Method Validation Studies How GLP Interacts With Guidance Documents

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1 Method Validation Studies How GLP Interacts With Guidance Documents Steven S. Kuwahara ROBERT GEORGE YOUNG/GETTY IMAGES GLP Forum addresses topics of interest associated with good laboratory practices. We intend this column to be a useful resource for daily work applications. The key objective for the column: Useful information. Reader comments, questions, and suggestions are needed to help us fulfill our objective for this column. Manuscripts or case studies submitted by readers are most welcome. Please send your comments and suggestions to column coordinator Steven Kuwahara at stevekuwahara@yahoo.com or to coordinating editor Susan Haigney at shaigney@advanstar.com. KEY POINTS The following key points are discussed in this article: This discussion addresses the need for analytical method validation in good laboratory practice (GLP) studies including stability studies Regulations clearly state that stability studies must be done and completed no later than the end of the GLP study. Responsibility for appropriateness of testing and completion of stability studies rests with test facility management. Stability of test mixtures containing active drugs with carriers is specifically addressed. Stability of the active drug alone does not answer this concern. GLPs apply to chemical procedures used to characterize the test article, to determine stability in mixtures, to determine the homogeneity and concentration, and to analyze specimens GLPs do not apply to development of chemical methods of analysis or to establish specifications of a test article The US Food and Drug Administration has defined stability-indicating assays and has clearly stated that these assays should be used in stability studies. These studies should also address freeze-thaw cycles, sample transport, and long-term storage. The statement that the validation data only needed to be available for review and not included with submissions was apparently taken by some to mean that the data did not need to be gathered until late in the clinical trial process. More recent GLP inspections by FDA under the biore- 20 Journal of GXP Compliance

2 Steven GLP S. Kuwahara FORUM search monitoring (BIMO) program have removed these expectations. Stress testing can help identify the likely degradation of products, which can in turn help establish the degradation of pathways and the intrinsic stability of the molecule and validate the stability indicating power of the analytical procedures Test method validation must be conducted at the level of non-clinical, GLP studies not at the later stage of phase 3 clinical trials When conducting a non-clinical safety study it is important to know that the material being studied will be stable over the course of the study, and to use validated stability-indicating tests. INTRODUCTION When dealing with the good laboratory practice (GLP) regulations and guidance documents issued by the US Food and Drug Administration, the quality unit worker is sometimes confronted with interesting conclusions that may or may not have been intended by the regulators. A situation of this type has arisen for analytical method validation studies because of the interaction among the GLP regulations and guidance documents related to the issue of stability studies. THE REGULATIONS The process starts with the GLP regulations. 21 CFR (b)(1) states, The stability of each test or control article shall be determined by the testing facility or by the sponsor either before study initiation or concomitantly according to written standard operating procedures, which provide for periodic analysis of each batch. This paragraph clearly states that stability studies must be done and completed no later than the end of the GLP study. This may be combined with 21 CFR 58.31(d) (2), which states, Testing facility management: Assure that test and control articles or mixtures have been appropriately tested for identity, strength, purity, stability, and uniformity, as applicable. The responsibility for assuring that the stability studies have been done is clearly placed with the management of the test facility. The stability of test mixtures is of special concern in GLP, and this is emphasized in 21 CFR (a, 2)(3) as follows: Mixtures of articles with carriers: For each test or control article that is mixed with a carrier, tests by appropriate analytical methods shall be conducted: To determine the stability of the test and control articles in the mixture as required by the conditions of the study either: (i) Before study initiation, or (ii) Concomitantly according to written standard operating procedures which provide for periodic analysis of the test and control articles in the mixture. This regulation ensures that stability tests are performed on the test article mixture used when drugs are fed to animals in non-clinical safety studies. This requirement is interesting in that some sponsors have stability data on the pure active pharmaceutical ingredient (API), but lack data that would reveal the effect of animal feed on the stability of the API. In some cases, the lack of data is brushed off with the statement that the test article mixtures do not represent the drug product when taken by the patient. However, the point of the requirement is not the drug product, but rather the material as administered to the animals. If the test mixture results in a loss of stability of the API, any resulting safety study will be compromised. The worst case might be a situation where the API reacts with a feed component to produce a toxic material that generates a false impression of the toxicity of the product. GUIDANCE AND INTERPRETATIONS The Center for Drug Evaluation and Research s (CDER) Good Laboratory Practice Regulations: Questions and Answers document (4) concerning the GLPs states the following: Q. Do the GLPs apply to all of the analytical support work conducted to provide supplementary data to a safety study? A. The GLPs apply to the chemical procedures used to characterize the test article, to determine the stability of the test article and its mixtures, and to determine the homogeneity and concentration of test article mixtures. Likewise, the GLPs apply to the chemical procedures used to analyze specimens (e.g., clinical Summer 2009 Volume 13 Number 3 21

3 chemistry, urinalysis). The GLPs do not apply to the work done to develop chemical methods of analysis or to establish the specifications of a test article. Chapter 48 of the Compliance Program Guidance Manual (CPGM) (5) states, III.C.8. Test and Control Articles (21 CFR b.) Characterization and Stability of Test Articles (21 CFR ) The responsibility for carrying out appropriate characterization and stability testing may be assumed by the facility performing the study or by the study sponsor. When test article characterization and stability testing is performed by the sponsor, verify that the test facility has received documentation that this testing has been conducted. 1) Verify that procedures are in place to ensure that [c] the stability of test and control articles is documented. This responsibility for verifying that the stability studies and other testing activities have been completed or are in progress is emphasized by an FDA 483 in a warning letter issued to WIL Research Laboratories (6), which was acting as a contract testing facility. The letter states, The response also indicates that, as a contract laboratory, WIL Research cannot be held responsible for sponsor conducted activities (analyses of dosing formulations) which are not under WIL Research s direct control. This response does not satisfy the requirements as set forth in Section 58.31(d) of the good laboratory practice regulations which requires that testing facility management shall assure that test and control articles or mixtures have been appropriately tested. Specifically, when analytical tests for homogeneity, concentration, and stability of dosing formulations are performed by another laboratory, it is the testing facility s responsibility to assure that such tests are performed. The importance of conducting good stability studies is further emphasized in the warning letter issued to the Institute for Human Gene Therapy (7). The citation states the following: The vector XXX was used in study YYY 25 months after the preparation date. The study protocol specified that expiration date of the test articles was ZZZ months from the date of preparation. There is no documentation to support the stability of the vectors stored longer than ZZZ months. A memorandum dated January 27, 2000, written by the former Associate Director of the Transitional and Clinical Research Program, states the rate of decay of infectivity is actually very slow, with an approximate WWW. Thus it is likely that the adenoviral vectors have retained a great deal of activity even after VVV months of storage. This memorandum would indicate that the animals who received a vector stored for 25 months would have been given a dose of vector from 52.2% to 65.6% below the vector dose specified in the protocol. This reduced dose of vector could result in an under-estimation of toxicity in the animal studies used to establish the dose of vector to be administered to human subjects. We note that the specific lot of this vector used in the clinical studies to treat human subjects was stored for only two months prior to its administration. Although this vector was the same for the monkey study and the human study, the lots were not prepared at the same time, and the stability of the stored lots could be very different. Finally, section III of the Draft Guidance, Analytical Procedures and Methods Validation, Chemistry, Manufacturing, and Controls Documentation (8) defines a stability-indicating assay as a validated quantitative analytical procedure that can detect the changes with time in the pertinent properties of the drug substance and drug product. A stability-indicating assay accurately measures the active ingredients, without interference from degradation products, process impurities, excipients, or other potential impurities. Assay analytical procedures for stability studies should be stabilityindicating, unless scientifically justified. Other guidance documents refer to the GLPs and contain elements of a method validation. The Guidance for Industry, Internal Radioactive Contamination-Development of Decorporation Agents (9) references following GLP for certain studies and states, Bioanalytical methods for determination of active moiety and metabolite(s). Sponsors should provide the rationale for selecting metabolites for analysis and describe the range of the standard curve and curve fitting techniques. They should also identify the upper and lower limits of quantification (ULOQ/LLOQ) and describe the accuracy, precision, and selectivity of analytical methods at these limits. In addition, sponsors should 22 Journal of GXP Compliance

4 Steven GLP S. Kuwahara FORUM address sample stability during freeze-thaw cycles, sample transport, and long-term storage. These requirements also make it clear that validated methods are expected, even if the studies are GLP and not good manufacturing practice (GMP) studies. Furthermore ICH Q2A, Text on Validation of Analytical Procedures (10) states, The objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose. One would expect that this demonstration would be desired for any analytical procedure that is employed regardless of the regulatory status of the product. Statements in guidance documents related to stability studies support the need for validation of stability-indicating tests as was stated in the definition (8). The Guideline for Industry: Quality of Biotechnological Products: Stability Testing of Biotechnological/Biological Products (11) states, At the time of submission, applicants should have validated the methods that comprise the stability-indicating profile, and the data should be available for review. The statement that the validation data only needed to be available for review and not included with submissions was apparently taken by some to mean that the data did not need to be gathered at all, as inspections were not expected until late in the clinical trial process. More recent GLP inspections by FDA under the bioresearch monitoring (BIMO) program have removed these expectations. The need for stability-indicating assays was also indicated in the main guidance documents related to stability studies (12). The section covering stress testing states, Stress testing of the drug substance can help identify the likely degradation products, which can in turn help establish the degradation pathways and the intrinsic stability of the molecule and validate the stability indicating power of the analytical procedures used. This statement also gives the need to validate the ability of the test to be stability-indicating. ICH Q1A (R2)(12) also shows the relationship between stability studies and method validation studies. In the guideline on method validation (10), the specificity of a test method is given as an element of method validation and is defined as follows, Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc. The question that is sometimes asked is What degradants? The stability study guidance document (12) makes it clear that the stress studies will show what the expected degradants are. In turn, this indicates that the stress testing should be conducted before a full method validation may be completed. DISCUSSION The regulations clearly indicate that stability studies need to be conducted on the drug product and drug product mixtures employed in non-clinical safety studies. This requirement has been supported by internal guidance given to FDA personnel and by citations in warning letters. While FDA communications have said that the stability studies only need to cover periods long enough to verify the stability of the product over the course of the non-clinical studies, the need for the stability studies has been maintained. In response to the question of how the stability studies should be conducted, there are clear guidance documents that describe the procedures. These documents show the need for stability-indicating assays, and, in turn, state that stability-indicating assays are validated assays. When all of these requirements are combined, they lead to the conclusion that test method validation must be conducted at the level of non-clinical, GLP studies, and not at the later stage of phase 3 clinical trials. The point may be addressed in a series of statements. First, when conducting a non-clinical safety study it is important to know that the material being studied will be stable over the course of the study. The consequences of not having this condition have been demonstrated (7). Second, when conducting a stability study, it is necessary to use stability-indicating tests. It would not make any sense to conduct a stability study with tests that could not measure stability related changes. Third, a stability-indicating test is a validated test. Any test should be known to be suitable for its intended purpose. Otherwise, why would one want to use it? Finally, given the costs of non-clinical testing and stability studies, it would not be sensible to base them on the foundation of test methods that may be unsuitable. Summer 2009 Volume 13 Number 3 23

5 REFERENCES 1. FDA, Code of Federal Regulations, Title 21, Part 58, Section 105(b), U.S. Government Printing Office, April 1, FDA, Code of Federal Regulations, Title 21, Part 58, Section 31(d), U.S. Government Printing Office, April 1, FDA, Code of Federal Regulations, Title 21, Part 58, Section 113(a)(2), U.S. Government Printing Office, April 1, CDER, FDA, DHHS, Good Laboratory Practice Regulations: Questions and Answers, 1981, Re-issued FDA, CDER, Compliance Program Guidance Manual, No , Chapter 48, Bioresearch Monitoring, part III, C. 8. Issued: 02/21/ FDA, Warning Letter, WIL Research Laboratories, Inc., Reference No. 99-HFD , issued 11/26/ FDA, CBER/OCTMA, Warning Letter, Institute for Human Gene Therapy, Reference No. CBER , Issued 7/3/ FDA, CDER, CBER, Guidance for Industry, Draft Guidance, Analytical Procedures and Methods Validation, Chemistry, Manufacturing, and Controls Documentation, August FDA, CDER, Guidance for Industry, Internal Radioactive Contamination Development of Decorporation Agents, March FDA, CDER, CBER, ICH Q2A, Guideline for Industry, Text on Validation of Analytical Procedures, March ICH, Guideline for Industry Quality of Biotechnological Products: Stability Testing of Biotechnological/Biological Products, ICH Q5C, July FDA, CDER, CBER, ICH Revision 2, Guidance for Industry: Q1A(R2) Stability Testing of New Drug Substances and Products, November GXP ARTICLE ACRONYM LISTING API Active Pharmaceutical Ingredient BIMO Bioresearch Monitoring CPGM Compliance Program Guidance Manual FDA US Food and Drug Administration GLPs Good Laboratory Practices GMP Good Manufacturing Practice ICH International Conference on Harmonisation ABOUT THE AUTHOR Steven S. Kuwahara, Ph.D., is principal consultant at GXP BioTechnology LLC ( in Sunnyvale, CA. Steve has more than 30 years of experience supervising quality control departments dealing with drugs, biologics, HCT/P, and nutraceuticals. He has supervised animal facilities and testing laboratories that operated under GLP rules and also those operating under GMP and ISO standards. He may be reached by at or com and by phone at Journal of GXP Compliance