Enhance B Cell Workflow Efficiency for Antibody Development

The development of immunotherapeutics has seen a marked increase in recent years1, with the most robust method for monoclonal antibody (mAb) discovery being the use of immune B cells in hybridoma studies. The significance of immune based therapies lies in the manipulation of the host’s own immune system to fight off disease. Taking immunized animals as a source, extracted plasma cells, such as B cells, can be fused with myeloma cells to generate immortalized hybridomas that can produce antibodies indefinitely2. These antibodies will be targeted to the infective agent the animal is treated with. The main drawback with this method of antibody production is the generation of hybridomas, which are time consuming, costly, require labor intensive processes to develop and are inefficient; around 1 in 5000 B cells survive fusion into hybridomas3. Characterization of B cells earlier in the hybridoma development workflow can avoid the potential pitfalls of this technological approach, ensuring those B cells taken through costly development pipelines are as robust and high performing as possible. September 25, 2023 Keywords or phrases: Immunotherapeutics, Plasma B Cells, Myeloma Cells, Antibodies, Hybridomas, Biotherapeutics, CellCelector Flex, iQue, Octet For further information, visit www.sartorius.com/cellcelectorConventional methods for characterization of B cells usually: In this application note, we describe solutions to the above challenges by using Sartorius platforms, focusing on the • Requires several days and is inefficient, costly and a laborious process • Uses complex protocols for screening and enrichment of B cells • Demands use of sophisticated procedures to asses B cell performance and yield • Requires multiple technological platforms and skilled personnel CellCelector Flex, at key stages of B cell workflows. Methods The following methods outline the processes for the isolation, enrichment and characterization of B cells using the CellCelector Flex, the iQue® Advanced Flow Cytometry Platform and the Octet® BLI Label-Free Detection system. CellCelector One-Day B-Cell Screening and Isolation Workflow Cell Loading (10 min) Single Cell Screening (15 min) Secretion Assay (2 – 3 hr) Hand cell seeding of single cell suspension in CellCelector nanowell array Plasma B cell Identification of single cell nanowells Assay with plate capture coating Automated detection of secreting cells (hits) Plasma B cell Reporter cell Antigen-expressing reporter cell assay Cell Transfer (1 hr) Export of hits to PCR plates for downstream processing Plasma B cell Report (15 min) Generation of the experiment documentation Antigen bead Bead-based assay Figure 1: Schematic of CellCelector Flex B cell workflow and nanowell based secretion assays. These can be used with the CellCelector Flex platform to rapidly evaluate and characterize B cell clones and their production of biologics. The CellCelector B cell workflow supports several types of cellular assays. Isolating and characterizing primary B cells A six-well plate with 60,000 nanowells per well was primed with 100% ethanol and centrifuged for 2 min at 700 x g to remove residual air microbubbles formed in the nanowells. Ethanol was removed and plates washed with phosphate-buffered saline (PBS). The nanowells were washed with PBS four more times and with culture media IMDM plus 15% fetal bovine serum (FBS), HEPES, Sodium Pyruvate, Pen-Strep-Glut, MEM-NEAA, 2-ME and Hybridoma Fusion and Cloning Supplement three times. A suspension of B cells (60×103 cells/mL), goat anti-human IgG Polystyrene beads (1.2×106 beads/mL), goat anti-human IgG-AlexaFluor (AF) 647 (5 μg/mL), biotinylated-EGFR (5 μg/mL) and Streptavidin AF488 (5 μg/mL) was prepared in culture media. One milliliter of the suspension was dispensed dropwise across one well of the six-well plate and cells settled for 5 mins at room temperature (RT) then were centrifuged for 5 mins at 300 x g. Cells were incubated for 4 h at 37˚C, 5% CO₂. Imaging, identification of IgG expression (AF647+) and antigen binding (AF488+) was performed on the CellCelector Flex platform. Cells were picked using the Single Cell Picking Module and dispensed into a well of a 96-well plate. Confirmation of B cell clone antibody expression and functional analysis Binding was performed on CHO cells transfected with EGFR. CHO cells were suspended in 10 mL of High Glucose DMEM culture media at 1×106 cells/mL. Cells acclimated to the new media for 3 h at 37˚C, 5% CO₂, 95% humidity. For 10 mL of CHO cells, a total of 10 μg of vector DNA was prepared with 10 μL of 3 mg/mL of PEIMAX™ (Polysciences Inc) in a total of 0.5 mL of culture media. Cells were incubated for 3 h before adding sodium butyrate to a final concentration of 5 mM and incubated overnight. The following day, CHO cells were centrifuged at 2000 rpm for 2 mins, supernatant removed and resuspended to 1×106 cells/mL in PBS + 2% FBS. To a V-bottom assay plate, 50 μL of CHO suspension (50,000 cells/well) and 50 μL of test antibody were added 2CellCelector Flex (Figure 1) compared to days or weeks to each well and incubated for 1 h at 4˚C. CHO cells were washed twice with 150 μL PBS + 2% FBS and 50 μL of secondary solution (5 μg/mL goat anti-human-Alexa Fluor 647 and 2.5 μg/mL 7AAD in PBS + 2% FBS) was added and were incubated for 1 h at 4˚C. CHO cells were washed twice with 150 μL PBS + 2% FBS and resuspended in 70 μL of PBS + 2% FBS and run using the iQue® Advanced Flow Cytometer. Data were analyzed using iQue Forecyt® software. Confirmation of B cell mAb affinity and kinetics using BLI The Octet® BLI instrument was used to determine affinities of EGFR antibodies. Prior to the binding measurements, the sensor tips were pre-hydrated for 10 min in Octet kinetics buffer. Streptavidin (SA) biosensors were used to load biotinylated anti-human Fc antibody (Invitrogen) followed by one baseline step of 60s in Octet kinetics buffer. Antibodies were captured by the anti-human Fc antibody and submerged in Octet kinetics buffer for 60s prior to being placed in wells containing EGFR at concentrations ranging from 1.5625 to 100 nM for 10 mins and followed by 10 mins of dissociation time in Octet kinetics buffer. Antibodies and antigen concentrations were all prepared in Octet kinetics buffer. SA biosensors were used once without regeneration. For data evaluation, Octet Analysis Studio was used. The kinetic rate constants, association rate constant (ka, M−1s−1), dissociation rate constant (kd, s−1) and the equilibrium rate constant (KD, M) were determined using a 1:1 Langmuir model. Plasma Cell Characterization and Isolation Using the CellCelector Flex Isolation of SIVmac239 SOSIP.664-specific plasma cells was conducted using the CellCelector Flex, which allowed isolation of antigen-specific Ab-secreting cells. The CellCelector Flex was housed in the CellCelector Incubator FlowBox at 37˚C with 5% CO₂. Nanowells were coated with streptavidin overnight and then incubated with biotinylated SIVmac239 SOSIP.664 trimer for 1 h. Then overlaid with processed bone marrow aspirates from Indian rhesus macaques (RMs) that had developed a neutralizing Ab response against SIVmac239. These aspirates were placed in medium containing a fluorescently labeled mAb against rhesus IgG and incubated at 37˚C for 12 h. Localized fluorescent halos were identified surrounding SIV-specific plasma cells because of their secreted OSIP-specific Abs. These plasma cells were then picked using the Single Cell Picking Module and placed into lysis buffer for later Ig amplification. Results Determining the yield of B cell clones for subsequent selection of high producers requires robust and effective assays. Using several proprietary nanowell based secretion assays (Figure 1), the CellCelector Flex platform provides several solutions for quick and easy yield determination during the early stages of a cell selection workflow. Determining clonal yield earlier in the workflow saves time and money that would otherwise be spent on expansion and development of hybridomas with undesirable characteristics, reducing B cell workflows to a matter of hours using the under conventional methods. Automated identification and analysis of B cell clones using CellCelector Flex Characterization and isolation of B cells for downstream analysis and selection for hybridoma production is a vital component in mAb development workflows. It is important that the methods used are robust, efficient and provide the best performing B cell clones. Data collected by Matochko et al.⁵ using the CellCelector Flex highlights the utility of this system when identifying B cell clones with the attributes required, in this case, high yield production of antibodies that bind to EGFR (Figure 2). Initial clones that are secreting antibodies can be identified quickly and efficiently using the CellCelector software highlighting three wells in the image (Figure 2A). By identifying antibody production using the bead-based secretion assay (Figure 2G), two of the three wells are found to contain antibodies that bind to EGFR (Figure 2B), and an overlay image (Figure 2C) confirms wells positive for secretion of antibodies that bind EGFR. These cells are targeted by the CellCelector Flex and harvested, leaving no remaining cells in the well after picking (Figure 2E, F). The whole process takes a few hours to complete, exemplifying the fast, efficient and accurate qualities of the CellCelector Flex in B cell clone development. Confirmation of B cell clone antibody binding using the iQue® and Octet® Identifying and collecting clones with the right characteristics can be effectively performed on the CellCelector at the beginning of the B cell workflow. Confirmation analysis and investigation of the binding affinity of secreted antibodies can be determined on the iQue® Advanced Flow Cytometry Platform and the Octet® Bio-Layer Interferometry System. The iQue® Advanced Flow Cytometer can be used to highlight clones that produce high affinity EGFR binding antibodies, while also eliminating those clones producing antibodies with a low affinity for, or that do not bind to, EGFR. The graph in figure 3A shows a range of clone antibodies with high levels of binding to EGFR, such as B06, E08 and G04, with binding over 100-fold higher compared to control. Conversely, the graph also shows those clone antibodies with low to no binding to EGFR; B09, D08 and H01, as examples. To further investigate the affinity of the antibodies produced, the Octet® BLI system was used to analyze protein interactions in greater detail. The table in Figure 3B provides data from the Octet® BLI for the binding affinity of each antibody to EGFR previously analyzed on the iQue®. It clearly shows that those antibodies that presented very low binding in the iQue® study correlated with those found to not bind via BLI, while high affinity binders were confirmed to bind EGFR on both the Octet® and iQue® systems. 3A C E B D F Analyzing the kinetics of antibody binding provides insight into the mechanism of action of biologics and provide a greater insight into an antibody’s function. For example, samples B06 and G04 share similar binding over control when analyzed on iQue®, however, when analyzed further using BLI, we can determine that B06 has lower affinity for EGFR than G04 (KD - 4.4 nM and 0.9 nM, respectively, Figure 3B). Sensorgrams show further detail on the kinetics of binding for these two antibodies and also highlights a non-binding sample, H01, that was confirmed not to bind EGFR by both iQue® and Octet® analysis (Figure 3C). Sample B06 displays a high on rate (8.0E+05 1/Ms) but also a high off rate (3.5E-03 1/s), leading to a lower affinity than G04, with a lower on rate (2.8E+05 1/Ms), but also a lower off rate (2.6E-04 1/s), as the sensorgrams detail. This data is useful because although the two antibodies bind to EGFR, their affinities impart different physiological properties. B06 associates to EGFR quickly but then dissociates quickly too, providing a short-term effect, while G04 takes longer to bind, G Figure 2: Isolating and characterizing primary B cells using the CellCelector Flex. 52,000 Nanowells were scanned in brightfield and fluorescence to identify Nanowells containing B-cells that secrete antibodies (AF647+) and are positive for binding to EGFR (AF488+). (A) Image of fluorescence specific 647 nm emission (RED); two neighboring wells contain antibody secreting cells (ASC). (B) Image of fluorescence specific 488 nm emission (GREEN); one neighboring well contains an ASC specific to EGFR. (C) Overlay of images from 647 and 488 nm channels and (D) brightfield image used to pick an EGFR+ ASC. Brightfield images (E) before and (F) after an EGFR+ ASC is picked from Nanowell. Brightfield images demonstrate all contents within the Nanowell are picked. (G) Schematic of the antigen coated bead-based secretion assay (Orange antibodies are secreted from the cell and bind to the coated bead in grey, fluorescently conjugated antibodies in blue and green bind to the secreted antibodies, providing the fluorescent signal).5 but dissociates much more slowly, providing a more stable, longer-term effect. Plasma cell antibody characterization and isolation using the CellCelector Flex Simian immunodeficiency virus (SIV) infected RMs are a commonly used animal model for human immunodeficiency virus (HIV) infection, and mAbs have been shown to prevent and treat HIV through neutralization of viral particles. Due to low frequency of circulating B cells encoding neutralizing mAbs, it has been a challenge to isolate these cells. Pedreño-Lopez et al.6 have exemplified the use of the CellCelector Flex in the isolation of antibody secreting plasma cells from host animals (Indian RMs) for the development of HIV-neutralizing mAbs. To isolate SIVmac239 SOSIP.664-specific plasma cells, 24-well nanowell plates were coated with streptavidin overnight, then biotinylated SIVmac239 SOSIP.664 was added 45 the following day (Figure 4A). Bone marrow aspirates were obtained from both femora and both humeri of RM at different time points during SIV infection. To prevent false positives, total bone marrow cells were pre-incubated with different mouse anti-Fcγ receptor (FcγR) I–III Abs to block the FcγRs present on the surface of certain types of cells, including macrophages and dendritic cells. These cells were then resuspended in medium containing recombinant a proliferation-inducing ligand (APRIL), interleukin- 6 (IL-6), and mouse anti-monkey IgG fluorescein isothiocyanate (FITC) and added to the SIVmac239 SOSIP.664 plates. After a 12 h incubation period, the CellCelector was used to visualize fluorescent halos around single cells that had secreted SOSIP.664-specific antibodies (Figure 4B). These cells were picked and transferred into 96-well plates containing lysis buffer for subsequent PCR amplification. This technique allowed for the functional screening of 10 million bone marrow cells in less than a day. Figure 3: Characterization of 20 expressed clones. (A) Binding of antibodies to EGFR expressing CHO cells analyzed by iQue® Advanced Flow Cytometry. EGFR binding was determined using the geomean of antibodies to EGFR – CHO over parental CHO cells. Antibodies were tested at 50 nM in triplicate. (B) Affinity of antibodies to soluble EGFR determined using Octet® BLI. Antibodies were captured to an anti-human Fc antibody loaded on streptavidin biosensors and binding measured to EGFR titrated from 1.56 to 100 nM. Antibodies binding to cells > 3.5 - fold also bind to soluble EGFR. (C) Three representative sensorgrams of antibodies demonstrating high affinity (<1nM), moderate affinity (1 – 10 nM), and no binding to soluble EGFR.5 EGFR Binding (Geomean Fold Over Irrelevant) A04 B01 B06 B08 B09 B12 C09 C10 C11 D03 D08 E08 E09 E11 F04 F11 G04 G07 H01 H03 1 10 100 B A C Loading Sample ID: G04 Loading Sample ID: B06 Loading Sample ID: H01 mm mm mm Time (sec) Time (sec) Time (sec) 0.4 0.4 0.4 0.2 0.2 0.2 0 0 0 0 0 0 500 500 500 1,000 1,000 1,000 Antibody Name (Well ID) Affinity kon (1/Ms) kdis (1/s) KD (nM) A04 2.7E+06 1.8E-03 0.7 B01 3.3E+05 6.2E-04 1.9 B06 8.0E+05 3.5E-03 4.4 B08 8.4E+04 1.0E-04 1.2 B09 * B12 7.6E+04 5.9E-04 7.8 C09 * C10 * C11 2.1E+05 2.3E-03 11 D03 * D08 * Antibody Name (Well ID) Affinity kon (1/Ms) kdis (1/s) KD (nM) E08 2.0E+0.5 1.0E-03 5.1 E09 1.1E+0.5 1.0E-03 9.5 E11 * F04 1.2E+05 3.4E-04 2.8 F11 1.2E+06 1.3E-03 1.1 G04 2.8E+05 2.6E-04 0.9 G07 2.4E+05 1.1E-03 4.7 H01 * H03 1.7E+06 1.3E-03 0.8 * Not BindingA Mouse Anti-monkey IgG FITC Secreted Non-specific Ig Plasma Cell Biotinylated SIVmac239 SOSIP.664 Streptavidin B Secreted SOSIP-specific Ig Conclusions Developing new antibody based biotherapeutics for rare and hard to treat disease most often requires the manipulation of B cells through hybridoma technology. Current techniques for the identification and characterization of suitable B cells for the development of hybridomas are time consuming, expensive and inefficient, often requiring extensive manual manipulation. Using various Sartorius platforms with a focus on the CellCelector Flex, we have shown that B cells can be accurately identified, picked and characterized within a highly condensed workflow from days using conventional methods down to hours using the CellCelector Flex, the iQue® Advanced Flow Cytometer and the Octet® BLI Label-Free Detection System. The key advantages of using Sartorius systems over conventional methods are: Figure 4: Characterization and Isolation of Abs from the Bone Marrow of a SIVmac239-Infected Rhesus Macaque. (A) Schematic representation of the fluorescence-based platform. Nanowells were coated with streptavidin (yellow) and the SIVmac239 SOSIP.664 trimer (blue) was subsequently added. Total bone marrow cells were added to the wells in medium containing APRIL, IL-6, and mouse anti-monkey IgG FITC (green), which binds to the Abs secreted by the plasma cells. Left: SIVmac239specific Abs bind the SOSIP.664 trimer. Right: the secreted Abs do not bind SOSIP.664 and diffuse away. (B) CellCelector Flex image of plasma cells using bright-field view on the left and the fluorescent field on the right. The green cursor in the lower image shows the picking diameter of the capillary.⁶ • Automation and speed, the CellCelector flex provides the opportunity to screen, characterize and pick B cells within hours, rather than days using a single system • Simplicity, B cell performance and product yield can be established within the same well, while optional confirmatory analysis can be performed on the iQue® and Octet® • High efficiency, accuracy and automated protocols remove the need for intensive, inconsistent manual handling of biological material • Ease of use, one system is required to identify, isolate and characterize B cells The data presented in this application note highlights the advantages of employing the CellCelector Flex in B cell workflows for increased efficiency and shorter timescales in clinical and biotherapeutic research and antibody development. 6References 1. Kaplon, H., Muralidharan, M., Schneider, Z., and Reichert, J.M. (2020). Antibodies to watch in 2020. 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