Transduction of CHO-K1 cells using LVs and generation of trastuzumab-expressing cell pools, mini-pools and oligoclones

Day prior transduction, 5 × 10³ CHO-K1 cells were seeded in 96 well plate using DMEM/F12-FBS medium and incubated at 37 °C in 5% CO₂. After 16 h, transduction was performed by incubating LVs with cells in DMEM/F12 medium supplemented with 10 μg/mL of polybrene (Sigma-Aldrich, USA). For co-transduction, two different MOI ratios were used: (400:400) and (600:200) (MOI for LVs bearing LC: MOI for LVs bearing HC) (MOI-LC: MOI-HC). Eight hours post-transduction, the medium was replaced with fresh DMEM/F12-FBS medium and 0.6 mg/mL of G418 (selection medium). A second round of co-transduction was performed in the same conditions as outlined above. The cell culture supernatant was harvested at 72 h post-transduction and human IgG-expression was assessed by ELISA (antibody whole molecule).

To obtain cell mini-pools, after a second round of transduction, cells were cloned in 96 well plate (strategy 1 or early screening) (Fig. 2). The cells were seeded in 10 plates at 100 cells/well in DMEM/F12-FBS medium and 0.6 mg/mL of G418. The plates were incubated at 37 °C in 5% CO₂ during 10 days. The 10 cell mini-pools with the highest IgG expression levels, for each co-transduction ratio, were expanded to suspension culture in 25 cm² T-flasks using CP-CHO-CB5 medium supplemented with 1% FBS (CP-CHO-CB5-FBS) and 0.6 mg/mL of G418. T-flasks were incubated in vertical position at 37 °C in 5% CO₂ and shaking culture (120 rpm) (Infors HT, Switzerland). After 21 days under drug pressure in presence of low content of FBS (1%), cells were cultured in previously mentioned conditions without G418.

Strategies for obtaining trastuzumab-expressing CHO-K1 cells. MOI: multiplicity of infection. FBS: fetal bovine serum. CP-CHO-CB5-FBS: chemically defined, protein-free medium CP-CHO supplemented with 3 g/L of CB5 and 1% FBS. LC light chain, HC heavy chain

To generate cell pools, transduced cells were expanded to 24 well plate in DMEM/F12-FBS medium and 0.6 mg/mL of G418 (strategy 2) (Fig. 2). The plate was incubated at 37 °C in 5% CO₂ until cells reached up to 90% confluence. Then, cells were expanded to suspension culture in 25 cm² T-flasks in CP-CHO-CB5-FBS and 0.6 mg/mL of G418. T-flasks were incubated in vertical position at 37 °C in 5% CO₂ and shaking culture (120 rpm) (Infors HT, Switzerland). After 26 days under drug pressure in presence of low content of FBS (1%), cells were cultured as outlined above without G418.

To obtain cell oligoclones, the two cell mini-pools with the highest IgG expression levels were cloned in 96 well plates. The cells were seeded in 10 plates at 10 cells/well in DMEM/F12-FBS medium and were incubated at 37 °C in 5% CO₂. After 10 days, cell culture supernatant was removed to quantify human IgG-expression levels by ELISA. The oligoclones with the highest IgG expression levels in this stage, were expanded to 24 well plate in CP-CHO-CB5-FBS medium in static culture at 37 °C in 5% CO₂. Later, they were cultured in 25 cm² and 75 cm² T-flasks in shaking culture (120 rpm) (Infors HT, Switzerland).