Quality Control in mRNA Manufacturing

Due to the novel nature of mRNA platform materials, a fit-for-purpose supply chain must establish quality standards. These materials will require several in-process quality control tests, including capping efficiency, mRNA sequencing verification, content (mRNA and delivery system components), and physicochemical characteristics such as nanoparticle size distribution and PDI.

Purity

Unlike traditional vaccines, which are made from live cells and require a complicated production process, mRNA is generated in an utterly cell-free system. This means that the mRNA product can be produced more quickly than other biologicals and does not carry the risk of causing a severe immune response in patients.

However, generating mRNA from its DNA template and performing downstream processing under cGMP conditions remains challenging for manufacturers because these processes often need to be more scalable and cost-effective. Moreover, mRNA requires specific raw materials that are often expensive and not readily available, mainly if they are of GMP grade. As such, many mRNA manufacturing methods currently rely on many chromatographic steps to achieve purity acceptable for encapsulation into LNPs.

To streamline the mRNA manufacturing and purification processes, researchers have developed a novel analytical platform that uses liquid chromatography and mass spectrometry to monitor the characteristics of individual mRNA molecules quantitatively. This system identifies mRNA fragments using an isotopic labeling technology and automatically analyzes their structure using specialized software. The platform can provide rapid identification of critical quality attributes and a detailed understanding of the mRNA structure that can be used to develop new and improved processing methodologies. 

Efficacy

As mRNA vaccines have gained global attention, researchers focus on improving the technology’s manufacturing process to bring this new medicine to the market. This includes developing methods for testing mRNA medicines’ quality attributes, especially ensuring the 5′ capping and poly(A) tail are intact. The goal is to provide the correct sequence and accurate mRNA translation during its entry into the body’s cells, which is then converted into therapeutic proteins.

One of the main challenges is the generation of mRNA at a large scale under cGMP conditions. The specialized components required for the IVT reaction are costly and difficult to procure in large amounts from animal component-free and cGMP-grade sources. Furthermore, the unit operations needed to generate mRNA at the laboratory scale are often complicated and require time-consuming processes.

As a result, the industry is looking at continuous processing of IVT reactions to reduce operation times and enable on-demand production. Additionally, continuous processing can increase process integration, which may lead to reduced operating costs. In addition, using single-use systems and sourcing RNase-free assemblies and components can eliminate the need for complex and expensive purification steps. Consequently, the overall cost per dose of mRNA can be significantly reduced. Ultimately, this could make mRNA more affordable to patients and medical professionals.

Reliability

Messenger ribonucleic acid (mRNA) vaccines are an emerging technology platform for treating infectious diseases, cancer, inherited disorders, and other conditions. mRNA vaccines generate immunogenic responses with the potential to reduce morbidity and mortality compared with traditional bacterial, viral, and protein-based vaccines and provide significant cost savings for vaccine production and manufacturing.

The mRNA vaccine process is based on in vitro transcription (IVT) of an mRNA template generated from DNA, a capping analog, and T7, SP6, or T3 RNA polymerases. The resulting mRNA is then packaged into liposomes with a nucleotide triphosphate (NTP) cofactor and capped using the vaccinia capping enzyme. The mRNA and LNPs are then formulated into a vaccine emulsion and combined with a vector to deliver mRNA to target cells in the body.

While mRNA is a cell-free technology, the synthesis and purification of the product requires multiple unit operations and controls to ensure quality attributes are achieved. To this end, USP is working with an expert committee to develop a procedural chapter on the testing of mRNA vaccines.

This will help de-risk the mRNA vaccine manufacturing process early on by setting acceptance criteria through the lens of an established regulatory landscape. These criteria are based on the impact on patient safety, operator safety, facility cross-contamination potential, and other quality hurdles. The framework approach enables risk-based decisions to inform a robust, detailed, material, and process-specific quality control plan. This includes release testing of incoming materials for endotoxin and vendor-based tests for RNase, as well as a full range of unit operations to purify the mRNA for vaccine use.

Safety

Ensuring the quality of mRNA vaccines under continuously compressed timelines is a challenge for developers. The novelty of this new vaccine platform makes it difficult to identify critical quality attributes (CQAs), which can lead to product development delays and misalignment between regulatory agency expectations and mRNA vaccine production.

MRNA manufacturing begins with template DNA (deoxyribonucleic acid) containing the desired protein’s genetic code. This mRNA is then used as a starting point for in vitro transcription, where the dsRNA is translated into a mature molecule. Following this step, the mRNA is caped and purified. The purity of the mRNA is determined by eliminating endotoxins, immunogenic double-stranded RNA (dsRNA), contaminating DNA, RNA polymerase, secondary RNA structures such as hairpin contaminants, elemental impurities, and other biological degradation products.

To improve mRNA processing efficiency and reduce downstream processing times, USP is working with a panel of experts to develop methods that can be applied continuously. 

Additionally, by developing methods that can be used for multiple disease targets, this approach will accelerate the development of new vaccines and therapeutics and the speed of their commercialization. Developing these methodologies will help to establish a framework for vaccines and therapeutics that is both consistent and predictable from an analytical perspective.