Supplying lipids in the global response to COVID-19 and mRNA vaccine development

Biotage large scale purification systems have been helping pharmaceutical companies and their contract manufacturers develop and purify new active ingredients for the rapidly growing high-potency drug market for a number of years. In 2020, we collabo- rated with the specialty chemical company Croda on a project focused on the production of a potential new small-molecule used in COVID-19 vaccine formulations for global distribution.

Introduction

Moving from a traditional lab based synthetic process to more commercially orientated and efficient processes with a limited timeline is often a challenge. In 2020 Croda approached Biotage with just such a challenge relating to the growing and unprec- edented humanitarian health crisis that was unfolding relating to the spread of the COVID-19 virus. Croda needed fast reliable access to pure lipids for LNPs used in mRNA vaccines.

At the time, large scale production of mRNA vaccines was in its infancy, and there had never been a such a global demand for raw materials used for the therapeutic application on this scale. In a collaborative project, the Biotage team contributed their expertise in scaling up purification chromatography. With the help of the large scale Biotage Flash 400 purification platform, Croda successfully scaled up their processes from development to production in just four months. This not only resulted in a stable, commercial grade manufacturing process, it also saved Croda valuable time and reduced their solvent consumption compared to existing, traditional methods.

The application of a new high performance spherical silica, with 2x larger surface area compared to standard and traditional irregular shaped silica stationary phases enabled a significant increase in sample loading on the column, resulting in both a higher concentration of final product, and even less solvent usage. The stationary phases used in purification cartridges at lab scale was the same as that used in the larger 40Kg cartridges, so there were no technical or process surprises and everything proceeded as expected.

A quick refresher, lipids, lipid membranes, how viruses work and mRNA

A number of components are typically involved the delivery of drugs into the body and optimizing the chemical formulation of the drug for the target region in the body, or specific cells can be a fairly complex undertaking. So drug formulation strategies are a very important topic in drug discovery, and in essence, the similar principles apply for the biological or biochemical methods also. For example, a potent drug may be rendered useless if it cannot get to the target destination in the body.

It may be attenuated by degradation and the bodies natural defense mechanisms along its journey, so bioavailability is a very important consideration. From a chemical perspective, the target API or biological entity must have a pH and solubility profile that is compatible with the area of ingestion, travel and finally mechanism of action. These technical challenges, coupled with the fact that much of the supply chain for synthesis of these technologies was in its infancy, made made this the perfect storm of negative factors, during the early days of the pandemic. Looking in greater detail at the chemistry and science of these elements gives a better idea of the challenges faced by researchers.

Lipids and membranes

Lipids are interesting bifunctional molecules usually characterized by a hydrophilic head and a hydrophobic tail. The polar head group can be made of a number of different moieties but classically phospholipids are present in biological systems and found in every cell.
Since the tails are hydrophobic, lipids can form several interesting self-organized structures from micelles to bilayers. The bilayers are typically found in cell membranes and are a critical interface and control measure to separate the contents of the cell and the outside.

What would normally happen

DNA is the master copy of the cellular and genetic information in the biological cell and mRNA is created in a process called transcription. mRNA is a ribonucleic acid polymer which is a shuttle for the information stored in the DNA, via its sequence of bases. The mRNA moves from the nucleus into the cytoplasm, which contains ribosomes, which are the specialist cellular machinery for making proteins in a process called translation. By copying the master code into mRNA, a biological cell maximizes the fidelity of the genetic information from generation to generation, and therefore mRNA plays a vital role in the day to dat functioning and creation of proteins from DNA.

How viruses work

A virus is a master at hijacking these normal biochemical pathways. Viruses evolve and have become skilled in the art of crossing the usually impenetrable cell membrane and evading normal cellular defenses. They create surface proteins (spike proteins) which have properties that allow the virus to travel through the bilayer and getting into the cell.
Once into the cell the virus commands the cellular machinery for its alternative purpose, based on the coding in its DNA.

A new type of vaccine based on mRNA

In 1990, a University of Pennsylvania scientist Katalin Karikó first proposed using mRNA as an alternative to DNA-based gene therapy. The idea being that adding appropriate lengths of genetic information into a cell can make it perform in alternative ways, that we engineer or program, rather like what a virus would do.. But this concept was not without its problems, mRNA is difficult to work with and could only be synthesized in small amounts at that time. Additionally, mRNA quickly degrades in the body and was not stable enough to administer as a viable drug candidate.

However, recent technological and chemical advancements have allowed for the rapid development and testing of mRNA as candidates for COVID-19 vaccines. A key factor in this success is the lipid nanoparticle formulation that allows mRNA to reach its biological target once administered to a patient, thus solving one of the key problems identified earlier. Vaccine developers learned to combine mRNA with lipids to make lipid nanoparticle (LNPs). Wrapping payload mRNA in lipid can help it get it into a cell, but the next problem to solve was which lipids to make, and then, how to make the LNPs.

Lipid Nano Particles (LNPs)

A major drawback of current mRNA-lipid nanoparticle (LNP) COVID-19 vaccines is that they have to be stored at very low temperatures. There are now many studies aimed at
understanding the instability of these vaccines, with the aim of improving mRNA-LNP stability and therefore being able to increase the temperature conditions for storage. Currently there are over 150 mRNA candidates under development for various therapeutic areas including infectious diseases, genetic diseases, and oncological disorders. Recent approval of mRNA vaccine candidates vs COVID-19 has accelerated the growth of this emerging class of biologics but solving the key physio-chemical issues of the formulation components, particularly the chemistry of the LNP above remains the industries greatest hurdle.

Lipids and advances in LNP formulations

Liposomes based on self assembled lipids, similar to the micelles shown above, were early version of LNPs, and extremely versatile nanocarrier platforms because they could transport a variety of different (hydrophobic, hydrophilic) molecules. The next generation however, of LNPs are more complex internally and have enhanced physical stabilities. With an improved ability to control location and timing of drug delivery in the body (therefore solving some of the bioavailability issues we discussed earlier), LNPs are now used to deliver treatments for a variety of diseases. In an LNP, there are 4 main types of lipid, ionizable cationic lipids (which encapsulates negatively charged mRNA), PEGylated lipids (which helps control particle lifetime and size), Distearoylphosphatidylcholine (DSPC) (which is a structural phospholipid) and cholesterol (which contributes to structure). COVID vaccines from Pfizer/BioNTech, Moderna and CureVac all utilize lipid nanoparticle formulation.

Furthermore, there are many variables, size (of lipid, and mRNA), shape once wrapped up (and unwrapped), stiffness and chemistry factors i.e. surface charges / polarity. These lipids can be quite complex, so purification is often a challenge.

Purification of lipids using prepacked flash chromatography cartridges

Biotage has developed optimized pre-packed purification cartridges with stationary phases optimized for such separations, in sizes from 5g through to 40Kg which can facilitate sample purification on a scale that can support hundreds of thousands, even millions of doses of vaccine. These cartridges are designed as plug and play, and scale up seamlessly from lab scale systems designed to probe purification methods, all the way to the large scale purification platforms designed to support commercial production. There are hundreds, possibly thousands of lipids, but all are characterized and may be separated by similar physical properties and underlying hydro- phobic/hydrophilic characteristics and their overall interaction with both stationary and mobile phases in flash chromatography. Below are a few examples of how typical lipid molecules separate under conditions of both normal and reversed phase purification.
(Typical lipid purification strategies, top: normal phase, below: reversed phase).

During this process, the team at CRODA were also able to engineer out the use of more toxic solvents, as used in the original synthetic lab procedures, and replace them with fewer, and less toxic solvents optimized for this type of purification. The adoption of more efficient purification protocols also allowed the synthetic route to be further simplified and the total number of steps reduced.