(Image 1 by CordenPharma) LNP Assembly Technologies at our GMP LNP facility CordenPharma Caponago in Italy.
Nucleic acid-loaded Lipid NanoParticle (LNP) formulation manufacturing starts with the material preparation step. For that, the chosen lipids are dissolved in the organic phase, typically ethanol, and nucleic acids such as RNA types or plasmid DNA are dissolved in the acidic aqueous phase. Once both phases are prepared, the following step is to mix those by nanoprecipitation techniques including microfluidic systems, T-mixing, or customized mixing technologies. After producing formulations, a buffer exchange step is followed to adjust the pH to a neutral value and to help dilute the ethanol content in the nanoparticle suspension. For the next steps, which are purification and diafiltration, Tangential Flow Filtration (TFF) systems are used to remove residual solvents and increase the concentration of the formulation to the final drug product in the dedicated storage buffer. After a final bioburden reduction and sterile filtration, the bulk drug product is ready for shipment or Fill and Finish.
Although cGMP manufacturing of LNPs comprises straightforward unitary manufacturing steps, there are many key parameters to consider before starting clinical manufacturing when developing a process, performing a technology transfer, or producing a formulation.
For the first step of clinical manufacturing, lipids are carefully weighed and solubilized, with attention paid to the unique solubility characteristics of each lipid component. For small quantities, a Class C dispensing room is utilized, which is equipped with a Laminar Airflow hood rated at ISO 5. For larger quantities, stainless steel vessels are employed to maintain the integrity and quality of the lipids being processed (Image 2). In the same room, the aqueous phase containing the payload is prepared.
(Image 2 by CordenPharma) Four stainless steel vessels located in an ATEX Class C LNP
assembly room for organic phase preparation with a capacity of up to 30 liters.
They utilize CIP/SIP technology and have the capability to be expanded to accommodate
300 liters and more, depending on the project’s requirements.
Once material preparation is set, the assembly of the aqueous phase with the organic phase is next. During that moment, the pre-solved lipids and nucleic acids get mixed into a solution consisting of both organic solvent and acidic aqueous phase (Image 1). The aqueous phase volume percentage used in the formulation is usually 2 to 3 times more, compared to the volume percentage of the total lipid mixture. The resulting change in the polarity of the solution forces the formation of nanoparticles by nanoprecipitation.
Before starting clinical manufacturing, it is critical to explore the stability of the formulation when adjusting the balance between the pH of the formulation and the buffers to keep the drug loading and quality of the formulation as high as possible throughout all the steps of manufacturing. This is because even a small change in the pH can significantly cause re-ionization of the ionizable lipids after the formation of nanoparticles is complete. Eventually, that impacts the nanoparticle assembly step as well as each step of the process development. Therefore, investigating the operational parameters such as flow rate ratios, and choosing all the intermediates such as the dilution buffer type, strength, and ratio, together with storage buffer conditions, scalable mixing technology and stability of the formulation at different conditions, are all important to developing robust Process and Quality Control (QC) methods which lead to achieving the desired Target Product Profile (TPP) and understanding the behavior of the formulations upon scale-up.
Mostly, the importance of the balance between the pH and the buffers unravels during the setting up of a TFF method. This method involves multiple parameters that complicate the maintenance of the TPP values before and after the purification step. When establishing a TFF method, it is essential to focus on maximizing xRNA yield while ensuring that the chosen membrane type remains unblocked. Additionally, it’s important to maintain consistent particle size, polydispersity index (PDI), and encapsulation efficiency of the cargo. To do that, CordenPharma’s Drug Product Development team evaluates different membrane types, pore sizes (MWCO), TFF operational parameter optimization such as transmembrane pressure (TMP), process time, buffers etc. before manufacturing engineering batches to ensure seamless translation from process development to clinical studies (Image 3).
(Image 3 by CordenPharma) Repligen KTM1600® Tangential Flow Filtration system
(TFF) unit that enables UltraFiltration (UF) and DiaFiltration (DF), capable of processing
large and small-scale batches equipped with fully disposable flow-paths and
disposable recirculation vessel temperature control (5-25 °C).
The next step in clinical manufacturing involves moving the formulation to the bioburden reduction and sterile filtration stage (Image 4), after which the bulk drug product is ready for shipment or the fill-and-finish process. If the filling is done at our site, then the sterile filtration step is done at the filling area. Before proceeding, the R&D scientist team evaluates which membrane material and surface area are optimal for this step. They do this by assessing the maximum filtered volume during non-GMP studies to ensure that optimal conditions are utilized.
(Image 4 by CordenPharma) Bioburden reduction step
under grade A laminar flow at our GMP LNP facility.
The analytical team plays also a crucial role in drug product development by ensuring quality, safety, and efficacy through rigorous testing and characterization. They develop and validate analytical methods to assess chemical composition, physical properties, and biological activity. Stability testing is performed to determine shelf life and identify potential degradation pathways under various environmental conditions.
For LNP-based formulations, quality control measures should include an assessment of physical properties to detect potential aggregation, an evaluation of encapsulation stability to ensure cargo retention, and analysis of chemical stability, including RNA integrity, lipid integrity, and lipid content. Finally, the team ensures regulatory compliance by preparing analytical reports and supporting submissions to agencies.
Particle size and Polydispersity
Dynamic Light Scattering (DLS) coupled with MALS (Multi-Angle Light Scattering) technology by Malvern Panalytical Zetasizer Ultra is available at CordenPharma’s R&D Lab.
DLS is useful for assessing average particle size and polydispersity, allowing the detection of aggregation, which can occur due to storage conditions or improper handling. An increase in size and PDI often signals potential quality issues. When paired with zeta potential measurements, DLS provides further insights, as RNA release can lead to shifts in zeta potential. Similarly, premature PEGylated lipid detachment may reduce shielding, resulting in additional changes in zeta potential.
Drug Loading
For a standard and rapid assessment of RNA or DNA encapsulation efficiency, Ribogreen or Picogreen assays are commonly used at our R&D lab. These fluorescence-based assays selectively bind nucleic acids and exhibit increased fluorescence upon interaction, allowing for the quantification of unencapsulated RNA or DNA in the presence of an appropriate detergent to release encapsulated material.
Chemical Stability
While Ribogreen and Picogreen assays are commonly used for rapid assessment of RNA or DNA encapsulation efficiency, they have certain limitations. For example, if the RNA integrity is not high enough, there will be a direct impact on the potency of the formulation, so it is quite important to develop more precise and robust quantification at an industrial scale. In this case, we also employ Capillary Electrophoresis, which provides high-resolution analysis of RNA/DNA size, integrity, and purity, as well as UPLC and HPLC methods for accurate separation and quantification. These techniques offer high sensitivity, reproducibility, and compliance with regulatory requirements.
(Table 1 by CordenPharma) A snapshot from a case study: exploring the
stability conditions for a pDNA-LNP formulation Post-TFF.
As demonstrated in the example above (Table 1), storage conditions have a significant impact on nucleic acid stability, leading to rapid degradation that ultimately reduces formulation potency. To mitigate this risk, the scientific team employs rigorous quality control measures and optimized storage strategies to preserve the integrity and efficacy of the LNP formulation for the success of further planned studies. Their expertise ensures that formulations remain stable under appropriate conditions, minimizing potential degradation and maintaining therapeutic performance.
Lipid stability and composition are also critical factors in formulation development. A comprehensive analytical approach involves HPLC-CAD coupled with a Charged Aerosol Detector (CAD) to accurately quantify the individual lipid components within a given formulation at the R&D lab.
Multiple analytical instruments are available at the R&D development lab in CordenPharma Caponago, Italy. We are also making investments in 2025 to expand our scientific team and analytical capabilities, which currently consist of 28 scientists specializing in sterile drug product formulation and process development, analytical methods development and validation, scale-up, and cGMP manufacturing studies, all dedicated to supporting our clients with the complex modalities needed to bring their formulations to global commercial markets.
With a strong track record in producing parenteral sterile solutions and emulsions for aseptic filling, we offer a variety of delivery formats, including vials, pre-filled syringes, and ampoules. Our cutting-edge injectable facility, advanced equipment, and highly skilled personnel ensure the highest quality and safety standards. To streamline production, our clinical and commercial scale Fill & Finish facility supports seamless manufacturing, from cargo molecule encapsulation to final packaging, ensuring efficiency, reliability, and scalability. Additionally, to reduce supply chain risks, we provide integrated offerings across multiple sites, covering lipid production and final packaging, ensuring a seamless and efficient end-to-end manufacturing process.
Please find here the nanoparticle assembly and TFF systems available at CordenPharma’s cGMP LNP facility:
In terms of the main GMP LNP processing equipment, the following systems are in place:
Watch our LNP GMP Manufacturing video or contact us here for further information on LNP projects.
Dr. Serra Gürcan
Associate Director, Lipids & LNPs Platform, CordenPharma International
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