High-throughput clone screening and upstream process optimization for recombinant therapeutic peptide expression:...

Area of Science:

• Biotechnology and Bioprocessing
• Molecular Biology
• Metabolic Engineering

Background:

• Biosimilar teriparatide production requires efficient upstream process optimization.
• Microfermenter platforms offer high-throughput screening for bioprocess development.
• Metabolic stress in host cells can impact recombinant protein expression.

Purpose of the Study:

• To optimize the upstream process for high-throughput expression of biosimilar teriparatide using a microfermenter.
• To identify optimal Escherichia coli (E. coli) host strains, growth media, and induction strategies.
• To validate the scalability and efficiency of the optimized process through comparative analysis and scale-up.

Main Methods:

• Systematic investigation of key upstream parameters including E. coli host strains, growth media, and induction strategies.
• Application of a design of experiments (DoE) approach to assess parameter effects and interactions.
• Evaluation of five E. coli strains: BL21 (DE3), Lemo 21™ (DE3), Rosetta™ (DE3), Origami™ 2, and Shuffle® T7 (DE3).

Main Results:

• E. coli BL21 (DE3) and Origami™ 2 showed the highest growth rates (0.529 ± 0.05 h⁻¹ and 0.242 ± 0.05 h⁻¹, respectively).
• Galactose induction was identified as the most effective strategy for enhancing teriparatide expression by minimizing metabolic stress.
• Scale-up to a 1-L bioreactor achieved a biomass yield of 67.9 ± 1.0 g/L and 12.0 ± 0.6 g/L of inclusion bodies using E. coli BL21 (DE3).

Conclusions:

• The DoE-based microfermenter approach is effective for optimizing upstream processes for biosimilar teriparatide production.
• Optimized conditions using E. coli BL21 (DE3) and galactose induction enable cost-effective and scalable peptide production.
• The microfermenter platform provides consistent and scalable results, validating its efficiency for bioprocess development.