Best Practices for Quantification of Residual Host Cell DNA with Real-time PCR

Advantages of Real-time Quantitative PCR

Residual DNA from cell lines can persist during biologic drug manufacturing, which can cause adverse side effects in patients if problematic genetic material is transferred. Regulatory agencies have set the limits for this critical quality attribute (CQA) between 10-100 pg host cell DNA/dose, depending on the parental cell line and dosing regimen. Testing in-process samples from downstream purification and polishing steps for residual DNA (Table 1) can inform process improvements during the manufacturing process to help prevent delays with regulatory approvals. Over the past several years, this has been achieved with semi-quantitative and quantitative PCR methods using hybridization techniques and intercalating DNA dyes; however, these methods have limitations, such as non-specific signal detection, which can lead to inaccurate measurements of DNA concentration.

Introduction of fluoroprobe-based detection tools has significantly improved the sensitivity and specificity of real-time PCR quantification. To this end, USP General Chapter <509> “Residual DNA Testing” outlines use of probe-based DNA quantification as a validated method for testing recombinant therapeutic products produced in Escherichia coli (E. coli) or Chinese hamster ovary (CHO) cell lines. When employed in combination with other hot start PCR reagents, the sensitivity and accuracy of the resulting real-time PCR assay can readily detect femtogram levels of host cell DNA remaining in process samples or the final drug substances, instilling confidence that the drug manufacturing process has reduced DNA levels to the safest levels possible.

Table 1. Manufacturing steps to monitor host cell DNA levels.

Three Steps to Sample DNA Quantification

Prior to quantitative PCR, DNA extraction must be performed to remove PCR-interfering components and enrich the DNA in the sample. The extraction can be performed using centrifugation or magnetic-bead-based approaches. While magnetic bead-based extraction methods are amenable to automation, yield can be impacted due to the length of procedure, number of steps, and unwanted carryover of reagents. Cygnus Technologies’ DNA extraction procedure utilizes a novel DNA carrier to concentrate and recover trace amounts of residual DNA with high yield in an environment free from contaminating proteins, salts and detergents. Although centrifugation steps are necessary for this method, performing the extraction in a deep-well plate accommodates high sample throughput with a minimal number of washing steps to ensure sample purity but prevent loss of yield (Figure 1). This carrier-based extraction method results in reproducible and accurate recovery of CHO DNA (Figure 2).

Figure 1. Carrier-based DNA extraction using Cygnus Technologies’ D100T [T] or D100W [W] kit.

Following DNA extraction, the samples are prepared for DNA amplification via real-time PCR. Many quantitative real-time assays are available on the market; choosing an assay specific to the host species and existing equipment from a reputable vendor will allow for a seamless experience qualifying the assay. Cygnus Technologies’ AccuRes™ Quantification kits include a primer/probe mix that amplifies host cell DNA in a highly specific manner, as the FAM-labeled nucleic acid probe is quenched by BHQ-1™ until PCR extension. CleanAmp® dNTPs and Hot Start Taq DNA polymerase are included to prevent non-specific amplification before the reaction temperature elevates. Together, these reagents comprise a highly specific, state-of-the-art master mix that ensures sensitivity and robustness of the assay while allowing the PCR reaction to be prepared at ambient temperature.

Once the assay is completed as specified in the protocol, analysis is relatively straightforward and can be performed using standard qPCR instrument software. The threshold cycle (C_t) values of the kit standard DNA provided are used to construct a standard curve with values reported in pg/10µL host cell residual DNA (Figures 3-4). Using Cygnus Technologies’ CHO or E. coli assays, residual DNA as low as 0.6-0.7 femtogram per microliter can be measured, respectively. Subsequently, the concentration of host cell residual DNA can be extrapolated to report residual DNA with respect to the amount of drug product or dose.

Figure 2. Spike and recovery of CHO DNA extracted and amplified with Cygnus Technologies AccuRes Quantification kit (D1555W) versus Competitor manual extraction and qPCR protocol. Superior recovery of in-process samples (CEX, AEX) and drug substances (BDS) was observed with the Cygnus kit compared to the Competitor kit.

Figure 3. Standard curves for CHO assays run at specified concentrations. While the efficiency and sensitivity of AccuRes™ reagents is similar to Competitor on control CHO DNA, non-specific amplification of negative controls (arrow) is minimized due to inclusion of CleanAmp® dNTPs and Hot Start Taq Polymerase.

Figure 4. Broad linear range and specificity of CHO primer/probe in the presence of foreign DNA. Presence of E. coli DNA at varying concentrations does not impact the CHO DNA standard curve.

Qualification of Your DNA Quantification Assay

The reproducibility and sensitivity of the selected quantification assay must also be confirmed in the biopharmaceutical manufacturer’s laboratory to demonstrate its suitability to quantify residual DNA for product lot release. This in-house testing confirms that the assay can reproducibly detect trace levels of host cell DNA from the species of origin, and that these levels are below the required regulatory limits. While designing an in-house assay is an option to reduce cost, choosing a well-designed commercially available assay will ultimately save time and reduce overall cost. Cygnus Technologies’ AccuRes Quantitative Assay Kits also offer flexibility to choose the extraction method and analysis equipment, and the additional technical support of assay developers should the assay encounter any issues.

Contact our Technical Experts if your production platform requires a unique solution and a custom host cell DNA assay implementation.

References:

USP Chapter <509> Residual DNA Testing. 01 Dec. 2019. Printed 10 August 2021.

About Cygnus Technologies

Cygnus Technologies, part of Maravai LifeSciences, offers generic HCP ELISA Kits for 25 different expression platforms, residual Host Cell DNA kits, advanced orthogonal antibody coverage analysis services, HCP identification of in process samples and drug substances by AAE-MS™, generic assay qualification services, and expert process-specific antibody and assay development services.

For more information, contact us at techsupport@cygnustechnologies.com.