Quality Assurance
The methods used for the collection of serum from Veterinary inspected healthy animals and its subsequent handling prior to processing for use largely dictate the quality of the final product.
Tests for sterile filtered serum are listed below and are performed according to the guidelines listed in the Code of Federal Regulations issued by the Food and Drug Administration (FDA) and the United States Department of Agriculture (USDA).
Our Quality Control Department verifies that the complete manufacturing process does not impart any adverse qualities to products. The function of our Quality Control Department therefore is threefold:
1. A protocol for the analysis of both raw material and final product serves to authenticate the Standard Operational Procedures that embrace all aspects of manufacture.
2. Inspection and evaluation of process materials, for examples, bottles, caps or filters, to verify the physical and functional compliance together with their suitability for cell culture.
3. Monitoring of the clean-room facilities in which aseptic filling and other critical production processes are conducted. Other areas are also checked for compliance with their specified environmental condition.
Sampling
By adopting sampling plans in accordance with BS 6001 statistically representative samples are selected from all batches of sera. These batches are kept in quarantine whilst comprehensive testing is carried out.
When the testing regime is concluded and it is known that the batch has achieved all the pre-set quality requirements, it is given final product status. Only then is it made available for sale.
Sterility Testing
1.0 Bacteria, Yeasts and Fungi
In order to maximise the opportunity to detect aerobic and anaerobic organisms over a wide temperature range a filter membrane concentration method is employed in addition to the standard broth enrichment procedure. All samples must prove to be sterile before the batch is released for sale.
Samples of serum are subjected to the following protocol:
1.2 Membrane Filtration
Samples of serum are subject to membrane filtration that is followed by broth enrichment for a period of 21 days. During the test period the samples are monitored regularly. Any ambiguous result is resolved by the adoption of a double sampling plan to re-test the batch. A batch is only accepted if all the test samples prove to be sterile.
2.0 Mycoplasma
Large quantities of serum are required initially in order to conduct Mycoplasma testing effectively. Statistically representative bottles are sampled and the serum is ultracentrifuged to concentrate any mycoplasma elements that may be present and therefore maximise the opportunity of detection.
SLI employs traditional broth enrichment and also a cell culture staining technique to ensure there is no evidence of mycoplasma in the test samples.
References:
1. Barrile, M.F. and Kern, J. (1971) Proc. Soc. Exp. Biol. Med. 138:432
2. Ki Hara, K. et al (1981) IABS 243
3. Chen, T.R. (1977) Exp. Cell. Res. 104:255
4. Del Guidice, R.A., and Hopps, H.E. (1977) Mycoplasma Infection of Cell Cultures (Plenum Press N.Y., Eds. McGarrity, G.J., Murphy, D.J. and Nichols, W.W.) 57
General Notes
The test procedures adopted by SLI take into account the “points to consider” as advised by the FDA.
3.0 Bacteriophage
Bacteriophage testing of serum collected aseptically from a healthy donor animal is an accepted practice ensuring that by their absence correct collecting and processing techniques have been observed.
Bacteriophage is determined by plaque assay using a host sensitive strain of E. Coli (C300).
4.0 Bovine Viruses
All batches of bovine sera are tested to confirm the absence of bovine viruses which are capable of either cytopathic or haemadsorbing effects, e.g. Infectious Bovine Rhinotracheitis (IBR) or Parainfluenza 3 (PI3). A specific test procedure for the absence of Bovine Viral Diarrhoea (BVD) virus is also conducted. For all tests virus-free bovine cells are propagated in the presence of test serum. These cells are adsorbed with the test serum prior to each of the six passages. The flasks of cells are regularly examined to verify there is no evidence of virus-induced cytopathogenic effects (CPE).
At the conclusion of the passage procedure the cells are adsorbed with the Guinea Pig red blood cells at 36.5°C for 1 hour. The absence of haemaglutination indicates freedom from PI3 infection of the test serum.
Further cells are tested specifically for BVD by incubating them in the presence of BVD specific antiserum. Any binding is then detected by fluorescent antibody staining techniques.
Physical and Biochemical Analysis
pH: This is measured using standard techniques at 25°C for hydrogen ion concentration. A specific pH range characterises each product.
Osmolality: This test confirms that by conforming to recognised standards the serum has not been adulterated during the course of collection and processing. It is determined by freezing point depression analysis.
Haemoglobin: This is measured spectrophotometrically to verify that both the blood and the sera have been collected in accordance with the Standard Operating Procedures.
Total Protein A: A modified Biuret method is used to verify that the serum has been collected from pathologically normal animals.
Albumin: A photometric method using Bromocresol verifies that the serum is within normal limits.
Gamma Globulin: The species of animal together with the concentration of immunoglobulin is verified by precipitation in the presence of anti-species IgG.
Endotoxin: This test not only confirms the suitability of the test serum as a culture medium additive but it also corroborates that the appropriate collection and processing techniques have been employed. Samples are analysed using a sensitive limiting dilution Limulus Amoebocyte Lysate (LAL) gel clot method.
Functional Testing
Serum products that actively support cell growth are fundamental to our operation. Samples of raw material are checked for compliance to product specifications prior to being committed for batch production.
The biological performance of the product is assessed in a variety of functional tests using a carefully chosen selection of human and animal origin cell types. The exact range of tests is, however, dependent upon the material to be analysed.
The concentration of test serum in control medium together with the inoculation density of cells is reduced to as low as practical to assess the nutritional properties of the material in supporting cell growth.
Morphology, confluency, cell numbers, clone size, cloning efficiency and plating efficiency as appropriate, are used to monitor performance. Microscopic examination is undertaken to confirm that the cells have remained free from morphological changes during the culture period.
Sera Lab chooses not to initiate functional assays from cells maintained in logarithmic growth phase but rather from frozen stabilities. This eliminates the possibility of introducing minor variations between cultures which can occur from various sources during the handling and sequential passage of cell stock. Furthermore, by the adoption of regimes that avoid sequential passaging a more objective assessment of test material quality is obtained. The results from all test assays are assessed in relation to those achieved by the reference cultures performed in parallel.
Human Diploid Fibroblast Cells
These cells, which are very fastidious in their nature and are widely used in vaccine manufacture, are seeded at a density of 1000 cells per test and incubated at 36.5°C for 6 days. The morphology and numbers of cells produced are then assessed and related to those of the control cultures.
Human Epithelial Cells
HEp2 cells have been chosen to represent the requirements of established (immortal) cells that are epithelial in nature. These cells are inoculated at a density of 1500 per test and incubated at 36.5°C for 7 days before assessing their morphology and calculating the number of colonies produced.
Relative Plating Efficiency
BHK21 cells are chosen for this quality procedure on some serum types due to their requirement for attachment factors and the role that they play in vaccine manufacture. The cells are seeded into flasks at a rate of 200 cells per test and incubated at 36.5°C for 7 days.
The results are assessed by the number of colonies formed as a factor of the number of cells inoculated. This factor is then related to that obtained from the reference culture.
Relative Cloning Efficiency
Batches of sera are tested using two mouse myeloma cell lines and a human epithelial cell line. Additionally, Foetal Bovine Serum is tested against mouse hybridoma cells. Each of these cell types is seeded at a concentration as low as one cell per well in 96 well microtitre plates that are incubated at 36.5°C for 7 days. The number of wells with clones are counted and related as a percentage to the number of wells inoculated.