Physicochemical and biological evaluation of JR-131 as a biosimilar to a long-acting erythropoiesis-stimulating agent darbepoetin alfa.

Renal anemia is predominantly caused by a relative deficiency in erythropoietin (EPO). Conventional treatment for renal anemia includes the use of recombinant human EPO (rhEPO) or a long-acting erythropoiesis-activating agent named darbepoetin alfa, which is a modified rhEPO with a carbohydrate chain structure that differs from native hEPO. We have developed a biosimilar to darbepoetin alfa designated JR-131. Here, we comprehensively compare the physicochemical and biological characteristics of JR-131 to darbepoetin alfa. JR-131 demonstrated similar protein structure to the originator, darbepoetin alfa, by peptide mapping and circular dichroism spectroscopy. Additionally, mass spectroscopic analyses and capillary zone electrophoresis revealed similar glycosylation patterns between the two products. Human bone marrow-derived erythroblasts differentiated and proliferated to form colonies with JR-131 to a similar degree as darbepoetin alfa. Finally, JR-131 stimulated erythropoiesis and improved anemia in rats similarly to darbepoetin alfa. Our data show the similarity in physicochemical and biological properties of JR-131 to those of darbepoetin alfa, and JR-131 therefore represents a biosimilar for use in the treatment of renal anemia.

Host Cell Proteins: The Hidden Side of Biosimilarity Assessment.

One major concern with biosimilars is that small differences compared with reference products might lead to unforeseen immunogenicity, thus affecting patient safety and drug efficacy. Differences could be due to either post-translational modifications of the therapeutic protein and/or to traces of impurities from the manufacturing process. The results presented in this communication illustrate the efforts to assess "biosimilarity" of a biosimilar candidate to a reference product for a specific group of process-related impurities, the host cell proteins (HCP). Extensive characterization of HCP in the drug substance of a biosimilar candidate revealed the identity of HCP copurifying with the protein of interest and guided process development to improve overall HCP clearance in the downstream process. The data presented illustrate the challenge of matching the reference product on either quantitative or qualitative aspects of HCP impurities.

Heterocyclic organobismuth(III) induces apoptosis of human promyelocytic leukemic cells through activation of caspases and mitochondrial perturbation.

We have synthesized novel heterocyclic organobismuth compounds that have potent antibacterial properties. In this study, we examined their anticancer activity and addressed the cellular mechanisms involved. Heterocyclic organobismuth compounds showed anticancer activities in various human cancer cell lines. These compounds have particularly potent anticancer activities against leukemia cell lines. One of them, bi-chlorodibenzo [c,f][1,5] thiabismocine (compound 3), inhibited the growth of the human promyelocytic leukemia cell line HL-60 at a concentration of 0.22 microM. Low concentrations of compound 3 (0.22-0.44 microM) induced apoptosis, whereas at a higher concentration (>1.1 microM) it causes acute necrosis. During the apoptosis, caspase-3, -8, and -9 were activated but caspase-12 was not. A broad caspase inhibitor (z-VAD-fmk), and caspase-3 (z-DEVD-fmk) and caspase-9 (z-LEHD-fmk) inhibitors suppressed the compound 3-induced apoptosis, but a caspase-8 inhibitor (z-IETD-fmk) was less effective, suggesting that the caspase-8 activity only partially participates in the apoptosis. In the apoptotic cells, cytochrome c was released from mitochondria to cytosol and a loss of mitochondrial transmembrane potential (DeltaPsi(m)) was detected. Compound 3-induced apoptosis was associated with enhanced generation of intracellular reactive oxygen species (ROS). Pretreatment of the cells with N-acetyl-L-cysteine or catalase suppressed the apoptosis. On the other hand, buthionine sulfoximine enhanced the compound 3-induced collapse of DeltaPsi(m) and apoptosis. Taken together, these results indicate that compound 3 is a potent inducer of apoptosis, triggering a caspase-3-mediated mechanism via the generation of ROS and release of cytochrome c from mitochondria, suggesting a potential mechanism for the anticancer activity of compound 3.