Evaluation of low volume sampling devices for a pharmacodynamic biomarker analysis: Challenges and solutions.

Low volume sampling technologies have gained popularity as they are minimally invasive, reduce patient burden, enhance population diversity, and have the potential to facilitate decentralized clinical trials. Herein, we validated a Gyrolab assay to measure soluble Mucosal Addressin Cell Adhesion Molecule 1 (sMAdCAM-1) in dried blood samples collected using two low volume sampling devices, Mitra and Tasso-M20. This validated assay was implemented in a proof-of-concept study to compare three low volume sampling devices (Mitra, Tasso-M20 and TassoOne Plus) with serum collected via venipuncture from healthy volunteers receiving etrolizumab. We observed significantly higher concentration of sMAdCAM-1 in dried blood samples collected using Mitra and Tasso-M20 compared to serum in some paired samples, which was attributed to interference from the dried blood extraction buffer. To mitigate this interference, samples required substantial dilution into the appropriate buffer, which negatively impacted the detectability of sMAdCAM-1 with the Gyrolab assay. By employing the Quanterix single molecule array (Simoa), known for its superior assay sensitivity, the interference was minimized in the diluted samples. Both liquid blood collected in TassoOne Plus and dried blood collected using Mitra and Tasso-M20 demonstrated great concordance with serum for sMAdCAM-1 measurement. However, a bias was observed in Mitra dried blood samples, presumably due to the different sample collection sites in comparison with venipuncture and Tasso devices. Our study highlights the potential of low volume sampling technologies for biomarker analysis, and underscores the importance of understanding the challenges and limitations of these technologies before integrating them into clinical studies.

Evaluation of intra- and inter-individual variations in plasma belimumab concentrations in adult patients with systemic lupus erythematosus.

In this study, plasma belimumab concentrations were measured over the course of treatment in systemic lupus erythematosus (SLE) patients on belimumab therapy, and intra- and interindividual variations in plasma belimumab concentration were evaluated. A single-center prospective study was conducted at Oita University Hospital to evaluate trough plasma concentrations over the course of treatment in 13 SLE patients treated with intravenous belimumab. Plasma belimumab concentrations were measured by a validated ultra-high performance liquid chromatography with tandem mass spectrometry method. The median age of the patients was 40 (interquartile range: 35-51) years and the median weight was 51.8 (47.0-58.1) kg. A mean of 9.4 (range: 1-13) blood samples was collected per patient at routine visits. The mean (± SD) plasma belimumab concentration was 33.4 ± 11.9 µg/mL in the patient with the lowest concentration and 170.0 ± 16.6 µg/mL in the patient with the highest concentration, indicating a 5-fold difference between patients. On the other hand, the within-patient coefficient of variation ranged from 7.1% to 35.7%, showing no large variations. No significant correlation was observed between plasma belimumab concentration and belimumab dose (mg/kg) (Spearman's rank correlation coefficient = 0.22, p = .54). Examinations of trough plasma belimumab concentrations over the course of treatment in patients with SLE showed small intraindividual variation but large interindividual variation. Plasma belimumab trough concentration varied widely among patients administered the approved dose.

Mitigating target interference challenges in bridging immunogenicity assay to detect anti-tocilizumab antibodies.

Aim: An assay to detect anti-tocilizumab antibodies in the presence of high levels of circulating target and drug is needed for immunogenicity assessment in comparative clinical studies.Methods: An assay was developed and validated using a combination of blocking agents and dilutions to overcome target interference challenges.Results: No false-positive signal was detected in serum samples spiked with 350-500 ng/ml of IL-6 receptor. As low as 50 ng/ml of positive control antibodies could be detected in the presence of either 500 ng/ml of IL-6 or 250 µg/ml of the drug product. Assay also demonstrated high sensitivity, selectivity and precision.Conclusion: A robust, easy to perform immunogenicity assay was developed and validated for detecting anti-tocilizumab antibodies.

[Box: see text].

Application of newly developed and validated LC-MS/MS method for pharmacokinetic study of adagrasib and pembrolizumab simultaneously in rat plasma.

Non-small cell lung cancer (NSCLC) is a significant subtype of lung cancer, and poses a dangerous global threat. One of the current approaches of NSCLC treatment is a combination therapy of adagrasib and pembrolizumab. Accurate monitoring of these drug concentrations in biological fluids is critical for treatment efficacy. Since no method was reported for simultaneous estimation of these drugs, this study focuses on the development of a validated LC-MS/MS bioanalytical method for simultaneous quantification of Adagrasib and Pembrolizumab in rat plasma. The analytes were extracted from the biological matrix through liquid-liquid extraction techniques using acetonitrile as extraction solvent. The analytes were separated on a Waters X-bridge phenyl C18 column, with a mixture of acetonitrile: 0.1 % TFA in water (50: 50 v/v) as mobile phase at an isocratic flow rate of 1.0 mL/min with a runtime of about 5 min. Adagrasib (m/z 605.12 → 201.62), Pembrolizumab (m/z 146.32 → 85.15), and Sotorasib (m/z 561.59 → 218.92) were determined by recording the mass spectra through multiple reaction monitoring in positive mode. The method was validated according to USFDA guidelines. The results demonstrate satisfactory linearity with an r2 value of 0.9998 in the ranges of 40-800 and 10-200 ng/mL, accuracy with mean percentage recovery of 95.22-98.59 % and 96.98-98.57 %, precision indicated with %RSD ranged between 0.39-1.91 % and 0.85-9.03 % for Adagrasib and Pembrolizumab respectively, and other key parameters. The developed method can determine the pharmacokinetic parameters to indicate the efficacy and safety of the drugs, and also can quantify selected drugs simultaneously in biological samples.

Development of time-resolved fluoroimmunoassay with enhanced fluorescence reaction for the quantitation of atezolizumab in plasma.

Atezolizumab (ATZ) is a human monoclonal antibody, which has been granted multiple approvals from the US Food and Drug Administration (FDA) for the immunotherapy of different types of cancer. This study describes the prototype of a time-resolved fluoroimmunoassay (TRFIA) for the quantitation of ATZ in plasma. The assay involved the non-competitive binding of ATZ to its specific antigen [programmed death-ligand 1 (PD-L1) protein]. The immune complex formed on the inner surface of the assay plate wells was quantified by anti-human secondary antibody labeled with a chelate of europium-ethylenediaminetetraacetic acid. The enhanced fluorescence signal was generated by an enhanced fluorescence solution composed of thenoyltrifluoroacetone, trioctylphosphine oxide, and Triton X-100. The conditions of the TRFIA were refined, and its optimum procedures were established. The assay was validated in accordance with the immunoassay validation guidelines, and all the validation parameters were acceptable. The working range of the assay was 20-1000 pg mL-1, and its limit of quantitation was 20 pg mL-1. The assay was applied to the quantitation of ATZ in plasma samples with satisfactory accuracy and precision. The proposed TRFIA has significant benefits over the existing methodologies for the quantitation of ATZ in clinical settings.

Analytical and pharmacological consequences of the in vivo deamidation of trastuzumab and pertuzumab.

A liquid chromatography-tandem mass spectrometry method is presented for the quantitative determination of the in vivo deamidation of the biopharmaceutical proteins trastuzumab and pertuzumab at an asparagine in their complementarity determining regions (CDRs). For each analyte, two surrogate peptides are quantified after tryptic digestion of the entire plasma protein content: one from a stable part of the molecule, representing the total concentration, and one containing the deamidation-sensitive asparagine, corresponding to the remaining non-deamidated concentration. Using a plasma volume of 10 µL and a 2-h digestion at pH 7, concentrations between 2 and 1000 µg/mL can be determined for the various protein forms with values for bias and CV below 15% and without unacceptable in vitro deamidation taking place. A considerable difference between the total and non-deamidated concentrations, and thus a substantial degree of deamidation, was observed in plasma for both trastuzumab and pertuzumab. After a 56-day forced deamidation test 40% of trastuzumab and 68% of pertuzumab was deamidated, while trastuzumab and pertuzumab showed up to 47% and 35% of deamidation, respectively, in samples collected from breast cancer patients during treatment with a combination of both drugs. A good correlation between the non-deamidated concentration results and those of a receptor binding assay indicate a loss of receptor binding for both trastuzumab and pertuzumab along with the deamidation in their CDRs. Deamidated trastuzumab also lost its capability to inhibit the growth of breast cancer cells in a cell-based viability assay, suggesting a relation between the degree of deamidation and pharmacological activity.

Prevention of acute graft-versus-host disease in adult T-cell leukemia-lymphoma patients who received mogamulizumab before allogeneic hematopoietic cell transplantation.

Mogamulizumab (Mog) is effective against adult T-cell leukemia-lymphoma (ATL), but as we reported previously, Mog increases the incidence of severe acute GVHD when administered before allogeneic hematopoietic cell transplantation (allo-HCT). Here, we report the cases of two ATL patients who did not develop acute GVHD despite receiving Mog before allo-HCT. Case 1: a 63-year-old female who underwent allo-HCT from an HLA-matched donor 2 months after the last dose of Mog. Case 2: a 47-year-old male with ATL that relapsed 3 months after first allo-HCT. He received eight doses of Mog and underwent a second allo-HCT from a haploidentical donor 4 months after the last dose of Mog. Mog blood levels were measured and lymphocytes analyzed by mass cytometry. Mog blood levels measured before starting the conditioning regimens were low. A small proportion of regulatory T cells (Tregs) was detected before and shortly after allo-HCT. When using Mog before allo-HCT, it is important to consider the number of Mog doses and the interval from the last dose of Mog to allo-HCT. Analyzing Mog blood levels and Treg counts before and after allo-HCT should also be useful.

Population pharmacokinetic and exposure-response analyses of elotuzumab plus pomalidomide and dexamethasone for relapsed and refractory multiple myeloma.

PURPOSE: Elotuzumab plus pomalidomide/dexamethasone (E-Pd) demonstrated efficacy and safety in relapsed and refractory multiple myeloma (RRMM). The clinical pharmacology of elotuzumab [± lenalidomide/dexamethasone (Ld)] was characterized previously. These analyses describe elotuzumab population pharmacokinetics (PPK), the effect of Pd, and assess elotuzumab exposure-response relationships for efficacy and safety in patients with RRMM. METHODS: A previously established PPK model was updated with E-Pd data from the phase 2 ELOQUENT-3 study (NCT02654132). The dataset included 8180 serum concentrations from 440 patients with RRMM from 5 clinical trials. Elotuzumab PK parameter estimates were used to generate individual daily time-varying average concentrations (daily Cavg) for multi-variable time-to-event exposure-response analyses of progression-free survival (PFS) and time to the first occurrence of grade 3 + adverse events (AEs) in RRMM. RESULTS: Elotuzumab PK were well-described by a two-compartment model with parallel linear and Michaelis-Menten elimination from the central compartment (Vmax) and non-renewable target-mediated elimination from the peripheral compartment (Kint). Co-administration with Pd resulted in a 19% and 51% decrease in elotuzumab linear clearance and Kint, respectively, versus Ld; steady-state exposures were similar. Vmax increased with increasing serum M-protein. Hazard ratios (95% confidence intervals) for daily Cavg were 0.9983 (0.9969-0.9997) and 0.9981 (0.9964-0.9998) for PFS and grade 3 + AEs, respectively. CONCLUSIONS: The PPK model adequately described the data and was appropriate for determining exposures for exposure-response analyses. There were no clinically relevant differences in elotuzumab exposures between Pd and Ld backbones. In ELOQUENT-3, increasing elotuzumab daily Cavg prolonged PFS without increasing grade 3 + AEs.

Clinical Immunogenicity Evaluation of Eptinezumab, a Therapeutic Humanized Monoclonal Antibody Targeting Calcitonin Gene-Related Peptide (CGRP) for the Preventive Treatment of Migraine.

Background: Eptinezumab is a humanized monoclonal antibody that selectively binds calcitonin gene-related peptide and is indicated for the preventive treatment of migraine in adults. This analysis characterizes the immunogenic profile of eptinezumab using data from clinical trials of eptinezumab for migraine prevention. Methods: Immunogenicity data were collected from five studies that included 2076 patients with episodic or chronic migraine treated with eptinezumab at dose levels ranging from 10 to 1000 mg, administered intravenously for up to 4 doses at 12-week intervals. Anti-drug antibody (ADA) results were available from 2074 of these patients. Four studies were randomized, double-blind, placebo-controlled trials with ADA monitoring for up to 56 weeks; one was a 2-year, open-label, phase 3 safety study with ADA monitoring for 104 weeks. Patients who had a confirmed ADA-positive result at the end-of-study visit were monitored for up to 6 additional months. Development of ADA and neutralizing antibodies (NAbs) were evaluated to explore three key areas of potential impact: pharmacokinetic exposure profile (eptinezumab trough plasma concentrations), efficacy (change in monthly migraine days), and safety (rates of treatment-emergent adverse events). These studies included methods designed to capture the dynamics of a potential humoral immune response to eptinezumab treatment, and descriptive analyses were applied to interpret the relationship of ADA signals to drug exposure, efficacy, and safety. Results: Pooled across the five clinical trials, treatment-emergent ADAs and NAbs occurred in 15.8 and 6.2% of eptinezumab-treated patients, respectively. Highly consistent profiles were observed across all studies, with initial onset of detectable ADA observed at the week 8 measurement and maximal ADA frequency and titer observed at week 24, regardless of eptinezumab dose level or number of doses. After 24 weeks, the ADA and NAb titers steadily declined despite additional doses of eptinezumab. Interpretation: Collectively, these integrated analyses did not demonstrate any clinically meaningful impact from ADA occurring after treatment with eptinezumab. The ADA profiles were low titer and transient, with the incidence and magnitude of ADA or NAb responses declining after week 24. Development of ADAs and NAbs did not impact the efficacy and safety profiles of eptinezumab.

Bioanalytical Challenges due to Prior Checkpoint Inhibitor Exposure: Interference and Mitigation in Drug Concentration and Immunogenicity Assays.

Monoclonal antibodies (mAbs) are a leading class of biotherapeutics. In oncology, patients often fail on early lines of biologic therapy to a specific target. Some patients may then enroll in a new clinical trial with a mAb specific for the same target. Therefore, immunoassays designed to quantify the current mAb therapy or assess immunogenicity to the drug may be susceptible to cross-reactivity or interference with residual prior biologics. The impact of two approved anti-PD-1 mAbs, pembrolizumab and nivolumab, was tested in several immunoassays for cemiplimab, another approved anti-PD-1 mAb. The methods included a target-capture drug concentration assay, a bridging anti-drug antibody (ADA) assay and a competitive ligand-binding neutralizing antibody (NAb) assay. We also tested bioanalytical strategies to mitigate cross-reactivity or interference in these assays from other anti-PD-1 biologics. Both pembrolizumab and nivolumab cross-reacted in the cemiplimab drug concentration assay. This was mitigated by addition of antibodies specific to pembrolizumab or nivolumab. ADA specific for pembrolizumab and nivolumab did not interfere in the cemiplimab ADA assay. However, pembrolizumab and nivolumab generated a false-positive response in a target-capture NAb assay. Our results demonstrate that similar exogenous pre-existing anti-PD-1 mAbs (biotherapeutics) such as pembrolizumab and nivolumab are detected and accurately quantified in the cemiplimab drug concentration assay. However, once steady state is achieved for the new therapy, prior biologics would likely not be detected. Cross-reactivity and interference in immunoassays from previous treatment with class-specific biotherapeutic(s) pose significant bioanalytical challenges, especially in immuno-oncology.

A randomized, double-blind, single-dose study (LAVENDER) to assess the safety, tolerability, pharmacokinetics, and immunogenicity of a combined infusion of ABP 980 and pertuzumab in healthy subjects.

PURPOSE: ABP 980 (KANJINTI™) is a biosimilar to reference product HERCEPTIN® (trastuzumab RP). The goal of this study was to characterize the safety, tolerability, and immunogenicity of ABP 980 plus pertuzumab (PERJETA®) when co-administered in a single infusion bag in healthy subjects. METHODS: This randomized, double-blind, single-dose, 2-arm, parallel-group study (LAVENDER Study) evaluated an intravenous (IV) infusion of ABP 980 (6 mg/kg) plus pertuzumab (420 mg) combined in a single infusion bag relative to an IV infusion of trastuzumab RP (6 mg/kg) plus pertuzumab (420 mg) combined in a single infusion bag given over 60 min. The subjects were followed for 92 days post dosing. RESULTS: A total of 42 subjects were enrolled in the study and treated with investigational product. Due to an operational issue during dosing, the first 6 subjects enrolled in the study were replaced. A total of 36 randomized subjects, n = 18 for ABP 980 plus pertuzumab and n = 18 for trastuzumab RP plus pertuzumab, were treated. Resulting serum concentrations of ABP 980 and trastuzumab RP were similar. There were no serious adverse events, no deaths, and no cardiac disorders during the study. No subject developed anti-drug antibodies throughout the study. CONCLUSIONS: This study demonstrated the safety and tolerability of ABP 980 and pertuzumab admixture in a single infusion bag. The safety profiles and pharmacokinetic parameters of ABP 980 and pertuzumab were consistent with what is known for trastuzumab RP and pertuzumab. CLINICAL TRIAL LISTING: EudraCT 2018-002903-33.

The Posology of Dupilumab in Pediatric Patients With Atopic Dermatitis.

Dupilumab demonstrated efficacy with an acceptable safety profile in two randomized, double-blind, placebo-controlled, parallel-group, phase III trials in adolescents (12-17 years; LIBERTY AD ADOL) and children (6-11 years; LIBERTY AD PEDS) with atopic dermatitis (AD) treated for 16 weeks. Here, we present the pharmacokinetic profiles and exposure-response (E-R) relationships of dupilumab that guided the posology in these populations. A total of 251 adolescent patients with moderate-to-severe AD were randomized to subcutaneous dupilumab monotherapy every 2 weeks (q2w; 200 mg q2w, baseline weight < 60 kg; 300 mg q2w, ≥ 60 kg), dupilumab 300 mg every 4 weeks (q4w; non-weight tiered), or placebo; 367 children with severe AD were randomized to dupilumab q2w (100 mg q2w, baseline weight < 30 kg; 200 mg q2w, ≥ 30 kg), dupilumab 300 mg q4w, or placebo. Children received concomitant topical corticosteroids in addition to dupilumab, and loading doses were administered at the start of therapy. Mean dupilumab trough concentrations at week 16 for weight subcategories in each dosing regimen were compared with adult exposures for the approved dupilumab 300 mg q2w regimen. Positive E-R relationships were demonstrated between dupilumab trough concentrations and AD outcome measures across patient populations and regimens; no relationship was observed with treatment-emergent conjunctivitis. Based on these analyses, a weight-tiered posology was proposed for adolescents (200/300 mg q2w in patients 30-< 60 kg/≥ 60 kg) and children (300 mg q4w in patients 15-< 30 kg, 200 mg q2w in patients 30-< 60 kg) with moderate-to-severe AD.

Overall survival in metastatic melanoma correlates with pembrolizumab exposure and T cell exhaustion markers.

Trial data support an absence of an exposure-survival relationship for pembrolizumab. As these relationships remain unexamined in a real-world setting, we determined them in metastatic melanoma prospectively in an observational study. Translational objectives included identifying biomarkers of progressive disease (PD). Checkpoint blockade naïve patients receiving 2 mg/kg Q3W pembrolizumab had pharmacokinetic and clinical outcome data collected. Trough, a valid surrogate for drug exposure, was assessed using ELISA. T-cell exhaustion and chemokine markers were determined using flow cytometry. Geometric means of exposures and biomarkers were tested against objective response groups using one-way ANOVA. The cohort was split by the median into high versus low pembrolizumab exposure groups. Kaplan-Meier progression-free survival (PFS) and overall survival (OS) curves were estimated for high versus low exposure, compared using the log rank test. The high pembrolizumab exposure group (n = 14) experienced substantially longer median OS (not reached vs. 48 months, p = .014), than the low exposure group (n = 14). A similar positive exposure PFS relationship was found (median not reached vs. 48 months, p = .045). The frequency of TIM-3 expression on CD4+ T cells was significantly higher in PD (mean 27.8%) than complete response (CR) (13.38%, p = .01) and partial response (12.4%, p = .05). There was a near doubling of CXCR6 and TIM-3 co-expression on CD4+ T cells in PD (mean 23.3%) versus CR (mean 11.4, p = .003) and partial response (9.8%, p = .0001). We describe positive exposure-PFS and exposure-OS relationships for pembrolizumab in metastatic melanoma. TIM-3, alongside co-expression of CXCR6 and TIM-3 on circulating CD4+ T cells are potential bio markers of treatment failure.

Comparing singlet and duplicate immunogenicity assay in human plasma for pembrolizumab using Gyrolab®.

Aim: For decades, the traditional approach for ligand-binding assays has been to generate two measurements from adjacent wells on the plate. In recent years, scientists have investigated the true benefit of this 'duplicate analysis' by looking back at previously generated bioanalytical data with the conclusion that the benefits are negligible. Materials & methods: We demonstrated a method development approach to determine the best number of replicate measurements of an immunogenicity assay. We used an anti-pembrolizumab immunogenicity assay on Gyrolab® to challenge the traditional use of duplicate measurements as we compare it to singlet measurement and show a balanced design for assessing the cut-point in singlet. Results & conclusion: We introduced the concept of calculating the maximum drug tolerance during method development. In this method, we found no practical benefit for duplicate analysis and go further in recommending that singlet analysis should be considered the default for all ligand-binding assays.

Results of a Dose-Finding Phase 1b Study of Subcutaneous Atezolizumab in Patients With Locally Advanced or Metastatic Non-Small Cell Lung Cancer.

Intravenous (IV) atezolizumab is approved for non-small cell lung and other cancers. Subcutaneous (SC) atezolizumab coformulated with recombinant human hyaluronidase, a permeation enhancer for SC dispersion and absorption, is being developed to improve treatment options, reduce burden, and increase efficiency for patients and practitioners. IMscin001 (NCT03735121), a 2-part, open-label, global, multicenter, phase 1b/3 study, is evaluating the pharmacokinetics (PK), safety, and efficacy of SC atezolizumab. The part 1 (phase 1b) objective was determination of an SC atezolizumab dose yielding a serum trough concentration (Ctrough ) comparable with IV. Patients enrolled in 3 cohorts received SC atezolizumab 1800 mg (thigh) once (cohort 1), 1200 mg (thigh) every 2 weeks for 3 cycles (cohort 2), or 1800 mg (abdomen) every 3 weeks cycle 1, then cycles 2 and 3 (thigh) every 3 weeks (cohort 3). In subsequent cycles, IV atezolizumab 1200 mg every 3 weeks was administered until loss of clinical benefit. SC atezolizumab 1800 mg every 3 weeks and 1200 mg every 2 weeks provided similar Ctrough and area under the curve values in cycle 1 to the corresponding IV atezolizumab reference, was well tolerated, and exhibited a safety profile consistent with the established IV formulation. Exposure following SC injection in the abdomen was lower (20%, 28%, and 27% for Ctrough , maximum concentration, and area under the concentration-time curve from time 0 to day 21, respectively) than in the thigh. Part 1 SC and IV PK data were analyzed using a population PK modeling approach, followed by simulations. Part 2 (phase 3) will now be initiated to demonstrate that SC atezolizumab PK exposure is not lower than that of IV.

A robust enzyme-linked immunosorbent assay to measure serum ramucirumab concentrations.

Aim: Ramucirumab, an anti-VEGFR2 monoclonal antibody, has been approved for the treatment of metastatic gastric and colorectal cancer. An assay measuring ramucirumab serum concentrations was needed to investigate its pharmacokinetics and concentration-response relationship. Results: An ELISA was developed and validated according to the international guidelines for ligand-binding assays. Ramucirumab calibration standards ranged from 0.125 to 40 mg/l. Low, middle and high quality controls were spiked at 0.2, 4 and 8 mg/l, respectively. The limits of quantification were established to be 0.125 and 10 mg/l for LLOQ and ULOQ, respectively. No cross-reactivity with anti-VEGF or anti-EGFR was detected. Conclusion: This in-house-developed ELISA is sensitive, accurate, reproducible and suitable for pharmacokinetic studies of ramucirumab.

Clearing drug interferences in myeloma treatment using mass spectrometry.

OBJECTIVE: To explore the possibility of using a combination of a rapid MALDI-TOF-MS method (Mass-Fix) in conjunction with higher resolution LC-ESI-QTOF-MS (miRAMM) measurements to discriminate the IgG kappa M-protein from daratumumab, elotuzumab and isatuximab in myeloma patients. DESIGN & METHODS: 86 patients with an IgG kappa M-protein were spiked with therapeutic levels of the drugs and examined by Mass-Fix and miRAMM to establish the percent of cases that could be resolved by each method. The method was then applied to 21 samples from patients receiving one of the drugs. RESULTS: Mass-Fix was capable of resolving the t-mAb from M-protein for 87 percent of the spiked samples. For the cases unresolved by Mass-Fix, miRAMM was capable of resolving the remaining drug interferences. The 21 IgG kappa myeloma patients that were receiving the drugs were all resolved by Mass-Fix. CONCLUSION: This proposed algorithm allows use of a clinical available assay (Mass-Fix) while maximizing the number of cases that can accurately resolve the t-mAb from the M-protein.

A bridging immunogenicity assay for anti-cabiralizumab antibodies: overcoming the low assay cut point and drug tolerance challenges.

Background: To support the clinical studies of cabiralizumab, an immunogenicity assay for detecting anti-cabiralizumab antibodies is required. Results: Strategies were developed to overcome two major bioanalytical challenges: poor drug tolerance of the anti-drug antibodies assay and very low cut point observed in the screening and confirmatory assays. By using acid dissociation (400 mM glycine solution at pH 2.0), drug tolerance of 200 µg/ml drug was achieved for both the screening and confirmatory assays. Effects of biological matrix (disease state vs normal serum) and assay conditions (capture/detector reagent concentration, minimum required dilution, acid pretreatment) on assay cut points were systematically evaluated. Conclusion: A bridging immunogenicity assay for detecting anti-cabiralizumab antibodies in human serum has been successfully developed, validated and applied to clinical studies.

Ex Vivo Prediction of Comprehensive Coagulation Potential Using Simulated Blood Concentrations of Emicizumab in Patients with Acquired Hemophilia A.

INTRODUCTION: Emicizumab prophylaxis improves coagulation function in congenital hemophilia A, regardless of inhibitor presence. We recently reported that emicizumab enhanced the coagulant potentials, ex vivo, in plasmas from patients with acquired hemophilia A (PwAHAs) at diagnosis. However, coagulant effects of emicizumab in PwAHAs during the clinical course remain unclear. AIM: To assess ex vivo coagulant effects of emicizumab in PwAHAs during the clinical course. METHODS/RESULTS: Blood samples were obtained from 14 PwAHAs on (median) days 0 and 6 during a severe-bleeding phase, and days 27 and 59 during a reduced-bleeding phase with elevated endogenous factor VIII (FVIII) and decreased inhibitor titers. If administered a single dose of 3 or 6 mg/kg, or two doses at 6 mg/kg followed by 3 mg/kg, estimated plasma emicizumab concentrations (10/5/2.5, 20/10/5, and 30/15/7.5 µg/mL on days 0-7/30/60, respectively) could be used to represent potential changes, based on the half-life (T 1/2: ∼30 days). Emicizumab concentrations that covered maximum plasma concentrations of each dosage were used for spiking on day 0. Ex vivo addition of estimated emicizumab to PwAHA's plasma containing endogenous FVIII and/or inhibitor, without and with recombinant (r)FVIIa administration during immunosuppressive therapy, increased the calculated Ad|min1| values, assessed by clot waveform analysis, and their coagulant potentials were below normal levels. Rotational thromboelastometry revealed that ex vivo emicizumab addition resulted in the further improvement of coagulant potentials in whole bloods when combined with rFVIIa administration. CONCLUSION: Based on ex vivo and in vitro data, emicizumab has the potential to be effective in clinical situations for PwAHAs.

A Novel Total Drug Assay for Quantification of Anti-C5 Therapeutic Monoclonal Antibody in the Presence of Abundant Target.

SKY59 or RO7112689 is a humanized monoclonal antibody against complement protein C5 with pH-dependent C5-binding and neonatal Fc receptor-mediated recycling capabilities, which result in long-lasting neutralization of C5. We developed and validated a novel total drug assay for quantification of target-binding competent SKY59 in the presence of endogenous C5 in cynomolgus monkey plasma. The target-binding competent SKY59 was determined after complex formation by the addition of recombinant monkey C5 using goat anti-human IgG-heavy chain monkey-adsorbed polyclonal antibody as a capture antibody and rabbit anti-C5 monoclonal antibody (mAb) non-competing with SKY59 for detection. The total SKY59 assay was shown to be accurate and precise over the range of 0.05-3.2 µg/mL as well as be tolerant to more than 400 µg/mL of C5 (~ 3000-fold molar excess of target). We also developed and validated a total C5 assay, confirmed selectivity and parallelism, and verified the utility of recombinant monkey C5 for the total C5 assay as well as the total SKY59 assay. Furthermore, we used these validated methods to measure SKY59 and C5 concentrations in cynomolgus monkey plasma samples in a toxicology study. This total drug assay can be applied not only to other antibody therapeutics against shed/soluble targets when a non-competing reagent mAb is available but also for clinical studies when a reagent mAb specific for engineered Fc region on a therapeutic mAb is available.