Narasimha Murthy Bandaru, Scientist
Anna Moberg, Sr Manager, Biacore Applications and Consumables
Cell line development (CLD) involves generating cell lines to be used for applications including drug discovery, vaccine production, and gene function studies. The development of a cell line comprises several key steps aimed at creating stable, high-producing cell lines that yield a therapeutic molecule with the necessary quality and consistency (1).
This article focuses on development of surface plasmon resonance assays for high throughput screening and selection of promising cell clones, in this case, a campaign to produce a bispecific antibody (bsAb). The process begins with the transfection of host cells, commonly Chinese hamster ovary (CHO) cells, with genes encoding the therapeutic protein. These cells are then screened to assess multiple characteristics, such as expression level, and identify high-producing clones with the desired product profile (2).
Introduction
Cell line development (CLD) involves generating cell lines to be used for applications including drug discovery, vaccine production, and gene function studies. The development of a cell line comprises several key steps aimed at creating stable, high-producing cell lines that yield a therapeutic molecule with the necessary quality and consistency (1).
This article focuses on development of assays for high throughput screening and selection of promising cell clones, in this case, a campaign to produce a bispecific antibody (bsAb). The process begins with the transfection of host cells, commonly Chinese hamster ovary (CHO) cells, with genes encoding the therapeutic protein. These cells are then screened to assess multiple characteristics, such as expression level, and identify high-producing clones with the desired product profile (2).
Fig 1. Overview of cell line development titer screening process and where Biacore SPR titer assays are applied.
Biacore™ 8 series surface plasmon resonance (SPR) systems offer high-throughput screening and selection of the best-performing clones. The analysis can take place directly in supernatant from cell cultures, and the high sensitivity of Biacore systems allows your sample to be diluted up to 1000 times for low sample consumption.
Challenges of bispecific antibody production
The structural complexity and functional requirements of bispecific antibodies make a CLD campaign for a bispecific antibody inherently more challenging than a traditional monoclonal antibody (mAb) (3).
Bispecific antibodies have two binding sites that target different antigens, requiring the correct pairing of heavy and light chains with different specificities. This contrasts with monoclonal antibodies, which only have one type of heavy and one type of light chain.
When binding two distinct antigens, you must confirm that binding to one antigen does not negatively affect binding to the other antigen. Verifying the correct assembly and expression of multiple chains is also significantly more complex. The risk of chain mispairing and unequal chain expression is higher with bispecific antibodies and can potentially lead to non-functional or less effective products.
Risk of bispecific antibody mispairing highlights need for new analytical tools
The need to screen for correctly assembled bispecific antibodies adds an extra level of complexity, which requires new tools for high-throughput screening analysis (3). When screening common monoclonal antibodies, a Protein A assay is usually sufficient for the initial selection of clone productivity. Screening for a bispecific antibody is more complex. The two heavy chains may generate homodimers of each respective heavy chain, and the light chains may pair with the incorrect heavy chain. This increases the frequency of undesirable products or product-related impurities. Product-related impurities are unwanted substances that are similar in size and properties to the desired bispecific antibody and typically include aggregates, homodimers, half-mers, and mispaired fragments.
The best clone produces the highest titer of antibodies with correct pairing and assembly. A Protein A assay cannot make this differentiation so it must be combined with additional assays that distinguish between correctly and incorrectly assembled bispecific antibodies.
Role of surface plasmon resonance (SPR) systems
Surface plasmon resonance systems can quantify the amount of correctly assembled bispecific antibodies early in the development process, reducing your time spent on suboptimal cell lines. SPR analysis also detects product-related impurities down to the pico-molar scale, for purity and consistency between batches.
Biacore 8K+ SPR system is tailored for automated, high-throughput, high-sensitivity analyses. Biacore 8 series systems measure up to 16 interactions simultaneously through unattended runs. This allows for rapid screening and characterization of multiple cell lines and accelerates the identification of candidates with optimal binding properties.
Development of screening assays to determine total titer and correctly assembled antibody
This article describes how a Biacore SPR Protein A total antibody titer assay on a ready-to-use sensor chip can be combined with a confirmation assay to detect the correctly assembled bispecific antibody (Fig 2, Fig 3). The CLD campaign discussed in this article aimed to produce a bispecific antibody that contains two Fc-binding heavy chains plus one lambda and one kappa light chain. The lambda light chain binds to CD3 and the kappa light chain binds to CD20.
The total antibody titer assay
The total antibody titer assay is based on the interaction between cell culture samples and Protein A. Cell culture sample is injected over Sensor Chip Protein A and the total antibody titer is analyzed.
For the total antibody titer determination of samples, a screening assay was set up using a ready-to-use Sensor Chip Protein A to capture antibodies with Fc regions. This sensor chip features pre-optimized surface regeneration and allows multiple runs to be performed on the same sensor chip without compromising data quality. The assay workflow involves establishing a calibration curve to cover the expected titer range. Biacore Insight software streamlines assay development with a predefined concentration method for quick setup and an evaluation method for simplified data analysis to support high-throughput antibody titer screening with minimal manual intervention.
Fig 2. Schematic illustration of Biacore SPR total antibody titer assay using the ready-to use Series S Sensor Chip Protein A
Bispecific antibody confirmation assay
To confirm bispecificity of the antibodies, a screening assay was established using Sensor Chip CM5 with covalently coupled CD3 (first antigen) as the ligand. The assay leveraged the Dual command feature to sequentially inject the bispecific antibody sample (solution A), followed immediately by the protein L-like peptide (solution B) as the second antigen. This workflow includes the generation of a calibration curve to quantify bispecific titer. The setup supported confirmation of bispecific antibody dual antigen-binding capability with minimal manual intervention.
The confirmation assay is based on the interaction between CD3 covalently attached to Sensor Chip CM5 and the lambda light chain of the antibody—the CD3 binding arm (Fig 3). Cell culture sample is injected over the sensor surface and all antibodies containing CD3 binding arms bind to the sensor surface.
To identify the correctly assembled antibody, a second injection of a protein L-like peptide directly follows the first sample injection. During assay development, the interaction between CD3 on the sensor chip and the CD3 binding arm of the bispecific antibody was found to be weak, which meant that bound antibody dissociated quickly from the surface. This resulted in an underestimation of the binding of protein L-like peptide to the kappa light chain. To overcome this issue, the Dual injection command was used. The Dual command injects two solutions (solutions A and B) in immediate succession to minimize the dissociation of solution A before injection of solution B, which allows for assessment of the interactions with the two solutions specific to their targets.
Protein L-like peptide was chosen over CD20 for this assay due to the high cost of full-length CD20. The protein L-like peptide binds specifically to antibodies on the sensor surface with a kappa light chain, similar to CD20 binding to a kappa light chain.
Fig 3. Schematic illustration of Biacore SPR confirmation assay using CD3 sensor surface
Results
High-throughput screening of bispecific antibody CLD samples on Protein A surface determines the total titer
The robustness and reproducibility of the total titer assay were tested in a high throughput run of CLD samples using Biacore 8K+ system in combination with Sensor Chip Protein A (Fig 4a). The total titer screening assay demonstrated robust performance when analyzing twenty 96-well microplates of crude CLD samples.
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Fig 4. (a) Sensorgrams showing the relative response of crude samples (green), controls (orange), and calibration samples (blue) using Biacore 8K+ system with Sensor Chip Protein A. (b) The assay shows excellent linearity within the sample concentration range.
The assay showed excellent linearity within the sample concentration range of approximately 0.05 mg/L to 10 mg/L, confirming its suitability for accurately quantifying total titer in crude samples (Fig 4b).
Confirmation assay of CLD samples with CD3 and protein L-like peptide determines the correctly formed bispecific antibody
Thorough experimental testing of various approaches resulted in the development of a confirmation assay with covalently attached CD3 and protein L-like peptide as the most suitable option for high-throughput screening. Testing confirmed that CD3 coupling did not affect its function, while the protein L-like peptide effectively stabilized the complex, preventing the antibody from dissociating from the surface. To achieve optimal results, the protein L-like peptide should be injected immediately after the bispecific antibody, which is enabled by the Dual command feature.
Sensorgrams were generated from a confirmation assay screening of 144 CLD samples, selected based on results from the total titer assay (Fig 5a). The bispecific titer was quantified by plotting the response of the protein L-like peptide (report point Dual B binding late) on a calibration curve (Fig 5b).
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Fig 5. (a) Sensorgrams showing the relative response of bsAb culture samples (orange) and calibration samples (blue) using Biacore 8K+ SPR system with the confirmation assay. (b) The assay shows excellent linearity within the sample concentration range.
Application of SPR assays as a tool in a CLD workflow
The assays described here were applied to the screening and analysis of cell culture samples from multiple plates of CHO cell samples for total antibody titer and bispecificity to select high-performing clones.
Total titer and confirmation assays supported the selection of high-performing clones
Initial titer screening was conducted on 20 static-phase 96-well plates using the total titer assay (Fig 6a). The high-throughput screening narrowed down the number of clones to those with higher productivity. Next, the confirmation assay was applied to evaluate the production of correctly assembled bispecific antibodies. Deep-well plate screenings were performed which demonstrated the assay's ability to distinguish the correct bispecific format from product-related impurities (Fig 6b).
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Fig 6. (a) First titer screening from 20 static phase 96-well plates, measuring total antibody titer by Protein A binding on Biacore 8K+ system. (b) Titer screening from shaken 96-deep well plates, measuring total antibody titer (Protein A binding) and correct form of bsAb (CD3 and protein L-like peptide confirmation assay) on Biacore 8K+ system. (c) Titer screening of seven clones selected for fed-batch in shake flasks. SPR analysis using Protein A binding for total antibody titer and confirmation assay for correct bsAb assembly titer measured on Biacore 8K+ system. The total antibody titer was also measured using the Cedex-Bio IgG assay.
Overlaying the plots of both assays provides a comparison of the titer ratios of correctly formed bispecific antibody to total antibody expression, which supports the identification of the best clones for bispecific antibody production (Fig 6b).
Seven clones were selected for further analysis in shake flask fed-batch cultures. Titer screening was repeated for the fed-batch cultures of the seven selected clones (Fig 6c). The robustness of the titer assay was demonstrated by measuring the total titer of fed-batch clones with both Biacore total titer assay and Cedex-Bio IgG assay (Fig 6c).
Confirmation of bispecific antibody composition using liquid chromatography-mass spectrometry and high-performance liquid chromatography
The seven selected clones were analyzed using other techniques to confirm the results of the bispecific antibody screening assay presented here. Bispecific antibody pairing was analyzed by liquid chromatography-mass spectrometry (LC‑MS) (Fig 7a). High-performance liquid chromatography (HPLC) based purification of the fed-batch material was also performed to verify the composition of the bispecific antibody (Fig 7b). The results are considered to be in agreement with Biacore data which confirms that Biacore SPR assays can be used to select clones for further development.
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Fig 7. (a) The seven selected clones were purified with PreDictor™ MabSelect PrismA™ plates. Confirmation of the amount of correctly paired and mispaired bsAb by LC-MS with UV absorbance at 210 nm. Minor bsAb molecular species are not shown here. (b) HPLC analysis (percent area at 210 nm) of samples after MabSelect™ VL purification verifies bsAb composition of the seven clones.
Discussion
Here we describe a simple and robust method for determining total expression levels and correct assembly of bispecific antibodies in a CLD workflow using Biacore 8K+ system. Combining Protein A total antibody titer screening with a confirmation assay enables efficient selection of correctly assembled, high-performing clones from large sample sets, with the capacity to screen over 1000 samples in a single run.
The results presented here highlight the utility of this method for identifying clones with optimal productivity and correct assembly. The total titer assay provides accurate quantification across a broad dynamic range, while the confirmation assay verifies the production of correctly assembled bispecific antibodies. The ability to overlay data from both assays allows for direct comparison of titer ratios, which supports the selection of clones producing high levels of functional bispecific antibodies. This method also offers insights into product-related impurities, ensuring only clones producing high-quality antibodies are taken forward for further analysis.
These assays support high-throughput screening with a small sample volume and minimal manual intervention to identify clones expressing high titers of correctly assembled bispecific antibodies. Orthogonal methods, such as LC-MS and HPLC, are labor-intensive and require larger sample volumes. Biacore 8K+ systems shorten the time spent on initial screening and increase the chance of success.
This method also ensures that selected clones meet the criteria for dual specificity, addressing a key challenge in bispecific antibody development.
The workflow described here was developed for a bispecific antibody that targets CD3 and CD20, for a dual-action mechanism of recruiting immune effector cells via CD3 while specifically targeting and engaging cancerous B cells via CD20 (4, 5). This highlights the method’s clinical relevance in CLD for innovative therapeutics. The workflow can easily be modified for characterization of other bispecific antibodies.
Biacore 8K+ SPR systems offer an automated and integrated solution for efficient, high-throughput antibody screening, characterization, cell line development, and quality control of novel antibodies for clinical or research purposes.
References
- Ding M, Shen L, Xiao L, Liu X, Hu J. A cell line development strategy to improve a bispecific antibody expression purity in CHO cells. Biochem Eng J. 2021;166:107857. doi:10.1016/j.bej.2020.107857
- Castan A, Schulz P, Wenger T, Fischer S. Cell Line Development. Biopharmaceutical Processing. Elsevier; 2018:131-146. doi:10.1016/B978-0-08-100623-8.00007-4
- Segués A, Huang S, Sijts A, Berraondo P, Zaiss DM. Opportunities and challenges of bi-specific antibodies. International Review of Cell and Molecular Biology. Vol 369. Elsevier; 2022:45-70.
- Liu X, Zhao J, Guo X, Song Y. CD20 × CD3 bispecific antibodies for lymphoma therapy: latest updates from ASCO 2023 annual meeting. J Hematol OncolJ Hematol Oncol. 2023;16(1):90. doi:10.1186/s13045-023-01488-4
- Dabkowska A, Domka K, Firczuk M. Advancements in cancer immunotherapies targeting CD20: from pioneering monoclonal antibodies to chimeric antigen receptor-modified T cells. Front Immunol. 2024;15:1363102. doi:10.3389/fimmu.2024.1363102
Resources
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Article: How do scientists design and select effective bispecific antibodies?
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White paper: Biacore™ systems in discovery and early-stage development of biotherapeutic antibodies.
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White paper: Biacore™ surface plasmon resonance systems in late-stage development and quality control of biotherapeutic drugs.