Sequence Mutation Aanalysis of Proteins Expressed in Stable Cell Lines

Introduction

The development of stable cell lines for biotherapeutics is an irreversible process, and Sequence variants (SVs) resulting from unintended amino acid substitutions in recombinant therapeutic proteins have increasingly gained attention from the biopharmaceutical industry given their potential impact on efficacy and safety. For well-optimized production systems, such sequence variants are usually present at very low levels in the final protein product due to the high fidelity of DNA replication and protein biosynthesis processes in mammalian expression systems. However, their levels can be significantly elevated in cases where the selected production cell line is not fully optimized of the manufacturing process or has unexpected DNA mutations. Small amounts of SV of the protein products are prone to occur during cell line construction and storage, which may also cause changes in biological activity and generate human immune responses, thereby creating a huge potential risk. Therefore, it is very important to detect this natural mutation before large-scale production. Currently, mass spectrometry (MS) The application of advanced methods enables the highly sensitive detection of sequence variants (SV), such as the characterization of cell lines and recombinant proteins.

Fig. 1. Elucidation of the central dogma and typical error rate in each step. (Zhang A M, et al., 2020)

Our Services

Creative Proteomics has developed a complete set of mutation analysis services based on the de novo sequencing platform combined with bioinformatics tools, which can accurately and completely determine all mutations in protein products, even in the case that protein is heavily mutated. The sequence variation platform we developed that enables them to identify all amino acid sequence variation in a mixture by combining HPLC-UV/MS/MS characterization of peptide maps generated using multiple protease digestions with bioinformatics' SIEVE analysis software, and this method can easily detected the sequence variant. With our technology, we can detect heavily mutated proteins, and we can detect very low-level mutations due to translational errors. The methods we have developed that can detect sequence differences as low as 1 amino acid and can cover the whole of protein/antibody.

Our Technical Advantages

• 100% sequence coverage based on multiple digestions of different enzymes.
• Capable of detecting mutations at as low as 1%, some as low as 0.1% of the protein level.
• Capable of obtaining a semi-quantitative measurement of a mutation level.

Our sequence mutation aanalysis of proteins expressed in stable cell Lines services are widely used in the development of protein therapy drugs. At Creative Proteomics, highly skilled and dedicated scientific staff ensures that the most appropriate methods and technologies are selected for you. Please feel free to contact us if you have any special requirements about our services. We are looking forward to working with you.

References

1. Zhang A M, Chen Z W, Li M N, et al. (2020) A general evidence-based sequence variant control limit for recombinant therapeutic protein development. MAbs. 12(1):1791399.
2. Li W, Wypych J, Duff R J. (2017) Improved sequence variant analysis strategy by automated false positive removal. MAbs. 9(6):978-984.