The Swiss Army knife is the perfect combination of form and function, found in countless pockets, purses, and packs simply because of its innumerous uses. Scientists rely on analytical tools every day to make pivotal decisions throughout the drug discovery, development, and manufacturing process. While many biophysical analytical tools are suited for detecting structural changes, few can assess structure-dependent function in the same system.
This interactive resource outlines the power of label-free protein-protein and protein-small molecule interaction characterization. We invite you to browse our extensive resource library that has been organized by application area as well as workflow stage, making it easy to find the right information and advice for your area of expertise.
The Swiss Army knife is the perfect combination of form and function, found in countless pockets, purses and bag packs simply because of its countless uses. Scientists rely on analytical tools every day to make pivotal decisions throughout the drug development and manufacturing process. While many biophysical analytical tools are suited for detecting structural changes, few can assess structure-dependent function in the same system.
This interactive resource outlines the power of label-free biologics characterization by enabling you to browse our extensive resources to easily find the right information and advice for your application.
Click here to find out more about the power of label-free BLI assays
Label-free bio-layer interferometry (BLI) assays measure molecular binding interaction without the use of labels and provides data in real time. It is a precise and efficient approach for collecting a wide range of information related to the biomolecular interaction itself, such as analyte concentration, bio-similarity, specificity, potency, and selectivity of the molecular binding. Whatever the analyte – therapeutic proteins, monoclonal antibodies (mAbs), bispecific antibodies (bsAbs), and antibody-drug conjugates (ADCs) – they can be reliably characterized and quantified.
The fluidic-free biosensor design aims to simplify quantitation and binding kinetic assays by eliminating limitations caused by microfluidic flow, sample loading, flow channel cleaning procedures and sample purification. From drug discovery through development to manufacturing, label-free characterization of biologics is ideal for active analyte concentration measurements, residue analysis and screening for critical quality attributes (CQAs) early in cell line development (CLD) and bioprocess development workflows.
Protein-protein interactions play a key role in predicting the function of target proteins and the drug ability of molecules. To prevent non-specific and potentially detrimental interactions, proteins must discriminate between interaction partners. This is often achieved via molecular recognition or non-covalent interactions between protein surfaces. When developing new protein therapeutics, it is essential to understand the specificity of any protein-protein interactions occurring.
During discovery and development, the binding profile and kinetics of a vaccine target can provide critical insight into structure and function. Binding kinetics concern the rate constants of ligand association and dissociation, and the ratio of the two defines the equilibrium dissociation constant. Measuring the kinetics of a drug-protein interaction is extremely important as it reveals the time component of the interaction which correlates to its safety, clinical efficacy, and duration of action. Unlike traditional end-point methods such as ELISA that rely on expensive labeling schemes, BLI is label-free. That means it’s much faster to set up and avoids the risk of functional interference from the label itself. Furthermore, several tedious washing steps in ELISA make it difficult to distinguish the poor-expressing, strong binders, from the high-expressing, weak binders.
Off-rate ranking of antibodies is an important tool during antibody generation where real-time analysis matters. Kinetic analysis of antibody-antigen interaction is a crucial step in selecting high-affinity monoclonal antibodies (mAbs) for therapeutic applications. High-throughput off-rate ranking enables the selection of the highest affinity clones in primary screening assays. Antibodies that exhibit high off-rates may produce ambiguous binning profiles when used as saturating mAbs in an in-tandem assay format. This is where fluidic-free BLI technology has a notable advantage, the capability of measuring crude samples. This supports screening and ranking of unpurified hybridoma supernatants based on off-rate as the measurement is not dependent on concentration and is a better dimension of antibody binding kinetics.
Affinity ranking is a powerful tool for the initial screening of large panels of antibodies or biologics in general. The binding affinity describes the strength of binding of an analyte molecule to its ligand. Research scientists use a variety of methods to analyze biomolecular interactions. A common method for measuring binding is solid-phase enzyme-linked immunosorbent assay (ELISA), which provides end-point verification of the binding affinity. However, end-point assays, like ELISA, provide limited data on biomolecular binding characteristics and often miss very weak or transient interactions due to the required washing steps. A protein-protein interaction is a reversible process. The association rate (kon) of a molecular interaction is concentration dependent and the dissociation rate (koff), how quickly the molecules depart, is concentration independent. The binding affinity is the ratio of both. Therefore, only real-time kinetic analysis of a biomolecular interaction provides a complete binding profile also for weak interactions.
Potential leads are evaluated for a range of properties that can include selectivity, kinetic analysis, dose-response curves, potency, or toxicity. Antibodies optimized by protein engineering for increased efficacy and specificity, or reduced immunogenicity are just one example. This stage is known as lead optimization includes antibody production, humanization for non-human origins, affinity maturation, or Fc engineering. Humanization is a technique to reduce the immunogenicity of a therapeutic antibody initially derived from a non-human resource. The process replaces significant parts in the parental antibody with a human IgG sequence. Affinity maturation is the process by which antibodies gain increased affinity, avidity, and anti-pathogen activity. With repeated exposures to the same antigen, leads with relatively low, high nanomolar, target binding affinities can be enhanced to reach a desired subnanomolar affinity range. Fc engineering technology is conducted mainly to maximize the receptor-mediated functions of antibodies, including antibody-dependent cellular cytotoxicity (ADCC), in vivo half-life, and agonistic activity. Optimized leads will be further characterized to confirm efficacy and favorable molecular attributes as a therapeutic candidate.
Small molecules are typically defined as having a molecular weight of less than 900 Daltons, and make up approximately 90% of pharmaceutical drugs. Understanding how a small molecule binds to its intended target is critical and insights into these target interactions can reveal information about its mechanism of action and provide insights into strategies for improving the small molecule.
The affinity of an antibody refers to the strength with which the antibody binds to the antigen-binding site. Affinity ranking is used to kinetically rank and screen antibodies via kinetic rate constants or equilibrium dissociation constants. This is a powerful tool for the initial screening of large panels of antibodies.
Accurate protein quantitation is essential to protein studies in a multitude of research topics. A wide array of different methods have been developed to quantitate both complex mixtures of proteins as well as a single type of protein. Total protein quantitation methods comprise traditional methods such as the measurement of UV absorbance at 280 nm, Bicinchoninicacid (BCA) and Bradford assays, as well as alternative methods like Lowry or novel assays developed by commercial suppliers, which often provide a well-designed, convenient kit for each type of the assay. Individual protein quantitation methods include enzyme-linked immunosorbent assay (ELISA), western blot analysis, and more recently, mass spectrometry, among others.
Label-free quantification is a method of determining the relative amount of proteins in two or more biological samples, but unlike other quantitative methods, it does not use fluorophores or radio-labelling for the detection. The label-free quantitative proteomics approach provides a powerful tool to resolve and identify thousands of proteins from a complex biological sample. Furthermore, the active concentration of an analyte of interest can be used as criteria to optimize process parameters. Concentration measurements using label-free BLI technology can be also used for monitoring production batches in terms of residues such as CHO host cell protein or protein A.
In order to produce high yields of recombinant proteins for therapeutic applications stable producer cell lines need to be developed. Cell line development (CLD) involves the screening of thousands of clones to find those that are stable, produce high yields of bioproduct and have desired critical quality attributes (CQAs). Screening and process optimization activities are typically carried out on a small scale in bioreactor cultures to ensure results translate to downstream processes. Performance data is primarily based on cell growth, cell viability, metabolite analysis and product titer. Data is assessed over the entire culture process duration. Titer measurements utilizing label-free BLI analysis are used to select high-producing clones and to normalize the functional activity of these clones in crude matrices. In addition, several CQAs such as glycosylation profiles can be screened vin very early stages to prevent late failures in the development process.
Hybridoma technology relies on B cells that are matured in secondary lymphatic to produce mAbs specific to antigens of interest. This natural antibody maturation process provides a huge variety of antibodies altered in their variable region. Hybridoma screening is performed to identify those that only produce antibodies of appropriate specificity and present a selection of high-affinity tight binders.
Contaminations or impurities associated with the manufacturing process of biopharmaceuticals at a variety of stages are controlled by limit values, as this is critical for product safety. All impurities are typically present in low concentrations in complex matrices. Hence their detection and quantitation are quite challenging. During quality control (QC) their detection is key to ensure the safety of the biopharmaceutical product.
Glycosylation is an important post-translational modification that plays a key role in biotherapeutic function and efficacy. Analysis of the distribution and composition of N-glycans represents a critical attribute – of increasing importance – for manufacturing and regulatory authorities. Glycan profiling is a novel structural analysis method for the rapid, sensitive, and high-throughput generation of essential information about target glycans or glycoproteins.
Epitope binning is a term used to describe segmentation of a panel of monoclonal antibodies (mAbs) into bins based upon the antigen region, or epitope, bound by each antibody. This grouping is performed using cross competition assays, in which the competitive binding of antibody pairs to a specific antigen is characterized. If the antigen binding of one mAb prevents the binding of another, then these mAbs are considered to bind to similar or overlapping epitopes. Conversely, if binding of a mAb to the antigen does not interfere with the binding of another, then they are considered to bind to distinct, non-overlapping epitopes. Two criteria must be fulfilled in order to assign mAbs into the same bin. First, all mAbs in the same bin should block each other’s ability to bind the antigen. Second, all mAbs in the same bin should have similar blocking profiles when paired with other mAbs in the panel.
In early drug development, cross-competition assays are used to characterize hundreds of antibody clones and can be performed with hybridoma supernatants, phage lysates or purified samples. Because mAbs in different bins bind to distinct epitopes and display diverse functional characteristics, epitope binning studies can increase the likelihood of choosing a lead antibody with the desired biological activity. Cross-competition assays also are performed to identify mAbs that bind similar epitopes to a previously characterized mAb as in the generation of biosimilars or biobetters. These assays may also be useful in selecting reagents for sandwich or ELISA-type assays, such as those used for biomarker testing or pharmacodynamic assays, to identify good antibody pairs that bind to the antigen simultaneously.
Octet® systems are ideally suited to run cross-competition assays, with their combination of assay speed, versatility of assay design and parallel, independent biosensor format.
In the field of pharmacology, the biological activity or potency of a biotherapeutic reflects its mechanism of action, and the concentration needed to induce an effect. The potency of biomolecules cannot be quantitatively correlated. A reference standard is usually applied to determine the efficacy by comparison, typically using a cell-based bioassay, but also biosensor-based assays which measure the active concentration of the analyte.
Rapid and real-time results for the study of viral biology and development of antiviral therapies
In this exclusive interview, Dr. Rebecca DuBois, Associate Professor in the Department of Biomolecular Engineering at the University of California, Santa Cruz, outlines the advantages of using Sartorius' Octet® platform in her research to understand the molecular mechanisms of human viral infection and to develop new vaccines and antiviral therapies.
Immunogenicity testing, the measurement of an immune response to a therapeutic drug, is an integral part of drug development. The immune system may respond to drug administration in patients by producing anti-drug antibodies (ADA). ADA can alter the pharmacodynamics and/or the pharmacokinetics of the drug, so detecting them is essential during the development process. ADA can be produced in a wide range of concentrations and with a wide range of affinities. To accurately detect these polyclonal antibodies, the system must have exquisite sensitivity, withstand a wide range of free drug concentrations, and have minimal matrix effects. Automated immunoassays on Octet® BLI instruments provide a high level of sensitivity, tolerance to drug, and flexibility to detect both high and low affinity ADA by providing multiple protocols without any plate washing steps. Label-free, plate-based assays work across the many drug types in the market today such as antibodies, proteins, and peptides and can be used with both human and animal samples.
A robust and flexible analytical characterization portfolio for biotherapeutics
The "Swiss Army knife" solution
The Octet® R8 utilizes bio-layer interferometry (BLI) technology to enable the direct detection of specific proteins and other biomolecules and an advanced fluidics-free approach to protein characterization.
Click here to read 5-star reviews of the Octet® R8
A powerful addition to our lab. The striking feature that comes up time and again is that the sample is preserved so the ability to control experiments and analyze samples by other techniques is a huge advantage over injection-based flow systems. I can run multiple antibodies through the very same sample and be certain that the result is real and not just ruled out as an anomaly or error.” Luke Miles, Florey Institute of Neuroscience
“The Octet® R series is fast, easy to use, and the software is very user-friendly. Results can be obtained in a 3 - 4 hour run time, even with low-volume samples. I strongly recommend as it is very economical compared to other players in market. Plus, an efficient and prompt sales and service team to serve customers.” Vishal Mehrotra, Kashvi Life Sciences
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