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Next-Gen Cell Line Characterization: Swiftly Advancing Safer Biopharmaceuticals

November 12, 2024
Valentina Armiento

Cell lines are the cornerstone of manufacturing diverse pharmaceuticals, including monoclonal antibodies, recombinant proteins, gene therapy vectors, cell therapy products, and vaccines. Successful production of these biological modalities hinges on the development and maintenance of reliable, effective, and safe cell lines. To mitigate manufacturing risks, such as the potential presence of harmful contaminants and delays in delivering life-saving therapies, it is essential to conduct a detailed characterization of the cell lines to ensure their Critical Quality Attributes (CQAs).

Cell line characterization, grounded in scientific principles and stringent regulations, ensures that cell lines perform reliably and predictably over time and across different batches. Regular testing of their CQAs, such as identity, genetic stability, and purity, confirms they are free from contaminants and genetically unchanged. Adhering to international guidelines standardizes production processes and ensures compliance across different genomic regions. These regulations support reliable and effective production, enhancing public trust and fostering innovation in the biopharmaceutical industry.

Advanced Technologies: Enhancing Characterization of Cell Lines

Next-generation sequencing (NGS) is an advanced technology that enables comprehensive genetic analysis of cell lines. Unlike traditional methods, which rely on both in vivo and in vitro assays and often require extended timelines with limited sensitivity, NGS provides rapid and accurate cell line characterization while meeting stringent regulatory requirements.

Yet, to fully harness the potential of NGS in characterizing cell lines, it is essential to integrate robust data analysis and management. Genedata Selector offers a comprehensive, off-the-shelf solution for NGS-based cell line characterization. Powered by wizard-based guides called Playbooks, it unlocks the powerful NGS multi-attribute method (MAM). This method allows for multiple quality control (QC) readouts from one single assay, consolidating multiple analyses into one study or workflow and eliminating the need for separate assays. For instance, the Genedata Selector MAM Cell Line Characterization Playbook can analyze both short- and long-read NGS data to evaluate the identity of cell line clones, integration sites, the integrity of the relevant genes, and the detection of adventitious agents such as viruses and mycoplasma—all in one run.

Genedata-Driven NGS Assays for Cell Line Characterization

Traditional assays to characterize cell lines include Short Tandem Repeat (STR) profiling and isoenzyme analysis for identity, mycoplasma testing and sterility testing for purity, and karyotyping and Southern blot for stability. Phenotypic characterization is also an important aspect, often involving assays to assess cell morphology, growth characteristics, and specific marker expression. While traditional methods such as PCR and Sanger sequencing have been widely used, they often require multiple assays to achieve comprehensive results. However, NGS offers a more detailed and comprehensive approach, replacing or complementing traditional assays. NGS can provide whole genome sequencing for identity, metagenomic sequencing for purity, and copy number variation analysis for stability. NGS technologies are versatile and can be used for different purposes, replacing traditional assays as shown in Table 1.

Table 1. NGS applications for cell line characterization.

Genedata Selector streamlines these NGS-based assays by annotating reference cell lines with proprietary data, ensuring data security and integrity. It tracks samples, manages bioinformatics workflows, and analyzes outcomes. The platform also offers sample-centric decision support, providing tailored and automatically generated reports that indicate whether the analysis has 'passed' or 'failed', supporting the entire lifecycle of cell line characterization. This feature is suitable for both in-house use and regulatory submissions, ensuring compliance and full traceability, while also protecting intellectual property through in-house analysis.

Clone Selection

Early clone screening is crucial in developing cell lines, as it helps select those with the best growth, productivity, and stability.2 This process identifies clones with the ideal properties for producing high-quality biopharmaceuticals.3 For monoclonal antibodies and recombinant proteins, it is essential to select high-yield and stable clones to meet product demands. In gene and cell therapies, clone selection is used to precisely identify the genetic modifications and functional properties of the cell lines. Advanced techniques, such as single-cell cloning, high-throughput screening, and NGS, are employed to select clones that not only meet regulatory requirements but also ensure the efficacy and safety of the final product.

Whole genome sequencing (WGS) with NGS, when combined with ample coverage, enables the precise detection of genomic integration sites and ensures sequence integrity, thereby accelerating clone selection. However, the data analysis requires significant bioinformatics expertise. Genedata Selector simplifies and accelerates the analysis and management of WGS data. The Integration Site Verification (ISV) Playbook’s step-by-step user guide ensures a smooth analysis process. Automated analysis runs in the background and generates a report upon completion that documents and summarizes all the results and monitoring CQAs.

By integrating robust clone selection strategies, biopharmaceutical companies can enhance the reliability and consistency of their manufacturing processes, delivering safer and more effective therapies to patients. Furthermore, the establishment of cell banks based on the selected clones guarantees that high-quality characteristics are preserved and maintained throughout the production lifecycle.

Cell Banks: Ensuring Consistency and Safety

The cell banking process starts with the Research Cell Bank as the basis for creating the Master Cell Bank (MCB). Ensuring that only high-quality, contamination-free cell lines enter the GMP manufacturing step is essential. The MCB undergoes extensive testing for adventitious agents to confirm that the cell lines are free from microbial contaminants such as fungi, bacteria, adventitious viruses, and mycoplasma. From the MCB, the Working Cell Bank (WCB) is derived, which is used for routine production and undergoes regular quality control measures. The End-of-Production Cell Bank (EOP CB), sometimes called Post-Production Cell Bank (PPCB), is created from the WCB, and undergoes detailed mycoplasma analysis to serve as a reference for future production, ensuring long-term stability and consistency.

Mycoplasma contamination is a critical issue in cell line characterization and development, significantly altering cell physiology and compromising research outcomes. Contamination rates can reach up to 35%, highlighting the importance of regular screening and stringent quality control measures. Implementing advanced detection methods and maintaining rigorous aseptic protocols are essential to ensure the integrity of cell cultures used in research and biopharmaceutical production.4-5 In this context, untargeted NGS analysis can replace targeted PCR-based screening for reliable strain typing. Genedata Selector offers several Playbooks to leverage NGS-based biosafety assays. Among these, the designated Playbook for Mycoplasma detection enables users to run the detection assays in a quick, simple, yet robust manner.

Contaminations can originate from the raw materials of human or animal origin or be inadvertently introduced during the manufacturing process, necessitating risk assessments to evaluate viral risk factors according to the European Pharmacopoeia Chapter 5.1.7.6 Besides the adventitious agent detection (AAD), identity and genetic stability of cell lines are also extensively tested to ensure that the cell lines maintain their desired characteristics. The Genedata Selector MAM Cell Line Characterization Playbook enables simultaneous monitoring of these attributes.

The Regulatory Landscape

Establishing a well-characterized cell banking system and selecting optimal clones in a validated environment is essential for the compliance and quality of the manufacturing process in a cGMP setting, as required by global regulators. The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) has provided two guidelines, ICH Q5B and Q5D, to qualify cell lines. ICH Q5B focuses on analyzing the expression construct in cell lines to ensure genetic stability and integrity,7 while ICH Q5D provides guidance on the deriving and characterizing of cell substrates.8 Regulatory authorities also request next-generation characterization of genetic consistency in manufacturing cell lines, performed in accordance with ICH Q5B and Q5D. For eukaryotic expression systems in product license applications, genetic characterization involves verifying the product coding sequence either by nucleic acid testing or by analyzing the final protein product. To enhance genetic characterization capabilities, USP chapter <1042> suggests using NGS.9

Additional guidelines are provided by the World Health Organization (WHO), the European Medicines Agency (EMA), and the US Food and Drug Administration (FDA). These regulations allow scientists to consistently use cell lines and cell banks that maintain their genetic stability during long-term storage. This is particularly important for cell and gene therapies, as the precision and stability of cell lines directly impact the success of the treatments. Leveraging this regulatory framework, the Genedata Selector team has extensive expertise in developing tailored solutions for in-house validation of NGS-based assays.

Conclusion

Using NGS for cell line characterization offers numerous benefits, including thorough genetic analysis, high accuracy, and faster results. An integrated, in-house platform like Genedata Selector capitalizes upon these advantages by enabling GMP validation within a company’s own facilities. In contrast, attempting to validate NGS-based assays independently is a complex and challenging task, often resulting in fragmented validation efforts. Genedata Selector streamlines this process, providing a unified, comprehensive platform for a series of validated NGS-based assays. This approach ensures compliance with regulatory standards, reduces reliance on external services, increases control over the testing process, and ultimately improves return on investment.

Looking ahead to 2025, NGS-MAM is expected to play a crucial role in biopharmaceutical quality control, including cell line characterization. Genedata Selector facilitates global GMP rollouts across institutions of varying sizes. Its scalable and flexible platform can be tailored to meet the specific needs of different organizations, from contract development and manufacturing organizations (CDMOs) and small biotech firms to large pharmaceutical companies. By offering a robust and reliable solution for genetic characterization testing, Genedata Selector streamlines the consistent production of high-quality biopharmaceutical products worldwide, eliminating the need to implement multiple assays to characterize cell lines and ensure patient safety.

The application of NGS in combination with Genedata Selector represents a significant advancement in cell line characterization. These technologies not only increase the accuracy and efficiency of genetic stability testing but also pave the way for future innovations related to the 3Rs (Refine, Reduce, Replace), analytical process automation, and scalability in biopharmaceutical quality control. Embracing these advancements will be key to maintaining the highest standards of product quality and safety in the rapidly evolving biopharmaceutical field.

References

  1. Short tandem repeat profiling via next-generation sequencing for cell line authentication . Chen, Yi-Hsien. s.l. : The Company of Biologists Ltd | Disease Models & Mechanisms, 2023.
  2. Enhanced CHO Clone Screening: Application of Targeted Locus Amplification and Next-Generation Sequencing Technologies for Cell Line Development. Aeschlimann, Samuel H. s.l. : Biotechnology Journal, 2019.
  3. Advances in Mammalian Cell Line Development Technologies for Recombinant Protein Production. Lai, Tingfeng. s.l. : Pharmaceuticals, 2013.
  4. Development and Evaluation of a New qPCR Assay for the Detection of Mycoplasma in Cell Cultures. Carrillo-Ávila, José A. s.l. : Current Issues in Molecular Biology, 2023.
  5. Detection of Mycoplasma in cell cultures. Young, Lesley. s.l. : Nature Protocols, 2010.
  6. Ph Eur Chapter 5.1 .7- Viral Safety. Pharmacopoeia, European. 2008.
  7. ICH Q5B: Quality of Biotechnological Products: Analysis of the Expression Construct in Cell Lines Used for Production of r-DNA Derived Protein Products. International Council for Harmonisation (ICH), European Medicines Agency (EMA). 1996.
  8. ICH Q5D: Quality of Biotechnological Products: Derivation and Characterisation of Cell Substrates Used for Production of Biotechnological/Biological Products. International Council for Harmonisation (ICH), European Medicines Agency (EMA). 1998.
  9. Cell Banking Practices for Recombinant Biologics. Pharmacopeia, United States. s.l. : USP-NF, 2023, Vol. General Chapter 〈1042〉.