Standardization and Streamlining of Escherichia coli Cultivation for Bioactive Peptide Production Using a Quality by Design Approach

The production of recombinant proteins requires careful optimization of all bioprocess stages, starting from the selection of the expression system and cultivation conditions to the control of critical process parameters (CPPs) and critical quality attributes (CQAs). Current regulatory requirements (ICH Q8(R2), ICH Q9, FDA, EMA, EAEU) are focused on the implementation of the quality by design (QbD) concept, which ensures a systematic approach to development and quality management. One of the key tools of QbD is the use of design of experiments (DoE), which allows for the identification and optimization of parameters influencing protein yield and quality. This study discusses the application of DoE in the development of recombinant protein cultivation processes in E. coli, including proinsulin aspart and glargine and two peptides with molecular masses of up to 5 kDa. It is demonstrated that the use of DoE enables the establishment of relationships between CPPs and CQAs, reduces process variability, increases outcome predictability, and decreases development timelines by at least twofold. The obtained data confirm the effectiveness of integrating statistical methods into bioprocess development, contributing to enhanced standardization, reproducibility, and compliance with international quality standards.