Use of PCR Analysis for Sterility Testing in Pharmaceutical Environments

Abstract

To determine the sterility of pharmaceutical samples, highly conserved bacterial ribosomal DNA sequences were used in a PCR‐based assay. Finished products, raw materials, growth media, and diluents were artificially contaminated with different types of microorganisms. Samples were incubated for 24 h. After incubation, microbial DNA was extracted from enrichment broths using a Tris‐EDTA‐Tween 20 buffer containing proteinase K. Extracted DNA was added to Ready‐To‐Go PCR beads and eubacterial primers. Contaminated samples were found to contain the conserved 1.5 kilobase (kb) DNA fragment of the bacterial genome by using the PCR assay. None of the uninoculated samples was found to show the presence of the 1.5 kb fragment. PCR test results were compared with standard conventional methods. There was a 100% correlation between standard conventional methods and the PCR assay. However, the PCR‐based assay was completed within 27 h while conventional methods required 4–5 days. Rapid PCR analysis using a simple sample preparation reduced the time for sterility testing of pharmaceutical samples allowing optimization of risk assessment and implementation of corrective actions.

Development of next-generation sequencing-based sterility test

“…Several rapid microbial contamination detection methods have been developed so far, such as ATP bioluminescence, flow cytometry, nucleic acid amplification, solid phase cytometry and so on. However, these methods have certain limitations in sensitivity or accuracy [2][3][4].…”

“…Recently, the development of metagenomic NGS (mNGS) technology and rapid bioinformatics pipelines allows unbiased detection of pathogens in samples such as urine, cerebrospinal fluid, blood and so on [5][6][7][8][9], which may contain mixed populations of microorganisms. Compared with traditional pharmacopoeia methods, NGS technology have certain advantages, including (1) shorter detection period; (2) unbiased detection and identification of microorganisms; (3) higher sensitivity; (4) no need of cell culturing. However, there are still several challenges in applying NGS technology in sterility testing, such as the lack of systematic validation and bioinformatics pipeline for rapid data analysis that can reduce the bioinformatics processing time from days to several hours [10][11].…”

Preliminary evidence of a new microbial species capable of sustainable intracellular survival and transfer in mammalian cell lines

“…PCR screening assays provide fast, dependable and cost-effective methods for quality assessment, ultimately resulting in faster product release and product optimizationdetection can be based on generic primer sequences to maximize detection of difference bacterial strains, (Jimenez 2001;Lleo 2005). The highly conserved bacterial ribosomal DNA sequence has been employed in PCR-based assays to determine sterility of pharmaceutical samples, (Jimenez 2007). Nucleic acid amplification has been described as a significant improvement in technology for microbial research laboratories and microbial diagnostic industries, due to sensitivity and capacity to be automated, (Nocker 2008).…”

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“…PCR screening assays provide fast, dependable and cost-effective methods for quality assessment, ultimately resulting in faster product release and product optimization -detection can be based on generic primer sequences to maximize detection of difference bacterial strains, (Jimenez 2001;Lleo 2005). The highly conserved bacterial ribosomal DNA sequence has been employed in PCR-based assays to determine sterility of pharmaceutical samples, (Jimenez 2007). Nucleic acid amplification has been described as a PeerJ PrePrints | http://dx.doi.org/10.7287/peerj.preprints.209v2 | CC-BY 3.0 Open Access | received: 21 Jan 2014, published: 21 Jan 2014 significant improvement in technology for microbial research laboratories and microbial diagnostic industries, due to sensitivity and capacity to be automated, (Nocker 2008).…”