Because of the versatility of mass spectrometry and the already established analytical workflow for protein bio-products, high accuracy mass spectrometry methods have been developed and applied to characterisation of viral vectors, their purity determination, and the characterisation of genetic material. Furthermore, several studies are on-going to demonstrate the utility of “omics” methods for cell characterisation ⁹ and innovative technology such as charge detection mass spectrometry (CDMS) for industrial applications. ¹⁰ 

Adeno-associated virus (AAV) and Lentiviral vectors are the most commonly used viral vectors for cell and gene therapy^11,12. AAV are a family of non-enveloped parvoviruses that are non-pathogenic, replication-defective and package a single stranded viral DNA. They have emerged as the most popular gene transfer vehicle for in-vivo gene therapy, largely owing to their high infectivity and low-pathogenicity.

Lentiviral vectors (LVs) are potent tools for the delivery of genes of interest into mammalian cells and are commonly utilised for the treatment of monogenic diseases and adoptive therapies such as chimeric antigen T-cell (CAR-T) therapy. Lentiviral vectors are engineered to modify the viral genome to remove its pathogenic properties, while retaining the essential elements necessary for efficient gene transfer. The viral genes, responsible for replication and pathogenicity, are replaced with the therapeutic gene of interest, making lentiviral vectors safe and useful vehicles for gene delivery. 

Viral Vector Characterisation

AAV-based drug characterisation poses significant challenges owning to its size, structural complexity arising from the viral capsid and packaged viral genome. The AAV capsid comprises 60 subunits of a combination of 3VPs (VP1, VP2 and VP3). The relative ratio of VP1:VP2:VP3 stoichiometry is considered to be crucial to the potency, infectivity, and transduction efficiency of the vector as well as the VPs amino acid sequence and post-translational modifications such as glycosylation, phosphorylation, acetylation and amino deamidation. 
Mass spectrometry workflows developed for bio-products represent already accepted and unique analytical solutions for confirmation of the sequence of the VP proteins and determination and location of post-translational modifications.

Capsid characterisation is a regulatory requirement for both FDA and EMEA and the uptake of this technology in the sector is expected to increase exponentially as the number of products increases with more stringent regulatory requirements¹³. The capability to effectively characterise VPs by LC-MS is also crucial for knowledge-driven improvements of AAV products, such as mixed serotype capsids that can outperform treatments using single serotype AAVs¹⁴.

Native mass spectrometry has shown potential to be used for characterisation of intact AAV as an orthogonal method to size exclusion chromatography-multi angle light scattering (SEC-MALS), analytical ultracentrifugation (AUC) and electron microscopy (EM).13 While translational research is required before its implementation in industry, advantages in using MS include low sample requirements, rapid analysis and the capability to distinguish between sub-populations of full, partially full and empty capsid. 

Challenges in the development of HCP methods for viral vectors include (i) differences between alternative vector types and in some cases presence of helper viruses, (ii) the host cell expression system, (iii) different upstream and downstream processes and sequences, and (iv) potential for interaction of HCPs with the viral vector, genome or incorporation/encapsulation into such vectors. 15 Mass spectrometry studies for HCPs in viral vector products are still in their infancy. Nevertheless LC-MS has already been successfully utilised in comparative studies and to aid to process optimisation16.

Nucleic acids

Therapeutic oligonucleotides (OGNs) have emerged as therapeutics with the ability to modulate gene expression precisely and efficiently. As a new class of therapeutics, these molecules are under active development, including antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), aptamers and microRNAs (miRNAs). Among those, single-strand ASOs and double-stranded siRNAs are the most advanced OGN therapeutics. Modifications of the backbone and sugars have been introduced to increase in-vivo stability against nucleases, as well for therapeutic purposes. Mass spectrometry based methods coupled with ion pair chromatography have been extensively applied in both characterisation of oligonucleotides (identity, purity)17 and bioanalysis 18.

More recently, large RNA including mRNA has emerged as a new class of therapeutics. Two highly efficacious vaccines, based on mRNA sequences encoding, were recently approved for a modified version of the SARS-CoV-2 spike protein. While mRNA characterisation and purity assessment are a regulatory requirement, available analytical methods to characterize large RNA (>1000 nucleotides) are limited and their development is challenging due to size and poor stability. Determination of the purity of process impurities, 5’ capping status and 3’ poly(A) tail is also mandatory for regulatory purposes.

Next generation sequencing (NGS) and Sanger have historically been employed for identity confirmation of nucleic acids. Their availability in industry is, however, still limited, if compared with the demand. Mass spectrometry based sequencing approaches are parallelly rapidly expanding providing several advantages over NGS or Sanger that include simultaneous analysis of several modifications, relative stoichiometric information, and increased speed of analysis19.  Given the complexity of nucleotide MS fragmentation and the size of mRNA molecules, the favourite route for analysis of mRNA by MS is a bottom‐up approach based on the use of enzymes such as RNase T1 or RNase A20.

These mimic the proteolytic approach applied to peptide mapping of bio-products and has the potential to substitute NGS and Sanger measurements in the field. However, the application of mRNA LC-MS bottom-up workflows is still limited by the availability of reliable bioinformatic tools that to facilitate data analysis and increase throughput. However, as instruments and bio-information solutions become available, it is expected that mRNA sequencing by MS will have a vital role in mRNA characterisation as peptide mapping for protein identity.

Mass spectrometry coupled with ion pair chromatography methods have also been developed and are broadly utilised for 5’ capping status and the 3’ poly(A) tail determination 21. 

Conclusions

Mass spectrometry is a fast-evolving analytical platform technology commonly applied for protein bio-product chemical and structural elucidation. The rapid growth of cell and gene therapy, the complexity of those molecules and the versatility of mass spectrometry have increasingly prompted academia, industry, and regulators to look at mass spectrometric methods as potential solutions.

The uptake of MS based workflows in the sector, such as for AAV characterisation and HCP determination, has been facilitated by the knowledge from the protein biopharma sector.  However the limited availability of data represents a bottleneck in maximising the outcome of these investigations. The industrial sector could also benefit from the development of improved bioinformatics for mRNA identity by LC-MS. 

Finally, translational research applied to advanced applications such as CDMS, omics, native mass spectrometry and MAM approaches for gene therapy has the potential to significantly advance the analytics in the sector and process/product knowledge. 

European Biopharmaceutical Review, Summer 2024, pages 65-68. © Samedan Ltd

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