(T1430-05-26) Lipid Nanoparticle Impurity Analysis: Leveraging Mass Spectrometry to Monitor Impurities and Aldehyde in Raw Materials and Formulated Drugs in Regulated Environments

Purpose: Lipid nanoparticles (LNPs) are a multi-component biologic comprised of lipid species with varying properties impacting chromatography and detector response; they require stringent quality control to ensure raw materials are free of critical impurities that could be carried over to drug products and adversely impact their safety and efficacy. The purpose of this study was to evaluate the performance of a single quadrupole mass spectrometer in a reversed phase liquid chromatography mass spectrometry (RPLC-MS) workflow in terms of specificity and sensitivity to monitor reactive impurities such as aldehydes in LNP raw materials and their carry-over to formulated drug products.

Methods: Ionizable lipid ALC-0315 from vendors and empty/surrogate RNA-containing assembled LNPs were dissolved in methanol. 2,4-Dinitrophenylhydrazine (DNPH) solution was prepared in acetonitrile. Aldehyde derivatization was performed at DNPH and ALC-0315 molar ratio 13:1. Stressed studies were performed with hydrochloric acid, sodium hydroxide, and hydrogen peroxide. The samples were analyzed via a UV detector, a charged aerosol detector, and a single quadrupole MS.

Results: A single quadrupole mass detector offers an increase of up to 2 orders of magnitude in dynamic range and 3 orders of magnitude in sensitivity in the analysis of impurities associated with LNPs compared to conventional optical detectors. This was achieved by selectively filtering mass data to remove ambient solvent ions ≤ 240 m/z and ions related to the native compound. Taking ionizable lipid ALC-0315 as an example, the LOD was reduced from 8% to 0.04%, by removing ions in the chromatogram that represent the ALC-0315 species and its associated sodium adduct form (Figure 1). This combined process effectively provides a lipid impurity profile and enables detection of impurities below 0.05% in accordance with reporting threshold in USP < 1086 >. One reactive impurity identified in ionizable lipid ALC-0315 is an aldehyde (6-oxohexyl 2-hexyldecanoate). This aldehyde, which may be a process/product related impurity, can be derivatized using DNPH to enhance ionization efficiency for detection in MS. It is critical to monitor and control this reactive impurity to minimize its potential carry-over into formulated LNP products where it can react with mRNA payloads and form adducts that can reduce the efficacy of the mRNA. Empty LNPs (without mRNA) manufactured using ALC-0315, cholesterol, PEGylated-lipid, and distearoylphosphatidylcholine (DSPC) was derivatized with DNPH and its aldehyde content was analyzed. As shown in Figure 2, residual aldehyde was detected in the empty LNPs following DNPH derivatization. The derivatized aldehyde peak was only observed in the control containing the ionizable lipid when testing individual LNP components. These observations not only demonstrate that potential for synthesis related impurities such as aldehydes to be “carried-over” into the manufactured LNP products, but that they can be monitored in LNP raw materials and potentially formulated products through derivatization using the proposed LC-MS platform. To assess the risk of this aldehyde may pose to drug products, formulated LNPs were assembled with and without surrogate mRNA and incubated at room temperature over a 7-day period using ALC-0315 from three different vendors. Formulated samples were monitored for changes related to aldehyde content and LNP-adduct formation. As shown in Figure 3A, aldehyde content was observed to marginally increase over the 7-day stress study indicating a degree of short-term stability for the ionizable lipid in liquid formulation in the absence of mRNA. In contrast, as shown in Figure 3B, approximately a 40% reduction in aldehyde content was observed for each LNP containing the surrogate mRNA over the same 7-day period suggesting the surrogate mRNA was reacting with the aldehyde species. As shown in Figure 3C, the % mRNA-adduct peak for each sample was observed to increase over a 7-day time period. More importantly, the relative amount of mRNA adducts observed exhibited a positive correlation to aldehyde content with vendor C exhibiting the highest amount of mRNA-adduct followed by vendor B and A.

Conclusion: The proposed RPLC-MS platform consistently exhibited an impurity detection limit at 0.04% of the main species which increase users’ ability to detect or quantify critical impurities that could adversely impact drug product safety. For mRNA-vaccines, the oxidation of the ionizable lipid is of particular interest given its potential to produce a reactive aldehyde species that can form an adduct with the mRNA payload and reduce drug efficacy. The effectiveness of the proposed RPLC-MS platform to detect aldehydes was demonstrated with the detection of 6-oxohexyl 2-hexyldecanoate in raw material of an ionizable lipid (ALC-0315) following derivatization with DNPH. Evidence of “carry-over” of critical impurities such as aldehydes and the potential for adduct formation in formulated LNPs further illustrates the value MS displays in monitoring quality indicating species as part of synthesis, raw material, or formulation and stability studies related to LNPs. Collectively, the findings observed in this study demonstrate the utility of the proposed QC-friendly MS platform as a method to identify and monitor stability indicating attributes of LNP components for improved drug product quality.

Acknowledgements: Duanduan Han, Robert Birdsall, and Erin McAllister are employees of Waters Corporation.