Cell lines used for the production of biologic drugs are required to be fully characterized in accordance to the ICH guidelines (Q5D) by the regulatory bodies prior to their approval.
Moreover throughout the development, trial phases and production, master cell banks and working cell banks need to be monitored in order to verify their clonality, the stability of the recombinant cassette and to rule out the presence of adventitious agents such as viruses.
Similarly, gene and cell therapy products account in certain context the permanent genome modification of cells prior to grafting or injection. Therefore, transformation events and vectors are required to be characterized to evaluate the probability of side-effects of genome modification and to serve as long-term follow-ups for the stability of such modification.
Several studies and working groups have acknowledged next generation sequencing (NGS) as a powerful tool for the analysis of recombinant events as well as the safety evaluation for the absence of dangerous contaminations (being either microorganisms or unwanted cell lines).
IGATech dedicated more than 10 years in developing and delivering tools in the fields of next generation sequencing. During this time, we have refined our expertise and built a technology platform for the structural analysis of genomes and their modifications along with the analysis of challenging samples for the detection of subtle contamination levels.
The precise determination of a transgene integration site is required for a number of technological applications. Registration and protection of new biotechnology products, GMO application, and risk assessment in gene and cell therapy are some of the areas that require detailed integration information.
Integration site analysis can also be used as a cell line development screening method to select the most promising clones. Depending on whether a homogeneous (clonal) cell line or a heterogeneous population of transformation events is under analysis, specific methods and sequencing platforms (Illumina or Nanopore) can be applied to reach a complete picture of the insertional landscape of any given genetic background.
The high depth of coverage provided by NGS can identify thousands of independent copies of the same integration cassette or vector within a given specimen. This allows for the detection of low frequency de novo mutations that might have occurred during cell line propagation or subcloning. We have established efficient protocols for the targeted sequencing of a region of interest (ROI) at very high depth. Specialized bioinformatics tools are then used to detect mutations occurring at very low frequency, avoiding false positives caused by sequencing errors or PCR artifacts.
Regulatory bodies often require valid characterization of cell lines in order to assess their stability and monoclonality throughout the product process, from the master cell banks to working cell banks and bulk harvest. A complete cell line characterization is an important tool for both early stages of cell line development and subsequent assessment of its stability. Long-read sequencing can now provide high-quality cell line genome assembly to provide a solid base for cell line optimization, stability and clonality control using more cost- effective resequencing and "integrome screening" methods.
The study of metagenomes broadened the understanding of complex living ecosystems while deploying methodologies and bioinformatics tools for this purpose. Similarly, the analysis of complex matrices by deep sequencing have enabled the detection (and assignation) of any possible microorganism, even when present in non-quantifiable amounts. IGATech has extensive experience in the field of RNA-Seq, including using pre-amplification technologies for successful analysis of difficult samples with non-detectable amounts of RNAs. This is of particular relevance for adequate sensitivity in the analysis of raw materials, growth media, bulk harvest and finished products to rule out the presence of adventitious agents (virus, bacteria, fungi or mycoplasma).
Remember to include a hard copy of sample spreadsheet in the parcel