Khaled Yamout - Senior Director of Analytical Services and Quality Control, TriLink BioTechnologies.
As the template for all protein synthesis, mRNA serves as the intermediate that allows cells to use genetic information to respond to the dynamicity of life. When applied for therapeutic purposes, mRNA can produce proteins of interest on demand to treat previously undruggable diseases.
While mRNA therapeutics have been around for over five decades,1 this modality gained visibility during the COVID-19 pandemic, when massive collaborative efforts led to two rapidly developed and approved vaccines. The success of these vaccines has revitalized interest in developing drugs that use the mRNA platform. Developers are broadening their efforts beyond COVID-19, seeking to make vaccines against other infectious diseases, cancer treatments, and cell and gene therapies from mRNA-based building blocks. As of February 2023, as many as 140 clinical trials using mRNA were underway, with 70% developing vaccines against infectious disease, 12% addressing cancer, and the rest serving various cell and gene therapy applications.2
To improve the chances that these and future mRNA therapeutics will be approved, developers need state-of-the-art nucleic acid analytical techniques to ensure that drugs are of the highest quality. This article will discuss the latest trends and technologies in analytical testing that ensure quality and help streamline the development of new mRNA therapeutics.
The Advantages of mRNA Therapeutics
mRNA therapeutics offer several advantages, making them a favorable therapeutic modality. First and foremost, they are highly effective, safe, and well-tolerated. They do not need to enter the nucleus to be functional, offering a relatively high transfection efficiency and low toxicity profile compared to other biological drugs. In the cell, translation of mRNA molecules results in the sustained expression of the protein of interest that produces a targeted effect on a specific biological process, in contrast to the off-target side effects often seen with small molecule drugs.
Additionally, they are relatively simple biological molecules to produce in contrast with other biological drugs in development today. Compared to therapeutic antibodies, which may take several months to make a batch, a batch of therapeutic mRNA can often be made in a week. In this setting, where a whole class of therapeutics can be developed and manufactured rapidly, developers need the most streamlined analytics to enterprise on that massive potential.
The Components of mRNA Therapeutic Manufacturing
mRNA therapeutics are made of specific components that each play a role in the efficacy and duration of the therapeutic function. These resemble eukaryotic mRNA as it exists in nature: a single-stranded nucleic acid molecule with a cap on the 5’ end and a poly(A) tail on the 3’ end, with untranslated regions flanking a coding sequence. The different roles of each of the components are as follows:3
- The 5’ cap enables translation initiation, determines innate immunogenicity, and protects mRNA from degradation.
- The untranslated regions flanking the coding mRNA perform regulatory functions.
- The poly(A) tail determines half-life and translation efficiency and aids in transport within the cell.
- The open reading frame (ORF) codes for the therapeutic protein. It can be produced with modified nucleotides to reduce degradation and immunogenicity and enhance stability.
- The delivery vehicle, most commonly a lipid nanoparticle, contains the mRNA molecule and improves pharmacokinetic properties like cellular uptake and flow through the lymphatic system.
Analytical characterization of these components throughout the manufacturing journey ensures that drugs meet specific identity, content, integrity, purity, potency, and safety requirements.3
Before manufacturing can begin in earnest, manufacturers must make or obtain raw materials. These include modified and unmodified nucleotides, capping agents, plasmids, and enzymes. Subsequently, mRNA drug substance manufacturing is a straightforward process: plasmid DNA is linearized and then in-vitro transcribed via a one-step reaction using CleanCap® mRNA capping technology or a two-step in-vitro transcription reaction using enzymatic capping and multiple purification steps.4 From start to finish, the process takes about a week, even when scaled up substantially.
Because production occurs in a cell-free in-vitro reaction, mRNA therapeutics are not likely to contain safety concerns like cell-derived impurities or viral components found in other biologics. Instead, quality control (QC) testing focuses on the quality of the product and impurities in the form of residual reagents and partially formed molecules. By partnering with a CDMO that can expertly carry out all analytical testing necessary, developers can streamline the manufacturing of their mRNA therapeutic to continue advancing it toward regulatory approval.
Analytical Testing in Development and Manufacturing
Although they are simple to manufacture, mRNA therapeutics are large molecules that require significant characterization to reveal their chemical makeup.
Testing occurs throughout each phase of the manufacturing process. First, the raw materials must be well characterized before use to identify any impurities that might be present. By starting with highly pure materials, developers can significantly streamline production. Next, the efficiency of the plasmid DNA linearization must be evaluated. A high percentage of linear DNA is crucial to the manufacturing process designed to result in mRNA products of appropriate length. Throughout manufacturing, in-process tests can be established to track reaction yield and purification recovery. After manufacturing, a quality control team will perform a battery of tests to evaluate the purity, size, and concentration of the mRNA product, as well as each building block of the mRNA product, including the cap, tail, untranslated regions, and coding sequence.
Two recent QC trends indicate a growing focus on establishing the overall purity of the mRNA product and the quality of the Poly(A) tail. There are numerous assays available, and each tells developers something different. Not only is it inherently valuable to assess these parameters using multiple techniques, but regulatory bodies also tend to expect developers to characterize their products via multiple orthogonal methods. As is the case with therapeutic antibodies, assessing purity may soon require evaluation across four to five methods for satisfactory results. Along these same lines, developers must determine what to report about the presence, length, and stability of their poly(A) tails. Ultimately, they need assays to reveal each of these parameters for any given product.
Tailored Quality Control is Key
QC assays designed to assess therapeutic mRNA products may be based on a common platform but are often ultimately tailored to be construct-specific because each mRNA sequence is unique. For example, chromatography results will differ slightly based on mRNA sequence, which may change further if the product consists of modified nucleotides. Unique chromatography assays may be needed to evaluate different mRNA molecules.
Developers aiming to make an mRNA therapeutic using a designated coding sequence and elements common to the backbone can generate feasibility data to show that certain generic methods apply to the construct. This approach allows them to avoid having to start the development of QC assays from scratch each time. On the other hand, for any therapeutic product with unique backbone components (the poly(A) tail, cap, and untranslated regions), new QC assays must be developed to establish purity, identity, and more.
Developers may benefit from partnering with a CDMO with expertise in both leveraging generic assays based on a common mRNA backbone, and analytical development of QC assays designed to characterize the specific therapeutic construct.
Putting mRNA to the Test
A battery of about 14 tests assesses each new batch of mRNA. These include safety testing for endotoxin and bioburden, testing for mRNA concentration and residual contaminants such as residual DNA, residual proteins/enzymes, residual NTPs and capping agents, double-stranded RNA (dsRNA), and residual solvents. Moreover, a cell-free translation assay can establish the mRNA codes for the expected protein.
The poly(A) tail and cap structure are assessed using liquid chromatography-ultraviolet/mass spectrometry (LC-UV/MS). Purity can be assessed by capillary gel electrophoresis (CGE), ion pair reverse-phase high-performance liquid chromatography (IP-RP-HPLC), size exclusion chromatography (SEC), or anion-exchange chromatography (AEX).
Having multiple identity methods on hand is crucial, as regulatory bodies often ask that two orthogonal assays be done to evaluate a novel mRNA therapeutic. One way to determine mRNA identity is by sequencing, either Sanger or NGS. Another older method to establish identity is base composition, in which the mRNA is digested down to individual nucleotides to ensure that it has the correct number of each. A third method for determining identity is fingerprinting using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Despite this battery of tests, the field needs more methods to evaluate mRNA. Previously, assays to evaluate nucleic acids tended to be semiquantitative, such as gel electrophoresis and light absorbance. A major trend now is to hold new mRNA products to a higher standard.
As the field continues to evolve, the requirement for more sensitive and accurate quantitative analytical methods will continue. Leaders in the field continue to work on adapting analytical methods for the characterization of mRNA (e.g., using Circular Dichroism, small-angle X-ray scattering, FT-IR, and other spectroscopic techniques).5 In addition, scientists are working on developing various standards to quantitatively assess various portions of mRNA such as the poly(A)-tail and cap.
Bottlenecks in mRNA Manufacturing
The biggest bottlenecks in mRNA characterization to support mRNA manufacturing include acquiring specific standards and gaining access to high-resolution instruments. Another challenge for developers is that regulatory expectations and analytics that must be completed may differ depending on whether the therapeutic is classified as a vaccine, cancer drug, cell therapy, or has another therapeutic use. By partnering with a CDMO that can support development from start to finish, offering these resources and guidance on how to meet regulatory requirements, developers can avoid significant delays.
That said, developers can create their own bottlenecks when they delay gathering the analytical data for their new product to support an investigational new drug (IND) application. The most successful development programs engage in their chemistry, manufacturing, and controls (CMC) activities from day one. They communicate with their regulatory agent to determine what assays will be required in their IND. These development programs do the work as they go rather than playing catch-up later, saving them time, money, and stress.
By collaborating with a CDMO and using state-of-the-art analytical testing, developers can offer top-quality results in their IND and CMC to solidify their chances of success. Most of the information filed in the CMC is analytical testing, so reproducible tests with high data integrity that tell developers precisely what their product is will help streamline the filing process. Developers working with a CDMO should receive their procedure, protocol, and report for each method already packaged for regulatory consideration.
Ensuring Progress Through Analytical Testing
For mRNA therapeutic development and manufacturing, analytical testing is critical from beginning to end. Advanced nucleic acid testing is used throughout the production process to ensure that drugs are high-quality, effective, and safe. Analytical practices constantly evolve to better evaluate mRNA products and streamline therapeutic development.
Advanced nucleic acid testing facilitates development by aligning all efforts with regulatory expectations. As new therapeutics enter the development pipeline and eventually reach the market, we will continue to learn of the new and exciting therapeutic capabilities of this remarkable modality.
References
- Sahin U, Karikó K, Türeci Ö. mRNA-based therapeutics--developing a new class of drugs. Nat Rev Drug Discov. 2014;13(10):759-780. doi:10.1038/nrd4278
- Newton, W., mRNA vaccines: Four major clinical trial readouts to watch in 2023. Clinical Trials Arena. Feb 2, 2023. https://www.clinicaltrialsarena.com/features/mrna-vaccine[1]trials-to-watch/?cf-view
- John F. Cipollo, United States Pharmacopeia, Guidelines For mRNA Drug Product Manufacturing And Quality Control. Bioprocess Online. Sept 15, 2023. https://www. bioprocessonline.com/doc/guidelines-for-mrna-drug-product-manufacturing-and-quality-control-0001.
- Rosa SS, Prazeres DMF, Azevedo AM, Marques MPC. mRNA vaccines manufacturing: Challenges and bottlenecks. Vaccine. 2021;39(16):2190-2200. doi:10.1016/j. vaccine.2021.03.038.
- Veronique Allusion and Frank Weir. RNA Spectroscopy methods and protocols. Springer Protocols, 2020.
Publication Detail
This article appeared in American Pharmaceutical Review:Vol. 27, No. 1Jan/FebPages: 42-44
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