Key Words: bioequivalence and bioavailability audit; bioanalytical laboratory audit; LC/MS/ MS; FDA inspection; method validation.

Transcription

1 Current Challenges for FDA-Regulated Bioanalytical Laboratories Performing Human BA/BE Studies; Part III: Selected Discussion Topics in Bioanalytical LC/MS/MS Method Validation Frank Chow 1,, Arlene Ocampo 1, Paul Vogel 1, Steven Lum 2 and Nhan Tran 1 1 Lachman Consultant Services, Inc., 1600 Stewart Avenue, Suite 604, Westbury, NY 11590, USA 2 Perrigo, 71 Sultons Lane Piscataway, NJ 08854, USA Summary This article is the third of a three-part series that deals with current compliance issues/challenges for bioanalytical laboratories performing analysis for bioavailability (BA)/bioequivalence (BE) studies. Part 1 of this series discusses the application of key elements from the FDA Good Laboratory Practices (GLP) and the current Good Manufacturing Practices (cgmp) regulations as the framework for the implementation of sound Quality Systems in a bioanalytical laboratory to be in compliance with current regulatory expectations. Part 2 discusses recent Food and Drug Administration (FDA) inspection trends for bioanalytical laboratories and provides an overview of the recent FDA 483 observations related to LC/MS/MS method validation issued to bioanalytical laboratories. This current article provides a more in-depth analysis of the scientific and compliance aspects of different approaches for selected validation parameters of LC/MS/MS bioanalytical methods. Copyright r 2009 John Wiley & Sons, Ltd. Key Words: bioequivalence and bioavailability audit; bioanalytical laboratory audit; LC/MS/ MS; FDA inspection; method validation Introduction *Correspondence to: Frank Chow, Lachman Consultant Services, Inc., 1600 Stewart Avenue, Suite, 604, Westbury, NY 11590, USA. F.Chow@LachmanConsultants.com An important section of the BE regulations (21 Code of Federal Regulations (CFR) (a)) requires that the analytical method used in an in vivo BA or BE study to measure the Copyright r 2009 John Wiley & Sons, Ltd. Qual Assur J 2009; 12,

2 Current Challenges for FDA-Regulated Bioanalytical Laboratories 23 concentration of the active drug ingredient or therapeutic moiety or its active metabolite(s) in the body fluids or excretory products, or the method used to measure an acute pharmacological effect shall be demonstrated to be accurate and of sufficient sensitivity to measure, with appropriate precision, the actual concentration of the active drug ingredient or therapeutic moiety or its active metabolite(s) achieved in the body [1]. The FDA Guidance for Industry entitled Bioanalytical Method Validation [2] published in May 2001 was developed based on two workshops jointly sponsored by both industry and FDA in 1990 and 2000 [3,4]. In early 2006, another FDA/industry joint workshop was held in Crystal City, Virginia, to further discuss various technical and compliance issues with respect to such bioanalytical methods that could lead to a revision of the May 2001 FDA Guidance. The current FDA Guidance is applicable to bioanalytical procedures in general and delineates the validation requirements for both Chemical Assay and Microbiological/Ligand-Binding Assays. The Guidance does an excellent job providing the general considerations for determining the fundamental validation parameters including precision and accuracy, recovery, selectivity, sensitivity, stability and the calibration curve. In addition, it makes certain specific recommendations for method validation, but it does not go into sufficient detail in a number of areas including recovery, stability and selectivity. This leaves the execution and implementation open to interpretation and potential controversy. This article examines common approaches used by bioanalytical laboratories when validating LC/MS/MS methods, which have significant impact on the reliability and validity of the data generated in routine drug analysis in the production runs for BA/BE studies. Selected Discussion Topics in Bioanalytical Method Validation As indicated in Part 2 of this series, method validation issues for bioanalytical methods are among the most common FDA 483 observations, despite the existence of the May 2001 FDA Guidance and the ongoing discussions between FDA and the industry since at least the early 1990s. The frequency of FDA 483s can perhaps be attributed to the lack of clarity and details discussed in the current FDA Guidance in a number of areas including selectivity, recovery and stability. As the Guidance is intended to be a general document applicable to different types of bioanalytical methods including chemical and microbiological/ligand binding assays, it may not be the appropriate forum to address criteria for a specific method type. This article will discuss some common issues impacting the evaluation of selectivity, recovery, and stability of LC/MS/MS bioanalytical methods. Matrix effect Ion suppression/enhancement due to matrix effect has been one of the major unknown variables that could adversely affect the accuracy and precision of the assay results for biological samples using LC/MS/MS methods [5 12]. The ion suppression/enhancement is generally caused by the presence of co-eluting matrix extracts that could affect the abundance of the available gas phase charged ion to the mass spectrometer Atmospheric Pressure Ionization (API) source by: a) affecting the ability of the analyte droplets in the solution phase to be transferred to gas phase as charged ions and b) the extent of neutralizing the charged ions by charge stripping and/or charge transfer reactions [5,8]. The extent of matrix effect could vary from subject to subject due to the complexity and potential variability of the biological matrix that contains a large variety of endogenous substances, metabolites and, in some cases, the presence of co-administered drug(s). A validated method could be at risk when the actual study samples are tested since the biological samples from different subjects could have different matrix effects [9,10]. The composition of the biological matrix from each subject could be different from each other and

3 24 F. Chow et al. different from the blank matrix lots used for validation. Therefore, matrix effect can be considered as unseen interferences that could adversely affect the accuracy, precision, selectivity and specificity of the validated method. When the suppression levels are different between the calibration samples and the study samples, significant assay variability/bias could be introduced by this differential matrix effect [6]. While the current FDA Guidance on Bioanalytical Method Validation requires the absence of interfering matrix effect to be demonstrated to ensure that precision, selectivity, and sensitivity would not be compromised [2], there are no procedural details provided in the guidance. Hartmann et al. [12] have suggested from statistical considerations that relatively rare interference will remain undetected with high probability using at least five different sources of blank matrix. However, in the 2000 Conference report [4], it was suggested that even one source was deemed acceptable if LC/ MS or other hyphenated mass spectrometric methods are used. While the number of donor sources used may vary from laboratory to laboratory, many bioanalytical laboratories demonstrate the absence of significant matrix effect by testing spiked quality control (QC) samples at the lower limit of quantification (LLOQ) and high quality control (QC) concentration level using at least six different lots of donor s blanks and assay them against calibration standards similarly prepared with preestablished acceptance criteria for evaluation. Examples of acceptance criteria are provided as follows: 1) coefficient of variance (CV) of the QC results should not be more than720% and 2) the CV of the slopes of the calibration curves generated using each of the matrix lots should not be more than 15%. While this approach provides a good indication of the presence or absence of matrix effect, many argued that the results from testing these six blank lots may not be statistically sufficient to provide a high degree of assurance to support the conclusion of the absence of matrix effect for testing of samples from hundreds of subjects. Furthermore, this holistic approach can only provide the extent of relative but not absolute matrix effect [5,9]. The variability of the assay results observed can in fact be due to a combination of the variability of extraction recovery and matrix effect. Nevertheless, the above approach has successfully provided supporting evidence that the probability of the LC/MS bioanalytical method to have significant matrix effect related problems could be minimal. Matuszewski et al. has suggested that the matrix effect and its impact can be minimized through the inherent design of the bioanalytical method using the following approaches [5]: 1) changing and improving the sample extraction procedure and by eliminating undetected interferences; 2) performing the assay under more efficient chromatographic conditions; 3) evaluating and changing the HPLC-MS interface and the mechanism of ionization of analytes; 4) use of pure, stable isotope-labeled internal standards to ensure relative ionization efficiency of the drug and its internal standard would not be affected by the matrix effect. The argument is that the reduction of the potential occurrence of matrix effect may minimize the need for rigorous validation. Although the determination of absolute matrix effect is not required to establish the method validity, the extent of the absolute matrix effect should be fully investigated during method development to ensure the ruggedness of the method during testing of subject specimen samples. Furthermore, this information could prove to be valuable in the investigations of aberrant data, which may arise in the subsequent production runs. If the absolute matrix effect were large, one would expect higher assay variability during actual testing of hundreds of subject samples during a production run than the testing of a limited number of donor lots in validation. A review of the literature indicated that the absolute matrix effects could be determined using various experimental designs including post-column infusion [5]. These experiments should be performed during method development to obtain a full understanding of the level of

4 Current Challenges for FDA-Regulated Bioanalytical Laboratories 25 matrix effect to facilitate the design of the approach used during method validation. The major challenge facing most bioanalytical laboratories is not how to demonstrate the absence of significant matrix effect for a newly developed method, but is defending the absence of significant matrix effect for a previously validated method with no formal method validation data to support such a claim, particularly when the accuracy and validity of the bioanalytical method is being challenged during FDA inspection. There are two alternative approaches to address such challenges: 1) Perform additional experiments designed to demonstrate the absence of significant matrix effect as described above [5] and 2) Perform retrospective review of previous method validation data and all study data from various BA/ BE studies using this bioanalytical method. Examples of the latter approach used to generate supporting data during retrospective review to support the absence of significant matrix effect include the following: 1. If the internal standard used is a deuterated or otherwise labeled compound of the analyte and each control and subject sample is spiked with the internal standard during sample preparation, the laboratory can evaluate the response of the internal standards for the entire study to determine if excessive variability is observed. If variability is not excessive and is within the accuracy and precision of the method, then one could extrapolate this data to support the absence of significant matrix effect of the analyte since the chemical structure is essentially identical. The potential issues with this approach are as follows: 1) If the internal standard is added prior to the extraction of the sample, the variability observed could be due to the extraction recovery process. 2) The level of internal standard is generally much higher than the LLOQ used for the analyte and the matrix effect at the LLOQ level cannot be demonstrated by this approach. 3) The deuterated or otherwise labeled compound may be more labile than the analyte. 2. Collective review of all control sample data (QC and Calibration Standards) to determine if excessive variability of the analyte response from LLOQ to high QC level is observed. The major problem associated with this approach is the fact that most bioanalytical laboratories prepare the control samples using a pool of blank biological fluids made up of a number of individual donors and the extent of matrix effect from each individual blank lot could be masked. Therefore, one may need to further evaluate the relative response of calibration standards and quality control samples prepared using different blank matrix pool lots to demonstrate the absence of significant matrix effect. 3. Review method development and literature data to support the absence of matrix effect. However, since the level of matrix effect is dependent on the type of ionization mode (e.g. electrospray or API), the extraction methods, the configuration of LC/MS interface, the HPLC column and conditions used etc., any changes made to the method after it was initially developed could significantly change the level of matrix effect. Stability studies According to the current FDA Guidance and various publications [2 4,13,14], drug stability in the biological matrix must be positively evaluated, established and validated. While the use of freshly prepared stock solution of the analyte in the appropriate analyte-free, interference-free biological matrix is recommended in the May 2001 FDA Guidance for Bioanalytical Method Validation (Section D), many bioanalytical laboratories were cited by the FDA for using frozen standards/qc samples instead of freshly prepared standards/ quality samples in their stability studies.

5 26 F. Chow et al. The rationale of using frozen standard/qc samples is supported by the practices of many bioanalytical laboratories in preparing QC samples and calibration standards in bulk and storing them with study subject specimens in the freezer. This practice is based on the assumption that if the subject samples were to degrade, the control and calibration standards would degrade at the same rate and therefore the accuracy and precision of the results generated would not be affected since the area ratio of the analyte to the internal standard would remain the same. In other words, the laboratory only generates data for relative stability but not absolute stability. Nevertheless, there are some uncertainties associated with this assumption, namely 1) the bulk calibration standards and QC samples may not be prepared at the same time and stored under the identical conditions as the subject samples; 2) the rate of degradation of the spiked control samples and calibration standards that typically contain both organic and aqueous spiked solvents could be different than that of the subject samples which typically do not contain organic solvent; 3) if the degradation is extensive, it could lead to loss of sensitivity of the method in detecting analytes at the LLOQ level. Due to the potential uncertainties described above, most bioanalytical laboratories are now using freshly prepared calibration standards and control samples in generating absolute stability data except for Processed Sample Integrity/Stability or Reconstituted Sample Stability, which are discussed below. In typical day-to-day operations, most commercial bioanalytical laboratories prepare/ process a large number of subject samples in LC vials, store them in refrigerators and analyze them when the instrument is available. In order to support this staging duration, stability data for Processed Sample Integrity/Stability or Reconstituted Sample Stability would need to be generated. The common approach to generate these types of stability data is to inject the set of spiked calibration standards and QC samples after extraction and reconstitution and re-inject the same set again (or back-up set) at a later date. If the calibration curves and QC samples for both runs meet acceptance criteria, then the processed samples will be considered to be stable for this staging duration. However, many laboratories went a step further to utilize these Process Sample Integrity data to support the FDA recommended Post Preparation Stability (PPS), or what is commonly known in the industry as Autosampler Stability without additional studies. It should be noted that the generation of Post Preparation Stability data requires the use of freshly prepared standards (Section D 5 of the FDA Guidance). In order to use the Processed Sample Integrity data to support the Post Preparation Stability claim, the following conditions should be met: 1) The staging temperature of the processed sample pending injection is identical to the temperature of the autosampler s and, 2) the internal standard used in the sample solution will degrade at the same rate as the drug analyte, and if deuterated or otherwise labeled internal standard is used, there would not be any significant exchange reaction taking place, since in both cases, the resulting peak area ratios would not be constant. While the 2001 FDA Guidance allows partial validation to be performed as a result of any changes made to the validated method (Section B), the extent of the partial validation required is not defined in the guidance. Many bioanalytical laboratories would consider analyte stability an inherent property of the analyte and therefore would not need to be repeated when a bioanalytical method is changed, for example, from HPLC/UV to LC/MS/MS. This assumption is only valid if the laboratories have the data to show that the HPLC/UV method has the same level of specificity as the LC/MS/ MS method. In other words, the laboratory should have a high level of certainty that if degraded samples were tested by both methods, similar assay results would be obtained. While the need to demonstrate Stock Solution Stability is required by the FDA Guidance (Section D 4), using freshly prepared solutions, the detailed procedure and approach were not

6 Current Challenges for FDA-Regulated Bioanalytical Laboratories 27 provided. Many laboratories only demonstrate the stock solution stability at the highest concentration and if the internal standard used were the deuterated/labeled compound of the analyte, the solution stability of the internal standard would not be demonstrated. This approach creates the following potential problems: 1) Since the rate of degradation could be concentration-dependent, the stock solution stability should be performed using the solution with lowest and highest concentration of analyte; 2) even if the deuterated/labeled compound of the analyte is used as internal standard, the assumption that the chemical degradation would be the same may not be true. Moreover, if the isotope labeled internal standard is not stable, degradation could also occur as a result of proton exchange or other reaction mechanisms. Therefore, additional experiments may need to be performed to demonstrate the stability of deuterated/labeled internal standard solutions even though the chemical structure is the same as the drug analyte. Recovery According to the current FDA Guidance [2], the recovery of an analyte is defined as the extraction efficiency of an analytical process, reported as a percentage of the known amount of an analyte carried through the sample extraction and processing steps of the method. It is generally determined by comparing the detector response obtained from an amount of the analyte added to and extracted from the biological matrix to the detector response obtained for the true concentration of the pure authentic standards. Due to the complex nature of the biological matrix, 100% recovery is generally not expected. The method is considered suitable provided that the recovery data of the analyte and internal standard can be demonstrated to be consistent, precise and reproducible. The recovery also should not be concentration-dependent and, therefore, recovery experiments should be performed by comparing the analytical results for extracted spiked samples at different concentrations (typically low, medium and high, covering the expected range of the test samples) with unextracted standards that represents 100% recovery. However, as discussed under the section Matrix Effect, the results of recovery determined are a combined effect of recovery and ion suppression/enhancement due to matrix effect. The absolute recovery of the method cannot be determined unless the absolute matrix effect is determined. While the current FDA Guidance [2] requires that the recovery of both the drug analyte and the internal standard must be validated, many bioanalytical laboratories would not perform recovery validation for an isotope-labeled internal standard. This decision was made based on the assumption that since the chemical structures are the same, the recovery would be the same. However, as indicated earlier, if the labeled isotope internal standard is not stable, the integrity of the internal standard during sample processing could be questionable. This issue needs to be carefully investigated during method development and/ or validation. The use of on-line extraction systems to improve the sample processing efficiency is a very popular approach among the bioanalytical laboratories. While the determination of recovery/matrix effect with off-line extraction LC/MS/MS methods is a relatively simple process, it cannot be performed as easily when the method involves an on-line automated extraction system (e.g. Cohesive Turbulent Flow System) using a multi-column switching approach [15]. When this type of on-line system is used, there is no guarantee that the reference solution to establish 100% recovery is fully retained by the on-line extraction columns. In order to obtain accurate recovery data for these on-line systems, the experimental design could be customized as described in a poster publication by Magellan Laboratories [15]. Due to this difficulty in validating recovery for these on-line extraction methods, many past on-line extraction methods were developed and validated without addressing the recovery issue. As

7 28 F. Chow et al. a result, this issue has been raised during the FDA reviews and inspections. In order to demonstrate the accuracy and validity of the test data retrospectively when the validation data for recovery is lacking, the following approaches can be considered: 1. Collectively evaluating the consistency of the internal standard peak response for all runs to ensure they are within the inherent variability of the method. This could serve as indirect evidence that the recovery of the method is relatively consistent. 2. Similarly, collectively evaluating the consistency of the drug analyte response of the known control samples and calibration standards to ensure they are within the inherent variability of the method. 3. Collectively evaluating the peak response for the known LLOQ samples to ensure the sensitivity of the method was not compromised due to loss of recovery. This retrospective type of review would form the scientific basis to support the accuracy, precision and recovery of the method and therefore that it is suitable for its intended use. 1. The method must be thoroughly developed and optimized prior to validation. The method development data/records should be properly documented to support method validation and future method changes/ improvement/investigation. 2. A proper document change control system/ Standard Operating Procedure (SOP) must be in place. 3. All method validation work should be driven by written methods and written protocols, properly reviewed by QA and approved by laboratory management. This will provide a good link between the method used and the supporting method validation report. All reports and methods must be version controlled. 4. Any subsequent changes to the method must be managed by the proper change control procedure and the level of partial validation should be defined, justified and documented. 5. After partial validation and re-validation, new versions of the method validation report should be issued and properly linked to the new version of the method in use. Method development and method validation In many bioanalytical laboratories, the line between method development, method optimization and method validation is poorly defined. As a result, method development/optimization work can be performed during method validations or even during testing of study subject samples in production runs. This situation can be further complicated by the lack of a formal document change control program to monitor, track and document all these changes and additional information generated. Therefore, this lack of adequate documentation to support the validity of the bioanalytical method currently in use could become an FDA inspection issue. To avoid this potential compliance issue, the following quality system procedures need to be installed: Changes of regression model without formal validation The 2000 conference paper and the current FDA Guidance [2,4] suggested a sufficient number of standards to define adequately the relationship between concentration and response. It further stated at least five to eight concentration levels should be used for linear and maybe more for non-linear relationships. The Guidance also requires the use of the simplest regression model that adequately describes the concentration-response relationship and that the selection of weighting and use of complex regression equation should be justified. However, the Guidance does not provide any information on how many replicates should be analyzed for each level. Peter et al. have summarized the recommendations of

8 Current Challenges for FDA-Regulated Bioanalytical Laboratories 29 various workers on the use of various numbers of concentration levels and replicates ranging from four to eight concentration levels and three to nine replicates at each concentration level [16]. It was also suggested that the use of more replicates would allow the reliable detection of outliers and also a better evaluation of variance across the calibration range, which in turn facilitates the selection of the right statistical model for the evaluation of the calibration curve. While the need to support the use of regression type is clearly stated in the FDA guidance, the rationale to support the selection of regression type is not always documented in the method validation report and could result in an FDA 483 observation. In some bioanalytical laboratories, the validated regression type was changed during testing study subject samples without formal revalidation and simply justified by the fact that the resulting calibration curves are able to meet the acceptance criteria. While this argument may be scientifically justifiable, it could present a serious compliance issue during FDA inspection for the following reasons: 1. The laboratory did not perform adequate investigation to determine why the validated regression type failed in these study runs. 2. The laboratory did not follow the SOP to perform partial validation or to justify why partial validation was not required. 3. The laboratory failed to document this deviation. Conclusion This three-part series has provided a comprehensive review of various regulatory compliance and technical issues related to bioanalytical laboratories performing human BE/BA studies. The first article provides a detailed discussion on current FDA regulations applicable to the bioanalytical laboratories performing human BE/BA studies. It is the authors opinions, supported by recent FDA enforcement actions, that GLP regulations (designed to support animal studies) appear to be the minimum requirements for these laboratories. Other quality systems as required in GMP operation might need to be considered. The second article provides a detailed discussion and analysis of recent FDA inspection trends for bioanalytical laboratories performing human BE/BA studies. It is apparent from the second article that FDA enforcement actions against well-established bioanalytical laboratories have recently intensified and, as a result, many bioanalytical laboratories are in the process of enhancing their systems, procedures and controls to assure continued compliance. These articles provide additional technical discussion on selected method validation issues which, if not handled properly, could complicate the FDA inspection process and create significant compliance situations. References 1. Food and Drug Administration. Bioavailability and Bioequivalence Requirements, 21 CFR Part 320 (2006). 2. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Veterinary Medicine. Guidance for Industry: Bioanalytical Method Validation; May Shah VP, Midha KK, Dighe S, McGilveray IJ, Skelly JP, Yacobi A et al. Analytical methods validation: Bioavailability, bioequivalency, and pharmacokinetics studies. J Pharm Sci 1992; 81: Shah VP, Midha KK, Findlay JW, Hill HM, Hulse JD, McGilveray IJ et al. Bioanalytical method validation: A revisit with a decade of progress. Pharm Res 2000; 17: Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategy for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem 2003; 75: Avery MJ. Quantitative characterization of differential ion suppression on liquid chromatographic/

9 30 F. Chow et al. atmospheric pressure ionization mass spectrometric bioanalytical methods. Rapid Commun Mass Spectrom 2003; 17: Georgi K, Boos K-S. Control of matrix effects in bioanalytical MS-MS using on-line multidimensional solid phase extractions. LC/GC North Amer 2005; 23(4): King R, Bonfiglio R, Fernandez-Metler C, Miller-Stein C, Olah T. Mechanistic investigation of ionization suppression in electrospray ionization. J Am Soc Mass Spectrom 2000; 11: Weng N, Halls TDJ. Systematic troubleshooting for LC/MS/MS. Pharm Tech 2001; James CA, Breda M, Frigerio E. Bioanalytical method validation: A risk-based approach? J Pharm Biomed Anal 2004; 35(4): Karns HT. Validation and control of quantitative bioanalytical LC/MS methods. Amer Pharm Rev 2003; 6(1): Hartmann C, Smeyers-Verbeke J, Massart DL, McDowall RD. Validation of bioanalytical chromatographic methods. J Pharm Biomed Anal 1998; 17(2): Timm U, Wall M, Dell D. A new approach for dealing with the stability of drugs in biological fluids. J Pharm Sci 1985; 74(8): Kringle R, Hoffman D, Newton J, Burton R. Statistical methods for assessing stability of compounds in whole blood for clinical bioanalysis. Drug Info J 2001; 35: Niggebrugge A, Parker D, Ford L, Chilton A. A method for the determination of recovery and matrix effect in cohesive turbulent flow LC/MS online extraction systems. Poster presented at ASMS 2002 (American Society for Mass Spectrometry). 16. Peters FT, Maurer HH. Review: Bioanalytical Method Validation-How, how much, and why. Toxichem 1Krimtech 2001; 68(3): 116.