Expression, purification and biological activity assessment of romiplostim biosimilar peptibody.

BACKGROUND: Romiplostim is a peptibody analogue of thrombopoietin (TPO) which regulates platelet production. This molecule consists of two main parts: Peptide sequences which like wild type TPO, mimics stimulation of TPO receptor and IgG1Fc, (Peptide + Antibody = Peptibody). This drug is used in treatment of chronic Immune Thrombocytopenic Purpura (ITP). METHODS: In this project E. coli bacteria were transformed by a construct harboring peptibody fusion gene. This construct consisted of two repeated peptide sequences which have fused to Carboxyl group of IgG1Fc. Designed construct in E. coli host resulted in protein expression in cytoplasm as inclusion body. The inclusion bodies were separated, washed and after denaturation and solubilization, in the last stage the desired peptibodies were refolded and purified. The resulting peptibodies were characterized by SDS-PAGE and Western immunoblotting. The bioactivity were assessed in vivo using subcutaneous injection in mice. RESULTS: Results showed accurate molecules were produced and purified. Also, in vivo experiment showed significant increment (more than two fold) of platelets compared to control group. CONCLUSION: In this study laboratory scale production of recombinant romiplostim showed proper in-vivo bioactivity. This new approach in expression and purification of this recently introduced thrombopoietin receptor agonist drug may be followed by scale up of its production to response the chronic ITP patient's demand.

Milestones in understanding platelet production: a historical overview.

The discovery of thrombopoietin (TPO, also termed THPO) in 1994 was a major achievement in understanding the regulation of platelet production. In prior decades, physiological studies had demonstrated that platelets were produced from bone marrow megakaryocytes and that the megakaryocytes responded to thrombocytopenia by increasing their number, size and DNA ploidy. In 1958, it was proposed that a 'thrombopoietin' must exist that regulated this interaction between the circulating platelet mass and the bone marrow megakaryocytes. After over three decades of effort, TPO was finally purified by five independent laboratories. TPO stimulated megakaryocyte colony-forming cell growth and increased the number, size and ploidy of megakaryocytes. When the genes for TPO or TPO receptor were eliminated in mice, megakaryocytes grew and platelets were made, but at 15% of their normal number. A first generation of recombinant human (rh) TPO molecules [rhTPO and pegylated recombinant human megakaryocyte growth and development factor (PEG-rhMGDF)] rapidly entered clinical trials in 1995 and increased platelet counts in humans undergoing non-myeloablative chemotherapy but not in those undergoing stem cell transplantation. Antibodies developed against PEG-rhMGDF and development of these recombinant thrombopoietins ended. A second generation of TPO receptor agonists (romiplostim and eltrombopag) was then developed. Neither of these TPO receptor agonists demonstrated any significant untoward effects and both are now licensed in many countries for the treatment of immune thrombocytopenia. This review describes the significant experiments that have surrounded the discovery of TPO and its clinical development.

A strategic crossflow filtration methodology for the initial purification of promegapoietin from inclusion bodies.

A novel crossflow filtration methodology is demonstrated for the initial purification of the therapeutic protein, promegapoietin-1a (PMP), produced as inclusion bodies (IBs) in a recombinant Escherichia coli bioprocess. Two strategic separation steps were performed by utilizing a filtration unit with a 1000 kDa polyethersulphone membrane. The first step, aiming for separation of soluble contaminants, resulted in a 50% reduction of the host cell proteins, quantified by total amino acid analysis and a 70% reduction of all DNA, quantified by fluorometry, when washing the particulate material with a 10mM EDTA in 50mM phosphate buffer, pH 8. The second step, aiming for separation of particulate contaminants from solubilized IBs, resulted in a 97-99.5% reduction of endotoxin, used as a marker for cell debris, and was quantified by the kinetic turbidimetric LAL endotoxin assay. The overall PMP yield was 58% and 33% respectively for the two solubilizations investigated, guanidine hydrochloride and arginine, as measured by RP-HPLC. The scope was also to investigate the physical characteristics of the intermediate product/s with regard to the choice of IB solvent. Preliminary results from circular dichroism spectroscopy measurements indicate that the protein secondary structure was restored when arginine was used in the second step.

Evaluation of refolding conditions for a human recombinant fusion cytokine protein, promegapoietin-1a.

Conditions to obtain correctly folded PMP-1a (promegapoietin-1a), an engineered fusion IL-3 (interleukin-3) and thrombopoietin receptor agonist from recombinant Escherichia coli IBs (inclusion bodies), were defined to generate sufficient amounts of protein for evaluation as a potential therapeutic compound. Several ionic and non-ionic detergents, as well as the chaotrope urea, in combination with selected additives, were screened for their ability to dissolve IB protein and promote formation of monomeric, oxidized protein. Upon dissolution, soluble aggregates constituted 50-60% of total protein in detergent-solubilized IBs depending on the level of detergent used, whereas use of urea increased aggregation to approx. 70%. Subsequent addition of 5 mM cysteine or DTT (dithiothreitol) reduced the levels of aggregation, but never lower than approx. 20%. Refolds from detergent-solubilized IBs with or without organic modifiers characteristically produced multiple persistent misfolded species. However, the addition of a 12:1 molar excess of cystine (cystine/DTT) to urea-dissolved IBs containing DTT, followed by dilution, promoted the formation of correctly oxidized, disulfide-paired PMP-1a monomer with minimal misfolds present. Thus treatment of urea-dissolved proteins with thiol-group-containing additives and control of dilution, pH, protein concentration and order of addition were able to produce a maximum refold efficiency of 40-50% of correctly paired protein monomer.

A novel thrombopoietin-stem-cell factor fusion protein possesses enhanced potential in stimulating megakaryocyte proliferation and differentiation.

TPO (thrombopoietin) and SCF (stem-cell factor) are functionally related cytokines with overlapping but distinct haematopoietic effects. In the present study, a novel TPO-SCF fusion protein that combined the complementary biological effects of TPO and SCF into a single molecule was expressed in, and purified from, Sf9 [Spodoptera frugiperda (fall armyworm)] insect cells. The specific activity of rhTPO (recombinant human TPO)-SCF in megakaryoblastic Mo7e cell proliferation assays was 2.90+/-0.35 x 10(7) units/micromol, approx. 1.7 times as high as that of rhTPO. The specific activity of rhTPO-SCF in TF-1 cells proliferation assays was 7.10+/-0.95 x 10(6) units/micromol, approx. 1.2 times as high as that of rhSCF (recombinant human SCF). In a megakaryocyte-colony-forming assay using human peripheral-blood CD34(+) cells, the SCF moiety of rhTPO-SCF worked in a synergistic way to augment the colony number and exhibited a higher potential to stimulate megakaryocyte colony growth. According to the results of EMSA (electrophoretic mobility-shift assay) and semi-quantitative RT (reverse transcriptase)-PCR, the synergistic effects of the SCF moiety were also reflected in increased STAT5 (signal transducer and activator of transcription 5) DNA binding and enhanced up-regulation of p21 expression in Mo7e cells treated by rhTPO-SCF, suggesting that rhTPO-SCF could be more potent in promoting megakaryocyte proliferation and differentiation.

Expression, purification, and characterization of a novel recombinant fusion protein, rhTPO/SCF, in Escherichia coli.

Thrombopoietin (TPO) is the principal regulatory cytokine of megakaryopoiesis and thrombopoiesis and promotes all aspects of megakaryocyte development. Stem cell factor (SCF) is mainly a pleiotropic cytokine acting on hematopoiesis by promoting the survival and proliferation of hematopoietic stem cells and has a potent synergistic effect on megakaryopoiesis in the presence of TPO. Here, we report the construction, expression, and purification of a novel recombinant human thrombopoietin/stem cell factor (rhTPO/SCF) fusion protein, which consists of a truncated human thrombopoietin (1-157 a.a.) plus a truncated human stem cell factor (1-145 a.a.), linked by a peptide (GGGGSPGGSGGGGSGG). The TPO/SCF gene was cloned into the Escherichia coli expression vector pET28a and expressed in BL21(DE3) strain. The rhTPO/SCF constituted up to 6% of the total bacterial protein. Co-expression with E. coli chaperones, Trigger Factor (TF) and GroES/GroEL, and lowering cultivation temperature cooperatively improved the solubility of expressed rhTPO/SCF, resulting in about fourfold increase in the yield soluble rhTPO/SCF. The rhTPO/SCF was purified to homogeneity using anion exchange followed by metal affinity chromatography. Western blot analysis confirmed the identity of the purified protein. rhTPO/SCF stimulated a dose-dependent cell proliferation in both TF1 and Mo7e cell lines.

Safety and efficacy of transfusions of autologous cryopreserved platelets derived from recombinant human thrombopoietin to support chemotherapy-associated severe thrombocytopenia: a randomised cross-over study.

BACKGROUND: The increasing demand for platelet products, and concern over the transfusion-associated risks of alloimmunisation and infections, have motivated a search for improved methods aimed at keeping exposure to donor antigens to a minimum. Transfusion of thrombopoietin-derived autologous platelets might provide an alternative strategy. We aimed to compare the safety and efficacy of this strategy with that of transfusion with fresh allogeneic platelets in patients with severe chemotherapy-induced thrombocytopenia. METHODS: 20 patients with gynaecological malignancies were treated with two doses of 1.2 microg/kg recombinant human thrombopoietin. From day 12, we aimed to collect 50 units of platelets from these patients by plateletpheresis. Harvested platelets were cryopreserved in ThromboSol and 2% dimethyl sulfoxide (DMSO) for use in subsequent autologous transfusions. Patients then received carboplatin for up to six cycles. Patients were randomly assigned to group A (n=10), which received allogeneic fresh platelets at the first instance of severe thrombocytopenia (platelet count <15,000/microL) and then autologous cryopreserved platelets at the next, or to group B (n=10), which received first autologous and then allogeneic platelets. In subsequent cycles, all patients received autologous platelets while available. The primary endpoint was platelet count increment corrected for the number of platelets transfused and the patients' body-surface area. Analysis was by intention to treat. FINDINGS: Treatment with recombinant human thrombopoietin significantly increased platelet count (median 2.3-fold [range 1.5-3.3], p<0.0001) in all but one patient in group A. The median number of platelets collected per patient was 53 units (14-66) in two collections (one to three). There was no significant difference in the corrected platelet count increments (CCIs) between the 19 paired transfusions of cryopreserved autologous platelets and fresh allogeneic platelets (median 1-h CCI 15.7 vs 19.8, p=0.398; median 24-h CCI 13.0 vs 18.1, p=0.398). 14 of the 19 patients had a good response (1-h CCI >7.5) to their first transfusion of allogeneic platelets. By contrast, all patients had a good response to their first transfusion of autologous platelets (p=0.063). Moreover, no significant decrease in the CCIs (p=0.405) was seen over six cycles after autologous platelet transfusions (n=63). No transfusion reactions or any serious adverse event was recorded during autologous platelet transfusions. INTERPRETATION: Recombinant human thrombopoietin facilitated collection of multiple units of platelets, which could be cryopreserved and reinfused to counteract severe thrombocytopenia during multicycle chemotherapy. Transfusion of autologous cryopreserved platelets derived from recombinant human thrombopoietin can provide a viable strategy to minimise the risks of allogeneic platelet transfusions and provide a long-lasting supply of platelet support.

Is the thrombopoietin assay useful for differential diagnosis of thrombocytopenia? Analysis of a cohort of 160 patients with thrombocytopenia and defined platelet life span.

BACKGROUND: Thrombopoietin (TPO), the major hormone controlling platelet production, has been measured in thrombocytopenias with discordant results. The aim of our work was to assess the value of the TPO assay for differential diagnosis of thrombocytopenias in a large cohort of patients classified according to the results of their platelet isotopic study. METHODS: We measured TPO (R&D Systems) in serum of 160 thrombocytopenic patients referred to our department for platelet life span isotopic studies. We classified patients as follows: (a) idiopathic or autoimmune thrombocytopenia group (ITP; patients with increased platelet destruction and shortened platelet life span; n = 67); (b) pure genetic thrombocytopenia group (patients with decreased platelet production, normal platelet life span, and without bone marrow aplasia; n = 55); (c) bone marrow aplasia group (BM; patients with decreased platelet production, normal platelet life span, and bone marrow aplasia; n = 13). RESULTS: In patients with pure genetic thrombocytopenia, TPO (median, 55 ng/L) was not different from TPO in patients with ITP (median, 58 ng/L) or controls (n = 54; median, 51 ng/L). Only in patients with bone marrow aplasia was TPO significantly higher (median, 155 ng/L) and negatively correlated to the platelet count (r(2) = 0.5014). CONCLUSIONS: Although the median serum TPO is increased in thrombocytopenia with decreased platelet production from bone marrow aplasia, it does not differentiate patients with pure genetic thrombocytopenia from those with ITP.

[Eukaryotic expression, purification and analysis of the fusion protein of Epo-Tpo(C)].

OBJECTIVE: To further elucidate the function of Tpo(C) and create a new type of Epo. METHODS: Tpo(C) was linked to Epo to produce a fusion protein--Epo-Tpo(C). The fusion gene was expressed in CHO cell. After selection with G418, Epo-Tpo(C)-expressing cell lines was obtained. The supernatant had 15 U/ml Epo activity. The results of PCR and Southern Blotting indicated the fusion gene was fused into the genome. RT-PCR and RNA Dot Blotting analysis found the Epo-Tpo(C) mRNA. The supernatant was purified with Blue-Sepharose CL-6B. RESULTS: The rate of recovery was 94%. The in vivo activity tested by reticulocytes was 20% higher than ELISA. The half-life was about 12 hours while the Epo standard sample had half-life of 8 hours tested with ELISA in rat. CONCLUSION: The results proved that Tpo(C) could protect Epo and improve its function. The fusion protein had satisfactory effect in the model of rat renal failure.

Chromatography of recombinant proteins.

Variants of intact polypeptides/proteins ranging in mass from 6,500 to 70,000 Da were easily separated using reversed-phaseHPLC (rpHPLC) or affinity chromatography. A variant of rhlGF-I, where the racemization of a serine residue was detected in the intact molecule, was resolved from rhlGF-I within 25 minutes by rpHPLC. Other variants of rhlGF-I separated by this method include methionine sulphoxide at position 59, des Gly1, des Gly1Pro2, Glu for Asp substitution at position 20 and incorrectly folded IGF-I. For rhDNase (approximately 40 kDa), affinity chromatography was able to clearly resolve three different amino acids (Asn, Asp and iso-Asp) at position 74 of the intact glycoprotein. The presence or absence of O-linked sugars on Thr -37 of recombinant human thrombopoietin was rapidly demonstrated by rpHPLC. While the separation of these types of variants is essential, the demonstration of biological activity is critical for designing specifications that allow the administration of these proteins into humans. Once a correlation exists between the variant and its biological activity, control of the manufacturing process can be better achieved with analytical methodology.

Mpl: from an acute myeloproliferative virus to the isolation of the long sought thrombopoietin.

Mpl, the receptor for thrombopoietin (TPO), was isolated as a cellular sequence transduced by a new acute myeloproliferative virus. Human and murine c-mpl were subsequently cloned and Mpl was identified as a member of the growth factor receptor superfamily. For a time, Mpl remained an orphan receptor. Engineering of cell lines expressing c-mpl provided a sensitive tool for detecting the ligand of Mpl, and led to the molecular cloning of TPO, the long sought proliferation and differentiation factor for the megakaryocytic lineage. Afterwards, signal transduction by Mpl was studied, and the functional elements of the cytoplasmic domain responsible for cell proliferation and differentiation were identified. When studied in various human hematologic malignancies, Mpl expression was shown to be increased in 50% of the patients with acute myeloblastic leukemia (AML). In vitro treatment of AML cells by TPO led to proliferation, suggesting that TPO could contribute, at least in part, to abnormal growth of AML cells. A tremendous number of studies have followed the isolation of TPO, and have shown that TPO is the primary regulator of physiological platelet production. However, roles for Mpl and TPO in other lineages, especially in erythroid and immature hematopoietic progenitors, have also emerged from these studies.

Expression in Escherichia coli and purification of human thrombopoietin.

Human thrombopoietin (TPO) has been successfully overexpressed in Escherichia coli, with an expression level of about 12% of total cellular protein. The full-length TPO gene was subcloned into the prokaryotic expression vector pKK233-2 under the control of the inducible tac promoter. The recombinant protein was produced mainly in the form of inclusion body. By efficient renaturation and one-step purification, the recombinant protein was purified to homogeneity. The specific activity and yield of recombinant TPO can reach 2 x 10(4) units/mg and 2 mg/g of wet E. coli cells respectively.

[High expression of human thrombopoietin mutant fusion protein in E. coli].

OBJECTIVE: To obtain human thrombopoietin (Tpo)mutant cDNA encoding mature peptide N terminal 1 approximately 196 amino acids and investigate its expression in E. coli JM109. METHODS: Polymerase chain reaction and DNA recombination techniques were employed. PCR product was inserted into pUC19 vector and sequenced and then cloned into expression vector pMAL-c2. RESULTS: E. coli JM109 cells with plasmid pMAL -MBP/TpoM were induced by IPTG for 4-5 hours. SDS-PAGE analysis showed that MBP/TpoM molecular weight is about 63KD. Scanning analysis indicated that expressed protein accounts up to 37% of total E. coli proteins. CONCLUSION: The Tpo mutant of interest is successfully expressed in E. coli JM109 cells.

[Mpl ligand (thrombopoietin) and platelet regulation]. / Le ligand de Mpl (thrombopoïétine) et la régulation plaquettaire.

After 35 years of research, the physiological regulator of platelet production has been isolated and its gene cloned. This discovery originates from studies performed with the myeloproliferative leukemia virus (MPLV), a murine retrovirus which induces an acute myeloproliferative syndrome in adult mice. MPLV carries in its genome the v-mpl oncogene which corresponds to a truncated form the c-mpl proto-oncogene. c-mpl encodes a cytokine receptor (Mpl-R) belonging to the hematopoietin receptor superfamily. Among the hematopoietic cell lineages, Mpl-R is preferentially expressed on late megakaryocyte progenitors, megakaryocytes and platelets. The ligand for Mpl-R, called Mpl-L or TPO or MGDF or megapoietin, is a glycosylated hormone of 352 amino acids in human which comprises two domains: the N-terminus domain shares 50% similarity with erythropoietin and is responsible for the biological activity; the C-terminus part is required for secretion. Notwithstanding its major action on megakaryocytopoiesis and thrombocytopoiesis, Mpl-L also potentiates the action of other cytokines on several hematopoietic lineages. Mpl-L/TPO/MGDF, the homeostatic regulator of platelet production, might be a useful therapeutical cytokine to treat thrombocytopenia induced in patients by chemotherapy.

Protein characteristics of thrombopoietin.

Thrombopoietin (TPO) was purified from irradiated thrombocytopenic rat plasma. In the process of purification, some biochemical and biological characteristics were investigated. Rat plasma TPO was extremely hydrophobic and exhibited multiple peaks of activity on gel filtration. Both the low and high molecular weight fractions were separately subjected to further purification. Consequently, a rat TPO cDNA was cloned based on the amino acid sequences of purified rat plasma TPO. It revealed that each final purified rat plasma TPO was not a full-length form. In addition, rat hepatocytes and three rat hepatoma cell lines were found to produce rat TPO. Each native TPO derived from cultured cells was also partially purified, and hepatocyte-derived TPOs were shown to be heterogeneous in molecular weight. To study the structure of TPO, various recombinant TPO molecules were generated. Two disulfide bonds (Cys7-Cys151 and Cys29-Cys85) located in the N-terminal domain of TPO have an important effect on its biological activity. The human TPO muteins, sequentially deleted from the C-terminal, were expressed in COS-1 cells. TPO (1-151) was active, but TPO (1-150), which lacks Cys151, did not exhibit TPO activity. These findings indicate that the region essential for TPO activity is the N-terminal domain, which contains two disulfide bonds. Although the role(s) of the C-terminal domain is not clear at present, the potential N-glycosylation in the C-terminal domain is not directly required for exhibiting TPO activity.

The new generation of recombinant human hematopoietic cytokines.

In the past year, the most exciting development in the field of hematopoietic growth factors has been the identification of the platelet-inducing factor Mpl ligand. Administration of recombinant Mpl ligand may alleviate the potential for hemorrhagic complications following cancer therapies. Stem cell factor continues to be studied clinically in the mobilization of peripheral blood cells for transplantation.

TPO and IL-3 induce overlapping but distinct protein tyrosine phosphorylation in a myeloid precursor cell line.

TPO is a recently cloned cytokine which appears to play a central role in megakaryopoiesis and platelet production. It has been shown to be the ligand for c-mpl, a member of the hematopoietic receptor superfamily. We examined intracellular tyrosine phosphorylation events induced by TPO and compared them with events stimulated by IL-3, a pleiotropic cytokine which also has activity on the megakaryocyte lineage. The overall pattern of tyrosine phosphorylation stimulated by TPO and IL-3 in myeloid precursor cells revealed an overlapping but not identical pattern reflecting their distinct but partially redundant biological effects. We identify Shc and Vav as intracellular targets of TPO-induced tyrosine phosphorylation. Moreover, we demonstrate that the tyrosine kinase Jak2 is phosphorylated after TPO stimulation. Whereas phosphorylation of these proteins was induced by both cytokines, phosphorylation of Jak1 was induced only by IL-3 and not by TPO, distinguishing the signal transduction of the two cytokines on a molecular level.