Next Generation LNP Manufacturing

Ease of scalability

Producing COVID-19 vaccines for the entire world population rapidly demonstrated the microfluidic development scale up roadblock. Discovery formulations developed using microfluidics had to be scaled up using alternative higher throughput formulating technology with different operating parameters.   

With a growing demand for more mRNA-LNP based vaccines, proven to have high efficacy, there is currently a need to facilitate a faster and cheaper global deployment of a high-throughput manufacturing method. A GMP compliant approach is needed, from the earliest possible stage through to manufacturing, that is also tuneable in size to access various tissues or specific drug targets.  

Advanced cross flow mixing (AXF) from Micropore Technologies allows for seamless scaleup with consistent physics, mechanisms conditions and geometry across its equipment range. Ultimately Micropores AXF technology can be scaled up to a device with over 10 million pores with the potential for a throughput of up to 20 Liters per minute all this from a device that would still fit inside a briefcase. AXF allows controlled, low shear precision continuous flow mixing technology from nano to micro formulations for when you want to avoid roadblocks on your product development journey. It uses the same same shear, same physics and same technology from lab bench to manufacturing scale to enable scale up with confidence.  

Micropore technology address key concerns for adoption

• Expertise, availability of skilled personnel, associated risks, and the need for a reliable supply chain. 

Micropore is a technology provider with global experience in manufacturing all different types of vaccine modalities can further ensure a cost-effective, high-quality process. Partnering with Micropore will enable a stronger benchmark with in-depth expertise and the ability to leverage novel technologies will also help reduce risk and shorten timelines.

• There is a need to develop standard analytical methods for quality characterisation and reference material that lend insight into the mechanisms of stability or degradation of mRNA and LNPs containing it. 

The determinants of stability of mRNA in LNP formulations – what parts are predicated on the payload of mRNA and what portions are predicated on the lipid nanoparticles themselves or what portions are predicated on the freeze-drying cycle that’s used – can be related to its size and its secondary structure. Messenger RNA poses a unique manufacturing challenge because of its large size. Other RNA entities such as siRNA and guide RNA for clustered regularly interspaced short palindromic repeats (CRISPR) technology typically are produced using chemical synthesis, which can be performed in a relatively controlled environment. But mRNAs are larger, with complex three dimensional structures that aren’t yet fully understood. Malvern Zetasizer (DLS) enables particle size and stability measurements while the RedShiftBio Aurora (MMS) system enables secondary structure (HOS) determination.

• The need to move to larger scale manufacturing of mRNA-LNPs without an extensive setup or excess capacity to convert.

With mRNA vaccine production requiring relatively less space than other approaches, new facilities may be more feasible and affordable. Micropore technology can enable localised production of vaccines and thus accelerate access to a much larger population. In locations with limited or no infrastructure, the Micropore approach can be the shortest route to production and can reflect the exact needs of the organisation at minimal cost.

• The need for future vaccine manufacturing facilities to be closed and continuous processing, plus equipment connectivity and communication.

Micropore offers a minimal cost model achieved through the AXF advanced cross flow technology platform. The flexibility of mRNA-based vaccines when manufactured using this single piece of stainless steel equipment with no consumables means it requires the least capital investment. The scalability of production (from 200 μL to 1500 L/hr) reduces the facility design complexity and means that more doses can be manufactured in a continuous process which eliminates variability, compared to a batch-to-batch approach. As such, this vaccine modality combined with the Micropore mixing platform can be a robust starting point for production with low risk.

• The need to overcome current barriers of manufacturing process to be fully digitalised as regulatory authorities rely on data and parameters recorded during production for verification and approval.

Micropore cross flow technology demonstrates predicable scalability which is favourable especially for GMP manufacturing which means process controls can be introduced that are automated – process analytical technologies (PAT). Automated analysis of properties such as online particle size enables the option to automatically control any deviations in size and feed that back to control the pumps and optimise control to give the correct size again. This increases confidence in quality of production meaning throughput can be increased further.

Continuous Formation & Stabilisation of LNPs Minimises Risk of mRNA Degradation

Batch mode vs continuous mode manufacturing: While Impingement jet mixing (IJM) or T-mixers are the most widespread manufacturing method currently, high turbulent mixing combined with high pressure and shear stress effects can compromise LNP stability and affect overall performance of the product. The process also suffers from high batch variability and high wastage due to holding or prolonged processing steps.

Micropore Technologies offers Continuous Manufacturing: Micropore Technologies employs laminar flow mixing across a permanent stainless-steel membrane to produce reproducible, scalable LNPs. The outer dispersed phase is continuously mixed with the aqueous inner compartment to form LNPs. Size controlled uniform particles are generated in a continuous flow capacity of up to 1500 L/hour making this by far the fastest production rate in the LNP industry. This would translate to roughly 58,000 doses of vaccine every minute, an important capability when faced with the demands of global disease emergencies. 

Micropore Technologies: 2^nd generation Vaccine manufacturing Process

Micropore’s Membrane Technology: How it works

Micropore’s equipment is uniquely suited to the production of complex nanomedicines in a scalable manner – from small 200µl samples through to 1 billion doses.

Micropore technologies differs from conventional mixing LNP techniques by considering the process not as discrete or separate unit operations but as one single whole process.

Attributes:

• High efficiency, scalable and reproducible.

• Reduced manufacturing time and costs.

• Customisation for diverse LNP formulations.

• Tunable particle size with narrow size distribution (PDI).

The Micropore system comprises a membrane inside a housing. Lipid mixtures added from the top are mixed with the RNA/buffer solution from the side which enters the membrane to produce monodisperse particles.  The system operates at very low pressure – approximately 2 Bar. This allows the size of the particles created to be very predictable with any change in flow rate. T-mixer devices operate at much higher pressures – about 30 Bar.

The graphs above show comparison control curves for AXF vs. T-mixer.

Advanced Cross Flow (AXF) mixing

Micropore’s equipment is uniquely suited to the production of complex nanomedicines in a scalable manner – from mL batches up to tonnes.

A lipid/organic phase passes through the 100,000s of membrane pores into the flow of aqueous continuous phase that passes through the centre of the membrane tube. Gentle, laminar flow based mixing allows for good preservation of sensitive materials. Precision engineered equipment gives great size distributions even at scale, allowing precise targeting of distributions.

Process robustness and high encapsulation efficiency

Here we present data from a formulation prepared using the AXF Pathfinder 20.

The conditions used are listed below:

• Formulation ratio DDAB:DSPC:cholesterol:DMS-PEG2000, 40:10:48:2 mol%.
• Lipid concentration, 3.5mg/ml
• N:P ratio, 1:6
• polyA concentration, 0.046mg/ml
• Flow rate ratio 3:1
• Total flow rate 100mL/min
• continuous phase, Tris buffer, 10mM pH 7.4

The Pathfinder delivered highly repeatable, monodisperse LNPs that were 70nm in size with a narrow PDI of 0.13. The experiment was repeated in triplicate and samples were measured using the Malvern Zetasizer DLS system.

Encapsulation efficiency is about 97-98%. The AXF mini uses cross flow mixing which is a gentle mixing technique and prevents RNA degradation.

Process tunability and predictability

The two graphs above demonstrate the superior capability of the AXF mini to control the size of nanoparticles produced which can be critically important, especially for GMP manufacturing.

The plot on the right shows two formulations, plotted in red and green. Differences arise from e.g. concentration of DMG-PEG200 in the range 0.004 – 0.12 μmol which reduces size from 200 nm to 30 nm but both exhibit the same shape and therefore control. Although the PDI is still quite tight in both formulations but the formulation plotted in green is considerably larger (100-160nm) compared to the formulation in red (50-110nm). From this experiment you can quickly see it is possible to produce a 70nm particle by using a total flow rate around 100 ml/min. 

The plot on the left shows an experiment where the AXF mini was used to create two different formulations using two different flow rates. At 20ml/min the LNP size was 107 nm. When the total flow rate was increased to 200 ml/min, the LNP size reduced to 55 nm with a remarkably low PDI of 0.06.  

This data demonstrates the predicable scalability which is favourable especially for GMP manufacturing and regulators which means you can start to introduce process controls that are automated – process analytical technologies (PAT). Automated analysis of properties such as online particle size enables the option to automatically control any deviations in size and feed that back to control the pumps and optimise control and give the correct size again. This increases confidence in quality of production meaning throughput can be increased further. ATA Scientific offers several technologies to characterise LNPs.

Contact us for a demo today!