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

Fayaz S, Fard-Esfahani P, Golkar M, Allahyari M, Sadeghi S - Daru (2016)

Bottom Line: Designed construct in E. coli host resulted in protein expression in cytoplasm as inclusion body.The resulting peptibodies were characterized by SDS-PAGE and Western immunoblotting.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.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Department, Pasteur Institute of Iran, Tehran, 1316943551, Iran.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Comparison of platelet counts (103/μl) between recombinant Romiplostim Injected Group and Control Group on days 0, 4, 7 and 17. On day 0 Recombinant Romiplostim (50 μg/kg) and normal saline (0.2 mL/mice) were subcutaneously injected into case and control groups, respectively. Mean platelet counts were assayed on days 0, 4, 7, 17 through preorbital sinus sampling. As it is shown in the recombinant Romiplostim treated group, platelet value has increased nearly more than 2 folds to the control group on day 4, and it returned to day 0 level on day 17. Error bar (95 % CI): Platelet counts in 10 BALB/c mice in each case and control group
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Fig7: Comparison of platelet counts (103/μl) between recombinant Romiplostim Injected Group and Control Group on days 0, 4, 7 and 17. On day 0 Recombinant Romiplostim (50 μg/kg) and normal saline (0.2 mL/mice) were subcutaneously injected into case and control groups, respectively. Mean platelet counts were assayed on days 0, 4, 7, 17 through preorbital sinus sampling. As it is shown in the recombinant Romiplostim treated group, platelet value has increased nearly more than 2 folds to the control group on day 4, and it returned to day 0 level on day 17. Error bar (95 % CI): Platelet counts in 10 BALB/c mice in each case and control group

Mentions: Our data indicated that platelet value has increased approximately more than 2 fold in the recombinant Romiplostim treated group to the control group on day 4, and it returned to day 0 level on day 7 and 17 (Fig. 7). Other blood factors remained unchanged on days 0, 4, 7 and 17 (it is showed in attach file). As it was expected, platelet increase was transient and returned to the normal range after several days, it was proved in the previous study too [15]. So weekly injection in keeping platelet count is required. Laboratory scale production of Romiplostim may be followed by scale up of its production to response the chronic ITP patients’ demands.Fig. 7


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

Fayaz S, Fard-Esfahani P, Golkar M, Allahyari M, Sadeghi S - Daru (2016)

Comparison of platelet counts (103/μl) between recombinant Romiplostim Injected Group and Control Group on days 0, 4, 7 and 17. On day 0 Recombinant Romiplostim (50 μg/kg) and normal saline (0.2 mL/mice) were subcutaneously injected into case and control groups, respectively. Mean platelet counts were assayed on days 0, 4, 7, 17 through preorbital sinus sampling. As it is shown in the recombinant Romiplostim treated group, platelet value has increased nearly more than 2 folds to the control group on day 4, and it returned to day 0 level on day 17. Error bar (95 % CI): Platelet counts in 10 BALB/c mice in each case and control group
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4940717&req=5

Fig7: Comparison of platelet counts (103/μl) between recombinant Romiplostim Injected Group and Control Group on days 0, 4, 7 and 17. On day 0 Recombinant Romiplostim (50 μg/kg) and normal saline (0.2 mL/mice) were subcutaneously injected into case and control groups, respectively. Mean platelet counts were assayed on days 0, 4, 7, 17 through preorbital sinus sampling. As it is shown in the recombinant Romiplostim treated group, platelet value has increased nearly more than 2 folds to the control group on day 4, and it returned to day 0 level on day 17. Error bar (95 % CI): Platelet counts in 10 BALB/c mice in each case and control group
Mentions: Our data indicated that platelet value has increased approximately more than 2 fold in the recombinant Romiplostim treated group to the control group on day 4, and it returned to day 0 level on day 7 and 17 (Fig. 7). Other blood factors remained unchanged on days 0, 4, 7 and 17 (it is showed in attach file). As it was expected, platelet increase was transient and returned to the normal range after several days, it was proved in the previous study too [15]. So weekly injection in keeping platelet count is required. Laboratory scale production of Romiplostim may be followed by scale up of its production to response the chronic ITP patients’ demands.Fig. 7

Bottom Line: Designed construct in E. coli host resulted in protein expression in cytoplasm as inclusion body.The resulting peptibodies were characterized by SDS-PAGE and Western immunoblotting.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.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Department, Pasteur Institute of Iran, Tehran, 1316943551, Iran.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus