# QbD product development: rapid optimization and scale-up of PBAE-based siRNA delivery via DoE-guided microfluidics

## TL;DR

This study uses a QbD and DoE approach with microfluidics to optimize and scale up PBAE-based siRNA delivery nanoparticles for stable and efficient gene silencing.

## Contribution

A scalable QbD workflow for rapid optimization of PBAE-based siRNA delivery using microfluidics and DoE.

## Key findings

- High flow rates, N/P ratios ≥10, and a 1:3 flow rate ratio produced smaller, more stable nanoparticles.
- A polymer with balanced hydrophobic and hydrophilic side chains showed optimal stability and gene silencing.
- Optimized parameters were successfully scaled up while maintaining quality attributes.

## Abstract

Poly(β-amino ester) (PBAE)-based nanoparticles have emerged as promising carriers for RNA delivery, yet clear design rules linking formulation parameters to performance are still lacking. In this study, a Quality by Design (QbD)-guided and Design of Experiments (DoE)-driven approach was combined with high-throughput microfluidics to rapidly identify formulations with favorable physicochemical properties and consistent critical quality attributes (CQAs). Response Surface Modeling revealed that high total flow rates (TFR ≥ 10), nitrogen to phosphorus (N/P) ratios ≥10, and a Flow Rate Ratio (FRR) of 1 : 3 (buffer : ethanol) led to the formation of smaller, more stable particles. Among the polymers tested, a polymer candidate with a balanced composition of hydrophobic and hydrophilic side chains demonstrated optimal intraparticle stability and gene silencing performance. Notably, transfection efficiency depended strongly on formulation parameters beyond polymer type and N/P ratio, with flow rate ratio emerging as a key driver of gene knockdown kinetics. The lead formulation achieved ∼95% gene knockdown even after two weeks of storage at 4 °C. Scale-up production of the lead candidate confirmed the transferability of optimized Critical Process Parameters (CPPs) and preserved CQA profiles, validating the robustness of the design space. This study establishes a robust and scalable QbD-guided workflow for the development of microfluidically manufactured siRNA nanoparticles, enabling rapid optimization, reliable scale-up, and clinically relevant performance.

A DoE-screening on an automated microfluidic small-scale device enabled rapid identification of stable PBAE/RNA nanoparticles. Successful transfer of optimized process parameters to a large-scale device confirmed robust and scalable manufacturing.