Gene Therapy Analysis

Premier Thermo Fisher Scientific brands for AAV gene production & characterization workflows

These brands are ideal for developing end-to-end workflows for AAV gene therapy production and characterization.

AAV vector development and production workflow

It is important to understand the capsid identity because each AAV serotype has unique capsid proteins that transfer the genetic material to specific cells or organs. Depending on the therapeutic target area, the correct AAV serotype is chosen and identity and purity are monitored. All AAV capsids consist of three proteins (VP1, VP2, and VP3) that share high-sequence homology, and ensuring the identity and purity of these proteins are critical to viral infectivity and viral transfer. Given the importance of the serotype and the capsid protein composition, there exists a need for methods to identify and monitor the capsid and the capsid proteins through gene therapy development and manufacturing.

 Webinar: Gene Therapy, A New Analytical Playground, Existing Methods and Future Possibilities Columns for the AAV capsid analysis

Analysis of AAV primary sequence and post translational modification (PTM)

In addition to identifying AAV serotypes and associated capsid proteins (and resulting ratios), one must characterize capsid proteins at the primary sequence level including analysis of post translational modifications (PTMs). This sequence level characterization is essential ensure product quality and consistency.

Webinar: Developing analytics to support the next generation of gene therapies

Mass confirmation of empty capsids can be performed with high confidence using the Thermo Scientific Q Exactive UHMR Hybrid Quadrupole-Orbitrap mass spectrometer.

Among others, one important step post-upstream production of AAVs is to accurately determine the titre of the viral capsids. Size exclusion chromatography with florescence detection can be utilized to determine viral capsid titre following upstream purification.

Host cell proteins HCPs are low level process-related impurities derived from the host expression systems during biotherapeutic manufacturing, which can impact quality or safety, or compromise product stability.

Peptide analysis by LC-HRAM-MS offers orthogonal solutions for detection and monitoring of HCPs compared to immunological methods with the unbiased discovery of HCP impurities and subsequent quantitation. HRAM MS data combined with Thermo Scientific BioPharma Finder 4.1 software provides comprehensive HCP qualitative and quantitative analysis.

Empty capsids, which do not contain the gene therapy of interest, and partial capsids, those containing only a fragment of the gene of interest, are by-products of the AAV production and can impact product safety and efficacy. The amount of full, partial, and empty capsids, therefore, needs to be characterized and monitored through process development.

Due to immunogenicity risks of aggregates, this is a CQA that must be fully defined and monitored.  Aggregates are formed during production and storage. Size exclusion chromatography (SEC) methods provide an inexpensive approach to monitoring aggregation in a high throughput manner.

The purity of mRNA can be determined by mRNA sequence mapping.

The presence of a 5’ cap structure is essential for subsequent steps in the life cycle of mRNA in eukaryotic cells. Therefore, capping efficiency must be determined and monitored throughout development.

To maximize gene expression, the purity of mRNA must be determined, impurities characterized, and monitored throughout the process. This includes aborted sequences, double stranded RNA, and residual plasmid DNA.