Characterization of an Sf-rhabdovirus-negative Spodoptera frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system

Abstract

Cell lines derived from the fall armyworm, Spodoptera frugiperda (Sf), are widely used as hosts for recombinant protein production in the baculovirus-insect cell system (BICS). However, it was recently discovered that these cell lines are contaminated with a virus, now known as Sf-rhabdovirus [1]. The detection of this adventitious agent raised a potential safety issue that could adversely impact the BICS as a commercial recombinant protein production platform. Thus, we examined the properties of Sf-RVN, an Sf-rhabdovirus-negative Sf cell line, as a potential alternative host. Nested RT-PCR assays showed Sf-RVN cells had no detectable Sf-rhabdovirus over the course of 60 passages in continuous culture. The general properties of Sf-RVN cells, including their average growth rates, diameters, morphologies, and viabilities after baculovirus infection, were virtually identical to those of Sf9 cells. Baculovirus-infected Sf-RVN and Sf9 cells produced equivalent levels of three recombinant proteins, including an intracellular prokaryotic protein and two secreted eukaryotic glycoproteins, and provided similar N-glycosylation patterns. In fact, except for the absence of Sf-rhabdovirus, the only difference between Sf-RVN and Sf9 cells was SF-RVN produced higher levels of infectious baculovirus progeny. These results show Sf-RVN cells can be used as improved, alternative hosts to circumvent the potential safety hazard associated with the use of Sf-rhabdovirus-contaminated Sf cells for recombinant protein manufacturing with the BICS.

Keywords: Baculovirus; Insect cells; Recombinant protein production; Sf-RVN cells; Sf-rhabdovirus.

Fig. 1

Sf-rhabdovirus assays. Total RNA isolated from Sf-RVN cells at various passage levels (from P0 to P60) was assayed for (A) Sf-rhabdovirus- or (B) Sf ribosomal protein L3-specific RNAs by RT-PCR/nested PCR or RT-PCR, respectively, as described in Materials and methods. In parallel, total RNA isolated from the pellet obtained by ultracentrifuging Sf-RVN cell-free media at P60 was assayed for (C) Sf-rhabdovirus-specific RNA by RT-PCR/nested PCR, as described in Materials and methods. Total RNAs from Sf9 cells (Sf9) or the pellet obtained by ultracentrifuging Sf9 cell-free media (CFM) were used as positive controls and total RNA from S2R+ cells (S2) was used as a negative control. An additional negative control reaction was performed with no template (H₂O) and the lanes marked M show the 100-bp markers, with selected sizes indicated on the left.

Fig. 2

Mycoplasma assays. Sf-RVN and Sf9 cell extracts (−) were assayed for mycoplasma contamination using the PCR-based Universal Mycoplasma Detection Kit (American Type Culture Collection), as described in Materials and methods. A plasmid encoding an M. arginini rRNA target sequence was used as the positive control. Additional controls were performed using Sf-RVN and Sf9 cell lysates alone or spiked with the positive control plasmid (+) to determine if the lysate interfered with the assay. A negative control reaction was performed with no template (H₂O). The lane marked M shows the 100-bp markers, with selected sizes indicated on the left.

Fig. 3

Cell growth and morphology. Sf-RVN and Sf9 cells were seeded into shake flasks at a density of 1.0 x 10⁶ cells/mL in ESF 921 medium. Triplicate samples were harvested at various times after seeding and viable cell counts and diameters were measured with an automated cell counter, as described in Materials and methods. The Figure shows the (A) average viable cell densities, (B) diameters, and (C) doubling times measured in three independent experiments, as well as (D) phase contrast micrographs of Sf-RVN and Sf9 cells at a magnification of 10X. The error bars represent the confidence intervals (P<0.05).

Fig. 4

Cell viability after baculovirus infection. Sf-RVN and Sf9 cells were infected with an Sf-rhabdovirus-negative stock of AcP(−)p6.9hSEAP at an MOI of (A) 0.1 or (B) 5 pfu/cell. Triplicate samples were harvested at various times post-infection and viability was measured using an automated cell counter, as described in Materials and methods. The plots show the average percent viability measured in two independent experiments. The error bars represent the confidence intervals (P<0.05).

Fig. 5

Recombinant β-gal production. Sf-RVN and Sf9 cells were infected with an Sf-rhabdovirus-negative stock of BacPAK6-ΔChi/Cath at an MOI of 5 pfu/cell. Triplicate samples were harvested at various times post-infection, and clarified intracellular extracts were assayed for (A) β-gal activity, as described in Materials and methods. This plot shows the average results with error bars representing the confidence intervals (P<0.05). One set of extracts was also used to measure (B) total intracellular β-gal production levels by immunoblotting analysis, with (C) scanning laser densitometry to estimate relative immunoreactive band intensities, as described in Materials and methods.

Fig. 6

Recombinant hSEAP production. Sf-RVN and Sf9 cells were infected with an Sf-rhabdovirus-negative stock of AcP(−)p6.9hSEAP at an MOI of (A, B, C) 0.1 or (D, E, F) 5 pfu/cell. Triplicate samples were harvested at various times post-infection, cell-free media were prepared and assayed for (A, D) hSEAP activity, as described in Materials and methods, and the average results were plotted with error bars representing the confidence intervals (P<0.05). One set of cell-free media was also used to measure (B, E) total extracellular hSEAP production levels by immunoblotting analysis, with (C, F) scanning laser densitometry to estimate relative immunoreactive band intensities, as described in Materials and methods.

Fig. 7

Recombinant hEPO production. Sf-RVN and Sf9 cells were infected with an Sf-rhabdovirus-negative stock of AcP(−)p6.9hEPO at an MOI of 5 pfu/cell. Samples were harvested at various times post-infection and cell-free media were prepared and assayed for (A) total extracellular hEPO production levels by immunoblotting analysis, with (B) scanning laser densitometry used to estimate relative immunoreactive band intensities, as described in Materials and methods.

Fig. 8

N-glycosylation profiles. Sf-RVN and Sf9 cells were infected with an Sf-rhabdovirus-negative stock of AcP(−)p6.9hEPO at MOI of 3 pfu/cell and hEPO-His was affinity-purified from the cell free media, as described in Materials and methods. N-glycans were enzymatically released, recovered, permethylated, and (A) analyzed by MALDI-TOF MS, as described in Materials and methods, with molecular ions detected as [M + Na]⁺ assigned structures, annotated using the standard cartoon symbolic representations, numbered for simplicity, and (B) presented as percentages of total.

Fig. 9

Recombinant baculovirus production. Sf-RVN and Sf9 cells were infected with Sf-rhabdovirus-negative stocks of AcP(−)p6.9hSEAP or AcP(−)p6.9hEPO. The resulting progeny were harvested and titered by plaque assays, as described in Materials and methods. The resulting titers were plotted as the average viral titers obtained in three independent experiments, with error bars representing confidence intervals (P<0.05 or 0.001). and methods.