Production of an anti-TNFα antibody in murine myeloma cells by perfusion culture.

Infliximab is a mouse/human chimeric IgG1 monoclonal antibody which recognizes the proinflammatory cytokine, tumor necrosis factor α (TNFα), and inhibits receptor interactions, thereby decreasing inflammation and autoimmune response in patients. This monoclonal antibody has been successfully used to treat rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis. However, the high treatment cost limits patient access to this biotherapy. One alternative to this problem is the use of biosimilars. In this work, we describe the stable expression and physicochemical characterization of an anti-TNFα antibody. While infliximab is produced in recombinant murine SP2/0 cells, our anti-TNFα IgG antibody was expressed in recombinant murine NS0 myeloma cells. The best anti-TNFα antibody-expressing clone was selected from three clone candidates based on the stability of IgG expression levels, specific productivity as well as TNFα-binding activity compared to commercial infliximab. Our results indicate that the selected cell clone, culture medium, and fermentation mode allowed for the production of an anti-TNFα antibody with similar characteristics to the reference commercially available product. An optimization of the selected culture medium by metabolomics may increase the volumetric productivity of the process to satisfy the demand for this product. Further experiments should be performed to evaluate the biological properties of this anti-TNFα antibody. KEY POINTS: • An anti-TNFα antibody was produced in NS0 cells using perfusion culture. • A proprietary chemically defined culture medium was used to replace commercially available protein-free medium. • The purified anti-TNFα antibody was comparable to the reference marketed product.

A protein-free chemically defined medium for the cultivation of various micro-organisms with food safety significance.

AIMS: To develop a broadly applicable medium free of proteins with well-defined and reproducible chemical composition for the cultivation of various micro-organisms with food safety significance. METHODS AND RESULTS: The defined medium was designed as a buffered minimal salt medium supplemented with amino acids, vitamins, trace metals and other nutrients. Various strains commonly used for food safety research were selected to test the new defined medium. We investigated single growth factors needed by different strains and the growth performance of each strain cultivated in the defined medium. Results showed that the tested strains initially grew slower in the defined medium compared to tryptic soy broth, but after an overnight incubation cultures from the defined medium reached adequately high cell densities. CONCLUSIONS: The newly designed defined medium can be widely applied in food safety studies that require media with well-defined chemical constituents. SIGNIFICANCE AND IMPACT OF THE STUDY: Defined media are important in studies of microbial metabolites and physiological properties. A defined medium capable of cultivating different strains simultaneously is needed in the food safety area. The new defined medium has broader applications in comparing different strains directly and provides more reproducible results.

In Vitro Culture of Human Hematopoietic Stem Cells in Serum Free Medium and Their Monitoring by Flow Cytometry.

Hematopoietic stem cells can be isolated from human blood cells trapped in leukoreduction systems. The leukoreduction systems filters or chambers are usually discarded from routine blood or platelet donations in blood banks around the world. These CD34+ cells are a good source of normal stem cells and can be used as models to characterize the blood stem cells before and after culture in vitro. This chapter contains detailed methodologies for the isolation of stem cells from peripheral blood, the culture of these cells in a medium exempt of animal proteins and for the flow cytometry analysis of the resulting cell population for the characterization of their differentiation.

Adaptation of CHO cells in serum-free conditions for erythropoietin production: Application of EVOP technique for process optimization.

Mammalian cell cultures are the preferred expression systems for the production of biopharmaceuticals requiring posttranslational processing. Usually, cell cultures are cultivated in medium supplemented with serum, which supports cell proliferation, viability, and productivity. However, due to scientific and regulatory concerns, serum-free conditions are required in recombinant protein production. Cell lines that are intended for commercial recombinant protein production have to adapt to serum- or protein-free conditions early in their development. This is a labor- and time-consuming process because of the specific cell requirements related to their adaptation in new microenvironment. In the present study, a Chinese hamster ovary (CHO) cell line producing glycosylated recombinant human erythropoietin (rhEPO) was adapted for growth and rhEPO production in serum- and protein-free conditions. The physiology, growth parameters, and morphology of the CHO cells and rhEPO biosynthesis and structure were closely monitored during the adaptation process to avoid unwanted selection of cell subpopulations. The results showed that the CHO cells were successfully adapted to suspension growth and rhEPO production in the protein-free conditions and that the structure of rhEPO remained nearly unchanged. In addition, during rhEPO production in the protein-free suspension conditions, the agitation rate seem to be significant for optimal process performance in contrast to the initial cell concentration, evaluated through evolutionary operation method.

Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells.

Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing.