The preparation of international reference materials for biological substances

Jowttal

of Bioiogicaal

Standardization

( 1988)

16,

129-

137

The preparation of international reference materials for biological substances*

Philippe Sizarett

INTRODUCTION The problems and difficulties which are inherent in the assay of biological substances such as serum proteins, ’ hormones,2*3 enzymes, * live attenuated viral vaccines’ and clinical chemistry reagents,’ have been the subject of comment by various authors both from a technical aspect’ and from the planning and analysis point-of-view. 8 To add to the body of such information, the World Health Organization (WHO) has prepared a revised document relating to the preparation and establishment of International and other Standards and Reference Reagents for Biological Substances.” The first step in the sequence of events which ultimately leads to international acceptance of a definition of the concentration of the active principles in any particular biological material is firmly based on the concept of the expression of the content of the unknown (or ‘test’) biological material in terms of an appropriate existing national or international reference. In the absence of such a ‘yardstick’ it is necessary to establish a reference preparation of the material de nouo following a sufficiently extensive, well-designed, capably analysed collaborative study of a candidate preparation. The same approach also is necessary in the case of an existing standard the stocks of which are becoming depleted, and in this case also the ‘replacement’ preparation must be viewed as a candidate reference material (CRM). When a particular biological reference * Received for publication 24 September t Biologicals, World Health Organisation, 0092-1157/88/020129+09

$03.00/O

1987. 12 11 Geneva 0

27, Switerland.

1988 The International

Association

of Biological

Standardamon

129

P. SIZARET

preparation is intended for international standard status, its establishment (or that of a replacement) requires the express decision of the WHO Expert Committee on Biological Standardization (ECBS), the decision being reached only after careful consideration of the data generated in a collaborative study of the CRM. The ‘corporate memory’ of the ECBS and of the WHO Biologicals Unit (which is the Secretariat of the Committee) is both deep and extensive, and past experience has shown that many of the problems which arise during the assessment of a potential reference material are due to imprecise and insufficient knowledge of the steps necessary for the preparation, evaluation and ultimate establishment of a material as a definitive biological International Reference Material (IRM). The lack of such knowledge can result in years of wasted effort and the loss of valuable material simply because a CRM was not found to be unsuitable early in the study. There have been occasions when, at a late state in its evaluation, a CRM has been found to be possibly contaminated by pathogenic agents such as hepatitis B virus (Hep B) or human immunodeficiency virus (HIV), both of whch would expose the laboratory workers handling an IRM to unaccepable health risks. Other factors which make a CRM unsuitable involve such things as presentation in rubber-stoppered vials instead of in heat-sealed neutral glass ampoules, inconsistent filling with a coefficient of variation from ampoule-to-ampoule within a batch greater than l%, failure to achieve a stable preparation, e.g. because of excessive residual moisture in the ampouled material and/or high levels of oxygen in the atmosphere within the sealed ampoules. There are also examples of extensive collaborative studies which have resulted in useless data because the objective was misunderstood or because the study was not properly planned. It is also not unknown that, by sheer coincidence, two groups of scientists have undertaken simultaneously and independently the provision of preparations of the same substance for presentation to the ECBS as a CRM. In order to prevent (or at least minimize) such difficulties, it may be useful to review the steps and precautions necessary to achieve the successful preparation of international biological reference materials. Where possible, the various steps indicated below should follow each other in sequence. However, the time-factor may make strict adherence to the steps unrealistic and some flexibility is necessary. The proposed sequence should not be regarded as a rigid constraint but rather as a framework for guidance. A summary listing the information required prior to the establishment of an IRM is given in the Appendix. STEPS AND

RELATED

PRECAUTIONS

The main steps and related precautions for the successful preparation of IRM can be broadly described as follows. Recognition of the wdfor an international refwece material and abmination of its ultimate use There is no point in embarking upon a project to produce a reference material (RM) for which there is no demand. Following a decision by the WHO ECBS in 19861°, any IRM to be established will fall into either one of two categories, namely International Standards (IS), the activities of which in the vast majority of cases are expressed in (arbitrary international units (IU) per ampoule, or International Reference Reagents (IRR). Such reagents may be used either for qualitative (e.g. for typing strains of bacteria or viruses) or quantitive purposes and in the latter case their content is not

130

INTERNATIONAL

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MATERIALS

PREPARATION

defined in IU. The decision whether to allocate to a CRM the status of international Standard is the responsibility of the WHO ECBS, which considers a number of factors such as: 1. The importance of the CRM in public health. 2. The extensive characterization of the CRM by means of international collaborative studies. 3. The suitability of the CRM for the purpose for which it is intended having regard to the nature of the substances to be evaluated; this implies that, by means of relevant test systems, a CRM gives results which can be compared with those of other traded biological preparations having similar biological activities. Inasmuch, for example, as certain live attenuated vaccines (e.g. BCG vaccines) are derived from several strains having different growth characteristics and biological properties, there cannot be a single IS for all such vaccines. The conditions to be fulfilled for the results of biological assays to be valid and meaningful have been extensively reviewed by Jerne and Wood.” 4. The strict adherence to expressed technical requirements, especially those dealing with uniformity and stability. In this document consideration will be given only to the lyophilized IRMs which are derived from homogeneous liquid bulks and to which a content is ultimately assigned either in international or other units. Procuring bulk materials meeting the basic requirementsfor IRM Whenever it is possible, it makes good sense to secure several alternative biological materials in bulk form so that, if preliminary investigation shows that one material is unsuitable, other materials will still be available. The main criteria to encounter at this stage are: Non-patbogenicity . This criterion is to be considered essential especially (but not exclusively) if CRMs are derived from human blood or tissues, for in such cases contamination by agents such as Hep B virus, HIV etc. must be excluded. lo Care should be taken also in the case of hazardous chemical agents (e.g. sodium azide) which may be present in buffers or bulking agents. Interfering substances. Materials which are being considered for IRMs should not contain bacterial contaminants or contaminating substances (e.g. bacterial enzymes) capable of interfering in assay systems. Composition. CRMs must contain substances with biological activities similar to those in the products which will be assayed. However, achieving purity of an IRM should not be a major concern, especially if matrix effects are a problem and/or if the achievement of purity results in instability. Availability of sufiient quantity of bulk material. The amount of each bulk CRM should be sufficent (a) for the contents of each final container, upon reconstitution, to serve its purpose; thus both the volume and the concentration of the reconstituted solution must be adequate; (b) for a sufficient number of containers of the CRM to be available for at least ten years. From a practical point of view, and depending upon the foreseeable use, the number of final containers should range between 1000 and 5000 ampoules. All should be 131

P. SIZARET

prepared under similar conditions during one working session and stored at a constant at temperature, preferably at -2O’C or below. of a project The various aspects involved in the planning and analysis of studies of biological RMs have been extensively reviewed by Kirkwood et a1.8 Identifying proposed participants in a project is closely associated with the planning of the project for the preparation of an IRM. Participants will include laboratory scientists who will perform the various assaysand, most importantly, a statistician with adequate experience of similar studies; the statistician must be closely involved at the planning stage and in the preparation of the protocol of the collaborative study; later on, he will analyse the results of the study. Initially, the project should be circulated in the form of a draft protocol which is finalized after appropriate amendments are introduced following discussions with participants. It is recommended that forms for reporting unprocessed assay results be provided so that there is no ambiguity concerning the information participants are expected to provide. The following points also need to be addressed. Preparation

The need for a pilot study. A limited pilot study may be necessary, especially if some of the following points are not clear as might, for example, be the case if filling and lyophilization were to be carried out in a laboratory without previous similar experiences in preparing IRMs or if the product has not been previously studied and its characteristics were unknown.

1. Capability to achieve a sterile fill. 2. Capability to achieve a precisely controlled fill of final containers: Coefficients of Variation (CVs) of filling must be less than 1% and this may require weighing at regular intervals, e.g. every fiftieth ampoule, the amount of liquid which has been introduced. 3. Capability to seal ampoules under appropriate (0.0 l-O.03 mmHg) vacuum,’ or in the presence of an inert gas such as pure nitrogen, in order to avoid the subsequent deleterious influence of oxygen. l2 4. Capability to achieve a suitable residual moisture in the heat-sealed glass ampoules in order to maintain maximum stability. As a general rule, a residual moisture content in excess of 1% should be avoided since it may adversely affect the stability of a CRM. To achieve this it may be necessary to carry out a secondary desiccation over P205 before sealing. Even if pilot studies show that experimental batches of a CRM are stable, similar evidence must be provided for the actual CRM, since successful trial fills do not necessarily guarantee the success of the main fill, even if it is prepared by the same procedures. 5. Capability of correctly sealing the ampoules. Each container should be tested for leakage. ’ Pilot studies should also determine, by means of accelerated degradation tests, ” the stability of a CRM at various temperatures of storage.‘*,‘s As a general rule, it is inadvisable to embark on the production of a CRM which has been shown in pilot studies to lose more than 0.5% of acitivity/year at the proposed final storage temperature. Since pilot studies are only preliminary to the main fill and subsequent collaborative study, they need not involve several laboratories provided that the results are conclusive and reliable. 132

INTERNATIONAL

REFERENCE

MATERIALS

PREPARATION

Preparing a protocolfw the main colaborative study. In the preparation of a protocol for a collaborative study the following should be carefully considered. 1. The number of laboratories to be involved. The number of laboratories which should be involved in the collaborative study will vary in relation to considerations such as the number of experts available, geographic location, the need to complete the study in a reasonable length of time, etc. The usual number of laboratories ranges from five to twelve. 2. The necessity to organise the study in such a way that the reliability of results from individual laboratories can be ascertained. Since individual laboratories can be expected to perform unequally, the study should be organized in such a way as to allow for greater emphasis to be given to more precise values in the combination of relative potencies to mean. For a given laboratory, statistical weight can be determined from differences between results of coded replicates in individual assays. It is then possible to determine whether differences among Relative Potency estimates obtained from a group of independent assayscan be accounted for solely by assay errors, in which case Relative Potencies are said to be ‘homogeneous’. 8 3. Inclusion among test preparations of specimens of the type which will be assayed routinely after the CRM has been established. Test preparations which are assayed against CRMs in collaborative studies should not consist of aliquots of a few bulks from which CRMs are derived. Collaborative studies would in such cases be of little value for establishing the suitability of CRMs as calibrators for the assay of biological materials from various origins; furthermore, the number of preparations should ideally be no greater than the number which can be tested in a single assay.8 Within such assaysit is advisable that individual laboratories also test their own internal reference so that the comparability of assay results obtained by testing unknown preparations against individual calibrators and against the common CRM can be examined. 4. Selecting assay methods. Major common assay methods should normally be represented. However, it is not necessary for each laboratory to use each method ofassay. Much flexibility on technical aspects and the selection of reagents should be left to the participants since the goal is to determine how CRMs behave under normal conditions of use. 5. Setting deadlines for the receipt and the analysis of results. Setting deadlines for the mailing of results and their analysis helps to achieve the project within reasonable time limits. Furthermore, it is normal practice to ask participants to refrain from publishing any results until the final report has been submitted. 6. Informing the WHO ECBS. In order to avoid duplication of efforts, it is important to advise WHO officially of the study; this is best done in a letter accompanied by a project protocol forwarded through the appropriate organization e.g. Standardization Committee of the International Union of Immunological Societies (IUIS), International Committee for the Standardization of Haematology (ICSH), etc. to the WHO Chief of ‘Biologicals’, Avenue Appia, 12 11 Geneva 27, Switzerland. WHO should also be kept informed of progress at regular intervals, e.g. every two years. Carrying out the project Appropriate data on the precision of filling,

homogeneity,

sterility,

absence of 133

P. SIZARET

hazardous agent, oxygen content, residual moisture, etc. of the CRM should be obtained and provided as indicated above. Data on stability obtained by different assay methods generated by single laboratories are acceptable provided that the results are conclusive and reliable. On the other hand, data on the relative potencies of test preparations, including coded aliquots of the CRM, should be generated by the various laboratories involved in the main collaborative study. Ampoules of CRM should not bear any indication which makes possible the identification of the origin of the product, i.e. the identity of the manufacturer. Usual information on the label includes the reference number of the batch and the nature of the product (e.g. ‘anti naja horse serum’, ‘ thromboplastin’ etc.) It is premature at that stage to be more precise as the final status of the product is still undetermined. Analysis of the results of the study The analysis of the results of the study should take into account every relevant parameter as described by Kirkwood et a/.* The main points of importance are homogeneity, stability and utility for improving the comparability of assay results in participating laboratories. Preparation of a draft report of the study A draft report containing all relevant technical information should be prepared. The draft should be sent to the participants for comments and suggestions. A proposal to assign a unitage to the CRM, usually in the form of (arbitrary) units, should be made. The draft report should also include a copy of the proposed leaflet of instructions to users in which a statement should be made of any precautions to be observed, even though tests for the presence of hazardous agents have been negative. The draft report should then be amended in the light of comments. Submission of the CRM to the WHO ECBS A request to have the CRM established as an IRM should be sent through the appropriate body (ICSH, Standardization Sub-committee of the IUIS, etc.) to Chief of ‘Biologicals’, WHO, Geneva, Switzerland. It should contain a copy ofthe final report of the collaborative study and the originals of figures which will be needed for the preparation of the relevant WHO document. The letter should specify the number of containers which are offered to WHO, the requisite temperature of storage, the address of the custodian laboratory, and should indicate the willingness of donors to send the CRM to a different custodian laboratory should WHO so request. Pending a decision from WHO, it is perfectly legitimate for the custodian laboratory to make available, upon request, limited quantities of a CRM to manufacturers, national control laboratories, etc., it being made clear that such materials have no international status at that stage. Similarly, it is normal practice for the participants to submit for publication in a scientific journal a paper describing the study and its results. Here again, care should be exercised in qualifying the RM as Candidate Reference material. Similarly, proposed definitions of activities should be stated in (arbitrary) units, not in international units. DISCUSSION The elevation of a CRM to the IS or the IRR category is not always straightforward 134

as it

INTERNATIONAL

REFERENCE

MATERIALS

PREPARATION

may have important implications; IS have units which are definitive in the sense that WHO member states have a moral obligation to require the expression of potencies of those substances related to IS, which are either produced or imported, in international units. l6 Special consideration must be given to situations in which apposite characterization of a CRM has not been obtained and yet it is urgent to establish an International Reference, e.g. in order to avoid the use by various groups of different unitages. In this

case, the simplest solution for the ECBS may be to establish the material as a provisional IRM,

be it standard

or reagent.

There

may also be situations

in which

a product

initially considered as being only of research interest and having been established as an IRR then becomes

useful

for the diagnosis,

treatment

or prophylaxis

of diseases, and is

traded in international commerce. In such casesthe WHO ECBS may decide, provided suitable characterization is made available, to change the former status of a preparation which then may become an IS, the content being expressed in IUs. These are problems falling within the competence of the WHO ECBS. Acknowledgements I am much indebted to Dr P. J. Campbell and Dr D. I. Magrath (NIB%, Potters Bar, UK), Dr J. Lyng (Statens Serum Institut, Copenhagen, Denmark) and Dr T. B. L. Kirkwood (National Institute for Medical Research, the Ridgeway, Mill Hill, London, UK) for their critical reviews of this manuscript and their pertinent remarks, and to MS J. Brunt and L. St Maxens for their excellent typing. REFERENCES 1. Reimer CB. Standards and reference materials for analysing specific serum proteins. In: Skokie IL, Nakamura RM, Rippey JH, eds. Diagnostic Immunology: Technology Assessment and Quality Assurance. College of American Pathologists, 1983: 129-145. 2. Bangham DR, Cotes PM. Standardization and standards. Br Med Bull 1974; 30: 12-17. 3. Bangham DR. Assays and standards. In: Hormones and Blood, 3rd edition. Vol. 5. London: Academic Press, 1983. 4. Lauwers A. Les enzymes pharmaceutiques et leurs standards de reference. Cronache Farmaceutiche 1979; 6: 202-209. 5. Magrath DI, Seagroatt VA. The standardization of infectivity titrations of poliovaccines-A WHO Collaborative Study. J Biol Stand 1985; 13: 159-166. 6. Batty I. Progress in standardization: 4. Immunological reagents. Bull WHO 1876; 54: 123-128. 7. Campbell PJ. International biological standards and reference preparations. I. Preparation and presentation of materials to serve a standards and reference preparations. J Biol Stand 1974; 2: 249-258. II. Procedures used for the production of biological standards and reference preparations. J Biol Stand 1974; 2: 259-267. 8. Kirkwood TBL, Seagroatt VA, Smith SJ. Statistical aspects of the planning and analysis of collaborative studies on biological standards. J Biol Stand 1986; 14: 273-287. 9. WHO Guidelines for the preparation and establishment of international and other standards and reference reagents for biological substances. WHO Tech Rep Ser 1987; 760: 39-8 1. 10. WHO Expert Committee on Biological Standardization-Thirty-sixth Report. WHO Tech Rep Ser 1987; 745. Il. Jerne NK, Wood EC. The validity of the results of biological assays. Biometrics, December 1949; 273-299. 12. Petukhov VG, Osin NS. The determination of the oxygen content of ampoules and vials of medical biological preparations filled with an inert gas. J Biol Stand 1985; 13: 87-91. 135

P. SIZARET

13. Jerne NK, Perry WLM. The stability of biological standards. Bull WHO 1956; 14: 167-182. 14. Tydeman MS, Kirkwood TBL. Design and analysis of accelerated degradation tests for the stability of biological standards I. Properties of maximum likelihood estimators. J Biol Stand 1984; 12: 195-206. 15. Kirkwood TBL. Design and analysis of accelerated degradation tests for the stability of biological standards III. Principles of design. J Biol Stand 1984; 12: 2 15-224. 16. International Standards and units for biological substances. Resolution WHA 37.27 of the Thirty-Seventh World Health Assembly, Geneva, 7-17 May 1984. Resolutions and Decisions,

Annexes,

WHA

37/ 1984/REC/

1. Geneva

1984.

APPENDIX SUMMARY

INFORMATION

ASSIGNED

UNITAGES

Gene&

NEEDED

FOR

ESTABLISHING

INTERNATIONAL

REFERENCE

MATERIALS

WITH

information

Name of the substance for which a Reference Preparation is proposed. Reason why a reference material is needed: indicate whether for diagnosis or prevention or therapy of (which) disease. Assay systems currently used for the assay of similar materials. An opinion as to whether similar materials are likely, in the near future, to be included in National Pharmacopoeias and/or National Requirements for the Manufacture and Control of Biological Substances, and are such materials likely to become items of international commerce. Pilot

study

If a pilot study successful working (in %), and results and assay method Bulk

is performed, report information on the number of final containers prepared in one session, precision of fill (% CV), vacuum achieved, residual oxygen, residual moisture of accelerated degradation tests (% loss per year at the proposed temperature of storage used for its determination).

mutwiah

Number of bulks and corresponding references (in the case that several CRMs were prepared, provide information on each corresponding bulk material). Origin, nature, approximate degree of purity, nature of matrix, nature and concentration of stabilizers (if any). Indicate composition of each final bulk (number and volumes of all single preparations which were pooled, volumes of diluent, and, if appropriate, results of tests for the absence of pathogenicity of each single preparation and the test method used). Dates of sterility tests on final bulk(s) and result(s). Results of any other relevant test. FinaL products The following information should be provided for each CRM for which establishment as an IRM is requested. Filling, lyophilization, seahng and determination of the stability of the CRMs. Reference numbers of CRMs. Address of laboratory which filled, lyophilized and sealed the ampoules. Dates on which these operations were performed. Number of ampoules the content of which was weighted before sealing, the intervals at which weights were determined, and detailed results of weights, coefficient of variation (in %). Was secondary desiccation over P205 performed before sealing? Number of sealed neutral glass ampoules. Individual weights of ‘cakes’ from ampoules which were opened after lyophilization, and corresponding CV (in %).

136

INTERNATIONAL

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MATERIALS

PREPARATION

Number of sealed ampoules found to be satisfactory after inspection and testing for leakage. Number of sealed ampoules offered to WHO. If sealed under vacuum, the results of testing for vacuum. If not sealed under vacuum, indicate the gas under which sealing was done and the method used for determining the residual oxygen content and the result.* Number of ampoules tested for residual moisture, the method used and the results (in %).* Date of test for sterility and results. The way in which the linearity and parallelism of dose-response lines were established and relative potencies determined. Laborarory(ies) which produced the stability data and the assay method(s) that were used. Number of containers exposed, for how long, at what temperatures, and activity remaining in each container after exposure. Predicted loss of activity in % per year at -2O”C, +@C and +2O”C and, if it is differenr from -20°C at the requisite temperature of storage. Address of place of storage (if different from address of fill) and name of Custodian. Actual temperature of storage. Report of the collaborative

study

The results of collaborative studies may be provided journal or in any other appropriate way. The report

as a report submitted for publication in a scientific should contain the following information:

addresses of participating laboratories; number and nature of each CRM studied; a statement that each preparation was coded differently for each collaborating laboratory; the assay methods which were used and by which laboratories; for each assay method, the number of assays requested from each laboratory and number of assays actually carried out; the way in which the linearity and parallelism of dose-response curves were established and relative potencies determined; a summary of valid and invalid assays; for each (coded) laboratory the overall within-assay CV and overall between-assay CV; overall CVs of assay results when Relative Potencies of test preparations were expressed in relation to individual standards and in relation to the CRM; proposal for assigning a content to each ampoule of the CRM; etc. Leafit

of intwctions

Include Name

a copy

and affiliation

to users of the proposed

leaflet

of responsible

project

(typed) Title Date Signature

of intructions officer

. .

. .

. . .

.

to users

.

.

. .

.

.

.

,_..........................................................................................................

* This type of information is usually relevant to the stability important is rhe stability itselfas determined by either accelerated tests, or both.

of a CRM; degradation

however, what is ultimately tests, or long-term stability

117