Optimization and validation of DNA extraction and real-time PCR assay for the quantitative measurement of residual host cell DNA in biopharmaceutical products

Hu, Binwu; Sellers, Joshua R.; Kupec, J.; Ngo, Winnie; Fenton, S.; Yang, Tong‐Yuan; Grebanier, Alice E.

doi:10.1016/j.jpba.2013.08.027

Cited by 17 publications

(4 citation statements)

References 2 publications

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“…One method, which is the most commonly used method for host residual DNA quantification, is the extraction of total DNA from the sample prior to qPCR analysis. There are many different extraction methods and kits currently available for use [6][7][8]. Success of such a method is dependent on the efficiency of extraction of presumably very small amount of DNA present in purified drug substance.…”

Section: Resultsmentioning

confidence: 99%

A direct qPCR method for residual DNA quantification in monoclonal antibody drugs produced in CHO cells

Hussain

2015

Journal of Pharmaceutical and Biomedical Analysis

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Section: Resultsmentioning

confidence: 99%

A direct qPCR method for residual DNA quantification in monoclonal antibody drugs produced in CHO cells

Hussain

2015

Journal of Pharmaceutical and Biomedical Analysis

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“…A variety of methodologies exist that can be applied for precise host cell DNA quantification. The most widely used technology is quantitative polymerase chain reaction (qPCR) ( 14 – 16 ). This approach uses highly specific DNA primers to detect the host cell DNA ( 17 ).…”

Section: Introductionmentioning

confidence: 99%

Overcoming Biopharmaceutical Interferents for Quantitation of Host Cell DNA Using an Automated, High-Throughput Methodology

Lauro

Bowman

Smith

et al. 2022

AAPS J

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The rapid development of biologics and vaccines in response to the current pandemic has highlighted the need for robust platform assays to characterize diverse biopharmaceuticals. A critical aspect of biopharmaceutical development is achieving a highly pure product, especially with respect to residual host cell material. Specifically, two important host cell impurities of focus within biopharmaceuticals are residual DNA and protein. In this work, a novel high-throughput host cell DNA quantitation assay was developed for rapid screening of complex vaccine drug substance samples. The developed assay utilizes the commercially available, fluorescent-sensitive Picogreen dye within a 96-well plate configuration to allow for a cost effective and rapid analysis. The assay was applied to in-process biopharmaceutical samples with known interferences to the dye, including RNA and protein. An enzymatic digestion pre-treatment was found to overcome these interferences and thus allow this method to be applied to wide-ranging, diverse analyses. In addition, the use of deoxycholate in the digestion treatment allowed for disruption of interactions in a given sample matrix in order to more accurately and selectively quantitate DNA. Critical analytical figures of merit for assay performance, such as precision and spike recovery, were evaluated and successfully demonstrated. This new analytical method can thus be successfully applied to both upstream and downstream process analysis for biologics and vaccines using an innovative and automated high-throughput approach. Graphical Abstract

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“…Since the allowable limit for host residual DNA (hrDNA) is 10ng per daily dose, most manufacturers of biologic drugs often set the acceptance criteria of host residual DNA to as low as ≤1.0 pg of DNA per mg of drug under development where the daily dose is unknown. As a result, very sensitive method of DNA quantification, like quantitative real-time PCR (qPCR), is employed with [7] or without [8,9] DNA extraction. Recently, digital PCR has become available from different vendors in different formats [10,11] as an improvement over the qPCR [12,13] for absolute quantification of nucleic acids.…”

Section: Introductionmentioning

confidence: 99%

A Droplet Digital PCR Method for CHO Host Residual DNA Quantification in Biologic Drugs

Hussain

Bowers

2017

JAPLR

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The Chinese Hamster Ovary (CHO) cells are the preferred host for manufacturing therapeutic biomolecules in the biopharmaceutical industry. Host residual DNA (hrDNA) is an impurity and needs to be monitored in the purified drug to ensure purity and safety. Currently, real-time quantitative PCR (qPCR) based methods are widely employed for quantification of hrDNA, however, digital PCR technology promises higher assay sensitivity and precision. Here, we report a method where the protein drug is digested with a protease, the protease is denatured and the CHO primers and fluorescent-tagged probe from Bio-Rad Laboratories, Inc. and droplet digital PCR (ddPCR) mix are added to the reaction and nanoliter-sized droplets are generated. The droplets are then subjected to end-point PCR followed by analysis for fluorescence. Compared to qPCR, the ddPCR method shows increased sensitivity, with high precision and accuracy of determination. Additionally, the method eliminates DNA extraction step and the requirement of DNA standards in routine sample testing. The method was successfully applied to hrDNA quantification in several biologic drugs under development at Merck.

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Optimization and validation of DNA extraction and real-time PCR assay for the quantitative measurement of residual host cell DNA in biopharmaceutical products

Cited by 17 publications

References 2 publications

A direct qPCR method for residual DNA quantification in monoclonal antibody drugs produced in CHO cells

A direct qPCR method for residual DNA quantification in monoclonal antibody drugs produced in CHO cells

Overcoming Biopharmaceutical Interferents for Quantitation of Host Cell DNA Using an Automated, High-Throughput Methodology

A Droplet Digital PCR Method for CHO Host Residual DNA Quantification in Biologic Drugs

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