EXECUTIVE INTERVIEW - Precision NanoSystems Inc. (part of Cytiva) & SCIEX: Lipid Impurities Within mRNA-LNPs

New, exciting research shows a novel, developing multivalent flu vaccine against all 20 known influenzas (A and B virus subtypes/lineages). This could be a breakthrough as current flu vaccines only treat a few strains, and each year, it’s an educated guess for which flu strain will dominate. This prevalent research comes as a new class of impurities within mRNA drugs has been detected by advanced and highly specific analytical tests being conducted on existing mRNA-lipid nanoparticle (LNP)-based therapeutics and vaccines. These new impurities are typically undetectable by traditional mRNA purity analytical techniques based on gel electrophoresis. Given these findings, it is essential researchers and manufacturers have the tools and know-how to assess the full range of potential lipid impurities and differentiate them in order to ensure mRNA-LNP vaccines are effective and safe.

Drug Development & Delivery recently spoke with Kerstin Pohl, Senior Manager of Cell & Gene Therapy & Nucleic Acids at SCIEX, and Scott Ripley, General Manager of Nucleic Acid Therapeutics at Cytiva, to dive into the detection of these impurities and the impact it has on manufacturing.

Q: Could you tell me more about how new classes of impurities within vaccines development have been detected?

Scott Ripley: Although LNP (Lipid Nanoparticle) technologies have existed since the late 90s, the success of the two COVID-19 mRNA vaccines and subsequent rapid expansion of the LNP delivery technology have resulted in the evaluation of countless new lipid compositions and LNP designs. Therein, ionizable lipids, the enabling technology for LNPs, and proprietary PEGylated lipids have radically changed in design and complexity, yielding new classes of impurities in mRNA-LNP vaccines. Such impurities are increasingly detected as more detailed and sensitive analytical technologies, such as high-resolution mass spectrometry (HRMS), capillary electrophoresis (CE), and ion pairing high-performance liquid chromatography (IP-HPLC), are applied within the LNP field. Consequently, LNP drug manufacturers and regulatory agencies are increasingly expecting careful assessment for impurities.

Q: What would the consequences be for the effectiveness and safety of mRNA-LNP vaccines if researchers and manufacturers don’t understand or have the tools to assess and characterize potential impurities and address them?

Kerstin Pohl: In general, there are three main risks resulting from a failure to detect LNP impurities: changes in LNP structure and manufacturability, reduction of vaccine potency, and direct immunologic or toxicologic concerns. In the former, low lipid purity or mRNA purity equates to changes in the LNP composition, affording unstable or un-manufacturable drug product. In the second case, there is a growing acknowledgement specific lipid impurities directly react with the mRNA payload, thereby inactivating the vaccine. In the latter, incomplete or ineffective purification of lipids and mRNA can result in unexpected synthetic reagents and compounds making their way into LNP vaccines. These species can potentially elicit a range of immunogenic and toxicological effects.

Q: What is the future for advanced analytical tests in enabling the safe and effective development of innovative healthcare technologies and vaccines? How can they ensure the next flu vaccine, COVID-19 booster, and mRNA therapies are effective?

Scott Ripley: In contrast to the revolution in technologies enabling robust and high-capacity manufacturing of LNP-based drugs, such as Precision NanoSystem’s NanoAssemlr® platform, LNP analytics remains an adapting and evolving field. Fortunately, several key players in the lipid and LNP manufacturing space are recognizing the necessity of developing and adopting cutting-edge technologies and heavily investing in the area. Consequently, advanced analytics are being applied throughout the design, process development, and manufacturing stages of the next generation of LNP vaccines. The result will be threefold. Adoption of HRMS will yield higher potency due to the early identification of detrimental impurities. CE, IP-HPLC, and HRMS will enable unprecedented mRNA characterization affording lower batch-to-batch variability. Finally, field flow fractionation (FFF) – multi-angle laser light scattering (MALLS) systems will enable unparalleled analysis of intact LNP properties, such as size, polydispersity, mRNA loading, shape, and density, enabling a deep understanding of how composition impacts both manufacturability and potency. Overall, advanced analytics will ensure a faster and smoother path of LNP drugs from concept to the clinic.

Q: Where do these new lipid impurities come from, and how do they arise?

Kerstin Pohl: The new lipid impurity of key concern was first described by Dr. Meredith Packer and her team at Moderna in 2021 (Packer et al, 2021. Nat. Comm.). Therein, the mRNA payload in LNP was found to inactivate following the reaction with a lipid impurity resulting from the degradation of an ionizable lipid. Subsequently, the industry has discovered that in fact, all ionizable lipids will undergo a similar reaction, albeit at a wide range of rates. Mechanistically, impurities are likely caused by the oxidation of tertiary amine to N-oxide species.

Q: In your experience, how commonly do these impurities arise in LNPs for mRNA-LNP vaccines

Scott Ripley: The significant growth and diversity of applications for mRNA-LNP vaccines have resulted in a wide range of ionizable lipid structures whose susceptibility to oxidation varies significantly. Furthermore, some reagents employed in the synthesis of ionizables lipids may elevate N-oxide levels. Therefore, differences in vendors, manufacturing, storage, and other parameters can radically, and unpredictably, impact the N-oxide levels. In general, N-oxide levels above 1% abundance will result in the complete inactivation of an mRNA vaccine; however, even trace amounts can be problematic. Consequently, careful quantitation of N-oxide content in all ionizable lipid lots is recommended.

Q: What are the three key things manufacturers and researchers should do to optimize mRNA-LNP vaccine design and development, and stringent manufacturing controls to manage these impurities and ensure mRNA stability?

Kerstin Pohl: Three approaches to minimize the risk of mRNA adducts are (1) adopt careful analytics on incoming raw materials, (2) take a Quality by Design (QbD) approach to mRNA adducts in manufacturing processes to limit conditions by which N-oxides form and react, and (3) carefully monitor the mRNA-LNP vaccine for signs of adduct formation. In the former, the use of state-of-the-art HPLC-HRMS, such as the ZenoTOF 7600 system (SCIEX), by both the lipid vendor and LNP manufacturer, enabling the differentiation of problematic N-oxides from other impurities and the determination of levels, is recommended. In the second, limiting exposure to oxygen and heat as well as storing material under cryogenic conditions will limit N-oxide formation. In the latter, monitoring for signs of adduct formation on the mRNA payload by IP-HPLC or leveraging robust in vitro potency assays to identify inexplicable drops in potency will afford early recognition and hopefully illuminate the causative agent. Overall, analytics should be a key consideration throughout the LNP vaccine manufacturing process to mitigate these mRNA adducts as well as countless other challenges.