Nivolumab and ipilimumab in the real-world setting in patients with mesothelioma.

OBJECTIVES: Nivolumab (anti-PD-1) plus ipilimumab (anti-CTLA-4) is a new first-line treatment combination for patients with pleural mesothelioma. Nivolumab-ipilimumab improved the survival, however, 30.3% of the patients suffered from grade 3-4 treatment related adverse events (TRAE's) and TRAE's led to discontinuation in 23.0% of all patients. Here, we present the first real-world data of nivolumab plus ipilimumab in patients with malignant mesothelioma treated in two mesothelioma expert centers. METHODS: Clinical data of patients with mesothelioma treated with nivolumab and ipilimumab were prospectively collected. Clinical parameters were obtained every visit, CT scans were evaluated every 12 weeks and adverse events were assessed continuously during the treatment. Data on grade 2-5 TRAE's and activity (overall response rate (ORR), duration of response (DOR), disease control rate (DCR), median progression-free survival (mPFS) and median overall survival (mOS) were reported. RESULTS: Between January 2021 and August 2022, 184 patients were treated with nivolumab plus ipilimumab. The median follow-up was 12.1 months (95 %CI 11.1 - 13.1). Grade 3-4 TRAEs were seen in 27.7 % of the patients and 25.0 % discontinued immunotherapy treatment early because of TRAE's. ORR was 21.7 % (95 % CI 15.7-27.7), median DOR was 5.7 months (IQR 3.2-8.7) and DCR at 12 weeks 56.0 % (95 % CI 48.8-63.2). The mPFS was 5.5 months (95 %CI 4.1-6.9), mOS was 14.1 months (95 % CI 11.1-18.2). CONCLUSIONS: Nivolumab plus ipilimumab had an equal efficacy in a real-world comparable population but also a high risk of TRAE's, leading to discontinuation of treatment in 25% of the patients.

Trastuzumab-Emtansine and Osimertinib Combination Therapy to Target HER2 Bypass Track Resistance in EGFR Mutation-Positive NSCLC.

Introduction: EGFR tyrosine kinase inhibitor improved the survival of patients with metastatic EGFR mutation-positive (EGFRm+) NSCLC. Despite high response rates, resistance develops inevitably in every patient. In up to 13%, HER2 protein overexpression is found on progression. We hypothesized that dual blockade of EGFR and HER2 by osimertinib combined with trastuzumab-emtansine (T-DM1) could reinduce tumor responses. Methods: In this multicenter, single-arm, phase 1-2 study (NCT03784599), patients with EGFRm+ NSCLC, progressing on osimertinib and HER2 overexpression were included. Patients were treated with T-DM1 3.6 mg/kg (intravenously) every 3 weeks and osimertinib 80 mg once a day. Primary end points were objective response rate (ORR) at 12 weeks and safety. Responses were assessed every 6 weeks (Response Evaluation Criteria in Solid Tumors 1.1). Sample size was calculated using Simon's two-stage minimax design (H0 = 41%, H1 > 55%, 80% power, one-sided type I error 10%: a ORR 16 of 36 was needed to proceed to 58 patients). Results: From January 2019 to April 2021, 27 patients were enrolled. ORR after 12 weeks of treatment was 4% (1 of 27). Median progression-free survival was 2.8 months (95% confidence interval: 1.4-4.6 mo). Most frequent treatment-related adverse events of any grade were fatigue, diarrhea, and nausea, among these, grade 3 in four patients. There were no grade 4 or 5 therapy-related adverse events. Conclusions: TRAEMOS (Trastuzumab-Emtansine and Osimertinib) is the first trial combining T-DM1 and osimertinib in patients with EGFRm+ NSCLC to target HER2 overexpression at osimertinib resistance. Safety profile was favorable compared with cytotoxic chemotherapy; but treatment revealed limited efficacy. Further clinical evaluation of this regimen is not warranted.

Evanescent-wave cavity ring-down spectroscopy for enhanced detection of surface binding under flow injection analysis conditions.

The feasibility of liquid-phase evanescent-wave cavity ring-down spectroscopy (EW-CRDS) for surface-binding studies under flow-injection analysis (FIA) conditions is demonstrated. The EW-CRDS setup consists of an anti-reflection coated Dove prism inside a linear cavity (with standard or super-polishing of the total internal reflective (TIR) surface). A teflon spacer with an elliptical hole clamped on this surface acts as a 20 muL sized flow cell. The baseline noise of this system is of the order of 10(-4) absorbance units; the baseline remains stable over a prolonged time and the prism surface does not become contaminated during repeated injections of the reversibly adsorbing test dyes Crystal Violet (CV) and Direct Red 10 (DR10). At typical FIA or liquid chromatography (LC) flow rates, the system has sufficient specificity to discriminate between species with different surface affinities. For CV a much stronger decrease in ring-down time is observed than calculated based on its bulk concentration and the effective depth probed by the evanescent wave, indicating binding of this positively charged dye to the negatively charged prism surface. The amount of adsorption can be influenced by adjusting the flow rate or the eluent composition. At a flow rate of 0.5 mL/min, an enrichment factor of 60 was calculated for CV; for the poorly adsorbing dye DR10 it is 5. Super-polishing of the already polished TIR surface works counter-productively. The adsorbing dye Crystal Violet has a detection limit of 3 muM for the standard polished surface; less binding occurs on the super-polished surface and the detection limit is 5 muM. Possible applications of EW-CRDS for studying surface binding or the development of bio-assays are discussed.

Cavity ring-down spectroscopy for detection in liquid chromatography at UV wavelengths using standard cuvettes in a normal incidence geometry.

Liquid chromatography (LC) with cavity ring-down spectroscopy (CRDS) detection, using flow cuvettes (put under normal incidence inside the ring-down cavity), is demonstrated. Fresnel reflections are maintained within the capture range of a stable cavity of 4 cm length. This method circumvents the need for specific Brewster's angles and possible mirror degradation is avoided. The flow cuvettes are commercially available at low cost. At 355 nm (the frequency-tripled output of a Nd:YAG laser), the system surpasses the performance of conventional absorbance detectors; the baseline noise was 1.3 x 10(-5)AU and detection limits (injected concentrations) were between 40 and 80 nM for nitro-polyaromatic hydrocarbons with an extinction coefficient epsilon of 7.3-10.2 x 10(3)M(-1)cm(-1). The system was also tested at 273 nm, but in the deep UV the reflectivity of the currently best available mirrors (R>or=99.91%) is still too low to show a significant improvement as compared to conventional UV-vis detection.

Cavity ring-down spectroscopy for detection in liquid chromatography: extension to tunable sources and ultraviolet wavelengths.

In earlier studies, it was demonstrated that the sensitivity of absorbance detection in liquid chromatography (LC) can be improved significantly by using cavity ring-down spectroscopy (CRDS). Thus far, CRDS experiments have been performed using visible laser light at fixed standard wavelengths, such as 532 nm. However, since by far most compounds of analytical interest absorb in the ultraviolet (UV), it is of utmost importance to develop UV-CRDS. In this study, as a first step towards the deep-UV region, LC separations with CRDS detection (using a previously described liquid-only cavity flow cell) at 457 and 355 nm are reported for standard mixtures of dyes and nitro-polyaromatic hydrocarbons (nitro-PAHs), respectively. For the measurements in the blue range a home-built optical parametric oscillator (OPO) system, tunable between 425 and 478 nm, was used, achieving a baseline noise of 2.7 x 10(-6) A.U. at 457 nm, improving upon the sensitivity of conventional absorbance detection (typically around 10(-4) A.U.). An enhancement of the sensitivity can be seen at 355 nm as well, but the improvement of the baseline noise (1.3 x 10(-5) A.U.) is much less pronounced. The sensitivity at 355 nm is limited by the quality of the UV-CRDS mirrors that are currently available: whereas the ring-down times as obtained at 457 nm are around 70-80 ns for the eluent, they are only 20-25 ns at 355 nm. Critical laser characteristics for LC-CRDS measurements, such as pulse length and mode structure, are given and prospects for going to shorter wavelengths are discussed.

Automation of selective assays for on-line bioprocess monitoring by flow-injection analysis.

On-line analysis of one component in a complex media used for bioprocesses requires the application of selective tests such as enzymes assays. Because these assays are susceptible to interference by other medium components and have a limited detection range, automatic sample pretreatment is a prerequisite. The progress made with automatic sample pretreatment in flow-injection analysis makes this technique particularly suitable for on-line monitoring of bioprocesses. Moreover, newly developed software control systems may improve the necessary robustness of flow-infection analysis systems.

On-line control of an immobilized hybridoma culture with multi-channel flow injection analysis.

An immobilized hybridoma cell line was cultivated at controlled glucose and glutamine concentrations. On-line analysis of the substrates was carried out with a multi-channel flow injection analysis system. The analysis system also determined on-line the lactate and ammonium concentration. The substrate concentrations were controlled using an adaptive-control strategy. This strategy consisted of the estimation of the real-time concentrations and volumetric substrate consumption rates by an Extended Kalman Filter, and a minimum variance controller, which used the estimated parameters to set the feed rates of the substrates. The closed-loop control was used to start-up two cultures with either glucose or glutamine as control-substrate for the medium feed rate. The controller kept the concentration of the control-substrate constant by enhancing the medium feed rate simultaneously to the increasing volumetric consumption rate of the substrate. When glutamine was used as control-substrate, the glucose concentration remained relatively constant, whereas the glutamine concentration decreased during the start-up at a constant glucose concentration. This indicates that glutamine is consumed faster than glucose and will be a better control-substrate to avoid limitation during the start-up of a culture with the applied hybridoma cell line. During the colonization of the microcarriers, the yield of ammonium on glutamine decreased from 0.80 to 0.55 (mol mol-1), indicating a change in the glutamine metabolism. The yield of lactate on glucose stayed constant for both experiments. During long-term culture of more than 800 h, the controller kept both the glucose and glutamine concentrations constant at perfusion rates between 0.50 h-1 and 0.15 h-1. The medium, glucose and glutamine feed rate were independently controlled. Both the specific glutamine and glucose consumption rates remained constant for all perfusion rates, which was probably as a result of the constant concentrations. The specific monoclonal antibody production rate decreased with the perfusion rate decreasing from 0.40 h-1 to 0.20 h-1. The immobilized-cell concentration decreased only at the lowest perfusion rate. Both effects could not be explained directly by the increasing ammonium and lactate concentrations nor by the decreasing amino-acid concentrations.

Rational medium design for Bordetella pertussis: basic metabolism.

In current Bordetella pertussis media ammonium accumulates because of an imbalance in the nitrogen:carbon ratio of the substrates used, which is one of the factors limiting cell density in fed-batch cultures. The aim of this study was to map B. pertussis catabolic and anabolic capabilities, in order to design a medium that avoids ammonium accumulation, while substrates are metabolised completely. Besides the known dysfunctional glycolysis, B. pertussis also possessed a partially dysfunctional citric-acid cycle. Although ammonium accumulation was avoided by adding various carbon sources to medium with glutamate, nuclear magnetic resonance (NMR) showed excretion of acetate, acetoacetate and beta-hydroxy-butyrate, thereby reducing the biomass yield. Acetoacetate and beta-hydroxy-butyrate were also formed in Verwey, B2 and modified Stainer-Scholte medium. Electron microscopy in combination with NMR showed that cells early on in these cultures contained poly-hydroxy-butyrate (PHB) globules, which disappeared later during the culture, coinciding with the appearance of beta-hydroxy-butyrate and/or acetoacetate. No globules nor metabolite excretion was detected when lactate in combination with glutamate were used as substrates. Thus, metabolite excretion and ammonium accumulation were avoided, while the yield of 8.8 g C-mol-1 compared favourably with literature values, averaging 6.5 g C-mol-1. Optimisation of this medium for pertussis toxin production will be reported in a separate article.

Liquid chromatography-Fourier-transform infrared spectrometry.

Over the past years the coupling of liquid chromatography (LC) and Fourier-transform infrared spectrometry (FT-IR) has been pursued primarily to achieve specific detection and/or identification of sample constituents. Two approaches can be discerned in the combination of LC and FT-IR. The first and simpler approach is to use a flow cell through which the effluent from the LC column is passed while the IR spectra are continuously recorded. The second approach involves elimination of the LC solvent prior to IR detection using an interface which evaporates the eluent and deposits the analytes onto a substrate. This paper provides a general overview of flow-cell based IR detection and briefly discusses early solvent-elimination interfaces for LC-FT-IR. A more comprehensive description is given of interface systems which use spraying to induce rapid eluent evaporation, and which basically represent the state-of-the-art in LC-FT-IR. Finally, the interface systems suitable for reversed-phase LC are summarized and the perspectives of LC-FT-IR are discussed. The overview indicates that flow-cell LC-FT-IR has rather poor detection limits but can be useful for the specific and quantitative detection of major constituents of mixtures. Solvent-elimination techniques, on the other hand, provide much better sensitivity and enhanced spectral quality which is essential when unambiguous identification of low-level constituents is required.

Evaluation of phytic acid as a buffer additive for the separation of proteins in capillary electrophoresis.

The use of phytic acid to improve protein analysis by capillary electrophoresis (CE) is becoming more and more popular. Due to its size and number of negative charges (up to 12) it provides a high ionic strength combined with a low conductance resulting in an efficient decrease of wall adsorption for proteins. Because of its twelve acidic groups, phytic acid can be used as a buffer over a wide pH range (pH 2-11). The limited wall adsorption of proteins using phytic acid-containing buffers is observed for buffers with a pH of 5.5 and higher. With a monoprotic buffer, most of the investigated proteins show wall adsorption at the pH values studied. In case of a phytic acid buffer, wall adsorption is reduced by a factor of 2-4. The use of phytic acid both as a modifier and as a pH buffer results in more pronounced differences between the various protein mobilities compared with the use of monoprotic buffers. As a result this feature can be used to improve resolution in protein separations.

Laser-based non-fluorescence detection techniques for liquid separation systems.

Over the last two decades, the possibility to use lasers for detection purposes in column liquid chromatography (LC) and capillary electrophoresis (CE) received much attention in the analytical chemistry literature. Most attention has been devoted to laser-induced fluorescence. The present review covers developments on non-fluorescence techniques for LC and CE. The techniques considered are thermal lens spectrometry, photoacoustic detection, refractive index detection including refractive index backscattering, Raman spectroscopy and degenerate four-wave mixing (a special mode of transientholographic spectroscopy). The paper starts with an outline of the characteristics of lasers; it ends with an overall evaluation and a discussion of the perspectives of the techniques dealt with.

Laser-induced fluorescence detection at 266 nm in capillary electrophoresis. Polycyclic aromatic hydrocarbon metabolites in biota.

The separation of five phenolic polycyclic aromatic hydrocarbon metabolites (hydroxy-PAHs) has been performed by cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) using a 30 mM borate buffer (pH 9.0) containing 60 mM sodium dodecyl sulfate and varying concentrations of gamma-cyclodextrin (gamma-CD). A concentration of 12.5 mM gamma-CD was found to provide a baseline separation of the five hydroxy-PAHs. We applied conventional fluorescence and laser-induced fluorescence (LIF) detection, using a new, small-size, quadrupled Nd-YAG laser emitting at 266 nm. The best limits of detection, in the low ng/ml range, were achieved using LIF detection. For all analytes, linearity was observed up to ca. 100 ng/ml. As an application, conjugated pyrene metabolites in hepatopancreas samples from the terrestrial isopods Oniscus asellus and Porcellio scaber were separated and detected. Finally, flatfish bile samples from individuals exposed to polluted sediment or crude oil, which were part of an interlaboratory study, were analyzed by CD-MEKC with conventional fluorescence and LIF detection to determine the 1-hydroxypyrene concentrations.

Liquid-core waveguide technology for coupling column liquid chromatography and Raman spectroscopy.

The on-line coupling of liquid chromatography (LC) and Raman spectroscopy (RS) via an entirely plastic liquid-core waveguide (LCW) was optimized in terms of excitation wavelength of the laser, especially in relation to the fluorescence background, and the length of the LCW. Excitation at 632.8 nm (He-Ne laser) was found to be a good compromise between a wavelength long enough to strongly reduce the fluorescence background and, on the other hand, short enough to avoid (re)-absorption of laser light and Raman signals by H2O in LCWs of considerable length. This conclusion is supported by a theoretical discussion on the optimization of LCW lengths as function of the excitation wavelength for H2O and 2H2O. When using the He-Ne laser the optimum length is approximately 50 cm for H2O; this corresponds to a detection cell volume of 19 microl for an LCW of 220 microm I.D., which is fully compatible with conventional-size LC. The influence of an organic modifier, usually necessary for reversed-phase LC, on the free spectral window was evaluated. The potential applicability of LC-LCW-RS was shown for a mixture of adenosine 5'-monophosphate (AMP), guanosine 5'-monophosphate (GMP) and uridine 5'-monophosphate (UMP), utilizing an aqueous eluent without the addition of a modifier. Improved detectability was achieved by using the stopped-flow mode and applying a large-volume-injection procedure (injection volume: 200 microl). Under these conditions, the limit of identification for AMP, GMP and UMP was in the 0.1-0.5-mg/ml range.

Sub-second liquid chromatographic separations by means of shear-driven chromatography.

Utilizing the concept of shear-driven chromatography, we have been able to realize reversed-phase LC separations in flat rectangular nano-channels coated with a C8 monolayer and being as thin as 100 nm. At this scale, the separation kinetics are strongly enhanced, as is witnessed by the extremely short time (< 0.1 s) needed to separate a mixture of coumarin dyes. The observed plate numbers are still relatively small, because the experiments were conducted in ultra-short columns (< or = 1 mm) and under injection band width-limiting conditions.

Sucrose conversion by immobilized invertase in a multiple air-lift loop bioreactor.

A new bioreactor series within one vessel, the multiple air-lift loop reactor (MAL), is introduced. In the MAL, a series of air-lift loop reactors is incorporated into one vessel. From residence time distribution studies, it was shown that the three-compartment MAL behaves as a series of three ideal mixers. A continuously operated MAL, containing immobilized invertase as a model biocatalyst, was evaluated. The advantage of approaching plug flow by using a bioreactor cascade could be shown by comparing substrate conversion in the three-compartment MAL to that in a single vessel at the same overall dilution rate. This was done for two sets of experimental conditions, which were chosen by using a previously developed model. Intrinsic kinetic parameters of the immobilized enzyme, needed for the model calculations, were determined experimentally. Model calculations gave good approximations of the results. The model incorporates external mass-transfer resistance and diffusion and reaction in the biocatalyst beads.

Effect of low inoculation density in the scale-up of insect cell cultures.

The scale-up of insect cell cultures and the production of baculovirus with these cultures is dependent on the inoculation density applied. The effect of applying a low inoculation on the specific growth rate and on the duration of the lag phase was tested. Three different cell lines, HzAm1, Ha2302, and Sf21 were tested in a total of five cell line/medium combinations. Growth in suspension culture was examined, and data obtained were fitted with the Gompertz equation. A significant decline in specific growth rate with decreasing inoculation density was observed in all cell line/medium combinations, except for HzAm1. No critical inoculation density, below which no growth would occur, was found. In suspension culture in shake flasks, an inoculation density of 5 x 10(4) cells/mL is achievable, without severely influencing the overall growth rate. A lower inoculation density in suspension culture results in less steps in the scale-up process and might be a tool in bypassing the viral passage effect.

Hybridoma and CHO cell partitioning in aqueous two-phase systems.

The partitioning of mouse/mouse hybridoma cell line BIF6A7, mouse/rat hybridoma PFU-83, and CHO DUKX B11-derived cell line BIC-2 in aqueous two-phase systems (ATPSs) of poly(ethylene glycol) (PEG) and dextran was studied. The partitioning of BIF6A7 was investigated systematically by using a statistical experimental design. The aims were to identify the key factors governing cell partitioning and to select ATPSs with suitable cell partitioning for extractive bioconversions with animal cells. The influence of five factors, i.e., the poly(ethylene glycol) molecular weight (PEG MW), dextran molecular weight (Dx MW), tie-line length (TLL), pH, and the ratio of potassium phosphate to potassium chloride, defined as the fraction KPi/(KPi + KCl), on BIF6A7 cell partitioning was characterized by using a full factorial experimental design. The cell partitioning ranged from complete partitioning into the interface to an almost complete partitioning to the lower phase. In all cases less than 1% of the cells partitioned to the top phase. The potassium phosphate fraction had the largest effect on cell partitioning. Low potassium phosphate fractions increased the proportion of cells in the lower phase. To a lesser extent the other factors also played a role in the cell partitioning. The best partitioning for the BIF6A7 cell line was obtained in ATPSs with PEG MW = 35,000, Dx MW = 40,000, TLL = 0.10 g/g, pH 7.4, and KPi/(KPi + KCl) = 0.1, where 93% of the cells were present in the lower phase. The previously reported partitioning of BIF6A7 cells in ATPS culture medium, corresponded well with the current findings. The partitioning of mouse/rat hybridoma cell line PFU-83 and CHO cell line BIC-2 was studied in an ATPS culture medium with PEG 35,000, dextran 40,000, TLL = 0.12 g/g, and hybridoma culture medium. Both cell lines partitioned almost completely into the lower phase. Moreover, the PFU-83 cell line was able to grow in the ATPS hybridoma culture medium. This ATPS hybridoma culture medium therefore seems to be suitable for extractive bioconversions with a wide range of hybridoma cell lines, provided that their product can be partitioned into the upper PEG-rich phase.

Some aspects of room-temperature phosphorescence in liquid solutions.

Experimental requirements for room-temperature phosphorescence measurements in liquids (RTPL) are discussed. Phosphorescence quantum yields and triplet lifetimes of some brominated naphthalenes and halogenated biphenyls at 77 K in 2-methyltetrahydrofuran and at room temperature in hexane are reported and compared. Surprisingly the naphthalenes show only little loss in quantum yields in going from 77 K to room temperature. Sensitized phosphorescence is discussed as a means of expanding the analytical potential of RTPL. Results with a model system of benzophenone as a donor (analyte) and 1,4-dibromonaphthalene as an acceptor are presented.

Determination of the anticancer drug metabolite WR1065 using pre-column derivatization and diode laser induced fluorescence detection.

A liquid chromatographic (LC) procedure using alumina as stationary phase in both the pre- and the analytical column, is reported for the determination of WR1065, the active metabolite of the amino- and thiol-containing anticancer drug WR2721. After pre-column derivatization of the thiol group, the analyte is determined by LC with diode laser induced fluorescence detection in the near-infrared. Selective removal of excess label is achieved by means of column switching; it allows the detection of 5 x 10(-9) M WR1065 in water and 10-fold diluted, deproteinated plasma samples. The detection limit is determined by the derivatization reaction and not by the fluorescence detection of the labelled analyte. Endogeneous thiols do not interfere.