Innovations in Cell Line Development

New Innovations in Cell Line Development with the Life Sciences Companies of Danaher Visit DHRLS.CO 04 26 Scale up and Bioreactor Studies 12 Screening and Monoclonality Verification 34 Digital and Data Solutions 20 Media Development 42 Analytical Data Management Table of Contents Challenges of Cell Line Development Stable Transfection Challenges of Cell Line Development: Stable Transfection 4 5 A primary challenge when performing stable transfections during early cell line development is the cell culture and hands-on burden when having to perform and screen thousands of transfections. Extensive hands-on time is required to optimize vectors, cells, and conditions to achieve the best possible transfection efficiencies with high genetic integration. Transient transfections during research and development do not necessarily translate to larger scale success during CMC. This can be a time-consuming process, adjusting various parameters such as cell-vector ratios, reagent volumes, and conditions. Scaling up or scaling out could be cost- prohibitive in terms of space and reagent needs. If you are looking to accelerate your cell line development, experts at Danaher Life Sciences companies can work with you to identify solutions to optimize transfection and gain insights faster. Sequence-verified DNA, RNA, and CRISPR-Cas complex kits from IDT provide peace of mind about input sequence accuracy and precisely controlled editing. Custom plasmids from Aldevron can be used to eliminate bottlenecks in raw material supply. Because capacity is limited, innovative solutions must be adopted to increase capacity without physical laboratory expansion. Beckman Coulter Life Sciences offers laboratory automation solutions for cell culture, verified transfection protocols, and CRISPR-Cas system delivery with the Biomek i-Series Automated Liquid Handler. Automating cell viability measurements can also help to assess transfection success, with a Vi- CELL BLU Cell Viability Analyzer providing automated sample preparation, staining, and cell counting. Traditionally, scaling requires more people and more space. Limited budgets and risks involved in preclinical development often result in limits on hiring and expansion, making it incumbent on the industry to identify efficient methods for accelerating cell line development with a limited workforce. For cellular factories to live up to their promise, the process of large-scale cell line development needs to be made faster, more efficient, and less expensive. Challenges with implementing and scaling transfection methods Stable Transfection 6 7 Methods for stable transfections Stable Transfection Transfection of cells for stable expression is typically done using random genome integration. Transfection protocols introduce genetic elements into host cells via electroporation or heat shocks without a delivery method targeted to the genome. Since genes may not be specifically engineered for insertion into a specific genomic site, they may be delivered anywhere within the host cell genome. Downstream, this can create a considerable screening burden. Finding a cell line that can express a desired product at an acceptable level can be like “finding a needle in a haystack!” Engineering the insertion of elements into a host cell genome will change CLD steps downstream of transfections. In the near term, designing and ordering vectors carrying integration sites for a host cell genome is a start to alleviating some of the pain points associated with random integration. Self-inserting transposons can be coded and engineered with a corresponding transposase to target insertion into specific genomic sites, such as hotspots for gene expression. Leveraging CRISPRCas systems to directly edit genomes is a promising technology for streamlining stable transfection; however, off-target effects must be addressed or optimized before seeing widespread use. Delivery systems must also be optimized, as well as optimizing delivery success of multiple CRISPR plasmids or ribonucleoprotein into cells. 8 9 What does the evidence say? Stable Transfection Beckman Coulter Life Sciences and IDT Application Note Automated IDT Alt-R CRISPR/Cas9 Ribonucleoprotein Lipofection Using the Biomek i7 Hybrid Automated Workstation IDT genome editing and analysis application note Flourescently labeled tracrRNA provides efficient genome editing while allowing cellular microscopy and FACS analysis Download Now Download Now 10 11 Post-transfection cell line screening Cell Line Screening and Monoclonality in cell line development Stable transfections results in a mixed population of cells, with inconsistent integration of DNA into cellular genomes across a population. Only a fraction of the cells may have been successfully transfected. Successful integration of the new DNA and expression of the desired phenotype must be verified. NGS can be used to confirm placement within a genomic area, while quantitative PCR (qPCR) can be used to establish the level of expression of the knocked-in sequence. Screening methods for protein products include biolayer interferometry (BLI), highperformance liquid chromatography (HPLC), and Beckman Coulter Life Sciences ValitaTiter IgG titer assays. These screening processes can be time-consuming, even more so for large-scale, parallel production of cell lines, taking months to complete. It is usually necessary to screen thousands of culture plates to identify optimal expressing clones, adding significant time to cell processing. Hands-on culture of multiple cell lines in parallel and sample preparation and screening of cells from many culture flasks are recognized pain points in the screening process. Also, handling larger volumes of cell lines, with the various manipulations, analytical sample preparations, and extended culture times increases the risk of compromising data integrity. Data must be managed in a way that enables traceability and thorough reporting once cells have been verified for monoclonality. Assurance of monoclonality, that a cell line is derived from a single progenitor, is critical for cell-derived biotherapuetics. As regulations for clinical and cell line development become more stringent, and biologic drugs become more complex, it is required to show that each new cell line is derived from a single clone, thus avoiding heterogeneity in the drug product. Monoclonality of cell lines must be established and documented. Accepted evidence of monoclonality are images of the cell culture over time demonstrating that the final cells are derived from a single cell. Challenges of Cell Line Development: Screening and Monoclonality Verification 12 13 14 15 Beckman Coulter Life Sciences CytoFLEX SRT Benchtop Cell Sorter Learn More Setup is smart and simplified, using automation to establish and maintain the sort stream Remote support ready using BeckmanConnect for on demand access to our technical specialists, perform many routine maintenance procedures independently Capable of complex sort logic, including 4-way sorting, Mixed Mode sorting, and the ability to catch aborts and preserve precious cells Automation of the cell handling, screening, and clone selection processes can help scale productivity, increase throughput and reduce the time for this step of cell line development. Instrumentation can be used to achieve single-cell sorting, deposition into wells, clone analysis and documentation of clonal density. The Life Sciences companies of Danaher offer several solutions for cell screening and monoclonality verification. During colony screening, quantify IgG titer in less than 15 minutes using Beckman Coulter Life Sciences ValitaTiter IgG assays, which can be automated with a liquid handler and plate reader to screen clones for IgG production quickly and efficiently. Meet your monoclonality documentation goals by leveraging a variety of techniques. Limiting dilution protocols can be automated using the Beckman Coulter Life Sciences Biomek i-Series Automated Workstation, removing significant associate hands-on burden associated with this protocol. For more confidence that cultures are starting with a single cell, the Beckman Coulter Life Sciences CytoFLEX SRT Benchtop Cell Sorter allows for delivery of cells directly into individual wells of a microplate for culture. Automating image acquisition minimizes associate time manually capturing images. The Molecular Devices CloneSelect Imager provides a hands-off image capture solution, and can be integrated with the Beckman Coulter Life Sciences Biomek i-Series workstation to enable automated cell culture and monoclonality report generation. For complete automation of a colony picking and monoclonality verification workflow from cell culture to image capture, the Molecular Devices ClonePix 2 Mammalian Colony Picker is an all-in-one, hands-off instrument that performs cell culture, screens, and selects single cells to start new monoclonal colonies. These techniques all generate a significant amount of data. The IDBS Polar Biopharma Lifecycle Management platform works seamlessly with these instruments to provide the convenience and efficiency of an integrated process and data capture workflow, making achieving and reporting monoclonality easier by serving as a single point of data acquisition and management. Screening and Monoclonality Verification Considerations for increasing capacity for monoclonality verification 16 17 If you are ready to scale up monoclonality analysis and data reporting, experts at the Life Sciences companies of Danaher can work with you to create a custom setup ideal for your specific workflow, allowing you to reduce hands-on time, improve efficiency, and conveniently and accurately report data. What does the evidence say? Screening and Monoclonality Verification Molecular Devices Monoclonality Assurance Application Note CloneSelect Imager FL fluroesence feature & rapid day zero monoclonality assurance as efficient tools to boost targeted gene editing and cell line development IgG Quantification app note Beckman Coulter Life Sciences and Molecular Devices IgG Quantification for clone screening Application note IDBS Case Study: Cell Line Development Customer Success Story: streamlined cell line development Download Now Download Now Download Now 18 19 Media development in cell line development Cell viability, stability of long- term cultures, and correct conformation and folding of the final product are critical attributes of candidates for a master cell bank (MCB). Throughout the development of new cell lines, the composition of cell culture media remains an essential consideration for maintaining the cells in optimal condition for bioproduction. Successful media development during cell line development directly informs the success of upstream process development when scaling up to larger volumes. Every cell line is unique, with each clone having optimized product with specifically developed and chemically defined media. Whereas labs regularly use commercially available, “off- the-shelf” cell culture media for routine work, when used for bioproduction, such media may require extensive modification to effectively produce the desired biotherapeutic. Media development and optimization are necessary to drive efficient production of biotherapeutics in cells, regardless of the therapeutic modality. Although formulation of cell culture media is a strong determinant of successful biotherapeutic production, other desirable features of candidates for the MCB, such as clone robustness, are not necessarily tied to media composition. However, before a cell line can be included in the MCB, the FDA requires the cell line to have undergone 60 rounds of culture to establish its suitability for production. So, it is essential for the chosen culture media to be able to sustain clone robustness by maintaining a constant pH, supporting protein folding, and providing necessary nutrients for protein production. Challenges of Cell Line Development: Media Development 20 21 Culture media for use in bioproduction must be developed with scaling in mind. Careful consideration for cost, availability, and quality should be given to the choice of components, nutrients, and additives. Inconsistent reagent quality and supply chain interruptions will be detrimental to process development and commercial success. Optimizing cell culture media can be a daunting task when educated guesses and trial and error are used to govern decisions. The number of permutations of components and concentrations that can be investigated is time- consuming and expensive. For media optimization with reduced hands-on time, the Beckman Coulter Life Sciences ValitaCell ValitaTiter assays facilitate rapid IgG quantification to aid a full factorial DoE method. Cell viability can also be determined using the Beckman Coulter Life Sciences Vi- CELL BLU Cell Viability Analyzer, which offers sample processing with fully automated sample preparation and analysis. For more efficient and consistent sample dispensing, the Beckman Coulter Life Sciences Biomek i-Series Automated Liquid Handler can be integrated into workflows, providing walk-away convenience. To expedite your media development processes, contact our team of experts today. We’ll work together to help you find a solution that fits your specific needs While implementing a design of experiment (DoE) approach can help to reduce burden associated with media optimization, DoE does not mitigate the lack of predictive insights into developing bespoke media that best delivers the desired qualities in each candidate cell line. Without preliminary media studies, it can be difficult to define a starting point for media composition. Post-culture analysis of combinations generated by DoE is often also time-consuming and expensive. An assay that mimics the stresses induced by bioprocessing and key production pathways would be transformative for guiding data-driven, predictive decisions about clone stability under different media conditions. In addition to analyzing the effects of varying media components on cells and their products, such assays should measure nutrient depletion and metabolite production in spent media to provide useful insights for media development and optimization. The Life Sciences companies of Danaher provide a wide range of solutions to help ease the burden and eliminate trial and error and bottlenecks during media optimization for cell line development. Media Development Considerations for development, optimization, and analysis of culture media 22 23 What does the evidence say? Media Development Beckman Coulter Life Sciences Automated DoE Application Note A fully automated plate-based optimization of fed-batch culture conditions for monoclonal antibody-producing CHO cell line Download Now 24 25 Challenges of Cell Line Development: Scale up and Bioreactor Studies 26 27 Assessing the genetic and expression stability of monoclonal cell lines is essential for successful cell line development and to establish product integrity. More importantly, for regulatory compliance, clones must remain genetically and phenotypically stable throughout the manufacturing process for biotherapeutics. There are few established methods for predicting longterm clone stability after a cell line has been generated, and data from one successful cell line cannot be projected to another. To establish productivity and stability, cell lines from the master cell bank (MCB) are expanded over 80–120 rounds of cell culture with continual assessment. With three to five days between passages, stability studies could take up to one year to complete. Due to the number of rounds of cell culture required, stability studies can be laborious and time-consuming, particularly if automation is not employed. The lengthy culture time, which includes maintaining and passaging many vessels in parallel, increases the risk of contamination. Cells must be constantly analyzed and multiple critical quality attributes (CQAs) measured throughout this process. Attributes assessed include cell count, cell viability, gene copy number, antibody titer, media nutrient depletion, and other parameters. For many cell line development labs, assessing CQAs during this process requires dedicated instrument practitioners or sending cell samples to an analytical lab, both of which can increase costs and extend time to results. Shortening time to data can be a boon to efficient bioreactor studies, as eliminating undesirable clones earlier reduces the amount of hands- on work and materials required. However, faster data acquisition also creates a separate bottleneck: the volume of data that must be collected over the duration of the stability studies can become unwieldy, leading to potential data reporting errors. Data integrity during these studies is a primary concern, as clones selected during this study will be the final candidates for the MCB. Assessment of stability for novel cell lines Scale up and Bioreactor Studies 28 29 Automation of labor-intensive, repetitive steps can help reduce the hands-on time required for stability studies. CQAs used for selecting clones should be monitored during this period. An integrated platform that automatically captures and transfers critical data to a secure server for convenient access is essential for early elimination of poor-performing clones. Danaher companies provide various solutions to streamline your stability studies and data recording. At-line assaying and data acquisition are essential for early identification of the best-performing clones in bioreactor trials. IgG titer measurements with Beckman Coulter Life Sciences ValitaTiter Assay allow quick monitoring of the consistency of mAb titer over time as cultures are passaged. Understanding the effects of varying conditions on cell count and viability is essential for identifying clones that will flourish under different production stressors, but sample preparation and processing during this process can be time consuming. Implementing automation with an instrument such as the Beckman Coulter Life Sciences Vi-CELL BLU Cell Viability Analyzer reduces manual steps and allows quick identification of critical parameters that influence cell stability. Hands-on time can be further reduced with automation solutions, such as a Beckman Coulter Life Sciences Biomek i-Series Automated Workstation for analytical sample preparation and a Molecular Devices SpectraMax microplate reader for assay measurements, improving consistency and efficiency of many daily processes. Properly assessing the various factors that support the generation of the most stable clones requires collection and evaluation of vast swathes of data, which can be challenging to manage. An integrated solution that enables data acquisition and technology transfer, while facilitating regulatory compliance, is essential for effective scaling. The convenience and efficiency of an integrated process and data capture workflow can be achieved with the IDBS E-WorkBook platform and Polar Biopharma Lifecycle Management platform, which serves as a single source for all data management and provides an audit trail in compliance with FDA 21 CFR Part 11. Scale up and Bioreactor Studies Considerations for increasing efficiency of stability studies To find the right solutions to expedite your media development and optimization, contact Danaher Life Sciences experts, who will design a solution for your specific needs. 30 31 What does the evidence say? Scale up and Bioreactor Studies Beckman Coulter Life Sciences application note A fully automated plate-based optimization of fed-batch culture conditions for monoclonal antibody-producing CHO cell line IDBS Polar eBook Revolutionize your lab with a cutting-edge bioprocess platform to drive quality and efficiency Download Now Download Now 32 33 Challenges of Cell Line Development: Digital and Data Solutions Digital record keeping in cell line development Data integrity is a critical requirement throughout biopharmaceutical drug development and manufacturing, with data quality, accuracy and consistency being key reporting concerns. During chemistry manufacturing controls, cell line development requires tracking thousands of candidate cells and conducting analyses to determine the best candidates for a master cell bank (MCB). According to the FDA’s industry guidance for data integrity and compliance with cGMP data must be reliable and accurate; meaningful and effective strategies should be implemented to manage data integrity risks based on an organization’s process understanding. However, data integrity is at risk when data transcription and transfer are performed manually. Digital tools, such as laboratory information management systems (LIMS), manufacturing execution systems (MES) and electronic lab notebooks (ELNs) are commonplace in commercial labs, but they have their limitations when used in an industry as highly regulated and diverse as bioprocessing. For example, while LIMS may be useful for sample tracking within a study, some labs have recognized that these tools do not scale without a more comprehensive data management strategy. Also, critical information such as analytical instrument setup, method validation documentation, sample QC, and system calibration records are not captured. Similarly, MES and ELNs are useful for recording data, but may not provide contemporaneous data or alert operators to anomalies that require further investigation. These tools are unable to provide insights that inform process and operational improvement, so they do little to contribute towards speeding up time to market. 34 35 Although some organizations have created or contract in centralized repositories that improve accessibility and facilitate data capture and sharing, inefficient technology transfer due to data siloing between organizations remains an issue across the biopharmaceutical industry. Data export from or to centralized solutions from different vendors can be a challenging exercise, as having to harmonize information and reconcile data with different identifiers may add significant processing time. The difficulties associated with data sharing and reconciliation within and between organizations represent a major challenge in cell line development. Internally, different labs and functions within an organization must be able to share data easily for efficiency, consistency, and reproducibility. The ability to transfer technology seamlessly to external partners, such as contract development and manufacturing organizations (CDMOs), is of critical importance in manufacturing biotherapeutics. The success of the relationship with CDMOs hinges on transferring vital information required to manufacture the product using the exact processes to avoid provoking regulatory scrutiny. FAIR and ALCOA+ guidelines set the standards for criteria that organizations must meet for data infrastructure and data integrity, respectively. Implementing a strong data backbone with digital tools that facilitate data sharing and technology transfer, is a step in the right direction towards meeting these standards. To ease the path towards commercialization, it is important to ensure that systems chosen for data management and reporting have been designed taking into consideration FAIR and ALCOA+ principles. Talk Data to Me: Data Sharing and Technology Transfer Digital and Data Solutions 36 37 Enabling compliant data management Digital and Data Solutions Large organizations may opt for in-house solutions for systems integration, but the time, resources and cost required for long-term maintenance makes this out of scope for many companies. A single, digital solution that underpins an entire drug development workflow would help to connect labs and their processes, removing data silos and facilitating hand-offs from R&D to process development and from process development to CDMOs. Biopharmaceutical manufacturers would also benefit if the same system could execute processes and capture data, automating readout from equipment and transfer to a secure server. A single data backbone that allows for efficient execution of processes and data curation can help to achieve a more streamlined and comprehensive drug development process. Offerings from the Life Sciences companies of Danaher, such as the IDBS E-WorkBook LIMS Platform and IDBS Polar BioPharma Lifecycle Management platform, present a lifecycle management solution that accurately captures complete data for easy access, reuse, and reporting. These platforms securely manage biotherapeutic workflow progression, enabling automation and curation of a process-centric data backbone, helping organizations meet FAIR and ALCOA+ guidelines. 38 39 What does the evidence say? Digital and Data Solutions IDBS Case Study: Cell Line Development Customer Success Story: streamlined cell line development Download Now References 1. Data Integrity and Compliance With Drug CGMP https://www.fda.gov/media/119267/download 2. The F.A.I.R. Principles: A quick introduction. https://www.idbs.com/the-fair-principles-a-quick-introduction 40 41 Challenges of Cell Line Development: Analytical Data Management 42 43 Analytics and data management in cell line development Cell line development requires characterization of cells and cell products using various analytical assays to identify the best candidate for a master cell bank (MCB). The lines between cell line development and upstream process development are becoming increasingly blurred. Having more insights earlier in cell line development eases subsequent steps of process development. Satisfying increasing regulatory requirements for drug product documentation requires a variety of assays, many of which can be logistically difficult or time consuming to perform. Many factors, including capital investment, laboratory space, and qualified personnel, determine whether laboratories conduct analyses in-house or outsource to contract laboratories or core facilities. Due to the extensive lab footprint and specialized knowledge required for many conventional analytical instruments, mass spectrometry and HPLC are frequently outsourced to third-party services (off-line analytics). The associated turnaround times and lack of immediate insight into critical data can delay making datadriven, informed decisions. Challenges of Cell Line Development: Analytical Data Management 44 45 Due to the number of cell lines laboratories routinely culture and process daily, automation of sample handling is a key advancement to reduce or eliminate labor-intensive steps. Assays previously requiring large cell culture volumes to generate adequate test material can be done from multiwell plate volumes using just a small fraction of the sample. Therefore, automation of analytical sample preparation within a liquid handler performing routine cell culture duties is feasible, facilitating higher throughput assaying. Another challenge is manipulating the vast amounts of data that results from monitoring a multitude of clones. Analyzing drug products and detecting low-abundance proteoforms typically involves identifying individual charge variant components and correlating structural differences in a process that may take days and requires specialized knowledge. The resulting cell line CQA and protein product CQA data are usually collected by various instruments with no intercommunication, leaving many labs juggling data between spreadsheets. What the industry needs is a fully integrated setup that simplifies the interplay between sample handling, analytics, and data management. This automation of data handling would allow laboratories to screen many more clones and select the best, using data-backed decision making rather than preset parameters. Challenges of data acquisition and management in cell line development Analytical Data Management 46 47 The Life Sciences companies of Danaher provide various solutions for integrating analytics during cell development. For fully automated, rapid immunoglobulin G (IgG) titer measurements, you can perform sample preparation for the Beckman Coulter Life Sciences Valita Titer IgG quantification assay using a Beckman Coulter Life Sciences Biomek i-Series Automated Liquid Handler and read with an integrated Molecular Devices SpectraMax multi-mode reader. You can also easily automate analytical sample preparation associated with lengthy, time- consuming preparations. Quick assessment of critical quality attributes (CQAs) of drug products, such as charge variants and glycosylation, can be achieved using the SCIEX Intabio ZT system, which automates sample preparation for separation, mass identification, and proteoform quantification on a single platform, reducing the space burden of several large pieces of equipment. Current technology allows cell line development labs to bring difficult analytical methods inhouse, reducing or eliminating dependence on core facilities and thirdparty services. Automated equipment, routinely operable with minimal training, provide the convenience of directly reporting product CQAs in formats that meet regulatory standards. Analytical Data Management Solutions for integrated analytics and data management in cell line development To find the right solution to simplify your cell line development workflow and obtain the level of control you want for your analytics, contact experts from the Life Sciences companies of Danaher, who will design an analytics solution for your specific needs. 48 49 What does the evidence say? Analytical Data Management GeneData Expressionist Customer Case Study Assessing Developability of Novel Biotherapeutics Through Mass Spectrometry SCIEX Intabio ZT Application Note Direct and rapid multi-attribute monitoring of intact monoclonal antibodies by icIEF-UV/MS Download Now Download Now 50 51 Danaher Corporation Danaher is a global science and technology innovator committed to helping its customers solve complex challenges and improving quality of life around the world. Its family of world class brands has leadership positions in the demanding and attractive health care, environmental and applied endmarkets. With more than 20 operating companies, Danaher’s globally diverse team of approximately 81,000 associates is united by a common culture and operating system, the Danaher Business System, and its Shared Purpose, Helping Realize Life’s Potential Leica Microsystems We see a way to transform scientific discovery with a combination of cuttingedge microscopes and ground-breaking digital analysis. Leica Microsystems is one of the market leaders in microscopy and scientific imaging. The company develops highly advanced instruments for the analysis of microstructures and nanostructures – increasingly harnessing the power of machine learning, automation, and data analytics to provide greater insights to scientists than ever before. Learn more about Leica Microsystems Beckman Coulter Life Sciences We see a way to expedite the development of life-saving therapeutics through the use of innovative instruments and reagents. 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The company’s integrated hardware and software solutions enable start-to-finish automated workflows for cell line development, 3D biology and drug screening. Learn more about Molecular Devices IDBS We see a way to harness the power of data to unlock faster and smarter decisions in biopharma research, development, and manufacturing. IDBS provides purpose-built software solutions to address data management challenges prominent across the biopharma lifecycle and supply chain. IDBS’ groundbreaking technologies, including the world’s first BioPharma Lifecycle Management (BPLM) solution, streamline the capture, analysis, reporting and sharing of data to accelerate the next generation of life-changing therapeutics. Learn more about IDBS SCIEX We see a way to use the power of precision to drive scientific breakthroughs, achieve better outcomes and advance human wellness. SCIEX has led the field of mass spectrometry and capillary electrophoresis for over 50 years, pioneering innovative solutions for the precision detection of quantification of molecules. Our products and services have supported thousands of scientists and influenced life-changing research that has positively impacted the lives of people around the world. Learn more about SCIEX 52 53 About our Brands The Life Science Companies of Danaher For research use only. Not for use in diagnostic procedures. ©2024 DH Life Sciences, LLC. All Rights Reserved. ©2024 Beckman Coulter, Inc. All rights reserved. Beckman Coulter, the stylized logo, and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries. All other trademarks are the property of their respective owners. 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