Elsevier

Biologicals

Volume 28, Issue 3, September 2000, Pages 137-148
Biologicals

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

https://doi.org/10.1006/biol.2000.0250Get rights and content

Abstract

Chinese hamster ovary (CHO) cells have been widely used to manufacture recombinant proteins intended for human therapeutic uses. Retrovirus-like particles, which are apparently defective and non-infectious, have been detected in all CHO cells by electron microscopy (EM). To assure viral safety of CHO cell-derived biologicals, quantification of retrovirus-like particles in production cell culture and demonstration of sufficient elimination of such retrovirus-like particles by the down-stream purification process are required for product market registration worldwide. EM, with a detection limit of 1×106 particles/ml, is the standard retrovirus-like particle quantification method. The whole process, which requires a large amount of sample (3–6 litres), is labour intensive, time consuming, expensive, and subject to significant assay variability. In this paper, a novel real-time quantitative PCR assay (TaqMan assay) has been developed for the quantification of retrovirus-like particles. Each retrovirus particle contains two copies of the viral genomic particle RNA (pRNA) molecule. Therefore, quantification of retrovirus particles can be achieved by quantifying the pRNA copy number, i.e. every two copies of retroviral pRNA is equivalent to one retrovirus-like particle. The TaqMan assay takes advantage of the 5′→3′ exonuclease activity of Taq DNA polymerase and utilizes the PRISM™ 7700 Sequence Detection System of PE Applied Biosystems (Foster City, CA, U.S.A.) for automated pRNA quantification through a dual-labelled fluorogenic probe. The TaqMan quantification technique is highly comparable to the EM analysis. In addition, it offers significant advantages over the EM analysis, such as a higher sensitivity of less than 600 particles/ml, greater accuracy and reliability, higher sample throughput, more flexibility and lower cost. Therefore, the TaqMan assay should be used as a substitute for EM analysis for retrovirus-like particle quantification in CHO cell-based production system.

References (27)

  • D Vapnek

    Choosing a production system for recombinant proteins

  • UI Heine et al.

    Enhanced proliferation of endogenous virus in Chinese hamster cells associated with microtubules and the mitotic apparatus of the host cell

    J Gen Virol

    (1979)
  • F Hojman et al.

    Biological and molecular characterization of an endogenous retrovirus present in CHO/HBs-A Chinese hamster cell line

    Dev Biol Stand

    (1989)
  • Cited by (46)

    • A direct RT qPCR method for quantification of retrovirus-like particles in biopharmaceutical production with CHO cells

      2020, Journal of Pharmaceutical and Biomedical Analysis
      Citation Excerpt :

      However, it requires large sample volume (3–6 L), is labor intensive, time consuming, expensive, and has low sensitivity and high variability [8]. Quantitative real-time PCR based methods for RVLPs are available but require RNase digestion of samples followed by total nucleic acid extraction, then digestion with DNase followed by reverse transcription and PCR [8–10]. Although the qPCR is a very sensitive method, the efficiency of nucleic acid extraction impacts the sensitivity and usefulness of the qPCR-based quantification method.

    • Evaluation of infectivity and reverse transcriptase real-time polymerase chain reaction assays for detection of xenotropic murine leukemia virus used in virus clearance validation

      2015, Biologicals
      Citation Excerpt :

      Because RVLPs are noninfectious, an infectivity assay cannot be used directly to detect these particles. Real-time polymerase chain reaction (RT-PCR) assay has been used for the quantification of the RVLP from CHO cells [10]. In the biopharmaceutical industry, xenotropic murine leukemia virus (X-MuLV) is commonly used as a specific model virus in evaluating the removal of RVLP by purification processes.

    • Growth curve of murine xenotropic leukemia virus-related virus grown in Chinese hamster ovary cells

      2014, Journal of the Chinese Medical Association
      Citation Excerpt :

      Nowadays, the PCR assay is a popular method of detecting viruses, especially real-time PCR. Lute et al,10 Shi et al,11 and Wit et al 12 used the Taqman prober to detect XMulV particles in CHO cells for the purpose of evaluating viral clearance; the limit of detection in their experiments ranged from 1.0 to 1.5 viral RNA copies/μL. In this study, the SYBR Green fluorescence qPCR was used to detect virions, and the quantity of virions on different days was calculated using the 2−ΔΔCt method.

    • Specific detection of residual CHO host cell DNA by real-time PCR

      2007, Biologicals
      Citation Excerpt :

      qRTPCR have been applied to characterize and detect numerous bacteria, fungal and viral DNA [10–13]. In the biologics industry, qRTPCR has been used to detect viral load in protein samples [14–16]. However, to date, there are no published methods to specifically monitor host cell DNA contamination using this sensitive technique although it is available as a service provided by laboratories specializing in quality assurance [17].

    View all citing articles on Scopus
    f1

    To whom correspondence should be addressed. E-mail:[email protected]

    View full text