# Quality by design for transient RBD-Fc fusion protein production in Chinese hamster ovary cells

## TL;DR

This paper shows how optimizing cell culture conditions can boost production of a fusion protein in lab cells.

## Contribution

The study applies quality by design to optimize RBD-Fc fusion protein production in Chinese hamster ovary cells.

## Key findings

- Optimal conditions include 5-day culture duration, 34.4°C temperature, and a PEI-Max/pDNA ratio of 4.2:1.
- The optimized process achieved a protein yield of 47.78 mg/L with high purity and receptor binding.
- QbD tools help identify critical parameters for consistent and scalable protein production.

## Abstract

•Quality by design can be applied to the upstream process of the fusion protein (RBD-Fc) to increase the production yield.•Understanding the factors that affect product quality is essential for new findings to enhance process performance and maintain product quality.•The process parameters are optimized by surface response methodology to find the optimum point to enhance production performance.•A robust cell culture platform in the upstream process can ensure that critical quality attribute profiles are consistent from the early stages of laboratory-scale development to upscaling.

Quality by design can be applied to the upstream process of the fusion protein (RBD-Fc) to increase the production yield.

Understanding the factors that affect product quality is essential for new findings to enhance process performance and maintain product quality.

The process parameters are optimized by surface response methodology to find the optimum point to enhance production performance.

A robust cell culture platform in the upstream process can ensure that critical quality attribute profiles are consistent from the early stages of laboratory-scale development to upscaling.

Quality by design (QbD) is applied to the upstream process to maximize the RBD-Fc fusion protein production in CHO cells. The three factors (culture duration, temperature, and polyethyleneimine to plasmid DNA (PEI-Max/pDNA) ratio) were identified as critical process attributes based on risk analysis (FMEA) and further optimized by response surface to maximize the protein yields. Using a Box-Behnken design, the optimal conditions for RBD-Fc production were determined to be a culture duration of 5 days, a culture temperature of 34.4 °C, and a PEI-Max/pDNA ratio of 4.2:1 (w/w) with a predictive value of 48 mg/L (desirability of 92.8 %). The PEI-Max/pDNA ratio and its interaction with culture duration to express the highest yield (47.78 ± 2.30 mg/l). In addition, the purified CHO-produced RBD-Fc fusion protein was highly pure and strongly bound to its receptor, ACE2. Our finding demonstrated that the QBD tools can identify the critical parameters to facilitate scaling-up production.

Image, graphical abstract