Pre-study Validation Research Articles

Quantification of OATP1B1 endogenous metabolites coproporphyrin I and III in human urine.

Coproporphyrin (CP)-I and CP-III are the markers of organic anion-transporting polypeptides' (OATPs) activities, and they are porphyrin metabolites that originate from heme synthesis. Furthermore, CP-I and CP-III, which are OATP1B endogenous metabolites, have gradually attracted the attention of scientists and researchers in recent years. Previous studies have also observed CP-I and CP-III levels as clinical biomarkers for predicting OATP1B inhibition in drug-drug interaction studies. To establish an accurate ultra-high performance liquid chromatography-mass spectrometry method for the quantitation of CP-I and CP-III, we reviewed previous methodological publications and applied them to a clinical pharmacology study using a human urine matrix. We used 13.25 M formic acid as a working solution for internal standards (CP-I 15N4 and CP-III d8) to avoid isobaric interference. The calibration curve showed good linearity in the range of 1-100 ng/mL, with a correlation coefficient (R2) higher than 0.996 in each validation batch. Both the between-run and within-run assays achieved good precision and accuracy, and we found that both CP-I and CP-III were stable in the pre-study validation. The method exhibited suitable dilution integrity, allowing for the re-analysis of samples with concentrations exceeding the upper limit of quantification through dilution. Overall, the application of the described method in a clinical study revealed that it can be utilized effectively to monitor drug-drug interactions mediated by OATP1B.

ODP178 Development and Validation of a Noncompetitive Immunoassay Optimized for the Assessment of Pharmacokinetics of Biologically Active Efruxifermin in Humans

Abstract Efruxifermin (EFX) is a novel fusion biotherapeutic consisting of two modified human fibroblast growth factor (FGF21) variants tethered via short linkers to a hIgG1 Fc domain. EFX is under clinical development by Akero Therapeutics, Inc. for treatment of non-alcoholic steatohepatitis (NASH). Fusion to the IgG Fc moiety extends the circulating half-life of EFX, enabling once-weekly subcutaneous administration. In addition, point mutations in the FGF21 moiety of EFX, relative to native human FGF21, protect against degradation of the C-terminus by the endogenous endopeptidase fibroblast activation protein (FAP), and increase affinity for the FGF21 co-receptor b-Klotho, which together enhance in vivo PD activity of EFX. Accordingly, to support clinical PK a sensitive and specific assay is needed for determination of biologically active (i. e., C-terminal intact) EFX serum concentrations that is minimally cross-reactive with endogenous FGF21 or potential inactive metabolites of EFX. Moreover, the assay needs to have reliability with serum samples from NASH patients, in whom hyperlipidemia and insulin resistance are prevalent. To achieve the analytical aims, we undertook development of a noncompetitive immunosorbent assay design, wherein a biotinylated version of a rat mAb directed against EFX's intact C-terminus serves as the capture reagent, while an affinity purified chicken anti-EFX pAb labeled with Sulfo-TAG functions as the detection reagent. This novel ECLIA design permits measurement of an electrochemiluminescence signal for maximizing assay sensitivity. In brief, serum samples are diluted 1: 10 in buffer and added to the MSD plate coated with rat mAb. After incubation and washing, bound EFX is detected using an affinity purified chicken anti-EFX PAB labeled with SULFO-TAG. After washing and addition of read buffer, the signal responses are measured in a SQ120 reader and concentration in test samples is estimated by dose-interpolation using a weighted 5-PL curve fit with an EFX standard curve ranging from 10–2,000 ng/mL. The new assay allows measurement of serum EFX at concentrations as low at 20 ng/mL. Upon pre-study validation, the assay has met analytical performance criteria outlined in BMV regulatory guidances. We conclude the optimized ECLIA will be useful for reliable assessment of EFX PK in patients with NASH. Presentation: No date and time listed

A simple sampling method for quantification of hazardous volatile organic compounds in mainstream cigarette smoke: Method development and prestudy validation

Cigarette smoke can be fatal to human health, as it contains a plethora of hazardous compounds such as volatile organic compounds (VOCs). To assess the toxic effects of cigarette smoke, both qualitative and quantitative information about the VOCs in cigarette smoke are essential. However, sampling and analysis of cigarette smoke are not simple tasks and require complicated validation procedures. Here, we develop and verify a simple sampling method for the quantification of VOCs in mainstream cigarette smoke using three steps: direct collection of the mainstream cigarette smoke sample by gas-tight syringe, injection of the sample into a bag sampler for dilution, and extraction of VOCs into an adsorption-tube sampler for quantitation by thermal desorption-gas chromatography-mass spectrometry. This method has been employed successfully to quantify seven target VOCs (isoprene, methyl ethyl ketone, benzene, acrylonitrile, toluene, m-xylene, and styrene) in cigarette smoke from 3R4F reference cigarettes. The quantities of these major five VOCs (isoprene, methyl ethyl ketone, benzene, acrylonitrile, and toluene: average of 459 ± 646 ng mL−1), when measured from 3R4F samples based on our method, were comparable to or moderately higher than those measured based on the established standard methods (e.g., Health Canada Intense (462 ± 557 ng mL−1) and International Standardization Organization (356 ± 438 ng mL−1)). As such, we succeeded in reliably collecting and quantifying VOCs in cigarette smoke using a simple sampling method proposed in this research. The proposed method is thus recommended as an alternative to complicated and costly representative methods based on international guidelines.

A Simple Sampling Method for Quantification of Hazardous Volatile Organic Compounds in Mainstream Cigarette Smoke: Method Development and Prestudy Validation

A Simple Sampling Method for Quantification of Hazardous Volatile Organic Compounds in Mainstream Cigarette Smoke: Method Development and Prestudy Validation

A Simple Sampling Method for Quantification of Hazardous Volatile Organic Compounds in Mainstream Cigarette Smoke: Method Development and Prestudy Validation

A Simple Sampling Method for Quantification of Hazardous Volatile Organic Compounds in Mainstream Cigarette Smoke: Method Development and Prestudy Validation

Extended Stability of Sodium Phosphate Solutions in Polyvinyl Chloride Bags.

Sodium phosphate injection is used to treat moderate to severe hypophosphatemia. There have been no published reports documenting the physical compatibility or chemical stability of sodium phosphate injection in IV solutions. To evaluate the physical compatibility and chemical stability of 30 and 150 mmol/L solutions of phosphate, prepared from sodium phosphate injection, in 5% dextrose in water (D5W) and in 0.9% sodium chloride (normal saline [NS]) and stored in polyvinyl chloride (PVC) bags at 23°C or 4°C over 63 days. On study day 0, solutions of phosphate 30 and 150 mmol/L in D5W or NS were prepared in PVC bags and stored at 4°C and 23°C. On prespecified days during the 63-day study period, the concentrations of sodium and phosphate were determined, and admixture weight was checked to assess moisture loss during storage without a plastic overwrap. Chemical stability was calculated from the intersection of the lower 95% confidence limit of the degradation rate and the lower limit of acceptability (90%) for concentration remaining. The analytical methods for both sodium and phosphate were found to be precise (coefficient of variation averaging less than 1% for pre-study validation samples). Both sodium and phosphate retained more than 94% of the initial concentration over the 63-day study period. With 95% confidence, the time to achieve 90% of the initial concentration of both sodium and phosphate approached or exceeded the 63-day study period, regardless of temperature, concentration, or base solution. Sodium phosphate solutions at a phosphate concentration of 30 or 150 mmol/L in either NS or D5W retained more than 94% of the initial concentration of both sodium and phosphate over 63 days when stored at 23°C or 4°C. In compliance with United States Pharmacopeia General Chapter <797> recommendations, a beyond-use date of 14 days (with refrigeration) or 48 h (room temperature) may be applied. Extending the beyond-use date beyond these limits may be considered, if a validated sterility test is performed.

Wide Spread use of LC-MS in Bioequivalence Studies

Submit Manuscript | http://medcraveonline.com FDA guidance on analytical method validation, in one form or another, has been adopted universally as a standard procedure for validating bioanalytical assays used for pharmcokinetic, bioavailability, and bioequivalence studies intended for regulatory submission [2]. Before the first bioanalytical method validation workshop, there was lack of uniformity in conducting validation of bioanalytical methods and submission of data to regulatory agencies. The bioanalytical validation workshop in 1990 was the first major workshop dedicated to investigating and harmonizing procedures in method validation. The workshop was cosponsored by the American Association of Pharmaceutical Scientists (AAPS), the US FDA, the International Pharmaceutical Federation (FIP), the Health Protection Branch (HPB), and the Association of Analytical Chemists (AOAC). The conference focused on the requirements for bioanalytical method validation procedures to establish reliability of the analytical method, parameters to ensure acceptability of analytical method performance, method development (prestudy validation), and method application (instudy validation). The workshop defined important parameters for method validation e.g. accuracy, precision, selectivity, sensitivity, reproducibility, limit of quantification as well as addressing “how to” evaluate and determine these parameters. It was also clarified that it is not essential to have 100% recovery, but it is important that the recovery be reproducible. One of the most important outcomes of the first workshop was that it defined “the acceptance criteria for a run”. The recommendations of the first workshop were well received within the scientific community. These recommendations did not become did not become official until the draft guidance on bioanalytical method validation was published in January 1999 by the US FDA with the intention of seeking public opinion prior to finalizing the guidance. The second bioanalytical method validation workshop was cosponsored by the AAPS and the US FDA in January 2000 a year after the draft guidance went into effect and ten years after the first workshop took place. The main advances occurred in the field of mass spectrometry. Two issues were addressed in relation to LC-MS;

Development of an ultra high performance liquid chromatography method for determining triamcinolone acetonide in hydrogels using the design of experiments/design space strategy in combination with process capability index.

An ultra high performance liquid chromatography method was developed and validated for the quantitation of triamcinolone acetonide in an injectable ophthalmic hydrogel to determine the contribution of analytical method error in the content uniformity measurement. During the development phase, the design of experiments/design space strategy was used. For this, the free R-program was used as a commercial software alternative, a fast efficient tool for data analysis. The process capability index was used to find the permitted level of variation for each factor and to define the design space. All these aspects were analyzed and discussed under different experimental conditions by the Monte Carlo simulation method. Second, a pre-study validation procedure was performed in accordance with the International Conference on Harmonization guidelines. The validated method was applied for the determination of uniformity of dosage units and the reasons for variability (inhomogeneity and the analytical method error) were analyzed based on the overall uncertainty.

Pre-study and in-study validation of a size-exclusion chromatography method with different detection modes for the analysis of monoclonal antibody aggregates.

Size exclusion chromatography (SEC) with different detection modes was assessed as a means to characterize the type of bevacizumab aggregate that forms under thermal stress, quantitatively monitoring the aggregation kinetics. The combination of SEC with light-scattering (SEC/LS) detection was validated using in-study validation process. This was performed by applying a strategy based on a control chart to monitor the process parameters and by inserting quality control samples in routine runs. The SEC coupled with a differential refractive-index detector (SEC/RI) was validated using a pre-study validation process in accordance with the ICH-Q2 (R1) guidelines and in-study monitoring in accordance with the Analytical Target Profile (ATP) criteria. The total error and β-expectation tolerance interval rules were used to assess method suitability and control the risk of incorrectly accepting unsuitable analytical methods. The aggregation kinetics data were interpreted using a modified Lumry-Eyring model. The true order of the reaction was determined using the initial-rate approach. All the kinetic data show a linear Arrhenius dependence within the studied temperature range. The Arrhenius approach over-predicted the aggregation rate for 5°C, but provides an idea of the aggregation process and amount of aggregate formed. In any case, real-time stability data are necessary to establish the product shelf-life.

Stability assessment in ligand-binding assays: a critical parameter for data integrity.

Stability assessment in ligand-binding assays: a critical parameter for data integrity.

Statistical approaches for the determination of cut points in anti-drug antibody bioassays

Cut points in immunogenicity assays are used to classify future specimens into anti-drug antibody (ADA) positive or negative. To determine a cut point during pre-study validation, drug-naive specimens are often analyzed on multiple microtiter plates taking sources of future variability into account, such as runs, days, analysts, gender, drug-spiked and the biological variability of un-spiked specimens themselves. Five phenomena may complicate the statistical cut point estimation: i) drug-naive specimens may contain already ADA-positives or lead to signals that erroneously appear to be ADA-positive, ii) mean differences between plates may remain after normalization of observations by negative control means, iii) experimental designs may contain several factors in a crossed or hierarchical structure, iv) low sample sizes in such complex designs lead to low power for pre-tests on distribution, outliers and variance structure, and v) the choice between normal and log-normal distribution has a serious impact on the cut point.We discuss statistical approaches to account for these complex data: i) mixture models, which can be used to analyze sets of specimens containing an unknown, possibly larger proportion of ADA-positive specimens, ii) random effects models, followed by the estimation of prediction intervals, which provide cut points while accounting for several factors, and iii) diagnostic plots, which allow the post hoc assessment of model assumptions. All methods discussed are available in the corresponding R add-on package mixADA.

Large molecule run acceptance: Recommendation for best practices and harmonization from the Global Bioanalysis Consortium Harmonization Team.

The L1 Global Harmonization Team provides recommendations specifically for run acceptance of ligand binding methods used in bioanalysis of macromolecules in support of pharmacokinetics. The team focused on standard curve calibrators and quality controls for use in both pre-study validation and in-study sample analysis, including their preparation and acceptance criteria. The team also considered standard curve editing and the concept of total error.

Large molecule specific assay operation: recommendation for best practices and harmonization from the global bioanalysis consortium harmonization team.

The L2 Global Harmonization Team on large molecule specific assay operation for protein bioanalysis in support of pharmacokinetics focused on the following topics: setting up a balanced validation design, specificity testing, selectivity testing, dilutional linearity, hook effect, parallelism, and testing of robustness and ruggedness. The team additionally considered the impact of lipemia, hemolysis, and the presence of endogenous analyte on selectivity assessments as well as the occurrence of hook effect in study samples when no hook effect had been observed during pre-study validation.

Serum alpha1‐proteinase inhibitor concentrations in healthy dogs – method validation and determination of reference interval and intra‐individual variation

A chronic loss of canine α1 -proteinase inhibitor (cα1 -PI) into the gastrointestinal (GI) tract could change the systemic proteinase-proteinase inhibitor balance. Serum cα1 -PI concentrations have not been studied in dogs with well-defined GI diseases. To further evaluate serum cα1 -PI concentrations in dogs with GI diseases, the objectives of this study were to (1) analytically validate a previously developed fecal cα1 -PI immunoassay to determine serum concentrations, (2) determine a population-based reference interval (RI) and assess the clinical utility, (3) determine stability of serum cα1 -PI, (4) determine the intra-individual variation in healthy dogs, and (5) determine the clinically relevant magnitude of change of serum cα1 -PI. Prestudy validation of the (125) I-cα1 -PI immunoassay included linearity, spiking recovery, and intra- and inter-assay precision. A RI was calculated with samples of healthy dogs. Stability at -20°C was tested on 36 samples. Intra-individual variation was assessed using samples collected from 11 healthy dogs over a 12-week period. The cα1 -PI radioimmunoassay (RIA) was linear, accurate, precise, and reproducible. Serum cα1 -PI decreased by 11% after one year at -20°C. Analytical, intra-individual, inter-individual, and total variation were 6.4, 9.9, 9.0, and 25.3%, respectively. The RI for serum cα1 -PI was 732-1802 mg/L (n = 87); there were no differences between sex and age groups. The index of individuality was 1.31. As analytical variation was > ½ inter-individual variation, the minimum critical difference was not determined. The results of this study provide the basis for further evaluating serum cα1 -PI in dogs with GI disease. Using a population-based RI for serum cα1 -PI appears to be appropriate.

Practical Approaches to Incurred Sample LC-MS/MS Reanalysis: Confirming Unexpected Results

Incurred sample reanalysis (ISR) is an important step in assuring the quality of an LC-MS/MS bioanalytical assay and the integrity of bioanalysis conduct. A conventional ISR involves analysis of at least 20 samples taken from an in vivo study a second time using the method that was described in prestudy validation and employed in generating the initial study sample results. However, this practice is sometimes inadequate to confirm bioanalytical results that are unexpected. The present report discusses several additional exploratory activities that were performed to confirm the unexpected plasma concentration-time results of NVP-1, an investigational drug candidate, observed in the plasma samples collected from patients in a phase II trial. These approaches include (1) LC-MS/MS reanalysis of the study samples after multiple freeze/thaw cycles followed by a short-term benchtop storage, (2) evaluation of additional MS/MS transitions in LC-MS/MS, (3) employment of a different sample preparation procedure in LC-MS/MS, and (4) study sample dilution using plasma samples from healthy volunteers. These procedures are practical and can be readily implemented in the confirmatory LC-MS/MS bioanalysis of other small molecules.

Bioequivalence assessment of two formulations of ibuprofen.

Background:This study assessed the relative bioavailability of two formulations of ibuprofen. The first formulation was Doloraz®, produced by Al-Razi Pharmaceutical Company, Amman, Jordan. The second forumulation was Brufen®, manufactured by Boots Company, Nottingham, UK.Methods and results:A prestudy validation of ibuprofen demonstrated long-term stability, freeze-thaw stability, precision, and accuracy. Twenty-four healthy volunteers were enrolled in this study. After overnight fasting, the two formulations (test and reference) of ibuprofen (100 mg ibuprofen/5 mL suspension) were administered as a single dose on two treatment days separated by a one-week washout period. After dosing, serial blood samples were drawn for a period of 14 hours. Serum harvested from the blood samples was analyzed for the presence of ibuprofen by high-pressure liquid chromatography with ultraviolet detection. Pharmacokinetic parameters were determined from serum concentrations for both formulations. The 90% confidence intervals of the ln-transformed test/reference treatment ratios for peak plasma concentration and area under the concentration-time curve (AUC) parameters were found to be within the predetermined acceptable interval of 80%–125% set by the US Food and Drug Administration.Conclusion:Analysis of variance for peak plasma concentrations and AUC parameters showed no significant difference between the two formulations and, therefore, Doloraz was considered bioequivalent to Brufen.

Comparison of the quantitative performances and measurement uncertainty estimates obtained during method validation versus routine applications of a novel hydrophilic interaction chromatography method for the determination of cidofovir in human plasma

Method validation is essential to ensure that an analytical method is fit for its intended purpose. Additionally, it is advisable to estimate measurement uncertainty in order to allow a correct interpretation of the results generated by analytical methods. Measurement uncertainty can be efficiently estimated during method validation as a top–down approach. However, method validation predictions of the quantitative performances of the assay and estimations of measurement uncertainty may be far away from the real performances obtained during the routine application of this assay. In this work, the predictions of the quantitative performances and measurement uncertainty estimations obtained from a method validation are compared to those obtained during routine applications of a bioanalytical method. For that purpose, a new hydrophilic interaction chromatography (HILIC) method was used. This method was developed for the determination of cidofovir, an antiviral drug, in human plasma. Cidofovir (CDV) is a highly polar molecule presenting three ionizable functions. Therefore, it is an interesting candidate for determination by HILIC mode. CDV is an acyclic cytidine monophosphate analog that has a broad antiviral spectrum and is currently undergoing evaluation in clinical trials as a topical agent for treatment of papillomavirus infections. The analytical conditions were optimized by means of design of experiments approach in order to obtain robust analytical conditions. These ones were absolutely necessary to enable the comparisons mentioned above. After a sample clean-up by means of solid phase extraction, the chromatographic analysis was performed on bare silica stationary phase using a mixture of acetonitrile–ammonium hydrogen carbonate (pH 7.0; 20 mM) (72:28, v/v) as mobile phase. This newly developed bioanalytical method was then fully validated according to FDA (Food and Drug Administration) requirements using a total error approach that guaranteed that each future result will fall within acceptance limits of ±30% with a probability of 95% over a concentration range of 92.7–1020 ng/mL. A routine application of the cidofovir determination in two pre-clinical trials demonstrated that the prediction made during the pre-study validation was consistent by retrospective analysis of the quality control (QC) samples. Finally, comparison of the measurement uncertainty estimations calculated from the method validation with those obtained from the routine application of the method was performed, stressing that the estimations obtained during method validation underestimated those obtained from routine applications and that the magnitude of this underestimation was function of the cidofovir concentration. Finally, this new HILIC method is reliable, easily applicable to routine analysis and transposable at low cost in other laboratories.

Bioanalytical Method Requirements and Statistical Considerations in Incurred Sample Reanalysis for Macromolecules

Incurred sample reanalysis (ISR) is the most recent in-study validation parameter that regulatory agencies have mandated to ensure reproducibility of bioanalytical methods supporting pharmacokinetic/toxicokinetic and clinical studies. The present analysis describes five representative case studies for macromolecule therapeutics. Single ISR acceptance criteria (within 30% of the averaged or original concentration) and a modified Bland-Altman (BA) approach were used to assess accuracy and precision of ISR results. General concordance between the two criteria was examined using simulation studies. All five methods met the ISR criteria. The results indicated that thorough method development and prestudy validation were prerequisites for a successful ISR. The overall agreement between the original and reanalyzed results as determined by BA was within 20%. Simulation studies indicated that concordance between the ISR criteria and BA was observed in 95% of the cases. Dilution factors had no significant impact on the ISR, even for C(max) samples where 1:100 or higher dilutions were used. The current ISR acceptance criteria for macromolecules was scientifically and statistically meaningful for methods with a total error of 25% or less.

Strategies to minimize variability and bias associated with manual pipetting in ligand binding assays to assure data quality of protein therapeutic quantification

Bioanalytical laboratories require accurate and precise pipetting to assure reproducible and accurate results for reliable data. Two areas where pipetting differences among analysts lead to poor reproducibility are long term stability testing and sample dilution. The purpose of this paper is to illustrate the problems with manual pipetting, describe an automation strategy to mitigate risks associated with manual pipetting, and provide recommendations on a control strategy that properly monitors samples requiring dilutions. We determined differences among various manual pipetting techniques by analysts within a laboratory. To reduce variability in pipetting, a flexible modular liquid handling script was created on the Hamilton Microlab Star (HMS) to perform sample dilution, pre-treatment and plate loading. The script is capable of handling variable dilution factors. Additionally, two dilution controls were prepared and tested at concentrations of high and mid quality controls (QC). These same dilution controls were incorporated into both pre-study validation and in-study QCs to monitor dilution processing and assay performance. Variability of manual pipetting among 11 analysts was more negatively biased with increasing dilution. Forward and reverse pipetting delivering different volumes contributed to the discordance. The dilutional bias with manual pipetting was eliminated using the liquid handler. Total error of dilution controls was less than 20%. The in-study pass rate was 100%. Application of liquid handlers minimizes the variability and bias due to manual pipetting differences among analysts. The incorporation of dilution QCs serves a dual purpose to monitor the dilution process of the samples as well as the binding assay performance.

A risk-based analysis of the AAPS conference report on quantitative bioanalytical methods validation and implementation

The 3rd American Association of Pharmaceutical Scientists (AAPS)/Food and Drug Administration (FDA) Bioanalytical workshop in 2006 concluded with several new recommendations regarding the validation of bioanalytical methods in a report published in 2007. It was aimed to conciliate or adapt validation principles for small and large molecules and an opportunity to revisit some of the major decision rules related to acceptance criteria given the experience accumulated since 1990. The purpose here is to provide a “risk-based” reading of the recommendations of 3rd AAPS/FDA Bioanalytical Workshop. Five decision rules were compared using simulations: the proposed pre-study FDA and Total Error Rules, the rules based on the β-Expectation Tolerance and β- γ-Content Tolerance Interval and, finally, the 4-6-20 rule for in-study acceptance of runs. The simulation results demonstrated that the β-Expectation Tolerance Rule controls appropriately the risk. The β- γ-Content Tolerance Interval was found to be too conservative, depending on the objective, and to lead to a high rate of rejection of procedures that could be considered as acceptable. On the other side, the FDA and the AAPS/FDA workshop Total Error Rule, combined or not, did not achieve their intended objective. With these rules, the risk is high to deliver results in study that would not meet the targeted acceptance criteria. This can be explained because, first, there is confusion between the quality of a procedure and the fitness of purpose of the results it could produce and, second, between the initial performances of a procedure, for example evaluated during pre-study validation and the quality of the future results.