Manufacturing Strategies for mRNA Vaccines and Therapeutics

Adeno-associated viral (AAV) vectors are a well-known therapeutic approach in the field of viral delivery systems. However, AAV also has disadvantages. They cause immunogenicity and more common systemic side effects than other approaches. In addition, the production process is also very complicated. High titers are required if you are targeting gene therapy applications. Therefore, we also use a non-viral delivery system.

Things change if we move to non-viral delivery systems. And the whole process will be simplified.

mRNA technology

mRNA technology uses a non-viral delivery system. Picture this: mRNA sneaks into the cytoplasm of a cell, directing the production of a protein of interest. It can serve multiple roles—it can be a therapeutic guard, a preventive shield, an inducer of immune responses to vaccines, a replacement for defective proteins, or even a warrior that awakens an anti-tumor response. The role of mRNA doesn't stop there. Strong early results for three mRNA vaccines against SARS-CoV-2 have implications far beyond the current pandemic and bode well for similar approaches to fighting cancer, heart disease and other infectious diseases.

Delve into the world of mRNA manufacturing and you will be inspired. It's like a breath of fresh air in the biomedical field - very simple, scalable and fast. But what exactly is it that is getting people's attention? The speed with which it has progressed from lab development to clinical trials to official approval is almost astonishing. While this agility makes it a sought-after weapon in the fight against sudden infectious outbreaks and epidemics, its appeal doesn't stop there. It promises to lead to breakthrough therapeutic innovations, especially for diseases that until now have remained shrouded in mystery, seeking answers.

From Synthesis to Production: The mRNA Process

Let's get into the substance. mRNA does not follow traditional rhythms. It is produced synthetically in vitro by precise enzymatic processes. Contrast this with traditional in vivo protein expression. Remember the tedious cloning technique? With mRNA, that's all history. There is no need to expel cells or remove host cell proteins. Here's the kicker: This fluid process, with its consistent materials and equipment, offers unprecedented flexibility for GMP facilities. New protein targets on the horizon? They turn instantly and can be adjusted with just a few tweaks here and there.

make mRNA

So where does the process of creating mRNA-based therapeutics and vaccines begin? Typically, it starts with a pDNA template. This template is filled with DNA-dependent RNA polymerase promoter and mapping sequences for mRNA constructs. This pDNA construct is not just a minor player; it is an important player. It plays an important role. It is like a backbone, and its structure and cleanliness are essential for refined mRNA production.

In the field of bacteria alone, E. coli is usually in the lead. After careful purification, we obtained pDNA that is pristine, highly efficient, and circular. But it doesn't stay in the loop for long; it's changed. It has been linearized, making it an ideal manuscript for RNA polymerase etching of the desired mRNA. Why is there such a change? Linearization ensures that we avoid sneaky transcriptional readthroughs that might generate mRNA forms that we don't want - forms that need to be evicted. How do we achieve this goal? Submerged in a mixture of plasmid DNA and specific restriction enzymes, boiled in reaction buffer, and allowed to visualize within four hours at 37°C. Finally, we can add EDTA or use a temperature spike of 65 °C.

purified mRNA

The process was not without fragments. There are also residues—unwanted fragments such as restriction enzymes, DNA fragments, endotoxins, etc. One might consider the lab's favorite method of solvent extraction, but this is not feasible in a strict GMP manufacturing environment. Instead, technologies such as tangential flow filtration (TFF) and chromatography have emerged as saviors adept at removing these impurities.

In vitro transcription uses linearized pDNA as a DNA template, which is sculpted into mRNA. Examples include RNA polymerase and nucleotide triphosphates. Once etched, this nascent mRNA craves stability. The 5' cap is the answer, and there are two ways to do it: co-transcriptionally or enzymatically. Both co-transcription and enzymatic capping have their advantages and challenges, ensuring versatility in building perfect mRNA structures.

Deciphering the World of mRNA Purification

The art of mRNA privacy. Once the dance of in vitro transcription is over, we are left with only mRNA. But its PURIS purification strategies do not yet exist, each with its own unique advantages and challenges. Technologies such as Tangential Flow Filtration (TFF) and various chromatography

The phic method offers several ways to ensure that the mRNA is in its most pristine form.

Chromatography

Gain insight into the world of chromatography in mRNA purification. At the heart of purification lies complex chromatography. From the most popular reversed-phase ion pairing, to reliable anion exchange, and the intriguing affinity chromatography with poly(dT) capture, each method offers unique advantages for ensuring mRNA purity. However, like any scientific method, they come with their own set of challenges. But once the chromatography stage is over, the focus shifts to concentration and diafiltration, with the ultimate goal of reaching pristine purity.

Finalizing mRNA Packaging: Formulation and Delivery

The final step in the mRNA journey is transferring it to the ideal buffer for safe storage or formulation. This is where Tangential Flow Filtration (TFF) technology comes into play again, facilitating simultaneous purification, concentration and diafiltration. But the road to delivery is not without obstacles, especially when dealing with heavyweight mRNAs.

Scale up mRNA production

Scaling up mRNA production is no easy task. Key considerations include:

Traditional purification methods may not be applicable on a larger scale. Hazardous solvents that do not meet GMP standards can be replaced by TFF or chromatography.

Vigilance against RNases is essential to prevent mRNA degradation.

A custom delivery system is critical to the success of a vaccine or therapy.

Consider alternatives for filtering large mRNA complexes.

Supply chains, especially cold storage, can significantly impact costs. Therefore, a thorough stability assessment is crucial.

Prospects and Prospects

The mRNA technology wave, amplified by its role in COVID-19 vaccine development, is reshaping the vaccine landscape. Its agility and adaptability not only ensure a rapid response to the outbreak, but also provide pioneering gene therapy solutions to unmet medical challenges.

The advantage of mRNA is that it shifts the focus from scientific hurdles to process development. With the genetic blueprint of a pathogen in hand, vaccines can be quickly designed. However, for mRNA to really shine, innovative strategies must be paired with expertise. Safety, quality, efficiency and manufacturability are pillars that must be constantly evaluated and strengthened.

By addressing these challenges head-on, mRNA-based therapeutics have the potential to transform global health outcomes.