Residual Host Cell DNA and HCP Testing Criteria: Where the Numbers Come From and What to Check in Test Methods

Why Test

Biopharmaceuticals produced in cells may retain DNA and proteins from the production cells. ICH Q6B classifies host cell protein (HCP) and host cell DNA as process-related impurities derived from the cell substrate [1][9]. Residual DNA is controlled because of its potential tumorigenicity and infectivity [2][3], and HCP because it can affect the quality, efficacy and safety of the product [8][9].

Residual DNA: Where the Numbers Come From

Sources: [2][3][4][5]

• All of the figures above are recommendations, not a single rule that applies to every product. WHO provides that the acceptable limit for each product be set in consultation with the regulatory authority, taking into account the characteristics of the cell substrate, the intended use, and the effect of the process on the size, quantity and activity of the DNA [2].
• "About 200 bp" is the value the FDA guidance documents give as the size of a functional gene [3][4].
• If tumorigenic cells (e.g. HEK293) are used, more stringent limits may be needed [3][4].
• WHO states that some live viral vaccines that are difficult to purify may exceed 10 ng/dose, and that this is an exception to be discussed with the regulatory authority [2].

HCP: There Is No Uniform Numerical Limit

HCP guidance documents do not give a numerical limit; the acceptable level is set product by product according to clinical conditions such as dose and route of administration [9][10]. The European Medicines Agency (EMA) Q&A advises that HCP testing is normally included in the drug substance specification and that the upper limit be set at a clinically justified value [8]. Details of HCP test methods are covered in European Pharmacopoeia 2.6.34 and USP <1132> [8][9][10].

Test Methods

• Residual DNA: quantitative PCR (qPCR) is the most widely used method [7][11]. USP <509> provides a qPCR measurement method, and the extraction procedure that precedes it, for recombinant medicinal products derived from CHO and E. coli [6][7].
• HCP: enzyme-linked immunosorbent assay (ELISA), which measures many HCPs at once, is the standard test method [9][12]. Because ELISA antibodies do not recognize every HCP, liquid chromatography-mass spectrometry (LC-MS), which can identify and quantify individual HCPs, is used alongside it as an orthogonal method (a complementary method based on a different principle) [9][12].

What to Check When Choosing a Test Method or Kit

• Host specificity: check that the test matches the production cell (CHO, E. coli, HEK293, Vero, etc.) and does not react with DNA from other species [6][11].
• Sensitivity and limit of quantitation (LOQ): check that it is sufficiently below the target limit on a per-dose basis. The 1997 FDA document suggested a sensitivity at the 10 pg level [5].
• Extraction recovery: because DNA is extracted from protein-rich samples before measurement, extraction efficiency is checked by spike recovery [7][11].
• Antibody coverage: how broadly the ELISA antibodies recognize the HCPs of the process in question must be evaluated [8][9][10].
• Generic and process-specific assays: generic commercial kits are widely used in early development. If coverage is insufficient, a platform or process-specific ELISA is developed, and regulators typically expect this before entry into Phase 3 clinical trials [9][10].

Sources