Beyond Lipids: The Science of LNP Manufacturing and Fill Finish

Lipid nanoparticles (LNPs), composed of lipids and encapsulating active pharmaceutical ingredients (APIs), have gained considerable attention due to their ability to overcome bioavailability and stability limitations associated with traditional drug delivery methods. These tiny vesicles offer a protective environment for fragile compounds, enable controlled release of drugs, and enhance cellular uptake. LNPs are transforming the way treatments are delivered.

Manufacturing LNPs requires a multidisciplinary approach, combining expertise from pharmaceutical formulation, lipid chemistry, engineering, and quality control. From the selection of suitable lipids and APIs to the optimization of particle size and scale-up processes, numerous factors influence the successful manufacturing of LNPs. Furthermore, the challenges extend beyond the manufacturing stage, as the sterile filling process demands meticulous attention to detail for achieving and maintaining product integrity and safety.

In this blog, we will cover the basics of LNP manufacturing, including:

  • How LNPs are produced
  • How the manufacturing process can be optimized to achieve desired particle sizes distribution and encapsulation efficiency.
  • And, share challenges to scaling up an LNP production process

We will also dive into the process of sterilizing and aseptically processing these drug products while mentioning special modifications for shear-sensitive drug products and techniques to limit drug product loss.

Therapeutic delivery methods are constantly evolving, and advancements in LNPs to deliver payloads are revolutionizing modern medicine. Staying informed of manufacturing and sterile filling challenges is critical to developing an effective drug product formulation and successful fill finish strategy.

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How Lipid Nanoparticles are Created

LNPs are composed of four types of fats:

  • Cationic or Ionizable Lipids
  • Cholesterol (or structural lipids)
  • Phospholipids
  • And PEG-Anchored Lipids

Cationic or Ionizable lipids typically constitute 30 – 50% of the LNP, and they are charged so they become attracted to the payload and encapsulate them. If the LNP was created to deliver a negatively charged API, such as an oligonucleotide, then positively charged ionizable lipids would be used to surround the oligos, encapsulating them in a fatty barrier.

Cholesterol makes up about 20 – 50% of the LNP. This fat helps improve intracellular delivery and fill in the gaps between lipids, improving the stability of the capsule.

Phospholipids, constituting approximately 10-20% of the formulation, help package nucleic acids and stabilize the LNP. They are also thought to help facilitate the release of the nucleic acid payload into cells.

PEG-anchored lipids (or PEG-lipids) are located on the outside of the LNP and make up only 1 – 2% of the total lipid formulation. They are typically charged to repel other LNP capsules, preventing aggregation and reducing particle sizes. PEG-anchored lipids also help prevent phagocytic immune cells from ingesting and destroying the LNPs in vivo.

Figure 1: A lipid nanoparticle (right) is comprised of four types of lipids: cationic or ionizable lipids, phospholipids, PEG-anchored lipids, and cholesterol.

The proportion of these lipids has a significant impact on the LNP’s efficacy and stability, and the optimal formulation will need to be worked out in formulation development.

To encapsulate API in an LNP, a solution of each unique lipid is prepared in an alcohol (typically ethanol), then combined into one lipid formulation at the ideal proportions. This solution is gently mixed into a homogenous solution. At the same time, a separate aqueous solution of API is prepared, and the API concentration is measured.

The two solutions – the lipid formulation and the aqueous API solution – are then gently mixed together. Positive displacement pumps for each solution dispense each solution at a controlled flow rate to regulate the ratio. The mixing process is performed in a mixer that is designed to gently homogenize the two solutions in a process that reduces shear and maintains tight control of the internal flow rate.

By mixing the two solutions in this manner, the lipids will collect around the API payload and form protective capsules.

Figure 2: Microfluidic mixing method for producing LNPs around an API payload.

This process of manufacturing LNPs is called microfluidic mixing. Microfluidic methods use non-turbulent, laminar flow to provide a controlled but rapid mixing of the lipid formulation and the API payload. There are several mixers that are available for microfluidic methods of LNP production, and they each have unique architectures (see Figure 3).

Microfluidic mixing has several advantages and a few limitations:


  • It is easy to tune nanoparticle size by adjusting the formulations and flow rate.
  • The process is reproducible and offers consistent formulations across batches.
  • It is a promising method for scale-up. Commercial instruments are available to produce batches from 0.5 mL to 10 L.
  • It is easy to combine the process with downstream purification (for example, tangential flow filtration).


  • Only able to encapsulate tough, small molecules and specific bioagents (e.g. nucleic acid) that are tolerant to alcohol solvents
  • Two- phase encapsulation only – any biomolecules, such as proteins, cannot be encapsulated directly by this process
Figure 3: Microfluidic mixers have unique yet specific architectures that are designed to limit shear, provide rapid mixing, and maintain controlled flow rates. (a) T-junction mixer (b) Biofurcating mixer (c) Staggering mixer (d) Baffle mixer

There are several other methods that produce LNPs that won’t be discussed here but are listed below:

  • Impinging jet mixing
  • Continuous manufacturing of LNP
  • Thin film hydration and extrusion
  • Thin film hydration and extrusion sonication
  • Thin film hydration and high-pressure homogenization
  • Ethanol injection

Overcoming Challenges of Creating Lipid Nanoparticles

When particle size becomes a problem

Exceptionally large particles reduce delivery and efficacy of the drug product in vivo. They can also clog the syringe needle while administering the medication. Ensuring that LNPs remain within a desirable particle size range is critical to maintaining the efficacy of the drug product.

Large LNP capsules

For a drug to be effective, it must reach its intended destination. LNPs are vehicles for APIs, and larger LNPs have reduced cellular uptake, poor tissue penetration, and limited circulation time that all reduce the efficacy of the drug product. Larger LNPs are also observed to have poor stability compared to smaller capsules, because they are more prone to aggregation and cleavage.

Finally, the wider the size range of the manufactured LNP, the less predictable the outcome of the drug. Different size LNPs will hold different volumes of their API payload, and a large range of different size LNPs will lead to inconsistencies in drug delivery.

Controlling the particle size distribution of the LNPs that are produced is critical to enhancing the drug product’s stability and efficacy. While the ideal size range for LNPs may vary depending on the specific application, target tissue, and cargo being delivered, it is crucial to optimize the size and properties of LNPs to achieve the desired therapeutic outcomes and minimize potential drawbacks.


Large LNPs can affect particle size, and so can aggregation. When LNPs begin to collect and “stick” together, they form extraordinarily large particle sizes. These sizes can be large enough to clog the syringe needle while the drug product is being administered to a patient.

LNPs are typically created to have a charged outer membrane so they repel each other (like repels like), reducing aggregation. So, agglomeration is more likely to happen when the charge of the LNPs is close to zero.

Fortunately, LNPs range in size from 20 to 200 nanometers (or .02 to 0.2 microns), and sterilizing filters have pore sizes of 0.22 microns. Anything larger than 0.22 microns, including LNP aggregates, will collect on the filter and will not flow into the final drug product formulation. While this removes the concern for having large aggregates in the final drug product formulation, an excessive presence of sizable aggregates will cause the concentration of the LNPs drug product formulation to drop significantly after sterile filtration. Too many large aggregates can also clog the sterile filter and end filtration. Both situations put the project at risk and should be avoided by developing the formulation to reduce aggregation.

Particle dispersion index (PDI) is used to assess the presence of large aggregates that can pose a risk to a formulation project. During quality testing of the manufactured LNPs, the average particle size and the size dispersion between the particles are determined. Tighter dispersions indicate greater uniformity and fewer large aggregates. Conversely, wider dispersions suggests both very small and very large particles are present, including many larger aggregates. If the PDI is greater than 0.2, then dispersion is very wide, and process or formulation changes are needed to reduce particle sizes.

Figure 4: Particle dispersions of two sample LNP formulations. The larger the dispersion and PDI, the greater number of potentially problematic agglomerates in the formulation.

Controlling particle size

Variations in lipid composition, mixing methods, and processing conditions all affect particle size. Controlling these variables and optimizing the process is critical to developing a manufacturing process that will create reproducible results. Fortunately, many of these processes are easy to adjust using a microfluidics method to manufacture LNPs.

The LNP manufacturing process that includes mixing and solvent removal can take six hours to a day to perform, and both particle size and encapsulation can experience complications at various stages, whether it occurs early, late, or anywhere in between.

During development, samples will be pulled throughout the mixing process and analyzed (via HPLC) to measure the lipid composition, API concentration, and to measure particle size (via a zetasizer). Advanced microfluidic systems will have process analytical technology (PAT) associated with it. This technology offers real-time, non-destructive readings for all quality testing requirements that were mentioned and maintains constant monitoring without a need for pulling samples.

If particle sizes are too large or desired encapsulation is not achieved, there are several means to optimize the process to produce smaller, more uniform LNPs:

  • Tweak the lipid formulation. Adjusting the concentration of each component will affect the resulting LNP’s stability, likelihood of aggregation, and feasibility to form a capsule around a payload.
  • Change the concentration of the lipid formulation and API solution. Too high of particle concentrations can lead to clogging, while too low of concentration can result in insufficient collisions. Optimizing the particle loading concentration is critical to achieving desirable particle sizes.
  • Adjust the channel dimensions in the microfluidic mixer.  Smaller channels promote faster and more efficient mixing due to higher shear rates and reduced diffusion distances, which may lead to reduce particle sizes. However, too small of channels may lead to aggregation and increase particle sizes. This will need to be optimized for the process.
  • Modify the flow rate. Higher fluid velocity helps disperse particles, leading to greater mixing, but too high of flow rates can cause clogging or unstable flows and greater shear. This could cause aggregation and greater particle sizes.

Scale-up challenges

Producing uniform LNPs is a delicate process, where small variations in the formulation and mixing conditions can have a significant impact on the properties and performance of the LNPs that are created.

The greatest challenges to scaling up a LNP production are:

  • Process reproducibility – As the scale is increased, it becomes more challenging to control the mixing of the lipid formulation and API solution while maintaining tight control over flow rates and shear. Achieving homogeneity and effective mixing will also be more challenging as the process is scaled up.
  • Batch-to-batch consistency – Scaling up will likely lead to differences in raw materials, equipment, or process parameters that can lead to variations in particle size, encapsulation efficiency, and drug loading. Controlling and minimizing these variables is essential.
  • Manufacturing Equipment – Larger batch sizes will likely require new equipment to scale the process. This will involve a tech transfer to the new equipment, and potentially modifications or customization of the equipment to meet needs and maintain batch-to-batch consistency.
  • Regulatory Considerations – Scaling up LNPs production may trigger additional regulatory requirements and considerations. Safety, quality, and compliance with regulatory standards become even more critical at larger scales. Ensuring adherence to good manufacturing practices (GMP) and addressing regulatory expectations are key aspects of successful scale-up.

Addressing these scale-up challenges requires a combination of scientific understanding, process optimization, equipment selection, and rigorous quality control measures. Collaboration between researchers, engineers, and manufacturing experts is essential to overcome these challenges and successfully translate LNPs from lab-scale to cGMP production.

Sterilizing and Aseptically Filling LNPs

The process

Once LNPs are produced, the next step to the process is to formulate and fill the product into its final container so it can be administered to patients. This process involves three critical steps: buffer exchange (solvent removal), sterile filtration, and filling.

Buffer Exchange (Solvent Removal)

After the LNPs are successfully manufactured, they need to be separated from the alcohol (commonly used solvent for LNP manufacturing). The alcohol will permeate through the membrane and be replaced with the buffer solution via osmosis. This is performed via tangential flow filtration (TFF) or dynamic dialysis. During the TFF process, the LNP solution is circulated and passed over a membrane with desired molecular weight cut-off where a suitable buffer or solvent is exchanged with the formulation kept under a re-circulated phase while maintained a constant trans-membrane pressure (TMP).

While using the dynamic dialysis technique, the formulation is loaded into a dialysis membrane with a with desired molecular weight cut-off and placed into a dialysis tank that holds an exchange buffer (see Figure 5). The efficiency of dialysis is optimized by tweaking different mode of operation that includes single-pass mode, recirculation mode or a combination of both modes.    

Since most LNP drug products are temperature and shear sensitive, the product will likely be prepared in a jacketed mixing vessel, where desired temperature (between 6°C – 15°C) can be controlled using a temperature control unit. To minimize the shear stress on the formed LNPs, the buffer exchange process needs to be optimized to maintain the desired particle size distribution and encapsulation. The bulk formulation obtained post-buffer exchange will undergo quality testing to determine the concentration and quality of the LNPs and to confirm that the alcohol was removed from the product. If required further excipients will be added to meet the product target product profile (i.e. pH, osmolality etc.), and further in-process quality testing may occur before sterile filtration begins.

Figure 5: Dynamic Dialysis is performed by loading the LNP formulation into a dialysis membrane and placing this into a dialysis tank. Exchange buffer is then passed over the membrane, and alcohol is removed via osmosis.
Figure 6: Alcohol is exchanged with buffer during dynamic dialysis. Eventually all alcohol will be removed and be replaced with exchange buffer.

Sterile Filtration

The mixing vessel will have a dip tube in it to draw solution out of the mixing vessel and pump the product through two sterilizing filters. The first filter is referred to as the bioburden reducing filter and the second is considered the sterilizing filter. The product will filter into a second sterile vessel, likely also a jacketed mixing vessel, that is prepared with a dip tube and sterile connector to be hooked up to the filling line. The two filters undergo filter integrity testing prior to beginning any sterile filling activities.

Figure 7: Temperature sensitive drug product is formulated in and filtered into jacketed, glass mixing vessels to ensure the drug product formulation remains cool during activities performed in controlled room-temperature cleanrooms. Drug product will also be filled from these jacketed vessels.

Aseptic Filling

The sterilized drug product solution is connected to an aseptic filling line via a sterile connection. At Berkshire Sterile Manufacturing, all filling lines are within isolators. For isolator-based filling, the drug product solution is connected to the filling line through the isolator wall. The interior of the isolator will have already undergone sanitization via vapor hydrogen peroxide (VHP) to achieve a six-log reduction in bioburden levels. All product contact items are passed through a no touch transfer system (NTT), except for bulk vials, which are depyrogenated and introduced directly into the filling line post-sterilization.

Filling occurs in an isolator. If the product requires lyophilization, it will travel directly to the lyophilizer. Otherwise, filled drug product will exit through a RABs. Vials will exit through a dedicated exit RABs containing a capping system.

Release of the Batch

The visual inspection team will perform a 100% visual inspection and a subsequent acceptable quality level (AQL) inspection before the drug product is placed in a freezer (if applicable). During the interim, the drug product will be held in a 2-8°C or other non-frozen storage condition.

Once the drug product makes it past the visual inspection team, it will be placed in the released product chambers to await analytical quality testing, microbiology results, and a quality assurance review of the batch. If test results pass, all deviations are closed, and QA has thoroughly reviewed and approved of the completed batch record, QA will initiate a release of the drug product batch.

Managing shear-sensitivity

LNP drug products are often exceptionally shear-sensitive. They are likely to aggregate under physical stress, leading to large particle sizes, and downstream quality issue that affect the efficacy and safety of the drug product. Pump motions often cause damage and degradation, and the fill finish process needs to be adapted to reduce harmful shear forces.

A standard fill finish project will use a stir bar or overhead mixer to mix the formulation, a peristaltic pump to push drug product through redundant sterile filters, and a peristaltic or piston pump to fill drug product. These all produce shear forces that can damage and degrade LNP drug products. Shear testing can be performed to gauge the extent of the sensitivity and what changes need to be made in the process during mixing, filtering, and filling. For exceptionally shear-sensitive drug product, mixing can be slowed, or special low-shear mixing shafts and vessels can be employed. Filtration can also be slowed, or an alternate pump can be used, such as diaphragm pump, to reduce shear. And, finally, the sterile filling pump and its operation parameters can be optimized to reduce shear or a time-pressure pump can be deployed (like what we offer on our low loss fill line) to reduce shear.

Figure 8: The low loss fill process (pictured) at Berkshire Sterile Manufacturing reduces drug product loss to less than 30mL and uses a time-pressure pump to reduce shear.


API encapsulated in lipid nanoparticles offer numerous advantages, including improved bioavailability, stability, controlled release, and enhanced cellular uptake. Manufacturing these lipid-based vesicles requires a multidisciplinary approach, combining expertise from various fields such as pharmaceutical formulation, lipid chemistry, engineering, and quality control. Selecting suitable lipids and APIs, optimizing particle size, and anticipating scale-up challenges are crucial factors in successful LNP synthesis and drug product formulation.

The fill finish aspect of these drug products presents its own set of challenges. Sterilizing and aseptically processing LNPs require special considerations, particularly protecting the product from temperature and shear.

As therapeutic delivery methods continue to evolve, staying informed about manufacturing and sterile filling challenges is essential for developing effective drug product formulations and successful fill finish strategies. By keeping up with advancements and understanding the complexities involved in LNP manufacturing and sterile filling, we can harness the full potential of LNPs in delivering transformative medicines to patients.

By combining scientific knowledge, technological advancements, and meticulous attention to quality, the field of LNPs holds tremendous promise in shaping the future of medicine and improving patient outcomes.


Dr. Sarandeep Malhi, Senior Manager of Process Engineering and Formulation Development. (2023, June). Personal communication [personal interview]

Eygeris, Y., et. al. (2021). Chemistry of Lipid Nanoparticles for RNA Delivery. Accounts of Chemical Research, 55, 2-12.

Hou, X., Zaks, T., Langer, R. et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater 6, 1078–1094 (2021).

Menon, I., et. al. (2022). Fabrication of active targeting lipid nanoparticles: Challenges and perspectives. Materials Today Advances, 16, 1-19.

Schoenmaker, L., et. al. (2021). mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability. International Journal of Pharmaceutics, 601, 1-13.

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