Therapeutic monoclonal antibodies (mAbs) are rapidly taking over the treatment of many malignancies, and an astonishing number of mAbs is in development. This causes a high demand for quantification ...of mAbs in biomatrices both for measuring therapeutic mAb concentrations and to support pharmacokinetics and pharmacodynamics studies. Conventionally, ligand-binding assays are used for these purposes, but LC-MS is gaining popularity. Although intact (top-down) and subunit (middle-down) mAb quantification is reported, signature peptide (bottom-up) quantification is currently most advantageous. This review provides an overview of the reported bottom-up mAb quantification methods in biomatrices as well as general recommendations regarding signature peptide and internal standard selection, reagent use and optimization of digestion in bottom-up quantification methods.
•Sample pre-treatment is optimized for ipilimumab, nivolumab, and pembrolizumab.•Only easily available reagents are used, hence the method is applicable in many labs.•A short sample pre-treatment ...time is combined with a short UPLC-MS/MS run time.•UPLC-MS/MS results were comparable to ELISA results.
Ipilimumab, nivolumab, and pembrolizumab are immune checkpoint inhibiting monoclonal antibodies. Their efficacy has been proven to be correlated with clearance, and hence, bioanalytical assays to study their pharmacokinetics are of pivotal importance. We present the first kit-free sample pre-treatment procedure of only three hours for the Ultra-Performance Liquid Chromatography - tandem Mass Spectrometry (UPLC-MS/MS) simultaneous quantification of ipilimumab, nivolumab, and pembrolizumab in human serum. The conventional bottom-up sample pre-treatment steps for protein MS bioanalysis including pre-digestion purification, denaturation, reduction, alkylation, and digestion were optimized in terms of sensitivity and reproducibility. In the final, optimal sample pre-treatment procedure, samples were purified by protein precipitation with saturated ammonium sulfate solution, reduced with dithiothreitol, denatured with methanol, and digested with trypsin. The method was then validated according to European Medicines Agency (EMA) and the United States Food and Drug Administration (FDA) guidelines for bioanalytical method validation, and 4–6-20 acceptance criteria were applied. This method was selective, accurate, and precise within the range of 3–200 µg/mL for all analytes. The validated developed assay was applied to determine ipilimumab, nivolumab, and pembrolizumab concentrations in patient serum, and the results were compared to enzyme-linked immunosorbent assay (ELISA) results.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•Simultaneous quantification of abemaciclib and its active metabolites by UHPLC–MS/MS.•This method is selective, linear, accurate and precise.•This method is suitable for the analysis of mouse and ...human plasma samples.•Isomers of M20 and M18 were detected in mouse plasma.•M20 isomers were confirmed by HR-MS measurements.
Abemaciclib is the third cyclin-dependent kinase 4 and 6 inhibitor approved for the treatment of advanced or metastatic breast cancer. In humans, abemaciclib is extensively metabolized by CYP3A4 with the formation of three active metabolites: N-desethylabemaciclib (M2), hydroxyabemaciclib (M20) and hydroxy-N-desethylabemaciclib (M18). These metabolites showed similar potency compared to the parent drug and were significantly abundant in plasma circulation. Thus, M2, M20, and M18 may contribute to the clinical activity of abemaciclib. For this reason, an UHPLC–MS/MS method for the simultaneous quantification of abemaciclib and its active metabolites in human and mouse plasma was developed and validated to support further clinical or preclinical investigations on this drug. Samples were processed by protein precipitation with acetonitrile, followed by supernatant dilution and filtration. Chromatographic separation was performed on a Kinetex C18 column (150 × 2.1 mm ID, 2.6 μm) using gradient elution with 10 mM ammonium bicarbonate in water (eluent A) and in methanol-water (9:1, v/v, eluent B). This method was selective, linear, accurate and precise within the range of 1−600 ng/mL for abemaciclib, 0.5−300 ng/mL for M2 and M20, and 0.2−120 ng/mL for M18. Furthermore, stability of the analytes in human and mouse plasma samples in several conditions was demonstrated. Finally, this assay was successfully used in a preclinical pharmacokinetic study, where abemaciclib and its active metabolites were identified and quantified. Inter-species differences between human and mouse samples were encountered, especially in the formation of M20, where isomers of this compound were detected in mouse plasma, but not in human plasma. This was confirmed by high resolution-mass spectrometry (HR-MS) measurements.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
To support pharmacokinetic studies, a multiplex UPLC-MS/MS assay was developed and validated to quantify PD-L1 checkpoint inhibitors atezolizumab, avelumab, and durvalumab in serum.
A bottom-up ...sample pre-treatment procedure was developed to determine atezolizumab, avelumab, and durvalumab in serum. This procedure consisted of (1) precipitation of the monoclonal antibody with ammonium sulfate, (2) reduction with dithiothreitol, (3) denaturation with methanol, and (4) tryptic digestion of the protein. The unique signature peptides resulting after sample pre-treatment of the antibodies were measured using UPLC-MS/MS with a total run time of 11 minutes. The clinical application was evaluated by analyzing 114 atezolizumab patient samples.
The developed method was found to be accurate and precise for all three analytes over a concentration range of 3.00–150 µg/mL. No endogenous interference was present in serum samples. Cross-interference experiments showed no cross-analyte interference and acceptable cross-internal standard interference. In addition, no substantial carry-over was observed. The stable isotopically labeled signature peptides were most effective in compensating for matrix effects. Recovery based on back-calculated concentrations of calibration standards and quality control samples was found to be high. The analytes were stable for at least three freeze-thaw cycles, for 42 hours at processing conditions, for at least two days at 2–8°C in the final extract, for five days before re-injection analysis at 4°C, and long-term for at least 11 months at −70°C. The assay was tested for its applicability in clinical practice. For this purpose, 114 atezolizumab patient samples were measured.
A multiplex UPLC-MS/MS assay was developed and validated to quantify atezolizumab, avelumab, and durvalumab in human serum. The applicability of this method was demonstrated by the analysis of clinical atezolizumab samples. The method is suitable to support clinical pharmacokinetic studies involving atezolizumab, avelumab, or durvalumab.
•A bottom-up sample pre-treatment method was developed for three PD-L1 inhibitors.•In a validated range of 3.00–150 µg/mL, these PD-L1 inhibitors can be measured.•The method is accurate and precise without interference and carry-over.•Long-term stability was proven for at least 11 months at −70°C.•114 atezolizumab patient samples were measured to assess the clinical application.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•A novel LC-QTOF-MS assay for the measurement of anti-SARS-CoV-2 IgG1 antibodies in human serum was developed•SARS-CoV-2 S1 protein coupled to magnetic beads extracted anti-SARS-CoV-2 antibodies from ...serum before LC-QTOF-MS detection•A calibration range of 1.35–135 nM was achieved using ipilimumab as calibration standard
The aim of this study was to develop the first quantitative serological test for anti-SARS-CoV-2 antibodies in human serum with liquid chromatography - quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Other assays, mostly immunoassays, are only qualitative or semi-quantitative, and hence, actual antibody concentrations after SARS-CoV-2 infection are unknown. In our assay, anti-SARS-CoV-2 antibodies were isolated with spike protein subunit 1 (S1) coupled to magnetic beads. IgG1 signature peptide GPSVFPLAPSSK was selected for quantification using ipilimumab calibration standards and SILuMAb K1 as the stable-isotope labeled internal standard. The anti-SARS-CoV-2 IgG1 calibration range was from 1.35 to 135 nM. Inter-assay accuracies were between 98.8%− 107% with inter-assay precisions between 8.37%− 13.5% measured at 3 concentration levels on three separate occasions. Anti-SARS-CoV-2 IgG1 antibodies were quantified in PCR-positive patients with mild to severe symptoms. IgM signature peptide DGFFGVPR was detected in patients that recently recovered from COVID-19. A unique and quantitative LC-QTOF-MS method to quantify anti-SARS-CoV-2 IgG1 in serum was successfully developed and its clinical applicability has been demonstrated.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Aquatic animals use and produce sound for critical life functions, including reproduction. Anthropogenic noise is recognized as a global source of environmental pollution and adequate conservation ...and management strategies are urgently needed. It becomes therefore critical to identify the reproductive traits that render a species vulnerable to acoustic disturbances, and the types of anthropogenic noise that are most likely to impact reproduction. Here, we provide predictions about noise impact on fish reproduction following a two-step approach: first, we grouped documented effects of noise into three mechanistic categories: stress, masking and hearing-loss, and test which type of noise (continuous vs intermittent and regular vs irregular) was most likely to produce a significant response in each category with either a meta-analysis or a quantitative review, depending on data availability. Second, we reviewed existing literature to predict which reproductive traits would render fish most sensitive to stress, masking and hearing-loss. In step one, we concluded that continuous sounds with irregular amplitude and/or frequency-content (e.g. heavy ship traffic) were most likely to cause stress, and continuous sounds were also most likely to induce masking and hearing-loss. From step two we concluded that the vulnerability of a species to noise-induced stress will mainly depend on: (1) its potential to reallocate reproduction to more quiet times or locations, and (2) its vulnerability to masking and hearing-loss mainly on the function of sound communication in its reproductive behaviour. We discuss in which stages of reproduction fish are most likely to be vulnerable to anthropogenic noise based on these findings.
Graphic abstract
The operational sex ratio (OSR) and density are considered important factors affecting the strength of sexual selection. Although there is increasing evidence that OSR and density affect the ...potential for sexual selection, few studies have addressed whether this is realized in phenotypic selection and how the two factors interact. We manipulated OSR (three levels) and male density (two levels) in 36 experimental breeding populations of Gobiusculus flavescens—a fish with paternal care. We measured mating competition behavior, the opportunity for selection (I), and selection on four morphological traits in males. We found sexual selection on two male traits, with the strongest selection being 20% of I. As predicted from OSR theory, increasing female scarcity caused males to become more competitive, concomitant with an increase in I and selection on morphological traits. Model simulations of I based on random mating (I min ) and maximum mate monopolization (I max ) demonstrated that the potential for sexual selection was close to its theoretical maximum across the range of OSRs. However, male density and its interaction with the OSR did not affect sexual selection. We argue that a multifaceted approach, combining mating behavior and selection analyses, can help us to understand how ecological factors affect sexual selection.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NMLJ, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
Trained immunity, a functional state of myeloid cells, has been proposed as a compelling immune-oncological target. Its efficient induction requires direct engagement of myeloid progenitors in the ...bone marrow. For this purpose, we developed a bone marrow-avid nanobiologic platform designed specifically to induce trained immunity. We established the potent anti-tumor capabilities of our lead candidate MTP10-HDL in a B16F10 mouse melanoma model. These anti-tumor effects result from trained immunity-induced myelopoiesis caused by epigenetic rewiring of multipotent progenitors in the bone marrow, which overcomes the immunosuppressive tumor microenvironment. Furthermore, MTP10-HDL nanotherapy potentiates checkpoint inhibition in this melanoma model refractory to anti-PD-1 and anti-CTLA-4 therapy. Finally, we determined MTP10-HDL’s favorable biodistribution and safety profile in non-human primates. In conclusion, we show that rationally designed nanobiologics can promote trained immunity and elicit a durable anti-tumor response either as a monotherapy or in combination with checkpoint inhibitor drugs.
Display omitted
•We have developed a trained immunity-inducing nanobiologic therapeutic named MTP-HDL•MTP-HDL favorably accumulates in hematopoietic organs of mice and non-human primates•MTP-HDL nanotherapy induces trained immunity through bone marrow progenitors in vivo•MTP-HDL nanotherapy inhibits tumor growth and potentiates immune checkpoint inhibition
A bone marrow targeted nanobiologic platform that is designed to elicit trained immunity responses has the ability to reduce tumor growth and augment immune checkpoint blockade.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
To better understand spawning vocalizations of Norwegian coastal cod (Gadus morhua), a prototype eight-element coherent hydrophone array was deployed in stationary vertical and towed ...horizontal modes to monitor cod sounds during an experiment in spring 2019. Depth distribution of cod aggregations was monitored concurrently with an ultrasonic echosounder. Cod vocalizations recorded on the hydrophone array are analysed to provide time–frequency characteristics, and source level distribution after correcting for one-way transmission losses from cod locations to the hydrophone array. The recorded cod vocalization frequencies range from ∼20 to 600 Hz with a peak power frequency of ∼60 Hz, average duration of 300 ms, and mean source level of 163.5 ± 7.9 dB re 1 μPa at 1 m. Spatial dependence of received cod vocalization rates is estimated using hydrophone array measurements as the array is towed horizontally from deeper surrounding waters to shallow water inlet areas of the experimental site. The bathymetric-dependent probability of detection regions for cod vocalizations are quantified and are found to be significantly reduced in shallow-water areas of the inlet. We show that the towable hydrophone array deployed from a moving vessel is invaluable because it can survey cod vocalization activity at multiple locations, providing continuous spatial coverage that is complementary to fixed sensor systems that provide continuous temporal coverage at a given location.
Alternative reproductive tactics are characterized by the occurrence of discrete alternative morphs that differ in behavioural, morphological and physiological traits within the same sex. Although ...much effort has been made to describe the behaviour, morphology and physiology of such alternative morphs, less effort has been invested investigating how much overlap there is in the characteristics of such morphs in natural populations. We studied random population samples of the invasive Round Goby Neogobius melanostomus from five different localities in the river Rhine system in the Netherlands. We found two morphologically and physiologically distinct male morphs which likely represent alternative reproductive tactics. Almost all mature males under 9.35 cm total length had a gonadosomatic index > 3%, suggestive of a sneaker tactic, while nearly all males above 9.35 cm has a gonadosomatic index of < 3%, suggestive of a parental tactic. Cheek size and eye diameter alone were sufficient to distinguish the two morphs. Gonads had a different relationship with size in the two morphs, indicating separate growth trajectories. The gonad mass of sneaker morphs would be ca. 7.5 times as high as the gonad mass of parental morphs of the same total length after extrapolation. Few (9%) intermediates were found, suggesting that the expression of alternative reproductive tactics is determined before the first breeding season. This contrasts with studies on other goby species, which show evidence of plastic tactics that can be affected by social circumstances. We conclude that it is possible to distinguish two alternative male morphs in the Dutch Round Goby population using morphological measurements alone. Although behavioural observations are needed to provide conclusive evidence, the difference in GSI between these morphs indicates that these morphs reflect alternative reproductive tactics.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
