Dr. MARKUS JUZA, ALEXANDER KLAIBER, UWE ALBRECHT
CordenPharma Switzerland in Liestal, CH
Supercritical Fluid Chromatography production of a commercial lipid to support mRNA LNP formulation at CordenPharma Chenôve.
Synthetic lipids have recently been used with great success in mRNA lipid nanoparticles (LNP) formulations, which has greatly increased their demand. Chromatography still remains a standard unit operation in the purification of these lipids. However, traditional normal phase chromatography often uses large amounts of toxic solvents, including chlorinated ones. In this article we present three case studies demonstrating alternative methods to achieve greater sustainability and/or reduce both environmental impact and operational cost. These methods include aqueous/non-aqueous reversed-phase chromatography (NARP) as well as super critical fluid chromatography (SFC).
Lipid Nanoparticles (LNPs) became widely known in the COVID-19 pandemic by playing a major role in the development of SARS-CoV-2 vaccines (1). Nowadays, LNPs are one of the most commonly used tools for xRNA (2, 3) and xDNA delivery (4). LNPs usually consist of five components, ionizable lipids, phospholipids, cholesterols, pegylated lipids, and the payload, each of them adding unique properties to the structure of the LNP. Therefore, synthetic lipids as well as lipid conjugates have been gaining significant importance in recent years as drug target, therapeutic molecules, and liposomes.
Traditionally such molecules are purified by normal phase chromatography on silica using large volume of toxic solvents, including chlorinated solvents. As CDMO and producer of numerous lipids CordenPharma accepted the challenge of reducing organic solvent usage and developing greener processes. Ways to achieve this goal include aqueous/non-aqueous reversed-phase chromatography (NARP) (5, 6) and super critical fluid chromatography (SFC) that can be considered to supplant organic solvents (7) with more benign alternatives. In SFC, the mobile phase consists of a supercritical fluid like carbon dioxide replacing commonly used organic solvents.
SFC shows several benefits compared to HPLC. The polarity of supercritical carbon dioxide is similar to n-heptane, and it is miscible with most organic solvents, allowing to tune the polarity of the solvent. Due to its low viscosity and high diffusivity chromatographic processes are much faster as in HPLC, reducing run times significantly. Also, in economic regard SFC plays an important role by decreasing solvent costs. CO2 is non-toxic, non-flammable, and readily available, making SFC an environmentally benign alternative to other solvent-based chromatography methods.
Three case studies based on such alternative technologies for purification of lipophilic compounds will be presented replacing traditional organic solvents in order to achieve greater sustainability and/or to reduce both environmental impact and operational costs.
CordenPharma Graphic Figure 1 – Overview of different lipid types used as LNP Building Blocks.
Introduction to relevant lipids for LNP formulation
The lipids used in formulation each add unique properties to the LNP. They contribute to the stabilization, encapsulation, and delivery of the xRNA/DNA.
Ionizable lipids play an important part in particle formation. Their positively charged moieties electrostatically interact with the negatively charged backbone of the RNA, leading to encapsulation in a self-assembly process. It also allows for the endosomal release of the drug substance once introduced into the target cells (2).
Phospholipids are built of one or more fatty acids attached to a glycerol “head”. Their function is the formation of a stable bilayer structure underneath the PEG surface. Besides, they also balance out the non-bilayer propensity of the ionizable lipids. Cholesterols also contribute to the structure of LNPs. They stabilize the liposomes and prevent bilayer-leakage by interaction between its hydroxyl groups and the polar heads of the phospholipids. A main source for cholesterols in industrial application are extracts from animal sources (wool grease of sheep or animal tissues) which is a potential risk factor for animal-sourced contaminations like Transmissible Spongiform Encephalopathies (TSEs). Therefore, chemists at CordenPharma and the Otto-von-Guericke-University of Magdeburg developed non-animal origin cholesterol (8), which can be made through a short synthesis directly starting from plant-based material.
Last but not least, pegylated lipids have a vital part in the stability of LNPs. By formation of a protective hydrophilic coating, they increase storage stability, avoid recognition of the LNP by macrophages of the immune system, and decrease nonspecific binding to proteins. Therefore, they not simply increase storage stability, but also prolong in vivo circulation. Due to this “camouflage” ability, they are often referred to as “stealth” lipids (9).
The different classes of molecules present a variety of challenges regarding their purification. Phospholipids for example can often be purified via precipitation or crystallization. For others, like the molecules discussed in this article, a chromatographic approach is the method of choice.
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