(T0930-04-26) A New Class of Lipids Conjugated to Poly(ethylene glycol) Structural Isomers: An Efficient Approach to Evade Immune Recognition of Lipid Nanoparticles

Purpose: Lipid structures conjugated to poly(ethylene glycol) (PEG) are an important class of excipients. They are used in lipid nanoparticle formulations for vaccination against Covid-19 [1]. A major concern of PEG is the immune response of the patient’s body that can lead to the formation of anti-PEG antibodies (APAs). APAs can cause the loss of the functionality of the drug and can lead to side effects [2]. Copolymers of PEG and glycidyl methyl ether (GME) are a class of polymers that are structural isomers to PEG (Figure 1). The polymers can be tailored to exhibit similar physicochemical characteristics compared to PEG homopolymers. The randomized PEG (rPEG) structure was designed to impede the interaction with APAs [3, 4]. Conjugation of rPEG copolymers to lipid anchors leads to a new class of lipids. rPEG lipids promise the same functionality as known PEG lipids without their immunogenic potential.

Methods: rPEG copolymers were synthesized by anionic ring opening polymerization (AROP) and characterized by nuclear magnetic resonance (NMR), size exclusion chromatography (SEC), liquid chromatography (HPLC), and mass spectrometry (MALDI-TOF MS). rPEG copolymers were conjugated to rPEG lipids and characterized by NMR and HPLC. Cell-compatibility of the rPEG lipids was quantified in vitro by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Interaction of rPEG lipids with APAs was quantified by competitive enzyme-linked immunosorbent assay (ELISA). rPEG lipids were used in lipid nanoparticle (LNP) formulations. Size and zeta-potential of the lipid nanoparticles were characterized by dynamic light scattering (DLS) and laser Doppler anemometry. Loading of the lipid nanoparticle with mRNA was confirmed by agarose gel electrophoresis (AGE) and RiboGreen assay. Transfection efficiency of the rPEG-lipid formulation was quantified by luciferase assay in different cell lines

Results: Four copolymers from EO and GME with systematically altered comonomer ratio and molecular weight were synthesized. Molecular weight as determined by SEC and MALDI-TOF is in line with expectation from stoichiometry. Dispersity (D) as determined by SEC is lower than 1.15 confirming controlled AROP. rPEG polymers were conjugated to rPEG lipids. Purity as determined by HPLC was higher than 95% for all compounds. Cell viability for rPEG lipids as determined by MTS assay revealed viability on the same level than the PEG lipid references. ELISA assay confirmed significantly reduced interaction of rPEG lipids with APAs compared to their PEG lipid counterparts. Use of rPEG lipids in LNP formulations resulted in particle diameters between 100 and 140 nm. It was observed that the degree of polymerization is an important parameter for the particle size of the LNP: rPEG lipids with similar degree of polymerization than PEG 2k resulted in particles with similar characteristics as the PEG lipid reference formulation (Figure 2). AGE and RiboGreen assays revealed no significant differences between the rPEG lipid formulations and the PEG lipid reference formulations. Quantification of transfection efficiency by luciferase assay in different cell lines revealed transfection efficiencies in the same range than the PEG lipid reference formulations (Figure 3).

Conclusion: A new class of rPEG lipids with reduced immunogenic potential was established. Use of rPEG lipids in LNP formulations revealed a structure-function relationship. It was found that for a given comonomer ratio the degree of polymerization is key for controlling the functionality of the LNPs. The chemical structure was adjusted to obtain rPEG lipids with desired characteristics: significantly reduced APA binding, non-toxicity, and identical behavior in LNP formulations compared to the reference materials. The new class of rPEG lipids enable new drug products with reduced immunogenic potential and less side effects.

References: [1] L. Schoenmaker et al., mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability, International Journal of Pharmaceutics 2021, 601, 120586.
[2] Y. Ju et al.: Anti-PEG Antibodies Boosted in Humans by SARS-CoV-2 Lipid Nanoparticle mRNA Vaccine, ACS Nano 2022, 16(8), 11769.
[3] J. T. Huckaby et al.: Structure of an anti-PEG antibody reveals an open ring that captures highly flexible PEG polymers, Communications Chemistry 2020, 3(1), 124.
[4] T. Nguyen et al.: Structural determination of an antibody that specifically recognizes polyethylene glycol with a terminal methoxy group, Communications Chemistry 2022, 5, 88.

Chemical structures of the structural isomers PEG and rPEG

Characterization of LNPs by DLS and laser Doppler anemometry. Reference LNPs containing PEG lipids (R) were directly compared to their rPEG lipid containing counterparts (1 - 4). Experiments were carried out in triplicate and values are presented as mean values ± standard deviation.

LNP in vitro transfection efficiency. Reference LNPs containing PEG lipids (R) were directly compared to their rPEG lipid containing counterparts (1 - 4). Experiments were carried out in triplicate and values are presented as mean values ± standard deviation.