Predefined Acceptance Criteria Research Articles

In Vivo Viability of Platelets Stored in Whole Blood at 4°C

Background: Trauma surgeons have suggested that the optimal transfusion support for actively bleeding patients may be a 1:1:1 ratio of red cells, plasma and platelets (plts). Therefore, the only reason to give component therapy - rather than whole blood (WB) - for these patients may be because of the poor post-transfusion survivals of plts stored at 4°C. However, for trauma/surgical patients, even short plt survivals may be sufficient to allow the surgeon enough time to repair the injury. We report the "first ever" data on the post-storage viability of plts stored at 4°C beyond 3 days. In the studies to be reported here, plts were stored within WB for up to 22 days at 4°C.Study Design/Methods: Normal subjects donated a unit of WB that was stored at 4°C either for 12 days with mixing only at the end of storage or for 10, 15, or 22 days with end-over-end rotation of the WB throughout storage. After storage, a plt concentrate was prepared from the WB using standard centrifugation procedures. At the end of WB storage, the donor donated a 40 ml blood sample from which fresh plts were prepared. The autologous stored and fresh plts were radiolabeled with 111In or 51Cr, respectively, before simultaneous transfusion into their donor. Serial post-transfusion blood samples were collected to determine stored versus fresh plt recoveries and survivals. Our pre-defined acceptance criteria were: 1) stored plt recoveries should average ≥50% of the same donor's fresh plt recoveries; and 2) plt survivals should average ≥1 day.Results/Findings: When the WB was mixed only at the end of storage, plt yields in the WB were just 48% of baseline values with average plt counts of 4.6 ± 2.0 x 1010. This amount of plt loss was considered unacceptable (Table). In contrast, with continuous end-over-end rotation of the WB during storage, plt yields after 15 days were 76% of baseline values and plt counts averaged 7.6 ± 1.4 x 1010 (well within FDA guidelines that require 5.5 x 1010 plts/concentrate prepared from WB). Radiolabeled autologous plt recoveries averaged 27 ± 11% (49 ± 16% of fresh) and survivals averaged 1.2 ± 0.4 days (16 ± 1% of fresh); i.e., our pre-defined plt acceptance criteria were met. Storage for 22 days gave unacceptable results.Conclusion: Plt concentrates stored at 4°C for up to 3 days have been FDA approved for use in thrombocytopenic patients since the 1970's. These plts had autologous plt recoveries of 40 ± 5% and survivals of 1.0 ± 0.1 days (S.E.) (Br J Haematol 1976;34:403-419). The FDA has recently approved the use of 3-day 4°C stored apheresis plts for actively bleeding patients. Our data may suggest that plts stored within WB at 4°C for up to 15 days may provide similar post-storage plt viability as plt concentrates or apheresis plts stored for ≤3 days. Red cells and plasma derived from CP2D WB stored for 21 days at 4°C are already licensed. Therefore, for actively bleeding patients who need all three components (red cells, plasma, and plts), WB stored for up to 15 days may provide effective quantities of all the needed components.Table 1AUTOLOGOUS YIELDS, RECOVERIES, AND SURVIVALS OF PLATELETS STORED IN WHOLE BLOOD AT 4°CStorage Time (Days)NRotationWB PLT COUNTS x 1010Post-Storage Yield(% of Baseline)PLT RECOVERIES (%)PLT SURVIVALS (Days)BaselinePost-StorageFreshStored% of FreshFreshStored% of Fresh127Mixed at end of storage*9.4 ± 2.44.6 ± 2.048 ± 1250 ± 1522 ± 848 ± 298.3 ± 1.61.8 ± 0.623 ± 101010Continuously during storage**9.5 ± 2.17.2 ± 1.677 ± 1050 ± 1026 ± 751 ± 78.1 ± 1.11.3 ± 0.316 ± 41510Continuously during storage**10.0 ± 1.07.6 ± 1.476 ± 1055 ± 1127 ± 1149 ± 168.0 ± 0.11.2 ± 0.416 ± 1223Continuously during storage**12.9 ± 1.99.2 ± 0.773 ± 1556 ± 1214 ± 425 ± 57.7 ± 0.60.8 ± 0.310 ± 3Data reported as average ±1 S.D.*WB units were hand mixed only at the end of storage before plt counting.**WB units were continuously rotated end-over-end during storage. DisclosuresSlichter:Cellphire, Inc.: Research Funding; Cerus Corporation: Research Funding; Terumo BCT: Research Funding; Department of Defense: Research Funding; Genentech: Research Funding.

Utilizing Internal Standard Responses to Assess Risk on Reporting Bioanalytical Results from Hemolyzed Samples.

Bioanalytical analysis of toxicokinetic and pharmacokinetic samples is an integral part of small molecule drugs development and liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been the technique of choice. One important consideration is the matrix effect, in which ionization of the analytes of interest is affected by the presence of co-eluting interfering components present in the sample matrix. Hemolysis, which results in additional endogenous components being released from the lysed red blood cells, may cause additional matrix interferences. The effects of the degree of hemolysis on the accuracy and precision of the method and the reported sample concentrations from hemolyzed study samples have drawn increasing attention in recent years, especially in cases where the sample concentrations are critical for pharmacokinetic calculation. Currently, there is no established procedure to objectively assess the risk of reporting potentially inaccurate bioanalytical results from hemolyzed study samples. In this work, we evaluated the effect of different degrees of hemolysis on the internal standard peak area, accuracy, and precision of the analyses of BMS-906024 and its metabolite, BMS-911557, in human plasma by LC-MS/MS. In addition, we proposed the strategy of using the peak area of the stable isotope-labeled internal standard (SIL-IS) from the LC-MS/MS measurement as the surrogate marker for risk assessment. Samples with peak areas outside of the pre-defined acceptance criteria, e.g., less than 50% or more than 150% of the average IS response in study samples, plasma standards, and QC samples when SIL-IS is used, are flagged out for further investigation.

A high-performance liquid chromatography – tandem mass spectrometry – based targeted metabolomics kidney dysfunction marker panel in human urine

BackgroundPrevious studies have examined and documented fluctuations in urine metabolites in response to disease processes and drug toxicity affecting glomerular filtration, tubule cell metabolism, reabsorption, oxidative stress, purine degradation, active secretion and kidney amino acylase activity representative of diminished renal function. However, a high-throughput assay that incorporates metabolites that are surrogate markers for such changes into a kidney dysfunction panel has yet to be described. MethodsA high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for the quantification of ten metabolites associated with the Krebs cycle, purine degradation, and oxidative stress in human urine was developed and validated. Normal values were assessed in healthy adult (n=120) and pediatric (n=36) individuals. In addition, 9 pediatric renal transplant recipients patients were evaluated before and after initial dosing of the immunosuppressant tacrolimus in a proof-of-concept study. ResultsThe assay met all predefined acceptance criteria. The lower limit of quantification ranged from 0.1 to 1000 μmol/l. Inter-day trueness and imprecisions ranged from 91.4–112.9% and 1.5–12.4%, respectively. The total assay run time was 5.5 minutes.Concentrations of glucose, sorbitol, and trimethylamine oxide (TMAO) were elevated in pediatric renal transplant patients (n=9) prior to transplantation as well as before and immediately after initial dosing of tacrolimus. One month post-transplant urine metabolite patterns matched those of healthy children (n=36). ConclusionsThe LC-MS/MS assay will provide the basis for further large-scale clinical studies to explore these analytes as molecular markers for the patients with renal insufficiency.

An interlaboratory transfer of a multi-analyte assay between continents.

Alex has worked at GlaxoSmithKline for the past 15years and currently works within the bioanalytical and toxicokinetic group in the United Kingdom. Alex's role in previous years has been the in-house support of preclinical and clinical bioanalysis, from method development through to sample analysis activities as well as acting as PI for GLP bioanalysis and toxicokinetics. For the past two years, Alex has applied this analytical and regulatory experience to focus on the outsourcing of preclinical bioanalysis, toxicokinetics and clinical bioanalysis, working closely with multiple bioanalytical and in-life CRO partners worldwide. Alex works to support DMPK and Safety Assessment outsourcing activities for GSK across multiple therapeutic areas, from the first GLP study through to late stage clinical PK studies. Transfer and cross-validation of an existing analytical assay between a laboratory providing current analytical support, and a laboratory needed for new or additional support, can present the bioanalyst with numerous challenges. These challenges can be technical or logistical in nature and may prove to be significant when transferring an assay between laboratories in different continents. Part of GlaxoSmithKline's strategy to improve confidence in providing quality data, is to cross-validate between laboratories. If the cross-validation fails predefined acceptance criteria, then a subsequent investigation would follow. This may also prove to be challenging. The importance of thorough planning and good communication throughout assay transfer, cross-validation and any subsequent investigations is illustrated in this case study.

Significance of 'post bioanalysis phase' in bioanalysis: is GMP creeping into GLP?

“In the year 2011, during a vendor (CRO) qualification audit, I asked the analyst what is the reason for the failure of an analytical run? He responded “the root cause of analytical run failure is the non-compliance of QC samples to pre-defined acceptance criteria” ... Can this be a root cause? I thought. Although I was not convinced with the analyst’s response, but at the same time realized the significance of correct bioanalytical practices especially with regard to post bioanalysis phase.” Rationale interpretation of regulated bioanalysis implies intrinsic use of bioanalytical science, ‘quality’ aspects, regulatory requirements and good practices (GxP). With an increasing demand for reconstruction of bioanalysis using available documents on-site, laboratory data on accuracy and reproducibility, time-bound performance and continuous efforts to improve method efficiency, signify the importance of controlling quality and thoughtful human intervene using a robust quality management system (QMS). In addition to bioanalysis, this system also applies to drug substance and drug product analysis. As per current understanding, regulated bioanalysis can be categorized into four key phases: method development, method validation, bioanalysis (study sample analysis) and post bioanalysis, which includes reviews, hypothesis creation, reinvestigations and corrective actions and preventive actions (CAPA). Although these phases have evolved significantly in the last two decades, post bioanalysis phase has been a subject of intense scientific discussions at different forums. The issues related to the failure of analytical batches, pharmacokinetic repeats, conduct, failure and reporting of incurred sample reanalysis (ISR) have indeed highlighted the importance of this crucial phase. The expertise of rationale interpretation of regulated bioanalysis not only emphasizes the adequate use of an error-proof QMS but also implies prudent use of good quality practices (GxP; good laboratory practice [GLP]; good clinical practice [GCP]; good documentation practice [GDP] and good manufacturing practice [GMP]) for various bioanalytical phases by taking into account the scientific, quality or regulatory calls rationally based on the type and intensity of the analytical problems. The bioanalytical method validation and application phase of a validated method are well established, and are accepted as process-driven phases (standard operating procedures [SOPs], protocols, instructions and training, among others). On the contrary, similar method development phase, post bioanalysis phase demands a high degree of thinking and innovative approaches to hypothesize, investigate and resolve problems encountered by bioscientists in their day to day regimen. At the same time, it poses different level of challenges as most of the problems encountered in this phase are required to be resolved through detailed investigation, conclusion, CAPA and documentation within a stipulated time frame. From the historical front, the preregulations era in mid to late 1990s, bioanalysis was characterized by basically two phases, Significance of ‘post bioanalysis phase’ in bioanalysis: is GMP creeping into GLP?

Results of the “Evaluation of NGS in AML-Diagnostics (ELAN)” Study – an Inter-Laboratory Comparison Performed in 10 European Laboratories

Background: The invention of Next Generation Sequencing (NGS) has spurred research into human diseases, especially in the field of malignancy. In acute myeloid leukemia (AML), a plethora of novel alterations have been identified, including mutations in epigenetic regulator genes (e.g. IDH1, IDH2, DNMT3A), genes coding for proteins of the cohesin complex (e.g. SMC1A, SMC3, STAG2) and spliceosome genes (e.g. SF3B1, U2AF1). Although the diagnostic and prognostic implications of many of these alterations are not yet clear, there is increasing evidence that several of them might have major implications for understanding the disease biology or for patient-treatment. Thus, there is increasing need to reliably detect these mutations in large patient groups in clinically relevant time-frames and at an affordable cost. Due to the large number of genes to be screened, amplicon-based NGS represents an attractive detection method. Although, several assays have been reported, integrating different numbers of genes, it is currently unclear whether they really allow reliable detection of alterations in a reproducible way. Here we report our results from a round robin comparison of the detection of known AML-variants using a highly multiplexed, single tube assay coamplifying a total of 568 amplicons covering 54 entire genes or hot spot gene regions involved in leukemia (TruSight Myeloid sequencing panel; Illumina), with respect to the sensitivity, reproducibility and quantitative accuracy.Material and Methods: Ten European laboratories routinely involved in molecular AML diagnostics participated in this study. All groups performed two sequencing runs, each containing 8 samples. These samples were centrally aliquoted and distributed, the analyses were done in a blinded fashion. Six out of the 8 samples on each run were derived from a set of 9 samples composed of DNA isolated from the blasts of 18 different newly diagnosed AML patients mixed at a 1:1 ratio, with 50 ng of DNA being used for the library preparation. Three of these 9 samples were analyzed in replicate in separate runs by each group. The remaining two samples were a commercial test DNA containing 10 known single nucleotide variants (SNV) or insertion/deletion (InDel) alterations with defined variant allele frequencies (VAF) between 4 and 25% and DNA derived from the OCI-AML3 AML cell line (mutant for DNMT3A and NPM1). Sequencing was performed on MiSeq NGS systems (Illumina) using 2x151 bp-runs. Sequencing data were analyzed by all laboratories using the VariantStudio software (Illumina), with the threshold for mutation calling set at 3%.Results: Analysis of data quality indicated that 85% of the samples met the predefined acceptance criteria (>=95% amplicons with at least 500 reads/amplicon), the median coverage was 7379 reads/amplicon (range 0-47403 reads). Of the 9 mutations present in the positive control, 7 were called at least once in the two replicates by all labs, two mutations with a VAF of 5% were missed by 1 and 4 participants, respectively. Overall, the VAF calls for this sample showed a high level of accuracy across the participants (median coefficient of variation 5%, range 0-22.5%) as well as excellent intra- and inter-laboratory reproducibility (Fig.1). In total, the 9 primary leukemic samples contained 43 known variants in 19 genes, including all commonly mutated genes in AML, i.e. CEBPA, DNMT3A,RUNX1, NPM1, FLT3, WT1. For these samples, the sensitivity was 95.7%. Based on the entire data set (positive control and leukemic samples), the calculated sensitivity of the assay for known variants with an expected VAF>=5% was 93.3%. The rate of non-calls was slightly higher for InDels (14/179; 7.8%) than for SNVs (25/407; 6.1%; P=.47). Two 57-bp long insertions in FLT3 exons 14/15 were not called, which is expected due to the specifications of the assay (max. detectable InDel length <=25 bps). The standard deviation of VAF estimates for the primary leukemic samples was 1.7% with a mean CV of 0.094.Conclusions: This inter-laboratory comparison shows a high sensitivity and impressive quantitative accuracy of NGS-based characterization for known variants down to a minor VAF of 5%. Although additional optimization in individual parameters might be necessary, these initial results clearly indicate that rapid comprehensive molecular characterization of patient samples appears feasible, even in the clinical setting. [Display omitted] DisclosuresThiede:AgenDix GmbH: Equity Ownership, Research Funding; Illumina: Research Support, Research Support Other. Bullinger:Illumina: Research Support Other. Hernández-Rivas:Illumina: Research Support Other. Heuser:Illumina: Research Support Other. Preudhomme:Illumina: Research Support Other. Lo Coco:Illumina: Research Support Other. Martinelli:Illumina: Research Support Other. Schuh:Illumina: Research Support Other. Enjuanes:Illumina: Research Support Other. Lea:Illumina: Research Support Other. Schlesinger:Illumina: Employment.

Qualification of a differential scanning calorimetry method for biophysical characterization of monoclonal antibodies and protein vaccine antigens

Background: Analytical method qualification consists of a simplified evaluation of a subset of validation characteristics with a goal to demonstrate that an analytical method is scientifically sound and suitable for its intended use. In contrast to validation, analytical method qualification is performed without predefined acceptability criteria. Qualification may be performed as a prerequisite to method validation, or when an assay for product knowledge has not yet been established as a test for a critical product quality attribute. Methodology The focus of this study was the qualification of a differential scanning calorimetry method that is used to examine thermal stability and tertiary structure of protein vaccine antigens and monoclonal antibody products. The analytical parameter evaluated was precision, through assessment of the characteristics of repeatability and intermediate precision. Conclusion: The results demonstrated that the assay met the desired performance characteristics, and is suitable...

Risk Criteria in Occupational Environments: Critical Overview and Discussion

The current work can be seen as a starting point for the discussion of the problematic on risk acceptance criteria in occupational environments. Some obstacles to the quantitative acceptance criteria formulation and use were analyzed. A look to the long tradition of major hazards accidents was also performed. This work shows that organizations can have several difficulties in acceptance criteria formulation and that the use of pre-defined acceptance criteria in risk assessment methodologies can be inadequate in some cases. It is urgent to define guidelines that can help organizations in the formulation of risk acceptance criteria for occupational environments.

Validation and ease of use of a new pen device for self-administration of recombinant human growth hormone: results from a two-center usability study

Close adherence to the recommended treatment regimen is important for the success of recombinant human growth hormone therapy, although nonadherence can be common. Ease of use and safety during use/storage are among several important factors in the design of a growth hormone injection device intended for long-term use. This study was performed to validate the usability and assess the ease of use of a new pen device (SurePal™) that has been developed to support daily administration of the recombinant human growth hormone product, Omnitrope® (somatropin). The primary objectives of the study were to assess if study participants, representing intended users of the pen in clinical practice, were able to perform an injection procedure into an injection pad effectively and safely and disassemble the pen without receiving a needlestick injury. A total of 106 participants (61 adults and 45 children/adolescents) were enrolled at two study centers (one in the US, one in Germany). Results for both primary usability tasks met the predefined acceptance criteria, with >85% of participants successfully performing each task. All of the other tasks/handling steps assessed were also successfully performed by most participants, with high success rates reflected in the high proportion of participants who classified each task as “very easy” or “easy”. After a second use of the device, 87%–97% of participants rated it as “very easy” or “easy” to use. In summary, the new pen device is safe and easy to use for both adults and children, and will help to support effective, long-term daily administration of the recombinant human growth hormone product, Omnitrope®.

Quantification of the 5-lipoxygenase inhibitor zileuton in human plasma using high performance liquid chromatography–tandem mass spectrometry

Zileuton is an orally active, selective inhibitor of 5-lipoxygenase, which catalyzes the first step in the conversion of arachadonic acid into leukotrienes. Given the important role of leukotrienes in inflammation and cell signaling, multiple studies have investigated the efficacy of zileuton in the treatment of human disease. Examples of disease targets include asthma, ulcerative colitis, rheumatoid arthritis, and more recently, acne, ischemic/reperfusion injury, inflammatory pain, and sickle cell anemia. Zileuton is currently approved for the prophylaxis and chronic treatment of asthma. We report the development and validation of a sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of zileuton in human EDTA plasma. The range of reliable response was 3.05-20,000ng/mL in human plasma. The calibration curves had a correlation coefficient of r(2)>0.99. The intra-day precision was 3.4-5.3%. The inter-day precision ranged from 4.5% to 7.3% and inter-day accuracy from 100% to 107%. No matrix interferences, ion suppression/enhancement, or carry-over was observed. The assay met all predefined acceptance criteria and was subsequently employed to measure plasma zileuton concentrations in a clinical trial.

A Method for the Analysis of Behavioural Uncertainty in Evacuation Modelling

Evacuation models generally include the use of distributions or probabilistic variables to simulate the variability of possible human behaviours. A single model setup of the same evacuation scenario may therefore produce a distribution of different occupant-evacuation time curves in the case of the use of a random sampling method. This creates an additional component of uncertainty caused by the impact of the number of simulated runs of the same scenario on evacuation model predictions, here named behavioural uncertainty. To date there is no universally accepted quantitative method to evaluate behavioural uncertainty and the selection of the number of runs is left to a qualitative judgement of the model user. A simple quantitative method using convergence criteria based on functional analysis is presented to address this issue. The method permits (1) the analysis of the variability of model predictions in relation to the number of runs of the same evacuation scenario, i.e. the study of behavioural uncertainty and (2) the identification of the optimal number of runs of the same scenario in relation to pre-defined acceptance criteria.

Novel surgical performance evaluation approximates Standardized Incidence Ratio with high accuracy at simple means

BackgroundExcess adverse events may be attributable to poor surgical performance but also to case-mix, which is controlled through the Standardized Incidence Ratio (SIR). SIR calculations can be complicated, resource consuming, and unfeasible in some settings. This article suggests a novel method for SIR approximation. MethodsIn order to evaluate a potential SIR surrogate measure we predefined acceptance criteria. We developed a new measure – Approximate Risk Index (ARI). “Number Needed for Event” (NNE) is the theoretical number of patients needed “to produce” one adverse event. ARI is defined as the quotient of the group of patients needed for no observed events Ge by total patients treated Ga. Our evaluation compared 2500 surgical units and over 3 million heterogeneous risk surgical patients that were induced through a computerized simulation. Surgical unit's data were computed for SIR and ARI to evaluate compliance with the predefined criteria. Approximation was evaluated by correlation analysis and performance prediction capability by Receiver Operating Characteristics (ROC) analysis. ResultsARI strongly correlates with SIR (r2 = 0.87, p < 0.05). ARI prediction of excessive risk revealed excellent ROC (Area Under the Curve > 0.9) 87% sensitivity and 91% specificity. Discussion and conclusionsARI provides good approximation of SIR and excellent prediction capability. ARI is simple and cost-effective as it requires thorough risk evaluation of only the adverse events patients. ARI can provide a crucial screening and performance evaluation quality control tool. The ARI method may suit other clinical and epidemiological settings where relatively small fraction of the entire population is affected.

Clinical Development of Biosimilars: an Evolving Landscape

Biosimilars, or similar biological medicinal products, can provide a meaningful option for patients and physicians provided they deliver the therapeutic value of a reference product at a more modest cost. Unlike generic small-molecule drugs that require primarily the demonstration of pharmaceutical equivalence, the complex nature of protein therapeutics warrants a rigorous evaluation of both pharmaceutical and therapeutic equivalence to the reference product in an abbreviated clinical program. Furthermore, the lack of comprehensive structure-activity relationship data increases the burden on appropriately designed human clinical studies with predefined acceptance criteria to demonstrate the absence of clinically meaningful differences between the biosimilar and reference product. Although a number of biosimilar proteins have been approved, especially in Europe, issues on substitutability, extrapolation to other disease indications, and selection of reference standards and comparators, remains to be standardized at a global level.

Analytical validation of the Oncotype DX prostate cancer assay – a clinical RT-PCR assay optimized for prostate needle biopsies

BackgroundThe Oncotype DX® Prostate Cancer Assay is a multi-gene RT-PCR expression assay that was developed for use with fixed paraffin-embedded (FPE) diagnostic prostate needle biopsies containing as little as 1 mm of prostate tumor in the greatest dimension. The assay measures expression of 12 cancer-related genes representing four biological pathways and 5 reference genes which are algorithmically combined to calculate the Genomic Prostate Score (GPS). This biopsy-based assay has been analytically and subsequently clinically validated as a predictor of aggressive prostate cancer. The aim of this study was to validate the analytical performance of the Oncotype DX Prostate Cancer Assay using predefined acceptance criteria.ResultsThe lowest quartile of RNA yields from prostate needle biopsies (six 5 μm sections) was between 19 and 34 ng. Analytical validation of the process requiring as little as 5 ng of RNA met all pre-defined acceptance criteria. Amplification efficiencies, analytical sensitivity, and accuracy of gene assays were measured by serially diluting an RNA sample and analyzing features of the linear regression between RNA expression measured by the crossing point (Cp) versus the log2 of the RNA input per PCR assay well. Gene assays were shown to accurately measure expression over a wide range of inputs (from as low as 0.005 ng to 320 ng). Analytical accuracy was excellent with average biases at qPCR inputs representative of patient samples <9.7% across all assays while amplification efficiencies were within ±6% of the median. Assessments of reproducibility and precision were performed by testing 10 prostate cancer RNA samples over multiple instruments, reagent lots, operators, days (precision), and RNA input levels (reproducibility) using appropriately parameterized linear mixed models. The standard deviations for analytical precision and reproducibility were 1.86 and 2.11 GPS units (100-unit scale) respectively.ConclusionsThe Oncotype DX Prostate Cancer Assay, a clinical RT-PCR assay specifically designed for use with prostate needle biopsies, has been analytically validated using very limited RNA inputs. The assay requirements and analytical performance will provide physicians with test results from a robust and reliable assay which will enable improved treatment decisions for men diagnosed with early-stage prostate cancer.

From Adults to Children

According to the International Conference on Harmonisation guideline E11, pharmacokinetic (PK) bridging studies can be applied to support pediatric drug development. However, for PK studies in infants and children the sampling schedule needs to be optimized to minimize the number of blood samples per individual. The aim of this study was to describe how clinical trial simulations (CTS) based on adult data were used to select an appropriate sparse-sampling schedule for a future pediatric population PK (popPK) study. A popPK model for gadobutrol (Gadovist®) was developed using data from a phase I study in adults. This model was used for CTS to select the most appropriate sparse-sampling schedule that met predefined acceptance criteria. This sampling schedule was applied in a pediatric clinical phase I/III study. Non-linear mixed-effects modeling was used for PK modeling and simulations. An appropriate sampling schedule requiring only three blood samples per patient was selected and successfully applied in a pediatric study with a gadobutrol standard dose of 0.1 mmol/kg bodyweight. A popPK analysis was performed to determine individual PK parameters in the pediatric study population. A priori evaluation of selected sampling schedules by simulation from adult data provides a useful tool for efficient planning of pediatric studies.

A Simple Method for Validation and Verification of Pipettes Mounted on Automated Liquid Handlers

We have implemented a simple, inexpensive, and fast procedure for validation and verification of the performance of pipettes mounted on automated liquid handlers (ALHs) as necessary for laboratories accredited under ISO 17025. A six- or seven-step serial dilution of OrangeG was prepared in quadruplicates in a flat-bottom 96-well microtiter plate, manually using calibrated pipettes. Each pipette of the liquid handler (1-8) dispensed a selected volume (1-200 μL) of OrangeG eight times into the wells of the microtiter plate. All wells contained a total of 200 μL liquid. The absorbance was read, and the dispensed volume of each pipette was calculated based on a plot of volume and absorbance of a known set of OrangeG dilutions. Finally, the percent inaccuracy (%d) and the imprecision (%CV) of each pipette were calculated. Using predefined acceptance criteria, each pipette was then either approved or failed. Failed pipettes were either repaired or the volume deviation was compensated for by applying a calibration curve in the liquid-handler software. We have implemented the procedure on a Sias Xantus, an MWGt TheONYX, four Tecan Freedom EVO, a Biomek NX Span-8, and four Biomek 3000 robots, and the methods are freely available. In conclusion, we have set up a simple, inexpensive, and fast solution for the continuous validation of ALHs used for accredited work according to the ISO 17025 standard. The method is easy to use for aqueous solutions but requires a spectrophotometer that can read microtiter plates.

A low blood volume LC‐MS/MS assay for the quantification of fentanyl and its major metabolites norfentanyl and despropionyl fentanyl in children

Preterm and term neonates often require surgical procedures and analgesia. However, our knowledge about neonatal pharmacokinetics of fentanyl, the most commonly used drug for these procedures, and its metabolites is still incomplete. To facilitate pharmacokinetic studies of fentanyl and its metabolites in neonates and other children, we developed and validated an LC-MS/MS method based on minimally invasive, low blood volume sampling. LC-MS/MS was used for the simultaneous analysis of fentanyl, despropionyl fentanyl (DPF), and norfentanyl from dried blood samples (DBS) collected on filter paper. Positive ions were monitored using multiple reaction monitoring. Since the standard matrix for measuring fentanyl blood concentrations is plasma, the assay was developed and validated in plasma, whole blood, and then DBS. Our method was able to measure clinically relevant levels of fentanyl and its metabolites. In DBS, the lower limits of quantification were 100 pg/mL for fentanyl with a range of reliable response from 0.1 to 100 ng/mL (r(2)>0.99) and 250 pg/mL for both DPF and norfentanyl with a range of reliable response from 0.25 to 100 ng/mL (r(2)>0.99). In plasma and in DBS inter-day accuracy and precisions of fentanyl met predefined acceptance criteria and also indicated comparable assay performance in both matrices.

A high-throughput U-HPLC–MS/MS assay for the quantification of mycophenolic acid and its major metabolites mycophenolic acid glucuronide and mycophenolic acid acyl-glucuronide in human plasma and urine

Mycophenolic acid (MPA) is used as an immunosuppressant after organ transplantation and for the treatment of immune diseases. There is increasing evidence that therapeutic drug monitoring and plasma concentration-guided dose adjustments are beneficial for patients to maintain immunosuppressive efficacy and to avoid toxicity. The major MPA metabolite that can be found in high concentrations in plasma is MPA glucuronide (MPAG). A metabolite usually present at lower concentrations, MPA acyl-glucuronide (AcMPAG), has been implicated in some of the adverse effects of MPA. We developed and validated an automated high-throughput ultra-high performance chromatography–tandem mass spectrometry (U-HPLC–MS/MS) assay using liquid-handling robotic extraction for the quantification of MPA, MPAG, and AcMPAG in human EDTA plasma and urine. The ranges of reliable response were 0.097 (lower limit of quantitation) to 200μg/mL for MPA and MPAG and 0.156–10μg/mL for AcMPAG in human urine and plasma. The inter-day accuracies were 94.3–104.4%, 93.8–105.0% and 94.4–104.7% for MPA, MPAG and AcMPAG, respectively. Inter-day precisions were 0.7–7.8%, 0.9–6.9% and 1.6–8.6% for MPA, MPAG and AcMPAG. No matrix interferences, ion suppression/enhancement and carry-over were detected. The total assay run time was 2.3min. The assay met all predefined acceptance criteria and the quantification of MPA was successfully cross-validated with an LC–MS/MS assay routinely used for clinical therapeutic drug monitoring. The assay has proven to be robust and reliable during the measurement of samples from several pharmacokinetics trials.

A sensitive assay for the quantification of morphine and its active metabolites in human plasma and dried blood spots using high-performance liquid chromatography–tandem mass spectrometry

Opioids such as morphine are the cornerstone of pain treatment. The challenge of measuring the concentrations of morphine and its active metabolites in order to assess human pharmacokinetics and monitor therapeutic drugs in children requires assays with high sensitivity in small blood volumes. We developed and validated a semi-automated LC-MS/MS assay for the simultaneous quantification of morphine and its active metabolites morphine 3β-glucuronide (M3G) and morphine 6β-glucuronide (M6G) in human plasma and in dried blood spots (DBS). Reconstitution in water (DBS only) and addition of a protein precipitation solution containing the internal standards were the only manual steps. Morphine and its metabolites were separated on a Kinetex 2.6-μm PFP analytical column using an acetonitrile/0.1% formic acid gradient. The analytes were detected in the positive multiple reaction mode. In plasma, the assay had the following performance characteristics: range of reliable response of 0.25-1000 ng/mL (r(2) > 0.99) for morphine, 1-1,000 ng/mL (r(2) > 0.99) for M3G, and 2.5-1,000 ng/mL for M6G. In DBS, the assay had a range of reliable response of 1-1,000 ng/mL (r(2) > 0.99) for morphine and M3G, and of 2.5-1,000 ng/mL for M6G. For inter-day accuracy and precision for morphine, M3G and M6G were within 15% of the nominal values in both plasma and DBS. There was no carryover, ion suppression, or matrix interferences. The assay fulfilled all predefined acceptance criteria, and its sensitivity using DBS samples was adequate for the measurement of pediatric pharmacokinetic samples using a small blood of only 20-50 μL.

Limited Sampling Models to Predict the Pharmacokinetics of Nevirapine, Stavudine, and Lamivudine in HIV-Infected Children Treated With Pediatric Fixed-Dose Combination Tablets

Full 12-hour pharmacokinetic profiles of nevirapine, stavudine, and lamivudine in HIV-infected children taking fixed-dose combination antiretroviral tablets have been reported previously by us. Further studies with these formulations could benefit from less-intensive pharmacokinetic sampling. Data from 65 African children were used to relate area under the plasma concentration versus time curve over 12 hours (AUC) to plasma concentrations of nevirapine, stavudine, or lamivudine at times t = 0, 1, 2, 4, 6, 8, and 12 hours after intake using linear regression. Limited sampling models were developed using leave-one-out crossvalidation. The predictive performance of each model was evaluated using the mean relative prediction error (mpe%) as an indicator of bias and the root mean squared relative prediction error (rmse%) as a measure of precision. A priori set criteria to accept a limited sampling model were: 95% confidence limit of the mpe% should include 0, rmse% less than 10%, a high correlation coefficient, and as few (convenient) samples as possible. Using only one sample did not lead to acceptable AUC predictions for stavudine or lamivudine, although the 6-hour sample was acceptable for nevirapine (mpe%: -0.8%, 95% confidence interval: -2.2 to +0.6); rmse%: 5.8%; r: 0.98). Using two samples, AUC predictions for stavudine and lamivudine improved considerably but did not meet the predefined acceptance criteria. Using three samples (1, 2, 6 hours), an accurate and precise limited sampling model for stavudine AUC (mpe%: -0.6%, 95% confidence interval: -2.2 to +1.0; rmse%: 6.5%; r: 0.98) and lamivudine AUC (mpe%: -0.3%, 95% confidence interval: -1.7 to +1.1; rmse%: 5.6%; r: 0.99) was found; this model was also highly accurate and precise for nevirapine AUC (mpe%: -0.2%, 95% confidence interval: -1.0 to +0.7; rmse%: 3.4%; r: 0.99). A limited sampling model using three time points (1, 2, 6 hours) can be used to predict nevirapine, stavudine, and lamivudine AUC accurately and precisely in HIV-infected African children.