The means of measuring the biological activity of a new drug is of critical importance to its release. The process through which a manufacturer can achieve this measure should be clearly defined before pre-clinical studies are initiated. The quality and validation requirements of these studies are defined in a number of regulations but experience of interpretation of these guidelines is essential in establishing a regulatory-compliant assay.
A biological measurement of the activity of a drug is perhaps the most critical step in the series of tests required for product release for both clinical trials as well as the market and plays an important role in the stability assessment of drug candidates. All drugs require a clearly defined specification for release; wherever possible, this should be in place at the pre-clinical stage in a basic format and made more stringent throughout the product life cycle leading to a clearly defined set of release tests that constitutes the marketing authorisation. Some of these assays are generic and can be very simple, such as a measurement of the pH of the material or the product appearance. However, others are product-specific and can be problematic and require significant scientific insight and thorough validation; the cell-based potency assays fall into the latter category.
Biological drugs seldom lend themselves to enzymatic reactions as a measure of biological activity. Insulin is perhaps the only biological which measures potency by enzymic reactions, therefore in vivo or in vitro measures of activity are the most popular choice. Animal studies have a number of distinct disadvantages when considering product release within a Good Manufacturing Practice (GMP) environment, not withstanding the industry's ongoing commitment to reduce the use of animal experimentation. Fundamentally, animal studies are typically performed to ascertain the Good Laboratory Practice (GLP) level of quality, rather than that of the GMP level demanded by the industry for other release tests. Animal studies are costly and time-consuming and are known to have a degree of variability raising questions about their effectiveness.
• Animal studies in which a defined animal model demonstrates a measurable, physiological change in response to application of the drug.
• Cell-based assays that use a specified cell system, which on addition of the drug, demonstrate a measureable biological response.
• Enzymatic reactions where the biological activity of the drug can be measured by the accumulation of product following the chemical reaction facilitated by the drug.
Another method to measure the biological activity of a drug that is proving to be of interest to the majority of manufacturers is cell-based potency assays or bioassays. Cells are living entities, representing biological systems that possess many of the important in vivo characteristics that make them useful for measuring biological activity.
Cell-based potency assays possess a number of advantages over animal models. The most obvious is the cost, with cell-based assays typically reducing cost by 80 to 90 per cent when compared to equivalent animal studies. Initiation of cell-based assays can be achieved with greatly reduced time frames and the variation associated with animal studies is largely removed as the cells used are usually clonally derived and maintained within strict parameters. Therefore, the variability of response is reduced when compared to in-bred animal groups as well. For all these reasons and many more, cell-based potency assays have been the method of choice. However, these assays do require careful consideration when preparing them for use in the release of a drug product.
When considering a bioassay, the cells are universally regarded as the most important feature; without the cells responding in the characterised manner, the assay will never be of use for the release of the product. Cell-based potency assays typically start their life in the research and development section of a large pharmaceutical organisation or in an independent research organisation such as a University or Government Department. These facilities often do not comply to recognised quality system such as GLP or GMP and this presents a significant problem. Cells from these institutions may not have sufficient provenance to demonstrate the appropriateness of their usage for GMP and this can be a time-consuming endeavour to undertake. Ultimately, these tests will need to meet standard GMP requirements for product release for clinical trials (in the European Union) and for marketing authorisation in the rest of the world.
The next problematic feature that can hamper assay development and validation is the availability of information regarding the characterisation of the reference standard. Cell-based potency assays generally report a value relative to a standard drug batch, with every new batch measured against it. In this way each batch can be shown to be consistent with previous batches and the reference standard. The provenance of the reference standard is critical to every batch released and characterisation can be an arduous process. A successful approach employed by most manufacturers is to use one of the early production batches as the reference standard and obtain information during initial assay development. Issues can arise when, following the initial clinical trials, a number of process and formulation changes and improvements to the manufacturing process are implemented. This results in the reference standard no longer being an identical composition to the new batches of the product. Significant effects on the results of the bioassays can ensue and a new reference standard may be required to be established for the new product design. Whenever a new reference standard is created, a bridging study to show the comparability of the new and old molecules is required. This again can be a time-consuming and expensive work and may lead to project delays if not managed effectively.
The next major hurdle, once the cells and reference standards are in place, is the validation of the assay to an International Conference on Harmonisation (ICH) standard that will also meet GMP requirements of the regulatory authority. The ICH sets out specific guidelines for all tests to be followed if they are to be used for the release of drugs. To meet GMP requirements, appropriate specifications and control of critical reagents and equipment must also be given full consideration. For example, the incubators used to grow the cells as well as the reagents used in the culture of the cells all require qualification and control. Reagents, such as foetal calf serum, should be considered as critical and even plasticware must be kept as consistent as possible to ensure the desired performance of assay.
To meet all the specifications in the validation guideline, cell-based potency assay may present many obstacles and also require considerable time and resources. The ICH Q2 guidelines describe a bioassay as a "Quantitative test of the active moiety in samples of drug substance or drug product or other selected component(s) in the drug product". The validation characteristics which should be considered are accuracy, precision, repeatability, intermediate precision, specificity, limit of detection, linearity and range.
The scope of the validation should include a number of assay runs to be performed by several operators to provide sufficient data from which appropriate conclusions can be drawn. The validation process should also focus upon critical assay parameters in all robustness experiments. Overall, the aim of the validation data set is to provide evidence for the appropriateness of the assay to have utility in the release of product.
Many organisations find that they have insufficient in-house resources to undertake the task of performing validation or batch release testing of product using these complex biopotency assays and often consider outsourcing to a Contract Testing Organisation (CTO). CTOs typically have experience, quality systems and suitably qualified equipment for successful assay validation and release of product to a GMP standard. Responsibility for stability studies, where the stability of the biological potency of several batches of the drug is measured over time, can also be transferred to these organisations. A major advantage of transferring an assay to a CTO is the provision of a back-up facility providing a critical aspect of the expected business continuity programme that can be utilised in the event of problems arising with the in-house testing programme.
Transfer of an assay to a CTO requires several stages to be considered. Quality and technical agreements need to be prepared where the individual responsibilities of both parties are defined and identified. Experience has shown that the important part of the agreements relate to critical GMP requirements such as responsibility for Out of Specification Results and Deviations to Protocols and these requirements should be clearly defined. Following agreement on documentation, "proof of concept" studies are performed to demonstrate that the cells used in the assay grow and respond in a consistent manner with the original (expert) laboratory. GMP compliance requires that the operators are suitably trained and it is often considered to be prudent for operators from the expert laboratory to conduct training for analysts of the receiving laboratory. Upon successful completion of these studies, a formal protocol should be initiated to establish conclusively that consistent results can be obtained between the laboratories. A technology transfer will then be performed, if transferring a validated assay or a validation study can be performed at the CTO.
Cell-based potency assays are clearly seen as the way forward as methods to set the specifications for new drug products. These assays provide a clear and reliable indication of the activity of the drug and provide a consistent means of measuring this over time. These assays do however require a commitment of time and resources which may necessitate the use of outsourcing the work to a CTO. This endeavour should be carefully considered and the right partner to assist should be selected with care, ensuring that sufficient experience and resources are available. As biologic drugs become widely used, the demand for efficient release of drug to the market will also increase. Planning for this in a timely manner is a challenge to all manufacturers. However, the failure to do so will result in inevitable delays to release and eventually add cost to the bottom line.