By Erik Guetschow, Ph.D., Manager of Analytical Chemistry and Paul Kennedy, Ph.D., Vice President of Analytical Chemistry

Rapid advancements in RNA therapeutics have fueled a surge in vaccine development and other RNA treatments since mRNA vaccines were first approved during the COVID-19 pandemic. To deliver mRNA safely and effectively to cells, lipid nanoparticles (LNPs) have been designed to encapsulate the RNA to prevent its degradation. These LNP formulations require several components, including lipids such as phospholipids and cholesterol, lipid polymers typically containing polyethyleneglycol (PEG), and ionizable cationic lipids that help with endosomal release of the mRNA cargo within cells.

Quality of the individual LNP components is critical from the earliest phases of research through final drug product manufacturing. Some of these components are commodity products with well-established quality specifications. However, there is still some confusion when it comes to the identification and characterization of PEGylated lipids. In earlier iterations of LNP formulations, PEGylated lipids consisted of a PEG polymer coupled to a diacylglycerophosphoethanolamine (PE for short). More recently, research has focused on adding functional proteins, peptides, and other targeting agents to these polymers to allow site-specific drug delivery.¹ The increasing variety and complexity of these PEGylated components has led to issues that include lack of formalized nomenclature, confusion about what quality specifications are appropriate or necessary, and lack of standardized methods for appropriate characterization of identity and purity.

PEGylated Lipid Nomenclature

PEGylated lipids are typically synthesized by coupling the appropriately functionalized PEG polymer with the lipid component (Scheme 1). The PE lipid typically contains two equivalent acyl groups such as distearoyl (DS), dipalmitoyl (DP), or dimyristoyl (DM). Therefore, its naming is usually shorthanded to a three- or four-letter notation such as DSPE for a PE lipid with two stearoyl groups.

In some instances, the lipid end may be designated with a numbering system such as 18:0 or 14:0 instead of the letter shorthand. This is another way to represent fatty acid chains, with the number before the colon being the carbon chain length of the acyl group and the number after the colon being the number of double bonds in the chain. For example, in DSPE, the stearoyl chain contains 18 carbons and zero double bonds, therefore DSPE may also be represented as (18:0/18:0) PE or simply 18:0 PE.

It may also be noted that the PE lipid contains a chiral center (annotated in Scheme 1 with the asterisk). If this chiral center is fixed during the synthesis, this will be identifiable in the full lipid name with the use of the 'sn-glycero-' nomenclature or may be defined in the structure by use of the wedged arrow as in Scheme 1. Racemic versions should be denoted as rac-glycero- and when the stereochemistry is not known they may be denoted as simply -glycero- or -X-glycero-.  

A word of caution here is that some suppliers may use the wedged bond drawing even when providing the racemic version of the product. There have not been any reported studies comparing LNP performance when using a racemic form versus single enantiomer form of these lipid polymers, but this may be an important consideration when purchasing these materials.

The PEG polymer component is where a large amount of misunderstanding and confusion exists. Rather than a single molecular species, PEGs are polymers that contain a distribution of species differing by the monomer repeating unit mass. An example mass spectrum of DSPE-mPEG(2000) is shown in Figure 1. For this reason, PEG products are characterized by the approximate molecular weight of the PEG starting material. The size of the PEG is controlled during its synthesis and this size is designated in the name, either by including the approximate molecular weight of the PEG starting material in parentheses or in some cases by noting the number of repeat units in place of the n, particularly when the PEG is produced as a single species.

In Figure 1, the product was prepared from a PEG starting material that had an approximate molecular weight of 2000 amu. After coupling to lipids and other functional groups, the molecular weight of the final product may in fact be much larger than 2000 amu. Thus, it is important to be aware that this molecular weight designation is based on the original PEG starting material molecular weight rather than the final product molecular weight. In practical use, the PEGylated lipid is formulated based on the average molecular weight, which will most certainly be different from the approximate molecular weight included in the name of the product. Since the originating PEG starting material may vary in molecular weight from batch to batch, it is important that the average molecular weight (or most abundant observed molecular weight) is determined empirically by mass spectrometry for each batch. This level of information is often omitted from supplier data or never determined.

One additional observation when purchasing these products is the use of various shorthand notations in the names. For example, DSPE-mPEG(2000) includes an m in the name which is specifically used when the PEG terminates with a methoxy group. This may be omitted in some supplier nomenclature. Additionally, other shorthand such as the use of 2k may be used in place of 2000, the name may be flipped such as PEG(2000) DSPE instead of DSPE-mPEG(2000), or there is no molecular weight designation in the name. In this latter case, it is imperative to review vendor website details carefully to confirm the material meets your required specifications. Cayman has trained technical staff and a team of chemists that understand the chemistry and nomenclature of these products and are always available to support your research if there is any confusion during your purchasing process.

Quality Testing and Specifications

Since PEGylated products are polymers, analysis and characterization can be more challenging than for discrete molecules. Proper characterization of purity and identity are critical for robust manufacturing processes, consistent performance of formulated lipid nanoparticles, and are an FDA requirement to ensure safe and effective therapeutics. While common analytical techniques, such as thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), mass spectrometry (MS), or nuclear magnetic resonance (NMR) spectroscopy, can be used to characterize PEGylated lipids, there is significant variation among suppliers on what tests are used to qualify these molecules and how the testing is performed. In isolation, each of these tests provides some information about PEGylated lipid structure but it is only when combined that a complete understanding of product quality is achieved.

NMR spectroscopy

NMR spectroscopy is a useful tool for characterization of PEGylated lipids and can provide information on average number of PEG units and confirm the presence of functional groups used for conjugation or targeting (maleimide, amine, methyl, etc.). This can be achieved through relative integrations of signals corresponding to the head group, glycerol group, fatty acid, and PEG chain (Figure 2). For example, the average number of PEG groups can be determined by dividing the normalized PEG chain integral by four protons per PEG group. Although some information on product purity can be determined from NMR data, there is often a spectral overlap between impurities and product signals in the NMR that make accurate quantitation of impurities difficult without other analytical data to support the findings.

Figure 2. Representative zoomed ¹H-NMR spectrum for DSPE-PEG(2000)-DBCO with signals representing the DBCO group, glycerol, PEG chain, and fatty acid (acyl CH₂) labeled.

MS

Mass spectrometry data is critical to ensure the identity and molecular weight for PEGylated lipids (which may be reported as average molecular weight or simply by reporting the most abundant peak in the mass spectrum). When coupled with high-performance liquid chromatography, additional insight on the uniformity of PEG distribution and even the presence of impurities in the product can be gained. While it may be necessary to invest in appropriate mass spectrometry instrumentation to perform this testing in-house, suppliers should provide this information to ensure material quality from batch to batch. Depending on the PEGylated lipid structure, these compounds can be detected in positive or negative mode ionization and the molecular weight distribution may vary based on the PEG vendor and batch. Refer to Figure 1 above for an example spectrum showing the mass distribution of DSPE-mPEG(2000).

HPLC

While NMR and mass spectrometry provide some information on product purity, the gold standard is high-performance liquid chromatography (HPLC) coupled with a non-UV detector, such as charged-aerosol (CAD) or evaporative light scattering (ELSD). Although these detectors operate differently, they both allow for detection of compounds that do not absorb UV light. This is especially important for determining purity of PEGylated lipids as the product and common impurities, such as phospholipids and PEG polymers, do not strongly absorb UV light. By tailoring the front end HPLC separation, products can be evaluated based on hydrophobicity, size, or ionization. Purity of PEGylated lipids is especially important when using functionalized PEG lipids as starting materials to produce other more complex LNP tools such as peptide-conjugated PEG lipids.

Figure 3 shows the purity of DSPE-PEG(2000)-DBCO from two sources. The less pure product obtained from Source B contains a significant amount of a proposed DSPE-PEG(2000)-DSPE dimer, which does not contain the DBCO group necessary for peptide conjugation.² In our hands, this resulted in significant downstream issues during the production of the corresponding peptide conjugate.

Source A

Source B

Figure 3. Purity comparison of DSPE-PEG(2000)-DBCO from two sources with HPLC-ELSD. Source A (top trace) shows only a single peak for the expected product, indicating high purity. Source B (bottom trace) shows a variety of PEGylated and non-PEGylated impurities throughout the chromatogram, indicating low purity.

Conclusion 

Ensuring the quality and unambiguous identity of your chemical tools is a critical step towards achieving reproducible scientific results. It is understood that PEGylated lipid size is a critical determinant of LNP particle formulation and encapsulation efficiency.³ Variation in RNA cargo size may require optimization of your LNP formulation and having well characterized and high-quality components will help to ensure reproducibility and minimize development time.

References

1. Xu, X. and Xia, T. Recent advances in site-specific lipid nanoparticles for mRNA delivery. ACS Nanosci. Au 3(3), 192-203 (2023).

2. Hackbusch, S., White, S., and Du, M. Characterization of DSPE-mPEG raw materials from different vendors reveals differences in impurity profiles and polymer chain length polydispersity. bioRxiv 10.1101/2024.01.03.574088.

3. Schober, G.B., Story, S., and Arya, D.P. A careful look at lipid nanoparticle characterization: analysis of benchmark formulations for encapsulation of RNA cargo size gradient. Sci. Rep. 14(1), 2403 (2024).