Developments in biological standardization最新文献

Recounting the origination and development of the concepts and institutions to foster and control new and existing biological products is a fertile subject for historical review. It describes what was a necessary and functional activity that needed to be brought into being. The earliest examples of biologicals regulation were created by the eighteenth century inventors themselves and became progressively institutionalized by the creation of national control authorities which increased in number and sophistication during the past century. In 1948, the World Health Organization (WHO) was established and created biological standards and regulations as an advisory to member nations. This too has grown in sophistication and is now of central importance in the biologicals enterprise worldwide. The field of biological standardization and regulation can be viewed in its need and role to foster technological development and to assure that the products of that endeavour are worthy and are retained. In many respects, it is a tale of troubles, tragedies, and triumphs and this is recounted in the text.

Eukaryotic cells, in general, require serum for growth in vitro. Serum is a complex mixture of a large number of constituents, so the addition of serum introduces an ambiguous factor into cell cultivation. However, many commercially available sera are of a high uniform quality. Of these, foetal bovine serum is the most frequently used and is capable of supporting the growth of a wide variety of eukaryotic cells. However, with the identification of essential growth factors and nutrients required by different cells, several very effective serum-free media have been formulated. The use of these serum-free media is limited to a very narrow range of cells. Regulatory constraints generally make it impractical and uneconomic to alter existing biopharmaceutical production processes in which serum is used as a raw material.

Biological materials are variable by their very nature. Serum in particular is a complex mix of substances and may contain adventitious agents. Serum for pharmaceutical use is controlled on a batch to batch basis given that each batch may have differing properties. Control points for slaughterhouse obtained bovine and foetal bovine serum exist with the disease status and disease surveillance of the country of origin, veterinary inspection for clinical disease, collection methods and post collection quality control. With these control points many batches of commercially available serum are contaminated with viruses such as BVD. The use of donor serum provides a mechanism to extend Good Manufacturing Practice principles back to the farm environment giving the manufacturer the ability to increase control on production parameters in the donor animal such as disease and specific antibody status, genetic type and history, age, diet, treatments and origin of feed. As well as providing a secure supply of serum for manufacture, donor herds provide added traceability and give the opportunity for in vivo manipulation of the serum and specialised selection of the animals for specific applications with more consistent reproducibility of performance. The BSE crisis of the last 10 years has highlighted the added control that donor serum producers can exert: Many producers of donor serum applied ruminant feed bans on their animals ahead of nation-wide bans in their own countries. SPF donor herds can be set up in several ways and the purchaser should make themselves familiar with the methods used. SPF herds with accompanying seronegative status allow quality control testing of product free from the interference of specific antibodies. Such specific antibody freedom may also allow better growth of viruses for vaccine manufacture.

Human influenza viruses are routinely isolated and grown in a variety of mammalian cell substrates. However, influenza viruses for use as inactivated vaccine are still produced in embryonated eggs. Using a perfusion culture-based bioreactor process using serum-free medium, both human and equine influenza viruses of different types and subtypes could be produced to high titres. Classical DEAE-dextran microcarriers were found to be more suitable than polyester sponge carriers for virus production. In addition, MDCK cells grown in serum-free medium were further validated as the most suitable cell substrate compared to Vero and BHK-21 C13 cells for large scale virus production of influenza virus. Finally, to minimize potential contamination by adventitious agents, it was demonstrated that a new serum-free medium in which all animal-derived products are replaced by a plant extract, efficiently supports the growth of MDCK cells as well as the production of influenza virus in the presence of trypsin when using the perfusion bioreactor process.

This study was initiated with the isolation of influenza A and B viruses from clinical throat swabs in both fertile chicken eggs (egg) and MDCK cells, which were used in subsequent vaccine production in the above two hosts. On the basis of haemagglutination-inhibiting (HI) tests, immune mouse sera from mice vaccinated with MDCK cell-derived vaccines revealed antigenic similarities among H3N2 or B viruses isolated in MDCK cells or eggs. Similarly, antiserum prepared by immunization with egg-derived H3N2 vaccine showed equivalent antigenicity between homologous and heterologous (MDCK cell-derived) viruses. In contrast, antigenicity of egg-derived B vaccines was differed somewhat from that of MDCK cell-derived vaccines, suggesting the occurrence of antigenic change due to passaging in eggs. The time-course of immune responses based on HI titres indicated that MDCK cell-derived vaccines elicited extremely high antibody levels. Also, it was evident that antibody production by MDCK cell-grown H3N2 vaccine was very similar to that of vaccine prepared from egg-grown viruses. These results were comparable to those of plaque neutralization tests, although antigenic differences between egg- and MDCK cell-derived challenge viruses were confirmed in the test with antiserum to MDCK cell-derived vaccine. Consistent with HI-antibody production, the immunogenicity of MDCK cell-derived B vaccine appeared to be low by plaque neutralization test, while immune responses in mice which received egg-derived vaccines were significantly higher than that of the former. Furthermore, immune responses confirmed in mice immunized with B virus vaccines prepared in eggs revealed slight antigenic differences between two viruses derived from their respective hosts. Nevertheless, through evaluation of immune responses, MDCK cell-derived influenza vaccines may be useful when weak immunogenicity of B virus vaccine is improved.

Alternatives to the standard WHO neurovirulence test (nvt) in simians for live attenuated poliovirus vaccines are highly developed, at least for poliovirus type 3. The alternatives are MAPREC, a molecular biological assay, and transgenic mice that express the human cellular receptor for polioviruses (TgPVR mice). The MAPREC assay quantifies reversion of key mutations that may accumulate during vaccine manufacture. Collaborative studies organised by WHO showed that the assay is sensitive, robust and standardised. WHO International Standard and Reference Reagents are established. Samples that fail the MAPREC assay need not be tested for neurovirulence in monkeys. The assay is now being used by both manufacturers and national control laboratories to characterise virus seeds and to monitor the consistency of production at the molecular level. A regulatory decision-making model has also been developed in the TgPVR21 mouse line. The model has been found a valuable indicator of neurovirulence in a WHO collaborative study. These alternative methods are the fruits of a long-term basic research programme on the molecular biology of polioviruses and provide an excellent example of co-ordinated regulatory research.

Foot-and-mouth disease virus (FMDV) has been one of the pioneering viral systems in the development of synthetic peptides as vaccines. Protection against FMDV infection is associated with the induction of neutralising antibodies. Therefore, attempts have been made to identify peptides capable of eliciting protective humoral responses. Peptides based on a continuous, immunodominant B cell site on the capsid protein VP1 have been shown to confer limited protection in natural hosts. This probably reflects the difficulties in reproducing the immunogenicity of an entire viral particle by using a much simpler synthetic antigen, due to: (i) the polymorphism of the class II MHC; (ii) the adequate presentation to the immune system of the peptides, and (iii) the difficulties of achieving protection against a highly variable RNA virus, which may favour selection of virus antigenic variants. The improvement of FMD peptide vaccines, and the development of in vitro alternatives to in vivo immunogenic assays require further understanding of the immune mechanisms leading to protection against this important animal virus disease.