Exploring Parametric and Mechanistic Differences between Expi293FTM and ExpiCHO-STM Cells for Transient Antibody Production Optimization

Abstract

Rapidly producing drug-like antibody therapeutics for lead molecule discovery and candidate optimization is typically accomplished by large-scale transient gene expression technologies (TGE) with cultivated mammalian cells. The TGE methodologies have been extensively developed over the past three decades, yet produce significantly lower yields than the stable cell line approach, facing the technical challenge of achieving universal high expression titers for a broad range of antibodies and therapeutics modalities. In this study, we explored various parameters for antibody production in the TGE cell host Expi293F^TM and ExpiCHO-S^TM with the transfection reagents ExpiFectamine^TM and polyethylenimine. We discovered that there are significant differences between Expi293F^TM and ExpiCHO-S^TM cells with regards to DNA complex formation time and ratio, complex formation buffers, DNA complex uptake trafficking routes, responses to dimethyl sulfoxide and cell cycle inhibitors, as well as light-chain isotype expression preferences. This investigation mechanistically dissected the TGE processes and provided a new direction for future transient antibody production optimization.

Keywords: CHO cells; DNA uptake trafficking pathways; HEK cells; Kappa and lambda light chain; antibody production; transfection reagents; transient gene expression.

Figure 1

Optimizing the dilution buffer processes for the DNA complexation with ExpiFectamine™ and PEI for Expi293F^TM and ExpiCHO-S^TM. As described in Section 2.4, the target DNAs and the transfection reagents were diluted separately in different volumes of Opti-MEM^TM (mL/L cell culture volume per tube); prior to the mixing of the two components for the complexation formation. For Expi293F^TM, the expression titers were determined five days post-transfection. For ExpiCHO-S^TM, the expression titers were measured seven days post-transfection. (A) The Opti-MEM^TM medium dilution conditions for the DNA complexation with ExpiFectamine™ 293 and PEI (*, p < 0.05) in Expi293F^TM for antibody-A. The titers obtained under the conditions of 10 mL/L per tube were set as 100%. (B) The Opti-MEM^TM medium dilution conditions for the DNA complexation with ExpiFectamine™ CHO (**, p < 0.05) and PEI in ExpiCHO-S^TM for antibody-A. The titers obtained under the conditions of 10 mL/L per tube were set as 100%. (C) The pH effects of the dilution buffers for the DNA complexation with ExpiFectamine™ 293 (#, p < 0.05) and PEI (#, p < 0.05) in Expi293F^TM for antibody-A. The Opti-MEM^TM medium and other indicated dilution buffers in different pHs were used for diluting the DNAs and the transfection agents in 100 mL/L per tube. The titers obtained under the conditions of Opti-MEM^TM medium were set as 100%. (D) The pH effects of the dilution buffers for the DNA complexation with ExpiFectamine™-CHO (&, p < 0.05) and PEI (&, p < 0.05) in ExpiCHO-S^TM for antibody-A. The titers obtained under the conditions of Opti-MEM^TM medium were set as 100%.

Figure 2

Optimizing the ratios between PEI and DNA in Expi293F^TM and ExpiCHO-S^TM. As described in Materials & Methods, the target DNAs encoding antibody-A were incubated with PEI in various ratios and transfected into Expi293F^TM and ExpiCHO-S^TM cells. For Expi293F^TM, the expression titers were determined five days post-transfection. For ExpiCHO-S^TM, the expression titers were measured seven days post-transfection. The titers obtained for those with ExpiFectamine™ were set as 100% (n = 4 ± S.D., ##, p < 0.05)).

Figure 3

Optimizing the DNA complex formation time in Expi293F^TM and ExpiCHO-S^TM with ExpiFectamine™ and PEI. As described in Section 2.4, the target DNAs encoding antibody-A were incubated with either ExpiFectamine™ (Panel A) or PEI (Panel B) for various time points (−0.5 min: the transfection reagents and the DNAs were directly added into the cell culture without mixing; 0 min: the transfection reagents and the DNAs were mixed, but without incubation prior to the addition to cell culture; incubation time after mixing: 0.5 min, 1 min, 2.5 min, 5 min, 10 min, 15 min, and 30 min), and transfected into Expi293F^TM and ExpiCHO-S^TM cells. The highest titers with each transfection reagent in each cell host were set as 100% (n = 3 ± S.D., ***, p < 0.05).

Figure 4

Size measurement of the DNA: ExpiFectamine™CHO or PEI complex with DLS. DLS measurements for size determination were described in Section 2.8. ExpiFectamine™CHO alone, or PEI alone, or in complex with the target DNAs encoding antibody-A (DNA:PEI = 1:3.5; DNA: ExpiFectamine™CHO = 1 µg:3.2 µL) performed in Opti-MEM^TM medium (#, p < 0.05, ##, p < 0.05).

Figure 5

Expi293F^TM and ExpiCHO-S^TM cells responded differently to the endocytosis blockers. As described in Section 2.4, various endocytosis blockers were added to the cell culture of Expi293F^TM and ExpiCHO-S^TM prior to the transfection of the target DNAs encoding antibody-A complexed with ExpiFectamine™. Cell viability in ExpiCHO-S^TM (pane A), and Expi293F^TM (panel B) as well as expression titers (panel C) were determined. The titers for the mock-treated control in each cell host were set as 100% (*, p < 0.05, **, p < 0.05).

Figure 6

Co-transfecting the genes encoding cell cycle inhibitor p21 and p27 enhanced transient expression in Expi293F^TM and ExpiCHO-S^TM cells, with a bigger effect in ExpiCHO-S^TM. As described in Section 2.4, the target DNAs encoding antibody-A, antibody-B, and antibody C complexed with ExpiFectamine™ along with different amounts of plasmid DNAs encoding p21/p27 were transfected into Expi293F^TM (Panel A) and ExpiCHO-S^TM (Panel B) cells. The control titers without p21/p27 DNAs were set as 100% (n = 3 ± S.D, #, p < 0.05).

Figure 7

DMSO with increased concentrations could enhance more transient expression in ExpiCHO-S^TM cells than in Expi293F^TM cells. As described in Section 2.4, Expi293F^TM (Panel A) and ExpiCHO-S^TM (Panel B) cells were pretreated with different concentrations of DMSO prior to the DNA transfection for antibody-A. The control titers without DMSO treatment were set as 100% (n = 3 ± S.D., *, p < 0.05, #, p < 0.05).

Figure 8

Expression preferences for kappa and lambda light chain isotypes were detected in Expi293F^TM and ExpiCHO-S^TM. As described in Section 2.4, the target DNAs encoding common light chain (CLC) antibodies A, B, and C with a kappa light chain or CLC antibody-D, E, and F with a lambda light chain were transfected into either Expi293F^TM or ExpiCHO-S^TM cells. (A) Expi293F^TM produced high titers for CLC antibodies A-C with kappa light chain and for CLC antibodies D-F with a lambda light chain, yet ExpiCHO-S^TM only expressed well for CLC antibodies A-C with kappa light chain (#, p < 0.05). (B) Co-transfecting heavy chain (HC) of CLC antibody E with both kappa (LCK) and lambda (LCL) light chain into either Expi293F^TM or ExpiCHO-S^TM cells resulted in 1:1 kappa/lambda expression ratio in Expi293F^TM cells but 99:1 expression ratio in ExpiCHO-S^TM cells. (C) Mass spectrometry analysis of the kappa and lambda co-transfection in Expi293F^TM cells. (D) Mass spectrometry analysis of the kappa and lambda co-transfection in ExpiCHO-S^TM cells.