This article is sponsored by Inotiv. In this VIEWS article, Drug Discovery & Development spoke with Inotiv’s Matt Westfall, Senior Director for Proteomics, and Mike Heaven, Principal Scientist, to explore how sophisticated lipid nanoparticle (LNP) bioanalysis is revolutionizing RNA-based gene therapy.
Matt Westfall heads Inotiv’s proteomics group in Nashville. A long-time industry scientist and biochemist by trade, his team specializes in protein analysis using mass spectrometry. This encompasses two types: targeted proteomics, which he likens to an equivalent of an RT-PCR experiment for absolute quantification of specific proteins, and global proteomics, which offers a high-level view to examine broad protein pathway changes across different experimental conditions.
Mike Heaven, a principal scientist within Inotiv’s bioanalysis group in St. Louis, focuses on high-resolution mass spectrometry for LNP analysis. Having analyzed numerous large molecules, his lab partners with clients to understand their needs and desired assays. Through collaborations with clients, Inotiv has developed a comprehensive suite of assays for LNP characterization, quantitatively measuring every component of a lipid nanoparticle — from individual lipid levels and their ratios to the encapsulated functional RNA and its encapsulation efficiency.
Inotiv offers an all-encompassing approach from start to finish with complementary services across sites. Recognizing the need to measure both delivery mechanisms and efficacy, and noting the shift away from AAVs towards LNPs, Inotiv analytically qualifies the LNPs used as delivery systems. They also offer in vitro and in vivo model testing. Proteomic analyses can then quantify the delivered gene expression at the protein level, and measure efficacy via impact on protein pathways. This one-stop-shop aims to help companies progress from their initial therapeutic idea to an IND.
The Unique Value of LNP Characterization
Inotiv’s three broad high-resolution LCMS assays for formulation evaluation include a multiplexed assay for each lipid, cataloging nucleobase and ribose modifications, and measuring guide/Cas9 RNA ratios. For guide RNAs (oligonucleotides around 35 kilodaltons) and much larger functional RNAs (over a million daltons), they offer optimized assays. They also identify lipid and RNA impurities and determine the extent of RNA modifications, such as the percentage of uridines modified with N1-methylpseudouridine. Inotiv’s comprehensive, high-resolution data often reveal unanticipated information, which provides added value to the customer.
Westfall adds that Inotiv provides a manufacturing-level qualification of LNPs for biopharmaceutical companies, which has not been available previously. He also points out that one of the biggest issues in the field is the consistency of LNP manufacture, lipid composition, and RNA cargo.
“LNPs aren’t like most pharmaceuticals,” Heaven points out. “They’re typically a mixture of at least two RNAs and four lipids — sometimes more. So quality control is essential.”
Successful Applications and Translational Impact
It is important to ensure the quality of material entering model systems, Westfall emphasizes. Given that almost all gene therapies are targeted toward expression or modifying proteins, Inotiv’s ability to measure that protein and link it to the phenotypic outcome is crucial. Their platform can analyze various protein alterations, providing protein-level validation, not just sequencing results — confirming whether the gene therapy did what it was intended to do.
When examining editing efficiency or protein expression, “customers often don’t understand why one formulation performs better than another — even though they think they made them the same way,” Heaven says. Inotiv’s analysis reveals differences in LNP components, allowing them to quantify those components and help customers understand why one lot worked better than another.
Critical Factors in LNP Design
The lipid panel profoundly impacts how the LNP functions. Heaven emphasizes the need to consider both RNA encapsulation and the lipid’s toxicology profile, as the best in vitro formulation might not be the best for in vivo use. Inotiv helps customers evaluate multiple formulations and make informed decisions.
Westfall approaches it from the biology side. “What is your target tissue, organ or cell type? Can you get your LNP there? How will you determine delivery efficiency? How much do you need delivered?” He uses the example of hemophilia, where delivery to a very small percentage of hepatocytes in the liver to express a circulating protein might be enough, versus Duchenne muscular dystrophy, which potentially requires a much higher penetrance in muscle tissue. “Delivery is one of the biggest roadblocks in the industry,” he says.
Emerging Trends and the Future of LNPs
“LNPs have advantages over AAVs. They can carry larger payloads, provoke fewer immune reactions, allow for more precise targeting, and can be dosed multiple times,” Westfall says. As gene therapy evolves, the focus moves beyond just delivery to questions such as, “What’s the phenotypic outcome?” Inotiv utilizes global proteomics and spatial transcriptomics to study pathway alterations, identify biomarkers, understand mechanisms and identify both on-target and off-target effects using bioinformatics tools, which is crucial for safety evaluation.
“We also sequence the five-prime and three-prime ends of guide RNAs, which are often the most error-prone regions for random integration,” adds Heaven.
An Integrated Approach for Accelerated Development
Inotiv provides real-time actionable data from start to finish, qualifying the LNP composition and then using proteomics to confirm efficacy and quantify protein levels. “Very few platforms offer this full suite,” Westfall says, which allows them to tell clients, “You used a qualified lot, and we can confirm you made the right gene edit and it produced the desired result at the protein level.”
Spatial transcriptomics assesses tissue penetrance, while global proteomics examines on-target and off-target impacts, and the ability to measure protein-level outcomes is key, says Westfall, especially when antibody generation is challenging.
“The more we can partner with someone and support them end to end, the better,” Westfall says. “We position ourselves as a drug discovery partner to our clients, versus just a service provider.”
Visit Inotiv to learn more about Proteomics Services, and DMPK and PD.