Tackling Vaccine (Im)purity

Though COVID-19 has commanded our collective attention since 2020, seasonal influenza continues to impact the lives of millions. Can vaccines protect us against both?

Recently, Pfizer and BioNTech announced that their mRNA-based combination flu and COVID-19 vaccine has reached the clinical trial stage – a world first. A combined vaccine would simplify the immunization process, leading to greater vaccine uptake and prevention of both respiratory diseases.

However, this landmark clinical trial comes as a concerning class of impurities has been detected within lipid nanoparticle (LNP)-based mRNA vaccines.

In the event of impurity
 

Process-related impurities are highly dependent on the type of product in question. Impurities can also vary with the scale of the product, meaning that smaller preclinical batches might have a different impurity profile than batches at later stages, when manufacturing is either scaled up or scaled out. In any case, impurities can have numerous negative effects on vaccine quality. Some impurities degrade the stabilizing excipients in the vaccine product or interact with the drug substance itself, reducing shelf life. Others can trigger undesired immune responses in patients and even become life-threatening. As we race along at pandemic speeds to provide solutions for viral diseases, we need to rely heavily on analytical technologies to guide us through production.

LNPs encapsulate the target mRNA, ensuring protection of the fragile mRNA, its delivery, and sufficient uptake by the cells, resulting in a desired immune response. However, a study from Moderna, published in late 2021, detected efficacy loss in mRNA-LNP products (1). The scientists concluded that this loss was most likely caused by an impurity in a lipid species used in the LNP – more specifically, an oxidation of the tertiary amine of the ionizable lipid, known as an N-oxide. When N-oxides break down, they can form highly reactive aldehydes, which can attack the mRNA and result in an mRNA-lipid adduct with altered function. And though I cannot speak to the details of any impurities found in specific vaccines, the approved mRNA-LNP-based COVID-19 vaccines do contain ionizable lipids, which can bear the risk of containing these impurities.

One technique used to detect disruptions in mRNA integrity throughout development and manufacturing is capillary gel electrophoresis (CGE). The main principle is based on measuring molecular size differences, which allows analysts to verify the integrity of the mRNA and simultaneously detect potential degradation in products – usually smaller-sized impurities (for example, those derived from breakages of the mRNA). Because of its ability to resolve high molecular weight species, pharmaceutical companies often use CGE during manufacturing and as a release assay at later stages of production.

The challenge with this new class of impurities is that the mass difference is negligible. Essentially, you are comparing a 100–200 Dalton (Da) mass addition to a 1–2 Megadalton (MDa) mRNA – and such trivial changes will most likely go unnoticed by CGE.

It is vital to minimize the risk of introducing impurities into the manufacturing process. To that end, the screening of ionizable lipid raw materials must be more thorough, requiring methods that allow us to profile the structural components in a given material so we can differentiate between non-harmful and harmful impurities. And that’s why there is a shift toward mass spectrometry (MS) to enable detailed fragmentation of lipid species for structural elucidation. Most lipid raw materials will contain a wealth of very low abundance impurities, but only some of these components are critical for the quality of a final product. Understanding the difference is crucial. MS is widely used to detect impurities in raw materials, and can also detect process-related impurities in the final drug product to help ensure quality standards are met. The technique has proven to be incredibly efficient, especially for identifying impurities that affect the quality of next-generation vaccines, such as mRNA-LNP-based vaccines. 

Going back to MS, collision-induced dissociation (CID) is a common fragmentation mechanism for structural elucidation (and it is particularly helpful in biomolecule analysis, where it is used to break labile bonds). Unfortunately, CID cannot always provide unambiguous information about the nature of oxidation in lipids used in LNPs. The Moderna study suggests that oxygen incorporation can occur in multiple parts of the ionizable lipid, but it is harmful to the mRNA only when it occurs on a tertiary amine. The structural elucidation derived from CID is not always enough to enable the differentiation needed for this analysis.

A recent collaboration between SCIEX and Precision NanoSystems ULC to analyze ionizable lipid structures demonstrated the potential of a novel fragmentation technique called electron activated dissociation (EAD), which can help unlock the fragmentation range necessary to detect species that identify the functional groups undergoing oxidation. In the collaboration, EAD enabled the comprehensive fragmentation of the lipids to reveal the identity of critical impurities below 0.01 percent relative abundance (2). Identifying very low abundant N-oxides is important; it is estimated that even amounts of ~10 ppm can result in significant efficacy loss of the mRNA cargo.

EAD provides the right information, but it will be important to improve throughput and automate the interpretation of these workflows to accelerate the development of various LNP-based vaccines and therapeutics. There are hundreds of different ionizable lipids to choose from for drug formulation – all with different structures and potentially different susceptibilities to forming critical impurities. These lipids must be assessed thoroughly before they are used.

EAD-driven lipid and LNP analysis can help evaluate impurities early in the process – for instance, within a few weeks instead of one to two years down the line. This can help increase efficiency by focusing on the most promising carriers earlier and avoiding the discontinuation or change in the carrier formulation of a drug late in the development process. 

Considering regulations
 

Analytical procedures are subject to strict inspection. The Q2(R1) guidance document from the FDA indicates that the validation of an analytical procedure should take into account several criteria, including accuracy, repeatability, detection limit, quantification limit, and linearity. In addition, changes to the analytical procedure or to the content of a product could require another round of validation. Notably, the process and instrument parameters must be documented without exception. It is fair to say that manufacturers err on the side of caution and, especially for new modalities, seek guidance from regulators to ensure compliance. 

The COVID-19 vaccines are new products, partly based on new mechanisms, that were fast-tracked for approval. All good news – but the fear that something important might have been missed is deeply ingrained in the pharma industry; in the early ages of medicine, approval criteria were very loose and the understanding of risks was rudimentary. Today, every medicinal advance comes with it a need for analytical technology, complying with regulatory criteria, and increased understanding of chemical and biological mechanisms. 

Analytical technology will play its important role in ensuring proper development of future vaccines by detecting potentially harmful impurities at every step of the production process and by documenting the integrity and functionality of both the genetic cargo and its carrier. Though scientific proof of the safety and efficacy of these products can and must be provided, it will take time for new, next-generation products to be fully trusted and accepted. I’m hopeful that analytical science can help establish the legitimacy of mRNA-based vaccines and therapeutics in the public eye.