Bispecific Antibodies (BsAbs)
Bispecific antibodies (BsAbs) are innovative class of therapeutic proteins designed with two binding sites directed at either two different antigens or two different epitopes on the same antigen simultaneously. A growing number of BsAbs are being explored for applications in cancer treatment and beyond for non-oncology indications like autoimmune diseases, infectious diseases, and other conditions.
Compared to traditional monoclonal antibodies, BsAbs offer enhanced cytotoxic effects due to dual targeting and broader applications for several indications.
FDA approved and clinical pipeline candidate BsAbs are available in different formats and mediate their therapeutic function via different molecular mechanisms.
Common bsAb formats include single-chain variable fragment (scFv) based BsAbs, tandem scFvs, CrossMab, DARTs (Dual-Affinity Re-Targeting Antibodies) and Triomabs, which are trifunctional antibodies that can bind to two different antigens and engage T-cells. The bispecific T cell engager (BiTE) antibodies connect two scFvs that bind to a cancer cell and a T-cell.
Thorough characterization of BsAbs is challenging due to their diverse formats to ensure their safety and efficacy. This includes detailed analysis of their pharmacokinetics, pharmacodynamics, immunogenicity, and potential toxicities.
Sartorius addresses these challenges with advanced analytical techniques to ensure that BsAbs are developed with the desired quality and specificity.
Bispecific Antibody R&D Workflow
Featured Products for Bispecific Antibodies (BsAbs) R&D
Octet® Label-Free Protein Analysis Platforms
Octet® platforms, available using Biolayer Interferometry (BLI) and Surface Plasmon Resonance (SPR), measure protein-protein interactions in parallel, without the use of detection agents.
Minimize time, effort, and costs in the assay development of bispecific therapeutics with the high-throughput Octet® RH96 system:
- Real-time BsAb binding assay to both antigens and titer determination
- Characterization of affinity and binding kinetics
- Rapid screening of cell lines and identification of pools and clones with higher concentrations of bispecific antibodies
Explore Label-Free Protein Analysis Read Case Studies
iQue® High-Throughput Screening (HTS) Cytometry Platform
The iQue® HTS Cytometry Platform is a high-throughput, suspension cell and bead analysis platform for multi-attribute screening of different antibody formats. It is the platform of choice for cell phenotype and function analysis in a single well and provides features that help streamline workflows and shorten time-to-results. The iQue® 21 CFR Part 11 Software Module ensures easy transitions from research lab towards regulated laboratories.
Evaluate mechanism of actions of different types of bispecific antibodies, i.e. CD3xCD19 Bispecific T Cell Engager (BiTE) Antibodies and identify temporal changes in the tumor microenvironment.
- Antibody internalization
- Antibody-Mediated T Cell Activation
- Analysis of T Cellular Subset, Activation Markers
- Cytokine Profiling
- Evaluation of Target Cytotoxicity
Incucyte® Live-Cell Analysis System
The Incucyte® Live-Cell Analysis System is designed to efficiently capture cellular changes where they happen - in the incubator. It enables in-depth functional and phenotypic assessment of several cell types and cellular interactions such us immune cell activation, cell health and proliferation.
Incucyte® 21 CFR Part 11 Software Module offers comprehensive security and electronic record keeping functionality.
- Morphological and Spatial Analysis of Immune and Tumor Cells
- Immune cell induced cytotoxicity - Immune Cell Killing
- Quantification of Target and Effector Cell Numbers
- Cell proliferation, health, apoptosis,
- Antibody Internalization
CellCelector Fully Automated Cell Selection and Retrieval Platform
The CellCelector Flex Platform is a fully automated cell imaging and picking system developed for screening, selection, and isolation of single cells, clones, clusters, and adherent colonies. It can process thousands of cells in parallel to identify and select optimal clones.
During Molecule and Cell Line Development, The CellCelector Flex can be used to:
- Isolate and clone specific cell lines that produce BsAbs
- Ensure monoclonality with extensive reporting capabilities
- Select and pick any CHO clones producing BsAbs
- Culture clones in a fraction of the space using Nanowell array technology
Picus® 2 Electronic Pipettes
Picus® 2 pipettes ensure reliable, repeatable pipetting results and feature an unbeatable ergonomic design that is kind to your hand.
- Connectivity
- Reproducible Results
- Guided Workflows
Sartorius Bispecific Antibodies (BsAbs) Solutions Offer:
The developed BLI method for BsAb binding and ProA titer measurement offers an easy screening method and workflow that assesses BsAb interactions in a versatile, label-free, and easy-to-use format.
Combining Incucyte® and iQue® analysis in a single assay plate enables more informed experimental design, mechanistic insights, and rapid progress to actionable results. Compatibility of the iQue® and Incucyte® workflows was demonstrated using a model of immune cell killing. Once maximal killing had been observed with Incucyte Live-Cell Analysis, cells were dissociated and evaluated using the iQue® T-Cell Activation kit antibody panel for identification of T cell subsets.
Miniaturized volume requirements for the iQue® platform enable repeated non-perturbing supernatant sampling. Study results demonstrated by using a modified miniaturized iQue® assay protocol, it was feasible to use only 20% of samples and assay reagents, thereby reducing sample consumption and reagent cost while maintaining assay sensitivity, quality, and reproducibility.
Frequently Asked Questions
Traditional technologies for characterizing antibodies, such as Enzyme-Linked Immunosorbent Assay (ELISA) and Surface Plasmon Resonance (SPR), have several limitations. They are often time-consuming and limited in their ability to provide quantitative functional assessment of two interactions to one bispecific molecule. This makes it challenging to evaluate complex bispecific therapeutics. Furthermore, these methods may not be suitable for high-throughput applications, which are often required in bioprocessing or cell line screening.
The Octet® Biolayer Interferometry (BLI) platform addresses these limitations by providing quantitative functional assessment of two interactions to one bispecific molecule in high-throughput applications.
These platforms and reagents together provide a comprehensive analysis of immune cell activation and tumor cell death. They allow for the study of multiple aspects of immune cell-tumor cell interactions, including morphological changes, cell numbers, and cytokine release. The real-time data analysis capability of the Incucyte® platform enables educated selection of time points for flow cytometric determinations on the iQue® platform This leads to more informed experimental design and efficient use of resources.
Incucyte® Live-Cell Analysis Immune Cell Killing assay offers morphological insights of immune cell activation and tumor cell death. Determination of the target cell number provides an indication of the degree of cytotoxicity elicited by the immune cells.
Publications
Production and modification of bispecific antibodies against human immunoglobulin E
Summary:
The Vivaspin® centrifugal concentrators (specifically the Vivaspin® turbo 15 and Vivaspin® 20 models) were employed as a key protein purification and sample preparation tool during the downstream processing of the BiTE® antibody constructs.
The main purpose of using the Vivaspin® columns was to concentrate protein samples, to reduce the volume of eluate fractions collected from the Immobilized Metal Affinity Chromatography (IMAC) step. This was a necessary preparatory step before the subsequent size-exclusion chromatography (SEC) or gel filtration step.
Source:
DOI: 10.5282/edoc.30681
Vogel, Franziska 2022, LMU Munich
Production of stable bispecific IgG1 by controlled Fab-arm exchange
Summary:
In this study, Vivaspin® 6 centrifugal concentrators were utilized for both the controlled Fab-arm exchange (cFAE) process and buffer exchange.
The primary function of the Vivaspin® 6 columns was the removal of the reducing agent, 2-mercaptoethylamine·HCl (2-MEA), from the reaction mixture following the incubation period. This step was essential to terminate the reduction reaction and enable the re-oxidation of interchain disulfide bonds, a prerequisite for the formation of stable bispecific antibodies.
Source:
DOI: 10.4161/mabs.26233
Programmable half-life and anti-tumor effects of bispecific T-cell engager-albumin fusions with tuned FcRn affinity
Summary:
In this study, Vivaspin® 2 centrifugal concentrators were employed as a key tool for post-purification sample processing of recombinant albumin-fusion proteins.
Following affinity purification of the HSA fusion proteins (Albu-LiTE constructs) using CaptureSelect™ Human Albumin affinity matrix, the Vivaspin® 2 columns were used to:
- Concentrate the purified protein eluates
- Exchange the buffer into PBS (phosphate-buffered saline) for subsequent characterization and functional assays
A bispecific anti-PD-1 and PD-L1 antibody induces PD-1 cleavage and provides enhanced anti-tumor activity
Summary:
In this study, the Incucyte® Live-Cell Imaging system was used to assess the ability of the bispecific antibody CTX-8371 to bridge PD-1-expressing and PD-L1-expressing cells. Specifically, it enabled live imaging of Jurkat cells to visualize the formation of cell clusters over time. These imaging data provided direct visual evidence that CTX-8371 can bridge PD-1⁺ and PD-L1⁺ cells (in trans configuration), leading to sustained cell–cell engagement. This clustering was correlated with the proteolytic cleavage and loss of cell-surface PD-1, supporting the proposed mechanism of action for the bispecific antibody.
Combination therapy with a novel CD2-targeted costimulatory bispecific antibody overcomes limitations of CD3 T cell engager treatment for solid tumors
Summary:
In the study, Live Cell Analysis Instruments from Sartorius were used to track the growth of mKate2+ wildtype or CD58-KO HCC1954 cell lines by imaging each well every 4 hours over a 4-day culture period. This allowed for precise quantification of the effects of different treatments on the cancer cells, which was crucial in determining anti-tumor cytotoxicity
Source:
DOI:10.1080/19420862.2026.2684378
Full Article
Tumor - targeted bispecific antibodies effectively inhibit oncogenic pathways while minimizing toxicity
Summary:
In the study, Incucyte Human Fabfluor-pH from Sartorius was used to label bivalent and bispecific antibodies, allowing for the visualization and quantification of antibody internalization in cancer cells. This enabled the researchers to understand the internalization rates and dynamics of the bispecific antibodies, which was crucial for assessing their efficacy and mechanism of action
Source:
pmc13196744 | Bioz | AI Empowering Scientific Research
NI-3201 Is a Bispecific Antibody Mediating PD-L1-Dependent CD28 Co-stimulation on T Cells for Enhanced Tumor Control
Summary:
In this study, the Octet® system was used to quantify the monovalent binding affinity of the AI3 arm to human CD28. This was a critical step in characterizing the newly identified anti-CD28 clone. The affinity data obtained from the Octet® system provided quantitative evidence that the AI3 arm binds CD28 with moderate affinity.
Source:
doi: 10.1158/2326-6066.CIR-24-0298
Design of orthogonal constant domain interfaces to aid proper heavy/light chain pairing of bispecific antibodies
Summary:
In this study, the Octet HTX instrument was employed to assess the dual-binding functionality of bispecific antibodies (bsAbs) containing the engineered constant domain mutations. The Octet® system was used in a sandwich-binding format to verify that the bispecific antibodies could simultaneously bind to both of their target antigens (EGFR and IL-12). This was a critical quality attribute to confirm that the introduced constant domain mutations did not disrupt the antigen-binding functionality of the variable domains.
Source:
DOI: 10.1080/19420862.2025.2479531
Engineering Fc heterodimerization via asymmetric β-strand reconfiguration for bispecific antibody assembly
Summary:
In this study, the Octet RED96 instrument (Sartorius) was employed to measure the binding affinity and kinetics of the engineered Fc domain (bsFc-D) to key Fc receptors, specifically FcγRI and FcRn. The Octet® measurements provided quantitative evidence that the StrandLock engineering strategy did not compromise the core Fc-mediated functions of the antibody. Preserved binding to FcγRI indicated that effector functions (e.g., ADCC) were likely intact, while maintained pH-dependent FcRn binding supported normal in vivo half-life. These findings were critical for validating the biophysical and functional integrity of the engineered bispecific antibody platform.
Source:
pm42316245 | Bioz | AI Empowering Scientific Research
A B7-H3-Targeted CD28 Bispecific Antibody Enhances the Activity of Anti-PD-1 and CD3 T-cell Engager Immunotherapies
Incucyte® Live-Cell Analysis System: This was used to perform real-time, live-cell imaging to monitor T-cell mediated tumor cell killing (cytotoxicity). The system allowed the researchers to track and quantify the destruction of cancer cells by T-cells over time in the presence of their experimental therapeutic antibody.
Octet® Biolayer Interferometry: This system was used to measure the binding affinity (kinetics) of the bispecific antibody. Specifically, it was used to determine how strongly and rapidly the antibody bound to its targets, CD3 and CD28, on T-cells.
Cytotoxic activity and safety characterization of IMV-M™, a MUC16 × DR5 bispecific antibody
Summary:
In the study, the iQue® 3 Advanced Flow Cytometry System was used to analyze the binding of antibodies to cells and to assess the cytotoxicity of IMV-M against MUC16+ tumor cells. This enabled the study to demonstrate that multivalent clustering on MUC16 is required for IMV-M's cytotoxic activity.
https://doi.org/10.1093/abt/tbag021
Preclinical Comparison of Two CD3xB7H4 Bispecific Antibodies for 1 Solid Tumor Therapy Reveals CD3 Affinity-Dependent Potency and 2 Tolerability Tradeoffs
Summary:
In the study, iQue® 3 Advanced Flow Cytometry System from Sartorius was used to measure the binding of antibodies to cells and assess the binding to human whole blood cells. This system was crucial for accurately evaluating the potency and tolerability of the CD3xB7H4 bispecific antibodies, contributing significantly to the study's findings.
Source:
Bioz | AI Empowering Scientific Research
Combination therapy with a novel CD2-targeted costimulatory bispecific antibody overcomes limitations of CD3 T cell engager treatment for solid tumors
Summary:
In the study, the iQue® 3 Advanced Flow Cytometry System from Sartorius was used to determine the percentage of T cells that formed conjugates with tumor cells after treatment with bispecific antibodies. This was crucial for quantifying conjugate formation, which is essential for assessing the efficacy of the bispecific antibodies in the study.
Source:
DOI:10.1080/19420862.2026.2684378
Full Article
