Characterization of an Sf-rhabdovirus-negative Spodoptera frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system
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.
Since it was first described in the peer-reviewed literature in the early 1980's [2], [3], the baculovirus-insect cell system (BICS) has become a widely recognized and heavily utilized recombinant protein production platform [4], [5]. The advantages of the BICS include its flexibility, speed, simplicity, eukaryotic protein processing capacity, and ability to produce multi-subunit protein complexes. For nearly 30 years, the BICS was used mainly to produce recombinant proteins for basic research in academic and industrial labs. More recently, however, the BICS has emerged as a bona fide commercial manufacturing platform, which is now being used to produce several biologics licensed for use in human (Cervarix®, Provenge®, Glybera® and Flublok®) or veterinary (Porcilis® Pesti, BAYOVAC CSF E2®, Circumvent® PCV, Ingelvac CircoFLEX® and Porcilis® PCV) medicine [6]. In addition, the BICS is being used to produce several other biologic candidates, including potential vaccines against norovirus, parvovirus, Ebola virus, respiratory syncytial virus, and hepatitis E virus, which are in various stages of human clinical trials [6].
The insect cell lines most commonly used as hosts in the BICS are derived from the cabbage looper, Trichoplusia ni, or fall armyworm, Spodoptera frugiperda (Sf), and most biologics manufactured with the BICS are produced using the latter. The original Sf cell line, designated IPLB-SF-21, also known as Sf-21, was derived from pupal ovaries in 1977 [7]. Other commonly used Sf cell lines include Sf9, a subclone of IPLB-SF-21 [8], and its daughter subclones, Super 9 [9] and Sf900+, also known as expresSF+ [10].
Recently, Ma and coworkers [1] discovered that every Sf cell line tested, including Sf21 and Sf9 cells obtained from two reputable commercial sources, was contaminated with a novel rhabdovirus. This adventitious agent, which is now known as Sf-rhabdovirus, was discovered in a general effort to evaluate the biosafety of the BICS platform. A research group at Takeda Vaccines, Inc. independently confirmed the presence of Sf-rhabdovirus in the Sf9 cells used to produce their norovirus vaccine candidate [11]. In addition, we found that all our lab Sf-21, Sf9, and expresSF+ cell stocks, obtained from a variety of sources, are contaminated with this virus (data not shown). Thus, Sf-rhabdovirus appears to be a very common, if not universal, contaminant of the Sf cell lines commonly used as hosts for recombinant protein production in the BICS.
It is important to note that Sf-rhabdovirus is unlikely to be harmful for humans, as it cannot replicate in human or monkey cell lines [1]. Moreover, the only detectable phylogenetic relationship between Sf-rhabdovirus and previously recognized viruses was found in a short sequence, which was related to the L genes of insect and plant rhabdoviruses [1]. Therefore, the potential impact of Sf-rhabdovirus on biosafety of the BICS platform, if any, remains speculative at this time. Nevertheless, whenever adventitious agents are discovered in human biologics, the usual response is to take steps to remove them from the product. Ideally, this involves eradicating the adventitious agent from all stages of the manufacturing process. One notable precedent was the elimination of porcine circovirus from an attenuated live rotavirus vaccine and the Vero cells used to produce that vaccine [12], [13].
In this study, we characterized the relevant properties of an Sf-rhabdovirus-negative Sf cell line, Sf-RVN, as a potential alternative host for the BICS. We detected no Sf-rhabdovirus in these cells for 60 continuous passages in culture. Compared to Sf9 cells, Sf-RVN cells had virtually identical growth rates, sizes, morphologies, viabilities after baculovirus infection, and provided equivalent recombinant protein production levels and N-glycosylation profiles. Interestingly, Sf-RVN cells produced ∼5–10 fold higher levels of infectious baculovirus progeny. These results validate Sf-RVN cells as an alternative host that can be used to circumvent the potential safety hazard associated with the use of Sf-rhabdovirus-contaminated Sf cells for recombinant protein manufacturing in the BICS.
The isolation of Sf9 cells has been described [8] and the isolation of Sf-RVN cells will be described (Geisler, C., Maghodia, A.B., and Jarvis, D.L., submitted) elsewhere. Both cell lines were routinely maintained as shake-flask cultures at 28 °C in ESF 921 medium (Expression Systems, Woodland, CA).
Sf-rhabdovirus and mycoplasma detectionSamples of Sf9 and Sf-RVN cultures containing 1 × 106 cells were harvested and the cells were pelleted by low speed centrifugation. The cell-free supernatants were filtered through a 0.22 μm filter
Sf-RVN cells have no detectable Sf-rhabdovirusTotal RNA was isolated from Sf-RVN cell extracts at various passage levels and tested for the presence of Sf-rhabdovirus using RT-PCR/nested PCR, as described in Materials and methods. A strong amplification product of the expected size was observed when total RNA from Sf9 cells was used as a positive control for this assay (Fig. 1A), as expected [1]. In contrast, no products were observed when we used total RNAs isolated from Sf-RVN cells every five passages during the course of 60 sequential
DiscussionThe recent surge of regulatory approvals for the use of BICS-derived biologics in human and veterinary patients is a critically important milestone in the emergence of the BICS as a bona fide commercial biologics manufacturing platform [6]. However, the discovery of adventitious agents in the Sf cell lines used most frequently as hosts for baculovirus vectors [1] raised concerns about the safety of BICS-produced biologics. In this context, it is important to emphasize that there is no evidence
Conflict of interestA.B.M. and C.G. are employees and D.L.J. is the President of GlycoBac, LLC. Accordingly, all authors declare potential conflicts of interest as we expect GlycoBac will provide the new cell line reported herein as a commercial product, which will be of financial benefit to the company.
AcknowledgementsWe sincerely thank Dr. Hideaki Mabashi-Asazuma (University of Wyoming) for providing BacPAK6-ΔChi/Cath for use in this study. We also thank Dr. J.C. Gatlin (University of Wyoming) for constructive comments during the course of this study. This work was supported by Awards R43 GM102982 and R43 AI112118 from the National Institutes of Health, Institutes of General Medical Sciences and Allergy and Infectious Diseases, respectively. The content is solely the responsibility of the authors and does
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