The evolution of mass spectrometry for analysis of bio-products

BY DR MILENA QUAGLIA | SENIOR ASSOCIATE PRINCIPAL SCIENTIST FOR BIOLOGICAL SCIENCES

28th May 2024

 

 

Advancements in technologies and processes like mass spectrometry offer new ways for researchers to engage with new biologicals - but where does their future potential lie? In this recent article for European Biopharmaceutical Review Dr Milena Quaglia attempts to answer this question.

 

Analytical platform technology is racing to keep pace with the increasing complexity of biologicals. Mass spectrometry (MS) has had a pivotal role in characterisation of conventional biotherapeutics such as monoclonal antibodies, and more recently MS data positively contributed to the improved understanding of products and processes in cell and gene therapy.


Hereby, an overview on the advances in mass spectrometry to improve analytical workflows for regulatory approval of protein biopharmaceuticals will be discussed together with the key role that this technology will play in monitoring the critical quality attributes of cell and gene therapy products and the requirements for innovation in the field. 

 

Mass Spectrometry for Protein Biopharmaceuticals

 

Analytical methods incorporating liquid chromatography - mass spectrometry (LC-MS) played a pivotal role in advancing knowledge on protein biopharmaceuticals in discovery, development, regulatory approval, batch release and comparative studies.

 

Currently, mass spectrometry is considered an essential tool for characterisation of protein-based biopharmaceuticals because of the depth of information obtained, its versatility and accuracy. This is demonstrated by the inclusion of MS based data in almost all biologics license applications lately granted 1,2 

Intact protein analysis by LC-MS and peptide mapping by tandem mass spectrometry (LC-MSMS), confirmation of sulphur bridges and identification and location of post-translational modifications are today considered critical methods to fulfil the requirements for identity and chemical characterisation of protein bio-products as dictated by the ICH (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use) Q6B Specifications: test procedures and acceptance criteria for biotechnological/biological products.

 

Mass spectrometry also has a significant role in the determination of the purity of protein bio-products. This includes verification of co-eluting peaks and identification of product and process related impurities. The advances in chromatographic technology compatible with MS, including size exclusion chromatography and ion exchange chromatography, together with the advances in MS technology (increased accuracy, resolution, and dynamic range) have undoubtedly facilitated the widespread utilisation of MS for purity assessment purposes. Advances in mass spectrometry technology and bioinformatics have also enabled the development of MS based workflows for monitoring host cell proteins.

 

HCPs are residual proteins from the bio-product’s host cells and are recognised as a major class of process related impurities. A perceived potential concern around residual HCPs is their ability to induce an immune response and to impact product quality, activity and excipient stability.3 Guidelines around the monitoring and reporting of residual amounts of HCPs in a product are provided by regulatory authorities (FDA and EMEA) and ICH. Generally, Enzyme Linked Immunosorbent Assay (ELISA), gel-based methods combined with immunoblotting and mass spectrometry are the most used methods for monitoring and quantifying HCPs. Mass spectrometry is considered a laborious, but extremely powerful and orthogonal tool that enables the user to build an understanding of HCP profiles and clearance patterns throughout the process, identify potential problematic HCPs, monitor their adequate removal, and facilitate risk assessment.

 

The development of new methods for HCP by LC-MS is also faster and cheaper than the development of new immune-base assays. The integration of MS HCP quantitation with in-silico prediction of immunogenicity risk for specific HCPs by identifying ‘foreign’ epitopes in prevalent HCPs is of interest and has the potential to significantly progress the area 4. The recognition of the role of MS in monitoring HCPs in bio-products has been recently shown by the recently updated USP chapter 1132 which now includes quantification and identification of HCP by MS.

 

Over the past decade mass spectrometry has also been accepted in industry and by regulators as a valuable analytical platform for higher order structural analysis of protein bio-products, and an orthogonal method to the conventional circular dichroism (CD), Fourier Transform Infrared- Spectroscopy (FTIR), differential scanning calorimetry (DSC) and Nuclear Magnetic Resonance (NMR). Hydrogen deuterium exchange mass spectrometry (HDX-MS), for example, has the unique advantage to provide an insight of protein structural changes at amino acidic level (HDX-MS) with no limitation in protein size and with low sample consumption.5 This provides an important insight in optimisation of formulation and protein engineering strategies as well as supporting bio-similarity studies. 6

 

The availability of commercially available solutions for data analysis and automatised systems, rendered HDX-MS popular also in the development of screening workflows for the selection of lead monoclonal antibodies, development of vaccines and characterisation and optimisation of the binding site of antibodies vs target. Ion mobility spectrometry-mass spectrometry (IMS-MS) has also been applied to studies of protein bio-pharmaceuticals to elucidate the global structural changes of a protein in a native state or by monitoring its unfolding process. 7 The compatibility of IM-MS with existing MS workflows makes it an attractive tool for implementation in controlled industry settings, but the requirements of standardisation of data analysis solutions are delaying its up-take. 

 


In 2015 an analytical approach referred to as the multi-attribute method (MAM) has gained considerable interest in the biopharmaceutical industry. The multi-attribute method (MAM) is a liquid chromatography−mass spectrometry-based method that is used to directly characterise and monitor multiple product quality attributes and impurities of biotherapeutics, most commonly at the peptide level. It utilises high-resolution accurate mass spectral data which are analysed in an automated fashion and can be implemented in a Good Manufacturing Practice environment. It is believed that MAM has the potential to replace or supplement several conventional assays with a single LC-MS analysis. 8

 

Mass spectrometry and cell and gene therapy products

 

Cell and gene therapy is an exciting area of bio-pharmaceuticals that offers the potential to treat, prevent, or cure diseases for which there is currently no other therapeutic option – for example, rare diseases and cancers. Cell therapies involve altering cells outside of the body so that they can restore normal function when introduced back into the patient. Gene therapy, on the other hand, involves changing or restoring the genetic function of a cell by introducing new genetic material, typically delivered by a vector that targets specific patient cells in-vivo.

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 therapy11,12. 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. 

 

Viral Vector Impurities

 

Residual impurities from viral vector manufacturing must be controlled to ensure product safety and quality. These include residual host cell proteins, empty capsids, extraneous nucleic acid sequences and process-related impurities such as residual reagents used during manufacturing.

 

MS based methods have been developed and validated for the analysis of residual reagents and other species that are leachable from columns or materials used during the production and purification of viral vectors. Furthermore, in parallel to protein bio-products, mass spectrometry may be a key analytical tool for HCP determination in viral vector products.

 

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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