SC I H. Biological Assays and Tests

This section provides information and guidance concerning the biological assays and tests of the Pharmacopoeia and the standard preparations required for them.

1. Biological, including biochemical or immunochemical, methods are described for the determination of potency or other specific properties of certain substances and preparations where these properties cannot be adequately determined by chemical or physical means. The principle applied wherever possible throughout these methods is that of comparison with a standard preparation so as to determine how much of a sample being examined produces the same effect as a given quantity, the Unit, defined by the standard preparation. It is an essential condition of such methods that the tests on the standard preparation and on the sample, the potency or other property of which is being determined, shall be carried out at the same time and, in all other respects, under strictly comparable conditions.

2. Standard Preparations, as defined in the biological assays and tests of the Pharmacopoeia, are of two kinds: primary standards which are established, held and distributed by the appropriate international or national organisation and secondary (working) standards which are preparations the potencies of which have been determined by an adequate number of comparative tests in relation to the relevant primary standard.

3. A primary standard is a selected representative sample of the substance for which it is to serve as a basis of measurement. It is essential that primary standards shall be of uniform quality and as stable as possible. These conditions are usually ensured by providing the preparations in the dry state, dispensing them in sealed containers free from moisture and oxygen and storing them continuously at a low temperature and in the absence of light.

For the majority of biological assays of the Pharmacopoeia, the primary standards are the International Standards and Reference Preparations, established by the World Health Organization.

Laboratories in the United Kingdom may obtain these for the purposes of the biological assays described in the Pharmacopoeia from the National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, England.

In October 2005 the European Directorate for the Quality of Medicines & HealthCare (EDQM) was designated as the International Collaborative Centre for the establishment and distribution of WHO International Standards for Antibiotics (ISA) , replacing the National Institute for Biological Standards and Control (NIBSC) in this role. These standards may be obtained from the Sales Section, EDQM - Council of Europe, 7 allée Kastner, CS 30026, 67081 Strasbourg, France. Further information can be found on the EDQM website (www.edqm.eu).

For the assay of certain enzymes in the Pharmacopoeia the Standard Preparations, as defined in the appropriate method, are the primary standards established by the International Commission on Pharmaceutical Enzymes of the International Pharmaceutical Federation (FIP). These standards may be obtained from the Centre for Standards, Wolterslaan 12, B–9000, Ghent, Belgium or, where these standards have been established in co-operation with, and adopted as official preparations by, the European Pharmacopoeia Commission, they may be obtained from the EDQM address shown above.

4. As a measure of economy in the use of the primary standards it is recommended that working standards should be prepared and used in those biological methods of the Pharmacopoeia where the definition of the Standard Preparation is so worded as to permit this. However, in some instances the complexity or lack of precision of the method or difficulties associated with the preparation of a secondary standard render such a practice inadvisable and in any country in which a particular assay is controlled by law it may be necessary to obtain the approval of the appropriate authority for the use of working standards. The biological properties of the samples selected as working standards should conform as closely as possible to those of the primary standard and an assurance that these conditions have been fulfilled is usually obtained by comparing the behaviour of the two samples under varying conditions of comparative testing. In such tests a detailed study of the dose-response curves may indicate whether the sample selected to serve as a working standard is a suitable preparation. For the assay of certain biological materials in the Pharmacopoeia, for example oxytocin, European Pharmacopoeia Biological Reference Preparations have been established and are recommended for use as the working standards. Such preparations may be obtained from the European Pharmacopoeia Commission (address as above).

5. Wherever possible the primary standard is the International Standard, and the biological activity is expressed in International Units (IU). In other cases, where Units are referred to in the official assays and tests, the Unit for a particular substance is, for the United Kingdom, the specific biological activity contained in such an amount of the respective primary standard as the appropriate international or national organisation indicates. The necessary information is provided with the samples of the primary standard.

6. For enzymes in the Pharmacopoeia, where the assay is such that the stoichiometry of the reaction is known, for example, where the substrate is a synthetic ester, the activity is measured in microkatals or nanokatals. A microkatal is defined as the enzyme activity that under defined conditions, produces one micromole of the reaction product per second or alternatively consumes one micromole of the reaction substrate per second. For other enzymes in the Pharmacopoeia, where the reaction involved in the assay is more complex, for example, where the substrate is a naturally occurring macromolecule such as a protein, the activity is measured in Units as previously defined.

7. The methods of biological assay described in the Pharmacopoeia have been found satisfactory but will not necessarily be the best methods for use in all circumstances. In most instances they may be replaced by other methods if it can be shown that such methods are at least equally accurate and precise and provide a measurement of the same active principles.

8. Any estimate of potency derived from a biological assay is subject to random error due to the inherent variability of biological responses and calculations of error should be made, if possible, from the results of each assay and recorded with the potency estimate even when the official method of assay is used. Guidance on the design of assays, the statistical analysis of results and the calculation of potency is provided in general text 5.3 of the European Pharmacopoeia (Supplementary Chapter IV G). The methods described therein take account of the inherent random error but assume that systematic errors, for example, errors in weighing or dilution, will not represent a major source of variation in the potency estimates. Alternative assay designs and methods of calculation may be used provided that they are not less reliable.

9. Where an immunoassay, that is an assay procedure based on the reversible and non-covalent binding of an antigen by antibody, is described in the Pharmacopoeia for detecting or quantifying either an antigen or an antibody, the considerations described in Appendix XIV B apply in addition to the general points referred to above.