Development and validation of a novel reporter gene assay for determination of recombinant human thrombopoietin
Introduction
Thrombopoietin (TPO), also called the ligand of c-Mpl protein, is the major physiological regulator of proliferation and maturation of megakaryocytes as well as of platelet production [1], [2]. It is mainly produced by the hepar, while other organs including the spleen, lung, kidney, bone marrow, and brain produce hormones in small amounts [3], [4]. The entire human TPO gene is located on human chromosome 3q26.33-q27 and spans 6.2 kb containing six exons and five introns [5], [6]. The human TPO protein is composed of 353 amino acids, including a 21-amino acid signal peptide sequence [7]. The structure contains two distinct domains: a c-Mpl binding domain (residues 1–153) and a carbohydrate-rich domain (residues 154–332) [8]. The first 153 amino acids of human TPO have 23% sequence identity to human erythropoietin and contain four cysteine residues; amino acids 154–332 consist of a unique sequence that includes six N-linked and multiple O-linked glycosylation sites and is less well conserved across different species (Fig. 1A) [9], [10].
The product of proto-oncogene c-Mpl, as the cell surface receptor for TPO, is a member of the type I hematopoietic cytokine receptor family [11]. Binding to its receptor leads to homodimerization, which induces Janus kinase 2 (JAK2) activation. The phosphotyrosine sites on the receptors serve as docking sites, allowing the binding of various signaling molecules including SHC, GRB2, SOS, VAV, and CBL to initiate intracellular signaling [3]. The multiple signaling pathways activated by TPO includes JAK/signal transducer and activator of transcription (STAT), mitogen-protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), and phosphoinositide 3-kinase (PI3K)/ATK. JAK/STAT and MAPK/ERK signaling pathways lead megakaryocyte progenitors to proliferate and mature, and the PI3K/AKT pathway is important for the regulation of cell progression [12].
TPO is the chief hematopoietic cytokine to be identified, cloned, expressed, and purified [8]. Its receptor is expressed in a wide variety of tissues ranging from hematopoietic stem cells (HSCs), and progenitor cells to mature megakaryocytes [13], [14], [15]. Therefore, TPO may have potential therapeutic benefits in treating thrombocytopenia. In 2010, TPIAO® (developed by Sunshine Pharma) was approved by the National Medical Products Administration for the treatment of thrombocytopenia in patients with immune thrombocytopenic purpura and chemotherapy-induced thrombocytopenia, who have had an insufficient response to corticosteroids, immunoglobulins, or splenectomy [16], [17]. Full-length recombinant human thrombopoietin (rhTPO) was expressed by Chinese hamsters’ ovary cells and purified from the culture medium, which has similar biological functions as endogenous TPO.
Comprehensive evaluation of the biological activity of rhTPO is crucial for evaluating its clinical studies on safety and efficacy in human subjects. However, bioassay of rhTPO has not been recommended in different national/regional pharmacopoeia, although TPIAO® is approved for therapeutic use. The modification of 75Se selenomethionine assay is deemed to the gold standard method for determining TPO bioactivity, but the method has significant shortcomings including the use of a radioisotope and complex process of animal preparation [18], [19]. All sorts of in vitro bioassays have been developed containing ligand- and receptor-binding assays, and cell-based assays. Ligand- and receptor-binding assays can confirm nothing more than antigenic sites and binding activities. Cell-based assays showed that rhTPO has significant proliferative effects on the MO7e, UT-7/TPO, PMN, BF-TE22, 32D-mpl, and N2C-Tpo cell lines [20], [21], [22], [23], [24], [25], [26]. Nevertheless, the above-mentioned bioassay is time-consuming and tedious (88–160 h per experimental period) and has high inherent variability with a poor signal-to-noise ratio (SNR). Reporter gene assays (RGAs) are action-related mechanisms involved in vivo with less variability and higher sensitivity than other bioassays [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38]. The methods have been increasingly accepted by the biopharmaceutical industry for the quality control of products.
Herein, this RGA for rhTPO was developed based on Ba/F3 cell lines with stable transfection introduced into the c-Mpl gene and luciferase reporter gene controlled by sis-inducible element (SIE), gamma response region (GRR), and gamma-interferon activated sequence (GAS) [27]. Upon rhTPO binding to c-Mpl and then activating intracellular signaling cascades, cooperative interaction with transcription factors and STAT5 binding to SIE, GRR, and GAS led to the activation and expression of the downstream region of the luciferase gene (Fig. 1B). Determination of bioactivity of rhTPO was measured by relative luciferase unit (RLU). The novel RGA was developed and fully validated according to the (ICH) Q2 (R1) guidelines and the Chinese Pharmacopoeia 2020 edition with respect to method linearity, precision, accuracy, specificity, and robustness. The results provide valuable data in the research, development, quality control, and manufacture of rhTPO.
Section snippets
Cells and materials
The murine interleukin-3 dependent pro B (Ba/F3) cell line (HB-283TM) was purchased from the American Type Culture Collection (Manassas, VA, USA). The pGL4.26 [luc2/minP/Hygro] firefly luciferase reporter vector and Bright-GloTM luciferase assay systems were obtained from Promega (Madison, WI, USA). Mammalian expression vector pcDNATM3.1, anti-p-c-Mpl antibody and anti-luciferase antibody were purchased from Invitrogen (Carlsbad, CA, USA). GeneticinTM (G-418), Opti-MEMTM, RPMI 1640, fetal
Development of a stable rhTPO-Dependent reporter cell line
The SGG-luciferase containing plasmid and human TPO receptor plasmid was successfully constructed and transfected into Ba/F3 cells. Resistant clones were selected in RPMI 1640 medium containing 10% FBS of hygromycin B and G418. The cell lines were assessed for luciferase activity after stimulating of rhTPO. The starting concentration of rhTPO was 1500 ng/mL, and a serial dilution was made using a 1:4 dilution ratio. As shown in Fig. 2A, luciferase activities were dose dependently increased by
Discussion
TPIAO® from Sunshine Pharma was the first to market full-length rhTPO in the world, which had been approved in China for the treatment of thrombocytopenia. TPO, predominantly produced by the hepar, plays a major role in regulating megakaryocyte (MK) progenitor expansion and differentiation [41]. During the early and late phases of MK, TPO could stimulate the proliferation of MK progenitors and increase the ploidy in these cells. Pro-platelets are produced from polyploid MKs, which further
CRediT authorship contribution statement
Jie Yuan: Conceptualization, Formal analysis, Funding acquisition, Project administration, Investigation, Software, Writing - review & editing. Jia Li: Writing - original draft, Data curation, Methodology, Resources, Software, Validation. Lihua Yang: Methodology, Validation. Yunying Lv: Data curation, Validation. Chao Wang: Validation, Resources. Zheng Jin: Conceptualization, Funding acquisition, Project administration, Supervision. Xianpu Ni: Conceptualization, Project administration.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by MOST of China (Grant No. 2016ZX09101113), and Major technology and innovative research program of Shenyang (Grant No. Y19-4-020).
References (44)
- et al.
Thrombopoietin from beginning to end
Br. J. Haematol.
(2014) - et al.
Thrombopoietin: a potential diagnostic indicator of immune thrombocytopenia in pregnancy
Oncotarget.
(2016) - et al.
A network map of thrombopoietin signaling
J. Cell Commun. Signal.
(2018) - et al.
Recombinant human thrombopoietin: basic biology and evaluation of clinical studies
Blood.
(2002) - et al.
Native thrombopoietin: structure and function
Stem Cells.
(1998) - et al.
Thrombopoietin and the TPO receptorduring platelet storage
Transfusion.
(2000) - et al.
Thrombopoietin: The Novel Hepatic Hormone
News Physiol. Sci.
(2002) The biology of thrombopoietin and thrombopoietin receptor agonists
Int. J. Hematol.
(2013)Thrombopoietin: biology and clinical applications
Oncologist.
(1996)- et al.
Thrombopoietin receptor agonists: ten years later
Heamatologica.
(2019)