Real-time Quantitative PCR for Retrovirus-like Particle Quantification in CHO Cell Culture

Wit, Christina de; Fautz, C.; Xu, Yuan

doi:10.1006/biol.2000.0250

Cited by 46 publications

(29 citation statements)

References 19 publications

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“…The CHO retrovirus Q-PCR assay was first developed by de Wit et al (2000) to quantify the amount of CHO retrovirus in a dose-equivalent volume of harvest (Brorson et al, 2003b). Even though the assay has been previously shown to accurately measure CHO retrovirus titers in harvest samples or HCCF, this is the first report demonstrating that it can successfully be used to evaluate retrovirus clearance by a chromatography step in actual large scale manufacturing.…”

Section: Discussionmentioning

confidence: 99%

“…Viral RNA was extracted from samples using a QIAamp Viral RNA kit (Qiagen, Valencia, CA), according to vendor instructions as described (de Wit et al, 2000). Sample sizes were 70-100 mL (undiluted and 1:10 diluted HCCF, undiluted protein A pool).…”

Section: Real-time Quantitative (Q)-pcr and Q-pert Assaysmentioning

confidence: 99%

“…Real-time quantitative PCR assays to measure CHO retrovirus genomes were performed as described (de Wit et al, 2000). Primers and probe sequences were designed to amplify a fragment in the highly conserved pol region from the CHO type C retrovirus genome.…”

Section: Real-time Quantitative (Q)-pcr and Q-pert Assaysmentioning

confidence: 99%

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A Novel, Q‐PCR Based Approach to Measuring Endogenous Retroviral Clearance by Capture Protein A Chromatography

Zhang

Lute

Norling

et al. 2008

Biotech & Bioengineering

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Quantification of virus removal by the purification process during production is required for clinical use of biopharmaceuticals. The current validation approach for virus removal by chromatography steps typically involves time-consuming spiking experiments with expensive model viruses at bench scale. Here we propose a novel, alternative approach that can be applied in at least one instance: evaluating retroviral clearance by protein A chromatography. Our strategy uses a quantitative PCR (Q-PCR) assay that quantifies the endogenous type C retrovirus-like particle genomes directly in production Chinese Hamster Ovary (CHO) cell culture harvests and protein A pools. This eliminates the need to perform spiking with model viruses, and measures the real virus from the process. Using this new approach, clearance values were obtained that was comparable to those from the old model-virus spike/removal approach. We tested the concept of design space for CHO retrovirus removal using samples from a protein A characterization study, where a wide range of chromatographic operating conditions were challenged, including load density, flow rate, wash, pooling, temperature, and resin life cycles. Little impact of these variables on CHO retrovirus clearance was found, arguing for implementation of the design space approach for viral clearance to support operational ranges and manufacturing excursions. The viral clearance results from Q-PCR were confirmed by an orthogonal quantitative product-enhanced reverse transcriptase (Q-PERT) assay that quantifies CHO retrovirus by their reverse transcriptase (RT) enzyme activity. Overall, our results demonstrate that protein A chromatography is a robust retrovirus removal step and CHO retrovirus removal can be directly measured at large scale using Q-PCR assays.

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Section: Discussionmentioning

confidence: 99%

Section: Real-time Quantitative (Q)-pcr and Q-pert Assaysmentioning

confidence: 99%

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A Novel, Q‐PCR Based Approach to Measuring Endogenous Retroviral Clearance by Capture Protein A Chromatography

Zhang

Lute

Norling

et al. 2008

Biotech & Bioengineering

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“…This fluorescence is released by a fluorogenic (TaqMan) probe hybridised to the internal portion of the template DNA (de Wit et al 2000, Roberts et al 2000, Tang & Lightner 2001. As qRT-PCR requires a small amount of RNA template (Dhar et al 2002, Durand et al 2003, it is possible to collect a series of blood or biopsy samples and to monitor changes in viral concentration in individual prawns over time.…”

Section: Abstract: Penaeus Monodon · Gill-associated Virus · Quantitmentioning

confidence: 99%

Quantitative real-time RT-PCR demonstrates that handling stress can lead to rapid increases of gill-associated virus (GAV) infection levels in Penaeus monodon

Vega¹,

Degnan²,

Hall³

et al. 2004

Dis. Aquat. Org.

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Gill-associated virus (GAV) of the black tiger prawn Penaeus monodon has been implicated as a cause of periodic production losses in Australia since 1996. We report here the development of a real-time quantitative RT-PCR (qRT-PCR) for GAV. A dilution series of in vitro transcribed RNA was used to determine the sensitivity limit of the qRT-PCR and as a standard for GAV quantification. A linear relationship between cycle threshold (Ct) values and input RNA was obtained over a wide concentration range between 4.86 × 10 9 and 0.5 template copies per reaction, the latter being the test detection limit. The qRT-PCR was used to follow the progression of GAV levels in a group of 15 adult male P. monodon with chronic GAV infections that were super-infected by intramuscular injection of an inoculum containing high levels of GAV. By Day 9 post-injection, cumulative mortalities reached 100% (15/15) in the GAV-injected prawns and 40% (2/5) in placebo-injected prawns. Spermatophores were collected at the beginning, and together with other tissues, at the end of the trial. Prawns were also bled at regular intervals to collect circulating haemocytes. The qRT-PCR revealed that GAV loads increased significantly in haemocytes collected from both the control and super-infected prawns (p = 0.010). This increase was significantly higher in the super-infected prawns (p = 0.047). The rapid increase in GAV levels in super-infected P. monodon was expected. However, the increase in the control prawns was not, and indicates that repetitive bleeding and handling stress can stimulate GAV proliferation in chronically infected P. monodon. KEY WORDS: Penaeus monodon · Gill-associated virus · Quantitative RT-PCR (qRT-PCR)Resale or republication not permitted without written consent of the publisher Dis Aquat Org 59: 195-203, 2004 2003, Tang et al. 2002, Callinan et al. 2003. Sequence comparisons of viruses from many healthy and moribund P. monodon indicate that they are genetically inseparable and are now regarded as the same virus (Cowley et al. 2000 and unpubl. data).GAV is a positive-strand ssRNA nidovirus closely related to the yellow head virus (YHV) from Thailand in genome sequence and organisation (Cowley et al. 1999. GAV and YHV have recently been classified in a new genus (Okavirus) and family (Roniviridae) within the Nidovirales (Mayo 2002). Both viruses have rod-shaped enveloped particles and cause similar histopathology and cytopathology (Spann et al. 1995, 1997, Tang & Lightner 1999, Tang et al. 2002. GAV diagnosis based on clinical signs and histopathology is not particularly specific as other viruses can cause similar effects. More precise diagnostic methods include transmission electron microscopy (TEM), in situ hybridisation (ISH) (Tang et al. 2002) and RT-nested PCR (Cowley et al. 2000).There is no detailed quantitative data directly linking GAV infection levels to the manifestation of disease symptoms. However, histological and ISH data on healthy and moribund Penaeus monodon and P. esculentus experimentally infec...

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“…Recently, real time polymerase chain reaction (PCR) assay is greatly aimed as a new method to detect and quantify PCR products simultaneously (10). Real time PCR is already employed to detect bacterial, fungal and viral loads in recombinant therapeutics (11)(12)(13)(14)(15). The current study established a new highly sensitive real time PCR approach based on SYBR Green Chemistry to detect a very small quantity of the residual E. coli host cell DNA in the recombinant streptokinase and alfa interferon samples produced in the Pasteur Institute of Iran.…”

Section: Introductionmentioning

confidence: 99%

Novel Real Time Polymerase Chain Reaction Approach for Rapid Detection of the Residual Escherichia coli Genomic DNA in Biopharmaceutical Products Establishment of Real Time Polymerase Chain Reaction to Detect Residual gDNA

Farivar¹,

Mamnoon²,

Arzenani³

et al. 2014

Biotech Health Sci

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Background: Contamination of therapeutic recombinant proteins with residual host cell DNA must be controlled under the regulatory standards. Objectives: The current study established a new rapid, sensitive real time polymerase chain reaction (PCR) approach to measure the reliably of the residual Escherichia coli (E. coli) host cell genomic DNA in the recombinant streptokinase and alfa interferon preparations. Materials and Methods: In this assay, a specific primer pair was utilized to amplify a 115 base pair sequence inside the E. coli 16S rRNA using SYBR Green Chemistry. This method enabled the authors to detect a very small quantity of the residual genomic DNA, as low as 0.8 pg, in the protein-based drugs. This method can, therefore, offer a dependable way to quantitatively analyze the major contaminant of biopharmaceutical products, the host cell DNA, during the manufacturing process. Results: SYBR Green PCR master mix may contain a source of DNA contamination during its manufacturing process. Conclusions:The current study data showed that E. coli host cell DNA contamination in streptokinase and alfa interferon manufactured in the Pasteur institute of Iran is much lower than the safety limits suggested by the FDA.

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Real-time Quantitative PCR for Retrovirus-like Particle Quantification in CHO Cell Culture

Cited by 46 publications

References 19 publications

A Novel, Q‐PCR Based Approach to Measuring Endogenous Retroviral Clearance by Capture Protein A Chromatography

A Novel, Q‐PCR Based Approach to Measuring Endogenous Retroviral Clearance by Capture Protein A Chromatography

Quantitative real-time RT-PCR demonstrates that handling stress can lead to rapid increases of gill-associated virus (GAV) infection levels in Penaeus monodon

Novel Real Time Polymerase Chain Reaction Approach for Rapid Detection of the Residual Escherichia coli Genomic DNA in Biopharmaceutical Products Establishment of Real Time Polymerase Chain Reaction to Detect Residual gDNA

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