Reference-Standard Material Qualification

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.

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.

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

Reference standard program

Compendial. The use of compendial reference standards is preferred for a reference-standard program. Regulatory agencies will accept reference-standard materials from a pharmacopeial source and NIST without further qualification (1). NIST provides a certificate of analysis (CoA) that includes purity information and an expiration date. USP, however, labels its reference standards (assay reference standards to the nearest 0.1% and impurity reference standards to the nearest percent). In addition, USP reference standards are considered suitable for use up to one year after a new lot is released.

Wherever possible, therefore, compendial methods should be used to qualify reference standards. If the reference-standard program requires tests that are not captured in compendial methods (as is the case with organic impurities), then analytical test procedures must be developed and qualified. This can be an expensive process and may delay the process of stability or clinical programs.

Noncompendial. Applicants that use proprietary materials will find that primary standards are not typically available through compendial sources. As a result, noncompendial (secondary) reference standards require characterization and, thus, reference-standard development and qualification programs need to be implemented. Materials can be developed or purchased from chemical-supply companies for use as in-house secondary reference standards even when compendial reference standards are available. In such instances, the secondary reference standard should be qualified against the compendial reference standard.

Identification of impurities. Actual and potential degradation products should be isolated and identified during development of the reference standard. Typically, organic impurities are identified and confirmed using liquid chromatography–mass spectrometry (LG–MS); nuclear magnetic resonance (NMR) and inductively coupled plasma/mass spectrometry (ICP–MS) are used for inorganic impurities; and gas chromatography/mass spectrometry (LC–MS) is used for residual solvents.

It should also be determined whether enantiomeric or polymorphic forms exist. Each of these factors must be considered in the development of a comprehensive reference-standard material program. Qualification of a secondary source reference-standard material begins with obtaining a CoA, the synthesis pathway (if available), and a list of methods used in product manufacturing. This information can help analysts determine essential parameters for qualification. The identity of the material should be confirmed with a "fingerprinting" technique such as fourier transform infrared spectroscopy (FTIR) to a library source or by elemental analysis to confirm the molecular formula. Once identity has been established and confirmed, the quality of the material must be ascertained. Elemental analysis, titration, GC, or LC can be used for purity determination. Based on the results, the material may require further purification by distillation or recrystallization. Additional testing may be required to identify and quantify known or potential impurities that may have been overlooked during the manufacturer's assessment of the material.

For instances in which a reference-standard material is not available from a commercial source, the material must be synthesized. For APIs, the material may start out as a lot of drug substance with sufficient purity to be designated as the reference-standard material, or it may require further purification. Known impurities or degradants will require custom synthesis. Their purity requirements, hoewver, are generally not as stringent. Although there is no set guideline to characterize a reference-standard material, Figure 1 depicts a decision-tree approach involving broad range analytical techniques.

Figure 1: Decision-tree for reference-standard qualification. MS is mass spectroscopy; NMR is nuclear magnetic resonance; UV is ultra-violet; FTIR is Fourier Transform Infrared Spectroscopy; HPLC is high-performance liquid chromatography; KF is Karl Fischer; GC is gas chromatography; and LC is liquid chromatography. (FIGURE 1 IS COURTESY OF THE AUTHOR.)

The analytical procedures shown in Figure 1 are dependent on the evaluation of the development process. Minimal required tests for initial characterization are typically performed using the following tests:

• Organic impurity—HPLC with UV detection

• Metals impurity—ICP with MS detection or ICP with optical-emission spectroscopy detection

• Noncombustible impurities—residue on ignition

• Residual solvents—GC with flame ionization detection

• Water content—Karl Fischer titration

• Structural confirmation: hydrogen and carbon–13 NMR, LC–MS, or FTIR

• Empirical confirmation—C, H, N analysis

• Appearance confirmation—visual inspection.

Other tests may include chiral evaluation (HPLC with UV detection), melting point, differential scan calorimetry, and polymorph evaluation by X-ray powder diffraction. Different types of reference-standard materials and the qualification tests recommended are presented in Table II.

Table II: Types of reference-standard material compared with recommended test.

Initial qualification and requalification. Initial characterization of the reference standard should include a full suite of analytical tests. Requalification at subsequent points may include a reduced suite of analysis, depending on initial results. It is recommended that a three-tiered approach be adopted to avoid interruption in stability or clinical programs, as outlined below.

• Tier 1: The reference-standard material qualification program should be started at least one month before the stability or clinical program begins to allow for requalification and assignment of a new expiration date. This timeframe will also help to avoid delays in testing for subsequent programs due to an expired reference standard.

• Tier 2: At least two lots of reference-standard material should be placed in the qualification program three months apart. If the primary lot degrades, the secondary lot may be used during the interim period while a new, third batch is manufactured, characterized, and qualified.

• Tier 3: At least two storage conditions should be chosen: the intended storage condition and an alternative storage condition as a contingency. For example, if the intended storage condition is 2–8 °C then the reference standard should also be stored at –20 °C as a contingency. This may allow for an extended life of the reference standard if it is proven to be unstable for a long period of time at its anticipated storage condition. In addition, useful stability information may be ascertained if the contingency conditions samples are tested as well as the intended storage condition.

For the initial lot, an example requalification period may be 3, 6, and 12 months for the first year and annually thereafter. In this scenario, it is recommended that during development, the reference standard be assessed after 3 months at the intended storage condition and at an accelerated storage condition. Validation of the analytical method for organic impurities should occur after the full accelerated storage condition has been evaluated. The total length of the requalification program will depend on the intended life of the reference standard and the length of the stability and clinical programs. If the initial lot is proven to be stable for at least one year, then subsequent lots will require annual requalification only. In all study scenarios, a protocol is required to outline the reference-standard material, lot, storage conditions, frequency of test, analytical procedures, acceptance criteria, and reporting criteria.

Distribution and control. Reference-standard materials are often expensive to manufacture and are generally of limited supply. It is important, therefore, to consider how the material will be stored, distributed, and controlled. Once the storage conditions are ascertained, the reference-standard material should be monitored continually using a suitable environmental monitoring system. It is advisable to store the material in at least two different locations in case there is a prolonged excursion from the storage condition. The material should be stored in a secure environment with controlled access and distribution.

David Browne is manager of stability and pharmaceutical testing at Intertek Pharmaceutical Services, d/b/a QTI, 291 Route 22 East, Whitehouse, NJ 08888, tel. 908.534.4445, david.browne@intertek.com.

Submitted: Mar. 20, 2008. Accepted: Sept. 22, 2008.

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References

1. FDA, "Reviewer Guidance, Validation of Chromatographic Methods" (Rockville, MD), 1994.

2. USP 30–NF 25 General Chapter <11>, "Reference Standards," p. 1.

3. FDA, "Guideline for Submitting Samples and Analytical Data for Methods Validation" (Rockville, MD), 1987.

4. ICH, Q3A(R2) Impurities in New Drug Substances (Geneva, Switzerland), Oct. 25, 2006.

5. USP 30 –NF 25 General Chapter <467>, "Residual Solvents."

6. ICH, Q2(R1) Validation of Analytical Procedures: Text and Methodology (Geneva, Switzerland), Oct. 1994.

7. ICH, Q1A(R2) Stability Testing of New Drug Substances and Products (Geneva, Switzerland), Feb. 6, 2003.