For both the gel clot and the kinetic chromogenic assays, valid endotoxin assays require appropriate treatment by heat or dilution in order to avoid adverse effects of interfering substances in serum. Researchers should include a positive product control in each assay to confirm that any interference has been overcome by the heat or dilution treatment.

The osmolality test is designed to evaluate the electrolyte concentration in bovine serum. Chemicals that affect serum osmolality include sodium, chloride, bicarbonate, potassium, proteins, and glucose. Serum manufacturers should measure the osmolality of each serum batch to verify compliance with product specifications, using equipment calibrated with standards that are traceable to the National Institute of Standards and Technology. Osmolality and Osmolarity 785 describes how osmolality is determined by freezing-point depression of the bovine serum solution. Scientists use at least two standards to calibrate the instrument. The osmolality of each sample is calculated and related to the serum water content and is expressed as mOsmol/kg H2O.

The total protein level in serum is measured to verify animal age and compliance with product specifications. Biotechnology-Derived Articles—Total Protein Assay 1057 describes two procedures, the Biuret and Bradford methods, for determining protein concentration. The acceptable level of protein in serum should be assessed by the end user based on the intended application.

Each lot of serum should be tested for its ability to support in vitro growth of specific cell lines. Bovine sera are highly variable, and different lots may yield different results. Because of this variability, end users should characterize and standardize the cell lines that they will use for this type of testing. End users should design cell growth procedures that will help them check the efficacy of bovine serum in promoting cell growth. Serum manufacturers will benefit from monitoring growth promotion over several generations of subcultures to detect any evidence of cytotoxicity or changes in cell morphology. Different serum manufacturers use different cell types, and the growth studies and cell lines used by serum manufacturers also may differ from those applied by serum end users. When serum manufacturers evaluate the growth properties of a specific cell line in response to a specific lot of serum, they should take into account plating efficiency and/or growth promotion or some other functionality tests that qualify the serum lot for its intended use.

Plating efficiency at low cell density is a preferred method for analyzing the proliferative capacity and survival of single cells under optimal growth conditions. This procedure can reveal differences in the growth rate within the population and is capable of distinguishing between changes in growth rate (colony size) and cell survival (colony number). The growth kinetic is another important aspect in the design of cell-based experiments. Determining the growth curve of each cell line helps define optimal culture conditions, because variation in serum and other growth additives may influence growth parameters, which may affect the experimental outcome.

In the absence of specific tests designed for their particular products, serum users can refer to the functionality tests described in the proposed general chapter Fetal Bovine Serum—Quality Attributes and Functionality Tests 90 to determine whether a lot of serum is suitable for their application. This chapter provides guidance about how to perform growth promotion and plating efficiency tests.

Serum manufacturers should use an appropriate cell line for testing each lot of serum, and should perform growth studies through several subcultured generations to ensure that the serum has no cytotoxic effect on the cells. The choice of cell line depends on the intended use of serum. The cell growth and cytotoxicity assays should be performed on the final batch of serum after any viral inactivation step or any further processing.