Rationale
A common strategy for antibody‐drug conjugate (ADC) quantitation from in vivo study samples involves measurement of total antibody, conjugated ADC, and free payload concentrations using ...multiple reaction monitoring (MRM) mass spectrometry. This not only provides a limited picture of biotransformation but can also involve lengthy method development. Quantitation of ADCs directly at the intact protein level in native conditions using high‐resolution mass spectrometers presents the advantage of measuring exposure readout as well as monitoring the change in average drug‐to‐antibody ratio (DAR) and in vivo stability of new linker payloads with minimal method development. Furthermore, site‐specific cysteine‐conjugated ADCs often rely on non‐covalent association to retain their quaternary structure, which highlights the unique capabilities of native mass spectrometry (nMS) for intact ADC quantitation.
Methods
We developed an intact quantitation workflow involving three stages: automated affinity purification, nMS analysis, and data processing in batch fashion. The sample preparation method was modified to include only volatile ion‐pairing reagents in the buffer systems. A capillary size‐exclusion chromatography (SEC) column was coupled to a quadrupole time‐of‐flight high‐resolution mass spectrometer for high‐throughput nMS analysis. Samples from two mouse pharmacokinetic (PK) studies were analyzed using both intact quantitation workflow and the conventional MRM‐based approach.
Results
A linear dynamic range of 5–100 μg/mL was achieved using 20 μL of serum sample volume. The results of mouse in vivo PK measurement using the intact quantitation workflow and the MRM‐based approach were compared, revealing excellent method agreement.
Conclusions
We demonstrated the feasibility of utilizing nMS for the quantitation of ADCs at the intact protein level in preclinical PK studies. Our results indicate that this intact quantitation workflow can serve as an alternative generic method for high‐throughput analysis, enabling an in‐depth understanding of ADC stability and safety in vivo.
Polyploid species have long been thought to be recalcitrant to whole-genome assembly. By combining high-throughput sequencing, recent developments in parallel computing, and genetic mapping, we ...derive, de novo, a sequence assembly representing 9.1 Gbp of the highly repetitive 16 Gbp genome of hexaploid wheat, Triticum aestivum, and assign 7.1 Gb of this assembly to chromosomal locations. The genome representation and accuracy of our assembly is comparable or even exceeds that of a chromosome-by-chromosome shotgun assembly. Our assembly and mapping strategy uses only short read sequencing technology and is applicable to any species where it is possible to construct a mapping population.
Rationale
The in‐sample calibration curve (ISCC) approach of quantification utilizes the response of isotopologue ions from spiked‐in stable isotope labeled internal standard (SIL‐IS) to build a ...standard curve. The quantitative analysis of the study sample is achieved based on the response of selected monoisotopic analyte ion against the calibration curve. Although this methodology has been demonstrated to be feasible by unit and high‐resolution mass spectrometers, quantitation on high‐resolution mass spectrometer with product ions has not been tested. We tested the feasibility of this approach using product ions on an high‐resolution mass spectrometer equipped with an Orbitrap detector.
Methods
Using a proteomics workflow for sample preparation, two surrogate peptides were quantified from a complex matrix of protein digest from human peripheral blood mononuclear cells (hPBMCs). SIL‐IS was spiked in at different levels to construct calibration curves in a traditional manner. ISCCs were prepared using extracted ion chromatograms from isotopically resolved mass spectra and compared with traditional calibration curves.
Results
A linear response was observed with ISCC approach for at least two to three orders of magnitude in MS1 as well as targeted MS2 (tMS2). From protein digests, isobaric interferences were observed for endogenous peptides on the MS1 level; this was circumvented with product‐ion‐based quantitation where for one peptide, %CV for endogenous levels was more than 20% with ISCC but higher with the traditional calibration curve approach. For the second peptide, endogenous levels could not be determined in the traditional approach as calibrant levels did not bracket the lower end, and with the ISCC approach, isotopologues at abundances lower than the endogenous level allowed for quantitative assessments.
Conclusions
ISCC demonstrated improved precision across surrogate peptides from endogenous protein digests. In samples where endogenous analyte concentrations were low in abundance, ISCC rescued what would have been a non‐reportable result in a traditional bioanalytical assay as calibrant levels were not prepared at adequately low levels to bracket unknowns. ISCC using high‐resolution mass spectrometer is feasible and ideal compared to unit resolution mass spectrometers. High‐resolution mass spectrometer allows for isotopic resolution for analytes with > + 2 charge state and provides flexibility in quantification using multiple product ions. ISCC using high‐resolution mass spectrometer allows for simultaneous assaying of low abundance isotopologues, the signal acquisition of which is not constrained by limits in data acquisition or calibrant preparation as with other approaches but rather limited by platform sensitivity. In contrast to unit resolution mass spectrometers, these features offered by high‐resolution mass spectrometer could be especially useful for the drug discovery assay support where there is less lead time for assay development than for the assays to support the drug development studies.
Screening for cytochrome P450 (CYP) induction potential is routine in drug development. Induction results in a net increase in CYP protein and is assessed typically by measuring indirect endpoints, ...i.e., enzyme activity and mRNA in vitro. Recent methodological advancements have made CYP protein quantification by liquid chromatography-mass spectrometry in vitro induction studies more accessible and amenable to routine testing. In this study, we evaluated CYP3A4 concentration dependence of induction response for 11 compounds (rifampin, rifabutin, carbamazepine, efavirenz, nitrendipine, flumazenil, pioglitazone, rosiglitazone, troglitazone, pazopanib, and ticagrelor) in plated hepatocytes from two or three donors incorporating in the assessment all three endpoints. In addition, the time-dependence of the induction was examined over 1, 2, or 3 days of treatment. For most compounds, mRNA, enzyme activity, and protein endpoints exhibited similarity in induction responses. Pazopanib and ticagrelor were notable exceptions as neither protein nor enzyme activity were induced despite mRNA induction of a magnitude similar to efavirenz, pioglitazone, or rosiglitazone, which clearly induced in all three endpoints. Static modeling of clinical induction responses supported a role for protein as a predictive endpoint. These data highlight the value of including CYP protein quantification as an induction assay endpoint to provide a more comprehensive assessment of induction liability. SIGNIFICANCE STATEMENT: Direct, liquid chromatography-mass spectrometry (LC-MS)-based quantification of cytochrome P450 (CYP) protein is a desirable induction assay endpoint; however such application has been limited due to inefficient workflows. Here, we incorporate recent advancements in protein quantitation methods to efficiently quantify CYP3A4 protein in in vitro induction assays with 11 compounds in up to 3 donors. The data indicate induction responses from mRNA do not always align with those of protein suggesting assessment of induction liability is more complex than thought previously.
Multicellular tumor spheroids have been increasingly used by researchers to produce more physiologically relevant experimental environments. However, tracking of spheroid growth and treatment-induced ...volume reduction has not been readily adopted. Here, squamous carcinoma cells were seeded at different starting cell numbers with growth and reduction kinetics monitored using live cell imaging. Following the initial growth phase, spheroids were treated with auristatin as small molecule (MMAE) or as antibody-drug conjugate containing non-cleavable auristatin drug payload (033-F). Compared to cells in monolayers, 033-F had notably weaker potency against spheroids despite potency levels of MMAE being similar against monolayers and spheroids. Accumulation of released payload from 033-F was reduced in higher volume spheroids, likely contributing to the potency differences. Despite lowered potency towards spheroids with 033-F, spheroid volume was still readily reduced by 033-F in a dose-dependent fashion, with >85% volume reductions at the highest concentrations for all spheroid sizes. Additionally, the core of the larger spheroids showed more resiliency towards microtubule inhibition. Overall, this work highlights how various in-vivo 'features' such as tumor penetration, cell interactions, and increased resistance to therapeutics can be integrated into a spheroid model and tracked over time by automated imaging technology.
Permeability is a key factor driving the absorption of orally administered drugs. In early discovery, the efficient evaluation of permeability, particularly for compounds violating Lipinski's Rule of ...5, remains challenging. Addressing this, we established a high-throughput method to measure the experimental polar surface area (HT-EPSA) as an in vitro surrogate to measure permeability. Compared to earlier methods, HT-EPSA significantly reduces data acquisition time with enhanced sensitivity, selectivity, and data quality. In the effort of translating EPSA to human in vitro and in vivo passive permeability, we demonstrated the application of EPSA for predicting Caco-2 cell and human intestinal permeability, showing improvements over topological polar surface area and the parallel artificial membrane permeability assay for rank-ordering permeability in a proteolysis targeting chimera case study. The HT-EPSA method is expected to be highly beneficial in guiding early stage compound rank-ordering, faster decision-making, and in predicting in vitro and/or in vivo human intestinal permeability.Permeability is a key factor driving the absorption of orally administered drugs. In early discovery, the efficient evaluation of permeability, particularly for compounds violating Lipinski's Rule of 5, remains challenging. Addressing this, we established a high-throughput method to measure the experimental polar surface area (HT-EPSA) as an in vitro surrogate to measure permeability. Compared to earlier methods, HT-EPSA significantly reduces data acquisition time with enhanced sensitivity, selectivity, and data quality. In the effort of translating EPSA to human in vitro and in vivo passive permeability, we demonstrated the application of EPSA for predicting Caco-2 cell and human intestinal permeability, showing improvements over topological polar surface area and the parallel artificial membrane permeability assay for rank-ordering permeability in a proteolysis targeting chimera case study. The HT-EPSA method is expected to be highly beneficial in guiding early stage compound rank-ordering, faster decision-making, and in predicting in vitro and/or in vivo human intestinal permeability.
The successful prospective incorporation of in vitro transporter kinetics in physiologically based pharmacokinetic (PBPK) models to describe drug disposition remains challenging. Although ...determination of scaling factors to extrapolate in vitro to in vivo transporter kinetics has been facilitated by quantitative proteomics, no robust assessment comparing membrane recoveries between different cells/tissues has been made. HEK293 cells overexpressing OCT2, MATE1, and MATE2K or human kidney cortex were homogenized and centrifuged to obtain the total membrane fractions, which were subsequently subjected to liquid-liquid extraction followed by centrifugation and precipitation to isolate plasma membrane fractions. Plasma membrane recoveries determined by quantitation of the marker Na
/K
-ATPase in lysate and plasma membrane fractions were ≤20% but within 3-fold across different cells and tissues. A separate study demonstrated that recoveries are comparable between basolateral and apical membranes of renal proximal tubules, as measured by Na
/K
-ATPase and
-glutamyl transpeptidase 1, respectively. The plasma membrane expression of OCT2, MATE1, and MATE2K was quantified and relative expression factors (REFs) were determined as the ratio between the tissue and cell concentrations. Corrections using plasma membrane recovery had minimal impact on REF values (<2-fold). In vitro transporter kinetics of metformin were extrapolated to in vivo using the corresponding REFs in a PBPK model. The simulated metformin exposures were within 2-fold of clinical exposure. These results demonstrate that transporter REFs based on plasma membrane expression enable a prediction of transporter-mediated drug disposition. Such REFs may be estimated without the correction of plasma membrane recovery when the same procedure is applied between different matrices. SIGNIFICANCE STATEMENT: Transporter REFs based on plasma membrane expression enable in vitro-in vivo extrapolation of transporter kinetics. Plasma membrane recoveries as determined by the quantification of sodium-potassium adenosine triphosphatase were comparable between the in vitro and in vivo systems used in the present study, and therefore had minimal impact on the transporter REF values.
Myeloid cell leukemia 1 (MCL-1) is a BCL-2 family protein that has been implicated in the progression and survival of multiple tumor types. Herein we report a series of MCL-1 inhibitors that emanated ...from a high throughput screening (HTS) hit and progressed via iterative cycles of structure-guided design. Advanced compounds from this series exhibited subnanomolar affinity for MCL-1 and excellent selectivity over other BCL-2 family proteins as well as multiple kinases and GPCRs. In a MCL-1 dependent human tumor cell line, administration of compound 30b rapidly induced caspase activation with associated loss in cell viability. The small molecules described herein thus comprise effective tools for studying MCL-1 biology.
A flux dialysis method to measure unbound fraction (
) of compounds with high protein binding and other challenging properties was tested and validated. This method is based on the principle that the ...initial flux rate of a compound through a size-excluding dialysis membrane is proportional to the product of the compound initial concentration,
, and unbound dialysis membrane permeability (
). Therefore,
can be determined from the initial concentration and flux rate, assuming membrane
is known. Compound initial flux rates for 14 compounds were determined by dialyzing human plasma containing compound (donor side) versus compound-free plasma (receiver side) and measuring the rate of compound appearance into the receiver side. Eleven compounds had known
values obtained from conventional methods (ranging from 0.000013 to 0.22); three compounds (bedaquiline, lapatinib, and pibrentasvir) had previously qualified
values (e.g., <0.001)
estimated from flux rates and known
values did not meaningfully differ among the compounds and were consistent with previously published values, indicating that
is a constant for the dialysis membrane. This
constant and the individual compound flux rates were used to calculate
values. The flux dialysis
values for the 11 compounds were in good agreement with their reported
values (all within 2.5-fold;
= 0.980), confirming the validity of the method. Furthermore, the flux dialysis method allowed discrete
to be estimated for the three compounds with previously qualified
Theoretical and experimental advantages of the flux dialysis method over other dialysis-based protein binding methods are discussed.
Amorphous solid dispersion (ASD) is a widely employed formulation technique for drugs with poor aqueous solubility. Polymers are integral components of ASDs, but mechanisms by which polymers lead to ...the generation and maintenance of supersaturated solutions, which enhance oral absorption in vivo, are poorly understood. Herein, a diverse group of newly synthesized cellulose derivatives was evaluated for their ability to inhibit crystallization of enzalutamide, a poorly soluble compound used to treat prostate cancer. ASDs were prepared from selected polymers, specifically a somewhat hydrophobic polymer that was extremely effective at inhibiting drug crystallization, and a less effective, but more hydrophilic, crystallization inhibitor, that might afford better release. Drug membrane transport rate was evaluated in vitro and compared to in vivo performance, following oral dosing in rats. Good correlation was noted between the in vitro diffusion cell studies and the in vivo data. The ASD formulated with the less effective crystallization inhibitor outperformed the ASD prepared with the highly effective crystallization inhibitor in terms of the amount and rate of drug absorbed in vivo. This study provides valuable insight into key factors impacting oral absorption from enabling ASD formulations, and how best to evaluate such formulations using in vitro approaches.
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