Residual Host Cell DNA Assays
Manufacturers of vaccines and other biological products must ensure that final products derived from continuous mammalian cell lines contain acceptable levels of residual host cell DNA (HCD). The presence of HCD in the final product is of significant concern due to the potential transfer of activated cellular and/or viral oncogenes (particularly if the cell substrate is tumourogenic), the production of infectious viruses from viral DNA and aberrant gene expression by insertion of sequences into sensitive control regions of genes. Consequently host cell and vector derived DNA should be detected using a suitably sensitive analytical technique and quantitative data obtained.

Quantification of residual HCD is critical for manufacturers of biopharmaceuticals and forms part of the manufacturing validation and the purity tests for each clinical lot. Stringent guidelines stipulate the maximum amount of DNA that should be present in the clinical lot. For example a vaccine production using continuous non-tumourogenic cell line such as low passage Vero should be limited to a maximum level of 10ng of cellular DNA per dose. There may be instances where continuous cell line DNA is considered to pose a greater risk. For example if the cell contains retroviral proviral sequences then residual DNA limits of limits of 100pg per dose can be recommended. In many cases the maximal amount of residual DNA per dose should be set on a case-by-case basis dependant on the product.

It is possible to accurately quantify residual HCD using Applied BioSystems sequence detection TaqMan assays. In many cases highly accurate sensitivities of 1.3pg/ml are achieved (depending on the amplicon sequence of the HCD assay).

Vitrology offers residual HCD assays validated to ICH Q2 guidelines for the following cell substrates (Table 1), A second launch of residual DNA qPCR assays for NS0, and Vero cell substrates is expected in 2008.

Table 1

For cGMP compliant lot release testing the drug product requires a detailed qualification study with the relevant HCD assay. This study ensures that the HCD assay will meet the required specifications and may be customised depending on the drug product. During every routine lot release test multiple high quality assay controls are included to provide a high degree of confidence in the quantitative result. Firstly, each sample is treated to inactivate any interference from the test material matrix. Samples are diluted and spiked with two individual internal control DNAs to determine the optimal DNA extraction and qPCR test volume for the drug substance. A drug substance specific validation study is performed according to ICH guidelines Q2, to ensure each drug product lot release specification has supporting data for accuracy, precision, limit of detection, robustness and limit of quantification within the sample matrix.

ICH Q2 validation reports are available for each of Vitrology’s HCD qPCR assays and the following Ph.Eur. 2.6.21 compliant control system is performed during the testing of each client sample:

• Contamination control: Separate clean rooms for reagents, test samples, and positive controls. Each room and assay step contains a multi-step contamination control system.
• Nucleic Acid Extraction Interference: Prior to extraction each test sample is diluted and spiked with “extraction control DNA” that mimics low level residual HCD. This provides a measurement of the recovery of residual HCD.
• Nucleic Acid Amplification Interference: Prior to testing the sample and dilutions for the presence of “extraction control DNA ” by qPCR each test sample is spiked with a secondary “amplification internal control DNA ”.
• Optimisation of test sample volume: The results from the low level internal control qPCR assays determine the optimal sample concentration for testing with the residual HCD qPCR assay
• Standard curve positive control: Each assay contains a minimum of 5 log range standard curve of the HCD.
  

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