An anti-apoptotic HEK293 cell line provides a robust and high titer platform for transient protein expression in bioreactors

Abstract

HEK293 transient expression systems are used to quickly generate proteins for research and pre-clinical studies. With the aim of engineering a high-producing host that grows and transfects robustly in bioreactors, we deleted the pro-apoptotic genes Bax and Bak in an HEK293 cell line. The HEK293 Bax Bak double knock-out (HEK293 DKO) cell line exhibited resistance to apoptosis and shear stress. HEK293 DKO cells sourced from 2 L seed train bioreactors were most productive when a pH setpoint of 7.0, a narrow pH deadband of ±0.03, and a DO setpoint of 30% were used. HEK293 DKO seed train cells cultivated for up to 60 days in a 35 L bioreactor showed similar productivities to cells cultivated in shake flasks. To optimize HEK293 DKO transfection cultures, we first evaluated different pH and agitation parameters in ambr15 microbioreactors before scaling up to 10 L wavebag bioreactors. In ambr15 microbioreactors with a pH setpoint of 7.0, a wide pH deadband of ±0.3, and an agitation of 630 rpm, HEK293 DKO transient cultures yielded antibody titers up to 650 mg/L in 7 days. The optimal ambr15 conditions prompted us to operate the 10 L wavebag transfection without direct pH control to mimic the wide pH deadband ranges. The HEK293 DKO transfection process produces high titers at all scales tested. Combined, our optimized HEK293 DKO 35 L bioreactor seed train and 10 L high titer transient processes support efficient, large-scale recombinant protein production for research studies.

Keywords: HEK293 cells; Transient transfection; ambr; bioreactor; polyethylenimine; recombinant protein production.

Figure 1.

Comparison of the HEK293 Bax Bak DKO (HEK293 DKO) cell line to the parental HEK293 cell line. (A) Viability after exposure of the cell lines to 1 µM staurosporine to induce apoptosis. Using a flow constriction device (FCD) to assess sensitivity to shear stress: (B) total lysis after FCD, (C) viable cell density (VCD) before and after FCD, and (D) viability before and after FCD.

Figure 2.

Optimizing N:P ratio and DNA concentration for HEK293 DKO transient transfections: (A) Transfections were tested across N:P ratios of 5 to 12.5 and DNA concentrations of 0.75 to 1.5 µg/mL. Transfecting HEK293 and HEK293 DKO cells at the 30 mL tubespin scale with an N:P ratio of 7.5 and a DNA concentration of 1 µg/mL: (B) VCD and viability over the 7-day production cultures and (C) day 7 titers.

Figure 3.

Scaling up the HEK293 DKO seed train from a 1 L shake flask to controlled 2 L bioreactors. Bioreactor #1: pH setpoint of 7 with a deadband of ±0.03 and a DO setpoint of 30%. Bioreactor #2: pH setpoint of 7 with a deadband of ±0.4 and a DO setpoint of 60%. Passaging the 1 L shake flask and 2 L bioreactors every 3–4 days for 25 days: (A) VCD and viability, (B) glucose and lactate, (C) offline pH, and (D) pO₂. (E) Day 7 transfection titers from 30 mL tubespins.

Figure 4.

Scale up of the HEK293 DKO seed train from a 1 L shake flask to a controlled 35 L bioreactor. Passaging the 1 L shake flask and 35 L bioreactor every 3–4 days for 60 days: (A) VCD and viability, (B) glucose and lactate, and (C) offline pH. (D) Day 7 transfection titers from 30 mL tubespins.

Figure 5.

HEK293 DKO transient transfections in controlled ambr15 bioreactors compared to 30 mL shake flasks. (A) VCD and viability, (B) glucose and lactate, (C) osmolality and pH, (D) pO₂, and (E) titers over the 7-day production cultures.

Figure 6.

Scale up of HEK293 DKO transient transfections from a 30 mL tubespin to a 10 L wavebag. (A) VCD and viability, (B) glucose and lactate, (C) osmolality and pH, and (D) pO₂ over the 7-day production cultures. (E) Day 7 titers.