A Novel Mass Spectrometry Paradigm to Analyze Classical and New Biotherapeutic Modalities

LCGC SupplementsCurrent Trends in Mass Spectrometry
Volume 21
Issue 1
Pages: 17–20

Next-generation biotherapeutics are on the rise to address diseases in new and promising ways, and gene-based vaccines offer fast development timelines for combating future pandemics rapidly. At the same time, classical biologics (such as monoclonal antibodies [mAbs]) and new protein modalities continue to be developed to combat diseases such as cancer and chronic inflammatory disease. However, these new biopharmaceutical modalities are introducing unique analytical challenges in the detection and characterization of impurities in lipid nanoparticles (LNPs) for gene therapy and gene-based vaccine products, host-cell proteins (HCPs) in viral vector-based therapies, and post-translational modifications (PTMs) in protein therapeutics. These irregularities could impair the safety and efficacy of novel therapeutics. A new fragmentation approach—tunable electron-activated dissociation (EAD) tandem mass spectrometry (MS/MS)—offers a solution. The tunable energy enables detailed characterization, in an intuitive manner, of lipid impurities, glycopeptides, and other PTMs.

The promise of next-generation biotherapeutics to address a wide range of diseases is borne out in the sheer size of the cell and gene therapy (CGT) pipeline. As of the start of 2023, there were more than 2000 gene therapies and modified cell therapies, such as chimeric antigen receptor T-cell (CAR-T) treatments, in the development pipeline, along with another 800+ non-genetically modified cell therapies (1). In addition, new protein modalities— such as bispecific monoclonal antibodies, bispecific T-cell engagers (BiTES), peptibodies, and nanobodies—are showing promise for their ability to bind to multiple therapeutic targets simultaneously (2).

These new technologies, however, introduce a host of challenges. The lipid nanoparticles (LNPs) used to encapsulate CGTs and messenger RNA (mRNA) vaccines can contain lipid impurities that threaten the stability and safety of the final products (3). Viral vectors used as delivery vehicles can contain host-cell proteins (HCPs), which are proteins expressed by the packaging cells that can alter a therapy’s mechanism of action (4). Next-generation protein therapeutics, such as dimeric fusion proteins, add a whole new layer of complexity in analyzing impurities and post-translational modifications (PTMs), which can affect the stability, safety, and efficacy of protein therapeutics (5).

Traditional analysis methods, such as enzyme-linked immunosorbent assays (ELISAs), are limited in their sensitivity, coverage, and ability to identify impurities, and they can be time-consuming (6). The mass spectrometry (MS) technique collision-induced dissociation (CID) is more effective at detecting a range of impurities, but scientists often need complementary technologies to fully understand which impurities are present and the risks they present.

Alternative fragmentation techniques, such as tunable electron-activated dissociation (EAD), can complete the picture by filling in the information missed by CID fragmentation methods. EAD provides sophisticated detection and analysis of lipid impurities, HCPs, and PTMs in biotherapeutics. EAD is a novel MS dissociation technique that uses tunable electron energy. It automatically adjusts electron energies to suit the molecules that are being analyzed, whether they are singly charged lipids or multiply charged peptides. It is reagent-free, and can be modulated to optimize the fragmentation patterns and obtain a wide range of information. The advent of tunable EAD technology provides significantly increased sensitivity and speed of fragmentation over traditional electron-based fragmentation mechanisms (7). This ability to rapidly adjust the electron energy makes EAD compatible with fast, ultrahigh-pressure liquid chromatography (UHPLC), and allows flexibility for integration into many existing LC–MS workflows.

Managing HCP Impurities in Genetic Medicines and Other Protein Drugs

The importance of managing HCPs became evident during the development of Covid-19 vaccines. HCPs were associated with vaccine-induced thrombotic thrombocytopenia (VITT), a rare but severe side effect characterized by blood clotting in vital organs. VITT was seen primarily in patients who received adenoviral-based vaccines. Researchers who investigated the vaccine components concluded that adenoviral protein impurities may have stimulated the production of antibodies that attack platelet factor 4 (PF4), triggering coagulation (8).

Standard procedures for HCP analysis rely on ELISAs, which are effective in HCP quantification, but have several limitations. They can be cumbersome to develop, and so they have limited use for fast-tracked new modalities such as viral vector-based vaccines. Moreover, they often can fail to detect weakly immunogenic proteins, or changes in HCP composition.

Researchers are increasingly turning to MS for HCP analysis. MS platforms are highly flexible, and they are becoming more user friendly with minimal method development needed. More importantly, emerging MS technologies, such as EAD, provide significant gains in completing the characterization of new modalities, and can achieve more comprehensive characterization of HCPs.

Even more depth can be obtained by integrating sequential window acquisition of all theoretical mass spectra (SWATH-MS) with data independent acquisition (DIA). This technique allows researchers to identify and quantify all detectable analytes in a particular sample, assisting with identification of potential HCPs that were not known to be present. Furthermore, SWATH-MS with DIA can be used for any biotherapeutic, including complex gene therapies, and it can detect HCPs from different organisms simultaneously (6).

Managing Lipid Impurities in LNPs

Ionizable lipids are the main components of LNPs that form LNP-based therapeutics and vaccines because they have optimal chemical properties for complexing the genetic cargo and delivering it to the cell. However, ionizable lipids are susceptible to chemical alterations—predominantly site-specific oxidation that can cause the formation of reactive lipid species, generating mRNA-lipid adducts (3). Even a tiny amount of oxidized lipids can prevent the mRNA from functioning properly in triggering the expression of the encoded protein that creates the desired immune response (9). Early identification of these lipid impurities is essential to managing development timelines. Once researchers identify lipid impurities, they can perform additional purification or modify the synthesis process to mitigate the risks.

The risks of ionizable lipids in LNPs came to light during recent developments. One study of an mRNA product showed that a reactive impurity based on one of the lipid components in the vaccine was covalently binding the mRNA and altering its function. The researchers discovered abnormalities that occurred only in the presence of ionizable lipids, which turned out to be a cross-linking between the lipid and the mRNA itself that formed lipid adducts, likely caused by the oxidation of tertiary amine to N-oxide species, according to the study (10).

Oxidized lipids and lipid adducts are missed by traditional assays used for analyzing the purity of LNP-based therapeutics, demonstrating a major analytical challenge. While nuclear magnetic resonance (NMR) spectroscopy can be used to determine lipid molecular structure, it is expensive, and requires large quantities of pure sample, making it unsuitable for detecting low levels of impurities.

MS can be used to elucidate lipid structures via fragmentation with CID, which is efficient for breaking different types of fragile bonds. But this technique cannot reliably differentiate between oxidations occurring at different parts of the lipid. That is a problem for analyzing lipids used for mRNA encapsulation, as some species can cause the loss of mRNA function through adducts, while others do not. Therefore, there is a need for MS methods with sufficient elucidation power and dynamic range to distinguish between different types of oxidation in lipids at very low levels.

MS platforms with alternative fragmentation, such as EAD, can be used as an orthogonal analytical strategy, providing more rich fragmentation spectra and increased fragment coverage to elucidate structural information for complete lipid characterization (7). EAD technology has proven amply capable in clearly identifying and quantifying oxidized lipids attributed to a loss of mRNA function (9). The increased dynamic range and added sensitivity of EAD enables the detection of impurities at very low abundance, while at the same time offering better coverage of bond breakage, allowing researchers to identify the exact oxidation site. EAD comprehensively fragments lipids along the headgroup and fatty acid chains, enabling differentiation among several oxidized lipids.

For in-depth analysis of complex biological mixtures, EAD can deliver new information that can clarify molecular structures (7). In a recent study, EAD was used to characterize the structures of the ionizable lipids MC3 and ALC-0315 and related impurities present at low levels. The EAD data was so comprehensive it could be used to distinguish structural isomers of two MC3 oxidative impurities and determine the specific sites of oxygen incorporation (11).

Analyzing PTMs in New Protein Modalities

PTMs such as glycosylation and glycation in protein therapeutics are important to detect and manage because they can affect stability, safety and efficacy. Thus, the comprehensive characterization of N- and O-linked glycosylation and identification of advanced glycation end products (AGEs) in antibody-based therapeutics are essential for ensuring drug safety and efficacy (12,13).

Identifying and characterizing such PTMs is challenging, largely because of the time required for full glycan analysis and characterization. Collision-based tandem mass spectrometry (MS/MS) approaches, such as CID, when applied to glycopeptides, can cause a loss of labile glycan moieties, making it difficult to determine glycosylation sites, particularly for O-glycosylation (12). In addition, collision-based MS/MS approaches are ineffective for fragmenting AGEs, complicating the challenge of understanding the relationship between AGE content and product quality attributes such as color change (13).

EAD can be used along with CID to address these challenges in managing PTMs. EAD promotes superior glycopeptide analysis, given its ability to preserve the glycan structures in the fragments. Because labile glycans can be retained in the EAD fragments, accurate localization of glycosylation sites and differentiation of positional isomers of O-glycopeptides can be achieved (12).

The promise of this approach has been demonstrated with etanercept, which is a dimeric fusion protein consisting of two tumor necrosis factor receptor (TNFR)-Fc chains with three N-glycosylation and 13 O-glycosylation sites on each chain. High-quality EAD data elucidated the complex O-glycosylation profile on the peptide level and enabled the localization of O-glycopeptides containing up to seven O-glycans, and positional isomers of O-glycopeptides were differentiated in a nearly complete series of sequence ions (12).

In the case of glycation, EAD allows for the fragmentation of AGEs, which enables sequence identification, localization of moieties, and differentiation of positional isomers (12). These advantages enabled accurate quantification of AGEs in a study of time-course forced glycation samples of NISTmAb—a reference standard monoclonal antibody (mAb)—that was designed to exam- ine the cause of color change in a sample.

In this work, NISTmAb was thermally stressed in the absence or presence of glucose for up to 10 days. The samples incubated at an elevated temperature in the presence of glucose developed discoloration at day seven, demonstrating that the discoloration of protein therapeutics can be reproduced in the laboratory under harsh stress conditions, and enabling investigation of the cause of discoloration. The data revealed a correlation between color change and the relative abundance of AGEs, but not glycation (13).

The speed by which EAD can characterize PTMs is also notable. A recent study of bovine fetuin O-linked glycosylation used UHPLC and fast EAD to fully characterize 57 O-glycopeptides, and partially characterize another 22 O-glycopeptides. EAD reaction times could be as short as 10 to 30 milliseconds, without any loss of resolution. (7).


With nearly 3000 CGTs in the biopharma pipeline, and robust development of complex next-generation protein therapeutics, technologies that can improve and streamline the detection of impurities are in high demand. MS techniques, including CID, are effective in detecting and characterizing HCPs, PTMs, and impurities in LNPs, but they often cannot provide a full picture of which abnormalities are present and how much of a risk they pose.

EAD is a complementary MS technology that can fill in the gaps and help researchers better detect and characterize abnormalities. This new fragmentation approach uses tunable electron energy that enables both large and small molecule applications.

The ability to rapidly adjust electron energy makes EAD compatible with fast UHPLC, enabling the rapid analysis of complex compounds of varying abundances, molecular weights and chemistries. For detecting HCPs in viral vectors, MS with SWATH DIA improves the depth of comprehensive data analysis. Because EAD offers an increased dynamic range that enables the detection and characterization of lipid impurities at very low abundance, it can be used to detect and analyze ionizable lipids and lipid adducts. Finally, it can improve the identification and characterization of PTMs such as glycosylation and glycation in protein therapeutics to help determine their effect on product stability and safety.

As this technology continues to evolve, it will bring the benefits of electron-based fragmentation for structural elucidation and quantitative assays to more researchers, enhancing and accelerating the development of next-generation medicines.


(1) American Society of Cell + Gene Therapy, Citeline, Gene, Cell & RNA Therapy Landscape. American Society of Cell + Gene Therapy, 2022. https://asgct.org/global/documents/asgct_citeline-q4-2022-report_final. aspx (accessed 2022-03-13).

(2) Mollah, S. Frankenbodies: How (Mad) Scientists Are Bringing New Treatments to Life. Eur. Pharm. Manuf. March 3, 2021. https:// pharmaceuticalmanufacturer.media/ pharmaceutical-industry-insights/biopharma-news/frankenbodies-how-mad-scientists-are-bringing-new-treatments/ (accessed 2022-03-13).

(3) Packer, M.; Gyawali, D.; Yerabolu, R.; Schariter, J.; White, P. A Novel Mechanism for the Loss of mRNA Activity in Lipid Nanoparticle Delivery Systems. Nat. Commun. 2021, 12, 6777. DOI: 10.1038/s41467-021-26926-0

(4) Pohl, K.; Kofoed, T. Q&A: LC-MS to Reinvent Impurity Analysis in Gene-Based Drugs. Eur. Pharm. Manuf. February 7, 2023. https://pharmaceuticalmanufacturer.media/pharmaceutical-industry-insights/biopharma-news/qa-lc-ms-to-reinvent-impurity-analysis-in-gene-based-drugs/ (accessed 2022-03-13).

(5) Zhang, P.; Woen, S.; Wang, T.; Liau, B.; Zhao, S.; Chen, C.; Yang, Y.; Song, Z.; Wormald, M.; Yu, C.; Rudd, P. Challenges of Glycosylation Analysis and Control: An Integrated Approach to Producing Optimal and Consistent Therapeutic Drugs. Drug Discov. Today 2016, 21 (5), 740‒765. DOI: 10.1016/j.drudis.2016.01.006

(6) Krishnan, M.; Kofoed, T. In Conversation: A Smarter Way to Eliminate Host Cell Protein Contamination from Biologic Drugs. Eur. Pharm. Manuf. October 2, 2020. https://pharmaceuticalmanufacturer.media/pharmaceutical-industry-insights/in-conversation-a-smarter-way-to-eliminate-host-cell- protein/ (accessed 2022-03-14).

(7) Electron Activated Dissociation: A New Paradigm for Mass Spectrometry. SCIEX, 2021. https://sciex.com/content/dam/SCIEX/pdf/ brochures/zenotof-7600-whitepaper-ead.pdf (accessed 2022-03-14).

(8) Pohl, K. Ensuring the Purity of Gene Therapy Products and Vaccines. Genet. Eng. Biotechnol. News. December 7, 2022. https://www.genengnews.com/topics/genome-editing/gene-therapy/ensuring-the-purity-of-gene-therapy-products-and-vaccines/ (accessed 2022-03-14).

(9) Crowe, A.; Pohl, K. Overcoming Oxidation. The Analytical Scientist 2022. https://theanalyticalscientist.com/techniques-tools/overcoming-oxidation (accessed 2022-03-14).

(10) Pohl, K. Lipid Impurities in mRNA: Implications and Solutions. Technology Networks. https://www.technologynetworks.com/vaccines/blog/lipid-impurities-in-mrna-implications-and-solutions-369025 (accessed 2022-03-14).

(11) Distinguishing Oxidative Impurities from Ionizable Lipids Used in LNP Formulations Using Electron Activated Dissociation, SCIEX, 2022. https://sciex.com/tech-notes/biopharma/distinguishing-oxidative-impurities-from-ionizable-lipids-used-i (accessed 2022-03-14).

(12) Comprehensive Characterization of O-linked Glycosylation in Etanercept by Electron Activated Dissociation (EAD). SCIEX, 2022. https://sciex.com/tech-notes/biopharma/comprehensive-characterization-of-o-linked-glycosylation-in-etan (accessed 2022-03-14).

(13) Time-Course Study of Glycation and Advanced Glycation End Products (AGEs) in Protein Therapeutics Using Electron Activated Dissociation (EAD). SCIEX, 2022. https://sciex.com/tech-notes/biopharma/time-course-study-of-glycation-and-advanced-glycation-end-produc (accessed 2022-03-14).

Roxana McCloskey is Senior Global Marketing Manager of Protein Therapeutics at SCIEX. Kerstin Pohl is Senior Global Marketing Manager of Cell and Gene Therapy at SCIEX. Direct correspondence to: roxana.mccloskey@sciex.com

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