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Published on: June 24, 2019
Stop-Codon Readthrough in Therapeutic Protein Candidates Expressed from Mammalian Cells
Zhongqi Zhang1, Neelam Khanal1, Andrew B Dykstra1
1Process Development, Amgen Inc. Thousand Oaks, CA 91320, USA.
Abstract:
Stop codon readthroughs were examined in 48 recombinant therapeutic protein candidates produced from multiple clones of Chinese hamster ovary cells, using peptide mapping with LC-MS/MS detection. We found that stop codon readthrough is a common phenomenon occurring in most of these candidates, with levels varying from below the detection limit of ∼0.001 % to ∼1 %. The readthrough propensity depends on the stop codon being used, as well as the nucleotides surrounding it. The amino acids misincorporated into the stop position can be well-predicted by a third-base wobble mismatch and a first-base U/G mismatch during codon recognition, i.e., tyrosine or glutamine insertion for the UAA and UAG stop codons, and tryptophan, cysteine or arginine insertion for the UGA stop codon. Data shown in this report demonstrate the importance of optimizing the DNA sequence near the stop codon, and the importance of detecting stop codon readthroughs during the development of a therapeutic product.
Insights
Stop codon readthrough is common in therapeutic proteins, impacting product quality. Optimizing DNA sequences near stop codons and monitoring readthrough are crucial for biopharmaceutical development.
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.
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