In this white paper, we discuss the challenges and opportunities associated with nucleic acid encapsulation technologies with a particular focus on mRNA encapsulation. We also share some of our insights into the key requirements for our LNP toolbox and how this approach facilitates a low-risk transition from pre-clinical stages to cGMP production.
Introduction
Estimations for the global mRNA therapeutics market size (post- pandemic) range from $5.1 to $11.75 billion in 2023, and the market is expected to grow at a 9–17% compounded annual growth rate (CAGR) from 2024 to 2033. The driverDriver for this growth is the promise that mRNA-based drugs could be used beyond prophylactic vaccines in treatment modalities such as therapeutic vaccines (i.e. oncology and autoimmune), immunomodulators, as well as cell and gene therapies. In August 2024, there were 980 active studies of mRNA therapeutics to treat a range of diseases. However, a major challenge in realizing the promise of mRNA therapeutics is being able to develop methods for manufacturing and safe delivery.
Naked mRNA is easily degraded, resulting in low levels of protein production. Encapsulation of mRNA in lipid nanoparticles (LNPs) has been utilized to improve delivery and protect mRNA from extracellular degradation. However, the production and encapsulation of mRNA therapeutics in LNPs under current Good Manufacturing Practice (cGMP) is challenging, and the efforts to deliver drug product (DP) are often underestimated.
Successful LNP encapsulation relies on several interconnected requirements; these includinge enablers such as a robust quality system with an established supply chain, a team experienced with LNP manufacturing, as well as the associated activities of Quality Control and Quality Assurance. Collaborative workflows with integrated analytics support process development to deliver robust encapsulation technologies that can be transferred to cGMP manufacturing at clinical and commercial scales.
Figure 1
Enablers of an LNP Platform for cGMP manufacturing
Encapsulation of mRNA and other related nucleic acid therapeutics
Several nucleic acid modalities share characteristics such as good solubility in water, a negative charge, and an acidic nature, which allow similar technologies to be used for encapsulation.
Readily available and well-characterized systems have been used to ensure a safe and efficient delivery of encapsulated mRNA to billions of people worldwide during the COVID-19 pandemic. However, the encapsulation technologies used for COVID-19 vaccines have limitations regarding flow and working-concentration ranges. These limitations may lead to a “scale-out” rather than a “scale-up” to produce larger batch sizes. Fortunately, mixing technologies are now available that allow easier scale-up and increase process robustness by widening the working flow ranges. This allows higher working concentrations while using smaller volumes of buffer and solvent.
Table 1
Nucleic acid modalities can be encapsulated alone or combined to be delivered as therapeutics.
Manufacturing of Lipid Nanoparticles
LNP manufacturing requires adaptation to the payload to be encapsulated and the lipids to be used for the encapsulation. To allow for fast on-boarding of new encapsulation projects, Lonza developed a toolbox of manufacturing and analytical technologies allowing to assess potential Critical Process Parameters.
Development of an LNP Toolbox
To establish a manufacturing LNP toolbox, Lonza evaluated several encapsulation and downstream processing technologies. In addition, several formulation options were tested, including different storage matrices. Besides acceleration of the onboarding of new projects this approach also has the potential to reduce the risk of reworking formulations, which could result in delaying entire development programs by several months.
As part of the analytical toolbox's development, methods were established to assess quality attributes such as lipid quality, lipid composition, and lipid/payload concentration. Also, methods for determination of the N/P ratio (ratio of positively charged polymer amine groups to negatively charged nucleic acid phosphate groups) and lipid molar ratio were developed.
The Importance of Potential Critical Process Parameters (pCPPs)
The transfer from Research and Development to cGMP manufacturing requires information on process variables impacting the quality of the final product. For Phase I materials, pCPPs refer to process parameters that could influence the quality of the product (e.g. buffer flow rates and ratios, buffer types and concentration, inline dilutions (ILD)). They are potentially critical as the understanding of the manufacturing process and its impact on product quality is still evolving. An early definition of pCPPs is vital for both clinical and commercial manufacturing success, as it prevents delays, supports a smooth transition from trials to full-scale production, and aids in regulatory approvals by establishing strong process controls from the start.
As the drug development progresses into later stages (e.g., Phase II and III), more data is collected through process characterization and optimization studies. This helps to establish a clearer understanding of which parameters truly impact the product's Critical Quality Attributes (CQAs). Through strict risk assessments and process validation/characterization activities, a pCPP can be confirmed as a Critical Process Parameter (CPP) if it is demonstrated that variations in this parameter have a significant effect on the CQAs of the product. Once a pCPP is confirmed as a CPP, it must be tightly controlled, and its status is documented in regulatory filings (e.g., in the Chemistry, Manufacturing, and Controls (CMC) section of a New Drug Application (NDA) or Biologics License Application (BLA)).
An efficient determination of pCPPs can be achieved through a well-designed and executed Design of Experiments (DoE) approach to identify parameters with the greatest impact on the formulation and tailor the process to match product-specific requirements.
Case Studies
The following three case studies illustrate how Lonza’s LNP Toolbox provides the tools to identify and monitor pCPPs from early development:
Case Study 1: Lipids raw materials characterization/incoming goods control
Case Study 2: Process monitoring and LNP characterization
Case Study 3: Evaluating a LNP next generation mixing device for cGMP
Case Study 1: Lipids raw materials characterization / incoming goods control
In this case study, we explored how the quality of a lipid affects quality attributes of an mRNA/LNP formulation, and how an incoming goods control and initial characterization can influence the selection of a phase- appropriate lipid supplier.
To assess the quality of a lipid for an LNP formulations, Lonza uses (among others) affinity assays with LC-UV-CAD and/or LC-MS. In this case study, lipids from one supplier triggered an agglomeration between mRNA and lipid impurities (Figure 2, blue chromatogram), negatively affecting quality attributes of the product. As also shown later (Ffigures 7 and 8), the agglomerations also impacted stability and potency . By choosing another supplier for the lipid, the impurity profile (Figure 2, black chromatogram) was much improved, and lipid-impurity agglomerations were avoided.
These results demonstrate why it is critical to have a robust supply chain with a range of suppliers and availability for different lipid quality grade options. It also shows the importance of having compatible in-house equipment and analytics to assess different lipids and perform stringent income good controls of critical raw materials.
Figure 2
Quality assessment of mRNA/LNP formulations using IP-RP coupled with a UV detector for the analysis of payload
integrity and mRNA-Lipid aggregates.
Case Study 2: Process monitoring and LNP Characterization
The encapsulation process can be monitored across different process steps (Figure 3), helping to identify pCPPs. The process is supported by analytics at each stage, which are designed to identify potential issues. This approach is particularly pertinent for the downstream stages, but it can also be employed to identify issues with the payload affecting pCPPs.
To gain deeper insights, physico-chemical and biophysical parameters of an LNP need to be assessed. At Lonza, we routinely measure LNP particle sizes, particle distribution Polydispersity Index (PdI), and the Zeta Potential using Dynamic Light Scattering (DLS). Extended characterization studies include the measurements of the pKa, layer thickness, and additional particle morphology is investigated via Cryogenic Electron Microscopy (CryoEM). Although CryoEM is not required by regulators, it is a powerful tool providing LNP quality evidence for regulatory submissions. The in-depth physicochemical analysis is completed with bioassays to determine the LNP-DP safety and potency.
It is also important to determine how the LNPs behave when exposed to “stress” conditions. At Lonza, we can perform accelerated stability and forced degradation studies (up to 6 months) to determine the effects of temperature, pH, oxidation and mechanical stress. One example is the execution of freeze-thaw and hold-up studies to determine if CQAs are affected during the manufacturing process. Finally, we perform transport stability studies to determine the best storage matrix (e.g. screening different buffers).
Figure 3
Typical LNP process design workflow.
DPI – Drug Product Intermediate
IPM – In-process Monitoring
EtOH – Ethanol TFF – Tangential Flow Filtration
ILD – In-line Dilution
Case Study 3: Evaluating an LNP next generation mixing device for cGMP
In this case study, we evaluated various mixing technologies for their effectiveness in nucleic acid encapsulation. The choice of mixing technology is governed largely by costs, materials, staff expertise and facilities available during production (see table 2). Microfluidics and T-piece derived technologies are well-characterized and part of the Lonza encapsulation offering. These legacy technologies are complemented by the FDmiX system from Fluid Dynamix, for which we have shown benefits that result in improved and faster cGMP scale manufacturing.
Table 2
Mixing technologies for LNP encapsulation of nucleic acids available at Lonza.
The FDmiX encapsulation technology uses oscillation to achieve instantaneous, homogenous mixing of emulsions, such as lipids and nucleic acid payloads (Table 3).
This technology was designed from the beginning having cGMP implementation use as the main goal. This case study outlines discusses the steps required to confirm that the FDmiX achieves the process and quality requirements to be used in clinical and commercial manufacturing. Determination of the FDmiX technology operational ranges and final product quality attributes was performed by executing 34 encapsulation batches over a period of 3 days on different lipid formulations.
Table 3
Testing conditions and analytical panel used to assess the FDmiX technology for mRNA encapsulation.
Results from the flow rate assessment (Figure 4) demonstrate that particle sizes and encapsulation efficacy remain stable across a broad range of flow rates. Additionally, no changes were observed on further quality attributes such as mRNA integrity, mRNA and lipid content, lipid composition, pH and zeta potential. These results show that the FDmiX platform supports a large range of operational flow rates (up to liters per minute)), while ensuring a predictable scale-up from R&D scale to a cGMP environment, thus minimizing development timelines. In other words, upon identification of the best LNP composition and formulation at small volumes and low flow rates (R&D) it is possible to increase the flow rate to commercially relevant L/min directly without affecting the mRNA quality and LNP CQAs.
Figure 4
Particle size distribution (by Dynamic Light Scattering), and encapsulation efficiency, (determined by the Ribogreen assay), for LNPs manufactured using the FDmiX platform. Encapsulation was conducted at flow rates ranging from 50 to 1000 mL/min and for three different mRNA concentrations: 0.2, 0.5, and 0.8 mg/mL.
End product quality assessment included exact determination of particle size, PdI and morphological characterization by CryoEM. Results shown in Figure 5 demonstrate consistent particle diameter sizes (46–63 nm/median: 44–58 nm) across all flow rates and concentrations tested. Particle morphology is also homogenous with a high number of quality solid core particles at 86%, compared to a significantly low amount of biphasic split at 11% and biphasic dense comprising only 3% of the particles. These results, taken together with the mRNA stability, suggest that homogenous particles manufactured with the FDmiX may correlate with increased product stability.
Figure 5
Particle morphology (left) and particle size distribution (right) of LNPs manufactured using the FDmiX platform, as visualized by cryogenic electron microscopy (Cryo-EM).
Results from the freeze thaw studies (Figure 6) show stable particle size, mRNA integrity, encapsulation efficiency and mRNA content across freeze-thaw cycles. Importantly, the use of the FDmiX maintains mRNA integrity and provides long- term stability for the payload. In combination, these observations demonstrate that even at high flow rates, the mixing is gentle and not prone to degrade the nucleic acids by shear forces.
Particle stability was further determined by an accelerated stability study, the results of which results (Figure 7) demonstrate that particle size, PdI, mRNA integrity and encapsulation efficiency are stable at temperatures equal to or below 4–8 degrees, over the period of 6 months in which the study was conducted.
Particle stability was further determined by an accelerated stability study, the results of which (Figure 7) demonstrate that particle size, PdI, mRNA integrity and encapsulation efficiency are stable at temperatures equal to or below 4–8 degrees, over the period of 6 months in which the study was conducted.
Process advantages from the GMP-ready FDmiX are exemplified by the robust and linear (predictable) scale-up and scaledown across an extended range of flow rates from R&D scale (screening) at 10 mL/min to commercial manufacturing scale (> 1 L /min). The technology also allows the encapsulation of nucleic acid payloads and lipids at higher concentrations than legacy technologies, which, combined with L/min flow rates results in lower operating volumes, faster downstream and shorter time in facility. In short, our studies have demonstrated the FDmiX technology offers significant process advantages, and produces LNP-mRNA with increased stability, high particle homogeneity and consistent Critical Quality Attributes (CQAs).
Figure 6
Stability of LNPs manufactured using the FDmiX platform over 5 freeze thaw cycles.
Future trends
The current optimization of LNPs is focused on enhancing specificity and improving the safety profile. Innovations in this field include the modification of the LNP surface with affinity ligands, such as antibody fragments. We are also are aware of novel chemistries/modalities such as polymeric, polyplexes and dendrimer formulations to improve nanoparticles stability and decrease undesired immune reactogenicity. It is exciting to see the very promising results related to the enhancement of the stability of formulated drug products through spray drying and lyophilization techniques. The implementation of these innovations requires a comprehensive and systematic approach, with consideration given to the most fundamental aspects. Offering a future-proof toolbox well-equipped to integrate new chemistries is a key criterion when choosing a CDMO for LNP manufacturing.
Figure 7
Particle size and polydispersity index (top) and mRNA integrity and encapsulation efficiency (bottom) of LNPs
manufactured using the FDmiX platform after an accelerated stability study covering up to 6 months where LNP
bulk material was stored at 40°, 22°C, 2-8°C, -20°C and -80°C.
Conclusions
RNA-based therapeutics require delivery systems that are able to support their therapeutic objectives. The efficient encapsulation of nucleic acids at commercial scale requires a thorough development approach, starting at early stages. In light of the multitude of CDMOs offering LNP services, it is of paramount importance to collaborate with a partner that not only provides an array of encapsulation technologies but also possesses the requisite expertise to facilitate the onboarding of bespoke solutions. This must be complemented with an in-depth knowledge of the processes, which is only achievable through expert use of state-of-the-art analytical techniques and experience to navigate many interconnected process and quality requirements. At Lonza, we have extensive experience in stringent quality control of raw materials, as well as early definition of potential CPPs, as demonstrated by the methods described in this article. We also provide a selection of encapsulation technologies. Our approach is underpinned by established quality systems, scalable platforms and process know-how. Biopharma companies partnering with us can shorten their development timelines, reduceing risk, and thus improve their chances of achieving cGMP readiness and manufacturing success with LNP/mRNA-based therapeutics.
References
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