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Regulatory agencies and associations set standards for the integrity of manufactured and distributed FBS and bovine serum products. Standardization is essential for producing the highest quality and for preserving integrity of bovine serum products. Reputable serum companies will perform at least the minimum testing requirements and will report results on their Certificate of Analysis (COA).
When comparing COAs it is important to review and compare units of measure and specification parameters for each serum type that is received from your serum supplier. Reputable serum companies are expected to report ranges that are within industry acceptable specifications. COA’s or quality related documents should not be signed by the President, CEO, or Executive whose objective is to make a profit for the company.
Assurance that bacteria and fungi have been removed during manufacturing process. Serum filtered through 0.1um pore sized integrity tested filters, will remove bacteria and fungi and provide a product suitable for cell culture applications.
| Reference | Title | Description |
|---|---|---|
| EP 2.6.1 | Biologicals Tests-Sterility | Satisfactory result indicating that no contaminating micro-organism has been found. |
| USP <71> | Sterility Tests | If no evidence of microbial growth is found, the product to be examined complies with the test for sterility. |
| 21CFR 610.12 | General Biological Product Standards - Sterility | Verified to demonstrate that the test method can consistently detect the presence of viable contaminating micro-organisms |
| 9CFR 113.26 | Detection of viable bacteria and fungi except in live vaccine | No extraneous growth found |
| JP 4.06 | General Test - Sterility Test | No evidence of microbial growth found, product tested meets the requirement of the Sterility Test. |
Composition of serum. A high level of GGT is an identifying marker of NBCS. FBS shall not exceed 10 IU/mL. An independent profile can be determined by an inexpensive, large animal chemistry panel from a veterinary diagnostic facility to ensure validity of COA. Method for authenticating FBS can be found here.
| Reference | Title | Description |
|---|---|---|
| As Reported | Clinical Chemistry Analyzer | Used to determine concentration of metabolites, electrolytes, proteins in serum. Serum is mixed with reagents and measured through a colorimeter. The analyzer calculates the chemical concentration. |
| EP - Bovine Serum | Bovine Serum- composition | Expected range for: Cholesterol, alpha, beta and gamma globulin, albumin, creatinine, bilirubin, glucose, serum aspartate transaminase (sAST, formerly sGOT), serum alanine transaminase (ALT, formerly sGPT), phosphorus, potassium, calcium, sodium, and pH. |
Characteristic of bovine animal age. FBS has a very low gamma globulin fraction compared to other serum types.
| Reference | Title | Description | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Cellulose Acetate | Electrophoretic identification - Company specified | Separation of protein by charge. Decreasing area under the curve for each protein type with albumin being most concentrated to gamma globulin being the least. Gamma globulin increases with age of donor animal.
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| Capillary Electrophoresis | Electrophoretic identification - Company specified | Electrophoretic migration of the individual proteins based on charge to mass ratio at specific voltage, electrolyte composition and pH conditions. |
Gram negative bacterial contamination. Low endotoxin concentration indicates care in collection and processing of bovine blood, serum, and manufacturing. Endotoxin is not removed by filtration and can affect cell growth characteristics.
| Reference | Title | Description | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| USP <85> | Bacterial Endotoxins Test | Portions have been harmonized with EP and/or JP. Test to detect or quantify endotoxins from Gram-negative bacteria using amoebocyte lysate from the horseshoe crab. Three techniques: gel-clot- based on gel formation; turbidimetric-based on development of rabidity after cleavage of an endogenous substrate; and chromogenic technique-based on the development of color after cleavage of a synthetic peptide-chromogen complex. | |||||||||||||||||
| EP 2.6.14 | Bacterial Endotoxins | Test to detect or quantify endotoxins from Gram-negative bacteria using amoebocyte lysate from the horseshoe crab. Three techniques: gel-clot- based on gel formation; turbidimetric-based on development of rabidity after cleavage of an endogenous substrate; and chromogenic technique-based on the development of color after cleavage of a synthetic peptide-chromogen complex. 6 Methods:
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| EP 5.1.10 | Guidelines for using the test for bacterial endotoxins | Method A is no longer declares as the reference method, and all methods A to F can be used. Where the method is stated in the monograph, the use of another method must be validated. Reference is made to the use of alternative reagent to the LAL such as recombinant factor C. | |||||||||||||||||
| JP 4.01 | Bacterial Endotoxin Test | Test to detect or quantify bacterial endotoxin of gram-negative bacterial origin using an amoebocyte lysate prepared from blood corpuscle extracts of horseshoe crab (Limulus Polyphemus or Tachypleus tridentatus). Two types: Gel-clot, based on gel formation by the reaction of the lysate TS with endotoxins; and photometric, based on endotoxin-induced optical changes of the lysate TS including the turbidimetric technique. |
Identify species of origin in fetal bovine serum using antigen / antibody immunological technique
| Reference | Title | Description |
|---|---|---|
| Ouchterlony | Ouchterlony Double Immunodiffusion Company Specified | Round wells are cut in a layer of agar. The central well is filled with the serum and other wells are filled with positive and negative antibody control specimens. Immune complex forms with reagents are allowed to diffuse in the agar and meet at optimal portions. Result is a visible precipitation between the serum and the bovine antibody. |
| EP 2.7.1 | Immunochemical Methods | Based on the selective irreversible and non-covalent binding of antigens by antibodies. Formation of the antigen-antibody complex is detectible and may be measured. Immunoprecipitation Methods -Simple diffusion method -Single radial immunodiffusion (sRID) -Double diffusion method -Comparative double diffusion method Immunoelectrophoretic Methods - Crossed immunoelectrophoresis - Electroimmunoassay (rocket immune-electrophoresis -Counter-immunoelectrophoresis |
Lower Hgb indicates care during collection and serum processing. Higher Hgb may indicate extended time before separation of blood to serum or cell lysis due to freezing prior to processing. Higher Hgb indicates red and white blood cell lysis and may result in virus release. Three hemoglobin types oxy, met and carboxy.
| Reference | Title | Description |
|---|---|---|
| Fleming | A spectrophotometric method for the quantitative estimation of bilirubin in liquor amnii | Fleming AF, Woolf AJ Clnica chimica act; international j. of clinical chem. 12:1 (1965) pg 67-74 Describes method of spectrophotometric analysis of serum for hemoglobin. Measures oxy-hemoglobin. Abs576x115 - Abs623x102 - Abs700x39.1 = mg/dL |
| USP <90> | Fetal Bovine Serum-Quality Attributes and Functionality Test | Calculate concentration of hemoglobin in mg/dL by absorbance of the serum sample. Abs576x115 - Abs623x102 - Abs700x39.1 = mg/dL |
| Three Wavelength | Direct Spectrophotometry of Serum Hemoglobin: An Allen correction compared with a three-Wavelength Polychromatic Analysis | Hunter F, Morton G, Soutter L (1950) Am. J. of Clin Path 20(5):429-433 Estimates hemoglobin concentration by spectrometry. Polychromatic formula gives concentration estimates approximately 5% greater than those of the Allen correction. Abs415x1.68 - Ab380x0.84 - Abs450x0.84 mA = mg/L |
| Three Wavelength Allen Correction | A spectrophotometric method for quantitating hemoglobin in plasma or serum | Noe D, Weedn V, Bell W; Clin Chem (1984) May;30(5):627-30 Estimates hemoglobin concentration by spectrometry. Abs415x1.65 - Ab380x0.93 - Abs470x0.0.73 mA = mg/L |
Type of antibody. Generally, IgG greater than 300ug/mL is associated with serum other than FBS. Purity of FBS can be determine if GGT is less than 10 IU/L
| Reference | Title | Description |
|---|---|---|
| ELISA | Enzyme Linked Immuno Assay (ELISA) | ELISA is a sensitive assay for measuring bovine serum IgG. Quantity of IgG in the serum can be interpolated from a stand curve of the assay. Serum types will vary in IgG quantity based on age. |
| EP 2.7.1 | Immunochemical Methods | Formation of the antigen-antibody complex is detectible and may be measured. |
| Refractometry | Company Specified | Measures how light is refracted when it passes through serum. Refractive index is used to compare values of known samples. Used as an estimation of serum IgG |
A cause of cell culture contamination that present problems in cell cultures and is difficult to diagnose. Capable of passing through 0.2um pore size filters. Read more details
| Reference | Title | Description |
|---|---|---|
| Barile (Large Volume) | Isolation of Mycoplasma arginine from Commercial Bovine Sera and Its Implication in Contaminated Cell Cultures | Barile, MF and Kern J (1971) Proc. Soc. Exp. Biol. 138:432-7 Mycoplasma testing of sera, media, and bioproduct by direct culture method described by Large Volume Barile Method. |
| 21CFR 610.30 | General Biologicals Products Standards - Mycoplasma | Test is satisfactory for vaccine manufacture if none of the tests on the samples show evidence of the presence of Mycoplasma. Specific to live and inactivated viral vaccines |
| 9CFR 113.28 | Detection of viable bacteria and fungi except in live vaccine | If no growth is found in any test vessel, the serial or subserial meets the requirements of the test. |
| 9CFR 113.24 | Detection of extraneous viable bacteria and fungi in live vaccines | No extraneous growth found |
| GMP | Real Time PCR | Real-Time polymerase chain reaction (PCR) for detection of Mollicutes species. |
| EP 2.6.2 | Mycobacteria | At the end of the incubation time no growth of mycobacteria occurs in any of the test media, the preparation complies with the test. |
| USP <63> | Mycoplasma Test: A new Regulation for Mycoplasma Testing | The product complies with the test if growth of typical Mycoplasma colonies has not occurred. |
| JP Mycoplasma | Mycoplasma Testing for cell substrates used for the production of biotechnological/Biological products | Detection of mycoplasma should be evaluated, and detection limit determined and validated. |
Reflects electrolyte and solute concentration. Ensures material is not diluted with water. Read more details
| Reference | Title | Description |
|---|---|---|
| USP <785> | Osmolality and Osmolarity | Corresponds to the molality of an ideal solution containing nondissociating solutes and is expressed is milliosmoles per kilogram mOsm/kg of solvent. Osmolality of a solution is typically determined most accurately and conveniently by measuring freezing point depression. |
| EP 2.2.35 | Osmolality | Submultiple milliosmole per kilogram (mosmol/kg). Determined by the measurement of the depression of freezing point. |
Relative functionality of a lot to a control serum. Standard performance test, results may vary depending on methodology and other variables. Cell survival in serum to a control. Useful to assess long-term culture of cells to determine any toxic effect present in serum.
| Reference | Title | Description |
|---|---|---|
| USP <90> | Serum Functionality Test | Determine the functionality of specific lots of serum. Growth kinetics is measured by counting viable cells after 7 days of culture on three cell lines. Viable cell counts are determined on days 0, 1, 2, 4, and 7 for cell lag, log and stationary phase. Doubling time of the test sample should be no less than 90% of the doubling time of control. |
| DNA Synthesis | Growth Promotion DNA synthesis | Measures of synthesized DNA in the presence of a label. Proliferating cells contain more DNA than necrotic or apoptotic cells. |
| Metabolic | Growth Promotion Metabolic Activity | Measures metabolic activity of proliferating cells. |
| USP <90> | Cloning | Plating efficiency or colony formation at low cell density evaluated proliferative capacity and survival of single cells under assay growth conditions. Percent plating efficiency is expressed by counting the number of colonies in a defined area divided by the number of cells seeded, multiplied by 100. |
Serum is part of a physiological environment supporting cell viability. pH range also confirms the serum has not been altered. Read more details
| Reference | Title | Description |
|---|---|---|
| USP <791> | pH | Value given by a suitable, properly calibrated, potentiometric sensor and measuring system. Measuring system has traditionally been referred to as the pH meter. Measurement should be at room temperature. |
| pH Meter | pH | Measurement should be capable of a two-point calibration with accuracy to 0.01 pH. |
| EP 2.2.3 | Potentiometric Determination of pH | Potentiometric determination of pH is made by measuring the potential difference between 2 appropriate electrodes immersed in solution to be examined: One electrode sensitive to hydrogen ions the other is the reference electrode. |
Characteristic of bovine animal age. FBS has the lowest protein concentration of the serum types.
| Reference | Title | Description |
|---|---|---|
| Biuret | Biuret Method for the Determination of Total Protein in Serum ad Exudates. | Tietz, Norbert W.: Fund. Of Clin. Chem (1976) 302-304 Biuret method is based on the complexation of Cu2+ to functional groups in the protein's peptide bonds. Produces a violet-colored chelated product when the formation of Cu+2 of two peptide bonds occurs. Intensity of color is directly proportional to protein content. |
| BCA protein | Measurement of protein using bicinchoninic acid | Smith et.al, Anal Biochemck (1987) May15; 163(1):279 Capable of monitoring cuprious ion produced in the reaction of protein with alkaline Cu2+. Forms a deep purple complex with bicinchonic acid. Measures total protein concertation compared to a protein standard. |
| Chem Analyzer | Clinical Chemistry Analyzer | Colorimetirc (Biuret). Total protein is measured in g/dL. Interferences include Lipemia>1000, Hemolysis>650 Icterus>21 |
| Properties | Method | Range | Units |
|---|---|---|---|
| Bacteria and Fungi/Sterility | EP 2.6.1, USP <71>, 21CFR610.12 | Not Detected | N/A |
| Biochemical Profile | Chemistry Analyzer, photometric | As Reported | Various |
| Electrophoretic Pattern | Cellulose Acetate | Characteristic | N/A |
| Endotoxin | USP <85>, EP 2.6.14 | <10 | EU/mL or IU/mL |
| Hemoglobin | Three-Wavelength Polychromatic | <30 | mg/dL |
| Immunoglobulin (IgG) | ELISA | Various | ug/mL |
| Mycoplasma | Barile, MF and Kern J (1971) | Not Detected | N/A |
| Osmolality | USP <785> | Various | mOsm/kg |
| pH | USP <791> | Various | N/A |
| Protein-Total | Biuret | Various | g/dL |
| Virus Testing | 9 CFR 13.53, 113.46 and 113.47 | Tested | N/A |
| Virus Testing - | 9 CFR 113.43 | Not Detected | N/A |
| Cytopathic Agents | |||
| Virus Testing - Hemadsorbing | 9CFR 113.46 | Not Detected | N/A |
| Virus Testing- | 9CFR113.215, company specific | As Reported | N/A |
| Serum Neutralization |
| Properties | Method | Range | Units |
|---|---|---|---|
| Performance - Growth Promotion | Cell grown in culture | 75% growth to control | N/A |
| Performance - Cloning | Cells grown in culture | 75% growth to control | N/A |
| Serum Species Identification | Ouchterlony double immunodiffusion | Positive for Bovine, Negative for Equine and Porcine | N/A |
Viruses are generally not removed by filtration. BVDV exists and a common adventitious agent in bovine serum. Tests for animal viruses such as IBR that produce a cytopathic effect in the host cell. Evidence of cytopathic effect include: inclusion bodies, abnormal number of giant cells, or other cytopathology indicative of cell abnormalities attributable to an extraneous agent. Test detections hemagglutinin producing virus such as PI3. Hemagglutinin is dependent upon selective attachment of erythrocytes onto the monolayer surface of tissue cultured cells. Test to detect and quantify antibodies specific for BVDV type 1 and type 2. Gamma irradiation provides good log reduction of most viruses while maintaining serum functionality.
| Reference | Title | Description |
|---|---|---|
| 9CFR 113.46 | Detection of cytopathogenic and/or hemadsorbing agents | If specific cytopathology or hemadsorption attributable to an extraneous agent is found, the material under test is unsatisfactory and shall not be used to prepare biologicals products. |
| 9CFR 113.53 | Requirements for ingredients of animal origin used for production of biologics. | Serum used to prepare a biological product shall be tested as prescribed in 113.53. A lot of ingredient found unsatisfactory by any prescribed test shall not be used to prepare a biological product. |
| 9CFR 113.47 | Detection of extraneous viruses by the fluorescent antibody technique | Test for detection of extraneous viruses by the fluorescent antibody technique. Cells shall be tested for Bovine virus diarrhea virus (BVDV), Reovirus, rabies virus, bluetongue virus, bovine adenoviruses (group A and group B), bovine parvovirus, bovine respiratory syncytial virus. |
| 9CFR 113.215 | Supplemental Assay method for titration of bovine viral diarrhea virus neutralizing antibody. | In vitro assay method that determines the serum neutralizing (SN) antibody titer to bovine viral diarrhea virus (BVDV) in serum as part of potency requirements for veterinary vaccines. Applies to cytopatic strains of either genotype 1 or genotype 2. Terminology of 1:10, 1:20 etc. specifies 1 part plus 9 parts (liquid). |
| CHMP/BWP/457920 | Guidelines on the use of bovine serum in the manufacture of human biological medicinal products | Test for viral contaminants should be carried out prior to any steps taken to inactivate or remove viruses. Specific test for the following viruses should be considered: Bluetongue and related orbiviruses, bovine adenovirus, bovine parvovirus, bovine respiratory syncytial virus, bovine viral diarrhea virus, rabies virus, reovirus 3. |
| CVMP/743 | Requirements and controls applied to bovine serum used in the production of immunological veterinary medicinal products | Combination of general and specific test to be carried out should be capable of detecting viruses inducing viraemia and transplacental infection such as Bovine Adenovirus, Bovine viral diarrhea virus, parvovirus, bovine respiratory syncytial virus, reovirus, parainfluenza 3, IBR and those responsible for diseases exotic to Europe such as Bluetongue. Test should be carried out before any inactivation treatments. Secondary test should be performed after the inactivation treatment at which time no virus should be detected in the final serum batch. These tests could be omitted if no virus is detected before inactivation treatment. |
| EP 5.2.4 | Cell Cultures for the production of veterinary vaccines | |
| EP 5.2.5 | Substances of animal origin for the production of immunological veterinary medicinal products | |
| Serum Neutralization (SN) | Serum virus neutralization is a serological test to detect the presence and magnitude of functional systemic antibodies that prevent infectivity of a virus. |
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