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Review: Reference-Standard Material Qualification
Reference-Standard Material Qualification
The author reviews the types of reference-standard materials used in drug-product manufacturing, discusses current regulatory requirements, and outlines a reference-standard qualification program.

Pharmaceutical Technology
Volume 33, Issue 4, pp. 66-73

The US Food and Drug Administration defines a reference-standard material as a "highly purified compound that is well characterized" (1). The US Pharmacopeia (USP) defines reference-standard materials as "highly characterized specimens of drug substances, excipients, reportable impurities, degradation products, compendial reagents, and performance calibrators" (2). Scientists performing analytical testing use reference standards to determine quantitative (e.g., assay and impurity) as well as qualitative (e.g., identification tests) data, performance standards, and calibrators (e.g., melting point standards). The quality and purity of reference standards, therefore, are critical for reaching scientifically valid results.

Reference standards can be segregated into two groups: chemical and nuclidic (1). Chemical purity must be determined for both groups; nuclidic reference standards, however, also need to be evaluated for radionuclidic and radiochemical purity. This article addresss chemical reference standards only.

Types of reference-standard materials

Reference-standard materials can be broadly categorized as such:
  • Assays—used to determine potency for active pharmaceutical ingredients (APIs) and salts
  • Degradation products—used to identify and possibly to quantitate degradation products
  • Process impurities—used to identify and possibly quantitate process-related compounds
  • Resolution—used to determine assay performance or impurity method
  • Metabolites—used to identify and possibly to quantitate substances generated through a metabolic process.

The level of characterization depends on the intended use of the reference standard. For example, a reference standard used to determine potency requires full characterization and qualification. A reference standard used as a resolution component or identification requires less discerning analyses.

Sources of reference-standard materials

Reference standards can be compendial or noncompendial and are typically obtained from the following sources.

Compendial (primary):

  • Pharmacopeias such as the United States Pharmacopeia (USP), European Pharmacopoeia (EP), or Japanese Pharmacopoeia (JP)
  • Nationally recognized standard institutions such as the National Institute for Standards and Testing (NIST).

Noncompendial (secondary):

  • The user (custom manufactures or synthesizes the reference standard)
  • Contract manufacturer
  • Companies such as chemical suppliers.

Reference-standard materials that are synthesized by the user or supplied by a contract manufacturer or secondary company must be characterized (3). Both the reference standards and drug substance may be synthesized initially using the same process. The reference standard should be of the highest purity possible; the drug substance may require further purification to become a reference standard (additional purification steps used for a drug substance should be fully described and included in any regulatory filing).

Storage and impurity detection

Impurities classified as organic (process and drug related), inorganic, or residual solvents (4) can be introduced during the manufacturing process for the drug substance, drug product, or excipient and/or through storage of the material. Impurities should be controlled throughout the manufacturing process. Impurities that are process-related should be kept to a minimum to avoid degradation and unwanted pharmacological effects. Compounds that are susceptible to hydrolysis, for example, should be thoroughly dried to remove moisture and then stored in a desiccator. Reference standards that contain a high percentage of organic volatile impurities may experience purity changes over time as the solvents evaporate. Impurities within acetone, a Class 3 solvent, for example, are permissible up to 5000 ppm or 0.5%, according to USP and ICH guidelines (5). The amount of acetone present may change during storage because of its volatility and therefore may alter the reference standard's purity.

Another reason to limit impurities is demonstrated in the following scenario. Consider a reference standard that is 90% pure. The remaining 10% of impurities have to be identified and monitored through the life of the material. More analytical tests must be performed, and the probability of the purity changing during the review period increases. Then consider a reference standard with a purity of 99.9%, which has less need for additional characterization and potential degradation. Such a product can be monitored more effectively. In addition, this type of standard reduces the degree of systematic and random error from the combined analytical tests.

If the reference standard is in a salt form, the amount of salt present must be determined so that the purity can be corrected for content. Applying the molecular weight to the correction will not account for residual salt that may be produced during synthesis. If possible, it is recommended the reference standard be in a salt-free state to reduce the characterization tests required.

Organic impurities. Determination of organic impurities is the most challenging aspect of developing a suitable analytical method because these impurities are unique to the parent compound and because various degradation pathways can lead to various impurities. Actual and potential organic impurities that arise during synthesis, purification, and storage must be identified and quantitated. The synthesis of the reference standard should be evaluated to predict and identify potential impurities from raw materials. Potential degradation product also can occur as a result of storage. Short-term (forced degradation) and long-term (evaluation under accelerated conditions) stress testing, therefore, should be evaluated during development. The design of the long-term stress test depends on the intended storage condition.

The quantity of organic impurities present can be determined with high-performance liquid chromatography (HPLC) and ultra-violet (UV) detection. Degradation products and compounds related to the product can be evaluated by the area percent or from the relative response of the standard being used. The technique used to obtain this data will depend on the amount of impurities and related compounds present and the decomposition pathway of the reference-standard material.

To consider the impact on the purity evaluation using area percent versus relative response factor, the following scenario may be considered. If analysis shows an impurity at 0.05% and the relative response factor of the impurity is half of the standard (i.e., the amount of impurity present shows a 50% detector response compared with the equivalent amount of standard), then there could be 0.1% of actual impurity. This level may be insufficient to affect overall purity results. If there was 1% impurity based on area percent present, however, then there would be 2% of actual impurity that could affect overall purity.

The approach to determining the relative-response factor for each impurity is a more accurate process, but potential pitfalls should be considered. The relative-response factor approach requires additional development because the component needs to be isolated and the relative response factor must be determined. In addition, as the reference standard ages, new unknown impurities may be detected. The relative-response factor of these new impurities must be determined, and the method updated if the new unknown is significant enough to alter the purity. Much of this information may be ascertained during the development of the drug substance.

Impurities that arise from raw materials, synthesis, purification, and storage require careful consideration because they may not produce detector responses that are related to the reference-standard material. Quantitation by area percent would not be appropriate in such cases. Rather, the impurities must be isolated and identified so that an appropriate reference standard can be used, or a relative response factor determined. For example, if the reference-standard material is a salt, then the cation response would not be equivalent to the reference standard. In such instances, a specific reference standard is required for the cation, and a separate analytical method for quantitation may be needed.

Inorganic impurities. Inorganic impurities such as metals and noncombustible materials are typically evaluated using compendial procedures. If inorganic impurities are proven to be less than the reporting threshold at initial characterization, then further analysis is not required.

Residual solvents. The potential for residual solvents should be evaluated during development of the drug substance and can be estimated by reviewing the synthesis pathway. USP General Chapter <467> Residual Solvents details a generic procedure for this evaluation. Residual solvents, however, may be specific to the manufacturing process and require a specific test procedure. An additional specific test procedure may be required if the USP procedure is not suitable for the reference standard being evaluated, or if the solvents used during synthesis are not included in USP <467>. If residual solvents (previously referred to as organic volatile impurities, or OVIs, by USP) are proven to be less than the reporting threshold at initial characterization, further analysis is generally not required at subsequent intervals. If the amount of residual solvents present affects the purity, however, they should be evaluated at each requalification interval.

Regulatory requirements

The integrity of reference standards must be proven for products that are used in registration applications, commercial releases, stability studies, or pharmacokinetic studies. FDA requires reference standards to be of the "highest purity that can be obtained through reasonable effort" and to be "thoroughly characterized to assure the identity, strength, and quality" (3). This requirement is meant to ensure that the product being evaluated is accurately tested to determine the amount of API present and to classify and identify related substances, process-related impurities, and degradation products.

To fully understand the development of a reference-standard material program, the required method validation needs to be discussed. FDA requires noncompendial reference standards to be "of the highest purity" and asks that reference standards validate analytical methods (1). This raises the question, Which requirement should be met first: the qualification of the reference standard or its method validation? The answer is a compromise based on suitable parameters for the intended application.

Quantitative analytical procedures for impurities' content or limit tests for the control of impurities must be validated and suitable for the detection and quantitation of impurities as directed by the International Conference on Harmonization (ICH) (6). FDA cites "failure to submit well characterized reference standards" as a "common problem that can delay successful validation" (3). An insufficiently characterized reference standard may delay or prevent FDA approval of a drug product to market.

To ascertain the degree to which an analytical method is deemed suitable for its intended use, the validation parameters set forth in ICH Q2(R1) Validation of Analytical Procedures (6) stipulates the following criteria:

  • Specificity—evaluation of interference from extraneous components
  • Linearity—linear range of the method
  • Range—the interval between the lower and upper concentration amounts of analyte in the sample
  • Accuracy—a measure of the closeness of agreement between the value obtained and the theoretical
  • Precision—a measure of the closeness of agreement (degree of scatter) of the data values over a number of measurements (i.e., injection repeatability, analysis repeatability (multiple measurements, same analyst) and intermediate precision (multiple measurements, different days, different analysts), reproducibility (precision between different labs)
  • Detection limit—the lowest level the analyte can be detected
  • Quantitation limit—the lowest level the analyte can be quantitated
  • Robustness—effects of small changes in method parameters
  • System suitability testing—evaluation of the suitability of the equipment.

Table I: Types of reference-standard material compared with recommended qualification.

Not all parameters can be evaluated because a reference standard is required to perform quantitation. In this case, where the reference standard is the sample, the parameters validated are restricted. However, the method can be assessed for parameters applicable to evaluating the reference material. The analytical method is therefore qualified for use but not validated per ICH guidelines. Table I presents recommended qualification parameters compared with reference-standard material type. ICH also requires the reference material to be proven stable under the intended storage conditions for the intended use period (7). The reference-standard material program, therefore, must be designed so that the material is assessed at its intended storage condition over time.

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