Advances in genetics and bioengineering have enabled the development of gene therapy and with it, the unprecedented potential to treat diseases caused by recessive gene disorders, acquired genetic diseases, and some viral infections. Technologies for manufacturing and analysis of gene therapy products are evolving with continued efforts to be fully optimized.
Solutions from Thermo Fisher Scientific are evolving as well. Our robust assays are tailored for the analysis of gene therapy products to enable fast results with better accuracy and reproducibility to facilitate the development of your gene therapy product and accelerate its progression to market. There are several recombinant DNA technology protocols, in which the gene-of-interest or healthy gene is inserted into a vector, which can be a viral, plasmodial, or nanostructured.
Messenger RNAs (mRNA) therapies — which upon introduction to the target cell are translated into desired proteins — are being explored in the use of personalized immunotherapy.
Popular products for gene therapy analysis
Adeno-associated virus (AAV) vector gene therapy workflows
AAV production & purification
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
There are multiple strategies to develop AAV vectors, but typical steps include plasmid development and production, cell expansion, plasmid transfection, viral vector production, purification, and fill and finish. Transient expression of AAV in either adherent or suspension cell lines are most common in pre-clinical and early phase development. An alternative option for AAV production is the development of a stable producer cell line which helps ensure long-term stable supply of the viral vector.
For details, go to: AAV Production Workflows
Innovative chromatography solutions, specifically designed to improve the downstream purification of mRNA and viral vectors such as the Adeno-Associated Virus (AAV), lentivirus, and Adv5.
For details, go to: Cell and Gene Therapy Purification Solutions
AAV gene product and process characterization and quality control
FDA guidance for industry on characterisation requirements for gene therapy products, focus on structure and characteristics of the product, process related impurities, and product related impurities. Below are the optimized workflows utilizing leading Thermo Scientific instruments, consumables, and software for AAV product and process characterization
AAV Identity Confirmation
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.
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.
Mass confirmation of empty capsids can be performed with high confidence using the Thermo Scientific Q Exactive UHMR Hybrid Quadrupole-Orbitrap mass spectrometer.
|Analytical Solutions for Intact Capsid Mass Analysis |
|Glass Autosampler Vials||Vanquish Flex UHPLC Systems||Q Exactive UHMR Hybrid |
Quadrupole-Orbitrap Mass Spectrometer
|BioPharma Finder Software|
Alternatively, using charge-detection mass spectrometry (CD-MS) opens the door to analyzing these macromolecules with high mass heterogeneity, providing a means to quality control genome packaging in production of gene therapy products.
AAV Impurity Analysis
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.
Messenger RNAs (mRNA) therapy workflows
Because mRNA is transient in the body, synthetic mRNA products require modifications to protect the product from natural degradation. To ensure safety and efficacy regulatory agencies demand in-depth analytical characterization and criteria defined for purity/impurity monitoring (Critical Quality Attributes or CQAs). Impurities are identified during characterization, with acceptable levels defined, and monitored throughout manufacture and lot release.
Thermo Fisher Scientific utilize leading instrumentation, consumables, and software to provide optimized mRNA characterization and impurity analysis.
Analysis of mRNA Therapies
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.