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Defining a sample preparation workflow for advanced virus detection and understanding sensitivity by next-generation

Christopher J Wang1, Szi Fei Feng2, Paul Duncan2

  • 1Merck & Co., Inc., West Point, PA christopher_wang@merck.com.

PDA Journal of Pharmaceutical Science and Technology
|December 6, 2014
PubMed
Summary

Next-generation sequencing (NGS) offers a sensitive method for adventitious virus detection in biopharmaceuticals. This study validates a sample preparation workflow, demonstrating robust viral nucleic acid recovery for enhanced safety testing.

Keywords:
Cell substrate and virus bank characterizationVaccine development and manufacturingadventitious agent detectionnext-generation sequencingqPCR

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Area of Science:

  • Biotechnology
  • Virology
  • Genomics

Background:

  • Next-generation sequencing (NGS) is increasingly adopted for adventitious agent detection in the biopharmaceutical industry.
  • Robust sample preparation is crucial for isolating viral nucleic acids from diverse biological matrices.
  • Demonstrating recovery of spiked model viruses is essential for validating NGS methods.

Purpose of the Study:

  • To implement and assess the sensitivity of a specific sample preparation workflow for NGS-based virus detection.
  • To evaluate the performance of the Illumina MiSeq platform for viral detection.
  • To develop a theoretical model for estimating virus detection in cell lysates and vaccine harvests.

Main Methods:

  • Implementation of the Feng et al. sample preparation workflow.
  • Assessment of virus detection sensitivity using Illumina MiSeq platform.
  • Application of nuclease treatment to reduce host nucleic acid background.
  • Recovery of nucleic acids from model viruses spiked into cell lysates and vaccine harvests.

Main Results:

  • The implemented workflow demonstrated reliable recovery of viral nucleic acids from various sample types.
  • Nuclease treatment significantly enhanced sensitivity for target virus detection by reducing data complexity.
  • Model viruses spiked at defined levels were confidently detected using NGS.

Conclusions:

  • The validated sample preparation workflow is effective for adventitious virus detection via NGS.
  • NGS provides a sensitive and reliable method for ensuring the safety of biopharmaceutical products.
  • Nuclease treatment is a valuable adjunct for improving NGS sensitivity in complex samples.