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 in...

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Bibliographic Details
Published in:	Journal of pharmaceutical and biomedical analysis Vol. 232; p. 115402
Main Authors:	Kinne, Adam S., Tillman, Erik J., Abdeen, Sanofar J., Johnson, Derrick E., Parmer, Elijah S., Hurst, Jacob P., de Temple, Brittany, Rinker, Sherri, Rolph, Timothy P., Bowsher, Ronald R.
Format:	Journal Article
Language:	English
Published:	England Elsevier B.V 05-08-2023
Subjects:	Animals Efruxifermin Electrochemiluminescence Humans Immunoassay Immunoglobulin G Liver Cirrhosis - drug therapy Non-alcoholic Fatty Liver Disease - drug therapy Non-alcoholic steatohepatitis Pharmacokinetics Rats Recombinant Fusion Proteins - pharmacology Recombinant Fusion Proteins - therapeutic use T2DM Immunoassay EZ-link™ Sulfo-NHS-LC-Biotin RT FACS Electrochemiluminescence NASH ECLIA ECL mAb pAb SEC BSA MWCO PEG FAP EFX Non-alcoholic steatohepatitis Pharmacokinetics ADA HPLC Efruxifermin ELISA MSD
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Summary:	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 β-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. •Efruxifermin (EFX) is an IgG1 FC-FGF21 fusion protein for treating NASH.•An electrochemiluminescence immunoassay is reported for quantitation of bioactive EFX.•Bioactive drug is captured by a biotinylated mAb specific to intact EFX C-terminus.•Captured EFX is detected by an affinity purified chicken IgY to core FGF21 domain.•ECLIA enabled demonstration of dose-proportional EFX PK in patients with NASH.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0731-7085 1873-264X
DOI:	10.1016/j.jpba.2023.115402