Accelerate Your Antibody Screening
In the quest for therapeutic or analytical antibodies, it is crucial to carefully select antibodies based on their kinetics, affinity, specificity and biophysical attributes. Antibody screening plays a pivotal role in identifying cell clones that generate antibodies suited to a specific purpose, often challenging these antibodies with a known antigen. Sample matrices are often complex, e.g., hybridoma culture supernatants or phage display preparations.
This app note focuses on screening complete monoclonal IgG antibodies, recognizing that assay conditions and prerequisites may vary for other antibody types and fragments.
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Step-by-step processes for antibody screening for precise antibody selection How to optimize your assay conditions for various antibody types How to efficiently evaluation tools to enhance your antibody screening workflow
Antibody screening with Biacore systems In the search for therapeutic or analytical antibodies the selection of appropriate kinetics/affinity, specificity and biophysical properties is essential. Antibody screening aims to identify cell clones that produce antibodies appropriate for the purpose, by challenging the antibodies with known antigen. Sample matrices are often complex, e.g. hybridoma culture supernatants or phage display preparations. Obtaining kinetic information early in the screening process is often a significant advantage. Information that is generally sought includes: • Specificity — which clones produce antibodies with the desired specificity? • Binding characteristics — which clones produce antibodies with suitable kinetic and/or affinity properties? • Expression level — which clones produce sufficient amounts of antibodies? Screening can be performed on full size antibodies or parts thereof, such as Fab fragments or scFvs. This Application guide deals with screening of complete monoclonal IgG antibodies. Assay conditions and prerequisites might differ for other antibody types and fragments.3 Assay format Antibody screening can be run using two different assay formats: Format Description Antibody as ligand Antigen as analyte Samples to be screened are injected over a generic antibody-capturing surface. Antigen is then injected as analyte¹. The surface is regenerated by removing captured antibody and bound antigen. Antigen as ligand Antibody as analyte Antigen is immobilized or captured on the surface. Samples to be screened are then injected as analyte. The surface is regenerated by removing the bound antibody. (If reversible antigen capture is used, the surface may be regenerated by removing captured antigen and bound antibody). ¹ In some Biacore™ systems, the analyte injection is termed sample. Do not confuse this terminology with the generic use of sample to refer to the variable element in the assay, in thiscase the captured ligand. Screening types Information from antibody screens using Biacore systems may be obtained at different levels as summarized below. Screening type Description Report point-based screening Provides information on specificity and expression level from single point binding level measurements. Off-rate ranking Provides comparative information on the dissociation rate of the antibody-antigen complex. Kinetic screening Provides information on both the association and dissociation rates of antibody-antigen binding. Capturing the antibody as ligand is generally more straightforward with respect to surface preparation and assay development, but requires injection of antigen for each cycle in the screen. Using the antigen as ligand can involve additional work to establish immobilization and regeneration conditions. Screening using antigen as ligand is only recommended for assessment of specificity and/or expression level, for several reasons: • Antibody concentrations are usually not known, and are required for kinetic screening • Complete antibodies are usually bivalent, making evaluation of off-rate ranking and kinetic screening more difficult The two approaches differ in experimental setup and evaluation, and are considered separately in the following sections. Frequently, report point-based screening is used as an initial screen to eliminate non-producing clones. Off-rate ranking and kinetic screening are then used for more detailed characterization of the clones that produce potentially interesting antibodies.4 Tips for antibody screening Choose the assay format depending on your needs and preferences. Choose antibody as ligand if your aim is to obtain kinetic information. When using antigen as ligand, estimate how much you need to immobilize in order to obtain high enough responses from the injected antibody Check for non-specific binding from the sample matrix by injecting blank samples (sample matrix without antibody) and check for binding to both the active and reference surfaces A data collection rate of 1 Hz is usually sufficient. Using higher data collection rates increases file sizes without providing additional information. Establish that the assay is suitable for purpose using a few samples and controls before you start extended runs with many samples If you are using pooled sample positions in the microplate, use Predip with the injection to minimize sample dilution For off-rate ranking and kinetic screening, consider the use of blank cycles. For best performance include one blank cycle per sample. To increase throughput and reduce antigen consumption a blank cycle can be included at selected intervals and used for all samples. Note that this approach is generally less precise. General considerations Surface preparation Response levels in antibody screening are usually quite high, and it is not necessary to use high ligand levels. Specific considerations for screening approaches with antibody as ligand and antigen as ligand are given in the respective sections below (see Surfaces for antibody capture, on page 5 and Preparing the antigen surface, on page 8 respectively). Buffers HBS-EP+ (HEPES-buffered saline with 0.3 mM EDTA and 0.05% Surfactant P20, available from Cytiva) or similar is recommended as running buffer for antibody screening. Samples may be diluted if required using the same buffer. Precise matching of sample and running buffer is neither needed nor practicable for antibody screening work, since the report points used are placed after the sample injections. Sample preparation Samples for antibody screening are typically clarified material such as hybridoma culture supernatants or phage display preparations, used without further purification. To verify that there is no binding of the sample matrix to the surface a control experiment is recommended in which only the sample matrix (cell culture supernatant or corresponding matrix) is injected over the sensor surface. If there is non-specific binding from the sample matrix this can be reduced by diluting the samples with running buffer (typically 1:1). Addition of NSB Reducer (available from Cytiva) at 1 mg/mL can also help to reduce non-specific binding.5 Screening using antibody as ligand The general approach to screening with antibody as captured ligand is summarized below. Step Action 1 Dock a pre-immobilized sensor chip or immobilize the capturing molecule. 2 Run at least 3 startup cycles using buffer and regeneration to equilibrate the system. 3 Inject sample containing antibodies (for example, hybridoma culture supernatants or phage display preparations) over the active surface to capture antibodies from the sample. Crude samples can be injected directly, without pre-treatment. 4 Inject antigen as analyte over both the active and reference surfaces. 5 Evaluate the results. The experimental setup differs slightly according to whether the results will be evaluated from single report points or as off-rate or kinetic ranking. Details are given below. Surfaces for antibody capture Ready-to-use kits for preparing surfaces for capture of mouse and human antibodies and human antibody fragments are available from Cytiva (see the table below). Custom capturing molecules may also be immobilized on the sensor chip if required. In general, aim to immobilize about 5000 RU or less of the capturing molecule, depending on the expected antibody expression levels and the size of the antigen. Using higher levels can lead to non-specific binding from complex samples. Sensor chips preimmobilized with Protein A (MabSelect SuRe™), Protein G and Protein L are also available from Cytiva. Protein A and Protein G bind selectively to antibodies from different species and subclasses. Protein L binds specifically to antibodies containing kappa light chains, with a broader selectivity than Protein A and Protein G. Ordering information may be found on the Products pages at www.cytiva.com/biacore. Product name Intended for Mouse Antibody Capture Kit Mouse IgG, IgA, and IgM antibodies Human Antibody Capture Kit Human IgG antibodies Human Fab Capture Kit Human Fab fragments (kappa and lambda) Sensor Chip Protein A Antibodies according to the binding profile of Protein A Sensor Chip Protein G Antibodies according to the binding profile of Protein G Sensor Chip Protein L Antibodies according to the binding profile of Protein L6 Conditions for report point-based screening Recommendations for report point based screening are listed below. Use the predefined screening method if one is provided with your Biacore system. Parameter Recommended value Comments Sample injection (ligand capture) Flow rate 10 µL/min Higher flow rates consume more sample. Contact time 60 to 180 s Long enough to give confidently measurable response levels without compromising throughput. Pooling Not applicable Molecular weight Not required Concentration Not required Antigen injection (analyte) Flow rate Not lower than 30 µL/min Avoid mass transport limitations that can obscure differences in ligand levels. Injection type Low sample consumption Contact time 60 s Use longer contact times if antigen binding is slow. Dissociation time 120 s Include a dissociation time to provide a rough indication of antigen binding stability. Pooling Optional Analyte may be pooled to conserve microplate space and reduce antigen consumption. Molecular weight Not required Concentration Not required Use the same concentration in all cycles.7 Conditions for off-rate ranking and kinetic screening Recommendations for off-rate ranking and kinetic screening are listed below. Parameter Recommended value Comments Sample injection (ligand capture) Flow rate 10 µL/min Higher flow rates consume more sample. Contact time 60 to 180 s Long enough to give confidently measurable response levels without compromising throughput. Pooling Not applicable Molecular weight Not required Concentration Not required Antigen injection (analyte) Flow rate Not lower than 30 µL/min Avoid mass transport limitations. Injection type High performance Contact time 60 to 120 s Use longer contact times if antigen binding is slow. Dissociation time 180 s Use a dissociation time that is long enough to allow estimation of dissociation rates. Pooling Optional Analyte may be pooled to conserve microplate space and reduce antigen wastage. Molecular weight Not required Concentration Not required for off-rate ranking Molar concentration required for kinetic evaluation Use the same concentration in all cycles. Kinetic screening is performed with a single antigen concentration. Blank cycles Blank cycles are not necessary for report point-based screening. Include blank cycles for off-rate ranking and kinetics screening. Blank cycles consist of antibody capture followed by injection of buffer instead of antigen. Ideally, there should be a blank cycle for each sample. Throughput can be improved by using a general blank cycle at intervals (for example, every 5 or 10 sample cycles), using a representative clone for the antibody capture. This approach requires that the antibody capture cycles are fairly similar throughout the assay with respect to parameters such as non-specific binding and drift. Screening using antigen as ligand Antibody screening with antigen as ligand is performed with single injections of each antibody clone over antigen on the surface. The antigen may be covalently immobilized or captured. No control samples are included. A typical antibody screening experiment with immobilized antigen includes the following steps: Step Action 1 Immobilize or capture the antigen on the sensor surface. Assay development work may be necessary to establish suitable immobilization conditions. 2 Run at least 3 startup cycles using buffer and regeneration to equilibrate the system. 3 Inject samples containing antibodies (for example, hybridoma culture supernatants or phage display preparations) over both the active and reference surfaces. Crude samples can be injected directly, without pre-treatment. 4 Evaluate the results.8 Preparing the antigen surface If the antigen is to be immobilized covalently on the surface, assay development work will probably be required to establish immobilization chemistry and conditions as well as regeneration conditions. It is important that the immobilization and regeneration do not interfere with antibody binding. Scouting strategies for immobilization and regeneration conditions are described in the Biacore Sensor Surface Handbook. Antigens that carry a suitable tag can be captured on the sensor surface. For most tags, regeneration removes both bound antibody and antigen from the surface, so that fresh antigen is captured for each cycle. Capture of biotinylated antigens on streptavidin surfaces however involves such a high affinity interaction that regeneration can remove the bound antibody but leave the antigen intact on the surface. With this approach, antigen is captured once at the beginning of the screen. Ready-to-use kits and pre-immobilized sensor surfaces for capturing tagged antigens are available from Cytiva. Ordering information may be found on the Products pages at www.cytiva.com/biacore. Product name Intended for His Capture Kit Histidine-tagged antigens Sensor Chip NTA Histidine-tagged antigens Biotin CAPture Kit Reversible capture of biotinylated antigens Sensor Chip SA Permanent capture of biotinylated antigens Conditions for screening Recommended injection conditions for antibody samples are listed below. Parameter Recommended value Comments Flow rate 10 µL/min Higher flow rates consume more sample. Contact time 60 to 180 s Long enough to give confidently measurable response levels without compromising throughput. Dissociation time 0 A dissociation time is not needed since off-rate evaluation is not used.9 Evaluation tools and options Antibody screens can be evaluated with a range of options for displaying the screening results, according to requirements and to some extent personal preferences. Several of the options described in this section are implemented automatically by predefined evaluation methods in the system software. The table below lists the information provided by commonly used options. Display option Antibody as ligand Antigen as ligand Sensorgram display Quality control Quality control Plot of capture_level against cycle Expression level Not generally applicable. However, for screening using capture on CAP, NTA or anti-His surfaces, the capture level may provide a check on surface performance throughout the assay. Plot of stability_early against cycle Expression level/antigen binding capacity Expression level/antigen binding capacity Plot of stability_early against capture_level Antigen binding capacity adjusted for expression level Not applicable Plot of stability_early against stability_late Antigen binding stability together with expression level/antigen binding capacity Antigen binding stability together with expression level/antigen binding capacity Off-rate ranking Antigen binding stability Not recommended Kinetic screening Antigen binding kinetics Not recommended Note: The report points stability_early and stability_late refer to the analyte injection (antigen when antibody is used as ligand and vice versa). Sensorgrams Sensorgram display can identify any disturbed cycles. Disturbed cycles should be excluded from the evaluation. Disturbances may not be apparent in report point plots. Sensorgrams can be aligned to zero at the baseline before the capture injection or before the antigen injection, to provide different perspectives on the capture and binding behavior. Examples: Sensorgrams from screening with antibody as ligand Sensorgrams from screening with antigen as ligand10 Capture_level against cycle A plot of capture_level against cycle number gives a good indication of the relative expression levels of the different clones, on the assumption that non-specific binding of material to the surface can be ignored. Clones that do not produce significant amounts of antibody give capture levels close to background. These clones can be excluded from further evaluation if desired. Stability_early against cycle The report point stability_early is placed immediately after the antigen injection. A plot of stability_early against cycle number provides a combined indication of 2 parameters: • Expression level: clones that produce low levels of antibody will show correspondingly low levels of antigen binding • Antigen binding properties: antibodies that bind antigen slowly or weakly will show low levels of antigen binding even if the expression levels are high The plot of stability_early against cycle number does not distinguish between these causes. However, clones can often be excluded from further work on the basis of low levels of antigen binding, regardless of the cause. Examples:11 Stability_early against capture_level A plot of capture_level against stability_early can distinguish between clones that express low levels of antibody and antibodies that bind antigen weakly. Points for low expression levels lie close to the stability_early axis, while weak binders lie close to the capture_level axis. The same information may be obtained by adjusting the antigen response for the capture level in systems that support this function. Stability_early against stability_late The report point stability_late is placed just before the end of the dissociation time after the antigen injection. A plot of stability_early against stability_late gives an indication of antigen binding stability, as illustrated to the right. More detailed information can be obtained from off-rate ranking and kinetic screening. Examples:12 Off-rate ranking Off-rate ranking is evaluated by fitting the dissociation phase of single-concentration antigen injections to a 1:1 dissociation model. Blank subtraction should be performed before the data is evaluated. The model describes an exponential process that is independent of the starting concentration, so that antigen concentration is not required. Biacore systems that support off-rate ranking for screening purposes provide both graphical and numerical overview of the results. Example:13 Kinetic screening Kinetic screening is evaluated by fitting the association and dissociation phases of single-concentration antigen injections to a 1:1 binding model. Blank subtraction should be performed before the data is evaluated. A molar value for antigen concentration is required for the fitting. However, since the same antigen concentration is used for all cycles, an approximate value can be used to obtain relative kinetic parameters if the exact value is not known. Inaccuracy in the molar concentration will be reflected in the reported association rate constants. A plot of association rate constant ka against dissociation rate constant kd (often called an on-off rate chart) is valuable in visualizing the results of a kinetics screen. In this plot, antibody clones with the same affinity lie on the same diagonal. Clones with different rate constants but the same affinity are distributed along the diagonal. (The affinity constant is equal to the ratio of the rate constants for a 1:1 interaction.) Example, showing thumbnails and on-off rate chart:cytiva.com/biacore Cytiva and the Drop logo are trademarks of Global Life Sciences IP Holdco LLC or an affiliate. Biacore, MabSelect, and MabSelect SuRe are trademarks of Global Life Sciences Solutions USA LLC or an affiliate doing business as Cytiva. © 2020 Cytiva All goods and services are sold subject to the terms and conditions of sale of the supplying company operating within the Cytiva business. A copy of those terms and conditions is available on request. Contact your local Cytiva representative for the most current information. For local office contact information, visit cytiva.com/contact CY12852-29Nov20-HB