PCR Applications In Host Residual DNA Samples
Posted 14th April 2017 by Jane Williams
Host residual DNA (hrDNA) as an impurity in biologic drugs is currently quantified by DNA extraction followed by qPCR. Musaddeq Hussain developed a method for direct qPCR without using DNA extraction from mAb drugs. He gave us his view about the research and challenges of working in the field.
Developments and challenges in PCR over the last decades
Since the gene holds the central position, PCR has impacted a big part of biologic sciences from estimation of gene copies, transcription level, size changes, microbial diagnostics, oncogenes and cancer research to cloning and sequencing. Digital PCR seems to be the greatest development in PCR techniques. It has brought the detection sensitivity way down and has unhooked the reaction efficiency from the quantification allowing testing of cruder samples.
Nevertheless, there are still some challenges that PCR techniques are still facing. For instance, preparing the standard and putting together the PCR plate for the experimental run. Future techniques will need to develop a small bench-top liquid handler or auto dispenser that can be easily programmed and flexible for the changing demands and very accurate.
The impact of PCR in host residual DNA research
PCR in hrDNA research is applied to quantify the amount of hrDNA in protein drug samples. By working alongside the biopharmaceutical industry, hrDNA researchers developed bioprocess for manufacturing protein drugs including monoclonal antibody drugs in host cells. The next step in this field will be determining the host cell derived impurities in the purified drug out of safety concerns. So far, researchers have always followed the same procedure which extracts the total amount of DNA from the protein drug sample and then performed qPCR to quantify the hrDNA in the sample.
Most of the available extraction methods start with digesting the protein matrix with a protease (e.g. nuclease-free proteinase K) for efficient DNA extraction. A few years back, I started thinking about how I could add test sample directly to qPCR for DNA quantification and started looking and testing different proteases. These are needed to digest the drug protein into pieces small enough not to precipitate during PCR cycling and then the protease could be completely denatured by heating at high temp like 95 °C. Finding the right protease took some time but led to eliminate DNA extraction step prior to PCR for hrDNA quantification.
Musaddeq Hussain works as a Principle Scientist at Merck Laboratories. He took part in the 2nd qPCR & Digital PCR Congress: USA.
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