BioOutsource

Molecular Biology

Detection of Residual Host Cell DNA

Biotherapeutic products are typically derived from a complex production system involving a genetically modified host cell (Eukaryotic or Prokaryotic). Among the impurities which are to be eliminated during the purification process are the host cell derived constituents including DNA and protein. Regulatory authorities in Europe (EMEA) and USA (FDA) in recent years have recognised that the risk of DNA from continuous mammalian cell lines. A limit for DNA impurities of 100 pg/dose is often recommended.

Using the Applied BioSystems TaqMan DNA detection System it is possible to perform the quantitation of residual DNA with high precision and sensitivity (up to 4 pg/ml, dependant on target substrate). In TaqMan based assays, the detection of DNA is based on the amplification of small amounts of residual DNA in a PCR based amplification system.

For establishment of routine batch release testing an evaluation of the test material is normally proceduralised, as some low molecular weight components may inhibit binding and subsequent enzymatic reagents. . These matrix effects including inhibiting low molecular weight components or proteins have to be defined for each sample. This is performed via dilution, concentration and spike recovery experiments to determine if any pre-treatment is required in a feasibility study.

Once the pre-treatment procedure is established, a validation study is performed to show accuracy, precision, limit of detection, robustness and limit of quantification within the sample matrix according to the ICH guideline Q2.

BioOutsource offer the following validated Taqman® real time PCR assays to detect and quantify residual DNA sequences:

  • Chinese Hamster Ovary (CHO)
  • Human Embryonic Kidney (HEK) 293
  • Vero
  • Canine

Detection of Residual Host Cell Protein

The assays we offer to detect the presence of host cell proteins are based on ELISA kits supplied by Cygnus Technologies, Inc. We currently offer the following assays:

Chinese Hamster Ovary (CHO) – a generic assay for the detection of CHO Host Cell Protein.  The lower detection limit (DL) for this assay is reported to be in the region of 400pg/mL.

Human Embryonic Kidney (HEK) - a kit designed to measure HEK 293 HCP contamination.  The lower DL for this assay is reported to be less than 300pg/mL.

Quantitative fluorescent product enhanced reverse transcriptase (QFPERT)

The detection of adventitious retroviruses has always been critical for addressing the safety concerns associated with viral vaccines.  Assays for the enzymatic activity of reverse transcriptase are used as general methods for the detection of both known and unknown retroviruses.  One such test, the QFPERT test is an extremely sensitive real-time PCR based assay for the detection of reverse transcriptase (RT) activity packaged within extracellular retrovirus particles and is the first choice for the evaluation of the retroviral status of vaccines and other biological material.  BioOutsource have designed and validated a sensitive and specific real time PCR based assay capable of discriminating between the amplification signals generated by genuine positive signals that result from retroviral RT activity and those from RT like activity that can arise from DNA polymerases. 

Custom PCR Assays

BioOutsource can develop, optimise and validate any custom PCR assay you require. Please contact us for further information.