Those who think that liposomes and lipid nanoparticles (LNPs) are interchangeable terms for the same drug delivery technology would be wrong. Key differences exist not only in their target applications, but their actual morphology, composition and manufacturing process. In this interview with Dr. Jay Natarajan, one of Evonik’s formulation and manufacturing experts for LNPs and liposomes, some of these differences are reviewed.

Technically speaking, lipid nanoparticles are nanoparticles that contain lipids, making liposomes a type of lipid nanoparticle. However, in scientific literature, the term lipid nanoparticles or LNP is now used to describe a specific type of such particle, which is different from liposomes. What most differentiates liposomes from LNPs is their morphology, and by extension their composition. Liposomes have an entrapped aqueous volume, while LNPs do not. Instead, LNPs contain the lipid in the core of the particle, along with nucleic acids such as RNA or DNA. While LNPs can take a variety of forms, if you were to look closely at their most common structure, you would see a multi-layered core of contracted rings of lipids and nucleic acids, interspersed between lipid layers. Depending on the specific composition and ratios of individual lipid components within the LNP, you can generate very different morphological features compared to conventional liposomes

A typical LNP formulation consists of a few components, plus the payload, which is typically a nucleic acid sequence. The main functional component is the ionizable cationic lipid, which usually represents around 50% of the entire structure. They facilitate entrapment of the nucleic acid during formation, help to maintain a neutral charge while in circulation, and improve intracellular delivery into the cells. Then you have cholesterol, which comprises around 40% of the structure, and due to its hydrophobic nature helps to provide rigidity, formulation stability and support controlled release. You have a small PEG lipid component of around 1-2%, which helps to control and maintain particle size, and extend circulation times by preventing opsonization in the blood. The final 10% or so of the formulation is comprised of structural lipid components such as the DSPC and DOPE. These help to provide overall structural stability of the formulation, as well as stability during manufacturing and long-term storage.

The process technologies and steps used to manufacture liposomes and LNPs are different in the upstream manufacturing process, but similar in the downstream process steps. For conventional liposomes, you start with formation of the crude liposomes using processes such as solvent dilution. You then reduce particle size via extusion or other processes to attain the target size distribution range. Purification is then conducted via systems such as Tangential Flow Filtration (TFF). The final stage is then terminal sterilization. For LNPs, you start with the preparation of the lipid stock solution in solvents such as ethanol and nucleic acid in an acidic buffer. Particle formation and size reduction are then typically handled via the use of microfluidic mixing systems. This mixing step increases the total process volume, so you then need to reduce it via TFF to a more manageable level. To achieve a neutral buffer, you then need to perform the buffer exchange via diafiltration, and add a cryoprotectant. Then, as with normal liposomes, you will perform terminal sterilization to complete the LNP formulation.

A typical micro-mixing system involves two vessels containing lipids and the nucleic acid. Two sets of pumps then bring them together via a mixing unit such as a T or Y connector. The pumps need to be pulsation free, as any pulse can increase the size polydispersity of the LNPs and can affect the reliable encapsulation of the payload. We’ve noticed during scale up that a lot of current technologies can struggle with achieving a consistent overall flow rate during this manufacturing step. We expect micro-mixing equipment will continue to evolve, in line with the development of nucleic-acid therapeutics. Overall, we need to reach the point where we can operate at high flow rates to avoid long processing times and address intermediate stability concerns.

It’s really important to optimize the process, because many of the manufacturing steps can influence critical factors such as particle size and the percentage of encapsulation. Not all micromixing equipment is equal, and they can all generate very different results. Temperature, hold times and flow rates during the mixing process all need to be carefully calibrated, because this will have a significant impact on the final product. For example, an additional temperature control after can sometimes be required after initial particle formation, to increase the stability of the intermediate. Another key factor that needs to be taken into account is your sterile filtration and fill-finish process. For example, most filing processes typically occur at room temperature. So, if the LNP has very limited stability at room temperature, the time that it takes to perform fill-finish needs to be taken into consideration.

As we’ve just made clear, there can be significant differences in how to develop and manufacture LNP- or liposomal-based products. Specific core competencies are required for each drug delivery technology. A pharmaceutical company would gain significant versatility in being able to align themselves with a CDMO that has a strong track record in both areas. However, few such CDMOs exist that bring together all the necessary capabilities for formulation development, process development, method analytical development and testing, cGMP manufacturing and aseptic filling. As a CDMO leader for complex parenteral drug products, Evonik has dedicated itself to providing customers with an integrated portfolio of solutions for both LNPs and liposomal-based formulations. We've got more than 30 years of expertise with liposomal formulations and have been actively involved in the development and scale-up of multiple LNP-based products over the last 10 years, including some of the COVID-19 vaccines.

For more information on Evonik’s CDMO services and other biomaterials such as lipids and cholesterol, contact the expert team for a consultation via healthcare@evonik.com.