Electron Microscopy Services
BioReliance offers a comprehensive range of electron microscopy assays. Transmission electron microscopy (TEM) is used to detect the presence of adventitious agents in biological fluids or in cell substrates. TEM methods enable characterization of cellular morphology of cultured cells and can be used to visualize and differentiate virus particles in cell cultures according to their morphology. By examining biological fluids, virus particles can be quantified to estimate the viral load of the sample. It is essential that viral load is determined prior to validating the ability of a purification process to inactivate or remove viral contaminants. Morphology of virus particles, as defined by TEM, is one of the major criteria for classification of viruses. Quantitation of virus particles can be performed by thin section TEM or by negative stain TEM.
Sterility Assays
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
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
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.
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.
Co-cultivation Assays
Cell lines used in the manufacture of biopharmaceuticals are derived from a number of mammalian species and are known to contain retroviruses. In some cases, these retroviruses exist solely as proviral sequences that are not expressed, are expressed as noninfectious particles, or are expressed only at very low levels. Co-cultivation of cells at the limit of the in vitro cell age (end of production cells) or of cells from a master cell bank with susceptible cells lines allows for amplification of these retroviruses to enhance the ability to detect their presence.
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.
In vivo Virology assays
BioReliance offers in vivo assays for the presence of adventitious and species specific virus.
Viruses that do not cause cytopathic or other noticeable effects in a cell culture system can be detected by an animal system. The International Committee on Harmonisation (ICH) document on 'Viral Safety of Biotechnology Products Derived from Cell Lines of Human or Animal Origin' (Q5A), the U.S. Food and Drug Administration and the Center for Biologics Evaluation and Research (FDA/CBER) 1993 Points to Consider (PTC) for cell line characterization and the Notes for Guidance issued by the Committee for Proprietary Medicinal Products (CPMP) require that in vivo tests for the presence of adventitious viruses be performed during various stages of product manufacturing.
Retrovirology Assays
BioReliance has experience in working with both human and animal retroviruses. A range of quantitative and qualitative assays have been developed for the purpose of detection of retroviruses. Tests include detection of reverse transcriptase by enzymatic and PCR assays, detection of the viral genomes by PCR, observation of virus particles by electron microscopy and detection of infectious retrovirus by tissue culture based methods e.g. co-cultivation assays.
Mycobacterium Assays
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.
