In our molecular biology department a wide set of analytical services for a detailed characterization of DNA products is available. These methods may be applied for the determination of genetic stability for recombinant products and for the characterization of plasmid products as well. Additionally different methods for the detection of residual DNA are in place to sensitively determine minimal amounts of host cell DNA.

Genetic stability testing

The genetic stability of the expression construct in a recombinant cell line needs to be analyzed. The identity of the expression construct has to be verified, additionally it has to be shown that the expression construct remains stable during the entire production process. Therefore, cells coming from the Master Cell Bank are compared to those taken from the limit of the in vitro cell age (EPC or PPCB). The identity issues may be addressed by DNA sequencing and restriction enzyme analysis followed by Southern blotting. The identity is then confirmed by comparing the length of the resulting resulting fragments to the theoretical values.

Determination of copy number

The Southern blot analysis may also be used to quantitatively determine the copy number of the expression construct in the cell line. Therefore, a dilution series of a product specific PCR product is separated on an agarose gel as well. To determine the copy number per haploid genome of the integrated construct, background corrected signal intensities of specific restriction fragments are compared to the calibration row.

DNA sequencing

NewLab BioQuality offers a validated, GMP-compliant method for sequencing of DNA using a polyacrylamide gel based sequencing system (LiCOR^TM). This includes analysis in triplicate of each strand and the use of two different control DNA’s. The control DNA must fulfill acceptance criteria regarding the reading length and the maximum number of ambiguities. The system is also tested for its capability to detect single point mutations. The results from the of both DNA strands are aligned and manually edited referring to the original gel picture. The sequencing method is based on a NewLab internal validation study with an accuracy of ³ 99.9%.

Detection of residual DNA

NewLab BioQuality generally uses two highly sensitive, validated methods for quantifying residual DNA. These methods encompass the entire range of residual DNA from unspecific detection of total DNA to the highly specific detection of single target sequences. The methods used are:

• The Threshold^TM technology for unspecific quantitation of total DNA using the Threshold^TM system by Molecular Devices Corporation. The assay uses DNA binding proteins with high affinity for single-stranded DNA.
• The hybridization method for the detection of the specific DNA of defined origin using dot blots and hybridization of radio-labelled DNA probes. Hybridization uses random hexamers to generate representative probes covering the whole genome of the host cells.

NewLab’s wide-range of experience with sample purification and preparation enables us to analyze samples in many different matrices.