Cedex hires

An expression vector encoding the gene for S_dF_2P or HexaPro along with a DHFR selection marker was transfected into CHO-DG44 cells by electroporation using the MaxCyte STX® scalable transfection system (MaxCyte, Gaithersburg, MD) as previously described²⁸ . Transfected cells were cultivated in an Multitron shaker (Infors HT, Switzerland) set to 37 °C, 5% CO₂, and 80% relative humidity with a shaking speed of 130 rpm (orbital throw of 1 inch) in CDM4CHO medium with 6 mM L-glutamine. Forty-eight hours after transfection, methotrexate (MTX) was added to the culture to a final concentration of 100 nM. Viable cell density and viability for the culture was assessed every three to four days using the Cedex HiRes (Roche CustomBiotech, Indianapolis, IN). Once a week, the cells were centrifuged at 100 × g for 10 min and resuspended in fresh CDM4CHO medium with 6 mM L-glutamine and 100 nM MTX. When the viability of the pools recovered to ≥ 80%, the medium was replaced with ActiCHO P medium containing 6 mM L-glutamine and 100 nM MTX.

Two Chinese hamster ovary (CHO) cell lines, one human embryo kidney (HEK) 293 and one insect cell line (Trichoplusia ni BTI-Tn-5B1-4, High Five) were used for the animal cell culture experiments (Table 5).
Both CHO cell lines produce the antibody immunoglobulin G (IgG) and behave in the same manner as industrial mammalian production cell lines. HEK cells are also widely used, but tend to form larger aggregates [86 (link)]. High Five insect cells have similar morphological characteristics to CHO cells but are cultured without CO₂ based pH control and at lower temperatures. The applied cultivation conditions are summarized in Table 6. The effect of different clones, media and cultivation conditions should test the robustness of the methodology.
Cell density and viability were measured using a Cedex HiRes (Roche Custom Biotech) for the CHO and High Five cells, and with a NucleoCounter NC200 (Chemometec A/S, Allerod, Denmark) for the HEK293 cells. Metabolites (glucose, glutamine, ammonia), as well as the product IgG, were determined using a Cedex Bio (Roche Custom Biotech) and the corresponding analytic kits.

Viable cell counts (VCC), dead cell counts (DCC), and cell size were determined with the Cedex HiRes automatic picture Analyzer (Roche, Mannheim, Germany). The metabolites glucose (Glc), lactate (Lac), and ammonia (Amm) were measured with the Cedex Bio HT Analyzer (Roche). Concentration of the mAbs was measured by HPLC (Ultimate 3000, Dionex, Sunnyvale, CA, USA) with a protein A sensor cartridge (Applied Biosystems, Bleiswijk, Netherlands).

During the production process 1 ml samples were taken daily. Viable cell densities and viabilities were measured using a Cedex HiRes (Roche Innovatis, Switzerland). The pH (7.2) and pO₂ (60%) were measured and controlled by the Ambr®250 system. Offline pH measurements for offset calibration (for ΔpH > 0.05) were performed using a FiveEasy Plus pH meter FP20‐Micro (Mettler Toledo, USA) every 2 days.
Substrates (glucose, lactate, glutamine, glutamate), the produced mAb and total protein concentrations were analyzed during the production process and after the cell separation using the Cedex Bio (Roche, Switzerland). The DNA concentration in the supernatant was analyzed using the Nanodrop 2000 (Thermo Scientific, USA). Antibody bioactivity was determined in triplicates by an adherent mouse fibroblast (L929) cell (CLS Cell Lines Service, Germany; catalog number 400260) based assay using the tumor necrosis factor alpha (TNF‐α) under the presence of actinomycin D. L929 cell viability was analyzed using the cell titer‐blue assay (Promega, USA) after 24 h of treatment with the antigen and antibody. The produced antibody was diluted and used in low and high concentrations of 8 and 80 ng/ml, respectively. The antigen TNF‐ α was used in a fixed concentration, determined to result in around 20% L929 cell viability without addition of functional antibody.