Cell and Gene Therapy / AAV Analytics
Within the evolving, complex and competitive arena of cell and gene therapies, the development of advanced therapy medicinal products (ATMPs) has made significant progress with regard to the treatment of many diseases, including cancer as well as genetic and autoimmune disorders. The pharmaceutical industry is now beginning to pay particularly close attention to gene therapy medicinal products (GTMPs). The GTMP global market is expected to surpass the US$20 billion mark by 2025, based on an anticipated compound annual growth rate (CAGR) of up to 40 percent
Advanced therapy medicinal product (ATMP) is
a general term for three groups of medicinal products:
sCTMP: Somatic cell therapy medicinal product
TEP: Tissue engineered product
GTMP: Gene therapy medicinal product
Made up of DNA, genes are the basic units of heredity. In each cell of the body, they contain the instructions for specific molecules, usually proteins. The transition from gene to protein is a complex workflow.
Several common and rare diseases are caused by a deficiency in the correct expression of proteins during the intracellular processes of transcription and translation, whereby information from genes is used to create the proteins. GTMPs are active substances consisting of DNA/RNA and given to patients in order to add, repair, regulate, substitute or delete designated genetic sequences. In a classic scenario, a defective gene that causes a certain disease is replaced by an intact gene.
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Gene therapy AAV Approach
Gene therapy researchers have found modified viruses to be effective vehicles (“vectors”) for introducing therapeutic genes into cells. Adeno-associated viruses (AAV) play a unique role here and represent the most commonly used approach: most gene therapy vectors deliver therapeutic genes by integrating their genetic material into the DNA of the host cell, whereas episomal AAV-delivered genes are expressed transiently and do not permanently modify the genetic information of the cells. This results in very low immunogenicity and practically zero oncogenic potential. Hence, AAV-derived active substances are becoming the vector of choice for delivering gene therapies for a vast number of diseases; they have been investigated for the treatment of cancer as well as ocular, pancreatic and CNS conditions.
The fundamental benefit of using AAV particles as the primary “delivery system” for gene therapeutics is based in its favorable safety profile. AAV cannot replicate its genetic information without additional help by specific plasmids not present in the cells of the patient, which means it will not propagate in the body like other viruses. The genetic information introduced by AAV particles thus eventually disappears, which gives physicians precise control over how much AAV is administered. For these reasons, AAV is driving today’s therapeutic discoveries and has become the dominant form of gene therapy for genetic diseases. At the same time, regulatory demands are increasing, as authorities request extensive analytical characterization for the drug vectors (e.g. capsid loading, process impurities). This requires reliable, robust, GMP-compliant analytical methods and characterization protocols, and specific analytical assays need to be performed to assess vector productivity, vector purity, biological activity and safety.
PPS offers a broad range of analytical methods for measuring the strength/dose of the AAV vector, quantifying full/empty particles and illegitimate encapsulated DNA, and ensuring the identity, purity, potency and safety of the final product. Verifying whether the gene of interest is actually present in the vector is essential for monitoring the production process as well as for evaluating the right dosage for the patient. PPS uses the following techniques to determine vector loading:
- Analytical ultra centrifugation (AUC)
- SEC-MALLS, AF4-MALLS
- cGE, IEX
Quality control is critical along the entire production pathway.
We have established specialized testing packages for the characterization and quality control testing of AAV particles and constituent capsid protein assemblies. PPS uses this comprehensive portfolio to support the development and implementation of cellular and gene therapy programs, from basic research, to discovery and safety testing, to clinical and CMC testing. We provide GMP-compliant CMC testing to ensure the identity, potency, purity and safety of starting materials, intermediate products, vectors and final drug products, as well as support for manufacturing process development and validation.
PPS analytical services for AAV based products
- Mass spectrometry
- Peptide mapping
- Intact molecular weight
- Glycan profiling of viral envelope proteins
- Biophysical characterization (higher order structure analysis)
- Analytical ultracentrifugation for determining the ratio of empty vs. full capsids (AUC/DLS)
- Stability characterization methods such as DSC and DLS
- Analytical Testing
- LC-MS studies on whole virus species to characterize the viral proteome
- Anion exchange HPLC (e.g. separation of empty capsids from intact viral particles)
- ELISA and RTqPCR methods for transgene expression
- Amino acid analysis
- N-terminal sequencing
- icIEF – charge heterogeneity
- Rapid screening of viral proteome fingerprinting by chromatography (HPLC), MALDI mass spectrometry and gel electrophoresis (SDS-PAGE). (MALDI is useful for proteins up to around 150 KDa.)
- Digestion of isolated proteins followed by LCMS and/or MALDI-MS to give detailed information