Development of a Bioassay System for Human Growth Hormone Determination with Close Correlation to Immunoassay

Maimaiti, Maihulan; Tanahashi, Yusuke; Mohri, Zen‐Ichi; Fukui, Kenji

doi:10.1002/jcla.21527

Cited by 8 publications

(4 citation statements)

References 31 publications

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“…In the published literature, a few studies focused on the proliferation assay and the hGHR-LHRE reporter system in some cell lines, e.g., HEK293, Ba/F3, and CHO, but the analytical method has not previously been fully validated. Moreover, transient transfection is not suitable for determination the bioactivity of therapeutic rhGH or LAGH [18,21,22,23,27]. Here, we developed and validated an animal-free bioassay based on RGA for the determination of the bioactivity of rhGH-Fc.…”

Section: Discussionmentioning

confidence: 99%

A Cell-Based Strategy for Bioactivity Determination of Long-Acting Fc-Fusion Recombinant Human Growth Hormone

Yao

Fan

et al. 2019

Molecules

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The long-acting growth hormone (LAGH) is a promising alternative biopharmaceutical to treat growth hormone (GH) deficiency in children, and it was developed using a variety of technologies by several pharmaceutical companies. Most LAGH preparations, such as Fc fusion protein, are currently undergoing preclinical study and clinical trials. Accurate determination of bioactivity is critical for the efficacy of quality control systems of LAGH. The current in vivo rat weight gain assays used to determine the bioactivity of recombinant human GH (rhGH) in pharmacopoeias are time-consuming, expensive, and imprecise, and there are no recommended bioassays for LAGH bioactivity in pharmacopoeias. Therefore, we developed a cell-based bioassay for bioactivity determination of therapeutic long-acting Fc-fusion recombinant human growth hormone (rhGH-Fc) based on the luciferase reporter gene system, which is involved in the full-length human GH receptor (hGHR) and the SG (SIE and GAS) response element. The established bioassay was comprehensively validated according to the International Council for Harmonization (ICH) Q2 (R1) guidelines and the Chinese Pharmacopoeia, and is highly precise, time-saving, simple, and robust. The validated bioassay could be qualified for bioactivity determination during the research, development, and manufacture of rhGH-Fc, and other LAGH formulations.

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Section: Discussionmentioning

confidence: 99%

A Cell-Based Strategy for Bioactivity Determination of Long-Acting Fc-Fusion Recombinant Human Growth Hormone

Yao

Fan

et al. 2019

Molecules

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“…There are several isoforms of GH circulating in blood together with the binding proteins (growth hormone-binding protein) making the measurement of GH a complicated task. Cell culture proliferation bioassay, depending on the expressed GH receptor by these cell lines, had recorded the lowest detection limit (~0.5 ng/ml), and had the highest specificity for GH in spite of the non-specific interference by factors present in serum ( 7 , 8 ). Meanwhile, different immunoassays (RIA, IRMA, and ELISA) are generally used in clinical laboratories because of sensitivity, speed, and accessibility ( 5 ).…”

Section: Discussionmentioning

confidence: 99%

“…However until recently, a standard test was lacking to detect administrated rhGH ( 4 ) in spite of the many proposed assays for measuring GH levels in the blood of young patients ( 5 ) or abusing athletes ( 6 ). For in vitro bioassays, GH measurement depends on its proliferative effect on cultured cell lines which display its specific receptor ( 7 , 8 ) or through measuring the biological changes of hGH protein markers in the serum ( 9 ). Because of their affordability, clinical laboratories are still considering immunoassays for GH measurement in biological samples ( 10 ).…”

Section: Introductionmentioning

confidence: 99%

Exploiting Nanobodies in the Detection and Quantification of Human Growth Hormone via Phage-Sandwich Enzyme-Linked Immunosorbent Assay

et al. 2017

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BackgroundMonitoring blood levels of human growth hormone (hGH) in most children with short stature deficiencies is crucial for taking a decision of treatment with extended course of daily and expensive doses of recombinant hGH (rhGH or Somatropin®). Besides, misusing of rhGH by sportsmen is banned by the World Anti-Doping Agency and thus sensitive GH-detecting methods are highly welcome in this field. Nanobodies are the tiniest antigen-binding entity derived from camel heavy chain antibodies. They were successfully generated against numerous antigens including hormones.MethodsA fully nanobody-based sandwich ELISA method was developed in this work for direct measurement of GH in biological samples.ResultsTwo major characteristics of nanobody were exploited for this goal: the robust and stable structure of the nanobody (NbGH04) used to capture hGH from tested samples, and the great ability of tailoring, enabling the display of the anti-GH detector nanobody (NbGH07) on the tip of M13-phage. Such huge, stable, and easy-to-prepare phage-Nb was used in ELISA to provide an amplified signal. Previously, NbGH04 was retrieved on immobilized hGH by phage display from a wide “immune” cDNA library prepared from a hGH-immunized camel. Here, and in order to assure epitope heterogeneity, NbGH07 was isolated from the same library using NbGH04-captured hGH as bait. Interaction of both nanobodies with hGH was characterized and compared with different anti-GH nanobodies and antibodies. The sensitivity (~0.5 ng/ml) and stability of the nanobody-base sandwich ELISA were assessed using rhGH before testing in the quantification of hGH in blood sera and cell culture supernatants.ConclusionIn regard to all advantages of nanobodies; stability, solubility, production affordability in Escherichia coli, and gene tailoring, nanobody-based phage sandwich ELISA developed here would provide a valuable method for hGH detection and quantification.

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“…5 × 10 4 SW480 cells/well were seeded in 6-well plates (Costar), with 2 mL of complete medium. After 24 h of pre-incubation, cells were treated during 72 h with orlistat, lonidamine, or DON at synergistic concentrations, as stated before 16 , and with the maximum circulating concentrations of GH, insulin, or indomethacin, reported in healthy subjects [45][46][47] . The concentrations are found on Supplementary Table S1.…”

Section: Methodsmentioning

confidence: 99%

Pharmacological inhibition of tumor anabolism and host catabolism as a cancer therapy

Schcolnik‐Cabrera

Chávez‐Blanco

Domínguez-Gómez

et al. 2021

Sci Rep

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The malignant energetic demands are satisfied through glycolysis, glutaminolysis and de novo synthesis of fatty acids, while the host curses with a state of catabolism and systemic inflammation. The concurrent inhibition of both, tumor anabolism and host catabolism, and their effect upon tumor growth and whole animal metabolism, have not been evaluated. We aimed to evaluate in colon cancer cells a combination of six agents directed to block the tumor anabolism (orlistat + lonidamine + DON) and the host catabolism (growth hormone + insulin + indomethacin). Treatment reduced cellular viability, clonogenic capacity and cell cycle progression. These effects were associated with decreased glycolysis and oxidative phosphorylation, leading to a quiescent energetic phenotype, and with an aberrant transcriptomic landscape showing dysregulation in multiple metabolic pathways. The in vivo evaluation revealed a significant tumor volume inhibition, without damage to normal tissues. The six-drug combination preserved lean tissue and decreased fat loss, while the energy expenditure got decreased. Finally, a reduction in gene expression associated with thermogenesis was observed. Our findings demonstrate that the simultaneous use of this six-drug combination has anticancer effects by inducing a quiescent energetic phenotype of cultured cancer cells. Besides, the treatment is well-tolerated in mice and reduces whole animal energetic expenditure and fat loss.

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Development of a Bioassay System for Human Growth Hormone Determination with Close Correlation to Immunoassay

Cited by 8 publications

References 31 publications

A Cell-Based Strategy for Bioactivity Determination of Long-Acting Fc-Fusion Recombinant Human Growth Hormone

A Cell-Based Strategy for Bioactivity Determination of Long-Acting Fc-Fusion Recombinant Human Growth Hormone

Exploiting Nanobodies in the Detection and Quantification of Human Growth Hormone via Phage-Sandwich Enzyme-Linked Immunosorbent Assay

Pharmacological inhibition of tumor anabolism and host catabolism as a cancer therapy

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