Residual Host Cell DNA Quantitation Regulatory Compliance

PCR-based detection of host cell DNA

Companies that produce biopharmaceutical products must verify the quantity of host cell DNA impurities carried over through the purification process and in the drug’s final dosage to meet or exceed regulatory guidelines. Due to its high sensitivity, accuracy, specificity, dynamic range, high-throughput capability and consistent performance, PCR-based solutions are the most commonly used techniques for residual DNA quantitation.

Developed to meet the rigors of regulatory guidelines

To verify product quality, the amount of residual DNA in a drug's final dosage form must meet regulatory guidelines established by the various regulatory agencies. Each regulatory agency has specific guidelines for acceptable limits, ranging from 10 pg to 10 ng DNA per therapeutic dose depending on the product and therapeutic modality. The Applied Biosystems resDNASEQ Quantitative DNA solution was specifically developed to help meet these regulatory guidelines, such as the ones below:

DNA content in the final product should be less than 10 ng per therapeutic dose (WHO)
DNA content in the final product should be as low as possible, as determined by a highly sensitive method (FDA)
It is suggested that a method with a sensitivity of 10 pg be used to determine DNA levels (FDA)
An appropriately conducted clearance study for DNA removal may be an acceptable substitute for lot-to-lot testing (FDA)
USP (United States Pharmacopeia) general chapter <509>: Residual DNA Testing, details the application of quantitative PCR for measurement of residual host cell DNA levels during manufacturing of biopharmaceuticals

On-demand webinar: Validation guidance for residual host cell DNA testing of biologics

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On-demand webinar: Development & validation of quantitative real-time PCR for the detection of residual HEK-293 host cell DNA

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Designed for purpose, confirmed in validation, implemented globally

Validation of residual host cell DNA assays may vary by regulatory agency, but the general expectation is to meet validation requirements outlined in ICH Harmonised Tripartite Guideline Validation of Analytical Procedures Q2(R1) (ICHQ2R1).

Here we share validation data from our resDNASEQ host cell DNA quantitation kits, focusing on some of the most challenging validation parameters including sensitivity by limit of quantitation (LOQ), specificity, and accuracy.

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of assays for low levels of compounds in sample matrices and is used particularly for the determination of impurities and/or degradation products. For resDNASEQ kits, the LOQ is 1.5 pg/mL for mammalian DNA; and LOQ to 15 pg/mL for bacterial/yeast DNA.

Figure 2. Superior sensitivity compared to USP recommended assay. resDNASEQ CHO assay is 10 times more sensitive than CHO assay recommended by USP.

Assay accuracy is measured by spiking replicate samples with a known concentration and calculating recovery from a standard curve. Assay precision is defined and measured by percent coefficient of variation (%CV), which is calculated by dividing the standard deviation by the average Ct of the number of replicates.

Spike recovery for CHO cell line DNA

Table 1 demonstrates the 90% or greater recovery from a typical antibody drug substance test sample that was spiked with 100, 10, 1, or 0.1 pg CHO standard DNA in triplicate.

**Table 1.** High DNA recovery rate from a typical antibody drug substance
Spiked DNA quantity (pg)	Quantity recovered (pg)			Average recovery %	CV %
100	105	133	123	120	12
10	10.2	10.9	11.5	109	6
1	1.1	1.1	1.2	110	5
0.1	0.09	0.10	0.08	90	7.9

For accurate quantitation, the assay must detect only the target DNA and no other DNA present in the sample. An example is shown below where the resDNASEQ Human DNA assay is unaffected by the presence of other DNA.

DNA	Quantity	Ct
Human	3 ng	13.59
CHO	10 ng	29.27
E. coli	10 ng	31.20
MDCK	10 ng	31.51
NS0	10 ng	31.29
Pichia pastoris	10 ng	30.38

Table 2: resDNASEQ human assay highly specific to human DNA and unaffected by high concentrations of unrelated DNA.

Figure 3. NS0 assay demonstrating no cross reactivity with unrelated DNA. Even with a high amount of unrelated human DNA, the standard curve remains unaffected.

For the most accurate quantitation of residual host cell DNA, the assay results must be unaffected by the size of the DNA molecules present in the test sample. Shown below are results generated from analysis of a dilution series of high molecular weight and fragmented CHO standard DNA in several different matrices representing typical monoclonal antibody purification scenarios. The resDNASEQ assay maintained consistent performance even with fragmented DNA across the entire concentration range of the analysis.

DNA added	Matrix 1		Matrix 2		Matrix 3		Matrix 4		Matrix 5
DNA added	HMW	LMW	HMW	LMW	HMW	LMW	HMW	LMW	HMW	LMW
1 ng	122%	91%	122%	100%	95%	88%	103%	93%	85%	105%
0.1 ng	95%	94%	95%	108%	109%	86%	99%	107%	82%	110%
10 pg	91%	84%	91%	98%	96%	89%	84%	98%	86%	101%
1 pg	88%	82%	88%	87%	91%	79%	85%	82%	66%	101%
0.1 pg	91%	87%	91%	80%	100%	87%	82%	86%	76%	115%

Table 3: Spiked CHO resDNASEQ recoveries at 5 concentration levels, showing high molecular weight DNA (uncut) and low molecular weight DNA (Sau961). Matrices are M1: ion exchange, pH 7.5; M2: hydrophobic Interaction; M3: ion exchange, pH 6; M4: protein A eluate; M5: purified drug substance.

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