Reference standard material potency

The quantitative analysis is only as good as the reference material allows. The reference standard is used as the primary standard against which all determinations are made. Reference standard characteristics should be appropriately determined and documented for each batch. Copies of this documentation must be included with study records and must be available for inspection. The reference standard should be (1) of the highest purity obtainable, (2) independently characterized to establish identity, strength or potency, and purity, (3) in the most stable form, and (4) stored under suitable conditions. 

Reference standards are labeled such that their identity, potency, correct use, and correct storage can be assured at all times. Reference standard labeling must also ensure that laboratory personnel handling the material are properly informed of safety risks associated with potentially harmful chemicals. 

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. 

In vitro assays do not use any whole-cell or animal-based components. The fibrin clot lysis assay, as established for tissue plasminogen activators and described for alteplase in the USP, is an example of this type of potency testing [5]. By means of defined standard materials, a fibrin clot is formed and the time to complete lysis is characterized as measure of potency, compared to a reference standard with defined activity. The LAL-test is a well-established and internationally harmonized in vitro alternative to detect or quantify bacterial endotoxins, using Limulus amebocyte lysate (LAL) obtained from the aqueous extracts of circulating amebocytes of horseshoe crab (Limulus polyphemus or Tachypleus tri-dentatus) which has been prepared and characterized appropriately [5]. Two types of technique may be used for this test gel-clot techniques, which are based on gel formation and photometric techniques. 

For all analytical testing, standard substances are required as reference material. These standards exhibit defined quality, and serve for all types of identity, purity, degradation product and potency assays as reference quantity. In QC, reference and working standards are used. Reference standards are highly purified and extensively characterized by all appropriate physico-chemical, biochemical and immunochemical methods. Working standards are used in daily practice, and are calibrated against reference standard for routine use. 

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. 

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. 

GH assays now use mouse monoclonal antibodies, and some of these assays are able to discriminate GH variants. Most procedures use recombinant-derived GH for labeling with a tracer and calibration material. The latter is usually prepared gravimetricaUy and verified by comparison with an international reference preparation (IRP), such as the World Health Organization s (WHO s) international standard, IRP 80/505 human growth hormone recombinant (hGHr), which has a potency of 3.3IU/mg of r-hGH, or other standard preparations, such as WHO IRP 66/217 or 88/624. The assay diluent, however, can vary considerably from one assay to another and is a potential source of bias some commercial kits use phosphate-buffered saline, and others use horse, calf, or human serum. 

In view of the fluctuation in the proportion of PRL variants with different molecular masses and immunoreactiv-ities, PRL procedures should be calibrated against reference materials with imown international unit potency, such as the WHO first IRP 75/504, the second international standard (IS) 83/562, or the third IS 84/500 (http //www.iiibsc.ac.uk). Without such calibration, PRL results expressed in mass units cannot be compared from assay to assay. Despite the heterogeneity of PRL, immunoassays correlate well with bioassays and remain the standard method of measurement in routine practice. 

In 1960, at the general assembly of the International Pharmaceutical Federation (FIP), the obsolescence of various national pharmacopeial methods for assaying pharmaceutical enzymes was demonstrated. An international commission on pharmaceutical enzymes was created to deal with this unsatisfactory situation and develop improved assay methods and guidelines for the preparation of pharmaceutical enzyme reference materials. The Center for Standards has a coordination function in organizing collaborative enzyme assays between academic, industrial, and national pharmaceutical control laboratories and in distributing FIP pharmaceutical enzyme standards. Since 1960, many FIP assay methods and standard preparations have been adopted by national and international pharmacopeias, such as the European Pharmacopoeia. The ultimate goal is to provide official, preferentially nonempirical, standardized assay methods by which comparison of commercially available pharmaceutical enzymes is made possible. The most desirable situation would be an international uniformity of enzyme standards and assay methods, which would allow physicians and clinicians to unambiguously compare the potencies of commercially available enzyme products. 

If a lot change is required, experiments should be performed to qualify the new reference material. Standards prepared using the new lot should be compared against the previous lot within one run. A set of established VS should also be included in the run for assay acceptance. If differences are observed, the potency of the new reference lot should be adjusted to match the old lot. If existing reference material is depleted, a new lot should be qualified by comparison in multiple runs against a set of established VS. Sometimes there is a consistent bias of the VS results, indicating a systemic... 

The final ODs readouts at each concentration of the therapeutic protein can be fit into a curve-fitting equation. Most of the binding immunoassays can be fitted with the four-parametric fit. It is important to determine the best fit for the data. The standard curve typically consists of known concentrations of the purified protein of interest. When ELISA is used as a potency assay the potency of the therapeutic protein is calculated relative to the representative well-characterized lot of therapeutic protein (reference material) that is used to generate the standard curve for the assay. For potency assays, it is important to include the entire standard and test sample curves in the calculation. Testing the entire standard curve and sample curve for parallelism is an important prerequisite for the potency calculation. Having standard and appropriate control(s) on each assay plate is highly recommended. 

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