SM-102 System mRNA-LNP Formulation Standard Protocol

II. Standard Operating Procedures (SOP)

2.1 Preparation of Stock Solutions

2.1.1 Lipid Stock Solutions

• Procedure: Weigh SM-102, DSPC, Cholesterol, and DMG-PEG2000 separately. To accurately prepare a 10 mg/mL stock solution for each lipid, precisely weigh the required mass of the lipid (e.g., 50 mg) into a clean glass vial, and completely dissolve it in the corresponding volume of absolute ethanol (e.g., 5 mL). Vortex the mixture thoroughly until a completely clear and homogeneous solution is achieved. All prepared lipid stock solutions can be aliquoted, tightly sealed, and stored at -20°C for future use.
• Critical Precautions: SM-102 is an ionizable lipid and typically presents as a viscous liquid. During preparation, the weighing method should strictly be utilized for quantification to prevent pipetting volume errors. Furthermore, cholesterol has limited solubility in ethanol; its stock solution must be incubated and kept warm (>37°C) to maintain proper fluidity, and it should be transferred promptly upon use to avoid precipitation caused by cooling.

2.1.2 mRNA Stock Solution

• Procedure: Dilute the mRNA to a target concentration of 166.7 μg/mL using 100 mM sodium acetate buffer (pH 5.0).
• Critical Precautions: This specific concentration is precisely calculated based on the total lipid concentration and the optimized lipid/mRNA weight ratio. It is imperative to ensure that the mRNA is completely dissolved within the acidic buffer to guarantee its phosphate backbone is fully and uniformly negatively charged.

2.2 Formulation Calculations

To systematically prepare the target product, the volume of each component must be calculated according to the following critical steps:

• Determine Total Lipid Mass: For example, to prepare 1 mL of the final lipid mixture solution (with a total lipid concentration of 10 mg/mL), the required total lipid mass is exactly 10 mg.
• Determine Lipid Component Volumes: Based on the designated molar ratios and molecular weight conversions to mass concentrations, the precise volumes of each stock solution per milliliter of the lipid mixture are formulated as follows:
  • SM-102 (10 mg/mL): 572 μL
  • Cholesterol (10 mg/mL): 240 μL
  • DSPC (10 mg/mL): 127 μL
  • DMG-PEG2000 (10 mg/mL): 61 μL
  • Total volume: 1 mL
• Determine Required mRNA Mass and Volume: Under the specific condition of a lipid/mRNA weight ratio of 0.05, the necessary mRNA mass = 10 mg × 0.05 = 0.5 mg. Consequently, the required volume of the mRNA solution (at 166.7 μg/mL) = 0.5 mg ÷ 0.1667 mg/mL = 3 mL.
• Note: The chosen lipid:mRNA weight ratio directly impacts encapsulation efficiency. Researchers can prepare alternative weight ratios as substitute formulations, and corresponding adjustments should be systematically applied.

2.3 Mixing Processes

Three conventional methods are widely utilized to achieve rapid solution mixing: the pipette mixing method, the vortex mixing method, and the microfluidic mixing method. Notably, microfluidic devices possess the capability to mix rapidly in a highly controllable and repeatedly consistent manner, thereby generating homogeneous LNPs with exceptionally high encapsulation efficiency. In contrast, the pipette and vortex mixing methods might yield more heterogeneous LNPs, generally exhibit lower encapsulation efficiency, and are significantly more prone to operational variability.

2.3.1 Pipette Mixing Method

• Mixing: Aspirate 3 mL of the prepared mRNA solution (aqueous phase) and rapidly inject it into 1 mL of the lipid mixture solution (organic phase). Immediately use the pipette to rapidly pipette up and down, mixing vigorously for 20-30 seconds.
• Incubation: Allow the resulting mixed solution to stand and incubate at room temperature for 15 minutes.

2.3.2 Vortex Mixing Method

• Mixing: On a vortex mixer, vortex 3 mL of the mRNA solution (aqueous phase) at a medium speed setting. Subsequently, rapidly inject 1 mL of the lipid mixture solution (organic phase) directly into the actively vortexing solution. Continue to vortex the resulting dispersion for an additional 20-30 seconds.
• Incubation: Allow the resulting mixed solution to stand and incubate at room temperature for 15 minutes.

2.3.3 Microfluidic Mixing Method (Highly Recommended)

• Preparation: Separately aspirate the calculated 1 mL of lipid ethanol solution and 3 mL of mRNA-sodium acetate buffer, securely injecting them into their respective corresponding syringes.
• Critical Filter Step: Prior to loading, it is absolutely essential that all solutions be meticulously filtered through a 0.22 µm filter membrane (utilize PTFE filters for the lipid phase, and PES or MCE filters for the aqueous phase).
• Chip Pre-flushing: Separately aspirate blank buffer and pure ethanol, and connect them to the microfluidic chip. Run the instrument to completely purge any air and residual storage solution from the microchannels, ensuring the flow path is perfectly clear. This preliminary step is critical for guaranteeing ultimate mixing uniformity.
• LNP Synthesis: Set the system's total flow rate; the recommended initial starting value is 12 mL/min (comprising an aqueous phase flow of 9 mL/min, and a lipid phase flow of 3 mL/min). Note: Operators can strategically alter parameters such as the flow rate ratio and total flow rate to finely tune LNP properties, including particle size.
• Sample Collection: Discard the initial effluent from the startup stage (approximately the first 100-200 µL), and subsequently collect the product generated during the stable, continuous flow stage.
• Product Characteristics: The properly collected LNP solution should visually exhibit a uniform, light blue opalescence (characteristic of the Tyndall effect), which serves as a macroscopic indicator that nanoparticles have been successfully formed.

2.4 Purification and Storage

• Incubation: Following collection, incubate the mixed LNP solution at room temperature for a maximum of 15 minutes to facilitate and promote particle structure stabilization.
• Dialysis: Transfer the incubated LNP solution into a suitable dialysis device (e.g., a 10-20 kDa MWCO dialysis bag or cassette), and dialyze against a 1× PBS (pH 7.4) buffer for exactly 2 hours to efficiently remove residual ethanol and fully exchange the buffer system.
• Filtration: The post-dialysis LNP solution must then be meticulously filtered through a 0.22 µm sterile filter membrane to ensure sterilization and to thoroughly remove any potentially existing aggregates.
• Storage: The final filtered LNP samples can be safely stored at 4°C for short-term utilization. Should long-term preservation be required, it is strongly recommended to incorporate an appropriate cryoprotectant prior to freezing the samples at -80°C.