Development of a qualitative real-time PCR for microbiological quality control testing in mammalian cell culture production

Kleinschmidt, K.; Wilkens, Erik; Glaeser, Stefanie P.; Kaempfer, P.; Staerk, Alexandra; Roesti, David

doi:10.1111/jam.13387

Cited by 5 publications

(2 citation statements)

References 16 publications

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“…One of the primary advantages of PCR is its high sensitivity and specificity, which enables the detection of very low levels of target DNA or RNA. PCR is also a quick and straightforward technique, making it ideal for routine monitoring in a bioprocess [63]. However, PCR has some limitations, such as the possibility of false positive results, the need for meticulous sample preparation, and the possibility of cross-contamination between samples.…”

Section: Polymerase Chain Reactionmentioning

confidence: 99%

“…Non-culture-based techniques, such as PCR and immunoassay approaches, have higher LODs but are more rapid and specific. PCR is a highly sensitive and specific method for determining specific DNA fragments, making it helpful in spotting various microorganisms [63]. Depending on the type of the PCR analysis, this method can have a limit of detection as low as 9 genome copies/mL [68].…”

Section: Limulus Amebocyte Lysatementioning

confidence: 99%

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Methods and Analysis of Biological Contaminants in the Biomanufacturing Industry

et al. 2023

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The advent of bioprocessing has revolutionized the biomanufacturing industry, leading to the rise of biotherapeutics derived from biologic products such as chimeric antigen receptor (CAR) T-cells used for targeted cancer treatment and the Vero cell line for the production of viral vectors and vaccines. Despite these promising developments, most biologic products are characterized by fragile macromolecular structures that are heterogenous with a purity profile that varies with each batch making them susceptible to microorganism contamination. Regulatory oversight of biologic products is imperative to ensure adherence to good manufacturing practices and compliance with quality management systems. Current quality assurance protocols during production include monoclonality during cell line development, real-time monitoring of process parameters, flow cytometry for microbial monitoring, polymerase chain reaction, and immunoassay techniques to amplify DNA sequences related to bacterial or biological contaminants. FDA guidance recommends the implementation of process analytical technology within biomanufacturing production to measure critical quality parameters, which includes screening for potential biological contamination. Future advancements in bioprocess monitoring and control should capitalize on providing cheap, real-time, and sensitive detection. Biosensors, mass spectrometry, and polymerase chain reaction present robust, rapid, and real-time capabilities for multiplexed detection of contaminant analytes and have shown promise in meeting these needs. This review discusses the main biological contaminants of bioprocesses, European Union and FDA regulatory guidelines for monitoring and control within biologics production, existing methods and their limitations, and future advancements for biological contamination detection.

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Section: Polymerase Chain Reactionmentioning

confidence: 99%

Section: Limulus Amebocyte Lysatementioning

confidence: 99%

Methods and Analysis of Biological Contaminants in the Biomanufacturing Industry

et al. 2023

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Quantification of live Lactobacillus acidophilus in mixed populations of live and killed by application of attenuated reflection Fourier transform infrared spectroscopy combined with chemometrics

Toziou¹,

Barmpalexis²,

Boukouvala³

et al. 2018

Journal of Pharmaceutical and Biomedical Analysis

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Bacterial Analysis of the Whole Blood in Chinese Healthy Donors Using 16S rDNA–Targeted Metagenomic Sequencing

Zhang,

He,

Chen

et al. 2024

Canadian Journal of Infectious Diseases and Medical Microbiolog

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Background: The presence of bacteria in the blood of healthy individuals remains controversial. This study explored the comprehensive bacterial profiles and specific biomarkers in different components of healthy Chinese blood donors.Methods: A total of 5230 whole blood (WB) specimens were collected. Among them, 5200 random samples were pooled into 26 mixed samples for bacterial profile analysis. The remaining 30 random samples were divided into 4 groups based on components: WB, plasma, red blood cells (RBCs), and buffy coat (BC). Subsequently, the amplicons of the bacterial 16S rDNA V3–V4 fragments were sequenced to measure the diversity and composition of the bacteria using next‐generation sequencing.Results: The bacterial DNAs in the blood primarily originated from the Proteobacteria phylum. A total of 301 species of bacterial DNA were found in blood specimens, with 46 species being present among all groups. A significantly higher abundance of bacterial DNA was found in the plasma and RBCs compared to those in BC and WB. However, the plasma and RBC groups showed significantly higher species diversity and richness compared to the BC and WB groups. In addition, the WB group had a significantly different community structure and composition compared to the plasma and RBC groups but was similar to the BC group.Conclusion: The presence of bacterial DNA fragments was confirmed in blood from healthy Chinese donors. The bacterial DNA fragments enriched in plasma showed the highest diversity, followed by RBC, WB, and BC. These results provide a foundation for further research on the microbiome in the blood of healthy individuals.

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Development of a qualitative real-time PCR for microbiological quality control testing in mammalian cell culture production

Cited by 5 publications

References 16 publications

Methods and Analysis of Biological Contaminants in the Biomanufacturing Industry

Methods and Analysis of Biological Contaminants in the Biomanufacturing Industry

Quantification of live Lactobacillus acidophilus in mixed populations of live and killed by application of attenuated reflection Fourier transform infrared spectroscopy combined with chemometrics

Bacterial Analysis of the Whole Blood in Chinese Healthy Donors Using 16S rDNA–Targeted Metagenomic Sequencing

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