Genetic analyses of Per.C6 cell clones producing a therapeutic monoclonal antibody regarding productivity and long-term stability.

Genetic characterization of protein-producing clones represents additional value to cell line development. In the present study, ten Per.C6 clones producing a Rebmab100 monoclonal antibody were selected using two cloning methods: six clones originated from limiting dilution cloning and four by the automated colony picker ClonePix FL. A stability program was performed for 50 generations, including 4 batches distributed along the timeframe to determine specific productivity (Qp) maintenance. Four stable clones (two from limiting dilution and two from ClonePix FL) were further evaluated. The relative mRNA expression levels of both heavy chain (HC) and light chain (LC) genes were verified at generations 0, 30-35, and 50-55 of the stability program. At generations 0 and 30-35, LC gene expression level was higher than HC gene, whereas at generation 50-55, the opposite prevailed. A high correlation was observed between Qp and HC or LC mRNA expression level for all clones at each generation analyzed along the continuous culture. The mRNA stability study was performed at steady-state culture. The LC gene displayed a higher half-life and lower decay constant than HC gene, accounting for the higher observed expression level of LC mRNA in comparison to HC mRNA. Clone R6 was highlighted due its high Qp, mRNA expression levels, and mRNA stability. Besides the benefits of applying genetic characterization for the selection of stable and high-producing clones, the present study shows for the first time the correlation between Qp and HC or LC expression levels and also mRNA stability in clones derived from human cell line Per.C6(®).

A new CHO (Chinese hamster ovary)-derived cell line expressing anti-TNFα monoclonal antibody with biosimilar potential.

Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine that mediates the homeostasis of immune responses; its exacerbated production is associated with the pathogenesis of autoimmune and chronic inflammatory diseases. Anti-TNFα drugs have revolutionized the treatment of inflammatory conditions such as rheumatoid arthritis and Crohn's disease. Currently, a worldwide race is on stage for the production of biosimilars moved by patent expiration of monoclonal antibodies (mAbs), such as anti-TNFα adalimumab. Our goal was to develop the first stage of an adalimumab biosimilar candidate with potential for national production, through the generation of a productive and stable cell line and assess its functionality. The robotic system ClonePix was used for screening and isolation of colonies from transfected CHO-S stable pools plated in semisolid medium. Selected clones were expanded based on growth and productivity. Purified mAbs from different clones were tested for binding and functional activity. The binding affinity of the denominated adabut clones to TNFα and FcRγ did not differ statistically when compared to reference adalimumab. One functional activity assay demonstrated the antibody neutralization capacity of the cytotoxicity induced by TNFα in L929 murine fibroblasts. A second assay confirmed adabut as an antagonist of the TNFα activity by the inhibition of the cell adhesion molecule expression in HUVEC cultures. The binding and functional activity analyses performed with selected adabut clones in comparison to reference adalimumab represent an important status of "non-inferiority," part of the process required for a biosimilar development. We generated and selected high-quality adabut clones which mAbs may be further developed as the first in-house made Brazilian biosimilar, demonstrating a success case for our incipient biotechnology industry, or also modified as biobetters, thus representing an innovative strategy for the patients' welfare.

Biosimilars advancements: Moving on to the future.

Many patents for the first biologicals derived from recombinant technology and, more recently, monoclonal antibodies (mAbs) are expiring. Naturally, biosimilars are becoming an increasingly important area of interest for the pharmaceutical industry worldwide, not only for emergent countries that need to import biologic products. This review shows the evolution of biosimilar development regarding regulatory, manufacturing bioprocess, comparability, and marketing. The regulatory landscape is evolving globally, whereas analytical structure and functional analyses provide the foundation of a biosimilar development program. The challenges to develop and demonstrate biosimilarity should overcome the inherent differences in the bioprocess manufacturing and physicochemical and biological characterization of a biosimilar compared to several lots of the reference product. The implementation of approaches, such as Quality by Design (QbD), will provide products with defined specifications in relation to quality, purity, safety, and efficacy that were not possible when the reference product was developed. Actually, the need to prove comparability to the reference product by the biosimilar industry has increased the knowledge about the product and the production-process associated by the use of powerful analytical tools. The technological challenges to make copies of biologic products while attending regulatory and market demands are expected to help innovation in the direction of attaining more productive manufacturing processes.

Rebmab200, a humanized monoclonal antibody targeting the sodium phosphate transporter NaPi2b displays strong immune mediated cytotoxicity against cancer: a novel reagent for targeted antibody therapy of cancer.

NaPi2b, a sodium-dependent phosphate transporter, is highly expressed in ovarian carcinomas and is recognized by the murine monoclonal antibody MX35. The antibody had shown excellent targeting to ovarian cancer in several early phase clinical trials but being murine the antibody's full therapeutic potential could not be explored. To overcome this impediment we developed a humanized antibody version named Rebmab200, expressed in human PER.C6® cells and cloned by limiting dilution. In order to select a clone with high therapeutic potential clones were characterized using a series of physicochemical assays, flow cytometry, real-time surface plasmon resonance, glycosylation analyses, immunohistochemistry, antibody-dependent cell-mediated cytotoxicity, complement-dependent-cytotoxicity assays and quantitative PCR. Comparative analyses of Rebmab200 and MX35 monoclonal antibodies demonstrated that the two antibodies had similar specificity for NaPi2b by flow cytometry with a panel of 30 cell lines and maintained similar kinetic parameters. Robust and high producer cell clones potentially suitable for use in manufacturing were obtained. Rebmab200 antibodies were assessed by immunohistochemistry using a large panel of tissues including human carcinomas of ovarian, lung, kidney and breast origin. An assessment of its binding towards 33 normal human organs was performed as well. Rebmab200 showed selected strong reactivity with the tested tumor types but little or no reactivity with the normal tissues tested confirming its potential for targeted therapeutics strategies. The remarkable cytotoxicity shown by Rebmab200 in OVCAR-3 cells is a significant addition to the traits of stability and productivity displayed by the top clones of Rebmab200. Antibody-dependent cell-mediated toxicity functionality was confirmed in repeated assays using cancer cell lines derived from ovary, kidney and lung as targets. To explore use of this antibody in clinical trials, GMP production of Rebmab200 has been initiated. As the next step of development, Phase I clinical trials are now planned for translation of Rebmab200 into the clinic.