Downloadable Literature

BLItz™ brilliantly packs the power of Dip and Read™ label-free analysis into a personal assay system. Small enough to fit on the palm of your hand (about the size of this page) and ready to go right out of the box, it can go anywhere you do. BLItz is also cleverly priced so everyone can have their own little genius.

Membrane proteins govern the majority of input and output signals of cells and represent the largest class of pharmaceutical drug targets, making the analysis of their molecular interactions critical to mapping the interactome as well as drug discovery efforts. Due to their integration into a lipid bilayer, in-vitro characterization of molecular interactions of membrane proteins presents a unique challenge compared to soluble proteins. By capturing 150nm sized lipoparticles containing a targeted membrane protein on the surface of a biosensor, the Octet platform enables the kinetic analysis (k_on, k_diss and K_D) of membrane protein-analyte interactions. Due to the large size of the lipoparticle, the observed data trace is often inverted, requiring a flip during data processing. Using a membrane protein-antibody model system, data processing and analysis of the membrane protein-analyte interaction on a lipoparticle is discussed and validated.

The Hybridoma Laboratory is a part of the University of Florida Interdisciplinary Center for Biotechnology Research. The main service provided by this laboratory is development of new mouse monoclonal antibodies for investigators at the University of Florida. The laboratory performs ELISA and western blot screening of antibodies. In vitro production and purification services are also offered. The Octet QK has enabled several new services to be added such as antibody quantitation, affinity ranking and determination of antigen : antibody binding constants.

Detection of foodborne pathogens is the primary means of preventing contaminated foodstuffs from entering the market. While traiditional immunoassays are widely used, they tedious ad time consuming. The Octet RED96 instrument shown to be used in the detection of bacterial pathogens in multiple matrices, and is shown to be a viable platform for rapid and specific detection of various bateria strains while avoiding time consuming enrichment steps.

Brian Bullard et. al. R&D, KPL, Inc.

For cell line development, it is ideal to have a quick and robust method to accurately determine antibody titer from crude supernatant. We have incorporated the ForteBio quantitation assay into the Takeda San Francisco cell line development platform. The quantitation assay was shown to have high specificity, precision and accuracy as judged by correlation of Octet data to HPLC data. The antibody quantitation assay is used in our cell line development platform at the 96 well, fed batch shake flask and bioreactor stages to streamline the evaluation of clones.

Presented by Amy Bass, Research Associate, Takeda Pharmaceuticals, at the 2nd Annual Workshop on Label-Free Assays for Bioprocessing Ligand Binding and Antibody Characterization, August 2011Boston MA
http://www.fortebio.com/CHI_2011/

Bio-layer Interferometry (BLI) is an established technology for evaluating the kinetic properties of ligand binding reagents and protein therapeutics. There are also some practical utilities for using this platform to quantify biotherapeutics. Here we present a strategy and results for the development and qualification of an Octet assay for the quantitation of a humanized antibody therapeutic with reagents previously used in a validated ELISA. Octet qualification and ELISA validation results are compared, and samples analyzed by the ELISA in support of a toxicokinetic (TK) study are analyzed via Octet. From these results we draw conclusions about the practical quantitative use of BLI in both non-GLP and GLP environments.

Presented by Mark Dysinger, Senior Scientist, Pfizer Global Research & Development, at the ForteBio Second Annual Ligand Binding Assay workshop in May 2011 at the AAPS National Biotechnology Conference.

This talk presents the label-free, real-time monitoring capabilities of the Octet RED system for kinetic characterization of a variety of protein-protein interactions spanning low, medium and high affinity to identify lead candidates in biotherapeutics drug discovery. Kinetic information often reveals details of interactions missed in a purely affinity-based ELISA screens that are important in ranking clones. The use of the Octet RED system for off-rate screening of proteins in bacterial lysates will also be discussed.

Presentation by Dr. Ryan Case, Amgen

The concentration of recombinant human Factor IX (rFIX) in bioreactor harvest samples manufactured at Pfizer is monitored using an ELISA (Enzyme-Linked Immunosorbent Assay). Since the ELISA based methods are time consuming and require several reagents, an alternate method for analyzing rFIX was evaluated. A new method for quantitation of rFIX in harvested cell culture media utilizing the ForteBio Octet Bio-Layer Interferometer (BLI) was developed, optimized and validated for use in the Quality labs. The Octet instrument utilizes BLI, a label-free dip and read detection technology to quantify biomolecules in solution. The BLI method makes use of a four-parameter logistic standard curve of binding rate vs. rFIX concentration from which samples are quantitated. The evaluation indicated that the BLI method is comparable to the current ELISA method. The BLI method provides improved accuracy, precision, and robustness, with time and cost savings for the analysis of rFIX from cell culture media, in-process steps and final purified bulk drug substance.

This presentation was made by Dr. Martha Jackson of Pfizer at a workshop at the CHI Bioprocessing summit in Boston in August 2010. Beta testing for Host Cell Protein (HCP) was performed at two Pfizer sites as a blinded study using the ForteBio Octet HCP method. Each site performed the basic method performance parameters: specificity, assay range, method robustness, accuracy, recovery and sensitivity. Sample analysis was also performed to determine HCP removal, batch consistency and equivalence to current HCP analytical methods.

Anti-drug antibody (ADA) immunogenicity assessment is a critical component in demonstrating the safety and efficacy profile of a therapeutic biological drug. Double antigen bridging immunoassays are widely used to measure ADA, however, these methods can generate false negative results due to low sensitivity for the detection of low affinity ADAs and interference from high concentrations of biological drug in samples. The goal of this study was to develop a method that could detect ADA to a human therapeutic antibody in the presence of drug within the sample. Using Octet-QK and mock ADA samples we demonstrated that this method offered advantages over ELISA and ECLIA methods. We then investigated the capability of Octet RED to detect ADA in a pre-clinical toxicokinetic and tolerability study of CNTO X mAb and compared it to ADA detection by ECLIA, leading us to believe that the Octet biolayer interferometry platform offers much promise for the development of drug tolerant ADA immunoassays. Experimental results of our study will be presented. Presented at AAPS NBC 2010

Presented by Arnout Gerritsen of Genmab at Screening Europe, February 2010. Bio-Layer Interferometry (BLI) plays an important role in the antibody discovery and development processes at Genmab. Integrating the quantitation, kinetic and affinity output of the Octet RED384 and Octet QK systems with ActivityBase enables Genmab to perform high value screening within early antibody discovery.

Dr. Yasmina Abdiche, Sr. Principal Scientist at Rinat-Pfizer discussed the versatility of the Octet QK384 system in screening crude antibodies and in epitope binning. She described the Octet systems as multiplexed biosensors that move sensors to samples and open up many possibilities for assay formats. The Octet system allowed batch immobilization of antibody from crude media onto 96 biosensors at-once offline; after screening her antigen samples in solution, she could batch regenerate all 96 biosensors offline to screen 150 sups in just 2 hours. The Octet quickly discriminated between tight, medium and weak binders and picked up some weak binders missed by ELISA. For epitope binning, the Octet QK384 was able to analyze 52 antibody pairs in just 2 hours including time for online coupling of 4 antibodies on 16 biosensors. In comparison, a Biacore 2000 only analyzed 39 antibody pairs in 6 hours even when excluding time for coupling 3 antibodies to biosensors and instrument prep time.

Dr. Yasmina Abdiche of Rinat-Pfizer talked about her experiences using the Octet QK and Octet RED systems to explore blocking assays for biotherapeutic product development.

She summarized that the Octet system is a reliable and versatile biosensor that is simple to use and is especially well-suited to blocking assays that are valuable to drug discovery. She said that Epitope binnings from Octet matched those from SPR and that solution affinities from Octet also matched those from SPR.

Presentation from Jane McIninch Ph.D. at IBC Antibody Production Confernce March 2009. Content includes a comparison of the Octet to ELISA and HPLC showing good correlation with both assay types, but allowing a significant throughput improvement IgG titer determination.

Improving the Accuracy, Speed, and Reproducibility of Titer Determination for the Clone Selection Process
Presented at: IBC 08 Antibody Production, San Diego, CA

Keith A. Davis, Ph.D., Scientist
Global Biologics, Pfizer Inc
Accurate titer determination remains a challenge, for Cell Line Development, with less than desirable precision, accuracy, and throughput. A new technology was evaluated that provides a > 10x decrease in hands-on time and turnaround time vs. both HPLC and ELISA while providing accuracy and precision comparable to that of Protein A HPLC without the HPLC waste or maintenance.

Presented by Dr. Laura Lerner at Bioprocess International '06, San Francisco, CA

Glutathione-S-transferase (GST) is commonly fused to recombinant proteins to facilitate detection and purification, and to increase solubility. The anti-GST biosensor consists of a high affinity anti-GST antibody pre-immobilized on a ForteBio biosensor. In conjunction with the Octet System, the anti-GST biosensor provides a rapid and label-free method for GST-tagged protein quantitation and kinetic analysis. The high specificity of the antibody-based biosensor enables the direct quantitation of GST analytes in crude lysates, column eluents, cell lysates and cell culture supernatants, serving as an alternative to traditional time-consuming analytical methods.

Amine Reactive 2nd Generation (AR2G) biosensors enable kinetic characterization of macromolecular interactions between purified proteins and target analytes. The AR2G biosensor surface is amenable to a wide range of pH and salt conditions, providing robustness and flexibility during the development of regeneration conditions for higher throughput applications.

This technical note provides guidelines for biotinylating a protein of interest for use with Streptavidin biosensors.

This technical note outlines a protocol for developing and routinely running an assay to detect residual host cell proteins. The protocol may be applied using commercially available generic HCP antibodies, or, process-specific ones. ForteBio’s Octet platform provides a superior alternative to ELISA with improved precision in measurements, equivalent or better sensitivity and dynamic range, low manual intervention, rapid assay development enabled by label-free real-time monitoring, and fast time-to-results.

Protein G biosensors provide a rapid and direct method for quantification of many types of mammalian IgG from buffer, media or other complex matrices. Protein G, which is pre-immobilized onto the biosensor, binds to rodent species IgG and many other mammalian IgG with higher affinity than Protein A but does not bind IgM, IgD or IgA. Specificity for IgG makes Protein G biosensors useful for quantifying many species of IgG, including murine, goat, and bovine in the presence of other types of immunoglobulins or protein contaminants.

The six-histidine or penta-histidine peptide tag (HIS-tag) is a common peptide tag fused to recombinant proteins during cloning. Many tools have been developed enabling the tag to be used for easy detection and purification of tagged proteins. The Anti-Penta-HIS biosensor, for use on the Octet system, now provides a rapid, label-free method for quantification of HIS-tagged proteins. This biosensor comes with the highly specific Qiagen Penta-HIS antibody pre-immobilized on the surface and is ready to use for specific detection and quantitation of HIS-tagged proteins.

Small molecule kinetics can be measured on the Octet RED and Octet RED384 instruments. This technical note provides guidelines for developing small molecule kinetic characterization methods using SSA biosensors.
It includes procedures for:
• Immobilizing biotinylated target protein onto SSA biosensors
• Performing small molecule analysis experiments using the Octet RED384 and Octet RED systems
• Analyzing and interpreting the small molecule binding data, including differentiating meaningful data from artifacts

In some applications, particularly kinetic screening, it may be advantageous to assay several protein samples using the same ligand-coated biosensor. To accomplish this, the target protein must be dissociated from the ligand-coated biosensor, regenerating the biosensor so that it can be used in another assay. This technical note provides guidelines to develop a successful regeneration
protocol.

A label-free quantitation assay for 6XHis-tagged proteins can be developed using the Octet QK and Octet RED instruments and streptavidin biosensors. By taking advantage of the flexible design of the biosensor tray, nickel-activated Biotin-NTA or an anti- His-tag directed antibody can be batch-immobilized on streptavidin biosensors outside of the Octet QK or Octet RED instruments. These ligand-coated biosensors can be used to rapidly quantitate His-tagged protein(s).

This technical note provides a detailed protocol for isolating
antibodies from carrier protein and subsequently
biotinylating the purified antibodies.

Provides examples for determining and validating an appropriate regeneration protocol.

The new Quantitation Assay Development Module
enables quantitation assays to be modified for specific
needs. By changing the assay time (time interval at
which data is taken for each set of 1-8 sensors) and/or
the flow rate, the user can adjust the functional range
of the assay. In the example shown below the anti-hIgG
Fc biosensor assay has been changed from a functional
range of 1-100 µg/mL to a new functional range of
0.2-25 µg/mL.

The BLItz system enables rapid identification, quantitation and characterization of expressed protein from as little as 4 microliters of sample. Data is presented in the poster to illustrate the ease of incorporating BLI into a laboratory workflow.

ForteBio’s Octet Dip and Read™ assay platform provides fast, accurate and easy-to-use assay solutions for many bioproduction applications. Direct binding assays that detect proteins without the need for secondary reagents are complemented by multi-step assay methods, affording a large dynamic range from picograms/mL to milligrams/mL. Octet systems are currently utilized in various stages of biopharmaceutical manufacturing to replace HPLC and ELISA methods which suffer from long analysis times, lack of specificity, labor-intensive protocols and imprecision. This talk will highlight Octet assays to detect residual residual host cell protein contamination in CHO cells and residual protein A contamination in antibody preparations.

The Octet RED384 instrument uses biolayer interferometry (BLI) to measure molecular interactions and is well‐suited for label‐free screening of small molecule fragments. It is a 16‐channel robot friendly instrument that is compatible with 384‐and 96‐well plates, is capable of screening thousands of compounds per day by measuring the interaction of a compound with protein target that is attached to the tips of biosensors. This instrument measures interactions directly in a microtiterplate without the use of microfluidics, and generates binding profiles, responses, and kinetic constants that correlate with SPR including kon , koff , and KD.

The Immunogenicity Assay Kit allows low affinity ADA detection, sensitivity and drug tolerance. This kit delivers a direct assay method and a bridging format - two fast and flexible formats.

The Octet RED has
increased sensitivity due to a 10X decrease in system noise. In addition the
RED has a significant increase in data sampling rate, allowing for true parallel
analysis of up to 8 interactions with a 96 well plate walk away capability. To
demonstrate the performance of the instrument kinetic analysis of proteinprotein
and protein-small molecules will be described.

One of the challenges in developing immunoassays is to identify suitable antibody pairs that recognize
the same target to non-overlapping, non-interfering epitopes. Selection of the most robust antibody
pair requires evaluation for both specii city and ai nity, which can be time consuming and dii cult to
determine with current methods. The Octet System provides a rapid analytical method to screen antibody
pairs under label-free, real-time conditions.

Screening for expression of clones using the Anti-human IgG biosensor is quickly
determined in the quantitation mode.
Screening for receptor:ligand binding and subsequent affinity ranking using
Amine Reactive Biosensors in the kinetics mode.
Rapid screening of antibody clones for of -rate using Streptavidin biosensors in
the kinetics mode.
Presented at the Society of Biomolecular Screening in Seattle, WA, 2006

Octet QK - A Novel System for Measuring Protein Interactions in Antibody Development and Production

Glutathione-S-transferase (GST) is commonly fused to recombinant proteins to facilitate detection and purification, and increase solubility. The robust properties of the GST tag have established it as the standard platform for protein-protein interaction pull-down assays. The Anti-GST biosensor consists of a high affinity anti-GST antibody pre-immobilized on a Dip and Read biosensor. It provides a rapid and label free method for both quantitation of GST targets (Figure 1) and kinetic analysis of GST fusions and interaction partners (Figure 2). The high specificity of the antibody-based biosensor enables direct analysis of GST analytes in crude lysates, column eluents, cell lysates and cell culture supernatants, serving as a time-saving alternative to traditional analytical methods.

Anti-Mouse IgG Fc Capture (AMC) biosensors enable kinetic characterization of macromolecular interactions between mouse Fc-containing proteins and target analytes. Immobilization of mouse Fc-containing proteins is achieved through a factory immobilized anti-mouse Fc-specific antibody whose high-affinity for the mouse Fc domain provides the stable baseline required for demanding kinetics applications. Cost-effective regeneration of the biosensors and the ability to directly immobilize mouse Fc-containing proteins from crude matrices make the AMC biosensor extremely useful in high-throughput applications. Subtypes IgG1, IgG2a and IgG2b are recommended for use with AMC biosensors; IgG3 should be evaluated on a case-by-case basis.

Protein L biosensors provide a rapid and direct method for quantitating a broad set of kappa light chain containing immunoglobulins, including whole molecules, FAb fragments and single chain variable fragments, from buffer, conditioned media or complex matrices.

ForteBio’s Octet RED96 system is a multi-functional, label-free, real-time analysis instrument. It is ideal for rapidly measuring
concentration of proteins and other biomolecules, measuring kinetics and affinity, and screening protein-protein and proteinsmall molecule interactions. The Octet RED96 can be used for a wide range of analyses including IgG and other protein titer, bioprocess development, quality analysis, crude antibody screening,
epitope binning/mapping, ligand binding assays, small molecule and fragment screening and analysis, elucidating cell signaling mechanisms and infectious disease monitoring.

ForteBio offers a range of high-quality, custom assay development services to help customers develop specific assays for its Octet platform of instruments, including host cell protein (HCP) detection assays. ForteBio’s HCP assays allow Octet users to cost-effectively accelerate their research efforts and maximize the utility of their Octet system. Since no two HCP detection assays are identical, the custom services team will work closely with each customer to tailor each assay to the customer’s specifications using customer-defined capture reagents and analytes. All custom products are developed by ForteBio’s world-class product development group to deliver the best quality-controlled products possible for the Octet platform. Custom HCP detection kits contain biosensors and all the reagents required to quantitate customer-designated host cell proteins.

ForteBio Streptavidin (SA) biosensors are designed for immobilization of biotin labeled proteins for use in assaying protein:protein interactions using the Octet system. The Octet system supports applications for kinetics characterization and quantitation of analytes binding to the immobilized protein.

The TW384 microplate is a black, polypropylene, 384-well tilted bottom
plate can be used on
the Octet RED384 and Octet QK384 instruments, both of which accommodate the 384-microwell plate format. The TW384 microplate enables use of samples as small as 40 uL in Octet assays and reduces variability in the background signal, thus improving assay sensitivity, particularly beneficial for peptide, small molecule and fragment analysis.

The six-histidine or penta-histidine peptide tag (HIS-tag) is fused to recombinant proteins during cloning, and many tools enable using it for easy detection and purification of tagged proteins. The Anti-Penta-HIS biosensor provides a rapid, label-free method for quantification of HIS-tagged proteins using the Octet System. This biosensor comes with the highly specific Qiagen Penta-HIS antibody pre-immobilized on the surface and is ready to use for specific detection and quantitation of HIS-tagged proteins.

The Sidekick station is an accessory to ForteBio's Octet family of real-time label-free biomolecular interaction analysis instruments. Sidekick enables simultaneous and uniform loading of reagents onto all 96 biosensors in a biosensor tray. Target analyte and other reagents that do not require online signal monitoring can be loaded onto biosensors on the Sidekick, freeing the Octet instrument for other users. The Sidekick can be installed beside an Octet system and used in collaboration with it, or it can be used independently.

The datasheet provides technical information on the capabilities and performance of the Octet RED384 and the Octet QK384 instruments.

The Octet platform is the fastest growing label-free technology in the world. The platform brochure reflects these advances and lays out its broad assay applicability and its strengths. Starting with the cover page, the first four pages of the brochure distills the benefits of the platform, emphasizing its strengths in specific application areas. The last four pages provide essential, targeted, and, up-to-date Octet platform information in one concise handout.

Aminopropylsilane (APS) Biosensors are designed for hydrophobic or electrostatic immobilization of most proteins used in assaying protein:protein interactions.

ForteBio's Protein A Biosensors, in conjunction with the
Octet System, are designed for monitoring antibody
concentrations from crude lysates and cell culture
supernatants. Using Protein A Biosensors, the Octet
System supports applications from cell culture screening
to purification monitoring during the process
development and production of therapeutics.

Protein A Biosensors Package Insert

Amine Coupling Reagent Kit Product Insert

Super Streptavidin Biosensors Product Insert