Release date:2023/11/24 13:29:47

Polyethylene glycol (PEG) is a synthetic biocompatible polymer that is widely used in industries such as cosmetics, household cleaning products, chemical fibers, rubber, plastics, pesticides, metalworking and food processing. In addition, the attachment of PEGylated molecules to proteins, peptides, nucleic acids, nanocarriers or small molecules has become one of the most widely used technologies in the pharmaceutical industry for increasing the water solubility of drugs, providing protection for drug molecules or nanocarriers, and reducing immunogenicity.

As of 2023, the FDA and EMA have approved more than 40 PEGylated drugs, most of which are administered locally or systemically. The main functions of PEGs are to alter the physical properties of the drug (e.g., solubility), to improve pharmacokinetics (e.g., long-acting), and so on. When PEG-lipids are used in nanoparticle drugs (including liposome and lipid nanoparticle (LNP)-related drugs), PEGs also have the effect of preventing particle aggregation and stealthing the particles to avoid plasma clearance - however, it has been found that anti-PEG antibodies may invalidate this stealthing in clinical applications.

The two mRNA-lipid nanoparticle (LNP) COVID-19 vaccines that are currently widely used around the world both contain lower doses of PEG-lipids. The BioNTech-Pfizer vaccine, Comirnaty or BNT162b2, contains 50 μg of ALC-0159 per dose; the Moderna vaccine Spikevax or mRNA-1273, containing 117 μg of PEG2000-DMG per dose. Both ALC-0159 and PEG2000-DMG contain methoxy PEG (mPEG) with a molecular weight of approximately 2 kD. There have been a large number of reports on the side effects of these two COVID-19 vaccines, of which several mention that one of the most significant side effects, anaphylaxis, is likely to be associated with PEG-lipids. The immunogenicity of PEGs has become a serious problem for all PEGylated formulations, including PEGylated liposomes.


Figure 1. Structure of lipid nanoparticles (LNPs) and lipid components employed in Comirnaty & Spikevax .

Immunogenicity of PEGs

1. Healthy people who have never used PEGylated drugs also have anti-PEG antibodies in their bodies.

According to research studies[2], anti-PEG antibodies exist in approximately 25% of healthy blood donors. Even among patients without a history of treatment with PEGylated products, up to 42% have higher concentrations of anti-PEG antibodies. This phenomenon can be attributed to the frequent use of PEG-containing or PEG-coupled products in daily life, including personal care, beauty and household cleaning products (e.g., soaps, sunscreens, cosmetics), and commonly processed foods.

2. Anti-PEG antibody can accelerate blood clearance (ABC) of PEGylated drugs and impair the efficacy of PEGylated drugs.

Several studies have observed that when repeated injections of PEGylated drugs are given within a week, clearance is faster after the second dose compared to the first, a phenomenon that is attributed to the formation of anti-PEG antibodies at the time of the first dose administration. The initial dose of PEGylated liposomes initiates the immune system to produce anti-PEG IgM, which selectively interacts with the PEG molecules in the second dose of PEGylated liposomes, leading to complement activation and increased phagocytosis of the second dose of PEGylated liposomes by Kupffer cells in the liver. [3]

3. PEGylated drugs can be absorbed transdermally, thus generating anti-PEG antibodies in the human body.

It has been found that various conditions such as ulcerations, abrasions and skin tears may lead to localized inflammatory reactions and induction of immune responses in the presence of PEG. The penetration of PEG into the site of inflammation in contact with immune cells can further stimulate the production of anti-PEG antibodies. These pre-existing anti-PEG antibodies may compromise the therapeutic efficacy of PEGylated drugs.

Time- and dose-dependent induction of anti-PEG IgM & IgG by PEGylated LNP

Recently, a research paper entitled "Polyethylene glycol (PEG)-associated immune responses triggered by clinically relevant lipid nanoparticles in rats" was published in NPJ vaccines.[3] 

The team investigated the effect of LNP at different times and doses after administration on the induction of anti-PEG responses in a rat model, as represented by the LNP carrier of Pfizer Comirnaty®. The results showed that anti-PEG IgM was induced in a dose-dependent manner after the initial injection of LNP, and higher doses of PEG-LNP would induce longer-lasting and higher levels of anti-PEG IgM.

Interestingly, anti-PEG IgG was not induced even by an initial single injection of a larger dose of LNP, and repeated injections were required before anti-PEG IgG production was observed. These results demonstrate a dose-dependent induction of anti-PEG IgM and IgG in rats.

It has also been found that anti-PEG antibody, a non-thymus-dependent antigen, PEG-LNP induced isotype switching and immune memory, with rapid and longer-lasting anti-PEG IgM and IgG responses after repeated injections in rats. Two consecutive injections induced transient anti-PEG IgM production even at very low doses.

Most importantly, initial LNP injection accelerated blood clearance in rats with subsequent administration of the drug. After entering the circulation via intramuscular injection they preferentially accumulate in the reticuloendothelial system. Repeated injections of PEGylated LNPs resulted in their rapid clearance from the circulation through a phenomenon known as accelerated blood clearance (ABC).

Accelerated Blood Clearance (ABC) Phenomenon

The ABC phenomenon presents two distinct phases: the induction phase and the effectuation phase. [2]

In the induction phase, the biological system is primed, in a T-cell-independent manner, by promoting the proliferation and differentiation of specific B-cells (MZ-B, responsible for antibody production and cell-mediated immune response) in the splenic marginal zone , resulting in anti-PEG IgM production.

The subsequent effectuation phase is manifested from day 5 to day 21 after the initial dose, which closely correlates with the time course at which anti-PEG IgM is produced after the initial dose. Additional doses of PEGylated liposomes during the effectuation phase are rapidly processed by the complement component C3 fragments and cleared from the systemic circulation by Kupffer cells in coordination with anti-PEG IgM and the complement system (e.g., Figure 2).

​Once the PEG liposomes (first dose) reach the spleen, they bind to and cross-link surface immunoglobulins on reactive B cells in the marginal zone of the spleen, triggering the production of anti-PEG IgM independent of T cells. After administration of the second dose, if the anti-PEG IgM produced in response to the first dose is still present in the circulation, it binds to the PEG on the liposomes and subsequently activates the complement system, triggering a C3-conditioning effect that promotes enhanced uptake by Kupffer cells via complement receptor-mediated endocytosis.


Figure 2. Representation of the sequence of events leading from anti-PEG IgM induction to accelerated clearance of PEGylated liposomes. [2]

In addition, the magnitude of the ABC phenomenon was strongly correlated with the level of anti-PEG IgM production by splenic B-cells following an initial injection of a dose of PEGylated liposome. It was also found that although removal of the spleen from mice reduced the production of anti-PEG IgM, it failed to completely reverse the rapid clearance of PEGylated liposomes to normal control levels. This suggests that more than one serum factor or cell may be responsible for this phenomenon.

Factors affecting the ABC phenomenon against PEGylated liposomes

There are many factors affecting the ABC phenomenon, including animal species, lipid dose, injection interval, nanoparticle physicochemical properties, and type of encapsulated drug.

Variations in the sensitivity of the immune system due to species differences may contribute to the differences in the pharmacokinetics of PEG components in different species.The magnitude of the ABC phenomenon may also vary with animal species. For example, anti-PEG IgM generation and ABC phenomena were significantly increased in the minipig model compared to the mice model. Therefore, discussion of the ABC phenomenon induced by PEG-LNP in humans cannot be limited to animal model studies.

The fact that the ABC phenomenon was instead lower at higher PEG lipid doses may also be related to differences in pharmacokinetics in vivo in different species. It was found that higher initial doses of PEG lipids could lead to immune tolerance or inactivation of marginal zone B cells (MZ-B), resulting in slowed blood clearance.

In addition, since the production of anti-PEG IgM occurred 3-4 days after the initial administration, the ABC phenomenon was strongest when the interval between injections was 4-7 days. In contrast, clearance of the encapsulated drug was reduced only after an interval of more than 4 weeks between injections.

Complement (C) activation-related pseudoallergy (CARPA)

The interaction of the immune system with lipid nanoparticle therapies may result in a hypersensitivity reaction (HSR) or infusion reaction, uniformly referred to as complement activation-related pseudoallergy (CARPA). This phenomenon is also categorized as a non-IgE-mediated pseudoallergy caused by the activation of the complement system. HSR occurs directly upon the body's first exposure to a lipid excipient, including lipid nanoparticles, and the symptoms usually diminish or disappear with subsequent treatment. For this reason, this immune response is referred to as "pseudoallergy".


Figure 3. Mechanism of CARPA. [2]

Intravenous administration of lipid-containing therapeutic agents can trigger an adverse immune response through activation of complement via both classical and alternative pathways, leading to CARPA. CARPA usually causes cardiopulmonary reactions that exhibit a variety of symptoms, the degreeof which depends on the rate of infusion: slower infusion rates are associated with lower reactions.

Complement activation releases anaphylatoxins such as C5a and C3a and activates macrophages, basophils and mast cells via their specific receptors. These cells then secrete a variety of vasoactive inflammatory mediators, including tryptase, histamine, platelet-activating factor (PAF), and leukotrienes. The released mediators bind to receptors on autonomic effector cells (e.g., endothelial cells and smooth muscle cells), leading to their activation and the resulting CARPA phenomenon.

Factors affecting liposome-induced CARPA

Theoretically, binding of anti-PEG antibodies to liposomes, as well as complement activation, is proportional to the overall surface area of exposed blood. Morphologic evidence suggests that low-curvature oval, elongated, and irregular liposomes will result in a significant increase in the level of SC5b-9-terminal complement complexes. The low-curvature, oval, large-diameter liposomes allow the PEG antibody to bind more efficiently to the liposome surface, while also exhibiting more pronounced complement activation.

The degree of complement activation also correlates with liposome composition and surface charge. PEG liposomes with positive or negative surface zeta potentials have been reported to activate the complement system. LMVs prepared from negatively charged DSPG had more vasoactive effects than electroneutral LMVs composed only of DMPC, and these findings suggest that surface-charged liposomes activate the complement system more than uncharged liposomes.

In addition, cholesterol >45 mol% increases complement activation, and excess cholesterol is exposed as microcrystalline aggregates on the surface of the liposome membrane and therefore more readily interacts with naturally occurring anticholesterol antibodies.

Approaches for eliminating or reducing the ABC phenomenon and CARPA

Regulation of physicochemical properties

Surface charge, especially positive surface zeta potential, increased size, and PEGylation have all been associated with liposome-induced complement activation.PEG is uniformly distributed and homogeneous, with a diameter of approximately 100 nm, and possesses a slightly negative surface zeta potential (to prevent aggregation), which has been demonstrated to be desirable for liposomal preparations with minimal reactogenicity.

Use of alternative polymers

The use of, e.g., polyglycerol, polyvinyl alcohol, polyvinylpyrrolidone (PVP), or polyacrylamide (PAM) instead of PEG allows liposomes to have a longer circulation time, and is an effective way to minimize the ABC phenomenon. Surface modification of liposomes coupled with polyglycerol 760 to DSPE was reported to significantly attenuate anti-PEG and anti-polyglycerol antibody responses. The subunits of polyglycerol may spatially prevent the modified liposomes from binding to immunoglobulins on splenic B cells, thereby hindering B cell stimulation.

Insertion of gangliosides

Gangliosides act as siglec ligands (lectin-like adhesion proteins on macrophages) on reactive B cells, resulting in reduced B cell tolerance and anti-PEG IgM production. Therefore, liposomal membrane modification with clinically acceptable gangliosides might be worth further exploration.

Alterations in the Administration Regimen

Many studies have confirmed that preadministration of high doses of drug-free liposomes and/or prolonged time intervals between injections (3 weeks or more) can reduce the magnitude of ABC phenomenon. Induction of a rapid prophylactic response or immune tolerance is considered a promising approach to avoid hypersensitivity reactions triggered by liposomal formulations, and in a porcine model, pre-infusion of drug-free liposomes for a short period of time (15-30 min) resulted in a significant reduction or near elimination of allergic reactions produced by subsequent doses, thus rapid prophylactic response induction may help prevent liposome-induced CARPA.

Use of the Complement Inhibitor Factor H

Complement inhibitor factor H (FH) is a 155 kDa plasma glycoprotein that is considered a major inhibitor of the alternative pathway of complement activation, inhibiting complement activation in body fluids and on the surface of host cells (basement membranes and endothelial cells), impeding the production of the C3bBb convertase, stimulating the dissociation of the convertase and participating in the enzymatic deactivation of C3b along with protease factor I.


During the development of PEGylated therapeutic formulations, it is necessary to pre-identify and process circulating anti-PEG antibodies in patients, not only to predict the differences in immunogenic responses induced by PEG components in different individuals, but also to predict in advance the impact of the interaction of PEGs with pre-existing anti-PEG antibodies on the therapeutic efficacy. Accordingly, further understanding of the mechanisms underlying these responses, along with various techniques that can help in the avoidance of these responses can aid in the design of PEGylated liposomes and other lipid-based nanocarriers for future applications.

Biopharma PEG is a dynamic science company dedicated to supplying excipients for your LNP formulation development. We are capable of supplying small to large quantities of a rich selection of PEG derivatives and cholesterol (plant derived) with GMP & non-GMP standards for your mRNA vaccine R&D.

[1] Fang, E., Liu, X., Li, M. et al. Advances in COVID-19 mRNA vaccine development. Sig Transduct Target Ther 7, 94 (2022).
[2] Mohamed M, Abu Lila AS, Shimizu T, Alaaeldin E, Hussein A, Sarhan HA, Szebeni J, Ishida T. PEGylated liposomes: immunological responses. Sci Technol Adv Mater. 2019 Jun 26;20(1):710-724. doi: 10.1080/14686996.2019.1627174.
[3] Ibrahim M, Ramadan E, Elsadek NE, et al. Polyethylene glycol (PEG): The nature, immunogenicity, and role in the hypersensitivity of PEGylated products. J Control Release. 2022;351:215-230. doi:10.1016/j.jconrel.2022.09.031
[4] Wang H, Wang Y, Yuan C, Xu X, Zhou W, Huang Y, Lu H, Zheng Y, Luo G, Shang J, Sui M. Polyethylene glycol (PEG)-associated immune responses triggered by clinically relevant lipid nanoparticles in rats. NPJ Vaccines. 2023 Nov 2;8(1):169. doi: 10.1038/s41541-023-00766-z.
[5] Abu Lila AS, Uehara Y, Ishida T, Kiwada H. Application of polyglycerol coating to plasmid DNA lipoplex for the evasion of the accelerated blood clearance phenomenon in nucleic acid delivery. J Pharm Sci. 2014 Feb;103(2):557-66. doi: 10.1002/jps.23823.

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