The Scale-Up Challenge Specific to LNPs
Scale-up is a challenge in pharmaceutical manufacturing generally, but it is a particularly acute challenge for lipid nanoparticle products. For a conventional tablet, scaling from a 1 kilogram development batch to a 100 kilogram GMP batch involves larger equipment with similar operating principles, and the scale-up relationships, though not always straightforward, are well understood from decades of industrial experience.
For LNPs manufactured by microfluidics, the situation is different. Microfluidic devices work by mixing two streams, an organic phase containing the lipids and an aqueous phase containing the nucleic acid payload, at controlled flow rates and flow rate ratios in a microchannel. The particle size and size distribution depend critically on the mixing characteristics within the channel, which are determined by the fluid dynamics at the specific flow rates and channel geometry used. Changing the scale of the process means changing the equipment, which changes the fluid dynamics, which can change the product.
Microfluidics Scale-Up: The Key Variables
| Scale Variable | Effect on LNP Properties | Scale-Up Strategy |
| Total flow rate | Higher flow rates increase mixing efficiency but can increase shear stress on particles | Maintain flow rate ratio (FRR) constant; scale total flow rate with batch size using parallel channels or larger devices |
| Flow rate ratio (FRR) | Primary determinant of initial particle size: higher aqueous:organic ratio gives smaller particles | Keep FRR constant across scales; validate that target particle size is achieved at each scale |
| Mixing geometry | Different microfluidic chips produce different mixing regimes; particle size can differ between chip types | Qualify each chip type at target scale; use manufacturer scale-up data as starting point |
| Lipid concentration in organic phase | Higher lipid concentration can increase particle size and polydispersity | Optimise lipid concentration at development scale; confirm at GMP scale before batch manufacture |
| mRNA concentration in aqueous phase | Affects N:P ratio and encapsulation efficiency | Keep N:P ratio constant; calculate mRNA concentration to maintain N:P at target |
| Post-mixing dilution and buffer exchange | Buffer exchange by tangential flow filtration (TFF) affects particle stability and formulation pH | Validate TFF parameters (transmembrane pressure, cross-flow rate) at each scale; monitor particle size through TFF |
Tangential Flow Filtration: The Step That Trips Up Scale-Up
After LNP formation by microfluidics, the product is in an organic solvent-containing medium that needs to be replaced with the final formulation buffer, and the product needs to be concentrated to the target dose concentration. Tangential flow filtration (TFF) is the standard approach for both buffer exchange and concentration of LNP products.
TFF passes the LNP suspension across a semipermeable membrane under a controlled transmembrane pressure, with smaller molecules (solvents, unencapsulated nucleic acid, buffer components) passing through the membrane while the LNPs are retained and concentrated. The process parameters, transmembrane pressure, cross-flow velocity, number of diafiltration volumes, and membrane molecular weight cut-off, all affect both the efficiency of the process and the quality of the retained LNP product.
At development scale, TFF is typically conducted in small-volume hollow fibre cartridges. At GMP manufacturing scale, larger cartridges or multiple units in parallel are used. The transition can introduce unexpected changes in shear stress on the particles and in the effective membrane area available for the batch size, affecting product quality and yield. TFF parameters must be re-optimised and validated at each manufacturing scale.
Process Analytical Technology in LNP Scale-Up
Process analytical technology (PAT) tools are particularly valuable in LNP manufacturing scale-up because they allow critical quality attributes to be monitored in real time rather than measured only at batch release. In-line or at-line dynamic light scattering can detect particle size changes during the mixing or TFF steps before they result in an out-of-specification batch. In-line fluorescence monitoring using intercalating dyes can provide a real-time indicator of encapsulation efficiency during the formulation process.
The FDA has actively encouraged PAT adoption in pharmaceutical manufacturing through its process validation guidance, and for complex products like LNPs where batch failure is expensive and the consequences of size distribution changes on clinical performance are significant, the investment in PAT capability during scale-up is well justified.
Ardena’s LNP Scale-Up Capabilities at Oss
Ardena’s GMP nanomedicine manufacturing facility in Oss operates microfluidics equipment capable of producing LNP batches at both development and GMP clinical scale. The site has TFF capability for buffer exchange and concentration, with process development experience in optimising TFF parameters for LNP products. Scale-up from development batches to GMP clinical batches is managed within the same facility, with the same formulation team involved at each scale.
