Accuracy and sensitivity of residual DNA detection by QPCR is not predicted by target copy number

Abstract

A major issue in the use of mammalian cell culture in biopharmaceutical manufacturing is the removal of process related impurities, such as residual host cell DNA, during the product purification process. To ensure that sufficient DNA removal is achieved during purification, it is essential to have an accurate and sensitive assay for host cell DNA. The quantitative polymerase chain reaction (QPCR) is widely used for this purpose; however, the extent to which the choice of QPCR gene target can have an impact on final results requires further understanding. In the present study, we examined the relationship between the genomic copy number of eight different Chinese Hamster ovary (CHO) gene targets and the sensitivity and accuracy afforded by those targets in a residual host cell DNA QPCR assay. We also evaluated the use of each gene target for accurate measurement of residual DNA clearance using in-process purification samples from two CHO production cell lines. Our results revealed a correlation between gene target abundance and the potential sensitivity for use in a QPCR assay. However, we found that higher copy number gene targets do not provide the highest measurement or reveal the largest clearance of residual host cell DNA from purification samples. These findings suggest that different DNA sequences may clear or degrade at differential rates and highlight unexpected considerations that must be made in the choice of QPCR gene target when designing QPCR assays.

Assessing Analytical Similarity of Proposed Amgen Biosimilar ABP 501 to Adalimumab

“…Residual DNA was analyzed by quantitative polymerase chain reaction and fluorescence detection, as previously described [ 26 ].…”

Investigation of a failed DNA assay leads to identification of an unexpected contaminant in a commonly used chromatography buffer

“…The TaqMan® qPCR method for residual host cell DNA quantitation has been described previously 13 . Briefly, all test samples were diluted, if necessary, to the desired protein concentration or desired volume as indicated in nuclease‐free water and digested with Proteinase K (Promega) at 60°C for 2–24 h. DNA was extracted from samples using standard chaotropic salt (sodium iodide) and alcohol precipitation protocols.…”

“…The TaqMan ® qPCR method for residual host cell DNA quantitation has been described previously. 13 Briefly, all test samples were diluted, if necessary, to the desired protein concentration or desired volume as indicated in nuclease-free water and digested with Proteinase K (Promega) at 60 C for 2-24 h. DNA was extracted from samples using standard chaotropic salt (sodium iodide) and alcohol precipitation protocols. Extracted DNA pellets were resuspended in nuclease-free water and the entire volume of recovered DNA was measured by qPCR as total DNA analysis using the ABI QuantStudio 7 running QuantStudio Real-Time PCR Software (version 1.1).…”

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

“…The optimized extraction method includes one incubation step and one alcohol precipitation step and can efficiently purify DNA from our biopharmaceutical intermediates and final products at a recovery close to 100% at a wide concentration range. The sensitivity of a real-time PCR assay is generally dependent on both the copy number of the target gene and the specificity of the primer and probe sequences [3]. Primer and probe sequences targeted to mouse and CHO repetitive interspersed nuclear elements showed consistently higher sensitivity [4] than those targeted to the housekeeping GAPDH gene or the highly-conserved 18S gene.…”