Reference standard material stability

Reagents and reference materials Likely will change but should have some documentation on early characterization Continue to screen for optimal reagents Lot no. and history (notebook reference) Evaluate different reagents and identify critical reagents Determine if sufficient quantities are available and their stability for later bioanalytical needs Include C of A for reference materials in assay validation documents Keep records of source and lot no. Use optimized capture/ detection reagents Use characterized reference standard from final manufacturing process with Cof A Record all lot nos. and sources... 

At a minimum, documentation of the characterization and stability of a standard, such as a certificate of analysis (Co A) and/or a certificate of stability (CoS), is typically available from the suppliers. The certificate should be obtained and recorded. The quantity of reference standard is typically limited in commercial kits designed for research use, and it is not uncommon that the reference material values may differ substantially between lots and manufacturers [16]. Novel biomarkers rarely have established gold standards against which their potency and abundance can be calibrated. A comparison of available sources can be useful, and when validating an assay for advanced applications it is desirable to plan ahead to obtain and reserve a sufficient supply of the same reference material. The example in Fig. 6.5 compares three reference standard curves, each prepared from a concentrated stock solution from a commercial supplier, an in-house reference standard, and a commercial kit, respectively. The instrument responses (optical density, OD) were highest with the standard from the commercial stock, the lowest with the kit, while the in-house reference standard response was intermediate. In this case, either the same commercial stock or the in-house reference standard can be used throughout the clinical study. 

Use of Complex Standards for Cell Proteins and Two-Dimensional Electrophoresis. One of the more widely-used cell lines chosen as a reference standard is the lymphoblastoid cell line GM607, derived from a normal individual and available from the Human Genetic Mutant Cell Depository, Camden, NJ 08103. This cell line may be grown in defined media, labeled with a radioactive tracer, and reproducibly separated in a 2-DE system. Heat shock proteins may readily be isolated and visualized from this cell line, as shown by Anderson et al. (42). For serum, a reference preparation for serum proteins is available as a certified reference material prepared and assayed by the College of American Pathologists (CAP) and by the U. S. Centers for Disease Control. A widely available human serum standard is that provided by the National Bureau of Standards as SRM 909. If sufficient interest from the user community is evident, a full electrophoretic characterization of this material can be included in the documentation. If the amount of selected standard proteins loaded on a gel is known, "relative" quantification of similar proteins could be obtained. In addition, the National Bureau of Standards could serve as an impartial evaluator of potential national standards (e.g. molecular weight standards, "tie-point" proteins, and Isoelectric point standards) to assess suitability and stability. 

Potency studies should be performed at appropriate intervals as defined in the stability protocol, and the results should be reported in units of biological activity calibrated, whenever possible, against nationally or internationally recognized standards. Where no national or international reference standards exist, the assay results may be reported in in-house derived units using a characterized reference material. 

The quality control department as a whole will also have other duties, such as to establish, validate, and implement all quality control procedures, to evaluate, maintain, and store the reference standards for substances, to ensure the correct labelling of containers of materials and products, to ensure that the stability of the active pharmaceutical ingredients and products is monitored, to participate in the investigation of complaints related to the quality of the product, and to participate in environmental monitoring. All these operations should be carried out in accordance with written procedures and, where necessary, recorded. 

Reference Material. In contrast to small molecules where reference standards are well characterized and certified standards are often commercially available from sources such as the U.S. Pharmacopeia, the European Pharmacopoeia, or the World Health Organization (WHO), proteins are often not as rigorously characterized and their purity may vary from supplier to supplier. Variation in posttransla-tional modifications such as glycosylation and deamidation may be present. Thus, the proteins can vary in their potency and immunoreactivity. As the reference compounds are used as standard calibrators, validation sample and QC sample variation of the reference will have a profound impact on the assay performance. Therefore, it is important to clearly document the source of the material and to characterize the proteins as thoroughly as possible. Comparability between lots or sources should be evaluated if possible. If the analyte is not a new drug entity, the innovator company is typically the most reliable source of authentic material. As stated for the reagents, stability of the reference compounds is an issue that has to be ensured. 

USP<11> USP Reference Standards To ensure that the Reference Standards maintain the properties determined at the initial evaluation, USP maintains a Continued Suitability for Use Program. Abbreviated protocols use the stability-indicating methodology employed in the initial characterization of the material to confirm the consistency of attributes such as appearance, chromatographic purity, or volatiles content... 

Materials, methods and equipment - lists the batch numbers that will be used, if known, reference standard lot numbers and the method references. Sample age and uniformity is important to the transfer and will be described here. For example, for a product which typically degrades on stability, assure that the protocol takes this into account by having both labs test similarly aged products, for example do not use the release results from the originating lab and compare to 6-month stability samples at the receiving lab. Equipment should be described. 

The WHO Expert Committee for Biological Standards (ECBS) assesses and if appropriate approves proposals for materials to be recognized as WHO International Biological Standards or Reference Reagents. The criteria reflect the suitability for purpose of the material, stability, reproducibility, and, if freeze-dried, the residual moisture (see Section II for details). There is no requirement for sterility but any microbial contamination should not cause interference in the assay system in which the reference material is to be used, which may include cell culture or in vivo assays. 

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