The quantitative analysis of human immunoglobulin G1 (hIgG1) by mass spectrometry is commonly performed using surrogate peptides after enzymatic digestion. Since some limitations are associated with this approach, a novel workflow is presented by hybridizing ligand binding assay (LBA) with liquid chromatography-high-resolution mass spectrometry (LC-HRMS) for hIgG1 quantification directly at the intact protein level. Different hIgG1s, including a [C]-labeled version used as internal standard, were immuno-enriched from rat serum with a fully automated platform based on streptavidin coated tips and a biotinylated mouse anti-hIgG capture antibody targeting the fragment crystallizable region followed by overnight deglycosylation prior to LC-HRMS analysis. The proposed quantitative workflow utilized extracted ion chromatograms (XICs) from the nondeconvoluted full-scan MS spectrum. The assay was validated in terms of selectivity, sensitivity, accuracy/precision, carry-over, dilution linearity, and reproducibility. Consistent data between the conventional approach based on surrogate peptide analysis and our proposed workflow were obtained in vitro and in vivo with the advantage of a less extensive sample pretreatment. Multiplexing capabilities for simultaneous quantification of different hIgG1s within the same spiked sample were also exemplified. Altogether our results pave the way not only for the thorough application of intact hIgG1 quantification by LBA-LC-HRMS but also as a generic quantitative analytical method for other hIgG isotypes or next generation biotherapeutics.

A series of 5-aryl-2-amino- i midazo t hia d iazole (ITD) derivatives were identified by a phenotype-based high-throughput screening using a blood stage Plasmodium falciparum ( Pf ) growth inhibition assay. A lead optimization program focused on improving antiplasmodium potency, selectivity against human kinases, and absorption, distribution, metabolism, excretion, and toxicity properties and extended pharmacological profiles culminated in the identification of INE963 ( 1 ), which demonstrates potent cellular activity against Pf 3D7 (EC 50 = 0.006 μM) and achieves “artemisinin-like” kill kinetics in vitro with a parasite clearance time of <24 h. A single dose of 30 mg/kg is fully curative in the Pf -humanized severe combined immunodeficient mouse model. INE963 ( 1 ) also exhibits a high barrier to resistance in drug selection studies and a long half-life ( T 1/2 ) across species. These properties suggest the significant potential for INE963 ( 1 ) to provide a curative therapy for uncomplicated malaria with short dosing regimens. For these reasons, INE963 ( 1 ) was progressed through GLP toxicology studies and is now undergoing Ph1 clinical trials.

For antibody drug conjugates (ADCs), the fate of the cytotoxic payload in vivo needs to be well understood to mitigate toxicity risks and properly design the first in-patient studies. Therefore, a distribution, metabolism, and excretion (DME) study with a radiolabeled rat cross-reactive ADC ([ 3 H]DM1-LNL897) targeting the P-cadherin receptor was conducted in female tumor-bearing nude rats. Although multiple components [total radioactivity, conjugated ADC, total ADC, emtansine (DM1) payload, and catabolites] needed to be monitored with different technologies (liquid scintillation counting, liquid chromatography/mass spectrometry, enzyme-linked immunosorbent assay, and size exclusion chromatography), the pharmacokinetic data were nearly superimposable with the various techniques. liquid extraction surface analysis coupled to micro-liquid chromatography-tandem mass spectrometry data proved that the lysine (LYS)-4(maleimidylmethyl) cyclohexane-1-carboxylate-DM1 (LYS-MCC-DM1) catabolite was the only detectable component distributed evenly in the tumor and liver tissue. The mass balance was complete with up to 13.8% 6 0.482% of the administered radioactivity remaining in carcass 168 hours postdose. LNL897-derived radioactivity was mainly excreted via feces (84.5% 6 3.12%) and through urine only to a minor extent (4.15% 6 0.462%). In serum, the major part of radioactivity could be attributed to ADC, while small molecule disposition products were the predominant species in excreta. We show that there is a difference in metabolite profiles depending on which derivatization methods for DM1 were applied. Besides previously published results on LYS-MCC-DM1 and MCC-DM1, maysine and a cysteine conjugate of DM1 could be identified in serum and excreta.