Evidence for more cost-effective surveillance options for bovine spongiform encephalopathy (BSE) and scrapie in Great Britain.

Transmissible spongiform encephalopathies (TSEs) are an important public health concern. Since the emergence of bovine spongiform encephalopathy (BSE) during the 1980s and its link with human Creutzfeldt-Jakob disease, active surveillance has been a key element of the European Union's TSE control strategy. Success of this strategy means that now, very few cases are detected compared with the number of animals tested. Refining surveillance strategies would enable resources to be redirected towards other public health priorities. Cost-effectiveness analysis was performed on several alternative strategies involving reducing the number of animals tested for BSE and scrapie in Great Britain and, for scrapie, varying the ratio of sheep sampled in the abattoir to fallen stock (which died on the farm). The most cost-effective strategy modelled for BSE involved reducing the proportion of fallen stock tested from 100% to 75%, producing a cost saving of ca GBP 700,000 per annum. If 50% of fallen stock were tested, a saving of ca GBP 1.4 million per annum could be achieved. However, these reductions are predicted to increase the period before surveillance can detect an outbreak. For scrapie, reducing the proportion of abattoir samples was the most cost-effective strategy modelled, with limited impact on surveillance effectiveness.

Selective reaction monitoring of negative electrospray ionization acetate adduct ions for the bioanalysis of dapagliflozin in clinical studies.

Dapagliflozin (Farxiga), alone, or in the fixed dose combination with metformin (Xigduo), is an orally active, highly selective, reversible inhibitor of sodium-glucose cotransporter type 2 (SGLT2) that is marketed in United States, Europe, and many other countries for the treatment of type 2 diabetes mellitus. Here we report a liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical assay of dapagliflozin in human plasma. A lower limit of quantitation (LLOQ) at 0.2 ng/mL with 50 µL of plasma was obtained, which reflects a 5-fold improvement of the overall assay sensitivity in comparison to the previous most sensitive assay using the same mass spectrometry instrumentation. In this new assay, acetate adduct ions in negative electrospray ionization mode were used as the precursor ions for selective reaction monitoring (SRM) detection. Sample preparation procedures and LC conditions were further developed to enhance the column life span and achieve the separation of dapagliflozin from potential interferences, especially its epimers. The assay also quantifies dapagliflozin's major systemic circulating glucuronide metabolite, BMS-801576, concentrations in human plasma. The assay was successfully transferred to contract research organizations (CROs), validated, and implemented for the sample analysis of pediatric and other critical clinical studies. This assay can be widely used for bioanalytical support of future clinical studies for the newly approved drug Farxiga or any combination therapy containing dapagliflozin.

Estimation of the sensitivity of various environmental sampling methods for detection of Salmonella in duck flocks.

Reports of Salmonella in ducks in the UK currently rely upon voluntary submissions from the industry, and as there is no harmonized statutory monitoring and control programme, it is difficult to compare data from different years in order to evaluate any trends in Salmonella prevalence in relation to sampling methodology. Therefore, the aim of this project was to assess the sensitivity of a selection of environmental sampling methods, including the sampling of faeces, dust and water troughs or bowls for the detection of Salmonella in duck flocks, and a range of sampling methods were applied to 67 duck flocks. Bayesian methods in the absence of a gold standard were used to provide estimates of the sensitivity of each of the sampling methods relative to the within-flock prevalence. There was a large influence of the within-flock prevalence on the sensitivity of all sample types, with sensitivity reducing as the within-flock prevalence reduced. Boot swabs (individual and pool of four), swabs of faecally contaminated areas and whole house hand-held fabric swabs showed the overall highest sensitivity for low-prevalence flocks and are recommended for use to detect Salmonella in duck flocks. The sample type with the highest proportion positive was a pool of four hair nets used as boot swabs, but this was not the most sensitive sample for low-prevalence flocks. All the environmental sampling types (faeces swabs, litter pinches, drag swabs, water trough samples and dust) had higher sensitivity than individual faeces sampling. None of the methods consistently identified all the positive flocks, and at least 10 samples would be required for even the most sensitive method (pool of four boot swabs) to detect a 5% prevalence. The sampling of dust had a low sensitivity and is not recommended for ducks.

Selecting the correct weighting factors for linear and quadratic calibration curves with least-squares regression algorithm in bioanalytical LC-MS/MS assays and impacts of using incorrect weighting factors on curve stability, data quality, and assay performance.

A simple procedure for selecting the correct weighting factors for linear and quadratic calibration curves with least-squares regression algorithm in bioanalytical LC-MS/MS assays is reported. The correct weighting factor is determined by the relationship between the standard deviation of instrument responses (σ) and the concentrations (x). The weighting factor of 1, 1/x, or 1/x(2) should be selected if, over the entire concentration range, σ is a constant, σ(2) is proportional to x, or σ is proportional to x, respectively. For the first time, we demonstrated with detailed scientific reasoning, solid historical data, and convincing justification that 1/x(2) should always be used as the weighting factor for all bioanalytical LC-MS/MS assays. The impacts of using incorrect weighting factors on curve stability, data quality, and assay performance were thoroughly investigated. It was found that the most stable curve could be obtained when the correct weighting factor was used, whereas other curves using incorrect weighting factors were unstable. It was also found that there was a very insignificant impact on the concentrations reported with calibration curves using incorrect weighting factors as the concentrations were always reported with the passing curves which actually overlapped with or were very close to the curves using the correct weighting factor. However, the use of incorrect weighting factors did impact the assay performance significantly. Finally, the difference between the weighting factors of 1/x(2) and 1/y(2) was discussed. All of the findings can be generalized and applied into other quantitative analysis techniques using calibration curves with weighted least-squares regression algorithm.

A novel and cost effective method of removing excess albumin from plasma/serum samples and its impacts on LC-MS/MS bioanalysis of therapeutic proteins.

We have developed an innovative method to remove albumin from plasma/serum samples for the LC-MS/MS quantitation of therapeutic proteins. Different combinations of organic solvents and acids were screened for their ability to remove albumin from plasma and serum samples. Removal efficiency was monitored by two signature peptides (QTALVELVK and LVNEVTEFAK) from albumin. Isopropanol with 1.0% trichloroacetic acid was found to be the most effective combination to remove albumin while retaining the protein of interest. Our approach was compared with a commercial albumin depletion kit on both efficiency of albumin removal and recovery of target proteins. We have demonstrated that our approach can remove 95% of the total albumin in human plasma samples while retaining close to 100% for two of three therapeutic proteins tested, with the third one at 60-80%. The commercial kit removed 98% of albumin but suffered at least 50% recovery loss for all therapeutic proteins when compared to our approach. Using BMS-C as a probe compound, the incorporation of the albumin removal approach has improved both assay sensitivity and ruggedness, compared to the whole plasma protein digestion approach alone. An LC-MS/MS method was developed and validated based on this new approach for the analysis of BMS-C in monkey serum. This assay was successfully applied to a toxicological study. When the albumin removal method was used in another clinical LC-MS/MS method, the sensitivity improved 10-fold to 50 ng/mL LLOQ comparing to a typical pellet digestion method.

Innovative use of LC-MS/MS for simultaneous quantitation of neutralizing antibody, residual drug, and human immunoglobulin G in immunogenicity assay development.

Immunogenicity testing for antidrug antibodies (ADA) faces challenges when high levels of the drug are present in clinical patient samples. In addition, most functional cell-based assays designed to characterize the neutralizing ability of ADA are vulnerable to interference from endogenous serum components. Bead extraction and acid dissociation (BEAD) has been successfully applied to extract ADA from serum samples prior to conduction of cell-based assays. However, in the BEAD, certain amounts of the drug and endogenous serum components (so-called residual drug and serum components) from serum samples are carried over to final BEAD eluates due to formation of protein complexes with ADA or nonspecific binding with the beads. Using current enzyme-linked immunosorbent assay (ELISA)-based ligand-binding assays, it is difficult to evaluate the residual drug, which is complexed with excessive amounts of ADA and endogenous serum components in the BEAD eluates. Here, we describe an innovative application of LC-MS/MS for simultaneous detection of the residual human monoclonal antibody drug and endogenous human IgG and the neutralizing antibody positive-control (NAb-PC) in the BEAD eluates. In this study, the low levels of the residual drug and human IgG in the BEAD eluates indicate that the BEAD efficiently removed the high-concentration drug and serum components from the serum samples. Meanwhile, the NAb-PC recovery (∼42%) in the BEAD provided an acceptable detection limit for the cell-based assay. This novel application of LC-MS/MS to immunogenicity assay development demonstrates the advantages of LC-MS/MS in selectivity and multiplexing, which provides direct and fast measurements of multiple components for immunogenicity assay development.

Feasibility assessment of a novel selective peptide derivatization strategy for sensitivity enhancement for the liquid chromatography/tandem mass spectrometry bioanalysis of protein therapeutics in serum.

RATIONALE: Sensitivity is one major challenge limiting the application of liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods for bioanalysis of proteins. A novel selective peptide derivatization (SPD) strategy was proposed to improve assay sensitivity. The main concept of the SPD strategy is to selectively derivatize surrogate peptides of the target protein in the digests, while not derivatizing the abundant background peptides, thereby improving the separation of target peptides during sample extraction and chromatography, and increasing the sensitivity. Additional benefits may help improve sensitivity include (1) increased ionization efficiency; (2) improved fragmentation pattern; and (3) increased sample extraction recovery of target peptides. METHODS: Feasibility assessment of the SPD strategy was conducted using BMS-986012, a monoclonal antibody, as the model protein, and with malondialdehyde (MDA) to selectively derivatize the arginine-containing surrogate peptide SLIY in tryptic-digested monkey serum samples. RESULTS: The decreased polarity and basicity of the MDA-derivatized peptide SLIY (MDA-SLIY) helped improve its separation from the background peptides during solid-phase extraction (SPE) and chromatography. The recovery of MDA-SLIY was 36.1-44.2%, which was ~3-fold higher than the recovery of peptide SLIY (11.9-16.1%). There was no significant ion suppression for MDA-SLIY. Overall, SPD improved the sensitivity ~5-fold. SPD methodology was then successfully applied to the development of a sensitive LC/MS/MS assay for BMS-986012 in monkey serum. CONCLUSIONS: This work demonstrates the feasibility of the SPD strategy for sensitivity enhancement. SPD can provide a simple, cost-efficient, and antibody-free sample preparation approach to improve sensitivity.

Estimation of the impact of vaccination on faecal shedding and organ and egg contamination for Salmonella Enteritidis, Salmonella Typhiumurium and monophasic Salmonella Typhimurium.

Salmonella Enteritidis (SE) and, to a lesser extent, Salmonella Typhimurium (ST) are associated with egg-related outbreaks in people. Recently, monophasic strains of ST (mST; lacking one phase of the flagellar antigen) have been described, and they have officially been classified as variants of ST and thus may contribute to human exposure to contaminated eggs. Currently used vaccination programmes are licensed for use against biphasic variants of ST, and their efficacy against mST has not yet been investigated. In this study, the effectiveness of four vaccination programmes currently in use in the UK poultry industry was evaluated against challenge with one SE strain, one ST strain and two mST strains. A Bayesian model was used to estimate the impact of vaccination on the rate of faecal shedding and on egg contamination. For the majority of vaccine/challenge strain combinations, there was little or no effect of vaccination on the proportion of birds shedding Salmonella for either biphasic or monophasic strains. However, vaccination was effective at reducing egg contamination. A significantly lower proportion of eggshells were positive for the vaccinated birds compared with non-vaccinated birds, including the mST strains (vaccination resulted in a 55% and 21% reduction for the two mST strains). Calculated across all strains, the estimated rate of positive egg contents was lower in vaccinated birds (Bayesian median was 0.13% for vaccinated birds versus 0.27% for non-vaccinated birds). For both vaccinated and unvaccinated birds, there was also an apparent difference in the proportion of positive organs between strains, with the SE strain resulting in a lower proportion of positive organs at post-mortem examination compared with the other strains.

Assessment of Survival Kinetics for Emergent Highly Pathogenic Clade 2.3.4.4 H5Nx Avian Influenza Viruses.

High pathogenicity avian influenza viruses (HPAIVs) cause high morbidity and mortality in poultry species. HPAIV prevalence means high numbers of infected wild birds could lead to spill over events for farmed poultry. How these pathogens survive in the environment is important for disease maintenance and potential dissemination. We evaluated the temperature-associated survival kinetics for five clade 2.3.4.4 H5Nx HPAIVs (UK field strains between 2014 and 2021) incubated at up to three temperatures for up to ten weeks. The selected temperatures represented northern European winter (4 °C) and summer (20 °C); and a southern European summer temperature (30 °C). For each clade 2.3.4.4 HPAIV, the time in days to reduce the viral infectivity by 90% at temperature T was established (DT), showing that a lower incubation temperature prolonged virus survival (stability), where DT ranged from days to weeks. The fastest loss of viral infectivity was observed at 30 °C. Extrapolation of the graphical DT plots to the x-axis intercept provided the corresponding time to extinction for viral decay. Statistical tests of the difference between the DT values and extinction times of each clade 2.3.4.4 strain at each temperature indicated that the majority displayed different survival kinetics from the other strains at 4 °C and 20 °C.

Fully validated LC-MS/MS assay for the simultaneous quantitation of coadministered therapeutic antibodies in cynomolgus monkey serum.

An LC-MS/MS assay was developed and fully validated for the simultaneous quantitation of two coadministered human monoclonal antibodies (mAbs), mAb-A and mAb-B of IgG4 subclass, in monkey serum. The total serum proteins were digested with trypsin at 50 °C for 30 min after methanol denaturation and precipitation, dithiothreitol reduction, and iodoacetamide alkylation. The tryptic peptides were chromatographically separated with a C18 column (2.1 × 100 mm, 1.7 µm) with mobile phases of 0.1% formic acid in water and acetonitrile. Four peptides, a unique peptide for each mAb and two confirmatory peptides from different antibody domains, were simultaneously quantified by LC-MS/MS in the multiple reaction-monitoring mode. Stable isotopically labeled peptides with flanking amino acids on C- and N-terminals were used as internal standards to minimize the variability during sample processing and detection. The LC-MS/MS assay showed lower limit of quantitation (LLOQ) at 5 µg/mL for mAb-A and 25 µg/mL for mAb-B. The intra- and interassay precision (%CV) was within 10.0% and 8.1%, respectively, and the accuracy (%Dev) was within ±5.4% for all the peptides. Other validation parameters, including sensitivity, selectivity, dilution linearity, processing recovery and matrix effect, autosampler carryover, run size, stability, and data reproducibility, were all evaluated. The confirmatory peptides played a critical role in confirming quantitation accuracy and the integrity of the drugs in the study samples. The robustness of the LC-MS/MS assay and the data agreement with the ligand binding data demonstrated that LC-MS/MS is a reliable and complementary approach for the quantitation of coadministered antibody drugs.

Simultaneous oral therapeutic and intravenous ¹4C-microdoses to determine the absolute oral bioavailability of saxagliptin and dapagliflozin.

AIM: To determine the absolute oral bioavailability (F(p.o.) ) of saxagliptin and dapagliflozin using simultaneous intravenous ¹4C-microdose/therapeutic oral dosing (i.v.micro + oraltherap). METHODS: The F(p.o.) values of saxagliptin and dapagliflozin were determined in healthy subjects (n = 7 and 8, respectively) following the concomitant administration of single i.v. micro doses with unlabelled oraltherap doses. Accelerator mass spectrometry and liquid chromatography-tandem mass spectrometry were used to quantify the labelled and unlabelled drug, respectively. RESULTS: The geometric mean point estimates (90% confidence interval) F(p.o) . values for saxagliptin and dapagliflozin were 50% (48, 53%) and 78% (73, 83%), respectively. The i.v.micro had similar pharmacokinetics to oraltherap. CONCLUSIONS: Simultaneous i.v.micro + oraltherap dosing is a valuable tool to assess human absolute bioavailability.

The synthesis of a carbon-14 labeled pegylated Adnectin™ for placental transfer studies in guinea pigs.

Adnectins™ are novel fibronectin-based proteins containing domains engineered to bind to targets of therapeutic interest. The molecular weights of adnectins are less than conventional monoclonal antibodies but larger than traditional small molecules. Until now, there has been no information on the placental transfer of adnectins. To assess placental permeability to adnectins in pregnant guinea pigs, a radiolabeled adnectin, ATI-1072, bound to polyethylene glycol through a [(14) C]Maleimide linker, was synthesized from [1,4-(14) C]Maleic acid. This publication describes the synthesis and analysis of PEG-[(14) C]Maleimide-adnectin ([(14) C]ATI-1072).

Practical and efficient strategy for evaluating oral absolute bioavailability with an intravenous microdose of a stable isotopically-labeled drug using a selected reaction monitoring mass spectrometry assay.

A strategy of using selected reaction monitoring (SRM) mass spectrometry for evaluating oral absolute bioavailability with concurrent intravenous (i.v.) microdosing a stable isotopically labeled (SIL) drug was developed and validated. First, the isotopic contribution to SRM (ICSRM) of the proposed SIL drug and SIL internal standard (IS) was theoretically calculated to guide their chemical synthesis. Second, the lack of an isotope effect on drug exposure was evaluated in a monkey study by i.v. dosing a mixture of the SIL and the unlabeled drugs. Third, after the SIL drug (100 µg) was concurrently i.v. dosed to humans, at T(max) of an oral therapeutic dose of the unlabeled drug, both drugs in plasma specimens were simultaneously quantified by a sensitive and accurate SRM assay. This strategy significantly improves bioanalytical data quality and saves time, costs, and resources by avoiding a traditional absolute bioavailability study or the newer approach of microdoses of a radio-microtracer measured by accelerator mass spectrometry.

Calculation and mitigation of isotopic interferences in liquid chromatography-mass spectrometry/mass spectrometry assays and its application in supporting microdose absolute bioavailability studies.

A methodology for the accurate calculation and mitigation of isotopic interferences in liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays and its application in supporting microdose absolute bioavailability studies are reported for the first time. For simplicity, this calculation methodology and the strategy to minimize the isotopic interference are demonstrated using a simple molecule entity, then applied to actual development drugs. The exact isotopic interferences calculated with this methodology were often much less than the traditionally used, overestimated isotopic interferences simply based on the molecular isotope abundance. One application of the methodology is the selection of a stable isotopically labeled internal standard (SIL-IS) for an LC-MS/MS bioanalytical assay. The second application is the selection of an SIL analogue for use in intravenous (i.v.) microdosing for the determination of absolute bioavailability. In the case of microdosing, the traditional approach of calculating isotopic interferences can result in selecting a labeling scheme that overlabels the i.v.-dosed drug or leads to incorrect conclusions on the feasibility of using an SIL drug and analysis by LC-MS/MS. The methodology presented here can guide the synthesis by accurately calculating the isotopic interferences when labeling at different positions, using different selective reaction monitoring (SRM) transitions or adding more labeling positions. This methodology has been successfully applied to the selection of the labeled i.v.-dosed drugs for use in two microdose absolute bioavailability studies, before initiating the chemical synthesis. With this methodology, significant time and cost saving can be achieved in supporting microdose absolute bioavailability studies with stable labeled drugs.

Esterase inhibitors as ester-containing drug stabilizers and their hydrolytic products: potential contributors to the matrix effects on bioanalysis by liquid chromatography/tandem mass spectrometry.

RATIONALE: Esterase inhibitors are widely used to stabilize ester-containing drugs in biological matrices for quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays. These co-existing inhibitors could cause matrix effects on bioanalysis and jeopardize the assay performance. We therefore developed an LC/MS/MS methodology to monitor the fate of inhibitors and evaluate their matrix effects, which is described in this study. METHODS: Human plasma containing 20 mM of diisopropylfluorophosphate (DFP), paraoxon, eserine, phenylmethylsulfonyl fluoride (PMSF) or 2-thenoyltrifluoroacetone (TTFA) was extracted by liquid-liquid extraction (LLE) and analyzed by an LC/MS/MS assay for BMS-068645 (a model drug) with additional pre-optimized selected reaction monitoring (SRM) transitions using positive/negative electrospray ionization (ESI) mode for each inhibitor. Hydrolytic products were characterized by product ion or neutral loss scan LC/MS/MS analysis. The matrix effect contribution from each inhibitor was evaluated by post-column infusion of BMS-068645. RESULTS: In the extracted samples by LLE, SRM chromatograms revealed the presence of paraoxon, eserine and TTFA with peak intensity of >2.50E08. Three DFP hydrolytic products, diisopropyl phosphate (DP), triisopropyl phosphate (TP) and DP dimer, and one PMSF hydrolytic product, phenymethanesulfonic acid (PMSA), were identified in the extracted samples. In post-column infusion profiles, ion suppression or enhancement was observed in the retention time regions of eserine (~10% suppression), paraoxon (~70% enhancement) and DP dimer (~20% suppression). CONCLUSIONS: The SRM transitions described here make it possible to directly monitor the inhibitors and their hydrolytic products. In combination with post-column infusion, this methodology provides a powerful tool to routinely monitor the matrix effects-causing inhibitors, so that their matrix effects on the bioanalysis can be evaluated and minimized.

Challenges for accurate and prompt molecular diagnosis of clades of highly pathogenic avian influenza H5N1 viruses emerging in Vietnam.

Forty-six chickens and 48 ducks were sampled from four Vietnamese poultry premises in 2009 infected with H5N1 highly pathogenic avian influenza (HPAI) clade 2.3.2 and 2.3.4 viruses, which also differed by cleavage site (CS) sequences in their haemagglutinin (HA) genes. All clinical specimens (n=282), namely tracheal and cloacal swabs plus feathers, were tested by five Eurasian reverse-transcriptase AI RealTime polymerase chain reaction (RRT-PCR) methods. Bayesian modelling showed similar high sensitivity for the validated H5 HA2 RRT-PCR and a new modified M-gene RRT-PCR that utilizes lyophilized reagents. Both were more sensitive than the validated "wet" M-gene RRT-PCR. Another RRT-PCR, which targeted the H5-gene CS region, was effective for clade 2.3.4 detection, but severely compromised for clade 2.3.2 viruses. Reduced sensitivity of the H5 CS and "wet" M-gene RRT-PCRs correlated with mismatches between the target and the primer and/or probe sequences. However, the H5 HA2 RRT-PCR sensitively detected both clade 2.3.2 and 2.3.4 viruses, and agreed with N1 RRT-PCR results. Feather testing from diseased chicken and duck flocks by AI RRT-PCRs resulted in the most sensitive identification of H5N1 HPAI-infected birds. Evolution of new H5N1 HPAI clades remains a concern for currently affected Asian countries, but also for more distant regions where it is important to be prepared for new incursions of H5N1 HPAI viruses. Genetic evidence for adamantane resistance and sensitivity was also observed in isolates from both clades.

Biomarker Assay Validation by Mass Spectrometry.

Decades of discussion and publication have gone into the guidance from the scientific community and the regulatory agencies on the use and validation of pharmacokinetic and toxicokinetic assays by chromatographic and ligand binding assays for the measurement of drugs and metabolites. These assay validations are well described in the FDA Guidance on Bioanalytical Methods Validation (BMV, 2018). While the BMV included biomarker assay validation, the focus was on understanding the challenges posed in validating biomarker assays and the importance of having reliable biomarker assays when used for regulatory submissions, rather than definition of the appropriate experiments to be performed. Different from PK bioanalysis, analysis of biomarkers can be challenging due to the presence of target analyte(s) in the control matrices used for calibrator and quality control sample preparation, and greater difficulty in procuring appropriate reference standards representative of the endogenous molecule. Several papers have been published offering recommendations for biomarker assay validation. The situational nature of biomarker applications necessitates fit-for-purpose (FFP) assay validation. A unifying theme for FFP analysis is that method validation requirements be consistent with the proposed context of use (COU) for any given biomarker. This communication provides specific recommendations for biomarker assay validation (BAV) by LC-MS, for both small and large molecule biomarkers. The consensus recommendations include creation of a validation plan that contains definition of the COU of the assay, use of the PK assay validation elements that support the COU, and definition of assay validation elements adapted to fit biomarker assays and the acceptance criteria for both.

The impact of BCG strains and repeat vaccinations on immunodiagnostic tests in Eurasian badgers (Meles meles).

Bacille Calmette-Guerin (BCG) is a potential tool in the control of Mycobacterium bovis in European badgers (Meles meles). A five year Test and Vaccinate or Remove (TVR) research intervention project commenced in 2014 using two BCG strains (BCG Copenhagen 1331 (Years 1-3/ BadgerBCG) and BCG Sofia SL2222 (Years 4-5). Badgers were recaptured around 9 weeks after the Year 5 vaccination and then again a year later. The Dual-Path Platform (DPP) Vet TB assay was used to detect serological evidence of M. bovis infection. Of the 48 badgers, 47 had increased Line 1 readings (MPB83 antigen) between the Year 5 vaccination and subsequent recapture. The number of BCG Sofia vaccinations influenced whether a badger tested positive to the recapture DPP VetTB assay Line 1 (p < 0.001) while the number of BadgerBCG vaccinations did not significantly affect recapture Line 1 results (p = 0.59). Line 1 relative light units (RLU) were more pronounced in tests run with sera than whole blood. The results from an in_house MPB83 ELISA results indicated that the WB DPP VetTB assay may not detect lower MPB83 IgG levels as well as the serum DPP VetTB assay. Changes in interferon gamma assay (IFN-γ) results were seen in 2019 with significantly increased CFP-10 and PPDB readings. Unlike BadgerBCG, BCG Sofia induces an immune response to MPB83 (the immune dominant antigen in M. bovis badger infection) that then affects the use of immunodiagnostic tests. The use of the DPP VetTB assay in recaptured BCG Sofia vaccinated badgers within the same trapping season is precluded and caution should be used in badgers vaccinated with BCG Sofia in previous years. The results suggest that the DPP VetTB assay can be used with confidence in badgers vaccinated with BadgerBCG as a single or repeated doses.

Biological Matrix Supply Chain Shortages: More Matrices Are Now Rare-the Case for Surrogate Matrices.

The COVID-19 pandemic has strained the biological matrix supply chain. An upsurge in demand driven by numerous COVID-19 therapeutic and vaccine development programs to combat the pandemic, along with logistical challenges sourcing and transporting matrix, has led to increased lead times for multiple matrices. Biological matrix shortages can potentially cause significant delays in drug development programs across the pharmaceutical and biotechnology industry. Given the current circumstances, discussion is warranted around what will likely be increased use of surrogate matrices in support of pharmacokinetic (PK), immunogenicity, and biomarker assays for regulatory filings. Regulatory authorities permit the use of surrogate matrix in bioanalytical methods in instances where matrix is rare or difficult to obtain, as long as the surrogate is appropriately selected and scientifically justified. Herein, the scientific justification and possible regulatory implications of employing surrogate matrix in PK, immunogenicity, and biomarker assays are discussed. In addition, the unique challenges that cell and gene therapy (C>) and other innovative therapeutic modalities place on matrix supply chains are outlined. Matrix suppliers and contract research organizations (CROs) are actively implementing mitigation strategies to alleviate the current strain on the matrix supply chain and better prepare the industry for any future unexpected strains. To maintain ethical standards, these mitigation strategies include projecting matrix needs with suppliers at least 6 months in advance and writing or updating study protocols to allow for additional matrix draws from study subjects and/or re-purposing of subject matrix from one drug development program to another.

Strategy and its implications of protein bioanalysis utilizing high-resolution mass spectrometric detection of intact protein.

Currently, mass spectrometry-based protein bioanalysis is primarily achieved through monitoring the representative peptide(s) resulting from analyte protein digestion. However, this approach is often incapable of differentiating the measurement of protein analyte from its post-translational modifications (PTMs) and/or potential biotransformation (BTX) products. This disadvantage can be overcome by direct measurement of the intact protein analytes. Selected reaction monitoring (SRM) on triple quadrupole mass spectrometers has been used for the direct measurement of intact protein. However, the fragmentation efficiency though the SRM process could be limited in many cases, especially for high molecular weight proteins. In this study, we present a new strategy of intact protein bioanalysis by high-resolution (HR) full scan mass spectrometry using human lysozyme as a model protein. An HR linear ion-trap/Orbitrap mass spectrometer was used for detection. A composite of isotopic peaks from one or multiple charge states can be isolated from the background and used to improve the signal-to-noise ratio. The acquired data were processed by summing extracted ion chromatograms (EIC) of the 10 most intense isotopic ions of octuply protonated lysozyme. Quantitation of the plasma lysozyme was conducted by utilizing high resolving power and an EIC window fitting to the protein molecular weight. An assay with a linear dynamic range from 0.5 to 500 µg/mL was developed with good accuracy and precision. The assay was successfully employed for monitoring the level of endogenous lysozyme and a potential PTM in human plasma. The current instrumentation limitations and potential advantages of this approach for the bioanalysis of large proteins are discussed.