The Role of CHO Cells in Antibody Production
Chinese hamster ovary (CHO) cells dominate the landscape of therapeutic antibody production, with nearly 90% of monoclonal antibodies (mAbs) and Fc-fusion proteins produced using this system. CHO cells offer a combination of scalability, adaptability, and a robust safety record, making them an indispensable platform for biologic drug development. However, host cell proteins (HCPs), impurities derived from CHO cells during production, remain a critical quality concern. These impurities can compromise drug safety, efficacy, or stability, posing challenges for regulatory compliance and manufacturing efficiency.
A recent paper by Tzani et al. reviews recent advancements in analytical techniques, including ribosome profiling (Ribo-seq) and mass spectrometry (MS), which have provided new insights into the presence of host cell microproteins.1 These tools not only enhance HCP detection but also provide actionable data for improving cell line engineering and bioprocess optimization.
Microprotein Impurities
Microproteins are typically defined as peptides under 100 amino acids. Using Ribo-seq, researchers have identified thousands of short open reading frames (sORFs) encoding microproteins in CHO cells. Notably, mass spectrometry analysis of commercial mAbs and Fc-fusion proteins has detected microproteins, albeit at low concentrations. While their impact on product safety or efficacy remains unclear, their discovery highlights a gap in traditional HCP detection methodologies.
Impact on Bioprocess Development
Microprotein abundance can vary with environmental factors, such as bioreactor temperature or nutrient availability, suggesting that upstream processes may influence their presence in final drug products. Monitoring microprotein levels throughout the manufacturing process provides opportunities for process optimization, including cell culture condition refinement and targeted cell line engineering to reduce impurity levels.
Related: What is Antibody Manufacturing Process Optimization?
Enhancing Cell Line Engineering with Ribo-Seq
Ribo-seq offers high resolution in studying translation events in CHO cells, identifying both canonical and non-canonical ORFs. This insight facilitates targeted cell line engineering strategies to minimize problematic HCPs. For instance, genome editing tools such as CRISPR/Cas9 can now be directed at newly identified sORFs to eliminate microprotein expression altogether.
Non-AUG Translation Initiation
Approximately 56% of newly identified ORFs begin at non-AUG start codons, challenging traditional models of translation initiation. These alternative start sites may produce microproteins that evade conventional purification methods. Understanding these mechanisms provides a pathway to designing more robust CHO cell lines optimized for therapeutic protein production.
Applications in Vector Design
The discovery of upstream ORFs (uORFs) in regulatory regions of CHO cell mRNAs offers potential for controlling gene expression more precisely. Synthetic uORFs could be engineered into expression vectors to regulate the translation of therapeutic proteins, improving their stability, glycosylation patterns, or yield.
Related: CHO-K1 Stable Cell Line Generation
Advanced Analytical Tools for HCP Detection
While traditional ELISA methods are valuable for quantifying total HCP levels, their limited antigen coverage necessitates complementary approaches. MS-based methods, particularly when paired with updated proteomic databases, have significantly advanced the identification and quantification of individual impurities.
Data-Independent Acquisition (DIA)
Emerging MS techniques, such as DIA, provide greater sensitivity and reproducibility in detecting low-abundance proteins like microproteins. These approaches, combined with spectral libraries generated through machine learning, promise to further enhance the resolution of HCP analysis in antibody drug products.
Bioinformatics Integration
Advanced bioinformatics tools are essential for managing the complexity of Ribo-seq and MS datasets. These tools facilitate accurate mapping of ribosome-protected fragments to genome annotations, reducing false positives and enabling more comprehensive impurity profiling.
Implications for Bioprocess Optimization
The discovery of microproteins and other sORF-encoded impurities underscores the importance of integrating upstream and downstream process development.
Upstream Process Modifications
Environmental factors, such as temperature shifts or media composition, influence microprotein expression during cell culture. Adjusting these variables can help reduce the burden of impurities entering downstream purification processes.
Downstream Process Enhancements
By analyzing HCP populations at each stage of downstream processing, manufacturers can refine their workflows to achieve higher clearance rates. This iterative approach ensures that even low-abundance impurities are effectively removed, improving final product quality.
Future Directions: Functional Insights into Microproteins
Machine learning models trained on Ribo-seq and MS data can predict antibody candidates with desirable properties, such as high stability or specificity. These computational approaches, combined with experimental validation, offer a pathway to accelerate antibody discovery and production.
Future research with alternative inhibitors or broader conditions could uncover additional sORFs. This knowledge will inform the next generation of cell lines and bioprocessing technologies, driving efficiency and innovation in antibody drug production. By leveraging cutting-edge analytical tools and a deeper understanding of CHO cell biology, the industry aims to overcome challenges in impurity detection and product optimization, ensuring the continued success of antibody therapeutics.
At Biointron, we are dedicated to accelerating antibody discovery, optimization, and production. Our team of experts can provide customized solutions that meet your specific research needs. Contact us to learn more about our services and how we can help accelerate your research and drug development projects.
References:
Tzani, I., Kelly, P., Strasser, L., Zhang, L., Clynes, M., Karger, B. L., Barron, N., Bones, J., & Clarke, C. (2024). Detection of host cell microprotein impurities in antibody drug products. Nature Communications, 15(1), 1-17. https://doi.org/10.1038/s41467-024-51870-0
