Decisions Upstream Have Major Effects Downstream: Streamlining mRNA Therapeutic Development

mRNA therapeutics have enormous potential. As seen with the COVID-19 vaccines, they are effective for inoculation against infectious diseases, but also hold promise as personalized cancer vaccines and for treatment of rare diseases. A major advantage of these therapeutics is their relatively fast development that does not utilize chicken embryos or cell cultures like protein-based vaccines. mRNA therapeutics are a new technology, and no one biopharma company has complete expertise in the field. 

Development of an mRNA therapeutic requires a broad range of knowledge—from plasmid design to in vitro transcription (IVT) to effective lipid formulations that are safe for human use. In addition to a deep knowledge of development techniques, it can be challenging to scale up production to manufacture a product to the quantities and quality often necessary for a therapeutic. The Life Sciences companies of Danaher have the expertise to help labs and manufacturers develop their mRNA therapeutics, from designing and synthesizing optimal DNA starting material to going all the way to the fill-finish phase of production. 

From plasmid to purification: developing an IVT protocol

A major concern when setting up an RNA therapeutics pipeline is the production of consistent and pure mRNA during the in vitro transcription phase. The starting material is typically the number one source of variability in the entire workflow, and making good choices upstream in the development pipeline can simplify downstream processes. If using plasmid from a master cell bank, several considerations must be made. Intrinsic qualities of the DNA, like codon optimization and GC content, are important to assess during the plasmid design process to ensure optimal transcription efficiency. Additionally, the antibiotic resistance genes typically necessary for E. coli-based propagation must eventually be removed to meet regulatory standards. This step can be eliminated when using a backbone with non-antibiotic resistance selection, such as Aldevron’s NanoPlasmid vector, easing downstream purifications and regulatory pressures. Beyond the starting DNA, use of high-quality IVT enzymes, offered by Aldevron, and pure, RNase-free NTPs, offered by Cytiva, are critical for robust transcription. 

Another key consideration during IVT is the method of 5’-mRNA capping. The cap is either added co-transcriptionally, with capping components in the same reaction as the NTPs, or enzymatically after IVT. There are pros and cons to each method. Co-transcriptional capping does not require the additional step that enzymatic capping does, but this method is generally more expensive. It is necessary to consider time, costs, and desired purity when choosing a strategy. While customers are currently split roughly 50/50, they are trending toward a reagent-based cap instead of an enzymatic cap. 

These considerations are necessary for robust mRNA production, but also for the reduction of contaminants. Double stranded RNA (dsRNA) by-products are a major concern. dsRNA is similar in structure to viral genomes and can trigger a robust immune response. While this contaminating dsRNA can be removed, it is typically more challenging to purify away than contaminants like unreacted reagents, due to its similarity in size and structure to mRNA. Optimization of IVT to minimize the amount of dsRNA is critical. Strategies to do this are currently being pursued, primarily by use of engineered polymerases during IVT. There are polymerases designed for efficiency and yield, but primarily what the industry is moving toward is polymerase optimization for the reduction of impurities including double-stranded RNA. 

Optimization for reducing impurities and to improve yield is important, and the proper selection of analytics throughout the IVT step will always be critical. 

Validated analytical methods are critical to ensure that there aren’t potential leftovers or contaminants from the IVT reaction or purification. Historically, dot blots have been used to assess the presence of dsRNA, but this method is generally not considered sensitive enough for the application. Presently, size exclusion chromatography (SEC) and ELISA-based methods are being used to increase the sensitivity of the double-stranded RNA assay to meet the needs of the industry, with cell-based immuno-assays as being developed as a future method for assessment.

Consistency is Key: Lipid Nanoparticle Encapsulation

Once the mRNA has been captured, polished, and analyzed, it is encapsulated into lipid nanoparticles (LNPs). Analytical techniques like uHPLC-CAD and dynamic light scattering provide the sensitivity necessary to assess the quality and consistency of novel or custom lipids. This quality control is important because the methods used by some vendors to assess unreacted lipids are not always sufficient for a product like an LNP. It is crucial to use of a reputable vendor or partner with a company, like Precision Nanosystems, that has deep expertise in LNP development and can provide proper analytics. Working closely with a vendor will ensure that specifications are met and is critical for successful drug development partnerships. 

Beyond lipid choice, achieving consistent and application-appropriate stoichiometry during encapsulation is important. Analytical techniques to assess lipid and mRNA quality should be used after encapsulation to determine the product’s therapeutic viability. For example, a large ratio of LNPs that do not contain RNA can trigger an immune response without providing any therapeutic benefit. Using sensitive methods to measure mRNA content is critical. Instruments like the SCIEX PA 800 Plus Pharmaceutical Analysis System and BioPhase 8800 System can assess the purity and integrity of mRNA and determine if LNPs are empty or full.     

Benefits of Working with A Knowledgeable Partner

Given the complexity of developing an mRNA therapeutic, collaboration with companies that possess expertise across IVT, purification, LNP encapsulation, and fill-and-finish processes is immensely beneficial. Working with Danaher can also help developers avoid the trap of developing quickly in areas of skill, while neglecting areas where they have less expertise. Danaher Life Sciences has the experience to know how to best set up individual steps in a pipeline to meet a client’s needs, looking up and down a process to determine decisions upstream that will affect downstream outcomes in unexpected ways.

RNA therapeutics have enormous potential to dramatically alter the medical landscape—from helping governments rapidly respond to the next infectious disease outbreak to changing how we treat cancer. Danaher Life Sciences provides end-to-end solutions to aid in the development of these therapeutics and simplify a complicated process. Learn more and get in touch with one of our experts today at lifesciences.danaher.com and cytivalifesciences.com. 

Written by Dave Sokolowski , Global Workflow Manager, Nucleic Acid TherapeuticsSenior Director, Global Head of Cell Culture R&D and Services at Cytiva.

About: Dave is an experienced biopharma professional with many years of industry experience. Dave joined PALL Corporation as an R&D engineer supporting the development of acoustic technologies, before moving into Project Management, and then into Global Product Management. He is an integral part of the cross-functional team between Cytiva, PALL, and PNI, created to design an integrated mRNA workflow.

Prior to his professional career, he acquired a B.S. in Microbiology from the University of Massachusetts, Amherst, which inspired him to join the UMass Med School PhD program investigating the post-transcriptional regulation of mRNA through RNA-binding protein interactions. Prior to completing his PhD, Dave chose to refocus his career towards scientific technology development, and instead earned a B.S. in Mechanical Engineering from UMass Amherst.

© 2023 DH Life Sciences, LLC. All Rights Reserved.

© 2023 Aldevron, LLC. 

© 2023 Cytiva. 

Cytiva and the Drop logo are trademarks of Life Sciences IP Holdings Corp. or an affiliate doing business as Cytiva.

© 2023 Precision NanoSystems. 

© 2023 DH Tech. Dev. Pte. Ltd. 

For Research Use Only. Not For Use in Diagnostic Procedures.