Final Product Testing

Serum suppliers test the final product against written specifications and provide certificates of analysis (CoA) for each lot of serum produced. As no pharmacopoeial monograph exists for serum, specific tests performed and reported on the CoA may follow existing test protocols found in the United States Code of Federal Regulations, or the United States, European or other pharmacopoeias, e.g. tests for sterility and mycoplasma. Where no guidance exists, such as for growth promotion testing, serum suppliers have developed their own testing methodologies or may perform client-specific assays relevant to the client's cell line and/or manufacturing regime. Table 4.3 lists the most common release tests along with the general methods that may be employed.

The desired ranges for the physiochemical parameters, pH, osmolality, appearance and physi-ochemical profile, are typically based upon historical ranges supplied by the serum producer. While the results provide information that the lot of serum being evaluated is similar to previous lots, it has been this author's experience that serum users rarely use this information as part of their selection criteria for serum, other than to record that testing has been performed, and rely most heavily on the content, performance and safety profile for the product.

In regard to the safety testing of the serum product, of special concern is viral screening for adventitious agents. Testing for viruses in animal sera by serial passage of susceptible cells with

FINAL PRODUCT TESTING Table 4.3 Example of tests and methods used in releasing final serum products.

Test

Method

Physicochemical characteristics

Appearance pH

Osmolality

Physicochemical profilea Identity and content Identity

Species identity Total protein

Haemoglobin concentration

Bovine IgG content

Safety testing

Sterility

Endotoxin

Detection of mycoplasma Adventitious viral agents

Virus antibody test Functionality Growth performance Productivity

Visual inspection

Potentiometrie

Depression of freezing point

Automated clinical systems, multiple assays

Serum electrophoresis band pattern Ouchterlony (double immuno-diffusion assay) Biuret reaction method Colorimetric determination Enzyme-linked immunosorbent assay

Membrane filtration

Kinetic chromogenic limulus amebocyte lysate (LAL) assay Hoechst stain (DNA fluorochrome stain) and/or broth/agar cultivation Cell culture isolation, fluorescent antibody staining, haemadsorption, cytopathic effects and/or polymerase chain reaction Measurement of neutralizing antibody in serum

Multiple-passage cell growth, clonal growth and/or plating efficiency Viral expression, protein expression aA list of chemical parameters tested can be found in Table 4.2.

the test sera is still the industry and government standard (FDA 2001; EMEA2002a; 2003), although other testing such as RT-PCR (reverse transcription polymerase chain reaction) may be employed. Cell lines such as VERO, and another primary or continuous cell line of the same species as the sera being tested, are serially passaged over a period of approximately 3 weeks. At regular intervals, the cell monolayers are observed for cytopathic effects (CPE) and haemag-glutination. After the last passage, cells are also screened for specific viruses by fluorescent antibody staining.

Serial passage in cell culture has many disadvantages:

• viruses may be non-cytopathic in cell culture;

• the limit of detection may not be sufficient to detect low level contamination in large serum batches. This is aggravated by the small size of evaluation samples compared with the total lot size. Low viral titres not detected as part of routine viral screening may be amplified during large-scale manufacture.

• The fluorescent antibodies may not detect all viral strains.

Serum that is reported negative on supplier CoA has been proven to contain live virus when assayed by a combination of RT-PCR and serial passage in cell culture (Yanagi et al. 1996). In another study, detection of bovine polyoma virus using susceptible cell cultures as a substrate took 4 to 7 weeks, which is approximately twice the length of time of the most common testing regimes (Schuurman et al. 1991). It should be assumed, regardless of viral testing results on a CoA, that all commercially available lots of sera that have not been treated with a viral inactivation or removal process contain some unknown level of virus.

PCR (polymerase chain reaction) has been shown to be an extremely sensitive test for detecting viral nucleic acid, but alone it is unable to distinguish between infectious viral contamination and nucleic acid contamination. Combining PCR with culture amplification can positively identify infected lots of serum (Yanagi et al. 1996; Schuurman et al. 1991). However, much validation of the appropriate probes and methodologies will be required before PCR is accepted as a routine product acceptance testing method, but it may be useful as an in-house screening tool. Validated, more sensitive methods of viral detection may create the quandary that more serum may end up being rejected even though similarly infected lots of serum are most likely used in manufacturing today. With the limitations of serum supply and the need to address safety concerns regarding viral transmission via medicinal products, methods to inactivate or remove contaminating viruses are required. This will be addressed in Section 4.8.

United States Department of Agriculture/Australian Quarantine and Inspection Service/ New Zealand Ministry of Agriculture and Forestry

Documentation on the origin of the batch is retained. Confirmation that the processing of the raw serum occurred on a specific date at a specified controlled facility is documented.

The certificate of origin documents that the raw serum was collected at USDA/AQIS/MAF approved slaughterhouses.

The origin of the batch, including the USDA/AQIS/MAF number of the abattoir for collection and date of collection, is documented in the raw material batch records for the serum sub-lots.

The device history record is the term used tor the batch record.

A health certificate contains a signed affidavit confirming the status of the country of origin, the source and health of the animals and the status of the sites of collection. However, it should be noted that these certificates are not obtained routinely for all batches of material, rather upon specific request.

Figure 4.2 Overview of traceability measures employed.

Some sera are sold as being specifically tested for a particular use, e.g. for insect or hybridoma cells. This may be misleading, as no growth assay can replace growth evaluation in a user's own laboratory with their cells, process and equipment. It is also relevant to point out that the presence of certain components that may be required for a given cell line might not be tested for specifically, such as oestrogen or platelet-derived growth factor.

As part of the CoA, serum origin information is listed along with a declaration of the inspection and health status of the animals, and a declaration of the bovine spongiform encephalopathy (BSE) status of the country of origin. Figure 4.2 provides a schematic presentation of the information deposited and retained by either the raw serum collector or the serum manufacturer to ensure traceability of serum of United States, Australian or New Zealand origin.

When raw serum is received by the serum manufacturer from the raw serum vendor, assurance of the country of origin and that the abattoirs are government registered and approved is provided on a certificate of origin (COO). As stated on the COO, the raw serum vendor retains documentation to confirm the following information on the collection of the whole blood:

• name of collection facility (abattoir);

• location of collection site, city, state;

• government approved establishment number;

• date of collection and amount collected.

This information is archived by the serum manufacturer as part of the raw material batch record.

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