eBook: Cell-Line Development CQAs A
C Analyzing Critical Quality Attributes to Enable Efficient and Rapid Cell Line Development High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Assessment of IgG Cell Culture and Production Media Optimization Monoclonal and Bispecific Antibody Titer Other CQA Screening and Evaluation Bioassays for Functional Characterization Summary and References
Introduction Cell line development (CLD) is the engineering of a cell line, often mammalian, to produce a therapeutic biomolecule or biologic. It allows scientists to tailor cultures depending on the applications and desired properties of the targeted product. A typical CLD process revolves around five key stages: gene cloning and transfection, clone selection, media cultivation and expansion, cell line evaluation and characterization and cell banking (Figure 1). The process includes the screening of thousands of clones to find those that are stable, produce high yields of the bioproduct and exhibit desired critical quality attributes (CQAs)1. During the process, cells must be monitored for viability, morphology, density and monoclonality. In addition, product-related attributes such as titer, glycosylation levels, propensity to aggregate and ability to bind to target need to be monitored early in the process to minimize product failure downstream. Due to the high number of clones screened upstream, analytics that enable high-throughput screening at the various stages of the process are desirable. In this document, we describe how Sartorius analytical technologies fulfill critical needs along the entire span of the CLD workflow. 1 2 3 4 5 Gene Cloning and Initial Clone Selection Clone Selection and Confirmatory Analytics Cultivation and Media Optimization Cell Line Evaluation and Characterization Cell Banking
High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Assessment of IgG Cell Culture and Production Media Optimization Monoclonal and Bispecific Antibody Titer Other CQA Screening and Evaluation Bioassays for Functional Characterization Summary and References
High-Throughput Image-Verified Cloning Cell lines used for the development and manufacturing of biotherapeutics must ensure monoclonality not only to avoid future setbacks, but also as a required part of the regulatory process. Isolating high-throughput nanowell-based image-verified cloning technology (HT-NIC) using the CellCelector system is a technique that generates clones in a single cloning round, while providing robust in-process image-verified monoclonality proof. Due to this integrated monoclonality and viability assessment of clones, as well as industry-leading outgrowth rates after clone transfer into 96- or 384-well plates, the CellCelector single-cell cloning technology represents the next generation of single-cell cloning approaches. It goes far beyond traditional methods and provides a superior alternative to limiting dilution, fluorescence- activated cell sorting (FACS), or single-cell printing techniques. This patent-pending method has been developed and validated in collaboration with ProBioGen AG and other CellCelector customers. With the CellCelector single-cell and colony picking platform, you can effectively assess and verify your clones before deciding which ones to expand. In less than one week, you will obtain monoclonal, viable and productive colonies from your pool of single cells. Instead of relying on large quantities of plates to produce a winner you can now use actual data to reliably predict the future of your clones. This saves consumable and media costs, incubator space and valuable time by avoiding missteps, additional cloning rounds and unnecessary procedures. HT-NIC method CellCelector Cloning method Figure 2: Nanowell plates allow cells to be cultured monoclonally Figure 3: CellCelector Nanowell-based single-cell cloning workflow Figure 4: CellCelector HT-NIC 100% monoclonality compared to traditional CLD limiting dilution techniques Figure 5: CellCelector HT-NIC results compared to traditional CLD picking techniques High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Assessment of IgG Cell Culture and Production Media Optimization Monoclonal and Bispecific Antibody Titer Other CQA Screening and Evaluation Bioassays for Functional Characterization Summary and References The HT-NIC method is based on the CellCelector nanowell plates. These plates are available in different formats, featuring thousands of nanowells at the bottom of each well. For a 24-well plate this results in 4,000 nanowells per well or 100,000 nanowells per plate. Cells inside the nanowells are efficiently separated from each other. Despite the local separation, the cells in the nanowells are covered by the same medium and thereby growth-promoting cellular crosstalk can occur (Figure 3). All cells in the pool will contribute to the outgrowth of the cell line, while maintaining their monoclonality. This leads to high outgrowth rates of single cells, even for cell lines that are extremely difficult to cultivate.
High-Throughput Image-Verified Cloning High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Assessment of IgG Cell Culture and Production Media Optimization Monoclonal and Bispecific Antibody Titer Other CQA Screening and Evaluation Bioassays for Functional Characterization Summary and References
High-Throughput Image-Verified Cloning After monoclonality has been documented, cells are incubated for 3-6 days, resulting in 20- 75 cells per clone. Unlike the traditional methylcellulose-based approach, the CellCelector nanowell-based method allows colony growth in liquid media, which is shared by the isolated colonies. After the cells have grown into single-cell clones, the nanowell plate is scanned again and monoclonal, viable clones are automatically selected and transferred into 96- or 384-well plates for further analysis and upscaling. In a conventional single-cell cloning workflow, single cells are seeded in 96-well plates making reliable automated single-cell detection difficult as cells are often settled at the very edge of the well. Thus, the monoclonality status of a given clone at Day 0 is usually checked manually or retroactively once the clone has grown. This requires searching for a single cell within a well area that is more than 100 times larger than the surface occupied by a cell. With the CellCelector HT-NIC approach, the cells are separated and clearly visible within 200 µm large nanowells and can therefore be identified reliably and automatically by the software. Identification is possible just after seeding and even when cells are in contact with the nanowell border. In limiting dilution, a well-established method for single-cell cloning, the monoclonality is based on statistical properties, which depend on the average cell number seeded per well according to the Poisson distribution. To limit the percentage of polyclonal wells, cell density for seeding is reduced to 0.2 cells per well. Further, only ~16% of the wells are monoclonal, while the other wells stay empty. Thus, more than twenty-five 96-well plates need to be seeded to start with 400 monoclonal wells. There is the possibility to increase cell density and thereby double the number of monoclonal wells, but this comes with a high risk for polyclonal wells. The CellCelector HT-NIC cloning method provides 100% monoclonal wells by automating the identification of single-cell nanowells, tracking their growth into clones, and transferring the grown clones into 96-well plates without cross-contamination. Importantly, the process is reliable independent of sample preparation, cell type, or the cell density used for seeding into the nanowell plate. A comparison of the CellCelector HT-NIC method and limiting dilution shows the significant increase in efficiency for obtaining monoclonal wells with HT- NIC (Figure 4). To obtain 400 monoclonal wells using the limiting dilution method, twenty-six 96-well plates (for 0.2 cells/well seeding density) or thirteen 96-well plates (for 0.5 cells/well seeding density) need to be used. The same number of monoclonal wells is reached within just one well of the CellCelector nanowell plate using the HT-NIC method. High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Assessment of IgG Cell Culture and Production Media Optimization Monoclonal and Bispecific Antibody Titer Other CQA Screening and Evaluation Bioassays for Functional Characterization Summary and References
High-Throughput Image-Verified Cloning Cell seeding is performed similarly to conventional cell culture plates. After seeding the cells, they are randomly captured inside the nanowells following the classic Poisson distribution. Automated scanning of the wells, followed by an automated identification of all nanowells containing a single cell, provides a robust and documented image-based monoclonality proof. Depending on the number of cells seeded, 400-600 single cells are captured per well and can be analyzed. Seeding multiple wells allows you to start with up to ~14,000 single cells captured in individual nanowells- all within just one nanowell plate. Cell seeding into a nanowell plate Monoclonality screening (Day 0) Clone growth assessment (Day X) Automated clone ranking and selection Automated transfer of selected clones for further expansion in a 96- or 384-well plate High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Assessment of IgG Cell Culture and Production Media Optimization Monoclonal and Bispecific Antibody Titer Other CQA Screening and Evaluation Bioassays for Functional Characterization Summary and References
High-Throughput Image-Verified Cloning 100% 100% 80% 60% 40% 30% 20% 16% Empty Wells Monoclonal Wells Polyclonal Wells 0% Limiting Dilution 0.2 cells/well Limiting Dilution 0.5 cells/well CellCelector HT-NIC High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Assessment of IgG Cell Culture and Production Media Optimization Monoclonal and Bispecific Antibody Titer Other CQA Screening and Evaluation Bioassays for Functional Characterization Summary and References Percentage of Wells
High-Throughput Image-Verified Cloning High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Assessment of IgG Cell Culture and Production Media Optimization Monoclonal and Bispecific Antibody Titer Other CQA Screening and Evaluation Bioassays for Functional Characterization Summary and References
High-Throughput Image-Verified Cloning The limiting dilution method leads to only 10%-20% outgrowth. Other traditional methods like FACS single cell sorting or single cell printing provide more single cells but cannot support strong outgrowth rates. The high shear stress usually associated with flow cytometry and the depositing of single cells in a large volume of fresh media leads to uncertain outgrowth at rates between 30% and 64% (Figure 5). Also, these methods require specialized and expensive cloning media with external growth factors and are unsuitable for difficult-to-grow cell lines. 100% 80% Percentage of Wells 60% 80% 85% 64% 100% 90% 40% 20% 16% 12% 30% 21% 30% Monoclonal Wells Monoclonal Wells with Viable Clones 0% LD 0.2 cells/well LD: Limited dilution LD 0.5 cells/well FACS Single Cell Printing CellCelector HT-NIC High-Throughput Image-Verified Cloning Assessment of IgG Cell Culture and Production Media Optimization Monoclonal and Bispecific Antibody Titer Other CQA Screening and Evaluation Bioassays for Functional Characterization Summary and References Measurement of Cell Count and Viability
Measurement of Cell Count and Viability Throughout the entirety of the CLD workflow, it is vital to monitor the density and viability of cell cultures. From the early stages of clone selection, it is important to choose clones that are healthy, with a favorable growth speed. Later in the workflow, density and viability measurements are used to monitor cell expansion and to ensure optimal growth conditions are selected. The iQue® Advanced Flow Cytometry Platform can be used in conjunction with the iQue® Cell Count and Viability Kit for fast and accurate determination of cell counts, from both 96-and 384-well plates. The kit comes with a template which is imported into the iQue® Forecyt® software, leading to instantaneous cell count and viability readouts from acquired plates. Figure 6 demonstrates the high reproducibility of this method for measuring viable cell counts (VCC) during CLD, as well as it's comparability to another method. This makes it a powerful tool to aid decision making for selection of optimal clones and conditions. Figure 6: Accuracy assessment of viable cell counting with iQue® High-Throughput Image-Verified Cloning Assessment of IgG Cell Culture and Production Media Optimization Monoclonal and Bispecific Antibody Titer Other CQA Screening and Evaluation Bioassays for Functional Characterization Summary and References Measurement of Cell Count and Viability
Measurement of Cell Count and Viability A) Cell Count Validation B) Mini-pool Cultivation C) Clone Cultivation 100 20 VCC [10⁵ cells/mL] 90 Deviation [%] 10 40 40 20 20 Deviation [%] Deviation [%] 0 80 0 0 −20 70 N = 72 −10 −20 −20 −40 −60 60 −40 −80 Other iQue® Other iQue® High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Cell Culture and Media Optimization Monoclonal and Bispecific Antibody Titer Other CQA Screening and Evaluation Bioassays for Functional Characterization Summary and References Assessment of IgG Production
Assessment of lgG Production It is important to find a highly productive cell line as early as possible in the CLD workflow to avoid resource waste during the costly and time-consuming scale-up processes. Typically, this is achieved by using techniques such as enzyme-linked immunosorbent assay (ELISA) to determine the IgG production per well of the cell culture plate. This method is limited in that it provides a bulk measurement of IgG in the well and, therefore, cannot distinguish between the highly productive clones and fast-growing clones that produce low levels of IgG. The iQue® Advanced Flow Cytometry Platform with iQue® Human IgG Titer & Viability Kit can overcome this limitation, as this technique provides simultaneous quantification of the IgG titer, cell density and viability. Together, these metrics are combined to provide a readout for IgG production per viable cell. This, combined with high-throughput acquisition by the iQue® (20 minutes to read a full 384-well plate) can improve the speed and quality of hits generated during early clone selection. Figure 7 displays how clones ranked differently when we incorporated IgG production per viable cell into the measurement, leading to a more robust screen for high producing cells. Figure 7: Example of simultaneously screening for lgG titer and cell health attributes High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Cell Culture and Media Optimization Monoclonal and Bispecific Antibody Titer Other CQA Screening and Evaluation Bioassays for Functional Characterization Summary and References Assessment of IgG Production
Assessment of lgG Production High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Assessment of IgG Production Monoclonal and Bispecific Antibody Titer Other CQA Screening and Evaluation Bioassays for Functional Characterization Summary and References Cell Culture and Media Optimization
Cell Culture and Media Optimization Sartorius' Ambr® 15 Cell Culture system is a robust platform for media optimization and has been shown to provide improved results when compared to shake flask or shaking plate cultures. This is primarily due to the high level of automation combined with reliable and independent process control for pH and DO. The industry standard Ambr® 15 Cell Culture automated microbioreactor system for mammalian cell culture can screen up to 48 × 15 mL per experiment, thus offering considerable advantages for screening of cell lines and media when running intensified processes, particularly in terms of costs. For ease of assessing CQAs, specifically titer determination, the Ambr® 15 can be integrated with the Octet® Biolayer Interferometry (BLI) platform (Figure 8). Octet® instruments offer CLD scientists a platform for the rapid analysis of antibody titer that enables a quick selection of optimal clones. With ready-to-use biosensor surfaces, such as Protein A and G, combined with the automation-ready Octet® RH16 instruments or high-throughput Octet® RH96 instruments, organizations can save significant full time equivalent (FTE) cost over comparative technologies, such as ELISA and HPLC. Moreover, the time to results on the Octet® platform should allow for many more projects per year compared to titer determination using either HPLC or ELISA (Figure 9, Table 1). for high producing cells. Table 1: Value comparison of Octet® BLI System, ELISA and HPLC for titer analysis Figure 8: Ambr® 15 and Octet® BLI Workflow Figure 9: Octet® BLI System titer screening workflow compared to a typical HPLC workflow High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Assessment of IgG Production Monoclonal and Bispecific Antibody Titer Other CQA Screening and Evaluation Bioassays for Functional Characterization Summary and References Cell Culture and Media Optimization
Cell Culture and Media Optimization 1. Ambr® data interface license allows an external computer or shared location to be informed when samples from a bioreactor are produced Mapped drive 2. Octet® Analyst directly imports bioreactor sample information (well mapping, timestamp, or bioreactor information) to Octet® BLI Discovery Software 4. Feedback of Octet® analytical data to Ambr® Software for integrating Octet® and Ambr® data 3. Octet® Analysis Studio Software automatically exports Octet® data to Ambr® 15 file formats after data analysis Export directory Import directory High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Assessment of IgG Production Monoclonal and Bispecific Antibody Titer Other CQA Screening and Evaluation Bioassays for Functional Characterization Summary and References Cell Culture and Media Optimization
Cell Culture and Media Optimization Hydrate biosensors 10 minutes Load biosensor, run reference, standard and unknown samples 4-5 minutes Buffer preparation 15-20 minutes Rinse column 5-10 minutes Sample injection and peak analysis 30-60 minutes Column equilibration 5-10 minutes System washing 10 minutes Total time required: ~60-70 minutes/sample Total volume of sample: ~25-100 µL/sample Quantitation of protein of interest in complex solution (prefiltering of sample required) Total time required: ~15 minutes for minimum 6 samples Total volume of sample diluent: ~200 µL/sample Rapid quantitation of protein of interest in complex solution (filtering not required) High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Assessment of IgG Production Monoclonal and Bispecific Antibody Titer Other CQA Screening and Evaluation Bioassays for Functional Characterization Summary and References Cell Culture and Media Optimization
Cell Culture and Media Optimization High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Assessment of IgG Cell Culture and Production Media Optimization Other CQA Screening and Evaluation Bioassays for Functional Characterization Summary and References Monoclonal and Bispecific Antibody Titer
Monoclonal and Bispecific Antibody Titer Fc-fusion proteins represent a promising class of biotherapeutic drugs. They include peptides, signaling proteins, or other analytes fused with the Fc domain of IgG, which helps to prolong the stability of the protein binding partner. In one study, the CLD group at Biogen IDEC needed a robust assay for the measurement of Fc-fusion protein in crude cell culture supernatants. The group had historically used HPLC for protein quantitation during screening and selection of promising mammalian clones at every scale-up step, from 96-well microplates to 3L-bioreactors. They wanted to replace the HPLC method due to its low throughput, cumbersome sample processing and long run times with a reliable, higher-throughput alternative. The 16-channel Octet® BLI instrument was evaluated due to its many advantages. Octet® BLI systems can analyze crude samples, allowing users to bypass time-consuming sample pre-processing. In addition, 96 samples could be analyzed in less than 30 minutes, expediting screening that took more than 19 hours to complete by HPLC. A higher throughput, automated screening workflow with significantly reduced analyst involvement was achieved via integration of the Octet® BLI system and a PerkinElmer (formerly Caliper Life Sciences) Sciclone robot5. Bispecific antibodies (bsAbs) have grown rapidly in recent years as one of the main classes of therapeutic antibodies. At an early stage of CLD, screening of cell lines expressing bsAbs is a major challenge due to the complexity of expressing multiple chains and the existence of many diverse bsAb formats. ELISA is one of the methods that can be used for bsAbs function evaluation, but it is both labor- and time-intensive. Analytical methods such as SDS-PAGE, CE-SDS and RP-HPLC can help to determine the bsAb purity in cell-line screening. However, all these methods require protein A (ProA) purification, which can be time-consuming. During CLD, thousands of pools (none clonal-derived cell lines) and clones (clonal-derived cell lines) are evaluated. Therefore, there is high demand for a simple and high-throughput method for the functional assessment of two or more interactions of complex bispecific therapeutics. In comparison with other methods, the high-throughput Octet® BLI systems allow for rapid screening of pools and clones expressing the target bsAb, without the need for ProA purification. This assay consists of capturing the bsAb by each antigen sequentially using the correspondent antigen loaded onto the biosensor surface. This fluidics-free and microplate-based assay format offers an easy screening method and workflow to assess bsAb interactions in a versatile, label-free, and easy-to-use format. High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Assessment of IgG Cell Culture and Production Media Optimization Monoclonal and Bispecific Antibody Titer Bioassays for Functional Characterization Summary and References Other CQA Screening and Evaluation
Other CQA Screening and Evaluation Lead selection through target binding and off-rate ranking 10, 11 Relative glycan screening 12, 13 Figure 10: Workflow of glycan screening on the Octet®BLI System High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Assessment of IgG Cell Culture and Production Media Optimization Monoclonal and Bispecific Antibody Titer Bioassays for Functional Characterization Summary and References Other CQA Screening and Evaluation
Other CQA Screening and Evaluation High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Assessment of IgG Cell Culture and Production Media Optimization Monoclonal and Bispecific Antibody Titer Bioassays for Functional Characterization Summary and References Other CQA Screening and Evaluation
Other CQA Screening and Evaluation Titer Analysis Sialic Acid Content vs. Titer Time (seconds) Titer/ Quantitation Protein titer Mannose Content vs. Titer Relative glycan screening Glycan Screening Time (seconds) Protein titer Octet® GlyS / GlyM Kit Octet® Analysis Studio Quantitation biosensor* Crude or purified protein High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Assessment of IgG Cell Culture and Production Media Optimization Monoclonal and Bispecific Antibody Titer Bioassays for Functional Characterization Summary and References Other CQA Screening and Evaluation Binding Binding Mannose Content Sialic Acid Content
Other CQA Screening and Evaluation High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Assessment of IgG Cell Culture and Production Media Optimization Monoclonal and Bispecific Antibody Titer Other CQA Screening and Evaluation Summary and References Bioassays for Functional Characterization
Bioassays for Functional Characterization During antibody characterization, it is important to characterize whether a candidate is likely to translate to a therapeutically-efficacious product. This can be done by conducting in vitro assays to assess the antibody function against potential mechanisms of action (MoAs). For example, the iQue® Human Natural Killer (NK) Cell Killing Kit is used to measure the ADCC activity of test antibodies in a 96- or 384-well format. Target cells are incubated with NK cells and test antibodies, before cell and supernatant samples are analyzed using the iQue® System. Cells are labelled using a membrane integrity dye and antibody cocktail, such that target cell death can be quantified and CD3-CD56+ NK cells can be phenotyped and assessed for their activation marker expression (CD16 and CD25) and ADCC activity (CD16 expression). Qbeads® are included to allow simultaneous evaluation of supernatant cytokine concentrations (Granzyme B and IFNγ). Together, this provides a comprehensive assessment of antibody in vitro ADCC activity. The iQue® and associated kits can also quantify other key MoAs of therapeutic monoclonal antibodies, for example antibody-dependent cellular phagocytosis (ADCP), as measured using the iQue® Human ADCP Kit. Similarly, to the NK assay, the iQue® ADCP assay measures the effect of test antibodies in a target and immune cell co-culture assay, this time quantifying the co-localization between phagocytic immune cells and antigen-positive targets as an indicator of ADCP activity. Some antibodies, for example antibody-drug conjugates (ADCs), rely heavily on mechanisms such as antibody internalization to exert their cytotoxic capabilities. ADCs release their payload from linkers when triggered by acidic conditions or proteases inside cells, leading to cell death. This can be quantified using the iQue® Human Antibody Internalization Kit, which combines analysis of cell viability with a fluorescent probe-based measurement of target antibody internalization into the acidic lysosomal and endosomal pathways. Carrying out a range of in vitro functional assays provides a broad picture of the potential MoAs of a novel antibody, which can inform downstream and pre-clinical processes. High-Throughput Image-Verified Cloning Measurement of Cell Count and Viability Assessment of IgG Cell Culture and Production Media Optimization Monoclonal and Bispecific Antibody Titer Other CQA Screening and Evaluation Bioassays for Functional Characterization Summary and References
Summary and References In this eBook, we have described a variety of Sartorius' bioanalytical platforms designed to facilitate early analysis of CQAs in the CLD workflow. These solutions enable scientists to gain insights into product quality early in development and avoid costly setbacks further downstream. From the CellCellector and the Ambr® 15 platforms for single-cell cloning and culture optimization, to the Octet® BLI and iQue® platforms for clone characterization, Sartorius simplifies the CLD process and helps you identify the winning clones with confidence.
Biosensor For more information or to get in touch with one of our Sartorius Lab Instruments GmbH & Co. KG Otto-Brenner-Strasse 20 37079 Goettingen Phone +49 551 308 0 Sartorius Corporation 565 Johnson Avenue Bohemia, NY 11716 Phone +1 631 254 4249 Toll-free +1 800 635 2906
For further contacts, visit www.sartorius.com Specifications subject to change without notice. Copyright Sartorius Lab Instruments GmbH & Co. KG. Status: 05 | 2023
