Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Glycosylation of thrombospondin type 1 repeats

In the course of mutagenesis of the forth thrombospondin type 1 repeat (TSR4) of the axonal guidance protein F-spondin, one mutant designated ΔDS2,3-TSR4 missing two out of three cystein bridges was found to lack the disaccharide Glcβ1,3-Fuc-O. This protein was demonstrated to be secreted as efficiently as wild type TSR4. In gel filtration experiments unusual features of folding could not be observed. Analysis of the protein by mass spectrometry indicated that the module had acquired an additional glycosylation. Using a combination of tandem CID MS and digestion with glycosidases, it was demonstrated that the tetrasaccharide corresponds to a biantennary NeuNAcα2,3Galβ1,3[NeuNAcα2,6]GalNAc-O- glycan. This carbohydrate structure is also known as the disialyl-T-antigen. The glycan could be demonstrated to be attached exclusively to the same residue (Thr-601) that carries the disaccharide Glcβ1,3-Fuc-O in wild type TSR4. ΔDS2,3-TSR4 is consequently an experimental proof that one residue can be modified with two different types of glycosylation. Since the disaccharide Glcβ1,3-Fuc-O is transferred in the Endoplasmatic Reticulum and the disialyl-T-antigen is added in the Golgi, ΔDS2,3-TSR4 has undergone a glycosylation shift involving different cellular organelles. In order to identify the enzymes that recognized the module and modify the Thr-601 residue ΔDS2,3-TSR4-derived peptides were screened in an in vitro assay. It was found that GalNAc T1 and GalNAc T3 are capable to add GalNAc moieties. GalNAc T1 was found to glycosylate as well Thr-601 and Ser-596. GalNAc T3 was identified to be specific for Thr-601, thus mimicking the situation observed in vivo. The role of this transferase could be further confirmed in vivo by co-expression experiments of GalNAc T3 together with ΔDS2,3-TSR4 in CHO-K1 cells. These experiments resulted in significant increase of the disialyl-T-antigen on glycopeptides of ΔDS2,3-TSR4. The results further demonstrated that after initiation of mucin-type Oglycosylation by a glycosyltransferase such as GalNAc T3 the carbohydrate is extended to the tetrasaccharide of a disialyl-T-antigen type in various cell types. Interestingly the same O-glycan was also identified in the rat axonal guidance protein F-spondin. Fspondin had previously been described to be C-mannosylated on tryptophans and to contain the disaccharide Glcβ1,3Fuc-O-Ser/Thr. Using low energy CID tandem MS in combination with glycosidases and specific sugar-binding lectins, it was possible to map the disialyl-T-antigen to a peptide located in the N-terminal reelin/spondin domain. Fspondin is therefore the first protein known to contain C-mannosylation, the disaccharide Glcβ1,3Fuc-O-Ser/Thr, N-glycosylation and also the tetrasacchride NeuNAcα2,3Galβ1,3 [NeuNAcα2,6]GalNAc-O-. In order to study the glycosylation of thrombospondin type 1 repeats of F-spondin in more detail, the fourth (TSR4) and four consecutive thrombospondin type 1 repeats (TSR1-4) were expressed in the mammalian cell line HEK-EBNA. In order to facilitate purification, the proteins were expressed as fusion proteins designated TSR4fchis and TSR1-4fchis. The proteins were purified in high amounts and C-mannosylation as well as the modification with the disaccharide Glcβ1,3-Fuc-O were examined by LC-MS. It was found that the TSR4fchis and TSR1-4fchis proteins carried C-mannosylation on their tryptophans and Glcβ1,3-Fuc-O on their serines or threonines in the consensus sequence C1X2–3S/TC2X2G. Quantification of the amount of glycosylation on the TSR4fchis protein revealed that the first tryptophan in the WXXW motif is almost quantitatively C-mannosylated, the second tryptophan shows partial C-mannosylation and the threonine in the consensus sequence C1X2–3S/TC2X2G was predominantly modified with the disaccharide Glcβ1,3-Fuc-O. In order to have non-glycosylated TSRs, TSR4fchis and TSR1-4fchis were expressed in the methylotrophic yeast Pichia pastoris. TSR4fchis could be successfully expressed and purified in amounts comparable to correctly folded TSR4 expressed in E. coli. It was found that this host is negative for Cmannosylation and O-fucosylation. However, indirect evidence indicated, that TSR4fchis expressed in yeast had undergone hyperglycosylation of the fusion protein. Therefore a direct comparison of TSR4fchis from HEK-EBNA and TSR4fchis from P. pastoris was not possible

Review of the Technical, Toxicological, and PKPD Considerations for Conducting Inhalation Toxicity Studies on Biologic Pharmaceuticals—The Outcome of a Cross-Industry Working Group Survey

The inhaled route is still a relatively novel route for delivering biologics and poses additional challenges to those encountered with inhaled small molecules, further complicating the design and interpretation of toxicology studies. A working group formed to summarize the current knowledge of inhaled biologics across industry and to analyze data collated from an anonymized cross-industry survey comprising 12 inhaled biologic case studies (18 individual inhalation toxicity studies on monoclonal antibodies, fragment antibodies, domain antibodies, oligonucleotides, and proteins/peptides). The output of this working group provides valuable insights into the issues faced when conducting toxicology studies with inhaled biologics, including common technical considerations on aerosol generation, use of young and sexually mature nonhuman primates, pharmacokinetic/pharmacodynamic modeling, exposure and immunogenicity assessment, maximum dose setting, and no observed adverse effect levels determination. Although the current data set is too small to allow firm conclusions, testing of novel biologics remains an active area and is likely to remain so for molecules where delivery via the inhaled route is beneficial. In the future, it is hoped others will continue to share their experiences and build on the conclusions of this review to further improve our understanding of these complex issues and, ultimately, facilitate the safe introduction of inhaled biologics into clinical use

Ask the Experts: biologics by LC–MS

no abstract availabl

Bioanalysis spotlight on Hybrid LBA/LC–MS Commentary: Carsten Krantz

Commentary on a survey condcuted by Bioanalysis Zone and to be published on their websid

2016 White Paper on Recent Issues in Bioanalysis: Focus on Biomarker Assay Validation (BAV) (Part 2 – Hybrid LBA/LCMS and Input from Regulatory Agencies)

This year marked the 10th anniversary edition of the Workshop on Recent Issues in Bioanalysis (10th WRIB), which was held in Orlando, Florida from April 18-22, 2016. Nearly 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations (CROs), and regulatory agencies worldwide were in attendance. In addition to the 3 focused and sequential workshop days, there were 6 advanced training sessions throughout the week providing attendees multiple choices to combine core workshop days and training, thereby maximizing their learning process in many fields of drug development expertise. As with prior WRIBs, this 10th edition was specifically designed to facilitate sharing, reviewing, discussing and agreeing upon scientific and regulatory approaches to address the most recent issues in both small and large molecule bioanalysis, biomarkers, and immunogenicity. The areas of interest included LCMS, hybrid LBA/LCMS as well as LBA/cell-based approaches

Development of an automated, interference-free, immunoaffinity-based mass spectrometric assay for quantification a therapeutic monoclonal antibody in human sera

Hybrid ligand binding-mass spectrometry assays are increasingly being applied to quantitative measurement of proteins in biological matrices. These assays combine the high specificity and enrichment capabilities of selective affinity reagents with the unmatched specificity and unequivocal analyte identification provided by mass spectrometry. Often these assays involve initial analyte enrichment at the protein level. Although these assays can work well, they are limited by the lack of appropriate well-characterized affinity reagents and, as with some immunoassays, may suffer from interferences caused by off-target protein binding, autoantibodies and anti-immunoglobulin antibodies. To address such problems, the method known as stable isotope standards and capture by anti-peptide antibodies (SISCAPA) was developed, which involves the use of well characterized, high affinity antibodies to enrich proteotypic peptide surrogates of the target protein followed by their identification and quantitation by mass spectrometry. The SISCAPA method involves tryptic digestion of the proteins in the sample matrix which destroys unwanted antibodies and other interfering proteins. Here we report the development of an automated, SISCAPA-based two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) assay for measurement of total levels a monoclonal antibody currently in clinical development. An affinity purified polyclonal antibody specific for a peptide in the complementarity determining region (CDR) of the antibody was used to enrich the surrogate peptide from trypsin-digested human plasma, followed by 2D-LC-MS/MS quantitation. The assay was compared to a standard ligand binding assay currently being used for the antibody quantitation. The data indicate that the peptide-based SISCAPA-2D-LC-MS/MS assay is a valuable alternative to specific protein quantification assays devoid of target or binding protein interference

Quantitative analysis of factor P (Properdin) in monkey serum using immunoaffinity capturing in combination with LC-MS/MS

Factor P (Properdin) is an endogenous protein found in the serum of human and other animals. As the only known positive regulator of the complement system, factor P plays a key role in innate immune defense against many diseases. Quantification of factor P in serum samples is important in understanding the pharmacodynamics (PD) of drug candidates. However, developing a suitable quantification method is challenging because of the endogenous nature of the protein. In the present work, a sensitive and selective method combining immunoaffinity capturing and LC-MS/MS has been developed and validated for the first time for quantitative analysis of factor P in monkey serum. True monkey serum was used for the preparation of calibration standards and QCs. The method was linear and rugged over a dynamic range of 125 to 25000 ng/mL using 50 uL sample volume. The intra- and inter-run precision (%CV) of the QC sample results were within 7.2% (CV) and the accuracy (%Bias) within ±16.8% across all QC concentrations evaluated. Other validation parameters, including stability under various conditions, extraction recovery and matrix effect, all met the acceptance criteria. The validated method was successfully implemented for the quantitative analysis of factor P in monkey serum samples in support of a preclinical PD study to assess response of a drug candidate. Factor P (Properdin), Immunoaffnity capturing, LC-MS/MS, monkey seru

Use of a generic LC-MS/MS assay to characterize atypical PK profile of a biotherapeutic monoclonal antibody

Background: The fully human monoclonal antibody mAb123 which binds to and neutralizes chemokine motif ligand-21 (CCL21) displays a faster clearance in cynomolgus monkey compared to typical IgG kinetics. Two variants of LC-MS/MS assays were developed to better understand this rapid clearance, previously established by ELISA. Results: Excellent correlation of LC-MS/MS PK data with ELISA data confirmed the rapid clearance of mAb123, indicating that this is an intrinsic property of the molecule. In one study, two animals tested positive for anti-mAb123 antibodies, in one animal these antibodies resulted in lower serum concentrations of mAb123. The LC-MS/MS assay indicates that the underlying reason for this apparent rapid disappearance of mAb123 from serum is due to catabolism, rather than interference of anti-mAb123 antibodies in the ELISA assay. Conclusions: The data illustrate that in cases of unexpected results from ligand binding assays, application of orthogonal bioanalytical techniques such as LC-MS/MS can add value