A Novel Bioassay for the Activity Determination of Therapeutic Human Brain Natriuretic Peptide (BNP)

Abstract

BackgroundRecombinant human brain natriuretic peptide (rhBNP) is an important peptide-based therapeutic drug indicated for the treatment of acute heart failure. Accurate determination of the potency of therapeutic rhBNP is crucial for the safety and efficacy of the drug. The current bioassay involves use of rabbit aortic strips, with experiments being complicated and time-consuming and markedly variable in results. Animal-less methods with better precision and accuracy should be explored. We have therefore developed an alternative cell-based assay, which relies on the ability of BNP to induce cGMP production in HEK293 cells expressing BNP receptor guanylyl cyclase-A.Methodology/Principal FindingsAn alternative assay based on the measurement of BNP-induced cGMP production was developed. Specifically, the bioassay employs cells engineered to express BNP receptor guanylyl cyclase-A (GCA). Upon rhBNP stimulation, the levels of the second messager cGMP in these cells drastically increased and subsequently secreted into culture supernatants. The quantity of cGMP, which corresponds to the rhBNP activity, was determined using a competitive ELISA developed by us. Compared with the traditional assay, the novel cell-based assay demonstrated better reproducibility and precision.Conclusion/SignificanceThe optimized cell-based assay is much simpler, more rapid and precise compared with the traditional assay using animal tissues. To our knowledge, this is the first report on a novel and viable alternative assay for rhBNP potency analysis.

A rapid reporter assay for recombinant human brain natriuretic peptide (rhBNP) by GloSensor technology

“…The reporter assay showed high precision with intra-assay CV being 8.3% and inter-assay CV being 14.1%; high accuracy with 80%, 100% and 120% recovery rate being 99.2%, 102.4% and 99.0% respectively; and great linearity with R 2 of linear fitting equation being 0.99. Given the specificity of reporter assay was consistent with ELISA assay, which has been validated in former study [11] , it is unnecessary to evaluate it again. Besides, no significant difference was found in results of reporter assay and 293GCAC3-based ELISA assay (paired T test, p = 0.630).…”

“…Given the well-characterized pathways activated by BNP, quantification of cGMP in cells exposed to rhBNP has been explored as attractive alternative assay. Indeed, several types of cGMP-involved assays for rhBNPs have been reported in recent years, including the measurement of cGMP in human umbilical vein endothelial cells (HUVEC) or PC12 cells by radioimmunoassay, as well as a modified cell line 293GCAC3 by ELISA assay, which was developed by our previous work [11] .…”

“…As rhBNP is used more and more widely in clinic, it is imperative to improve the quality standards of relevant pharmaceutical products. In previous study, we developed a 293GCAC3 cell-based ELISA assay to replace rabbit aortic strips test (RAST), which simplifies the experiment and improves accuracy and precision, and this cell-based assay has been widely applied within the industry in China [11] . But ELISA procedure is still very tedious, with repeated washing of plate, which surely would bring variation in measurements.…”

Regulatory science accelerates the development of biotechnology drugs and vaccines by NIFDC

“…Compared to the traditional rabbit aortic strip method, this new method can lower the coefficient of variation from 66% to 12%. 3 The technology has been incorporated into the WHO Collaborating Centre promotion program.…”

Development of a robust reporter-based assay for the bioactivity determination of anti-VEGF therapeutic antibodies

“…The reporter gene assay (RGA) has been widely adopted as a tool in developing potency assays for biologics based on its advantages of reflecting regulation of signal pathways as mode of action (MoA) [19][20][21][22]. VEGF signal pathway has been extensively explored over the past decades.…”