External quality assessment in the absence of proficiency testing: A split-sample testing program experience.

In the field of laboratory medicine, proficiency testing is a vehicle used to improve the reliability of reported results. When proficiency tests are unavailable for a given analyte, an alternative approach is required to ensure adherence to the International Organization for Standardization (ISO) 15189:2012 standard. In this study, we report the results of a split-sample testing program performed as an alternative to a formal PT. This testing method was based on recommendations provided in the Clinical and Laboratory Standards Institute (CLSI) QMS24 guideline. Two different laboratories measured, in duplicate, the heparan sulfate concentration in five samples using ultra-performance liquid chromatography and tandem mass spectrometry. The data analysis to determine the criterion used for the comparability assessment between the two laboratories was based on Appendix E of the QMS24 guideline. Mean interlaboratory differences fell within the maximum allowable differences calculated from the application of the QMS24 guideline, indicating that the results obtained by the two laboratories were comparable across the concentrations tested. Application of the QMS24 split-sample testing procedure allows laboratories to objectively assess test results, thus providing the evidence needed to face an accreditation audit with confidence. However, due to the limitations of statistical analyses in small samples (participants and/or materials), laboratory specialists should assess whether the maximum allowable differences obtained are suitable for the intended use, and make adjustments if necessary.

Using 1.5 mm internal diameter columns for optimal compatibility with current liquid chromatographic systems.

This article describes the use of a new prototype column hardware made with 1.5 mm internal diameter (i.d.) and demonstrates some benefits over the 1.0 mm i.d. micro-bore column. The performance of 2.1, 1.5 and 1.0 mm i.d. columns were systematically compared. With the 1.5 mm i.d. column, the loss of apparent column efficiency can be significantly reduced compared to 1.0 mm i.d. columns in both isocratic and gradient elution modes. In the end, the 1.5 mm i.d. column is almost comparable to 2.1 mm i.d. column from a peak broadening point of view. The advantages of the 1.5 mm i.d. hardware vs 2.1 mm i.d. narrow-bore columns are the lower sample and solvent consumption, and reduced frictional heating effects due to decreased operating flow rates.

Safety risk management for low molecular weight process-related impurities in monoclonal antibody therapeutics: Categorization, risk assessment, testing strategy, and process development with leveraging clearance potential.

Process-related impurities (PRIs) derived from manufacturing process should be minimized in final drug product. ICH Q3A provides a regulatory road map for PRIs but excludes biologic drugs like monoclonal antibodies (mAbs) that contain biological PRIs (e.g. host cell proteins and DNA) and low molecular weight (LMW) PRIs (e.g., fermentation media components and downstream chemical reagents). Risks from the former PRIs are typically addressed by routine tests to meet regulatory expectations, while a similar routine-testing strategy is unrealistic and unnecessary for LMW PRIs, and thus a risk-assessment-guided testing strategy is often utilized. In this report, we discuss a safety risk management strategy including categorization, risk assessment, testing strategy, and its integrations with other CMC development activities, as well as downstream clearance potentials. The clearance data from 28 mAbs successfully addressed safety concerns but did not fully reveal the process clearance potentials. Therefore, we carried out studies with 13 commonly seen LMW PRIs in a typical downstream process for mAbs. Generally, Protein A chromatography and cation exchange chromatography operating in bind-and-elute mode showed excellent clearances with greater than 1,000- and 100-fold clearance, respectively. The diafiltration step had better clearance (greater than 100-fold) for the positively and neutrally charged LMW PRIs than for the negatively charged or hydrophobic PRIs. We propose that a typical mAb downstream process provides an overall clearance of 5,000-fold. Additionally, the determined sieving coefficients will facilitate diafiltration process development. This report helps establish effective safety risk management and downstream process design with robust clearance for LMW PRIs.

Commutability of possible external quality assessment materials for progesterone measurement.

BACKGROUND: The commutability of control materials used for external quality assessment (EQA) programs is of great importance. Evaluating the commutability of control materials is crucial to assess their suitability for EQA programs. METHODS: Forty-eight individual patient serum samples, commercial EQA samples, human serum pools (HSPs), commercially available sterile filtered charcoal stripped serum (CS) and swine serum were analyzed using the isotope dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) comparative method and six immunoassays for progesterone. The commutability was assessed according to the EP14-A2 guideline and the difference in bias approach, respectively. RESULTS: According to the EP14-A2 guideline, HSPs and CS were commutable for all the tested immunoassays, while swine serum showed positive matrix effects in some assays. Based on the difference in bias approach, a large number of inconclusive and noncommutable results appeared. CONCLUSIONS: The commutability of the processed materials varied depending on which evaluation approach and criterion was applied. Noncommutability of the EQA materials was observed. And HSPs and CS were possible commutable candidate control materials according to the EP14-A2 guideline.

Evaluation, identification and impact assessment of abnormal internal standard response variability in regulated LC-MS bioanalysis.

Internal standard (IS) plays an important role in LC-MS bioanalysis by compensating for the variability of the analyte of interest in bioanalytical workflow. Due to the complexity of biological sample compositions and bioanalytical processes, a certain level of IS response variability across a run or a study is anticipated. However, an extensive variability may raise doubts to the accuracy of the measured results and also suggest nonoptimal analytical method. In this current paper, recent publications and guidelines regarding IS response in LC-MS bioanalysis were thoroughly reviewed with focus on the evaluation, identification and impact assessment of 'abnormal' IS response variability. A systematic decision tree was proposed to facilitate investigation into abnormal IS response variability after each run.

Peptide hormone analysis in diagnosis and treatment of Differences of Sex Development: joint position paper of EU COST Action 'DSDnet' and European Reference Network on Rare Endocrine Conditions.

Differences of Sex Development (DSD) comprise a variety of congenital conditions characterized by atypical chromosomal, gonadal, or anatomical sex. Diagnosis and monitoring of treatment of patients suspected of DSD conditions include clinical examination, measurement of peptide and steroid hormones, and genetic analysis. This position paper on peptide hormone analyses in the diagnosis and control of patients with DSD was jointly prepared by specialists in the field of DSD and/or peptide hormone analysis from the European Cooperation in Science and Technology (COST) Action DSDnet (BM1303) and the European Reference Network on rare Endocrine Conditions (Endo-ERN). The goal of this position paper on peptide hormone analysis was to establish laboratory guidelines that may contribute to improve optimal diagnosis and treatment control of DSD. The essential peptide hormones used in the management of patients with DSD conditions are follicle-stimulating hormone, luteinising hormone, anti-Müllerian hormone, and Inhibin B. In this context, the following position statements have been proposed: serum and plasma are the preferred matrices; the peptide hormones can all be measured by immunoassay, while use of LC-MS/MS technology has yet to be implemented in a diagnostic setting; sex- and age-related reference values are mandatory in the evaluation of these hormones; and except for Inhibin B, external quality assurance programs are widely available.

Optimization of XCMS parameters for LC-MS metabolomics: an assessment of automated versus manual tuning and its effect on the final results.

INTRODUCTION: Several software packages containing diverse algorithms are available for processing Liquid Chromatography-Mass Spectrometry (LC-MS) chromatographic data and within these deconvolution packages different parameters settings can lead to different outcomes. XCMS is the most widely used peak picking and deconvolution software for metabolomics, but the parameter selection can be hard for inexpert users. To solve this issue, the automatic optimization tools such as Isotopologue Parameters Optimization (IPO) can be extremely helpful. OBJECTIVES: To evaluate the suitability of IPO as a tool for XCMS parameters optimization and compare the results with those manually obtained by an exhaustive examination of the LC-MS characteristics and performance. METHODS: Raw HPLC-TOF-MS data from two types of biological samples (liver and plasma) analysed in both positive and negative electrospray ionization modes from three groups of piglets were processed with XCMS using parameters optimized following two different approaches: IPO and Manual. The outcomes were compared to determine the advantages and disadvantages of using each method. RESULTS: IPO processing produced the higher number of repeatable (%RSD < 20) and significant features for all data sets and allowed the different piglet groups to be distinguished. Nevertheless, on multivariate level, similar clustering results were obtained by Principal Component Analysis (PCA) when applied to IPO and manual matrices. CONCLUSION: IPO is a useful optimization tool that helps in choosing the appropriate parameters. It works well on data with a good LC-MS performance but the lack of such adequate data can result in unrealistic parameter settings, which might require further investigation and manual tuning. On the contrary, manual selection criteria requires deeper knowledge on LC-MS, programming language and XCMS parameter interpretation, but allows a better fine-tuning of the parameters, and thus more robust deconvolution.

Increasing clinical liquid chromatography/tandem mass spectrometry assay throughput using a full calibration curve generated by one injection from a single-tube calibrator.

Mass spectrometry (MS) generally delivers more accurate results than immunoassay (IA) for certain clinically relevant analytes, but IA is still the more prevalent methodology used by clinical laboratories because of barriers to MS adoption, such as lower throughput. Therefore, it is increasingly important to develop new strategies to increase LC/MS/MS throughput so that more accurate results can be delivered to patients and clinicians. METHODS: Throughput can be increased by reducing assay calibration time using a single-tube calibrator, a mix of isotopologues of the target analyte at different concentrations in a biological matrix, rather than a set of traditional, multiple-tube calibrators. One injection from a single-tube calibrator can generate a full calibration curve such that each calibration point is from the multiple reaction monitoring (MRM) signal corresponding to a specific isotopologue. RESULTS: In this study, a single-tube calibrator (five levels in one vial) and a set of multiple-tube calibrators (seven levels in seven vials) were used to measure the concentration of testosterone in 42 serum samples originally value assigned by the Centers for Disease Control and Prevention (CDC) reference method. The bias between the CDC reference method and the single-tube calibrator measurements and the multiple-tube calibrators measurements was +1.1% and - 5.5%, respectively. These results were within the CDC Hormone Standardization (HoSt) program bias acceptance criteria of ±6.4%. CONCLUSIONS: The results show that LC/MS/MS throughput can be increased using a single-tube calibrator because it reduces assay calibration time while delivering equivalent results to those generated using traditional, multiple-tube calibrators. The single-tube calibrator may also save cost to laboratories through reductions in consumable consumption, technician labor time, and inventory management, as well as to manufacturers because fewer vials would need to be manufactured, tested, stored, and shipped.

Very complex internal standard response variation in LC-MS/MS bioanalysis: root cause analysis and impact assessment.

Internal standards (ISs) are essential for the development and use of reliable quantitative bioanalytical LC-MS/MS methods, because they correct for fluctuations in the analytical response that are caused by variations in experimental conditions. Sample-to-sample differences in the IS response are thus to be expected, but a large variability often is an indication of nonoptimal sample handling or analysis settings. This paper discusses a number of cases of very complex variation of IS responses that could be attributed to analytical problems such as injection errors and sample inhomogeneity, and matrix-related issues such as degradation and increased ionization efficiency. A decision tree is proposed to help find the underlying root cause for extreme IS variability.

External Quality Assessment of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) assays.

The discovery that mutations of the CYP24A1 gene are a cause of idiopathic infantile hypercalcemia (IIH) has revived interest in measuring serum 24,25(OH)2D3. Several studies have also suggested that a high 25-hydroxyvitamin D3(25-OHD3):24,25(OH)2D3 ratio might provide additional diagnostic information in the investigation of vitamin D deficiency. Measurement of 24,25(OH)2D3 is necessarily restricted to laboratories with mass spectrometry methods although cross reactivity of the metabolite in immunoassays for 25-OHD is a potential cause of misleading results. The international External Quality Assessment (EQA) scheme for vitamin D metabolites (DEQAS) was set up in 1989. In 2013 DEQAS became an accuracy based EQA for 25-OHD with 'target values' assigned by the National Institute of Standards and Technology (NIST) Reference Measurement Procedure (RMP). A pilot scheme for serum 24,25(OH)2D3 was started in 2015 and participants were asked to measure the metabolite on each of the 5 samples sent out for 25-OHD. Inter-laboratory agreement was poor but this may reflect methodological differences, in particular different approaches to assay standardization. An important potential contribution to reducing variability among assays was the development by NIST of a 24,25(OH)2D3 RMP and its use in assigning values to SRMs 972a, 2973 and 2971, supported by the NIH Office of Dietary Supplements (ODS) as part of the Vitamin D Standardization Program (VDSP) effort.

Incurred sample reanalysis in AstraZeneca small molecule portfolio - what have we learned and where do we go next?

In this paper, experiences and learnings are shared from the 10-year application of incurred sample reanalysis (ISR) in support of the AstraZeneca small molecule portfolio. The conclusions from including ISR in every clinical bioanalysis study for a period of 5 years, generating ISR data from 550 studies, are shared. Our preclinical ISR approach is described and data generated using capillary microsampling demonstrate confidence in its routine application. The data demonstrate that ISR failures are very rare and the assessment can and should therefore be limited. Dialogue between the bioanalytical teams internally, as well as with the partner contract research organizations, is however critical for a successful bioanalytical method validation and to avoid any ISR failures.

Quality assessment of cellular and tissue-based products using liquid chromatography-tandem mass spectrometry.

We are currently conducting clinical research on cell sheets for cartilage regeneration. One issue with the future use of chondrocyte sheets as cellular and tissue-based products is quality assessment. Currently, chondrocyte sheets are evaluated using invasive methods that cannot be performed on every sheet produced. We report here on our liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique that allows the noninvasive assessment of every sheet using only 50 µl of culture medium. We found that LC-MS/MS could be used to confirm cell sheet viability through the measurement of glucose and glutamine uptake, to estimate extracellular matrix production by measuring serine consumption, to estimate cell kinetics by measuring cytidine and uracil concentrations, and to estimate melanoma inhibitory activity level by measuring pyridoxal concentration. LC-MS/MS may be useful for the noninvasive assessment of products to be used in regenerative medicine.

Qualitative assessment of extractables from single-use components and the impact of reference standard selection.

The advent of single-use bioprocess systems used for the delivery, storage or manufacture of biopharmaceuticals has introduced a new potential source for extractables and leachables (E&L) as these systems are comprised of polymeric materials. Several industry working groups, the FDA and USP have issued guidance and draft guidance on E&L analyses for a variety of applications. These documents typically indicate that mass spectrometry should be applied for discovery of E&L's but provide little guidance as to the exact analytical methodology which should be used. We investigated the extractable profiles of a model single-use bioprocessing system consisting of a single-use bioprocess bag, connector tubing, and a hydrophilic disk filter including filter housing. Extractions were performed in water, ethanol, ethanol/water (50:50) and saline solutions. Extracts were analyzed using a stepwise analytical methodology including a variety of screening and mass spectrometry methods We then used this model system to demonstrate the use of recursive feature finding to automatically detect unique extractables followed by statistical filtering to focus on differentially present extractables which were above the analytical evaluation threshold (AET). We further show the significant affects of standard selection on the number of compounds determined to be above AET when reducing liquid chromatography-mass spectrometry (LC/MS) data. A relative response factor database consisting of 14 structurally diverse commercially available polymer additives was used to arrive at an LC/MS identification threshold. The results of this study demonstrate that significant care should be taken when selecting standards for LC/MS analysis to avoid under reporting of extractables and leachables.

Monoclonal Antibodies Production Platforms: An Opportunity Study of a Non-Protein-A Chromatographic Platform Based on Process Economics.

Monoclonal antibodies currently dominate the biopharmaceutical market with growing sales having reached 80 billion USD in 2016. As most top-selling mAbs are approaching the end of their patent life, biopharmaceutical companies compete fiercely in the biosimilars market. These two factors present a strong motivation for alternative process strategies and process optimization. In this work a novel purification strategy for monoclonal antibodies comprising phenylboronic acid multimodal chromatography for capture followed by polishing by ion-exchange monolithic chromatography and packed bed hydrophobic interaction chromatography is presented and compared to the traditional protein-A-based process. Although the capital investment is similar for both processes, the operation cost is 20% lower for the novel strategy. This study shows that the new process is worthwhile investing in and could present a viable alternative to the platform process used by most industrial players.

Baseline Assessment of 25-Hydroxyvitamin D Assay Performance: A Vitamin D Standardization Program (VDSP) Interlaboratory Comparison Study.

The Vitamin D Standardization Program (VDSP) coordinated an interlaboratory study to assess the comparability of measurements of total 25-hydroxyvitamin D [25(OH)D] in human serum, which is the primary marker of vitamin D status. A set of 50 individual donor samples were analyzed by 15 different laboratories representing national nutrition surveys, assay manufacturers, and clinical and/or research laboratories to provide results for total 25(OH)D using both immunoassays (IAs) and LC tandem MS (MS/MS). The results were evaluated relative to bias compared with the target values assigned based on a combination of measurements at Ghent University (Belgium) and the U.S. National Institute of Standards and Technology using reference measurement procedures for the determination of 25(OH)D2 and 25(OH)D3. CV and mean bias for each laboratory and assay platform were assessed and compared with previously established VDSP performance criteria, namely CV ≤ 10% and mean bias ≤ 5%. Nearly all LC-MS/MS results achieved VDSP criteria, whereas only 50% of IAs met the criterion for a ≤10% CV and only three of eight IAs achieved the ≤5% bias. These results establish a benchmark for the evaluation of 25(OH)D assay performance and standardization activities in the future.

Assessment of intra-particle diffusion in hydrophilic interaction liquid chromatography and reversed-phase liquid chromatography under conditions of identical packing structure.

A recently developed stripping protocol to completely remove the stationary phase of reversed-phase liquid chromatography (RPLC) columns and turn them into hydrophilic interaction liquid chromatography (HILIC) columns with identical packing characteristics is used to study the underlying mechanisms of intra-particle diffusion in RPLC and HILIC. The protocol is applied to a column with a large geometrical volume (250×4.6mm, 5µm) to avoid extra-column effects and for compounds with a broad range in retention factors (k" from ∼0.6 to 8). Three types of behavior for the intra-particle diffusion (Dpart/Dm) in RPLC versus HILIC can be distinguished: for nearly unretained compounds (k"<0.6), intra-particle diffusion in HILIC is larger than in RPLC; for compounds with intermediate retention behavior (k"∼0.9-1.2), intra-particle diffusion in HILIC and RPLC are similar; and for well retained compounds (k">1.8), intra-particle diffusion in RPLC is larger than in HILIC. To explain these observations, diffusion in the stationary phase (γsDs) and in the stagnant mobile phase in the mesopore zone (γmpDm) are deduced from experimentally determined values of the intra-particle diffusion, using models derived from the Effective Medium Theory. It is demonstrated that the larger intra-particle diffusion obtained for slightly retained compounds under HILIC conditions is caused by the higher mesopore diffusion in HILIC (γmp=0.474 for HILIC versus 0.435 for RPLC), while the larger intra-particle diffusion obtained for strongly retained compounds under RPLC conditions can be related to the much higher stationary phase diffusion in RPLC (γsDs/Dm=0.200 for RPLC versus 0.113 for HILIC).

In Vitro Assessment of Guanylyl Cyclase Activity of Plant Receptor Kinases.

Cyclic nucleotides such as 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) are increasingly recognized as key signaling molecules in plants, and a growing number of plant mononucleotide cyclases, both adenylate cyclases (ACs) and guanylate cyclases (GCs), have been reported. Catalytically active cytosolic GC domains have been shown to be part of many plant receptor kinases and hence directly linked to plant signaling and downstream cellular responses. Here we detail, firstly, methods to identify and express essential functional GC domains of receptor kinases, and secondly, we describe mass spectrometric methods to quantify cGMP generated by recombinant GCs from receptor kinases in vitro.

High-throughput determination of vancomycin in human plasma by a cost-effective system of two-dimensional liquid chromatography.

Therapeutic drug monitoring (TDM) is one of the most important services of clinical laboratories. Two main techniques are commonly used: the immunoassay and chromatography method. We have developed a cost-effective system of two-dimensional liquid chromatography with ultraviolet detection (2D-LC-UV) for high-throughput determination of vancomycin in human plasma that combines the automation and low start-up costs of the immunoassay with the high selectivity and sensitivity of the liquid chromatography coupled with mass spectrometric detection without incurring their disadvantages, achieving high cost-effectiveness. This 2D-LC system offers a large volume injection to provide sufficient sensitivity and uses simulated gradient peak compression technology to control peak broadening and to improve peak shape. A middle column was added to reduce the analysis cycle time and make it suitable for high-throughput routine clinical assays. The analysis cycle time was 4min and the peak width was 0.8min. Compared with other chromatographic methods that have been developed, the analysis cycle time and peak width for vancomycin was reduced significantly. The lower limit of quantification was 0.20µg/mL for vancomycin, which is the same as certain LC-MS/MS methods that have been recently developed and validated. The method is rapid, automated, and low-cost and has high selectivity and sensitivity for the quantification of vancomycin in human plasma, thus making it well-suited for use in hospital clinical laboratories.

Rapid and sensitive LC-MS/MS method for the determination of auraptene in rat plasma and its application in a pharmacokinetic and bioavailability study in rats.

A simple, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of auraptene, a constituent isolated from Fructus aurantii with potential to combat Alzheimer's disease, in rat plasma. Rat plasma samples were pretreated by protein precipitation with methanol. The analytes were separated by a Waters Sun Fire C18 column (50 mm x 2 mm, 5 µm) and eluted with 1:1000 methanol and formic acid/water (v/v) mobile phase with a flow rate of 0.5 mL/min. Multiple reaction monitoring was used to monitor the transition of the deprotonated auraptene molecule with an m/z of 299.3 [M+H](+), to the product ion with an m/z of 162.9 [M+H](+). Progesterone, with an m/z of 315.2→ 96.9 was used as an internal standard. The limits of detection and of quantification of auraptene in the rat plasma were 1 and 5 ng/mL, respectively. The method was linear in the concentration range of 20- 2000 ng/mL with coefficient correlation of 0.9956. After auraptene (100 mg/kg, p.o.) administration, the maximum plasma concentration and the time taken to reach maximum concentration were 1719.5 ± 384.3 g/mL and 108.0 ± 25.3 min, respectively. The elimination half-life was 108.0 ± 25.3 for auraptene (100 mg/kg, p.o.) and 3.0 ± 0 min for auraptene (2 mg/kg, i.v.). The oral bioavailability was about 8.5%.

Quantification of active infliximab in human serum with liquid chromatography-tandem mass spectrometry using a tumor necrosis factor alpha -based pre-analytical sample purification and a stable isotopic labeled infliximab bio-similar as internal standard: A target-based, sensitive and cost-effective method.

The therapeutic monoclonal antibody Infliximab (IFX) is a widely used drug for the treatment of several inflammatory autoimmune diseases. However, approximately 10% of patients develop anti-infliximab antibodies (ATIs) rendering the treatment ineffective. Early detection of underexposure to unbound IFX would result in a timely switch of therapy which could aid in the treatment of this disease. Streptavidin coated 96 well plates were used to capture biotinylated-tumor necrosis factor -alpha (b-TNF-α), which in turn was used to selectively extract the active form of IFX in human serum. After elution, IFX was digested using trypsin and one signature peptide was selected for subsequent analysis on liquid chromatography-tandem mass spectrometry (LC-MS/MS). The internal standard used was a stable isotopic labeled IFX bio-similar. The assay was successfully validated according to European Medicines Agency (EMA) guidelines and was found to be linear in a range of 0.5-20µg/mL (r(2)=0.994). Lower limit of quantification for the assay (<20% CV) was 0.5µg/mL, requiring only 2µL of sample. Cross-validation against enzyme-linked immunosorbent assay (ELISA) resulted in a high correlation between methods (r(2)=0.95 with a ρc=0.83) and the accuracy was in line with previously published results. In conclusion, a sensitive, robust and cost-effective method was developed for the bio-analysis of IFX with LC-MS/MS by means of a target-based pre-analytical sample purification. Moreover, low volume and costs of consumables per sample promote its feasibility in (pre)clinical studies and in therapeutic drug monitoring. This method should be considered as first choice due to its accuracy and multiple degree of selectivity.