Developments in biological standardization最新文献

In many cases, the viral safety evaluation of biological products does not derive solely from direct testing for the presence of contaminants, but also from the demonstration that the manufacturing process is able to inactivate/eliminate them. This is achieved by the voluntary addition of a virus load at various steps of the process and the evaluation of viral reduction during the subsequent steps. The major difficulty for such viral validation studies is less to calculate a reduction factor for each step than to identify clearly and demonstrate the contribution of each in-process parameter to the reduction. Consequently, the first approach consists of the identification of all the parameter which may influence viral reduction. The design of the viral validation needs to take this inventory into account and control experiments must be carried out in parallel with the main spiking experiment, i.e. mainly to: (i) hold controls with and without the biological intermediate product; (ii) control experiments with each individual inactivating/partitioning parameter; (iii) control experiment without stabilizer if necessary. In addition to these process controls, cytotoxicity and interference experiments will allow the use of each in vitro infectivity assay for the testing of processed samples to be validated. For a viral inactivation step, the kinetics of inactivation will be studied and the data will show: (i) a progressive decrease of the viral load over time. If the decrease is too rapid to plot the kinetics, the direct relation between the inactivation and the inactivating parameter has to be demonstrated in complementary experiments; (ii) the reduction obtained when in-process limits are used and (iii) the different phases of inactivation when they exist. Moreover, it is pertinent to evaluate for each treatment the margin of safety derived from the treatment time on the one hand and the strength of the inactivating parameter on the other.

The development of media free of serum and animal or human protein is of utmost importance for increasing the safety of biologicals produced for therapy and vaccination. The main drawback associated with the use of serum or animal-derived substances for animal cell technology is the potential introduction of contaminants (adventitious agents) into the process and thus potentially into the final product. This fact led to an increased effort to replace serum-containing with serum-free media. In most cases, these media are supplemented with purified proteins, peptones, or hydrolysates, mainly of animal or human origin. Although such serum-free media are more defined than serum-containing media, the risk of the introduction of viruses by using animal-derived substances is still present, signifying that only a complete replacement of animal-derived substances by non-animal-derived products leads to a relatively safe serum-free medium. The potential replacement of these animal/human-derived substances by those of non-animal origin (e.g. plant origin) will be discussed. In several examples, the potential of serum-free media free of any animal-derived component in supporting cell growth and production of biologicals will be presented. In this context, the risk of using non-animal-derived substances in serum-free media for animal cell technology will be discussed with respect to classically used cell culture media.

Growth factors have a wide variety of actions in living systems, providing a range of potentially quantifiable responses for measurement of their biological activity. The biological activity has to be assessed by bioassay as it cannot be predicted from physicochemical data alone. Bioassay systems range from in vivo responses to changes in receptor binding and in early components of signal transduction pathways. The most commonly used systems are based on the measurement of responses of immortalized cell lines, which although not as functionally relevant as in vivo assays, are easier to use. Most growth factors have multiple actions on multiple targets, and can show differential changes in their different activities, so use of the biological activity measured in one bioassay system to predict biological activity in another system must be rigorously validated. Since the bioassay systems are themselves inherently variable, measurement of the growth factor's activity must be made relative to a common, stable, reference preparation to permit valid inter-assay and inter-laboratory comparisons.

Chemokines are mediators of inflammation and trafficking of cells of the immune system including a pivotal role in the recruitment and activation of leukocytes. Due to their involvement in a variety of disease processes, chemokines are potential therapeutic targets. The use of chemokines as pharmaceuticals will require that the folded state and the association properties of the protein are well characterized. In this report, we describe the utility of nuclear magnetic resonance spectroscopy as a tool to study these aspects of chemokine structural properties.

For a biological assay to be useful for quality control it should fail bad lots, pass good lots, and estimate relative potency with high accuracy and precision. To fail a lot we rely most heavily on the test for parallelism. For the parallelism test and other preliminary tests as well as for inference, appropriate estimates of assay variation are crucial. Location effects on 96 well plates and serial dilution of samples using multichannel pipettes make it difficult to obtain good estimates of assay variation. This paper develops the use of a split-block design and analysis where blocks are reasonably consistent regions of a plate; this approach removes some location effects, allows other location effects to be treated as assay variation and provides appropriate measures of assay variation. Randomization, even within the split-block design, is difficult without robots to reduce the likelihood of procedural errors. There are hardware, software, and validation obstacles to implementation of robots in the bioassay laboratory. More generally, validation of a bioassay should be reported on log relative potency and must address between- and within-assay variation. When between assay variation is not small, the usual weighted approach to combining relative potency estimates (which ignores between-assay variation) is inappropriate; a simple sampling average and standard deviation is a better solution.

To achieve better standardization of influenza vaccines, an ELISA immunocapture assay was developed for N2 neuraminidase quantification. This sensitive and highly specific assay was successfully applied to vaccine preparations produced in embryonated hens' eggs from 1992 to 1997 and to antigenically related viral suspensions produced in MDCK cells. A study of the neuraminidase activity of prototype A/H3N2 strains stored at 4 degrees C showed the gradual development of enzymatic instability from 1994 onwards, accompanied by antigenic modifications of the antigen. As the phenomenon was also more pronounced with the recombinant, the question arose of the standard of immunity provided when such viruses are used for vaccination. The antibodies inhibiting neuraminidase activity in vaccinated subjects were monitored in parallel using both complete virus and purified N2 NA. The study revealed the existence of an interference phenomenon which resulted in the titre of the N1 antibodies being overestimated. The interference was due to anti-HA antibodies impeding access to the substrate at the enzymatic site by steric hindrance.