Detection of adventitious agents using next-generation sequencing
| Autor: | Katherine W. Klinger, Robert J. Mattaliano, Stephen L. Madden, Brenda Richards, Robert J. Pomponio, Sherry Cao, Claire A. Davies, Mark Plavsic, Adam Palermo |
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| Rok vydání: | 2014 |
| Předmět: |
DNA
Bacterial Microorganism Cell Culture Techniques Pharmaceutical Science Computational biology CHO Cells DNA sequencing Biopharmaceutics chemistry.chemical_compound Bioreactors Cricetulus Limit of Detection Virology Bioreactor Animals Humans Illumina dye sequencing Bacteriological Techniques Biological Products biology Bacteria Chinese hamster ovary cell High-Throughput Nucleotide Sequencing Reference Standards biology.organism_classification chemistry Cell culture DNA Viral Viruses RNA Viral Drug Contamination DNA |
| Zdroj: | PDA journal of pharmaceutical science and technology. 68(6) |
| ISSN: | 1948-2124 |
| Popis: | Next-generation sequencing has been evaluated at Genzyme as a means of identifying bioreactor contaminants due to its capability for detection of known and novel microbial species. In this approach, data obtained from next-generation sequencing is used to interrogate databases containing genomic sequences and identities of potential adventitious agents. We describe here the use of this approach to help identify the causative agent of a bioreactor contamination. We also present the results of spiking experiments to establish the limits of detection for DNA viruses, RNA viruses, and bacteria, in a background of Chinese hamster ovary cells, a cell line used for production of many human therapeutics. Using Illumina sequencing-based detection, all of the viruses included in this study were detected at less than 1 copy per cell, and bacteria were detected at 0.001 copy per cell. Thus, next-generation sequencing–based detection of adventitious agents is a valuable approach that can fill a critical unmet need in the detection of known and novel microorganisms in biopharmaceutical manufacturing. LAY ABSTRACT: Because biological products are manufactured in cells, the living environment must be kept sterile. Any introduction of microorganisms into the culture vessel may affect the growth and other biological properties of the cells or contaminate the product. It is therefore important to monitor the culture for such contaminants, but many methods can only detect a specific microorganism. In this study, we show that next-generation sequencing–based detection is a sensitive and complementary approach that can potentially detect a wide range of organisms. |
| Databáze: | OpenAIRE |
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