As monoclonal antibodies (mAbs) have grown into a leading therapeutic class over the past thirty years, the pharmaceutical industry has had to contend with the structural and chemical complexity of recombinant proteins, which are approximately three orders of magnitude larger than small molecules. A battery of analytical assays to ensure mAb quality and safety has since become standard practice, with each assay targeting a different critical quality attributes.
In contrast, Multi-Attribute Method (MAM) is a single assay that is used to address one or more quality attributes. First proposed by Rogers et al1, MAM typically consists of a bottom-up or middle-down liquid-chromatography-mass spectrometry (LC-MS) technique designed to fully characterize the sequence or intact mass and chemical modifications of recombinant proteins. A single MAM analysis can be used for product identity, glycan profiling, charge heterogeneity determination, as well as surveying for the presence of impurities or sequence variants. While developing a comprehensive MAM analysis is challenging, the time and cost savings can be considerable. With increasingly mature data analysis software, cheaper and more widely available high-resolution mass spectrometers (e.g. Waters BioAccord system) and increasing evidence of their reproducibility2, MAM techniques have entered mainstream acceptance.
Advanced therapy medicinal products (ATMPs) represent a leap in complexity over mAbs as they consist of modalities such as viral vectors, tissues and cells. Adeno-associated viruses (AAVs), a leading class of viral vectors, are over an order of magnitude larger than mAbs with masses approaching 5 MDa. AAVs have far more complex structures and compositions than mAbs, comprising (i) a protein capsid with 60 copies of three different but homologous proteins, and (ii) an encapsulated single-stranded DNA payload of at least 3000 nucleotides.
This complexity increases the analytical burden required to ensure the quality and safety of AAV products. As with all proteins, AAV capsid proteins often undergo posttranslational modifications (PTMs) (e.g. phosphorylation, oxidation, deamidation and truncations), some of which are known to influence AAV infectivity3. In addition, the stoichiometry of the three different types of capsid proteins, commonly known as VP 1 – 3, also influences AAV infectivity4. There is also considerable variability in the nucleic acid payload, with many viral particles containing truncated payloads, packaged host cell genomic DNA, or being completely empty. Such empty or partially filled capsids are viewed as unwanted contaminants that need to be quantified and eliminated.
Developing a bottom-up LC-MS/MS-based MAM for AAV analysis requires careful consideration. AAV preparations often have low titers because their production hosts (often HEK293 cells) are not yet fully optimized. Hence, careful sample preparation is required to ensure unbiased results and acceptable signal intensities. In addition, certain peptide sequences, e.g. VP1 and 2 N-termini, are present at 10-fold lower abundance relative to total capsid proteins5, hence these regions tend to be poorly covered. To mitigate these pitfalls, sample denaturation, alkylation, digestion, and cleanup can be automated to improve the consistency and quality of peptide digests. Nanoflow liquid chromatography (Nanoflow-LC) is also particularly useful and can be implemented to increase peptide coverage. Nanoflow-LC utilizes low flow rates to increase the concentration of analytes in eluting peaks, enhancing ionization efficiency and improving the detection of low abundance peptides such as those from VP1 and VP2.
Middle-down LC-MS approaches address denatured, intact AAV capsid proteins, and have proven useful as capsid proteins are small enough (~70kDa) for PTMs such as oxidation and phosphorylation to be well-resolved using the latest high-resolution Q-TOF and Orbitrap mass spectrometers6. The main challenge in this approach is the chromatographic separation of the highly homologous VP1-3 proteins as insufficient separation compromises sensitivity, resolution, and accurate estimations of capsid protein stoichiometry. Studies have found that mobile phase composition is key to achieving this, with strong eluting solvents (e.g. isopropyl alcohol) and low levels of modifiers such as trifluoroacetic acid being essential for good peak shape7. Reversed phase columns are often be screened on a case-by-case basis for each AAV serotype, and columns with altered selectivity (e.g. diphenyl chemistry) can sometimes facilitate the resolution of minor sequence variants and truncated forms of AAV capsid proteins.
Multi-dimensional liquid chromatography can be used to increase the analytical power of middle-down LC-MS. Zhijie Wu et al.8 used 2D-LC-MS with anion exchange chromatography in the first dimension to resolve full and empty capsids, followed by reversed-phase LC-MS in the second dimension to assess the masses of intact viral capsid proteins. Based on an Agilent 1290 system, this efficient approach automatically transferred fractions between dimensions, increasing peak capacity and reduced sample loss as compared to an offline fraction collection. This enabled quantification of full/empty capsid ratio, as well as capsid protein stoichiometries, PTMs and truncations specific to the full capsid fraction in a single assay and may prove highly useful for in-process testing.
Finally, with advancements in mass spectrometry, top-down LC-MS can be implemented despite the considerable mass of AAV virions. Large biological molecules such as AAV virions pick up a high, variable number of charges when ionized in an electrospray, causing the sample’s mass spectrum to be compressed into a charge envelope that can be difficult or impossible to deconvolute using conventional high-resolution mass spectrometers. Charge detection mass spectrometry (CDMS) overcomes this problem by measuring the charge state of each ion based on the induced charge as each ion passes through a metal tube9. With this information, the mass spectrum of the sample can be deconvoluted on a per-ion basis, enabling very large structures such as plasmids, genomes or whole virions to be measured.
Two recent papers by Tobias P. Worner et al.10 and Lauren F. Barnes et al.11 show powerful applications of CDMS to distinguish full, partially filled, and empty capsids, and to quantify payload truncations. One of the main advantages of a top-down approach is that sample complexity retained, in contrast to middle-down or bottom-up approaches which inevitably destroy information during denaturation or digestion. In addition, little to no sample preparation is required in a top-down approach, minimizing the likelihood of artifacts.
The future of MAM for viral vectors looks bright, with new technologies such as CDMS and continuing improvements in LC-MS technology promising to improve analytical capabilities further. Improvements to analytical methods will allow for a better understanding of product quality, opening the doors to refining manufacturing methods and better determination of critical quality attributes. Establishing strong analytical assays early reduces the discovery of issues with identity and purity during late-phase process development or at the point of technological transfer to GMP manufacturing. These mistakes result in failed runs and repeat development cycles, increasing the time to market. In a world where more life-saving cures are being discovered, it is key to ensure high-quality drugs with well-defined properties are found in the clinic.
Thanks for reading our article! Stay tuned for the next article in our series of thought leadership pieces which address cost reduction and efficiency improvements in the manufacturing of cell, nucleic acid and gene therapy products.
- Rogers, R. S. et al. Development of a quantitative mass spectrometry multi-attribute method for characterization, quality control testing and disposition of biologics. in MAbs vol. 7 881–890 (Taylor & Francis, 2015).
- Millán-Martín, S. et al. Inter-laboratory study of an optimised peptide mapping workflow using automated trypsin digestion for monitoring monoclonal antibody product quality attributes. Analytical and Bioanalytical Chemistry 412, 6833–6848 (2020).
- Zhong, L. et al. Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression. Virology 381, 194–202 (2008).
- Popa-Wagner, R. et al. Impact of VP1-Specific Protein Sequence Motifs on Adeno-Associated Virus Type 2 Intracellular Trafficking and Nuclear Entry. Journal of Virology 86, 9163–9174 (2012).
- Wörner, T. P. et al. Adeno-associated virus capsid assembly is divergent and stochastic. Nat Commun 12, 1642 (2021).
- Hale, W., Colangelo, C., Hegedus, R. & Garceau, N. Characterization of Viral Vector Particles Using the Agilent 6545XT AdvanceBio LC/Q-TOF. Agilent Technologies 5994–1980EN, (2020).
- Liau, B. LC/MS of Intact Adeno-Associated Virus Capsid Proteins for Rapid Confirmation of Product Identity. Agilent Technologies 5994–2434EN, (2021).
- Wu, Z., Wang, H., Tustian, A., Qiu, H. & Li, N. Development of a Two-Dimensional Liquid Chromatography-Mass Spectrometry Platform for Simultaneous Multi-Attribute Characterization of Adeno-Associated Viruses. Anal. Chem. 94, 3219–3226 (2022).
- Keifer, D. Z., Pierson, E. E. & Jarrold, M. F. Charge detection mass spectrometry: weighing heavier things. Analyst 142, 1654–1671 (2017).
- Wörner, T. P., Snijder, J., Friese, O., Powers, T. & Heck, A. J. R. Assessment of genome packaging in AAVs using Orbitrap-based charge-detection mass spectrometry. Molecular Therapy – Methods & Clinical Development 24, 40–47 (2022).
- Barnes, L. F. et al. Analysis of AAV-Extracted DNA by Charge Detection Mass Spectrometry Reveals Genome Truncations. Anal. Chem. 95, 4310–4316 (2023).