Romiplostim Amgen, BLA # David Frucht, M.D. Product Quality Reviewer Division of Monoclonal Antibodies Office of Biotechnology Products David Frucht, M.D. Product Quality Reviewer Division of Monoclonal Antibodies Office of Biotechnology Products

2 Cellular Target: TPO Receptor (Mpl) Thrombopoietin (TPO) : –Constitutively synthesized in the liver, released into circulation; degraded following uptake –Binds the TPO receptor (c-Mpl, murine myeloproliferative leukemia proto-oncogene) –Interacts with TPO receptor dimers, leading to activation of the JAK/STAT and MAPK pathways –Activation of STAT and MAPK pathways leads to cellular proliferation and differentiation, respectively –Promotes the viability and growth of megakaryocyte colony- forming cells, increasing platelet production Thrombopoietin (TPO) : –Constitutively synthesized in the liver, released into circulation; degraded following uptake –Binds the TPO receptor (c-Mpl, murine myeloproliferative leukemia proto-oncogene) –Interacts with TPO receptor dimers, leading to activation of the JAK/STAT and MAPK pathways –Activation of STAT and MAPK pathways leads to cellular proliferation and differentiation, respectively –Promotes the viability and growth of megakaryocyte colony- forming cells, increasing platelet production TYK2 JAK2 STAT5STAT5 STAT5STAT5 TPOTPO

3 Product History: TPO Receptor Agonists Two prior therapeutic protein products: –Recombinant human TPO, identical amino acid acid structure to full- length endogenous TPO –Pegylated human recombinant megakaryocyte growth and development factor (PEG-MDGDF), truncated TPO, contains the first 163 amino acid residues coupled to polyethylene glycol Sustained thrombocytopenia, associated with immunogenic responses that cross-reacted with TPO, in some healthy subjects receiving PEG-MGDF Development of both products was discontinued, despite evidence that this class of products promoted platelet production in humans Ongoing effort to develop TPO receptor agonists that do not induce immunogenic responses that cross-react with endogenous TPO Two prior therapeutic protein products: –Recombinant human TPO, identical amino acid acid structure to full- length endogenous TPO –Pegylated human recombinant megakaryocyte growth and development factor (PEG-MDGDF), truncated TPO, contains the first 163 amino acid residues coupled to polyethylene glycol Sustained thrombocytopenia, associated with immunogenic responses that cross-reacted with TPO, in some healthy subjects receiving PEG-MGDF Development of both products was discontinued, despite evidence that this class of products promoted platelet production in humans Ongoing effort to develop TPO receptor agonists that do not induce immunogenic responses that cross-react with endogenous TPO

4 Romiplostim: A New TPO Agonist A homodimer of 59kD single-chain subunits Each subunit consists of a human IgG1 Fc domain, linked at its C- terminus to a peptide chain containing two thrombopoietin receptor binding domains; termed a “Peptibody” by Amgen Receptor binding domain has no homology with TPO A homodimer of 59kD single-chain subunits Each subunit consists of a human IgG1 Fc domain, linked at its C- terminus to a peptide chain containing two thrombopoietin receptor binding domains; termed a “Peptibody” by Amgen Receptor binding domain has no homology with TPO

5 Romiplostim: Mechanism of Action TYK2 JAK2 STAT5STAT5 Cell membrane TPO Receptor (Mpl) romiplostim Romiplostim: Higher binding affinity than human TPO Displaces TPO on human platelets Leads to TPO receptor, JAK2, Stat5 phosphorylation Increases megakaryocyte colony formation in vitro Romiplostim: Higher binding affinity than human TPO Displaces TPO on human platelets Leads to TPO receptor, JAK2, Stat5 phosphorylation Increases megakaryocyte colony formation in vitro STAT5STAT5

6 Romiplostim: Manufacturing Produced in E. coli Purified using standard bioprocessing technology Produced in E. coli Purified using standard bioprocessing technology