Virus susceptibility of Chinese hamster ovary (CHO) cells and detection of viral contaminations by adventitious agent testing

“Chinese hamster ovary (CHO) cells are one of the main cell lines used in the production of recombinant therapeutics (Wurm, 2004; see Grillberger et al., 2009 for examples of proteins produced in CHO cells).”

“…1980s, the first recombinant medicinal products from mammalian cell cultures have become available (for review see Grillberger et al., 2009) and since then, production of therapeutics in cell lines has become prevalent, with more than 165 biopharmaceuticals now approved world-wide (Walsh, 2006).”

“…et al. (2008), Marsh and Bron (1997) Sindbis virus Potts (2008) Gorchakov et al. (2008), Sanz et al. (2007) Rubella virus — Malathi et al. (2001) Japanese encephalitis virus — Tsai et al. (2007), Su et al. (2002) Eastern equine encephalitis virus — Petrakova et al. (2005) Venezuelan equine…”

“(2007) , Chu and Whittaker (2004) Govorkova et al.”

“…(2007) Paramyxoviridae Parainfluenza virus 1/2/3 Potts (2008) Nishio et al. (2005), Shimokata et al. (1981), De et al. (1991) Simian virus 5 Potts (2008) He et al. (2002) Mumps virus Wisher (2005), Potts (2008) McCammon and Riesser (1979) Bovine respiratory syncytial virus Potts (2008) — Sendai…”

“Although experimental infection of CHO cells with Porcine circovirus, a member of the Circoviridae, has been shown (Misinzo et al., 2006) , this virus was not included in the present screen, as the previously published experiments showed no productive replication in CHO cells and no substantial safety concerns are the…”

“…Potts (2008) Gorchakov et al. (2008), Sanz et al. (2007) Rubella virus — Malathi et al. (2001) Japanese encephalitis virus — Tsai et al. (2007), Su et al. (2002) Eastern equine encephalitis virus — Petrakova et al. (2005) Venezuelan equine encephalitis virus — Petrakova et al.…”

“Chinese hamster ovary (CHO) cells are one of the main cell lines used in the production of recombinant therapeutics (Wurm, 2004; see Grillberger et al., 2009 for examples of proteins produced in CHO cells).”

“(2006) — Theiler’s mouse encephalitis virus — Son et al. (2008), Takano-Maruyama et al. (2006) Foot and mouth disease virus — Vagnozzi et al. (2007), Carrillo et al.”

“…al. (2002) Eastern equine encephalitis virus — Petrakova et al. (2005) Venezuelan equine encephalitis virus — Petrakova et al. (2005) Arteriviridae Porcine reproductive and respiratory syndrome virus — Shanmukhappa et al. (2007) Retroviridae Foamy virus — Li et al. (2002) ds RNA Reoviridae…”

“…Bovine coronavirus Francis (2003) — Murine hepatitis virus — Matsuyama and Taguchi (2000) Flaviviridae Yellow fever virus — Patkar and Kuhn (2008) Dengue virus — Ng et al. (2007) West Nile virus — Widman et al. (2008), Puig-Basagoiti et al. (2007) Tick borne encephalitis virus — Goto…”

“…(1985), Nettleton and Rweyemamu (1980) Circoviridae Porcine circovirus 1 Misinzo et al. (2006) — ds DNA Adenoviridae Adenovirus Condon et al. (2003) Hosel et al. (2003) Poxviridae Modified vaccinia virus ‘Ankara’ — Najera et al. (2006) Herpesviridae Pseudorabies virus Nixdorf et al. (1999) Slivac…”

“…et al. (2006) Foot and mouth disease virus — Vagnozzi et al. (2007), Carrillo et al. (2007), Garcia-Briones et al. (2006) Bovine enterovirus — Smyth et al. (2002) Porcine enterovirus — Knowles et al. (1979) Togaviridae Semliki Forest virus Marsh and Bron (1997) Kiiver et al. (2008), Marsh…”

“Marsh M, Bron R. 1997.”

“…— Najera et al. (2006) Herpesviridae Pseudorabies virus Nixdorf et al. (1999) Slivac et al. (2006) Murine gammaherpes 68 — Gillet et al. (2006) Herpes simplex virus 1 — Conner et al. (2005) Iridoviridae Frog virus 3 — Chinchar et al. (2003) Berting et al.: Virus Susceptibility of CHO Cells…”

“(2007), Carrillo et al. (2007), Garcia-Briones et al. (2006) Bovine enterovirus — Smyth et al. (2002) Porcine enterovirus — Knowles et al. (1979) Togaviridae Semliki Forest virus Marsh and Bron (1997) Kiiver et al. (2008), Marsh and Bron (1997) Sindbis virus Potts (2008) Gorchakov et al. (2008), Sanz et al. (2007) Rub…”

“We thank Barbara Potts for raising our interest in the subject and for initial discussions.”

“In the case of CVV and more so VSV, the ability to reproduce on CHO cells appeared to improve after the first 7 1 days incubation, possibly indicating an adaption of the virus to growth in CHO cells, as higher titers were detected more consistently in the 14d CHO cell culture supernatant (Fig.”

“(1981), De et al. (1991) Simian virus 5 Potts (2008) He et al.”

“…(2003) — Murine hepatitis virus — Matsuyama and Taguchi (2000) Flaviviridae Yellow fever virus — Patkar and Kuhn (2008) Dengue virus — Ng et al. (2007) West Nile virus — Widman et al. (2008), Puig-Basagoiti et al. (2007) Tick borne encephalitis virus — Goto et al. (2003) St. Louis…”

“…Yellow fever virus — Patkar and Kuhn (2008) Dengue virus — Ng et al. (2007) West Nile virus — Widman et al. (2008), Puig-Basagoiti et al. (2007) Tick borne encephalitis virus — Goto et al. (2003) St. Louis encephalitis virus — McLean et al. (1978) Caliciviridae Vesivirus 2117 Oehmig et al.…”

“Bunyamwera virus — Salanueva et al. (2003) Northway encephalitis virus — McLean et al.”

“…McLean et al. (1978) Caliciviridae Vesivirus 2117 Oehmig et al. (2003), Genzyme (2009) — Picornaviridae Encephalomyocarditis virus Potts (2008) Jia et al. (2008) Coxsackie virus B-3 Zautner et al. (2006) — Theiler’s mouse encephalitis virus — Son et al. (2008), Takano-Maruyama et al. (2006) Foot…”

“…De et al. (2005) Epizootic hemorrhagic disease virus Rabenau et al. (1993) Aradaib et al. (1998) Reovirus 1/2/3 Nims (2006), Potts (2008) Gaillard and Joklik (1985) Avian reovirus — Chen et al. (2008), Chulu et al. (2007) Rotavirus — Lopez et al. (2006) ss DNA Parvoviridae Mice minute virus…”

“…— Slosaris et al. (1989) Pneumonia virus of mice — Weir et al. (1988) Rhabdoviridae Vesicular stomatitis virus Potts (2008) Connor et al. (2006) Rabies virus — Kallel et al. (2002) ss (þ) RNA Coronaviridae Bovine coronavirus Francis (2003) — Murine hepatitis virus — Matsuyama and Taguchi…”

“…et al. (2008), Puig-Basagoiti et al. (2007) Tick borne encephalitis virus — Goto et al. (2003) St. Louis encephalitis virus — McLean et al. (1978) Caliciviridae Vesivirus 2117 Oehmig et al. (2003), Genzyme (2009) — Picornaviridae Encephalomyocarditis virus Potts (2008) Jia et al.…”

“(2007) , Chu and Whittaker (2004) Govorkova et al.”

“Carrillo C, Lu Z, Borca MV, Vagnozzi A, Kutish GF, Rock DL. 2007.”

“Viral translation is coupled to transcription in Sindbis virus-infected cells.”

“References Aradaib IE, Wilson WC, Schore CE, Mohammed ME, Yilma TD, Cullor JS, Osburn BI.”

“…(2008) Jia et al. (2008) Coxsackie virus B-3 Zautner et al. (2006) — Theiler’s mouse encephalitis virus — Son et al. (2008), Takano-Maruyama et al. (2006) Foot and mouth disease virus — Vagnozzi et al. (2007), Carrillo et al. (2007), Garcia-Briones et al. (2006) Bovine enterovirus — Smyth et…”

“Junin virus - Ellenberg et al.”

“…circovirus 1 Misinzo et al. (2006) — ds DNA Adenoviridae Adenovirus Condon et al. (2003) Hosel et al. (2003) Poxviridae Modified vaccinia virus ‘Ankara’ — Najera et al. (2006) Herpesviridae Pseudorabies virus Nixdorf et al. (1999) Slivac et al. (2006) Murine gammaherpes 68 — Gillet et al.…”

“…Bron (1997) Kiiver et al. (2008), Marsh and Bron (1997) Sindbis virus Potts (2008) Gorchakov et al. (2008), Sanz et al. (2007) Rubella virus — Malathi et al. (2001) Japanese encephalitis virus — Tsai et al. (2007), Su et al. (2002) Eastern equine encephalitis virus — Petrakova et al. (2005)…”

“(2007) Garnick (1996) , Nims (2006 ) Zoletto (1985 , Nettleton and Rweyemamu (1980) Viral contaminations of CHO cell-derived bulk harvests are rare, but have occurred.”

“…et al. (2006) — ds DNA Adenoviridae Adenovirus Condon et al. (2003) Hosel et al. (2003) Poxviridae Modified vaccinia virus ‘Ankara’ — Najera et al. (2006) Herpesviridae Pseudorabies virus Nixdorf et al. (1999) Slivac et al. (2006) Murine gammaherpes 68 — Gillet et al. (2006) Herpes simplex…”

“…(1979) Togaviridae Semliki Forest virus Marsh and Bron (1997) Kiiver et al. (2008), Marsh and Bron (1997) Sindbis virus Potts (2008) Gorchakov et al. (2008), Sanz et al. (2007) Rubella virus — Malathi et al. (2001) Japanese encephalitis virus — Tsai et al. (2007), Su et al. (2002) Eastern equine…”

“Reovirus 1/2/3 Nims (2006), Potts (2008) Gaillard and Joklik (1985) Avian reovirus — Chen et al. (2008), Chulu et al. (2007) Rotavirus — Lopez et al. (2006) ss DNA Parvoviridae Mice minute virus Garnick (1996), Nims (2006) Zoletto (1985), Nettleton and Rweyemamu (1980) Circoviridae Porcine circovirus 1 Misinzo et al.…”

“…et al. (2006) Newcastle disease virus — Slosaris et al. (1989) Pneumonia virus of mice — Weir et al. (1988) Rhabdoviridae Vesicular stomatitis virus Potts (2008) Connor et al. (2006) Rabies virus — Kallel et al. (2002) ss (þ) RNA Coronaviridae Bovine coronavirus Francis (2003) — Murine hepatitis…”

“…et al. (2003), Genzyme (2009) — Picornaviridae Encephalomyocarditis virus Potts (2008) Jia et al. (2008) Coxsackie virus B-3 Zautner et al. (2006) — Theiler’s mouse encephalitis virus — Son et al. (2008), Takano-Maruyama et al. (2006) Foot and mouth disease virus — Vagnozzi et al. (2007),…”

“…(2003) St. Louis encephalitis virus — McLean et al. (1978) Caliciviridae Vesivirus 2117 Oehmig et al. (2003), Genzyme (2009) — Picornaviridae Encephalomyocarditis virus Potts (2008) Jia et al. (2008) Coxsackie virus B-3 Zautner et al. (2006) — Theiler’s mouse encephalitis virus — Son et al.…”

“…Poxviridae Modified vaccinia virus ‘Ankara’ — Najera et al. (2006) Herpesviridae Pseudorabies virus Nixdorf et al. (1999) Slivac et al. (2006) Murine gammaherpes 68 — Gillet et al. (2006) Herpes simplex virus 1 — Conner et al. (2005) Iridoviridae Frog virus 3 — Chinchar et al. (2003) Berting…”

“Bovine viral diarrhea virus (BVDV), Coxsackievirus B3 (CVB3), Murine hepatitis virus (MHV), Pseudorabies virus (PRV) and Porcine parvovirus (PPV) did not induce a CPE in CHO cell culture (Fig.”

“…virus — Son et al. (2008), Takano-Maruyama et al. (2006) Foot and mouth disease virus — Vagnozzi et al. (2007), Carrillo et al. (2007), Garcia-Briones et al. (2006) Bovine enterovirus — Smyth et al. (2002) Porcine enterovirus — Knowles et al. (1979) Togaviridae Semliki Forest virus Marsh and…”

“(1993) Aradaib et al. (1998)”

“…(2006) ss DNA Parvoviridae Mice minute virus Garnick (1996), Nims (2006) Zoletto (1985), Nettleton and Rweyemamu (1980) Circoviridae Porcine circovirus 1 Misinzo et al. (2006) — ds DNA Adenoviridae Adenovirus Condon et al. (2003) Hosel et al. (2003) Poxviridae Modified vaccinia virus ‘Ankara’ —…”

“(2008) Coxsackie virus B-3 Zautner et al. (2006) — Theiler’s mouse encephalitis virus — Son et al.”

“…(2009) — Picornaviridae Encephalomyocarditis virus Potts (2008) Jia et al. (2008) Coxsackie virus B-3 Zautner et al. (2006) — Theiler’s mouse encephalitis virus — Son et al. (2008), Takano-Maruyama et al. (2006) Foot and mouth disease virus — Vagnozzi et al. (2007), Carrillo et al. (2007),…”

“We thank Barbara Potts for raising our interest in the subject and for initial discussions.”

“…Carrillo et al. (2007), Garcia-Briones et al. (2006) Bovine enterovirus — Smyth et al. (2002) Porcine enterovirus — Knowles et al. (1979) Togaviridae Semliki Forest virus Marsh and Bron (1997) Kiiver et al. (2008), Marsh and Bron (1997) Sindbis virus Potts (2008) Gorchakov et al. (2008), Sanz et…”

“Although experimental infection of CHO cells with Porcine circovirus, a member of the Circoviridae, has been shown (Misinzo et al., 2006), this virus was not included in the present screen, as the previously published experiments showed no productive replication in CHO cells and no substantial safety concerns are ther…”

“2010 Wiley Periodicals, Inc. Potts (2008) Bunyamwera virus — Salanueva et al. (2003) Northway encephalitis virus — McLean et al. (1978) Orthomyxoviridae Influenza A/B virus Kumari et al. (2007), Chu and Whittaker (2004) Govorkova et al. (1999) Arenaviridae Junin virus — Ellenberg et al. (2007)…”

“…hemorrhagic disease virus Rabenau et al. (1993) Aradaib et al. (1998) Reovirus 1/2/3 Nims (2006), Potts (2008) Gaillard and Joklik (1985) Avian reovirus — Chen et al. (2008), Chulu et al. (2007) Rotavirus — Lopez et al. (2006) ss DNA Parvoviridae Mice minute virus Garnick (1996), Nims (2006)…”

“…et al. (2005) Venezuelan equine encephalitis virus — Petrakova et al. (2005) Arteriviridae Porcine reproductive and respiratory syndrome virus — Shanmukhappa et al. (2007) Retroviridae Foamy virus — Li et al. (2002) ds RNA Reoviridae Bluetongue virus Potts (2008) De et al. (2005) Epizootic…”

“(2008) Coxsackie virus B-3 Zautner et al. (2006) — Theiler’s mouse encephalitis virus — Son et al.”

“…(2008) He et al. (2002) Mumps virus Wisher (2005), Potts (2008) McCammon and Riesser (1979) Bovine respiratory syncytial virus Potts (2008) — Sendai virus — Nishio et al. (2006) Newcastle disease virus — Slosaris et al. (1989) Pneumonia virus of mice — Weir et al. (1988) Rhabdoviridae Vesicular…”

“Further, there is perceived species barrier protection with the use of hamster-derived cells, reducing the potential risk of transfer of contaminating adventitious agents to humans (Berting et al., 2010; Swiech et al., 2012).”

“In addition, CHO cells have been demonstrated to display reduced susceptibility to human viral infections [11], which represents an additional advantage over cell lines of human”

“Third, CHO cells are less prone to virus infection than other mammalian production cell lines and are therefore regarded as a safe host for the production of human therapeutics (Berting et al., 2010).”

“From a safety perspective, many viral entry genes are not expressed in CHO cells and there is a species barrier that minimizes risk of transferring infectious agents to humans (325, 326).”

“For example, diverse viruses have been identified as contaminants of Chinese hamster ovary cells, which are widely used in biologics manufacturing [46].”

“As opposed to BHK, this cell line derived from an inbred (captive) female Chinese hamster (Cricetulus griseus) [84] is not permissive for many viruses [85–87].”

“In addition to its ability to produce glycoproteins with post-translational modifications compatible to humans [1], and its refractory nature to human viruses [2], the availability of well-established gene amplification systems for CHO cells coupled with its ability to adapt and grow in serum-free suspension culture m…”

“As pointed out in the introduction, one of the reasons for the extensive adoption of CHO cells in the biopharmaceutical industry is their reduced susceptibility to human viruses [103].”

“Current adventitious agent testing (AAT) assays are effective for the detection of viruses which are able to replicate in CHO cells (Berting et al., 2010).”

“Among them, CHO cells are the preferred host due to the advantages that they offer in terms of growth [63,64,65], productivity [3], specific culture mediums [66], safety [67], and product quality [68].”

“A general concern in cell-based manufacturing of recombinant proteins, including vaccines, is the potential contamination of the cell culture with viruses, which has had severe consequences for patients and manufacturers [1,2].”

“To ensure the safety of biological therapeutics, regulatory guidance requires adventitious agent testing of the bulk harvest [10].”

“MMV is a notorious contaminant of rodent cell lines important for the biopharmaceutical industry [67].”

“contamination in CHO cell cultures, with some incidents affecting bulk harvests from production runs (7).”

“Among mammalian cells, CHO cells most often represent the mammalian cell of choice for their ease of use, reduced susceptibility to viral infection, and familiarity with biologics regulators.(30,31) However, due to growing demand and development of DTE recombinant proteins such as bispecific antibodies, fusion protein…”

“MMV, a common virus in laboratory mice that can replicate in CHO cell lines [16], has a D10 value of approximately 2.”

“For instance, it was shown that CHO cells possess reduced susceptibility to certain viral infections (Berting et al., 2010).”

“…the risk of introducing adventitious agents, such as control of the raw material source, testing for adventitious agents in raw materials and the bulk harvest at appropriate stages during the manufacturing process, and implementation of virus-inactivation and removal steps (Berting et al., 2010).”

“…agent testing is mandated for the prevention of contamination by viruses able to replicate in CHO cells and has led to the detection of five incidents in the past 20 years caused by murine minute virus, reovirus, Cache Valley virus, epizootic hemorrhagic disease virus and vesivirus 2117 [18 – 23].”

“[i] = [î] + "c with "c ∼  (0, 2 c ) (2) where "c is a standard normal random variable with relative standard deviation (RSD) c∕[î] chosen between 0.”

“CHO cells are refractory to viral infection from most human pathogens [46] and can produce glycopro te ins that a re both bioac t ive and nonimmunogenic in human hosts [47].”

“CHO cell lines are widely used because of their attractive attributes, including protein modification and glycosylation, invulnerability to several human pathogenic viruses, serum ‐ free adaptation, and scale ‐ up capabilities (Berting et al., 2010; Omasa et al., 2010).”

“CHO cells are permissive to multiple types of viruses such as Adenovirus, Parainfluenza virus, Bovine respiratory syncytial virus, Vesicular stomatitis virus, Reovirus, and Bluetongue virus [14].”

“These results were consistent with the virus susceptibility of CHO cells reported by Berting et al. (2010).”

“In particular, CHO cells have low susceptibility to many human virus families [2] such as influenza and HIV, which greatly reduces the threat of contamination to the supply of critical drugs [3].”

“…posttranslational modifications similar to human proteins, which are offered by the CHO cellular machinery in the secreted target proteins, makes CHO the preferred production host over other mammalian systems in the biopharmaceutical industry (Berting et al., 2010; Spier, 1989).”

“42,54 This resistance, while advantageous for ensuring the safety of biotherapeutics, poses unique limitations for effective viral-vector-based transgene delivery.”

“For example, CHO cells are susceptible to many different viruses belonging to a dozen or so families based on existing studies [26–28].”

“Finally, they have lower susceptibility to human viruses, making them an ideal host for biopharmaceutical production [6].”

“The intention of the latter was to understand the value of each of the test methods in detecting potential adventitious agents, similar to a more limited study earlier (34), as well as to provide”

“Approximately 70% of all biotech products are produced by Chinese Hamster Ovary (CHO) cell lines [11] which are typically more resilient than other alternative expression systems [12].”

“These can include Adenovirus, Vesicular stomatitis virus, Reovi-rus, Parainfluenza virus, Bovine respiratory syncytial virus, and Bluetongue virus (Berting et al. 2010).”

“In addition, CHO cells have reduced susceptibility to human viral infections, which is an additional advantage compared to human-derived cell lines [3].”

“…hamster ovary (CHO) cells are widely used as mammalian hosts for the production of recombinant biopharmaceutical proteins, particularly glycoproteins that require human-like post-translational modifications (PTMs) (Berting et al. 2010; Butler and Spearman 2014; Walsh 2018; Zhu and Hatton 2018).”

“In addition to its ability to produce glycoproteins with post-translational modifications compatible to humans [1], and its refractory nature to human viruses [2], the availability of well-established gene amplification systems for CHO cells coupled with its ability to adapt and grow in serum-free suspension culture m…”

“68,118 Lastly, some medium risk viruses complete the full life cycle, but do so poorly.”

“…derivatives are a popular cell–substrate for the production of biotherapeutic protein products due to the restricted susceptibility of the cell line to infection by human viruses [56] and easy scale-up to produce proteins with human-like glycosylation and other post-translational modifications [3].”

“Most documented events in CHO-derived products involved mouse minute virus, reovirus, Cache Valley virus, epizootic hemorrhagic disease virus, and vesivirus 2117, usually traced to raw materials like animal serum or media components [1,3–5].”