Noncompetitive immunoassay optimized for pharmacokinetic assessments of biologically active efruxifermin.

Efruxifermin (EFX) is a homodimeric human IgG1 Fc-FGF21 fusion protein undergoing investigation for treatment of liver fibrosis due to nonalcoholic steatohepatitis (NASH), a prevalent and serious metabolic disease for which there is no approved treatment. Biological activity of FGF21 requires its intact C-terminus, which enables binding to its obligate co-receptor ß-Klotho on the surface of target cells. This interaction is a prerequisite for FGF21 signal transduction through its canonical FGF receptors: FGFR1c, 2c, and 3c. Therefore, the C-terminus of each FGF21 polypeptide chain must be intact, with no proteolytic truncation, for EFX to exert its pharmacological activity in patients. A sensitive immunoassay for quantification of biologically active EFX in human serum was therefore needed to support pharmacokinetic assessments in patients with NASH. We present a validated noncompetitive electrochemiluminescent immunoassay (ECLIA) that employs a rat monoclonal antibody for specific capture of EFX via its intact C-terminus. Bound EFX is detected by a SULFO-TAG™-conjugated, affinity purified chicken anti-EFX antiserum. The ECLIA reported herein for quantification of EFX demonstrated suitable analytical performance, with a sensitivity (LLOQ) of 20.0 ng/mL, to support reliable pharmacokinetic assessments of EFX. The validated assay was used to quantify serum EFX concentrations in a phase 2a study of NASH patients (BALANCED) with either moderate-to-advanced fibrosis or compensated cirrhosis. The pharmacokinetic profile of EFX was dose-proportional and did not differ between patients with moderate-to-advanced fibrosis and those with compensated cirrhosis. This report presents the first example of a validated pharmacokinetic assay specific for a biologically active Fc-FGF21 fusion protein, as well as the first demonstration of use of a chicken antibody conjugate as a detection reagent specific for an FGF21 analog.

Critical reagent inventory management system and web portal specifically optimized for supporting external clients.

High-quality critical reagents are essential for the establishment of robust ligand binding assays to support regulated bioanalysis. To ensure consistency in assay performance over the lifetime of a project, a well-defined set of processes is needed for critical reagent life cycle management. Moreover, contract research organizations must support reagent life cycle management for diverse global clients. To address these needs, the authors designed and implemented a customized inventory management system, known as LCM+. This software solution provides external clients with efficient, secure access via a web portal to their critical reagent information, pertinent documentation and inventory tracking. Hence, the authors believe that LCM+ can serve as a useful prototype to aid the design of future inventory management systems for optimal management of critical reagents.

FN3-based monobodies selective for the receptor binding domain of the SARS-CoV-2 spike protein.

A phage library displaying 1010 variants of the fibronectin type III (FN3) domain was affinity selected with the biotinylated form of the receptor binding domain (RBD, residues 319-541) of the SARS-CoV-2 virus spike protein. Nine binding FN3 variants (i.e. monobodies) were recovered, representing four different primary structures. Soluble forms of the monobodies bound to several different preparations of the RBD and the S1 spike subunit, with affinities ranging from 3 to 14 nM as measured by bio-layer interferometry. Three of the four monobodies bound selectively to the RBD of SARS-CoV-2, with the fourth monobody showing slight cross-reactivity to the RBD of SARS-CoV-1 virus. Examination of binding to the spike fragments and its trimeric form revealed that the monobodies recognise at least three overlapping epitopes on the RBD of SARS-CoV-2. While pairwise tests failed to identify a monobody pair that could bind simultaneously to the RBD, one monobody could simultaneously bind to the RBD with the ectodomain of the cellular receptor angiotensin converting enzyme 2 (ACE2). All four monobodies successfully bound the RBD after overexpression in Chinese hamster ovary (CHO) cells as fusions to the Fc domain of human IgG1.

Peptide inhibitors targeting Clostridium difficile toxins A and B.

Clostridium difficile causes severe hospital-acquired antibiotic-associated diarrhea due to the activity of two large protein toxins. Current treatments suffer from a high relapse rate and are generating resistant strains; thus new methods of dealing with these infections that target the virulence factors directly are of interest. Phage display was used to identify peptides that bind to the catalytic domain of C. difficile Toxin A. Library screening and subsequent quantitative binding and inhibition studies showed that several of these peptides are potent inhibitors. Fragment-based computational docking of these peptides elucidated the binding modes within the active site. These antitoxin peptides may serve as potential lead compounds to further engineer peptidomimetic inhibitors of the clostridial toxins.