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Ensuring Genetic Stability in CHO Cell Lines: NGS and MAM

January 20, 2025
Valentina Armiento

The pharmaceutical market is experiencing rapid growth in biologics, including vaccines, monoclonal antibodies (mAbs), bi- and multi-specifics, and antibody-drug conjugates (ADCs). Chinese Hamster Ovary (CHO) cell lines are used in the production of 70% of all biologics1 due to their high productivity and ability to thrive in suspension cultures. Characterizing and confirming the genetic stability of these cell lines is therefore critical and requires rigorous testing methods and adherence to strict regulatory protocols. We explore the best practices for performing stability testing on CHO cell lines and the benefits of next-generation sequencing (NGS) and the multi-attribute method (MAM) in this process.

The Power of NGS in Genetic Stability Testing

Characterizing and confirming the genetic stability of CHO cell lines is crucial for their successful use in biopharmaceutical production. By employing rigorous testing methods and adhering to strict regulatory requirements, manufacturers must ensure the consistency, safety, and efficacy of their products.

Traditional methods for testing genetic stability, such as PCR, Sanger sequencing, karyotyping, fluorescence in situ hybridization (FISH), and Southern blot analysis, are time-consuming, labor- and resource-intensive, and costly. In some cases, they provide only qualitative assessments open to subjective interpretation. Therefore, conclusive testing typically requires using multiple distinct assays to assess genomic stability.

NGS stands out as the most accurate method for genetic stability testing today. From host cell line development to the master cell bank clone, NGS provides molecular insights on the sequence level, enabling the tracking of genetic stability throughout the entire process. It facilitates comprehensive sequencing of the entire genome, identifying even the smallest genetic variations that could impact the production of biologics. The precision of NGS ensures that any genetic drift or mutations within CHO cell lines are detected early, allowing for timely interventions.

Enhancing NGS with MAM

MAM, initially developed for mass spectrometry, is now being integrated into other technologies, including NGS. MAM is designed to simultaneously detect, analyze, and monitor multiple Critical Quality Attributes (CQAs) in the same sample. The current trend of integrating MAM with NGS streamlines the testing process and provides a holistic view of the genetic stability and functional attributes of CHO producer lines. Implementing MAM can lead to a better understanding of CHO cell lines during the production process and consistent monitoring of both the genetic and phenotypic stability. It can replace traditional methods to monitor genetic stability and detect variations that may not be readily identified using conventional techniques. Once the NGS instruments, wet lab protocols, and analyses are set up in-house, the NGS-MAM approach can also lead to significantly lower resource requirements, reduce timelinesas much as fivefold, and lower costs, all the while still delivering high-quality results and decision support. 

The Challenge of In-House NGS-MAM Implementation

Setting up NGS-MAM for in-house CHO cell line development and stability testing presents several significant challenges. These include establishing a robust bioinformatics and data analysis infrastructure. A team with deep expertise in both NGS and complex data analysis is typically required. Additionally, integrating NGS-MAM into existing workflows and scaling it up requires careful coordination with other laboratory systems. Finally, the entire process, from sample preparation to data analysis and result reporting, must be validated to meet regulatory standards. 

Despite these challenges, the combination of NGS and MAM remains the most efficient and accurate approach for genetic stability testing in CHO cell lines today. The benefits, including enhanced accuracy and increased efficiency, make it a highly valuable investment for many biopharmaceutical companies.

A Comprehensive Solution for NGS-MAM

Genedata Selector® offers a unique solution to tackle these challenges, making implementation and maintenance of NGS-MAM significantly easier and more cost-efficient. It automates the simultaneous analysis of multiple NGS assays and provides a validated platform that meets regulatory requirements. The automation not only simplifies the testing process but also ensures scalability and efficiency. Once up and running, Genedata Selector makes NGS-MAM more efficient, reproducible, and reliable. It also frees up bioinformaticians’ time so they can focus on other projects. 

To fully realize the MAM potential of NGS, seamless integration and interpretation of complex data is essential. For genetic stability testing, the Genedata Selector platform provides an end-to-end solution for data processing and analysis using whole genome sequencing (WGS) with NGS. It includes a wide range of tools for developing and analyzing different cell line characterization assays within a single, integrated environment. Genedata Selector offers extensive capabilities for tracking NGS samples, managing diverse bioinformatics workflows, analyzing outcomes, and automatically generating standardized reports for regulatory submissions. It also maintains connectivity between raw and processed NGS data and tracks all assay parameters as well as reference libraries. By using Genedata Selector, cell line development (CLD) groups can achieve a holistic view of the CHO clone genome, which enhances the accuracy and precision of genetic stability assessments.

Manufacturers must ensure the stability, safety, and efficacy of their products by employing rigorous testing methods and complying with regulatory requirements.

Navigating Regulatory Standards with Next-Generation Technologies

To comply with global regulatory standards, including those set by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH), it is essential to adopt advanced technologies such as NGS in the context of CHO cells. The ICH Q5A(R2) guideline highlights the importance of ensuring the genetic stability and viral safety of cell lines, and NGS offers a comprehensive analysis of the gene of interest (GOI) and its flanking regions. This advanced method provides detailed insights into structural variants and potential recombination events, addressing the limitations of traditional techniques and ensuring genetic stability and integrity in manufacturing cell lines in a single, integrated assay.

The Most Efficient Way to Ensure Genetic Stability of CHO Cells  

Genedata Selector leverages advancements in NGS and bioinformatics to simplify and enhance genetic stability testing throughout the CLD process, clone selection, bioprocess development, and master cell bank (MCB) characterization. With whole-genome sequencing, this end-to-end platform provides a high-resolution, base-by-base view of the entire CHO cell genome, including any integrated exogenous genes and corresponding vector elements. This eliminates the need for primer design and reduces subjective interpretation, providing a more accurate and comprehensive analysis.

The platform automates the analysis and visualization of genetic elements, generating detailed reports that meet regulatory standards. It confirms the nucleotide sequence of the gene of interest (GOI) and flanking regions, detecting single nucleotide polymorphisms (SNPs), insertions, and deletions, thus eliminating primer bias and ensuring a complete sequence of the inserted genes. Genedata Selector also determines gene copy number by comparing the sequencing coverage of the inserted regions to reference genes in the CHO genome. Furthermore, it identifies the precise sites of integration within the CHO genome, including chromosome number and coordinates, and assesses the genetic integrity and structural arrangement of the vector expression cassette by analyzing chimeric reads and identifying vector-vector junctions. This approach provides insights into structural variants and potential recombination events, validated for precision, accuracy, linearity, and range, ensuring reliable results.

Validating Multiple NGS Assays

Genedata Selector offers a validated platform for many assays, covering genetic stability testing thoroughly. Genedata’s dedicated Quality Assurance (QA) team, with extensive experience from close collaboration with customers, ensures that the platform consistently meets the highest standards of quality and reliability. The software also supports additional validation needs for novel workflows, if needed. This allows companies to focus on their primary objectives without the burden of developing and maintaining a home-grown NGS infrastructure and the lengthy validation of individual NGS assays while leveraging state-of-the-art technology for their genetic stability testing. 

Summary

Genedata Selector marks a significant leap forward in genetic stability testing for CHO cell lines and Adventitious Agent Detection (AAD) by simplifying and streamlining the in-house implementation and maintenance of NGS-MAM. This cutting-edge platform automates the testing process, enhances scalability, and provides comprehensive insights. Its ability to simultaneously validate multiple assays ensures quicker, more reliable, and more robust testing while guaranteeing compliance with regulatory requirements. Looking ahead, NGS-MAM is expected to become the method of choice for genetic stability and AAD testing since it offers a more reliable and faster approach to meet regulatory requirements.

References

  1. Tihanyi, B.; Nyitray, L. Recent Advances in CHO Cell Line Development for Recombinant Protein Production. Drug Discovery Today: Technologies. Elsevier Ltd December 1, 2020, pp 25–34. doi.org/10.1016/j.ddtec.2021.02.003.