rPEG lipids: Risk-free delivery

The researchers followed up with extensive experiments. Over several weeks, they used GME and EO as starting materials to synthesize various new types of rPEG polymer structures. The research duo then brought the new polymer into contact with antibodies. And, as predicted, there was no interaction with the antibody at comparable concentrations.

The randomly distributed GME molecules’ side chains prevented the antibodies from binding to the rPEG. The Mainz researchers’ discovery could be very significant for the pharmaceutical industry. PEG lipids are among the most important ingredients of the lipid nanoparticles (LNPs) that are used to package mRNA active ingredients. LNPs are tiny spherical ferries that transport the active ingredient through the body into the cells. These delivery vehicles are needed because mRNA is extremely unstable and requires protection.

LNPs are similar to drops of fat in water. These nanometer-scale droplets of fat comprise a carefully designed combination of various lipids. The PEG chains are located at the surface of the LNP. They stabilize the particle and avoid recognition by the immune system. The hydrophobic fatty acid chains of the lipid protrude inwards. The mRNA is located inside the LNPs.

“After we realized that rPEGs can be used to prevent the formation of antibodies to PEG, we approached industry,” says Frey. That’s when he got in touch with Evonik. He knew that during the peak phase of the COVID-19 pandemic Evonik researchers had successfully produced important components for the vaccines from BioNTech/Pfizer—including customized specialty lipids. “When I told the Evonik researchers about our rPEG, they were immediately enthusiastic,” Frey says.

The Evonik researchers took on the task of conjugating the rPEG from Mainz to lipids and using the resulting compounds to develop functional LNPs. “We had the huge advantage that we could tap into existing resources in Hanau and Dossenheim for the synthesis and characterization of customized lipids,” says Dr. Thomas Endres, who heads the project at Evonik. He formed a project team that brought together experts from several units—especially from the synthesis laboratory in Hanau and the formulation and cell culture laboratories in Darmstadt.

The initial aim was to link the rPEG from Mainz with fat-soluble molecule segments to a lipid. Erich Kraus, a member of the lab team in Hanau, tested various conditions in small glass reactors. He varied the reaction temperature and the concentration of the individual reagents and the solvents, removed unwanted byproducts, and isolated the product.

“The first trials were sobering, with a yield of only about 30 percent of the desired rPEG lipid,” reports Dr. Ulrich Klöckner, who heads the work in Hanau. It quickly became evident that the functional end of the rPEG molecule had to be modified to increase chemical reactivity. “Our colleagues in Mainz solved that problem very quickly,“ Klöckner adds. Using the modified molecule, the team in Hanau is now reporting yields of more than 70 percent. This is also important for potential industrial production, because a chemical process is cost-effective and sustainable only if a large proportion of the raw materials is converted into the desired end product. Currently, the researchers in Hanau are working to make the production process of the rPEG lipid even more efficient.

Many chemical products are produced by the ton. Quantities of LNPs and mRNA-based vaccines or medications are minute in comparison. The COVID-19 vaccine Comirnaty from BioNTech/Pfizer contains only 30 micrograms of mRNA. One vaccination dose has a volume of 0.3 milliliters. A 300-liter container would be big enough to hold vaccine doses for millions of people.

At the Evonik lab in Darmstadt, Melanie Liefke uses the rPEG lipids and other components to produce the LNPs that are needed for further tests. Sitting at the laminar flow cabinet, she uses pipettes to precisely add doses of various liquids—rPEG lipids, three other lipids, and mRNA—to a thimble-sized plastic cap. Thanks to her experience and skill, she can produce functional LNPs in the desired sizes. “You have to mix the components quickly and gently, because the mRNA is very sensitive,” she explains.

Well-structured LNPs are formed during the controlled mixing. This is partly because the mRNA, the rPEG lipid, and the other components assemble automatically, explains the molecular biologist Dr. Anne Benedikt, the head of the cell culture laboratory in Darmstadt. “This is because the material is self-organizing,” she says. “For example, the fatty acids avoid water, and we can predict how the units assemble driven by their electric charge.” The real challenge in handling mRNA lies in the fact that it can be degraded if the work is not performed in an ultraclean environment, she adds. “That’s because we humans have enzymes on our skin that quickly degrade foreign mRNA—it’s our innate protection system,” Benedikt explains. Researchers who work with mRNA in a laboratory therefore need to make sure the environment is ultraclean and free of these enzymes.

The LNPs are not only produced but also tested in Darmstadt. The team is investigating the interaction of LNPs with living cells. Katrin Häfner, a senior scientist at the cell culture laboratory, points to a transparent plastic microplate under the laminar flow cabinet. The panel has indentations that are filled with a liquid. In some the liquid is rather orange, in others it’s purple. “In this test we determine the highest concentration of LNPs that the cells can tolerate,” she says.

Next door, researchers are investigating whether the LNPs are successfully delivering mRNA into the target cells. To do this, they are using special test systems that emit a dim light if the mRNA has been successfully transferred and if the transfer has been followed by the production of the target protein. The light is detected by a sensor. The better the carrier system performs, the brighter the light.

Endres, the project leader, is proud of what has been accomplished so far. “We have demonstrated that rPEG can be used to produce fully functional LNPs that deliver mRNA effectively under cell-culture conditions,” he says. Moreover, this was accomplished quickly. “We went from the initial idea to a successful proof of concept in only one year,” he explains, adding that research and development in the pharmaceutical sector is complex and time-consuming, often requiring many years of work.

Endres says that the beauty of the technology lies in the similar properties of rPEG and PEG. “rPEG blends seamlessly into industrial production processes,” he adds. Other research groups all over the world are trying to replace PEGs with other water-soluble macromolecules in order to avoid antibody formation. However, these macromolecules were chemically so different from PEG that development of completely new production processes would be needed.

Another factor that promoted speed was the complementary expertise from both partners. “Together we cover the entire value chain from basic research to lipid production on a commercial scale under quality oversight,” Endres says. The University of Mainz and Evonik also want to take the next step together. The LNPs made with the rPEG lipids must undergo further tests before the researchers can start thinking about applications in humans. If these tests are also successful, Evonik can offer pharmaceutical companies the rPEG lipids as a product and the LNPs based on them as a formulation option for mRNA active ingredients. According to Endres, “This would be an ideal addition to our portfolio.”

During the peak phase of the COVID-19 pandemic, the magazine Der Spiegel devoted a long feature article to lipid nanoparticles and mRNA therapies. The article even speaks of a “magic bullet.” Thomas Endres doesn’t want to go quite that far. However, he’s convinced of one thing: that rPEGs have the potential to make future mRNA medications better with an improved safety profile.

Read on the same topic: mRNA-technology: Facts and figures, Lipid nanoparticles for the medicine of the future,

Photos: Robert Eikelpoth (10), private, Nathalie Zimmermann, Stefan Wildhirt / Evonik Industries AG

Infographics: Maximilian Nertinger

Illustration: Oriana Fenwick / Kombinatrotweiss with photo by Foto- und Bilderwerk