Bioproducts obtained from fisheries and aquacultures, like any biotechnological products, must be extensively characterized at development and processing stages to define strict specifications, in terms of physicochemical properties, biological activity, immunochemical properties, purity, impurities and contaminants. Bioproducts need to meet certain predefined acceptance criteria. The main aspects that need verification concern the consistency of manufacture and the stability and safety of the bioproduct.

For example, in the case of medicinal products for human use (including biopharmaceuticals, EMEA's guidelines require extensive characterization to be done in the development phase as well as following considerable process modifications. Ideally, this characterization will include comparison with a reference standard. and/or the bioproduct's natural equivalent, if feasible. Manufacturers should also present suitably characterized in-house reference materials at the time of submission. These reference materials are intended for analytical (physicochemical and biological) assays of production lots.

A range of analytical methods are employed to characterize the physicochemical properties: composition, physical properties, and primary structure (desired secondary, tertiary etc. structure is commonly confirmed based on biological activity). This set of techniques aims to uncover any changes that may occur in the structure of the desired bioproduct during manufacture or storage, or as a natural part of the bioprocess. An overview of the common approaches is given in Table 3.3.2 (more 3.3.3).

Heterogeneity that occurs during manufacture and/or storage affects the quality of the bioproduct. This heterogeneity should have a consistent pattern, with consistency in product activity, efficacy and safety. Protein heterogeneity that is inherent to the bioprocess is often due to alternative post-translational modifications. What is important in this case is for the manufacturer to demonstrate that the resulting mixture of forms is safe (e.g. immunologically safe) and active, and that the pattern of heterogeneity is consistent. Any deviations from the pattern defined at the preclinical and clinical stages require additional testing.

Biological properties (i.e. biological activity) are assessed to demonstrate that the bioproduct has the ability/capacity to exert its desired biological effect. The manufacturer should provide a valid bioassay for this purpose. The most common types of bioassays and the endpoints they use are outlined in Table 3.3.3.

The quantitative measure of biological activity is the (i.e. potency) of the bioproduct (versus mass in the case of physicochemical assays). The potency reflects the biological properties of the bioproduct. When the bioproduct is to be used as a drug, its activity in the biological assay should correlate with the expected clinical response. The units of activity measured in the biological assay should be calibrated against an international or national reference standard, or if such does not exist, against an in-house one. For bioproducts with complex higher order structure, the biological activity may be more informative than the physicochemical properties. There are only limited cases when physicochemical tests may replace a biological assay (Table 3.3.4 ).

EMEA requires the choice of relevant quantitative assay (biological and/or physicochemical) for the purpose of batch release to be justified by the manufacturer.

Testing the immunological properties is essential when the bioproduct is an antibody (affinity, avidity and immunoreactivity) or a protein drug. In the case of antibodies, the tests are typically binding assays to purified antigens and defined regions of antigens are used. In the case of protein drugs, immunochemical assays are often applied (e.g., ELISA, Western-blot) to demonstrate the protein's identity, homogeneity or purity, or to quantify it. If the bioproduct's immunochemical properties are among the criteria for lot release, these properties should be made available.

Another important property of bioproducts that needs to be tested is their absolute and relative purity. The relative purity is defined based on specific activity, i.e. units of biological activity per milligram of product. Since it largely depends on the method of choice, EMEA's guidelines require that the purity of the drug substance/product be tested using a combination of analytical assays.

As pointed out above, bioproducts can include several molecular entities or variants, e.g. owing to intrinsic or manufacture/storage-related heterogeneity. Such variants are not considered impurities, but product-related substances, when they are an anticipated part of the desired product and their properties do not deviate from its specifications. Product-related substances should meet pre-defined individual and/or collective acceptance criteria, as appropriate. For the purpose of lot release, purity should be determined based on an appropriate set of appropriately selected methods.

The manufacturer is also required to assess the impurities, which are defined as components that do not have properties comparable to those of the desired product with respect to activity, efficacy and safety. Impurities may be either process- or product-related (Table 3.3.5); of known structure, partially characterized or unidentified.

EMEA requires that acceptance criteria (individual and/or collective) be set for impurities (product-related and process-related), as appropriate, based on data obtained from lots used in preclinical and clinical studies and manufacturing consistency lots. For the purpose of lot release, impurities should be reported based on an appropriate subset of analytical methods. Examples of analytical procedures which may be employed to test for the presence of impurities are listed in more 3.3.4.

Contaminants are defined as all adventitiously introduced materials not intended to be part of the manufacturing process (1). For example, contaminants include chemical and biochemical materials (e.g., microbial enzymes), and/or microbial species. Contaminants must be strictly avoided and/or controlled based on suitable in-process acceptance criteria or action limits. Action limits do not apply to certain contaminants, e.g. viruses or mycoplasma, so the strategies proposed in ICH Harmonised Tripartite Guidelines "Quality of Biotechnological / Biological Products: Viral Safety Evaluation of Biotechnology Derived Products Derived from Cell Lines of Human or Animal Origin" and "Quality of Biotechnological/Biological Products: Derivation and Characterisation of Cell Substrates Used for Production of Biotechnological/Biological Products" should be considered. The control of contaminants is, naturally, also essential in food safety (more 3.3.5.).

Another essential property of bioproducts is their quantity. A typical quantitative measure is protein content. The methods used for quantitative analysis are usually physicochemical ones. If the obtained quantity values directly correlate with the values obtained in the biological assay, it is possible to use measurement of quantity instead of the measurement of biological activity in the manufacturing processes.