Procho5 medium

Suspension adopted CHO-S cells were provided (Invitrogen, CA, USA). Cells were maintained in ProCHO5 medium (Lonza AG, Verviers, Belgium). The medium was supplemented with 4mM L-glutamine (Invitrogen, CA, USA), 2mM PenSterp (Invitrogen, CA, USA), and anti-clumping agent (Invitrogen, CA, USA). The cells were kept in a humidified incubator at 37°C with 5% CO2 atmosphere. The cells were cultivated either in T-flasks or shaken in glass bottles. The cells were sub-cultured twice a week at a density of 3×10⁵ cells/ml. Trypan blue exclusion method was used to evaluate cell number and viability.

The human embryonic kidney (HEK) 293, HEK293T (ATCC) cell line and mouse NIH-3T3 cell (ATCC, Manassas, VA, USA) line were cultured in DMEM (Invitrogen, Waltham, MA, USA) supplemented with 10% FBS (BioWhittaker) at 37 °C in a 5% CO₂ environment. ExpiCHO cells (Thermo Fisher, Waltham, MA, USA) were cultured in ProCHO5 medium (Lonza) and incubated with agitation at 31 °C and 4.5% CO₂. HEK293E cells were cultured in an EX-CELL 293 serum-free medium (Sigma, St. Louis, MO, USA) and incubated with agitation at 37 °C and 4.5% CO₂. Expi293F cells (Thermo Fisher, Waltham, MA, USA) were cultivated in an Expi293™ Expression Medium (Thermo Fisher, Waltham, MA, USA) at 37 °C and 8% CO₂ on an orbital shaking platform with the shaking speed based on shaking diameter and flask size.

Cell lines were cultured in a shake flask (SF, 125 mL format, Corning) on an orbital shaker (125 rpm, 2.5 cm pinch, Celltron, Infors) at 37 °C/5% CO2 humidified conditions. Cells were passed twice per week in ProCHO-5 medium (Lonza) supplemented with 4 mM L-glutamine (Gibco) and 0.1% Pluronic® F-68 (Gibco)at 0.5 × 10⁶ viable cells/mL. Starting from these cultures, the fed-batch (FB) evaluation test was initiated. The cells were seeded at 0.5 × 10⁶ viable cells/mL in fresh medium in a 125 mL SF format and incubated under the conditions described above. The FB cultures were monitored for 14 days on viable cell density, viability (LUNA™ cell counter, Logos Biosystems), glucose, and lactate concentration (Biosen S-Line, EKF Diagnostic). A flat feed procedure was followed from day 4 onwards. A daily volume of 2% (v/v) Cell Boost™ 7a and 0.2% (v/v) Cell Boost™ 7b (Cytiva) was added to the FB cultures. Additionally, a 4 g/L glucose concentration was maintained in the FB cultures by adding an additional glucose stock solution from day 4 onwards (Sigma). From day 5 onwards the recombinant protein expression levels were determined by Bio-Layer Interferometry (BLI) analysis (Octet RED96e, Sartorius) using protein A biosensors and an Ig standard as reference.