Developments in biological standardization最新文献

Development of combination vaccines is a time consuming and expensive exercise with significant technical, clinical, regulatory, manufacturing and marketing challenges, which is likely to result in a rationalisation of production worldwide.

Infection with bovine viral diarrhoea virus (BVDV) and other viruses is frequent in the bovine population. In utero infection leads to virus and antibody contamination of foetal and other serum used in cell culture production. The use of contaminated cells for vaccine production may result in contaminated vaccines, which may lead to seroconversion or disease in the vaccinated animal. Contaminated serum or cell cultures may also interfere with the diagnosis of viral infections. Methods for the detection of BVDV and other viruses in serum, cell cultures, seed viruses and vaccines at the CVB-L, and the frequency of detection are described. Reasons for continued use of serum in cell culture production, and the risks of using serum, are discussed.

This paper surveys the legislation and policies governing serum supply in force in New Zealand at present, and outlines the regulatory controls, both direct and indirect, associated with the production of animal sera. Direct controls are concerned with the welfare of the donor animals and the production and processing of the product. Controls are also applied to preserve New Zealand's animal health status through import controls of animals and animal products. The Animal Health Surveillance Programme also monitors the animal health status of New Zealand. The level of assurances and integrity represented in this regulatory system has led to an increase in recent years in the demand for animal-derived materials that are free from major animal diseases.

Anti-lymphocyte globulins (ALG) are immunosuppressive agents of animal origin currently used in clinical organ transplantation and for the treatment of severe aplastic anaemia. The potency of each batch is tested in vivo using primates as recipients for allogeneic skin grafts. The two in vitro methods commonly used are (i) a cytotoxic assay and (ii) the rosette inhibition assay, both of which are evaluated by microscopy. In addition to animal welfare aspects, these methods require considerable experience, are difficult to validate, and the information as to the biological potency of the sera is questionable. The aim of our study is a better characterization of the biological properties of ALG in order to subsequently define an in vitro alternative for the potency test in monkeys. Several antibody specificities directed against functional molecules on T-cells, B-cells, NK-cells, macrophages as well as non-lineage specificities can be identified in competition assays with monoclonal antibodies. The cytotoxic capacity of ALG in the presence or absence of complement as well as DNA-fragmentation characteristic for apoptosis can be analysed by flow cytometry using propidiumiodide- (PI) incorporation. Immunoprecipitation of cell lysate with ALG and subsequent incubation with radioactive ATP (kinase-assay) shows specific bands which seem to be identical between different batches of one product.

Most animal species respond with high antibody levels to polysaccharide antigens after they have been covalently linked to a protein carrier, converting a T-cell independent to a T-cell dependent antigen. This chemical modification has enabled the development of glycoconjugate vaccines for Haemophilus influenzae type b, Neisseria meningitidis, and multivalent Streptococcus pneumoniae. This new generation of vaccines can be well characterized physically and chemically to ensure consistent vaccine manufacture. Such analytical tests provide an alternative to animal models for Quality Control Laboratories; biological models can be difficult and costly to develop and use on a routine basis. If animal tests are used, they need to be refined, defined, and validated for their intended purpose.

Growth of continuous cell lines for preparing biopharmaceuticals in the absence of animal serum has been attempted by many organizations to improve process and product quality, prevent exposure to adventitious agents, and reduce costs. Literature surveys suggest that substantial academic studies on serum-free medium have been pursued for many decades, with varying levels of success for different cell types and cell lines in terms of achieving cell growth while retaining cell function. Industrial research proceeded for at least three decades. Recent work with CHO cells and with some hybridomas has been successful in providing the basis for serially propagating cells on a large scale in suspension in the total absence of serum, while preserving the ability to prepare biopharmaceuticals. In some cases, this can be achieved not only without serum, but also without the use of other animal-derived proteins.

Tissue infectivity in BSE has been comprehensively investigated in cattle with natural BSE and during the incubation period in an experimental pathogenesis study in which cattle were challenged orally with infected cattle brain from natural cases. In natural cases of BSE in cattle, infectivity has been found only in the CNS, (the brain, the spinal cord and retina). No infectivity has been found in about 50 other tissues including bone marrow, clotted blood, buffy coat, serum or foetal calf serum. In the pathogenesis study in which clinical disease was first detected at 35 months post-infection (39 months of age), infectivity has not been found in blood or any assayed component of blood. Experimental parenteral challenge of cattle and mice in three separate experiments with (i) a pool of five brains, (ii) a pool of five spleens and (iii) a pool of lymph nodes from five cattle is incomplete. However, whereas the brain has transmitted disease to both species (in cattle even when diluted about one million times) neither the spleen pool nor the lymph node pool has transmitted disease to either, although the cattle study is incomplete. These experiments have also shown that cattle can detect about 1000 times less infectivity/g than can mice. No infectivity has ever been detected in the blood or any component of blood in natural scrapie of sheep and goats, natural BSE of cattle or experimental BSE of cattle.

Although eclipsed in recent years by immunoassays and molecular biology techniques, bioassays remain a vital research tool for cytokine biology. Like any type of biological system, cytokine bioassays require meticulous technique to obtain accurate and reproducible results. However, these minor difficulties are more than compensated for by their exclusive detection of biologically active molecules, a feature as yet unmatched by other assay methods.

Biosensor and electrochemiluminescent (ECL) assays are replacing enzyme-linked immunosorbent assays (ELISAs) at Schering-Plough as immunoassays of choice to monitor cytokine levels and detect anti-cytokine antibody responses during cytokine therapy. These new assays provide increased sensitivity and a better correlation with biological assays. Biosensor assays using the BIACORE 2000 (BIACORE, Uppsala, Sweden) are being adopted to support preclinical and clinical trials for the detection of antibodies capable of binding to IL-10 and IL-4. Significant advantages when using a biosensor assay are that real-time and label-free detection permit increased throughput and direct detection of binding interactions which enables detection of low affinity antibodies that are not detected by ELISA. The ECL assays using the ORIGEN Analyser (IGEN, Gaithersburg, MD) that we have implemented to replace existing ELISAs for quantification of serum IL-10 and serum interferon alfa levels are more sensitive and less subject to matrix effects. Data obtained during the validation of these assays are described.