abstract

Abstract

In this technical note, the ZenoTOF 7600 system was used to sensitively quantitate the ionizable lipid ALC-0315 and structurally elucidate its related impurities from lipid nanoparticles (LNPs) in plasma. Due to its inherent sensitivity, a triple quadrupole mass spectrometer (TQMS) is typically used for targeted bioanalytical quantitation. However, collision-induced dissociation (CID)-based lipid fragmentation is insufficient for their complete structural characterization. The ZenoTOF 7600 system is capable of sensitive, targeted quantitation and can utilize a complimentary fragmentation mode (electron-activated dissociation; EAD), which is effective for confident metabolic identification (MetID) (1-3) and the structural elucidation of lipids (4-9), thereby adding unique value for analytical versatility.

To demonstrate the quantitative performance of the ZenoTOF 7600 system for bioanalysis of ALC-0315, quantitation curves with lower limits of quantitation (LLOQs) were determined using either a precursor-to-fragment or precursor-to-precursor high-resolution MRM (MRM^HR), and the amount of ALC-0315 from formulated LNPs diluted into plasma was then estimated. Impurities related to ALC-0315 at relative abundances as low as 0.02% were observed from the LNPs and some of the structures of these impurities were elucidated using EAD (Figure 1).

Figure 1. Versatility of the ZenoTOF 7600 system to support quantitation and characterization workflows for analysis of the ionizable lipid ALC-0315 from LNPs. Upper right panel: Representative peak areas of the LLOQ of ALC-0315 diluted into plasma from precursor-to-fragment MRMHR (m/z 766.72 to 510.49) or precursor-to-precursor MRMHR (766.72 to 510.49). The precursor-to-precursor MRM provided the greatest sensitivity. Bottom right panel: Identification of the N-oxide impurity of ALC-0315 from LNPs diluted in plasma. Three distinct fragment ions from EAD of m/z 187.16, 443.40, and 709.66 not observed by CID were used to isolate the site of oxygen incorporation to the tertiary amine to confirm N-oxidation.

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results1

Key features of ionizable lipid analysis on the ZenoTOF 7600 system

• Through MRM^HR acquisition, the ZenoTOF 7600 system can detect and quantitate ionizable lipids at sub ng/mL levels from formulated LNPs
• The simultaneous acquisition of full product ion spectra during quantitative analysis allows verification of other components within LNPs, such as the PEGylated lipid and structures related to the ionizable lipid
• EAD coupled with the Zeno trap allows for confident structural elucidation of low-level impurities associated with the ionizable lipid, demonstrating its usefulness in supporting MetID studies

introduction

Introduction

LNPs have emerged as safe and effective delivery vehicles for oligonucleotide-based therapeutics such as the siRNA-based Onpattro as well as messenger RNA (mRNA)-based vaccines such as Spikevax and Comirnaty (10). This has spawned investment into new precision genetic medicines by designing novel lipids to fine-tune properties like targeted delivery, biodegradation, and endosomal escape. LNPs typically comprise ionizable lipids, helper lipids such as distearoylphosphatidylcholine (DSPC), cholesterol, and PEGylated lipids (Figure 1). For example, the LNPs used in the Pfizer/BioNTech COVID-19 vaccine contained ALC-0315, DSPC, cholesterol, and PEG2000 conjugated lipid.

With the administration of novel exogenous materials, like the ionizable lipids used in LNPs, it is necessary to track their biodistribution and metabolism products to assess their safety and efficacy profiles. TQMSs are used for quantification studies to support bioanalytical assays using an MRM approach. However, some lipids may not fragment well by traditional CID fragmentation techniques nor produce diagnostic product ions to distinguish them from other interfering ions within biological matrices. In addition, it can be challenging to identify the structures of compounds formed from degradation or metabolism using a CID-based fragmentation alone. It has been shown that EAD-based fragmentation can produce information-rich spectra that can confidently elucidate structures of ionizable lipids and pinpoint sites of modifications within their related impurity structures (11-13).

In this study, we assessed the ability of the ZenoTOF 7600 system to quantify ALC-0315 in plasma. This instrument includes a Zeno trap device that enhances MS/MS sensitivity to drive quantitative sensitivity. In addition, the instrument can provide complementary fragmentation via EAD to aid in structural characterization. Both aspects of the instrument were used to quantify and provide in-depth characterization of impurities related to ALC-0315 to demonstrate its utility in supporting LNP metabolism studies.

results2

Materials and methods

Materials: ALC-0315 was purchased from Broadpharm (Cat #BP-25498) or Caymen Chemical (Cat #34337). DSPC (Cat #850365), cholesterol (CAT # 700100), and 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (PEG2000-PE) (CAT# 880150) were from Avanti Polar Lipids. The lipid components were diluted in EtOH before formulation. The nucleic acids, gWiz-GFP plasmid DNA (pDNA), and DasherGFP mRNA were from Aldevron. The nucleic acids were prepared in citrate-buffered saline at pH 4.25. All solvents were LCMS grade and obtained from Burdick and Jackson. Human control plasma was from SCIEX.

LNP Formulation: To produce LNPs, lipids were mixed at a molar ratio of ALC-0315Cholesterol: PEG2000-lipid 46.3:9.4:42.7:1.6. LNPs were formulated with a NanoAssemblr Ignite microfluidic device from Cytiva using a 10 mL/min flow rate and a 3:1 mixing ratio of aqueous citrate buffer, which contains the nucleic acid, to EtOH, which contains the lipid mixture. Following formulation, LNPs were diluted with phosphate-buffered saline and a buffer exchange was performed using 100kDa molecular weight cut-off ultracentrifugal filters.

Sample preparation: ALC-0315 and LNPs were diluted into plasma. Samples were extracted by protein precipitation by adding 100% ACN, followed by benchtop centrifugation for 10 minutes. Supernatants were collected, and 2 mL were injected into the LC column for LC-MS/MS analyses.

Chromatography: Extracts were analyzed on a ZenoTOF 7600 system using an Exion UHPLC. Samples were separated with a Peptide BEH C18 column (150 x 2.1 mm; 1.7 µm, 130 A, Waters, P/N:186003687). Chromatographic gradient details are shown in Table 1. The autosampler and column oven temperatures were 15 °C and 70 °C, respectively. The mobile phase flow rate was 0.5 mL/min, and the total run time was 27 min. Mobile phases A and B consisted of 10 mM ammonium acetate in water/MeOH/ACN (15/30/55; v/v) and 10 mM ammonium acetate in ACN/MeOH (60/40; v/v), respectively

Table 1. Chromatographic gradient (flow rate = 500 µL/min)

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Mass spectrometry: All analyses were performed using the ZenoTOF 7600 system with an OptiFlow Turbo V ion source. Data for the ALC-0315 standard curve and the quantitation of ALC-0315 from formulated LNPs were acquired with TOF-MS and highresolution MRM (MRM^HR) using either precursor-to-precursor (m/z 766.7 to 766.7) or CID-based precursor-to-fragment (m/z 766.7 to 510.5) transitions. (Instrument parameters are shown in Table 2.) The external calibration curves were derived from three separate injections for each concentration. The amount of ALC-0315 from LNPs was estimated from these curves. Impurities related to ALC-0315 from LNPs were identified from a data-dependent acquisition (DDA) experiment, termed IDA, after initial dilution into MeOH Table 1. Chromatographic gradient (flow rate = 500 µL/min) (Table 3). An MRMHR target list using CID- or EAD-based fragmentation for these impurities was then incorporated into an MRM^HR method for impurity analysis of ALC-0315 after diluting the LNPs into plasma (Table 4).

Table 2: ZenoTOF 7600 system parameters for quantitation by MRMHR

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Table 3: ZenoTOF 7600 system parameter settings for IDA

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Data processing: Quantitation data were processed using the Analytic module within SCIEX OS software. The Explorer module of SCIEX OS performed structural characterization of ALC-0315-related impurities from EAD and CID fragment ion spectra.

results3

Results and discussion

Figure 2. Fragmentation of ALC-0315 by CID. Selected fragment ions at high relative abundance were overlayed on the structure of ALC-0315. Due to the inefficiency of CID-based fragmentation, a precursor-to-precursor MRM transition was used to maximize sensitivity. Note the lack of diagnostic fragments generated using CID, as shown in the chemical structure.

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Figure 3. ALC-0315 interferences from plasma matrix. TOF MS analysis of ALC-0315 diluted into plasma shows the presence of ions with similar molecular weights to ALC-0315. The highlight blue box shows a surrounding 0.7 m/z window to represent a typical quadrupole mass analyzer isolation window containing these major interfering ions. This demonstrates the ability of the TOF mass analyzer to provide added selectivity.

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Quantitation in biological matrices by LC-MS/MS traditionally relies upon the specificity of precursor-to-fragment MRM experiments using a TQMS. ALC-0315 does not fragment efficiently by CID even after optimizing instrument parameters (Figure 2); consequently, it was investigated whether the specificity from HRMS could improve quantitative analysis. For example, the TOF MS spectrum of ALC-0315 diluted into plasma showed an interference from a prominent ion in the matrix observed at m/z 766.650, which is close to the precursor ion of ALC-0315 observed at m/z 766.728 (Figure 3). The high-resolution mass analyzer on the ZenoTOF 7600 system can distinguish this signal, which otherwise would interfere with a TQMS system. The ZenoTOF 7600 system offers high sensitivity MS/MS via the Zeno trap, low detector noise compared to other HRMS systems, and the ability to analyze ions within a narrow extraction window, and we explored whether these attributes could provide specific benefits from different acquisition modes. These included TOF MS to monitor the ALC-0315 precursor, MRM^HR of precursor-to-precursor ions, and MRM^HR monitoring the 510.486 m/z fragment ion. Figure 4 shows a dilution curve spanning 4 orders of linearity with high accuracies from the precursor-to-precursor MRM^HR experiment. This experiment yielded greater sensitivity with a 0.4 ng/mL LLOQ, which was 5x lower than precursor-to-fragment MRM^HR (Figure 1).

Figure 4. Quantitative calibration curve of ALC-0315 in plasma using precursor-to-precursor MRMHR transitions. A quantitation curve (top) was created from various dilutions of ALC-0315 in plasma across over 4 orders of linear dynamic range. The table (bottom) displays quantitative statistics demonstrating the quantitative performance of the ZenoTOF 7600 system

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Figure 5. Quantitation of ALC-0315 from LNPs diluted into plasma. Three different preparations of LNPs formulated with or without genetic material were diluted 1:6000 into plasma. The amounts of ALC-0315 from the LNPs were determined using the calibration curve equations from Figure 4.

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After establishing the quantitative methodology for ALC-0315, we applied this to formulated LNPs. LNPs were diluted in methanol and subjected to LC-MS analysis for initial assessment. All components of the LNPs were observed, including ALC-0315 and the PEGylated lipid (Figure 6). This indicated that a bioanalytical method could be developed for other exogenous materials like the PEGylated lipid in addition to the ionizable lipid using this methodology.

The amounts of ALC-0315 from three different preparations of LNPs diluted into plasma were measured to assess the quantitative performance of the ZenoTOF 7600 system. The LNPs were initially formulated with mRNA, pDNA, or they were empty. The peak areas of the ALC-0315 detected from these LNP preparations diluted 1:6000 are shown in Figure 5. The concentrations of ALC-0315 from 3 LNP preparations were calculated as 0.33, 0.23, and 0.22 mg/mL, based on the external calibration curve from Figure 4. These measurements estimated the original stock concentrations to range from 1.32 to 1.98 mg/mL by extrapolation. This is consistent with the amount of ALC-0315 initially used in the LNP formulation, allowing for some moderate sample loss, where 20-50% lipid loss from filtration (e.g., dialysis) is typical (14-15). A dilution of 1:6000 was 1000x higher than the LLOQ, demonstrating the sensitivity of the ZenoTOF 7600 system. Overall, this method is suitable for measuring trace amounts of material for thorough and sensitive biodistribution assessments.

Figure 6. TOF MS analysis of LNPs. TOF MS of LNPs using the ZenoTOF 7600 system was able to detect all the lipid components of the LNPs, including several low-level impurities related to ALC-0315

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During the initial analysis of the LNPs, we also observed several low-level impurities (Figure 6, inset with zoomed x and y axes). These analytes were noted and analyzed to assess the utility of the ZenoTOF 7600 system in supporting MetID studies using EAD. Previously, using positive ion mode LC-MS/MS, ALC-0315 was shown to undergo biotransformation via hydrolysis to m/z 528.4986 (16). We also observed a peak at m/z 528.4999 from the impurity profile. From the other impurities found, chemical formulae were deduced based on precursor masses listed in Table 4.

Table 4. Precursor ions and their masses from impurity profile experiment (Figure 6)

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The ZenoTOF 7600 system can perform low-energy electron impact fragmentation (electron impact excitation of ions from organics; EIEIO) using the EAD cell. EIEIO is especially useful for elucidating lipid structures because it generates significantly more fragment ions than other fragmentation techniques like CID (Figures 2 and 7) (8,9,11, 12). This technique was previously used to elucidate structures related to ALC-0315 (12) confidently.

Figure 7. Fragmentation of ALC-315 by EAD. Compared to the CID spectrum, EAD produces product ion spectra that denote more structural detail and can be used to better characterize this type of compound.

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EIEIO-based fragmentation was used on selected impurities of ALC-0315 after LNP dilution into plasma. Unique fragments of m/z 187.156, 443.397, and 709.656 were observed for an oxidative impurity of m/z 781.716 by EIEIO, which were not seen by CID. (Figure 1, lower right). These fragments were used to pinpoint oxygen incorporation to a specific, confined region surrounding the tertiary amine within the structure of ALC-0315, strongly suggesting that this impurity was the N-oxide of ALC-0315.

A more complex unknown impurity at m/z 610.540 was also observed. This molecular weight is consistent with the loss of one of the hexane-6,1-diyl bis(2-hexyldecanoate) (C₂₂H₄₄O₂) chains, and the addition of an unknown covalent addition with a molecular weight of 185.153 (Figure 8A). This molecular weight could be explained by two different chemical formulae based solely on MS accurate mass – C₉H₁₉N_3O or C₁₁H₂₁O₂, as shown in Figure 8B. Fragmentation by EAD resulted in a unique fragment of m/z 553.494 not seen by CID, which was 58.053 mass units less than the precursor molecular weight (Figure 8C). This loss could only be explained by a nitrogen-containing chemical formula of C₂H₆N₂ due to accurate mass measurement and the mass defect of molecular nitrogen. In addition, fragment ions matching losses relating to the chemical formulae of C₈H₁₉N₂ and C₉H₂₁N₂ were also observed in the EAD fragment ion spectrum. As a result, the adducted chemical formula was narrowed down to C₉H₁₉N₃O. Although it is difficult to derive the exact structure of this adduct, these data show that EAD provides valuable information that is not attainable by accurate mass measurements or by CID alone. This information could help corroborate the elucidation of other unknown lipid-adducted species from oligonucleotide or protein modifications.

Figure 8. Deduction of unknown chemical adduct to an ALC-0315 impurity from plasma using EAD. A unique impurity with a precursor m/z 610.54 was observed. Initial interpretation from a fragment ion spectrum revealed a loss of one of the hexane-6,1-diyl) bis(2-hexyldecanoate) chains of ALC-0315 and the addition of an unknown adduct of 185.153 MW (A), which could be represented by two different chemical formulae based on accurate mass (B). (C) Fragmentation by EAD uniquely produced a m/z 553.495 fragment ion that could only result from loss of a nitrogen containing moiety - C6H6N2. By process of elimination, the unknown adduct formula was determined to be C9H19N3O.

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The ZenoTOF 7600 system provides tremendous versatility to support in-depth and sensitive LC-MS/MS-based DMPK studies. The resolution, sensitivity, and dynamic range enabled a quantitation curve of ALC-0315 in plasma of over 4 orders of linearity with an LLOQ of 0.4 ng/mL using a precursor-to-precursor MRM transition. This experiment overcame the CID-based fragmentation inefficiency of ALC-0315 that would be encountered on a TQMS system while still providing the necessary specificity to support bioanalysis from the plasma matrix. In addition, EAD fragmentation provides definitive structural information on lipid impurities from LNPs. This information could help identify process-related impurities formed during LNP manufacturing and formulation or biotransformations to support MetID studies.

conclusion

Conclusion

• The sensitivity of the ZenoTOF 7600 system enables the low-level detection and quantitation of lipids related to LNPs in plasma to support quantitative bioanalysis in complex matrix
• Fragmentation of ionizable lipids and their related impurities using EAD results in information-rich product ion spectra even at low abundance after dilution into plasma
• Unique fragment ions of impurities related to ALC-0315 from EAD can be used to confidently identify these analytes and pinpoint sites of modification, demonstrating that this technique can support and benefit metabolism studies
• Sensitive quantitation paired with detailed structural characterization shows the versatility of the ZenoTOF 7600 system to support multiple workflows within DMPK

references

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