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Biosimilars have emerged as one of the fastest-growing development opportunities in the biopharmaceutical sector. However, biosimilar development presents challenges at every level, from selection of a manufacturing platform, to analytical assays, demonstrating comparability, to in vivo testing, clinical testing, market access, and post-marketing surveillance.
While the opportunity is immense, the risk involved with biosimilar development is still relatively high with large up-front investment required and possible failure of the drug during development stages. Biosimilars are molecules manufactured to emulate a marketed biologic drug in chemical composition and structure, and possess comparable pharmacologic activity, safety and efficacy. Unlike generic small molecule drugs, however, creating an exact copy of a therapeutic protein is impossible, as the manufacturing process is integral to the final drug product composition and by its nature can never be identical. As a result, regulatory agencies evaluate this category of biologics based on their level of similarity to, rather than the exact replication of, the innovator drug.
This poster will present some of the challenges encountered during the development of a biosimilar compound and will highlight the critical factors encountered. Case studies will be presented that illustrate why a particular factor is critical and what the implication was of not paying sufficient attention to this factor.

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　L-carnitine is required for many physiological roles as participating in β-oxidation. Human OCTN2 (organic cation/carnitine transporter 2; SLC22A5), and its rat ortholog Octn2 (rSlc22a5), are ubiquitously-expressed membrane proteins that are hypothesized to be sodium-dependent specific transporters of L-carnitine. Although not generally regarded as a drug transporter, its role in drug pharmacokinetics has been clearly shown, particularly in relation to renal excretion.
Mildronate, is a carnitine congener which acts to inhibit fatty acid oxidation and has been shown to be transported by rat Octn2. However, characterization of kinetics of transport has not been described.
　Aims: To correlate transport properties of Octn2 and OCTN2 and investigate mildronate transport by Octn2/OCTN2 in particular. Identify and examine drugs inhibiting L-carnitine uptake, and to perform a species specificity analysis of the rat and human orthologs.
　Methods and results: Cellular uptake assays were performed using CHO-K1 cell lines stably overexpressing OCTN2 or Octn2 to examine species differences between the two transporters. Uptake of L-carnitine was quantitated using radiolabelled compound, whereas the detection of mildronate was carried out using HPLC-MS. Among others amiloride, imatinib, mildronate, omeprazole, quinine, quinidine and vincristine were used to examine their influence on L-carnitine uptake.
　Conclusion: ?Similarly to OCTN2, Octn2 also transports mildronate with high potency. However, L-carnitine was found to be a lower affinity substrate for Octn2 than for OCTN2. Furthermore, many pharmacologically important drugs were shown to affect L-carnitine transport by Octn2/OCTN2, although several differences between rat and the human orthologs were also observed.

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BACKGROUND: Cryopreserved hepatocytes are extensively used in drug industry today. However, comparing with cryopreserved human hepatocytes the single animal hepatocytes particularly rodent hepatocytes are limited on small size of batch regarding what is needed for setting up large scale study. In addition, animal hepatocytes are less resistant to isolation and cryopreservation process, with a significant alteration both at viability and plateability. Hence, the aim of the present study was designed to (i) to prepare freshly pooled hepatocytes from several small animals for making large size batch of pooled and cryopreserved rodent hepatocytes; (ii) to validate the pooled and cryopreserved rodent hepatocytes by comparing with fresh and cryopreserved hepatocytes from single animal. (iii) to characterize the post-thawing cell quality and pre-qualification of cryopreserved cells
STUDY DESIGN AND METHODS: For producing large size batch, rat and mouse hepatocyte were isolated from several animals and pooled and then cryopreserved by using an optimized process in BPI. The post-thaw viability, yield and plateability as well as the functionality of cryopreserved hepatocytes were checked and compared with both fresh and small size batch of cryopreserved hepatocyte from single animal donor. The pooled cryopreserved hepatocytes were also pre-qualified according to application including prediction of metabolic clearance, evaluation of CYP induction and hepatocyte transporter uptake assays.
RESULTS: We have developed an optimized technique for preparing and freezing of large size lot of pooled hepatocytes from multiple animal donors like rat and mice. They retain their fresh hepatocytes ability to attach to a collagen I coated matrix (post-thaw plateability), thereby permitting their use for long-term plated assay, such as induction and sandwich cultures. The comparison study shows that metabolism activity is comparable between the fresh and pooled cryopreserved hepatocytes. As well, a good lot-to-lot reproducibility was observed. Furthermore, some pre-qualified applications like induction or transport on cryopreserved pooled cells shown an acceptable inducibility of cytochrome P450 and efflux activity with sandwich-cultured hepatocytes.
Conclusions: Our pooled cryopreserved hepatocytes from multi-animal donors and multi-animal species represent a good alternative for use of freshly isolated hepatocytes for drug studies.

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　We evaluated three kinds of human hepatocytes (HepG2 cells, HepaRG cells and primary human hepatocytes (PHH)) as materials for metabolite-induced hepatotoxicity testing, and developed assay methods for that. First, we conducted cytotoxicity tests in HepG2 cells, HepaRG cells and PHH using ATP as a cytotoxicity parameter. The three kinds of hepatocytes were treated with 16 compounds whose metabolites have been reported to be hepatotoxic. In order to judge whether observed cytotoxicities are caused by metabolites or parent compounds, cytochrome P450 inhibitor (1-aminobenzotriazole: ABT) or glutathione synthesis inhibitor (L-Buthionine-S, R-sulfoximine: BSO) was treated concomitantly with each compound. As results, toxicity of ticlopidine was reduced with ABT and that of cyclophosphamide was increased with BSO in HepaRG cells and PHH, but not in HepG2. These results indicate that the cytotoxicity tests using HepaRG cells and PHH could detect metabolite-induced toxicities, meanwhile it is considered that the assay method should be modified to improve detection sensitivity. To find indicators with higher sensitivity, we conducted comprehensive gene expression analysis in HepG2 and HepaRG cells treated with ticlopidine, cyclophosphamide and acetaminophen. As a result, we identified three gene biomarkers, heme oxigenase 1, p62 and sulfiredoxin 1. Changes in their expression levels were greater than those in ATP. The assay using human hepatocytes and these gene markers is useful for detection of metabolite-induced hepatotoxicity.

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Hepatic cell lines are widely used by pharmaceutical companies and have a key role in drug research and development. However, their relevance as hepatic models is mostly dependent on the banking of the initial cells which is also the first source for distribution of cells with highly stable properties.
Our aim was to set up a new strategy based on reprogramming towards stem cells of the parental HepaRG cell line, allowing establishment of a new indefinite source for differentiated HepaRG cells. We then used the “stem cell like” “Hepa-SC” to reconvert them into hepatic and other differentiation lineages.
As the first step, stem-like Hepa-SC cells were obtained by submitting parental progenitors to mechanical forces induced by shape constraint which favoured occurrence of stemness properties, including self-renewal, plasticity and pluripotentiality. These cells were subsequently stabilized by epigenetic factors able to modulate the methylation/acetylation status, and they were expanded to produce the master and working banks.
In a second step, we successfully demonstrated that Hepa-SC cells from the banks were able to lose their stem-like status and retrieve the hepatic lineage. This strategy has led us to obtain new cell lines called “Hepa-RP” which rapidly recover the unique characteristics of the parental cells, including a bipotent progenitor stage corresponding to a proliferative stage, and a differentiation stage leading to the organization of mature hepatocyte colonies with a polarized accumulation of F-actin at the biliary poles. Further analyses have revealed that Hepa-RP cells: i)- grow faster than HepaRG cells; ii)- express high levels of drug metabolism enzymes such as CYP3A4, as well as the glycolytic enzyme, aldolase B, and hepatic lipid metabolism enzymes such as APOA1, APOB, and APOC; iii)- exhibit functional transporter expression e.g. the canalicular MRP2 s efflux of the fluorescent MRP2-substrate, CDFA. Finally, incubation with specific substrates of CYP2B6, CYP3A4 and CYP1A2 has confirmed that the levels of these enzymes are the same in both Hepa-RP and HepaRG cells. By contrast, as expected, an analysis of Hepa-SC cells showed that these stem-like cells do not express any liver markers but expressed stem markers including OCT3/4 and Sox17.
In conclusion, we succeeded in producing stem-like cells, Hepa-SC, which constitutes an indefinite source for establishing new Hepa-RP cell lines that preserve all main hepatic characteristics of the parental HepaRG cell line. This technology ensures the sustainability unlimited supply of this robust and valuable cell line for use in ADME and toxicology assays.

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Humoral Immunity and Lymphocyte Immunophenotyping Control Background Data in Mainland
Lymphocyte immunophenotyping (LIP) of peripheral blood and T-cell dependent antibody response (TDAR) data from Mainland (ML, China and Cambodia) and Indonesian Island (IL) cynomolgus monkey infants (from 1 to 12-month old, the control groups from 10 to 14 pre and postnatal development studies) were retrospectively analyzed and compared for use as historical background data.
Blood samples for LIP were collected throughout the postnatal period and analyzed for total CD3+ T cells, CD3+CD4+ Helper T cells, CD3+CD8+ Cytotoxic T cells, CD3-CD20+ B cells, and CD3-CD16+ or CD3-CD159a+ Natural Killer (NK) cells. To evaluate TDAR, keyhole limpet hemocyanin (KLH) was injected twice into the infants, and primary and secondary IgM and IgG levels in serum were measured by ELISA.
IL monkeys had higher absolute counts and relative percentages of NK cell populations, and lower circulating B cell numbers when compared to ML monkeys. IgM elevated rapidly after the 1^st and 2^nd KLH doses, with peak IgM concentrations in IL 24-27% lower than that observed in ML. IgG elevated gradually after the 1^st dose and increased rapidly after the 2^nd challenge. The highest IgG levels after primary KLH challenge in IL was 24% lower than ML. After the 2^nd KLH dose, the highest IgG in IL was 39% higher than ML. The trends of IgM and IgG responses were similar between IL and ML.
In conclusion, there were distinct differences in NK and B-cell populations and IgM/IgG responses between infants of ML and IL origin, suggesting origin-specific differences in lymphocyte development and function.

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Although a T-dependent antibody response (TDAR) assay is generally recommended as the first-line immune function assay in nonclinical immunotoxicity evaluation, second-line assays such as delayed-type hypersensitivity (DTH) to measure cell-mediated responses can provide helpful additional information. In this study, male Cynomolgus monkeys were injected intramuscularly either once or twice with 1 mg Keyhole Limpet Hemocyanin (KLH) or twice with a commercially available tetanus vaccine (40 IU tetanus toxoid + 0.06 mg aluminum hydroxide). All animals were subsequently challenged by intradermal injections of the same antigen or aluminum hydroxide after 4, 6 and 8 weeks. Clinical reactions at the injection sites were scored 24, 48 and 72 h post challenge. Skin biopsies were taken on completion of the observation period after each challenge for standard histological examination and immunolabeling using CD3 (T lymphocytes), CD19 (B lymphocytes) and CD68 (macrophages) antibodies. Tetanus toxoid induced stronger clinical reactions than KLH, whereas aluminum hydroxide induced no clinical reaction. Perivascular mononuclear cell infiltrates, a histopathological finding consistent with a DTH reaction, were seen after all challenges with tetanus toxoid or KLH, but not with aluminum hydroxide. Immunohistochemistry evidenced the presence of T lymphocytes and macrophages within these infiltrates. These results suggest that tetanus toxoid adjuvanted with aluminum hydroxide can induce a consistent DTH response for use as a model of cell-mediated response in Cynomolgus monkeys.

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