Lipids for LNPformulations – Flash chromatographypurification options

To protect the mRNAand enable its effectiveness, pharma companies have worked with biotech companies to create more viable cell delivery options

COVID-19 caused accelerated research into developing vaccine options. Some of the created vaccines are based on mRNA, which, if not protected, easily degrade in humans before their therapeutic benefits can be realised.

To protect the mRNA and enable its effectiveness, pharma companies have worked with biotech companies to create more viable cell delivery options. The most widely used is lipid nanoparticle (LNP) encapsulation. This technology uses microfluidics to combine the vaccine, lipids, other excipients and adjuvants into small spherical particles called LNPs.

The lipid mixtures used typically contain one that is cationic, one that is PEG-based, a phospholipid, and one that is neutral, such as cholesterol. Due to the nature of these molecules, many purification techniques such as distillation or crystallisation are either extremely difficult or impractical to use; so, the industry has faced a new challenge at scale. Luckily, lipid molecules tend to be well suited to off-the-shelf purification methods and platforms, such as Biotage automated flash chromatography development systems (Biotage® Selekt) and scale up (Biotage® Flash 400) platforms for rapid scale up – by both normal-phase and reversed-phase methods.

Historically, detection by UV was hindered since these compounds have little UV absorbance. This problem required alternative detection techniques (evaporative lightscattering, (ELS)), or just collection by volume with post-purification analysis by TLC with staining or charring. Modern flash chromatography systems now utilise photo-diode array UV detection, which can be focussed in the absorption range for the targetted molecules, thus, enhancing their detectability.

Reversed-phase flash chromatography separates compounds based on their hydrophobicity differences through a partitioning mechanism as seen with the lipid chromatogram below where compound retention increases with the number of carbons in the molecule (Figure 1).

Normal-phase chromatography, on the other hand, separates compounds based on polarity differences using an adsorptiondesorption mechanism. Lipophilic compounds elute quickly and those that are more polar elute later. For lipid purification, this can be beneficial as fatty acid type compounds will only be marginally retained, while cholesterol and other more polar compounds will be retained better, (Figure 2).

These examples were performed a low scale (<100 mg) using small flash columns (10 gram), but the purification methods are easily scaled to 100s of grams to even kilogram-scale by using larger columns from Biotage up to 50 kg (Biotage® Flash 400L).

So, if you are involved in the world of lipid production and need to purify your lipids, consider your options. Depending on your goals, it is very likely either normal-phase or reversedphase flash chromatography will help you attain them.

Interested in learning more about fast-tracking purification? Learn more at www.biotage.com, contact: india@biotage.com