The emergence of Advanced Therapy Medicinal Products (ATMPs), including cell and gene therapy medicinal products (GTMPs), promises significant progress in the treatment, mitigation and potential cure of many diseases, including genetic diseases and cancer.
Gene therapy products aim to cure a disease by replacing or fixing a faulty gene or a mutated gene, or delivering a new gene where it is missing. However, these rapidly developing technologies are complex and very different to traditional biopharmaceutical products, which brings specific structural characterization challenges.
In the field of Gene Therapy, recombinant adeno-associated viruses (rAAVs), initially discovered as a contaminant of adenovirus preparations, are the most widely used delivery vehicle for therapeutic administration of transgenes due to their high transduction efficiency, high levels of gene expression and favorable safety profile.
As AAV capsid proteins are critical for viral infectivity and the AAV vector potency, complete characterization of the constituent Virus Particles (VPs) of AAV vectors, including their sequences and post-translational modifications (PTMs), is highly recommended. For example, it has been shown that deamidation of a number of Asparagine residues can have a significant impact on transduction activity and can also be an early factor in terms of loss of vector activity. In depth structural characterization of viral proteins is therefore essential to ensure AAV product quality and consistency.
AAV Gene Therapy Product Regulations
Specific regulatory guidelines have been developed outlining the analytical testing required to characterize GTMPs throughout the development process. These regulations take into consideration starting materials, intermediates, drug substance and drug product, with a clear requirement that “all components” are to be characterized.
By their very nature, AAV vectors are composed of different biochemical elements (namely protein and DNA components). It follows that viral capsid proteins need to be structurally characterized including determination of capsid loading, and process impurities such as Host Cell Proteins.
Regulatory guidelines recommend that primary, secondary and higher-order protein structure and post-translational modifications are assessed using “a range of orthogonal state-of-the-art techniques” including:
- Physicochemical properties – refractive index, particle/molecular size average and distribution, aggregation
- Viral protein identification, quantitation (total protein content), molecular weight and amino acid sequencing
- Viral protein peptide mapping, including identification of PTMs and sites of glycosylation
- Impurities, both product and process-related including HCP analysis
Characterization of Recombinant Adeno-Associated Virus Gene Therapy Products (rAAVs)
BioPharmaSpec offers specialized, custom designed testing packages for characterization of the Adeno-Associated Virus (AAV) particles and constituent capsid proteins. Our scientists use their extensive glycoprotein structural characterization experience to apply the below techniques to your gene therapy product.
Physicochemical Characterization Methods
Various chromatographic and electrophoretic techniques are used for identification and purity analysis of the viral proteins:
Mass Spectrometry Characterization Methods
A variety of mass spectrometric methods are used for protein ID, sequence confirmation, assessment of N- and C-termini, and assessment of post-translational modifications (including, amongst others, disulfide bridge and glycosylation) of the viral proteins:
- Intact molecular weight
- Confirmation of amino acid sequence using MS/MS and/or MSe
- Peptide mapping, including LC-MS analysis of PTMs
- Glycan profiling of viral envelope proteins (both N- and O-linked (if present))
- Quantitative monosaccharide and sialic acid analysis of glycans
- Protein ID from SDS gels
- Detection, characterization and quantitation of residual Host Cell Proteins and process- related impurities
Biophysical Characterization Methods (Higher Order Structure Analysis)
Zentriforce is BioPharmaSpec’s strategic partner in providing assessments of capsid loading, aggregate formation and particle size distribution for gene therapy products. Based in Heidelberg, Germany, Zentriforce offers a comprehensive range of analytical methods including:
- Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC)
- For quantification of empty, partially filled and full capsids (i.e. the ratio of DNA incorporation into the virus capsids). This method also provides information about sample purity regarding the amount of aggregates and fragments.
- Asymmetrical flow field flow fractionation (AF4)
- A suitable technique for sample purity determination in gene therapy products. The assessable size range ( 1 nm up to 1 µm) allows the quantification of small fragments as well as large aggregates within a single measurement.
- Dynamic Light Scattering (DLS)
- To test for aggregate formation, particle size distribution, particle titer as well as thermal stability.
Are you developing a gene therapy product?
Please contact our scientists today to discuss the most efficient characterization strategy for your AAV- based gene therapy product.