Developments in biological standardization最新文献

Transmission of viruses by animal sera represents a considerable risk for humans and animals particularly when the serum is used for the production of pharmaceutical products such as vaccines. Procedures applicable for inactivating large numbers of different viruses, both enveloped and non-enveloped, are therefore mandatory. For this purpose we have developed and validated UVC irradiation as the virus-inactivation procedure of choice for serum to be used in an industrial setting. Spiking experiments in foetal calf serum (FCS) were performed by independent contract laboratories and revealed constantly high clearance rates for various viruses such as bovine parvovirus, parainfluenza type III virus, bovine diarrhoea virus, foot-and-mouth disease virus and different forms of mycoplasmas. UVC-treated sera maintained their growth-promoting activities for various cell types (MRC-5, Vero, CHO). Conventional growth curves generated in the presence of 10% and 1% UVC-treated FCS differed only slightly from controls, indicating the lack of significant damage during UVC exposure. Experiments using a sensitive photometric-based acid phosphatase assay (APA), which correlates well with the more tedious cell counting procedure, confirmed these findings even in the presence of minimal serum requirements. UVC treatment of animal sera appears advantageous compared to currently recommended inactivation procedures, such as Gamma irradiation, for at least three reasons: (i) it possesses a high inactivation capacity for parvoviruses, a pathogen that cannot be destroyed easily by conventional methods; (ii) it causes no noticeable impairment in cell growth and (iii) it can be performed in a controlled manner at the production site.

Serum-free formulations may be << re-engineered >> to eliminate traditional protein constituents and replace their biological function with non-protein substitutes. Non-protein additives may also be obtained from animal sources. Nutrient formulations totally free of exogenous protein and containing no materials of animal origin may be designed for high density cell culture and biological production. Cell-culture medium production requires (i) strict vendor qualification and raw material specifications; (ii) scrupulous maintenance of media kitchen facility and equipment; (iii) monitoring of process water; (iv) air-handling systems and technical personnel; (v) clearly-defined manufacturing protocols to ensure correct formulation and dispensing and (vi) validated sanitization processes to guard against cross-contamination within a multi-use facility.

There are a number of problems associated with the development of standards suitable for use in the most commonly used assays to detect cytokines in biological fluids. These problems include: (i) the failure of some MoAbs used in immunoassays to detect all different <> of recombinant or natural material; (ii) the use of many different MoAbs, with different specificities, in different immunoassay kits, and (iii) the detection of non-active cytokines (fragments, inhibitors, receptor antagonists, etc.) in these immunoassays. As a result, it is possible to have biologically active material which is not detected in these immunoassays. Alternatively, biologically inactive material can be detected in these assays and is indistinguishable from biologically active material. In addition, the use of different antibodies with different specificities, affinities and avidities in different kits designed to detect the same biological materials results in markedly different sensitivities and specificities. Many of these same concerns can be raised for the use of bioassays for detection of molecules in biological fluids. The solution will not be simple (if possible at all). In most cases, the immunoassay kits are designed to detect <> material in biological fluids, but are made with MoAbs against recombinant material. Because of the markedly different specificities, affinities, etc. of the MoAbs in these kits, their standardization is possible only with a highly purified preparation of natural material. For the assay of recombinant materials, immunoassays should be specifically designed with the recombinant material in mind (i.e. the MoAbs made specifically against the recombinant material to be detected or shown to bind effectively with the recombinant material). Importantly, it should be made clear to investigators using different immunoassays that: (i) the reporting of biological material detected using immunoassays can only be made in units of weight (i.e. ng/ml); (ii) because of the detection of biologically active and inactive material using immunoassay kits these assays cannot be directly compared to bioassays or their results represented as <>; (iii) because of the difference in specificity and sensitivity of the different reagents used in different immunoassays, the results from different assays cannot be directly compared, and (iv) because of these same considerations, comparison of different > of materials within a single immunoassay is also not possible. The use of specific immunoassays for recombinant material in combination with bioassays and the use of cytokine standards, made from highly purified natural material, would help to standardize the results in this field.