Click here to learn more about our sterility testing services. Meeting USP/EP/CFR sterility testing requirements with a two media assay - PDF
The three principal methods of sterility testing are direct inoculation, membrane filtration, and direct transfer.
Three principal methods for sterility testing are used:
- Direct inoculation
In the direct inoculation method, the test articles are inoculated directly into tubes or bottles that contain an appropriate medium and are incubated for a specified period. The advantages of the direct inoculation method are that it:
- provides a means of sterility testing for materials that cannot be easily filtered
- consumes less product volume during the conduct of the bacteriostasis and fungistasis (B&F) testing
2. Membrane Filtration
In the membrane filtration method, the test article is passed through a membrane filter, which is designed to retain microbial contaminants while permitting the passage of liquid test articles and inhibitors out of the test system. After the test article passes through the filter, the membrane is rinsed with an appropriate sterile rinse fluid. The filter units are then inoculated with the appropriate sterile rinse fluid and generally are incubated for the same time as in the direct inoculation method. Advantages of the membrane filtration method include:
- Accommodation of large volume samples (up to 500 ml)
- Removal of inhibitory substances that inhibit the growth of microorganisms by rinsing the filter membrane with a suitable agent
3. Direct Transfer
BioReliance offers custom protocols for direct transfer method. Typically, the direct transfer method is used for solid dose forms, medical devices, ointments, and creams. For test articles produced by non-aseptic manufacturing processes, a bioburden (or microbial limits) assay should be performed. Often, it is necessary to evaluate a non-sterile test article for the presence of objectionable organisms, depending on the intended use of the material. A microbial limit approach is recommended for the evaluation for objectionable organisms.
Mycoplasma Detection Assays
Mycoplasma are among the most common cell culture contaminants.
At a minimum, mycoplasma can alter normal cellular processes, virus production rates, and lymphocyte proliferation. Therefore, testing for mycoplasma in manufacturing cell substrates is essential. Moreover, mycoplasma can either directly or indirectly contribute to human disease, which represents a further regulatory concern. The general mycoplasma test is performed by using a method that tests for the presence of agar-cultivable and non agar-cultivable (cell-dependent / cell-associated) mycoplasma. Agar and semi-solid broth are used for detecting agar-cultivable mycoplasma species. An indicator cell line is used to detect non-agar-cultivable mycoplasma species. BioReliance offers a variety of mycoplasma assays that are performed in accordance with US FDA, PTC, CFR, EP and JP regulations. The EP requires mycoplasmastasis testing. BioReliance also offers mycoplasma detection by PCR.
In Vitro Virology Assays
Cell lines, virus seeds and raw materials used in the manufacturing of biological products can potentially harbor adventitious agents, including viruses. The purpose of the in vitro assays for adventitious viruses is to detect viruses in master and working cell banks, master and working virus seeds, unpurified bulk harvest and end of production cells. In vitro assays to detect the presence of numerous species specific viruses in raw materials/additives and cell lysates are also available.
Residual Contaminant Assays
For residual DNA. The level of contaminating DNA should be measured using quantitative based methods. BioReliance performs residual DNA evaluation for a wide range of host species, CHO, mouse, human, E.coli, yeast, canine, Pichia, and Vero using quantitative PCR based technologies. The ability to detect to femtogram levels of DNA ensures that assays are in compliance to FDA and WHO recommendations. Includes Residual Host Cell Proteins.
• Our LAL assays exploit this clotting reaction by using amebocyte lysate to specifically target the endotoxin LPS, which is a component in the outer cell membrane of gram-negative bacteria.
• The United States Pharmacopeia (USP) describes two LAL techniques for the detection of endotoxins.The gel-clot method quantifies endotoxin by the ability of the endotoxin to clot LAL reagents. Serine protease, a component in the clotting enzymatic cascade, cleaves water-soluble coagulogen to form coagulin, a water-insoluble protein, thus forming a clot.
• Photometric technique is the second method addressed by the USP.
• In the chromogenic method, serine protease acts on a chromogenic substrate to produce a chromophore. The quantity of chromophore is measured by spectrophotometry. BioReliance offers assays that use either the gel-clot or chromogenic methods.
• The purpose of this study is to allow quantitative enumeration of mesophilic bacteria and fungi that may grow under aerobic conditions and to determine the absence of or limited occurrence of, specified microorganism that may be detected.
• This assay meets and exceeds current USP <61> and <62>, Microbial Enumeration Tests and Tests for Specified Microorganisms.
The purpose of this study is to detect the presence of Mycobacteria species in a test article containing little or no bioburden using direct inoculation method.
Virus Specific Assays
BioReliance performs quantitative PCR based tests designed to detect species specific and adventitious viruses in cell banks, virus seed stock, raw materials and final products. Each assay method is fully validated in accordance with ICH Q2 (R1) guidelines and EP 2.6.21. Assays are available for human viruses, respiratory viruses, porcine viruses, simian viruses, canine, murine and many more specific target PCRs. Development and validation services are available for emerging viruses.
9 CFR Testing
Many cell culture systems require additives derived from bovine or porcine origin such as fetal bovine or newborn calf sera, insulin, transferrin, various growth factors and trypsin. BioReliance offers In Vitro assays for the presence of bovine and porcine viruses as described in the 9CFR.