Stop-Codon Readthrough in Therapeutic Protein Candidates Expressed from Mammalian Cells

Area of Science:

• Biotechnology
• Molecular Biology
• Biopharmaceutical Development

Background:

• Recombinant therapeutic proteins are critical in modern medicine.
• Ensuring protein integrity and accurate termination is essential for efficacy and safety.
• Chinese hamster ovary (CHO) cells are widely used for producing these proteins.

Purpose of the Study:

• To investigate the prevalence and characteristics of stop codon readthrough in recombinant therapeutic protein candidates.
• To identify factors influencing stop codon readthrough.
• To highlight the implications for therapeutic protein development.

Main Methods:

• Peptide mapping using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
• Analysis of 48 recombinant therapeutic protein candidates derived from multiple CHO cell clones.
• Quantification of stop codon readthrough levels.

Main Results:

• Stop codon readthrough is a frequent event in therapeutic protein candidates, observed in most samples.
• Readthrough levels ranged from below 0.001% to approximately 1%.
• Readthrough propensity is influenced by the specific stop codon (UAA, UAG, UGA) and surrounding nucleotide sequences.
• Misincorporated amino acids (tyrosine, glutamine, tryptophan, cysteine, arginine) can be predicted based on codon and wobble mismatches.

Conclusions:

• Stop codon readthrough is a common and significant post-transcriptional event in CHO cell-expressed therapeutic proteins.
• Understanding the sequence context and codon recognition mechanisms is key to predicting and potentially mitigating readthrough.
• Detecting and controlling stop codon readthrough is vital for optimizing DNA sequences and ensuring the quality of therapeutic products during development.