Bioanalytical Chemistry Methods: Development and Validation
Duane B. Lakings, Ph.D., Principal, DSE Consulting
A lead candidate has been selected for preclinical development and definitive pharmacokinetic and toxicology studies are scheduled to begin. To support these research efforts, a bioanalytical chemistry method is required. First this method needs to be developed and then validated for each matrix and each species to be used in the preclinical research studies.
The purpose of this DSE White Paper is to provide information on the techniques, requirements, and documentation needed to successfully validate a bioanalytical chemistry method for the quantification of a drug candidate in a physiological matrix. These validation experiments may uncover concerns that need to be resolved before beginning the definitive preclinical pharmacokinetic and toxicology studies necessary to support an IND filing and later a NDA submission. Identifying and correcting these concerns as early as possible allows for a more timely and data productive drug development program.
Developing the Bioanalytical Chemistry Method
If not already defined as part of the lead developability assessment program, a bioanalytical chemistry method needs to be developed and characterized for the quantification of the drug candidate in physiological fluid matrices. This method should be available prior to the initiation of definitive preclinical pharmacokinetic and toxicology studies and can also support experiments in pharmacology, drug delivery, and drug metabolism. The first step in developing a bioanalytical chemistry method is to select the analytical technique. For a drug candidate with a molecular weight of <2000, instrumental methods such as HPLC or GC, with a variety of possible detectors such as UV, florescence, FID, ECD, NPD, MS, may be employed. Selection of columns, eluents, instrumental conditions that can resolve the drug candidate from potential interfering substances is an important consideration to be addressed. Also, the detection system should have sufficient sensitivity to be able to quantify the drug candidate at the low concentrations expected in physiological fluid specimens. A macromolecule (large peptide, protein, or oligonucleotide) may require an ELISA or RIA method. Selecting the appropriate antibodies for capture and detection and the detection system to be employed should be defined at this stage. Samples in assay diluent and in a physiological matrix and fortified over a large concentration range are assayed to assess the ability of a technique to produce an appropriate signal to detect the drug candidate and to determine the potential interference caused by the matrix. Also important during method development is the definition and evaluation of sample preparation procedures for isolating the drug candidate from matrix components.
The ability to quantify a drug candidate in a physiological fluid commonly depends on the matrix. For example, serum is a poor choice when a compound interacts with clotting factors. Freshly obtained blood samples, fortified with known amounts of the candidate, are processed to obtain plasma or serum, and then aliquots of each fluid are assayed. If the amount of candidate found is less than the amount added to the blood, the compound may be associating with the cellular material. Though, if the amount found is 1 to 2 times the amount added to blood, the compound has a relatively uniform distribution in blood and is apparently not adversely affected by the processing procedure. However, if the amount found is greater than 3 times the amount added, the experimental design needs to be checked. The matrix that gives the best recovery and has the least interference should be selected.
Validating the Bioanalytical Chemistry Method
Once a bioanalytical chemistry method has been developed, the next step is validation, according to guidelines listed by ICH, for each matrix (plasma, urine, bile) and species (rodent, dog, non-human primate, human) to which the method is to be applied. Validation experiments include assessments of specificity, linear range or range of reliable results (RRR), lower (LLQ) and in some cases upper (ULQ) quantification limits, precision, accuracy, absolute recovery, and stability. The procedures and tests, including acceptance and rejection criteria, to be used in the validation experiments are frequently defined and documented in a validation protocol prior to the actual experiments. Normally, assay validation is completed prior to the initiation of analyses of specimens and reflects how collected specimens are to be processed (the procedure used in sample preparation), the criteria for standards and quality control (QC) samples, and the technique employed to generate the standard curve and to calculate the concentrations of the drug candidate in QC samples and specimens.
Specificity
The specificity is assessed by evaluating the potential interferences from physiological matrix components from the different animal species. Samples, commonly five or more, from each species are analyzed neat (with no added compound) and fortified with known amounts of the drug candidate, and the results calculated using a standard curve prepared in assay diluent. The signals obtained from the neat samples indicate the level of interference from each matrix type and the calculated amounts in the fortified samples show the difference in absolute recovery from the matrix compared with the compound in buffer. Normal acceptance criteria for specificity are that the levels of interfering components present in the blank matrix are not greater than 20% of the lowest standard. If necessary, slight adjustments in the instrumental parameters may resolve the drug candidate from interfering matrix components.
Range of Reliable Results
The RRR is the standard curve range over which the detector response to the drug candidate in prepared samples can be reliably defined by an appropriate mathematical function (linear, quadratic, sigmoidal, etc.). The RRR is usually determined using at least six concentrations prepared in at least duplicate or a minimum of 12 unique concentrations. The lowest and highest concentrations in the RRR are, by definition, the lower (LLQ) and upper limit of quantification (ULQ), respectively. Extrapolation of the curve above the ULQ or below LLQ is not permitted when analyzing specimens to determine the concentration of the drug candidate.
Lower Limit of Quantification
LLQ is the lowest concentration that can be measured with a definite level of certainty. LLQ selection for validation may be based on: 1) an estimate of the limit of detection (LOD) obtained during method development, 2) the requirements of the experiment for which the assay is being validated, and/or 3) historical data on the characteristics of the drug candidate. The minimal requirements for assessing LLQ in physiological matrices include evaluations of both accuracy and reliability. Samples of matrix are obtained from at least five independent sources. Each sample is analyzed neat and fortified with the drug candidate at the projected LLQ. Fortification above and below the projected LLQ is recommended for those methods without historical data or where the projected LLQ may not be a reliable selection. The observed concentrations are determined from the standard curve, regardless of whether the result is above or below the LLQ. The accuracy at LLQ can be determined as the percent deviation (%Dev) of the calculated value (Found) to the theoretical value (Added) (%Dev = [(Found - Added) ÷ (Added)] x 100). The reliability of LLQ can be evaluated by comparing the observed concentrations in the five blank matrices with the observed concentrations in same matrices fortified at the projected LLQ. Using a t-test, or other appropriate statistical test, the results from the neat and fortified matrices are compared to determine the concentration that is statistically different from the blanks. The LLQ is a combination of the accuracy and reliability results. The fortified concentration that is statistically different from the blanks and has a %Dev of less than 20% is considered an acceptable definition of the LLQ.
Upper Limit of Quantification
ULQ is the highest concentration that can be determined reliably. For most analytical methods, ULQ is defined as the highest standard in the standard curve. However, ULQ may be evaluated for accuracy and reliability. For example, most immunological methods do not have a linear response over the standard curve range. In these cases, evaluation of both LLQ and ULQ may be considered necessary. The method of ULQ determination is similar to the techniques used to evaluate LLQ.
Accuracy and Precision
The accuracy and precision determine how close the measured concentration of a drug candidate is to a known amount and the variability of the measured concentration, respectively. The minimum requirements for evaluation of the accuracy and precision consist of the following. A minimum of three bulk, control samples of the specific matrix are prepared containing low (first quartile of the standard range), medium (second or third quartile), and high (fourth quartile) concentrations of the drug candidate. At least triplicate aliquots from each bulk control sample are analyzed on each of at least three separate days and the concentration of the drug candidate determined using the standard curves prepared and analyzed on each of the separate days. Accuracy is determined by calculating the percent relative recovery (% R) of found concentrations for each control sample level on the second and third validation days to the average found concentration, defined as 100%, for that same level on the first validation day. Normal acceptance criteria for accuracy are that the % R falls within ±15% of the day one validation results. The control samples are also used for determining the precision. On each validation day, the results for the at least triplicate determination of each control sample level are used to assess intra-day precision by calculating the averages, standard deviations (SD), and relative standard deviations (RSD). The RSD ranges define the intra-day precision with normal acceptance criteria being that the RSDs are less than 15%. Inter-day precision is assessed from the same analytical results by calculating a pooled average, SD, and RSD for the control sample levels assayed on the different validation days. Normal acceptance criteria for inter-precision day are that the pooled RSDs are less than 15%.
Absolute Recovery
The recovery of the drug candidate from the matrix during sample preparation needs to be defined so that reliability of the method can be ascertained. The absolute recovery can be assessed by a number of techniques. For example, the control samples used in accuracy and precision definition and calibration standards of the drug candidate and the internal standard (IS), if appropriate, at the same concentrations and prepared in an appropriate solvent for analysis are assayed and the absolute responses compared. The absolute recovery is the response for the drug candidate or IS in matrix divided by the response of the drug candidate or IS in solvent. If necessary the responses can be normalized for slight differences in concentration. Another possibility consists of preparing a standard curve in both matrix and an appropriate solvent. The difference in slopes for the two standard curves is a measure of the absolute recovery. Normal acceptance criteria for absolute recovery are that the recovery is uniform (not greater than ±15%) over the concentration range to be evaluated and is greater than 60%.
Stability
The ability to measure a drug candidate in a physiological matrix is not useful if the compound is unstable during collection, processing, storage, or sample preparation. The stability of a drug candidate in a matrix determines how long a specimen can be stored under specified conditions before bioanalytical results are considered unacceptable because of compound degradation. Stability studies are conducted to ensure that the drug candidate does not degrade in blood, during processing to obtain plasma or serum, during the time (hours, days, and weeks) and under the conditions (room temperature, refrigerated, frozen at -20˚C or -80˚C) that specimens may be stored until analyzed, and during sample preparation (dilution, liquidliquid or liquid-solid extraction, derivatization). The results ensure that measured concentrations in specimens reflect the amount of compound present at the time of collection. The following sections describe the minimum requirements to assess the stability of a drug candidate during assay validation.
Freeze-Thaw Cycle Stability
For specimens to be stored frozen from the time of collection to the time analysis, stability of the drug candidate to freezing and thawing is evaluated to ensure the compound does not degrade during these processes. The control samples prepared for accuracy and precision evaluations can be used to evaluate freeze-thaw cycle stability. Normally, at least two concentrations are evaluated and a minimum of three freeze-thaw cycles is studied. Normal acceptance criteria are that the relative recovery of the drug candidate is at least 90% after each cycle.
Ambient Temperature Stability
Specimens are maintained at room temperature from the time of collection until placed under appropriate storage conditions and after removal from storage until the time of sample preparation and analysis. Stability of the drug candidate under ambient temperature conditions is evaluated to ensure the compound does not degrade under these conditions. Again, the control samples prepared for accuracy and precision evaluations can be employed. Normally, at least two concentrations are evaluated and samples are stored at the desired temperature for a minimum of 4 hours with sampling at hourly intervals. Normal acceptance criteria are that the relative recovery of the analyte is at least 90% after 4 hours of storage.
Storage Stability
Specimens are normally stored frozen from the time of collection until the time of sample preparation and analysis. Stability of the drug candidate under storage conditions is evaluated to ensure the compound does not degrade under these conditions. As before, the control samples prepared for accuracy and precision evaluations can be used. Normally, at least three concentrations and two storage temperatures are evaluated. Control samples are stored at the desired temperatures for a minimum of 28 days with sampling at various intervals, for example 1, 2, 4, 7, 11, 14, 21, and 28 days. Normal acceptance criteria are that the relative recovery of the analyte is at least 90% of the initial concentration.
Chemistry Assay Method
The generated results from the validation experiments are used to prepare a detailed bioanalytical method assay procedure, which describes the reagents and materials; instruments and instrumental conditions; preparation of standards, QC samples, and specimens; calculations; and acceptance and rejection criteria
Reagents and Materials
The reagents and materials, including the drug candidate and internal standard (IS), if appropriate, are listed along with any information on the quality and source, if appropriate, of the items that are considered necessary for the successful conduct of the method. If appropriate, ‘or equivalent’ is commonly listed for items where substitution of another item of equal or superior quality may be used.
Instruments and Apparatus and Instrumental Conditions
The instruments and apparatus required for the performance of the method are listed along with any other information, such as specific makes or models of equipment, considered necessary for the successful conduct of the method. If appropriate, ‘or equivalent’ is listed for items where substitution of another item of equal or superior quality may be used.
If specific instrumental conditions are required for conducting the method, the conditions are listed in sufficient detail to guide the analyst in setting up and operating the instrument(s). For example, a method employing HPLC may have instrumental conditions listed on the column, guard column, eluent composition and preparation, flow rate, injection volume, detector, and retention times or volumes of the drug candidate and IS, if appropriate. Information on instrumental conditions considered critical for the success of the method is to be noted.
Preparation of Standards, QC Samples, and Specimens
Information on the preparation of standards, QC samples, if appropriate for the method, and specimens is provided in sufficient detail to guide the analyst in the sample preparation procedures. This information includes, where appropriate, detailed examples which list volumes of reagents, concentrations of standards, aliquots of unknowns, and conditions such as centrifuge speed and time, drying temperature and gas, filtration steps, etc. Information on standard and sample preparation procedures considered critical for the success of the method is to be noted.
Calculations
The calculations required to determine the concentration of the drug candidate in QC samples, if appropriate, and specimens are listed. These calculations may include, but are not limited to, standard curve generation and QC and specimen drug candidate concentration determination. If appropriate, information, such as chromatographic peak shape requirements, peak area ranges for acceptance, and interfering peak evaluation, on the techniques used to evaluate the suitability of the results for calculation can be included.
Acceptance and Rejection Criteria
The acceptance and rejection criteria, determined during assay validation, are listed and used by the analyst to determine if the results for standards, QC samples, and specimens are within the predefined criteria. This section is also to provide guidance to the analyst in determining the acceptability of the entire analytical run and on the evaluation or designation of potential degradation products or metabolites of the drug candidate.
Conclusion
Successful completion of the above experiments will validate and document in a bioanalytical chemistry assay method for use in evaluating the pharmacokinetics or toxicokinetics of a drug candidate in animal models and can provide necessary information for the later validation of the method in human physiological matrix specimens.
