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The Octet® HTX and RED384 Systems are the industry-leading platform for monitoring the binding of proteins and other biomolecules directly in real-time. With the ability to read up to 96 samples in parallel, the Octet system reduces titer determination and kinetic screening from hours to minutes. Integrated solutions from the Advanced Workflow Engineering Solutions (AWES) team can help you maximize the throughput and unattended run time of the Octet HTX and RED384 systems, with round-the-clock operation. Pick and choose a variety of possible configurations — each with a varying amount of components like robotic arms, plate hotels, incubators, and more — that best fit your unique research needs.

The Octet family of instruments is designed to meet a broad range of application and workflow requirements. Your local ForteBio representative can provide detailed information on system capabilities and our extensive library of Octet application solutions to help you choose the system that’s right for your lab.

Pioneer and Pioneer FE systems provide high quality kinetics (ka, kd) and affinity (KD) measurements in a single injection in a matter of minutes. They use surface plasmon resonance (SPR) technology to rapidly characterize a wide variety of biomolecular interactions from small molecule fragments to biologics, without compromising sensitivity. Pioneer systems provide rapid affinity data (KD) directly from primary screens, significantly reducing the time and cost associated with secondary screening while enabling the prompt identification of lead candidates.

The BLItz system 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. The BLItz system is also cleverly priced so everyone can have their own.

Cell-based assays that qualitatively measure the function of an interacting molecule under physiological conditions provide valuable information for drug discovery. Two approaches are commonly used in early drug discovery; target-based approaches use label-dependent molecular assays to measure the effect of compounds on a specific target protein in vitro, while phenotypic approaches use unbiased phenotypic assays to evaluate the effect of compounds on specific phenotype of cells, tissues or animals.

In this application note, we discuss the strategies for the development and validation of a potency assay using Octet systems. We have highlighted the Octet system’s ease-of-use and fast time to results by showcasing strategies for the development and validation of a method for evaluating the binding of an Fc gamma receptor III molecule to the widely characterized NISTmAb.

The Pioneer Next Generation SPR platform from ForteBio improves the efficiency of the characterization process over traditional SPR by determining the kinetics and affinity in a single step. The next-generation OneStep® gradient injection featured on the Pioneer platform dramatically increases the speed of affinity characterization while maintaining accuracy and high confidence in results. In this application note, we explore the utility of OneStep Injections in kinetic characterization of biologics in both research and drug discovery. 

The principal role of assay groups in drug discovery is to provide reliable methods, analysis, and data for confident decision-making about series progression. Particular assays are chosen to differentiate between affinity, specificity, cellular action, and most importantly mechanism of action. Increasingly, drug discovery faces challenges from harder-to-drug targets along with regulatory pressures to increase safety, efficacy, and to improve drug ADME (absorption, distribution, metabolism, excretion) properties. The responses to these challenges come through the creative use of both emergent and standard technologies, investing in new discovery approaches (e.g. fragments), and increasing exploration.

Monoclonal antibodies have long been an essential tool for cell and molecular assays and have moved into the clinic as part of a shift from small molecules into biologic treatments for disease. Antibodies are typically produced from a cell line that has been screened to ensure the protein is expressed with the desired post-translational modifications, target specificity and affinity, and at relatively high levels. Once this producing line has been identified, one can achieve higher titers by optimizing the culture media to increase cell growth and/or protein production.

The accurate determination of recombinant protein titer is critical to the selection of high-producing clones during cell line development and in optimization of bioreactor conditions during production of therapeutic proteins. Concentration measurements for these therapeutic proteins are often done using enzyme-linked immunosorbent assays (ELISAs), RP-HPLC or coomassie-stained SDS-PAGE gels. Although these techniques are prevalent, they are encumbered by drawbacks that include long assay times, extensive hands on and in some cases, low throughput. In addition, techniques such as ELISA tend to exhibit high variability, resulting in lower accuracy.

Fragment-based drug design (FBDD) has become an increasingly popular platform for the identification of lead candidates in drug discovery programs. The detection and characterization of fragment binding events is facilitated by sensitive biophysical technologies capable of detecting low affinity interactions of low molecular weight compounds. Over the last decade approaches such as nuclear magnetic resonance (NMR), X-ray crystallography, differential scanning fluorimetry (DSF), and surface plasmon resonance (SPR) have become core technologies in many pharma and biotech settings for the identification of these low-affinity fragment compounds. In particular, SPR-based biosensors have sufficient sensitivity and throughput to provide complete fragment screens on libraries of several thousand compounds in just a few weeks per target.  

Vaccines are biological preparations that contain agents resembling disease causing microorganisms, and can improve immunity against a specific disease. They are typically prepared from inactivated or weakened forms of the microbe or its toxins, or surface proteins. Classical vaccines against the influenza virus are developed in embryonated hen eggs and may include whole virus, split virus or a purified subunit with every component other than hemagglutinin (HA) or neuraminidase (NA) removed. The target molecule for the protective immune response triggered by vaccination is generally accepted to be the HA molecule; a glycoprotein found on the surface of the influenza virus. Measuring the vaccine potency or the biologically active components is critical to the determination of the vaccine's effective dose. In addition, the stability of the vaccine has major impact on its usage for immunization programs worldwide. Although real-time stability studies under different storage conditions is preferable, thermal stability testing using potency assays with samples subjected to heat or environmental stress conditions can be used as predicators of vaccine stability over time.

The Fc region of human IgG contributes to a number of beneficial biological and pharmacological characteristics of therapeutic antibodies. One of the most important is prolonging plasma halflife, due to its unique, pH-dependent interaction with the neonatal Fc-receptor (FcRn). Because altered FcRn binding can increase or decrease serum half-life of Fc-containing therapeutics, thereby impacting drug efficacy, FcRn binding interactions are increasingly being assessed at multiple stages of biologic drug development. FcRn-Fc activity and binding assays are performed as part of characterization studies to enhance overall product understanding and demonstrate comparability in the development of biosimilars. Commonly used in vitro methods for analysis of FcRn binding include ELISA, SPR, and bead-based proximity assays.

In this application note, we’ll describe best practices for getting started with the BLItz® system, as well as developing and running quantitation assays, sample and biosensor preparation, and data analysis.

The ability of therapeutic monoclonal antibodies to bind FcγRs can greatly impact their safety and efficacy. Induction of ADCC by an antibody depends on its binding affinity to both the target and to the FcγR. Therefore efforts to analyze and enhance Fc interactions with FcγRs have become an integral part of biotherapeutic development processes. Throughout drug development, antibodies are selected, engineered, and assayed for either improved or reduced binding to Fc receptors depending on their mechanism of action. Effector function is a critical component of biological characterization and SAR studies of lead candidates. The FDA requires data on Fc effector function for regulatory filings and potency lot release, and Quality Control groups routinely assay in vitro binding and kinetics of therapeutic candidates to Fcγ receptors in parallel with functional cell-based ADCC assays. Binding affinities of Fc gamma receptors to monoclonal antibodies can be determined in a high throughput and highy sensitive format using biosensor analysis on the Octet platform. Here we introduce the Octet platform as the technology of choice for analyzing Fc gamma receptor-IgG binding interactions. Assay formatting options and best practices for various biosensor formats on the Octet system will be discussed, as well as considerations for assay optimization, data acquisition, curve fitting and analysis of results.

The Octet HTX system is a powerful tool to perform high throughput cross-competition or epitope binning assays in a matter of a few hours. The Octet platform is amenable to all assay formats and allows the user to test multiple permutations of mAbs in the same walk-away assay. It is a highly versatile system that enables 96 discrete and independent binning interactions to be monitored in parallel, making it possible to assemble 96 different mAbs or biosensor chemistries. New features in Octet Data Acquisition and Analysis software provide templates and enable visualization of data, calculation of nm shifts at user-specified report points, flagging problematic data, data normalization and creation of two-dimensional matrices to resolve mAbs into different clusters or "bins". This application note reviews principles, methods and best practices for developing and performing cross-competition or epitope binning assays on the Octet systems.

Application Note 15 assesses the relative merits of Protein A High Pressure Liquid Chromatography (HPLC) and Protein A bio-layer interferometry using the Pall ForteBio Octet® RED96 system to determine MAb concentration in a complex feedstock. The assessment was performed within the context of determining a MAb breakthrough curve from a Protein A column loaded with a CHO culture feedstock. The authors find that HPLC and the Pall ForteBio Octet system can be used to generate comparable data, with the Octet system providing significant time savings in both sample preparation and in machine time.

This Application Note introduces large molecule kinetic analysis on the Octet platform, and discusses options, techniques and considerations for developing and performing successful kinetic characterization assays and interpreting results.

The Octet QK384 instrument was evaluated as an alternative to HPLC due to its many advantageous features and suitability for screening proteins in cell culture supernatant fluids. Octet systems have the ability to analyze crude samples, allowing users to bypass sample pre-processing. In addition, 96 samples could be analyzed in less than 30 minutes, expediting screening that took more than 19 hours to complete by HPLC. A higher throughput, automated screening workflow with significantly reduced analyst involvement was achieved via integration of the Octet system and a PerkinElmer (formerly Caliper Life Sciences) Sciclone robot. This document summarizes the performance of Biogen IDEC's Fc-fusion protein assay and highlights the excellent accuracy, precision, robustness and reliability of Octet assays.

The analytical group at Boehringer Ingelheim, Fremont, USA required a robust assay to measure the biological activity of an antibody fragment (Fab) molecule for in-process testing as well as stability and lot release testing in their Quality Control (QC) department. The group developed a working Fab activity assay on their Octet® RED system in less than one week to monitor Fab activity during process development.

Biopharmaceutical companies have enthusiastically adopted Pall ForteBio's Octet systems due to their broad utility in protein quantitation and functional characterization combined with enhanced throughput, decreased sample preparation requirements, and low cost of operation. This white paper describes the use of Octet instruments for protein quantitation, particularly in the areas of process development and quality control.

CaptureSelect affinity ligands provide a unique group of molecules that can be designed for almost any purification target, with as narrow or as broad specificity as is required.

Tips and tricks for obtaining the best possible kinetic data are presented, including optimization of target immobilization and analyte binding, along with regeneration strategies (if required) and sample plate and method design. Octet data analysis software options are also discussed, giving details of the analysis models and their applicability to data interpretation.

The Community Structure Activity Resource (CSAR, www.csardock.org) group is developing a database of high quality protein-ligand structures and the corresponding binding affinities. The data will be provided from in-house experiments and community collaborations. The proteins are generally well-studied structures that have been targeted in drug discovery projects. Current projects include CDK2, LpxC, and urokinase. The drug-like ligands for each of these targets consist of several series of compounds with a wide range of affinities. The Octet RED system has dramatically increased the data we can produce in a short time, turning it into the workhorse of the CSAR project.

Minimizing the variability of background signals is a key parameter to the success of demanding applications such as small molecule analysis1. By reducing the background variability, smaller positive signals can successfully be resolved, providing an increase in assay sensitivity. This application note demonstrates the use of a reference biosensor with biotinylated streptavidin for increasing signal-to-noise levels, thereby improving the detection of small molecules, including fragments.

This application note describes the use of the BLItz system to both streamline workflows and obtain rapid, direct quantitation of proteins in crude matrices.

This application note describes the use of the BLItz system for instant, specific protein detection in crude matrices, enabling access to realtime sample information during bioprocess development and production.

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 (kon, kdiss and KD) of membrane protein-analyte interactions.

ELISA-based assays and Octet platform quantitation assays share many similarities. Therefore, conversion of a pre-configured ELISA assay to the Octet platform may only require transfer of assay conditions. When assay conditions need re-optimization on the Octet platform, considerations are often similar to those employed in ELISA. The Octet platform's direct binding assay method is simple, fast and accurate; the multi-step methods offer high sensitivity and expanded dynamic range. The automated assay formats enhance time-to-results and walk-away time and reduce operating expenses

In this white paper, guidelines for preparing an instrumentation grant application will are provided for principal investigators interested in acquiring an Octet® system, the label-free BLI biosensor platform for biomolecular interaction analysis. Many resources are available on the topic of grant writing help and this white paper is designed to specifically help the author in justifying the need for the Octet system as it applies to the institution’s research. The structure of the application is modelled after the NIH Shared Instrumentation Grant (S10) program, but the recommendations are broadly applicable for other instrumentation grants.

Drug development and production is challenging. Avitide, based in Lebanon, New Hampshire, provides on-demand development and supply of high-performance affinity purification resins for the manufacture of biotherapeutic drug molecules. Molecule-specific affinity purification resins simplify complex downstream manufacturing processes that can require years of development and multiple steps that often sacrifice yield for purity. Using a proprietary discovery and development platform technology, Avitide enables its partners to rapidly advance candidate molecules to the clinic and achieve faster and more predictable bioprocess development timelines by providing one-step affinity purification solutions for a range of biomolecules including enzymes, novel scaffolds, multi-specific antibodies, gene therapies and vaccines—in just three months. The speed at which Avitide serves their growing client base is a key strategic advantage for their partners but presents a unique challenge in the R&D process.

Pall ForteBio’s Octet instruments utilize BLI technology to monitor biomolecular interactions in real time. They are an ideal replacement for ELISA and HPLC techniques for the quantification of antibodies and recombinant proteins and are especially suitable for product potency lot release assays. The plate-based and non-fluidic format also offers GMP users distinct advantages over comparative SPR based techniques. Octet systems provide higher throughput with the flexibility to run 2 to 96 samples simultaneously and better sample versatility, including the ability to analyze crude samples and more tolerance to diverse sample matrices. In addition, ease of use, low maintenance and high data precision speeds time to results throughout the drug development process. The Octet platform is particularly well-suited for GxP and QC laboratories.

Antibody and other protein therapeutics are a major focus in drug discovery pipelines today. The overall process for developing protein therapeutics encompasses target selection and validation, library screening to generate early candidates (‘‘hits’’), follow-up characterization for lead selection, lead optimization, and clinical candidate selection. During lead selection, molecules identified as hits are subjected to screening via multiple analytical methods to select a few candidates for progression to the next stage of the development process. This is typically followed by detailed characterization for confirmation of binding and functional activities via biochemical and biophysical analyses. 

Carson Cameron, MS, Analytical Development Sr. Research Associate, KBI Biopharma. Speaker presentation from Bethesda User Meeting, April 2017.

Victor Ayala, Ph.D., Cell Biology, Advanced BioScience Laboratories, Inc. Speaker presentation from Bethesda User Meeting, April 2017.

Mike Kowalski. Ph.D., Beckman Coulter. Speaker presentation from Bethesda User Meeting, April 2017.

Common approaches to the identification of hits from small molecules or fragments are via high-throughput screening (HTS) assays and/or surface plasmon resonance (SPR). The aims of this study were to validate the hits from a high-throughput (HT) SPR assay with a well-characterised HTS enzymatic assay against an undisclosed target.

Jason Goldstein, Ph.D., Annular Biotechnology, speaker presentation from Cambridge user meeting, May 17, 2016

Many of the antibodies published in biomedical research suffer from the lack of properly controlled studies into their reliability and reproducibility against specific antigens. In at least one past study, fewer than half of approximately 6,000 routinely used commercial antibodies recognized only their specified targets. Issues with improperly characterized antibodies prevented the replication of data in 47 of 53 preclinical studies. It is estimated that the resulting waste in labor and consumable costs are $350 million annually just for US researchers. Unlike large bio- pharmaceutical companies with the means to validate reagents, most Ab users are reliant on the documentation provided by commercial sources. The content of documents accompanying Ab batches is highly variable and often not corresponding to the supplied batch.

Mark Fisher, Ph.D., University of Kansas Medical Center, speaker presentation from Cambridge user meeting, May 17, 2016

Bio-Layer Interferometry offers users the ability to obtain quantitative and kinetic data for following the assembly of large protein complex systems. In this presentation, we expand the uses of this system by demonstrating that we can easily identify protein complex components attached to the BLI biosensor surface using Mass spectroscopy. From a structural standpoint we demonstrate that easily reversible complexes attached to BLI biosensor surfaces can also be deposited onto electron microscopy glow discharged copper grids and visualized using negative stain electron microscopy.

Colby A Souders, Ph.D., Scientist - Protein Engineering, Kanyos Bio, speaker presentation from Cambridge user meeting May 17, 2016

Immunoglobulin G (IgG) has an unusually long serum half-life in comparison to proteins of similar size. It is well-known that this phenomenon is due to IgG’s ability to bind the neonatal Fc receptor (FcRn) in a pH-dependent manner. FcRn binding properties can vary among IgGs, resulting in altered in vivo half-lives, and therefore it would be beneficial to accurately predict the FcRn binding properties of therapeutic IgG monoclonal antibodies (mAbs). Here we describe the development of an in vitro model capable of predicting the in vivo half-life of human IgG.

Phil Barish, Ph.D., VP Operations, AbSci, speaker presentation from South San Francisco, CA user meeting April 21, 2016

The Octet is a powerful platform for high-throughput quantitation of antibody products, including full-length antibodies, antibody fragments (Fabs), and Fc-fusion proteins. AbSci’s E. coli protein expression technology utilizes two homogeneously inducible promoters in combination with cytoplasmic expression to achieve high-yields of soluble antibody products. To perform high-throughput expression optimizations it is desirable to evaluate protein yields in crude cell lysates. Unfortunately, due to the presence of free heavy chain and/or free light chain in these E. coli lysates, it is difficult to ensure biosensor binding exclusively to fully assembled multimeric product. AbSci has developed a high-throughput protocol for quantitation of heterodimeric Fabs that combines single-step plate purification with the Octet to accurately quantitate yields of fully assembled Fab.

Stefan Kol, Ph.D., Protein Biochemist, DTU NNF Center for Biosustainability, speaker presentation from Amsterdam user meeting, October 22, 2015

Erythropoietin (EPO) quantification during cell line selection and bioreactor cultivation has traditionally been performed with ELISA or HPLC. As these techniques suffer from several drawbacks, we developed a novel EPO quantification assay. A camelid single domain antibody fragment directed against human EPO was evaluated as a capturing antibody in a label-free biolayer interferometry-based quantification assay. Human recombinant EPO can be specifically detected in Chinese hamster ovary cell supernatants in a sensitive and pH-dependent manner. This method enables rapid and robust quantification of EPO in a high-throughput setting. In addition, we are developing assays to quantify the human therapeutic proteins alpha-1 antitrypsin and C1 esterase inhibitor.

Thomas M Duncan, Ph.D., Associate Professor of Biochemistry & Molecular Biology, Upstate Medical University, presented at Bethesda user meeting October 29, 2015

F-type ATP synthase is a critical enzyme in cellular energy metabolism. Some bacterial ATP synthases can be auto-inhibited by the C-terminal domain (CTD) of its ε subunit, which undergoes a dramatic conformational change to 'jam the gears' of this rotary nanomotor enzyme. With the isolated catalytic complex (F₁-ATPase) from E. coli, the ε subunit becomes a dissociable inhibitor, and we used Bio-Layer Interferometry (BLI) to confirm that the inhibitory position of ε's CTD restricts dissociation of the subunit. Bias of ε's CTD toward inhibitory or non-inhibitory conformations is sensitive to specific ligands at catalytic sites on F₁-ATPase. With BLI to monitor interaction of F₁-ATPase with sensor-bound ε, different ligand conditions were used during the Association and Dissociation steps of the assay, which allowed us to observe kinetic effects of catalytic-site ligands on the conformational state of F₁-bound ε. As an example of the value of this indirect but sensitive method, it allowed us to conclude that conversion of ε's CTD to the inhibitory conformation occurs after hydrolysis of ATP at the catalytic dwell. This extension of BLI to monitor effects of small ligands on protein-protein interactions should be applicable to other enzyme or protein complexes.

Patricia Buckley, Ph.D., Research Microbiologist, US Army ECBC, Bethesda user meeting October 29, 2015

Cindy Benod, Ph.D., Research Associate, Houston Methodist Research Institute, Bethesda user meeting October 29, 2015

Nuclear receptors (NRs) are important ligand-dependent transcriptional factors. The orphan NR tailless (TLX, NR2E1), a transcriptional repressor, is a major player in neurogenesis and emerging evidence indicates that it is an excellent target to treat Neural Stem Cell (NSC) derived brain tumors and glioblastomas. Until recently, no chemical probes to validate TLX as a therapeutic target were available, and it was not clear whether TLX was druggable because of its uncharacteristic auto-repressed conformation. To assess TLX ligand binding capacity, we designed a screening strategy using the Octet Red 384 instrument to investigate the direct binding of small molecules to purified human TLX protein. As a result, we reported the first identified TLX ligands that bind to TLX and modulate its transcriptional activity. Based on this study, we could conclude that TLX is druggable. We are now devising strategies to chemically modify initial TLX ligands to obtain novel compounds with desirable biological activities in glioblastoma cells. The Octet Red 384 system is used to assess binding of the derivatives and as will be shown in the presentation a key instrument of our screening platform for TLX.

Jakob Wallner, Research Scientist, University of Natural Resources and Life Sciences, speaker presentation from Cambridge, MA Sep 22, 2015 user meeting

Liposomes are well established spherical bilayer structures mimicking cellular membranes. Their use in binding assays is attractive for various applications in pharmaceutics and biopharmaceutics, respectively. They are useful tools in drug discovery and drug screening, process development as well as in preclinical and clinical analysis. Their main application is seen in measuring the interaction of molecules, either in solution or preferential with biosensor technologies. Due to the attractiveness of biosensor based methods, there is a growing need for more efficient more simple and reliable methods for the intended purpose. Based on Pall ForteBio’s BLI platform we are establishing different assays to study protein/liposome interactions in more detail. The selected examples address assay development for the liposome approach, the use of different sensor types and individual protein aspects. Individual assays emphasis the binding kinetics of liposomes with different protein classes such as enzymes, antibodies and hormones. Each of these assays is aimed to be applicable in basic as well as in applied research.

Yuhong Du, Ph.D., Associate Director, Emory Chemical Biology Discovery Center, Emory University, presented at Cambridge, MA Sep 22, 2015 user meeting

The Emory Chemical Biology Discovery Center (ECBDC) aims to enable investigators to discover novel chemical leads targeted to disease-related proteins and processes for research tools and therapeutic agents. Equipped with two lines of independently integrated robotic system for high throughput screening (HTS) and image-based high-content screening (HCS), we have carried out a wide range of biochemical HTS and phenotypic HCS campaigns, including a large number of protein-protein interaction screens. The newly acquired Octet RED384 label-free biosensor system further expands our assay and HTS capabilities to a new dimension. Case studies will be presented to illustrate the applications of Octet RED384 system for monitoring protein-small molecule interactions.

Nazneen N. Dewji, Ph.D., CEO/Associate Professor Cenna Biosciences Inc./UCSD, presented at Cambridge, MA Sep 22, 2015 user meeting

Β-Amyloid (AΒ) accumulation in the brain is widely accepted to be critical to the development of Alzheimer’s disease (AD). Here we demonstrate that two small, non-overlapping water-soluble peptides, P4 and P8, from the PS-1 NH2-terminal domain, can substantially and specifically inhibit the production of total AΒ as well as AΒ40 and 42 in vitro and in vivo in the brains of APP transgenic mice. Using Bio-Layer Interferometry and confocal microscopy we provide evidence that the peptides effective in reducing AΒ give a strong, specific and biologically relevant binding with the purified ectodomain of APP 695. Finally, we demonstrate that the reduction of AΒ by the peptides does not affect the catalytic activities of Β- or γ-secretase, or the level of APP. P4 and P8 are the first reported protein site-specific small peptides to reduce AΒ production in model systems of AD. These peptides and their derivatives offer new potential drug candidates for the treatment of AD.

Mathieu Cinier, Ph.D., Scientific Director, Affilogic, presented at the Lyon 6/4 user meeting

Affilogic is a biotech company developing a new class of affinity ligands, Nanofitins^®, which are able to selectively bind many targets and have proven excellent tools for:

• Targeting (immunolocalization, in vivo neutralization): Therapeutic applications
• Capture (affinity chromatography, protein removal): Bioprocessing applications
• Detection (immunoassays, Western Blot): Diagnostic applications

These antibody-mimetics demonstrate superior pharmaceutical properties: high affinity and controlled specificity, excellent druggability (very stable > 65°C and highly soluble), fast generation process and cost-effective manufacturing in standard bacterial strains.

Dubois Herve, Ph.D., Analytical Scientist, Sanofi, presented at June 4, 2015 Lyon user meeting

A part of the process to select clonal cell lines is based on the productivity of the recombinant protein of interest. Using the Octet QK^e system, we developed a specific and rapid assay to measure the protein concentration in culture supernatant, including regeneration of SA biosensors. The presentation will describe the strategy used to develop this quantification.

Marc Turini, Ph.D., Development Scientist II, Beckman Coulter/Immunotech, presented at June 4, 2015 Lyon user meeting

Beckman Coulter Diagnostics develops clinical diagnostic products that help advance and optimize the clinical laboratory. Beckman Coulter's instruments, systems and tests help streamline processes to enhance efficiency, reduce costs and speed the delivery of results. For nearly 80 years, Beckman Coulter has been a global leader devoted to providing solutions to laboratories of all sizes, offering a broad portfolio of immunoassays. In order to deliver the highest sensitive immunoassays on the market, selection of best monoclonal antibodies is a crucial step in the development process. Generally we are looking for antibodies with the highest Kon and the lowest Koff to be as close as possible to antigen-antibody binding equilibrium. In this context, the Octet platform allows to characterize bound antibody epitopes and to determine binding kinetic constants of the analyte to the antibody providing important informations to expedite assay development. Octet technology also help us to identify potential cross reactivity and to reduce the number of antibody pairs to investigate. This presentation will give two different examples of monoclonal antibody selection using Octet system, lessons learned of this tool and comparison with results obtained by using conventional ELISA method to screen antibodies for sandwich assay.

Nestor Santiago, Ph.D., Scientist, BioKit, presented at June 4, 2015 Lyon user meeting

Selection of best antigen-antibody pair is crucial for the development of immunoassays, especially in the case of IVD reagents, since specificity and sensitivity, key determinants of a successful diagnostic test, rely on the nature of the antibody-antigen interaction. The integration of simple binding analyses using Octet platform in early stages of the research has allowed us to identify and select for further studies the best candidates to develop the final immunoassay.

Here we present a particular case in where the system in which an antibody is produced determines its specificity towards a certain antigen.

Jesper Pass, Principal Scientist, Novo Nordisk A/S, presented on May 7, 2015 Copenhagen user meeting

An automated workflow for high throughput generation of monoclonal antibodies (mAbs) has been established in order to meet the increasing demand for antibodies for therapeutic use and as research reagents. With an increase in the number of antibodies generated, early characterization is essential to ensure selection of the mAbs with the desired properties. The Octet HTX system has been implemented in the screening for specific binders and characterization of selected mAbs with regards to affinity and epitope binning. The instrument is further used for selection and prioritization of high expressing clones in production cell-line development. Examples of the use of the Octet HTX system in the mAb generation process will be presented.

Magnus M. Berglund, Ph.D., Senior Scientist, Swedish Orphan Biovitrum (Sobi), presented at May 7, 2015 Copenhagen user meeting

Swedish Orphan Biovitrum (Sobi) is dedicated to develop biopharmaceuticals for rare diseases. It is highly desirable to have candidate drugs that are functional in relevant animal species enabling studies in various disease models and on-target toxicology studies. Such early cross-species activity analyses are often hampered by the lack of purified and/or recombinantly produced target molecules. In addition, kinetic data in a relevant matrix (serum) is also desirable early in drug development.

SOBI002, is a 108 amino acid fusion protein with an N-terminal complement component 5 (C5) targeting Affibody^® domain linked to an albumin binding (ABD) domain. Using the superior matrix-insensitivity of the BLI technology and SOBI002 immobilized via amine-coupled human serum albumin (HSA) on AR2G sensors allowed kinetic studies of binding to exogenous human C5 in buffer but also to endogenous C5 in serum from various species diluted serially in buffer. The output kinetic parameters of such an experiment are not traditional k_on-, k_off- and K_D-values but rather expressed in relation to a dilution factor that can be translated into molar if the endogenous target concentration is known. It is likely that this method is applicable to many serum proteins. Other approaches to use BLI for kinetic analyses will also be discussed.

Stefan Kol, Ph.D., Protein Biochemist, DTU NNF Center for Biosustainability, presented at May 7, 2015 Copenhagen user meeting

Erythropoietin (EPO) quantification during cell line selection and bioreactor cultivation has traditionally been performed with ELISA or HPLC. As these techniques suffer from several drawbacks, we developed a novel EPO quantification assay. A camelid single domain antibody fragment directed against human EPO was evaluated as a capturing antibody in a label-free Bio-Layer Interferometry-based quantification assay. Human recombinant EPO can be specifically detected in Chinese hamster ovary cell supernatants in a sensitive and pH-dependent manner. This method enables rapid and robust quantification of EPO in a high-throughput setting.

Alison Clargo, Research Scientist, UCB, presented at the April 2015, Pall ForteBio User Meeting in London

UCB's proprietary antibody discovery technology exploits the highly efficient natural mechanisms of the immune system to generate high quality antibodies to targets of therapeutic interest. Over one billion B cells can be screened per project in order to discover a candidate molecule. Therefore, the need to implement well-designed assays to focus in on those antibodies of interest is essential. The Antibody Discovery group uses the ForteBio Octet RED384 for many applications in our workflow but currently the key purpose is to test the cross-blocking of antibodies to determine epitope bins. During candidate selection, we often want to find antibodies which bind to the same epitope with a different binding profile than the original antibodies. On the other hand, we have also been examining a panel of antibodies which bind to different epitopes, through a 'sequential binding' experiment, in order to build up an immune complex around a target molecule.

Laura Hook, Senior Scientist, GlaxoSmithKline, presented at the April 2015, Pall ForteBio User Meeting in London  

At Biopharm Discovery, GlaxoSmithKline we generate and optimise monoclonal therapeutic antibodies in order to fill a fast moving, sustained and differentiated pipeline of medicines. The versatility of the Octet RED system allows the Protein Analytics team to support multiple programmes in parallel throughout the discovery procress. This talk will present a case study of one such programme and demonstrate the variety of techniques employed that use the Octet RED system.

Frances Neal, Scientist I, MedImmune, presented at the April 2015, Pall ForteBio User Meeting in London

ForteBio's Octet RED384 is a label free system that uses Bio-Layer Interferometry (BLI) to understand biomolecular interactions in real time. The Octet RED384 system can be applied to evaluate a number of key characteristics of protein interactions to aid biologics discovery. These include: antibody and antibody fragment quantification from crude and purified samples; competition assays for characterisation of receptor - ligand neutralising antibodies or distinguishing different epitopes; and kinetic screens for antibody affinity and antibody fragment off rate ranking. Here we present examples for these different biologics applications, demonstrating the versatility of this system, and its utility in the discovery of biologic therapeutics.

Jonathan Lowther, Ph.D., Senior Scientist II, Immunocore Limited, presented at the April 2015, Pall ForteBio User Meeting in London

Immunocore prepares therapeutics that can re-direct the immune response to recognise and kill diseased cells. ImmTACs (immune-mobilising mono-clonal TCRs against cancer) are a new class of bispecific reagent based on soluble monoclonal T cell receptors. ImmTACs have been engineered to possess extremely high affinity for cognate tumour antigen presented on the surface of diseased cells as a peptide-human leukocyte antigen (pHLA) complex. In this way, ImmTACs overcome the problem of low affinity tumour-specific T cells imposed by thymic selection and provide access to the large number of antigens presented as peptide–HLA complexes. By introducing mutations within one or more of the six complementarity determining regions (CDRs), phage display can be used to generate mutant TCRs with higher affinity for cognate antigen compared to the wild-type TCR. In this presentation we show how Octet RED96 can be used to select TCRs from the phage display mutants with highest affinity and specificity for the cognate antigen.

Alessio Bortoluzzi, Postdoctoral Research Assistant, University of Dundee, presented at the April 2015, Pall ForteBio User Meeting in London

In our laboratory we are broadly interested in understanding and exploiting the ligandability of proteins beyond classical orthosteric ligand-binding sites. These include for examples solvent-exposed protein surfaces and interfaces involved in protein-protein interactions (PPIs). Fragment-Based Lead Discovery (FBLD) is a popular approach that is particularly well suited for identifying and mapping binding sites on proteins and for rapidly developing high-affinity small molecules against these sites. One of the critical steps in the FBLD process is the identification of bona-fide low-affinity hits for the protein of interest. To this purpose we developed a screening cascade that implements Bio-Layer Interferometry (BLI). This cascade relies solely on identification of protein-ligand binding, does not require functional assays, and thus can be performed against virtually any protein of interest, making it of general applicability. In short: the primary screen is performed in parallel against the whole fragment library with two orthogonal techniques: BLI and Differential Scanning Fluorimetry (DSF). All the hits identified in this initial step are then validated by ligand-observed NMR experiments. The interaction between the NMR-validated hits and the target protein is then further characterized biophysically and structurally, typically by ITC, X-ray crystallography and/or protein-observed NMR.

Jonathan L. McMurry, Ph.D., Associate Professor, Kennesaw State University, presented at the April 2015 Pall ForteBio User Meeting in Atlanta, GA

Recent studies have identified direct submicromolar affinity interactions of MAP kinases (MAPK) p38 and ERK with endothelial nitric oxide synthase (eNOS) via a pentabasic sequence in the autoinhibitory insertion of eNOS that resembles a MAPK binding motif. The neuronal isoform, which lacks the pentabasic motif, did not bind MAPK. In the present study, eNOS binding by another representative of the families of conventional MAPK, a c-Jun N-terminal kinase (JNKα1), was examined using optical biosensing. Similar to p38 and ERK, high affinity binding was measured with a 31 nM KD as determined by fit to a one-state global model. Rate constants were determined. kon was 4125 M-1s-1 and koff was 1.3 x 10-4 s-1. Immunoblotting identified phosphorylation at S116, contrasting with ERK, which phosphorylated S602, and p38, which phosphorylated both. Phosphorylation by JNK inhibited eNOS as measured by cytochrome C reduction assays. These results underscore the importance the newly discovered noncanonical MAPK binding site on eNOS as a regulatory element bound by an array of MAPK that phosphorylate different sites. Further, they contribute to the emerging paradigm of eNOS as a junction of multiple signaling pathways.

Krystal Cadle, Clemson University, Department of Genetics & Biochemistry, presented at the April 2015 Pall ForteBio User Meeting in Atlanta, GA

Spider silks, composed of proteins called spidroins, are an amazing biodegradable and biocompatible material that has numerous potential uses in medical and industrial fields. The process by which these spidroins form fibers, through the homodimerization of the C and N-terminal domains, is quite interesting. For the N-terminal domain (NTD) stable homodimerization is correlated with a pH-dependent conformational change. This study focuses on understanding the role certain amino acids play in this pH-dependent dimerization of the NTD. NTD variants with specifically chosen amino acid changes were examined using Trp fluorescence to determine the effect on NTD conformational change.

Patricia Buckley, Ph.D., US Army Edgewood Chemical and Biological Center, presented at the April 2015 Pall ForteBio User Meeting in Atlanta, GA

Current sensor platforms for detection and diagnosis of specific biothreat agents primarily depend upon the use of antibodies, and ensuring that the reagents used in these platforms are of the highest quality is essential for the soldier out in the field. To date, the selection of antibodies for inclusion in a final assay format has primarily relied on an antibody's performance in an enzyme-linked immunosorbant assay (ELISA) with little regard for quantifying the full spectrum of variables affecting antibody-antigen interaction. This prompted our lab to investigate the binding characteristics of available antibodies; thus providing critical information for assay development to increase the ability to quantify both known and unknown biological threats on future platforms.

Michael Sadick, Ph.D., Senior Manager, Large Molecule Analytical Chemistry, Catalent Pharma Solutions, presented at the April 2015 Pall ForteBio User Meeting in South San Francisco, CA

Characterization of IgG monoclonal antibody (mAb) therapeutic molecules requires, among other orthogonal assessments, measurements of the interaction of the Fc region of the mAb with all of the potential human IgG-Fc-binding cell-surface receptors. This list includes the low affinity CD16a and CD16b receptors, responsible for the majority of antibody-dependent cellular cytotoxicity function associated with mAb therapeutics (chiefly IgG1). Also included are the low affinity receptors, CD32a and CD32b/c (FcγRIIA and FcγRIIB/C, respectively), as well as the high affinity receptor,CD64 (FcγRI), also responsible for mediating multiple immune responses. Finally, it is important to assess the interaction of the Fc region of IgG mAbs with the 'neonatal' FcRN, which is known to have great impact on the in vivo half-life of a therapeutic mAb. To this end, we have successfully developed and established a platform for characterization of IgG/Fc receptor binding, utilizing a panel of appropriate Fc receptors and easy-to-use Octet instrumentation. Using Ni-biosensor tips and his-tagged recombinant receptors as the immobilized moieties for this panel, the analyte of interest (the mAb) is in no way derivatized, allowing objective analysis of the receptor panel with any mAb. With minimal additional optimization, the Fc receptor panel, established here, can be quickly adapted for pretty much any originator or biosimilar mAb.

Cindy Benod, Ph.D., Research Associate, Houston Methodist Research Institute, presented at the April 2015 Pall ForteBio User Meeting in South San Francisco, CA

Nuclear receptors (NRs) are important ligand-dependent transcriptional factors. Presently, no natural or synthetic ligand has been identified for a large group of so- called "orphan" NRs. The orphan NR tailless (TLX, NR2E1), a transcriptional repressor, is a major player in neurogenesis and emerging evidence indicates that it is an excellent target to treat Neural Stem Cell (NSC) derived brain tumors and glioblastomas. Until recently, no chemical probes to validate TLX as a therapeutic target were available, and it was not clear whether TLX was druggable because of its uncharacteristic Ligand Binding Domain auto-repressed conformation. While our homology models suggest that TLX could harbor a ligand binding pocket, the consequences of its unusual organization for small molecule development were unknown. To assess TLX ligand binding capacity, we designed a screening strategy using the Octet RED384 instrument to investigate the direct binding of small molecules to purified human TLX protein. As a result, we reported the first identified TLX ligands that bind directly to TLX and modulate its transcriptional activity. Based on this study, we could conclude that TLX is druggable.

Paul Stephen Marinec, Ph.D., NIH NRSA Postdoctoral Fellow, UCSF, presented at the April 2015 Pall ForteBio User Meeting in South San Francisco, CA

RAS proteins are critical components of signal transduction pathways in cells, regulating a myriad of essential functions from proliferation and gene expression to apoptosis and cell cycle arrest. Given their central role, it is not surprising that RAS mutations are frequently detected in many solid tumors. Indeed, aberrant RAS signaling is implicated in over 30% of all human cancers. Despite these proteins and their oncogenic potential being discovered over three decades ago, there is still not a single therapeutic in the clinic or in human trials that directly targets RAS. As such, RAS proteins have been termed "undruggable", and these shortcomings illustrate a fundamental gap in our knowledge of RAS biology and signaling.

Christian Demmler, Head of Bioassays, ProBioGen AG, presented at the March 2015 Pall ForteBio User Meeting in Berlin, Germany

Fc-receptor binding assays are used to mimic more complex and often primary cell-based assays like ADCC and ADCP known to be prone to high variations. These types of binding studies are not only crucial during drug candidate selection but also in the course of biosimilar development programs and are mandatory in many regulatory regions (EMA, FDA). Several of these assays were established on the Octet Red platform and can give a deep insight into the kinetics of Fc-FcR interactions.

David Bunka, Ph.D., Chief Technical Officer, Aptamer Group Limited, presented at the March 2015 Pall ForteBio User Meeting in Berlin, Germany

The Pall ForteBio Octet & Blitz systems allow quality control as well as selection, maturation and characterisation of aptamer populations through hit picking, affinity ranking and functional characterisation. We will present data to demonstrate the selection and maturation of target specific aptamers as well as demonstrating the use of an aptamer as targeted affinity purification reagent with a tailored release profile.

Francesco Reddavide, Ph.D. Student, Technische Universität Dresden, Zentrum für Innovationskompetenz B CUBE, presented at the March 2015 Pall ForteBio User Meeting in Berlin, Germany

Dresden GE abstract DNA-encoded chemical library (DECL) technology has emerged as a new avenue in the field of drug discovery. Combined with high-throughput sequencing, DECL selection experiments can provide not only many lead compounds but also insights into the structure-affinity relationship. We describe a Bio-Layer Interferometry approach to realize an automated high-throughput assay for the kinetic characterization of the interaction between DNA-conjugated small organic compounds (ligands) and protein targets (analytes). Importantly, this method can be applied to both single-pharmacophore DECLs and self-assembled dual-pharmacophore DECLs (fragment based approach).

Jean-Philippe Julien, Scientist, The Hospital for Sick Children Research Institute, presented at Dec 11, 2014 Toronto User Meeting and Workshop

Vaccine development against HIV-1 is a global health priority. Cleaved envelope glycoprotein (Env) trimers mediate HIV-1 entry into CD4+ target cells and are the only virally encoded antigens on the surface of HIV-1. As such, Env trimers are a major focus for vaccine design as the only potential targets of broadly neutralizing antibodies (bNAbs).

Trevor F. Moraes, Associate Professor, University of Toronto, presented at the Dec 11, 2014 ForteBio User Meeting in Toronto, Canada

The Moraes lab focuses on the structural and functional characterization of protein and ion translocation machineries within the membranes of pathogenic bacteria. We use primarily X-ray crystallography in combination with other molecular approaches to gain a detailed understanding of how these membrane protein complexes function. Herein we will describe using Bio-Layer Interferometry to analyze several bacterial surface membrane proteins interactions with their host proteins. We will also describe a new protocol for using BLI to detect the delivery of proteins to the cell surface of E. coli.

Alan Wong, Graduate Student, James Rini Lab, University of Toronto, presented at the December 11, 2014 ForteBio User Meeting in Toronto, Canada

Characterizing protein interactions is fundamental to understand the biological function of a protein. We have used the Octet RED96 instrument to characterize a number of different systems including protein-protein, antibody-antigen, lectin-carbohydrate and protein-lipid interactions. Interactions were characterized on samples ranging from crude tissue culture media for rapid qualitative testing to highly purified proteins for quantitative kinetic and affinity measurements. In this presentation I will describe our approaches with emphasis on both data collection and analysis.

Jesper Pass, Senior Scientist, Novo Nordisk A/S, presented at the October 16, 2014 ForteBio User Meeting in Amsterdam, Netherlands

An automated workflow for high throughput generation of monoclonal antibodies (mAbs) has been established, in order to meet the increasing demand for antibodies for therapeutic use and as research reagents.

The Octet RED384 system has been implemented at several stages of the mAb generation process, including screening for specific binders and characterization of selected mAbs with regards to affinity and epitope binning. The instrument is further used for selection and prioritization of culture supernatants for purification. Examples of the use of the Octet RED384 system in the mAb generation process will be presented.

Magnus M. Berglund, Senior Scientist, Swedish Orphan Biovitrum (Sobi), presented at the October 16, 2014 ForteBio User Meeting in Amsterdam, Netherlands

Swedish Orphan Biovitrum (Sobi) is dedicated to develop biopharmaceuticals for rare diseases. It is highly desirable to have candidate drugs that are functional in relevant animal species enabling studies in various disease models and on-target toxicology studies. Such early cross-species activity analyses are often hampered by the lack of purified and/or recombinantly produced target molecules.

SOBI002, currently undergoing phase I clinical testing, is a 108 amino acid fusion protein with an N-terminal complement component 5 (C5) targeting Affibody^® domain linked to an albumin binding (ABD) domain enabling prolongation of plasma persistence by targeting serum albumin. Using the superior matrix-insensitivity of the Octet technology and SOBI002 immobilized via amine-coupled human serum albumin (HSA) on AR2G sensors allowed kinetic studies of binding to exogenous human C5 in buffer but also to endogenous C5 in serum from various species diluted serially in buffer.

Qian Wu, XBiotech, presented at IBC Well Characterized Biologicals Nov 3-4, 2014

Nicole Duriga and Jason DelCarpini, Pfizer, presented at the March 2014 ForteBio User Meeting in Cambridge, MA

Antibody-drug conjugates (ADCs) pose a unique challenge for ligand binding assays (LBA) due to the dynamic and heterogeneous nature of ADCs. ADCs containing various drug-to-antibody ratios (DAR) may have different binding affinities for capture and/or detection reagents potentially leading to differences in detection and quantitation of various DAR species. Octet was used to screen a panel of monoclonal antibodies (mAbs) for binding to ADCs. mAbs that showed optimal binding were further evaluated as capture and/or detection reagents in plate based LBAs for both the quantitation of conjugated Ab (DAR ≥ 1) and total Ab (DAR ≥ 0). Several mAbs were further characterized with Octet for independence to DAR and epitope binding. Using Octet allowed for rapid screening of capture and detection reagents which accelerated our plate based assay development time in addition to improving our overall understanding of the plate based assay at the biophysical level. Knowledge of how the reagents interact with the ADC is essential for developing a robust assay.

Mark J. Schofield, Senior R&D Engineer, Pall Life Sciences, presented at the March 2014 ForteBio User Meeting in Cambridge, MA

Rapid, accurate and cost-effective quantitation of monoclonal antibodies (MAbs) is essential for bioprocessing. Here we assess the relative merits of Protein A High Pressure Liquid Chromatography (HPLC) and Protein A bio-layer interferometry using the Pall ForteBio Octet RED96 system to determine MAb concentration in a complex feedstock. We perform this assessment within the context of determining a MAb breakthrough curve from a Protein A column loaded with a CHO culture feedstock. In summary, we find that HPLC and the Pall ForteBio Octet system can be used to generate comparable data, with the Octet system providing significant improvements in assay cost, throughput, and sample preparation time.

Cindy Jan Choy, Post Doctoral Researcher, Washington State University, presented at the March 2014 ForteBio User Meeting in Seattle, WA

The rapid dilution of the enzyme-inhibitor complex assay to monitor the recovery of enzyme activity is a well-established assay to determine the reversibility of inhibition (e.g., rapidly reversibly, slowly reversible, irreversible).  Our laboratory has previously employed this method to ascertain the reversibility of inhibition for glutamate carboxypeptidase II (GCP II) targeting agents.  We previously employed this assay to determine the reversibility of inhibition for two potent GCP II targeting agents (CTT54 and CTT54.2).  Due to the tedious and time-consuming nature of the rapid enzyme-inhibitor dilution assay; we sought to develop a facile method to determine the reversibility of inhibition using biolayer interferometry (BLI).  During our studies, we noticed some interesting binding events, which will be presented.

Erik Procko, Senior Research Fellow, University of Washington, presented at the March 2014 ForteBio User Meeting in Seattle, WA

Computer-aided protein design has made remarkable progress in recent years, including the design of new protein structures and designing functional sites into existing nonfunctional proteins as scaffolds.  Rarely have the two been accomplished together; that is, the design of an entirely new and novel protein with a function of interest.  Using new computational methods, we have now purpose-built a protein with no identifiable sequence homologues in nature that binds an apoptosis regulator from Epstein-Barr virus associated with lymphoma.  Using bio-layer interferometry, the designed protein was found to have remarkable affinity and specificity; it binds the intended viral target with picomolar affinity but several orders of magnitude weaker affinity for related human apoptosis regulators.  When coupled with antibody-based delivery systems, the designed protein slows the progression of Epstein-Barr virus-positive lymphoma in mouse models.

Jeff Dantzler, Associate Scientist, Novo Nordisk Research Center, presented at the March 2014 ForteBio User Meeting in Seattle, WA

Nuwan Dinuka Abeydeera, Scientist, AM Biotechnologies, LLC, presented at the March 2014 ForteBio User Meeting in Houston, TX

Phosphorodithioate-substituted (PS2) aptamers are stable, chemically-synthesized oligonucleotides that demonstrate high specificity and affinity to biomolecular targets such as proteins and peptides. PS2 aptamers result from the chemical substitution of both non-bridging oxygen atoms with sulfur at a limited number of phosphodiester linkages, maintaining an achiral linkage. Aptamers modified with PS2 linkages typically have enhanced resistance to nuclease degradation and can also demonstrate significantly enhanced binding affinity.  We describe the use of Bio-Layer Interferometry (BLI) to rapidly screen aptamers containing PS2 substitutions to identify those with enhanced binding affinity. Results show that a single PS2 substitution in the right sequence location can increase binding affinity by a factor of 5 to 10 times, whereas multiple substitutions can enhance binding affinity by up to 600 times whilst retaining excellent specificity.  BLI protocols enabled rapid, parallel optimization and screening of numerous PS2 aptamer variants saving a significant amount of time and effort over other screening technologies while delivering excellent data. Additionally, BLI has also enabled multiple epitope recognition on a single protein target using an aptamer pair.

Charles Wartchow, Investigator III, Novartis, presented at the April 2014 ForteBio User Meeting on South San Francisco, CA

Protein-protein interactions (PPIs) are important regulators of pathological processes in oncology, inflammation, and infectious disease. Identifying validated low-molecular weight (LMW) compounds for PPI targets is more challenging than for more tractable targets like kinases and proteases because PPI targets most often lack well-defined pockets. Although PPI targets are more challenging, they contain "hot spots" where ligands can bind. An emerging strategy for identifying starting points for discovery efforts is fragment-based screening, where relatively large chemical space is sampled by a relatively small number of fragments. An integrated biosensor-based screening platform including a liquid dispenser, a robotic arm, and the ForteBio Octet RED instrument is an efficient tool for screening a thousand compounds per day. Successful PPI screening campaigns are challenging, and counter screens are required to remove false-positives that occur as a result of poorly-behaved compounds and contaminating substances. Workflow to address these issues and to identify validated hits is described for oncology targets eIF4E and Ras.

Mark Fisher, Professor, University of Kansas Medical Center, presented at the March 2014 ForteBio User Meeting in La Jolla, CA

This work demonstrates the ability of immobilized GroEL to detect the presence of structurally altered and or aggregated species of pharmacologically relevant proteins. Partitioning of the molten globule state of fibroblast growth factor (FGF-1) onto GroEL tips was readily observed and was virtually abolished with the FGF-1 stabilizer heparin was present. ATP addition diminished FGF binding demonstrating that the binding was specific. Heat stressed polyclonal IgG  was evaluated by the GroEL-BLI biosensor method and size exclusion chromatography. The BLI method was able to bind initial monomeric and dimeric species even before multimeric species were present. The stability of a IgG1 monoclonal antibody (mAb) with negible dimer content was also able to bind to the biosensor under very mild heating conditions.  In this latter case, transmission electron microscopy (TEM) analysis of tip released GroEL-mAb complexes confirmed that the interaction of the stressed mAb with GroEL was located at the GroEL binding site.  Taken together, these results indicate that the GroEL-biosensor-BLI method was able to detect conformationally altered and/or aggregated states of proteins and may potentially be useful as a rapid, stability-indicating biosensor assay for monitoring the structural integrity and physical stability of protein therapeutics.

John F. Kenney, President, Antibody Solutions, presented at the March 2014 ForteBio User Group in La Jolla, CA

Employing ForteBio's BLI technology platform, synthetic D-protein antagonists for VEGF-A were developed and optimized. The D-protein antagonists can bind VEGF-A with nanomolar affinity and block the interaction of VEGF-A with VEGFR2 receptor. The D-protein antagonists are more stable and less immunogenic compared to their L-protein counterparts. The antagonists can inhibit VEGF-A-induced VEGFR2 phosphorylation in human retinal endothelial cells.  Synthetic D-protein antagonists of VEGF-A show promise as new chemical entities with unique therapeutic properties.

James DelProposto, Research Associate, University of Michigan, presented at the March 2014 ForteBio User Meeting in La Jolla, CA

Computational Structure-Based Drug Design(SBDD) techniques, particularly docking and scoring are being widely used to assist in lead discovery and optimization. The effectiveness of SBDD is reduced by limited accuracy in both prediction of binding modes of ligands to their targets and binding affinity. The Community Structure Activity Resource (CSAR) Center is developing a large data set containing experimentally determined protein-ligand complex structures and accurate binding affinity data. For our first targets, we experimentally determined the dissociation constants (Kd) of CDK2, LpxC, and Hsp90 ligand complexes using the complementary techniques of the Octet RED, Thermofluor and Isothermal Calorimetry.

Kannan Alpadi, Research Associate, Baylor College of Medicine, presented at the March 2014 ForteBio User Meeting in Houston, TX

Modular transient protein complexes are crucial for a variety of biological processes such as membrane protein trafficking, autophagy and apoptosis. Most of these transient protein interactions are valuable drug targets. The assay format used to characterize transient protein complexes are limited by low expression, nonfunctional protein expression because of tagging, and smaller or bigger size of the protein. To better understand transient protein-protein interaction between the fission protein, dynamin and  fusion protein, syntaxin,  we used Octet biolayer interferometry technology. These two proteins control the spatiotemporally regulated antagonistic processes of membrane fusion and fission. By using BLI, we were able to demonstrate the binding specificity of dynamin and syntaxin, and also identify the domains responsible for binding.

Liu Liu, Assitant Research Scientist, University of Michigan, Comprehensive Cancer Center, presented at the Spring 2014 ForteBio User Meetings in Houston, TX and South San Francisco, CA

Chromatin regulators, such as many BRD family proteins have quickly become attractive targets for novel anti-cancer therapies. However, few tool compounds exist for detailed biological studies and target validation except the widely studied BET family proteins. Here, we designed and synthesized a small probe compound library against different BRD family proteins with compound molecular weight starting as low as 150 Da. Using a label-free assay, these compounds were quickly screened against a variety of BRD proteins. Hits were identified and further confirmed to have affinities ranging from tens to hundreds of micromolar. These small molecules serve as starting points of further medicinal chemistry modifications in order to obtain good tool molecules for various BRD proteins. Additionally, we designed and developed a new class of small-molecule inhibitors targeting BET family BRD proteins. These inhibitors are significantly more potent in various in vitro assays than both IBET-762, the compound developed by GSK and currently in early stage of clinical trials and JQ-1, the most widely used tool compound for BET family BRD proteins.

Dr. Liu Liu, Scientist, University of Michigan Presented at the Protein Society Meeting, Boston, July 2013    Dr. Liu Liu, scientist in the Department of Internal Medicine at the University of Michigan described Octet assays for the screening of hits and lead characterization of small molecule compounds in the drug discovery of bromodomain proteins. Chromatin regulators, such as many BRD family proteins have quickly become attractive targets for novel anti-cancer therapies. However, few tool compounds exist for detailed biological studies and target validation except the widely studied BET family proteins. Dr. Liu and his colleagues designed and synthesized a small probe compound library against different BRD family proteins with compound molecular weight starting as low as 150 Da. Using a label-free assay, these compounds were quickly screened against a variety of BRD proteins. Hits were identified and further confirmed to have affinities ranging from tens to hundreds of micromolar.

Sidharth Seth, Kyowa Hakko Kirin
Presented at the June 2013 Fortebio User Meeting in Princeton, NJ
ELISA is the typical method to determine the isotype subclass of monoclonal antibodies. However, an ELISA can take an entire workday to generate results. By contrast, the Octet® QKe is able to produce comparable results in a fraction of the time. To accomplish this, biotinylated antibodies specific to a human IgG isotype subclass (hIgG1, 2, 3, or 4) were captured using Streptavidin biosensors. The biosensor tips were then used to detect and determine the isotype subclass of monoclonal human IgG in cell media supernatant.

Dinuka Abeydeera, AM Biotechnologies LLC
Presented June 2013 at the Pall Fortebio User Meeting in Cambridge, MA
Phosphorodithioate-substituted (PS2) aptamers are stable, chemically-synthesized oligonucleotides that demonstrate high specificity and affinity to biomolecular targets such as proteins and peptides. PS2 aptamers result from the chemical substitution of both non-bridging oxygen atoms with sulfur at a limited number of phosphodiester linkages, maintaining an achiral linkage. Aptamers modified with PS2 linkages typically have enhanced resistance to nuclease degradation and can also demonstrate significantly enhanced binding affinity. We describe the use BLI to rapidly screen aptamers containing PS2 substitutions to identify those with enhanced binding affinity. Results show that a single PS2 substitution in the right sequence location can increase binding affinity by a factor of 5 to 10 times, whereas multiple substitutions can enhance binding affinity by up to 600 times whilst retaining excellent specificity. BLI protocols enabled rapid, parallel optimization and screening of numerous PS2 aptamer variants saving a significant amount of time and effort over other screening technologies while delivering excellent data.

Vishal Kamat, Regeneron Pharmaceuticals
Presented at the May 2013 ForteBio User Meeting, San Diego, CA
During conventional primary screening, monoclonal antibodies are characterized and grouped into different bins based on their binding affinity, specificity, species cross-reactivity, and cell based functional assays. However, when multiple antibodies demonstrate similar binding behavior with comparable binding affinities, selecting lead antibodies could be challenging. Identifying antibodies binding to distinct, non-overlapping epitopes would be critical in selecting lead therapeutic monoclonal antibodies with broader diversity. Epitope binning provides insight on the relative spatial distribution of the binding epitope on the surface of antigen and also reveals epitope diversity of different monoclonal antibodies. Our efforts to develop high throughput epitope binning assay using different label-free platforms and challenges faced during the development of this assay will be presented.

Greg Cantin, Pfenex Inc. Presented at the May 2013 ForteBio User Meeting, San Diego, CA Discarding the traditional, linear and iterative approach, Pfenex Expression Technology™, a Pseudomonas fluorescens-based expression platform, adopts a parallel, high throughput method for microbial strain development. Thousands of unique host strains and plasmid combinations are seamlessly integrated to enable rapid strain engineering for optimal protein production. For a typical screen, the assessment of both the expression level and the quality of the target protein is needed for each unique expression strain. Here, we present case studies outlining how BLI facilitates this high-throughput process.

Jason Goldstein, Immunochemistry and Cellular Development Team, Centers for Disease Control and Prevention Presented at the May 2013 ForteBio User Meeting, San Diego, CA Label free technology has provided insight into toxin studies and antibody development for improved diagnostics in infectious disease. We have established a reliable toxin assay for the discovery of novel mAbs used in the detection of Anthrax infection. Binding and kinetic data generated from assembly of Lethal Toxin (Ltx,; a potent virulence factor in disease progression) was elucidated with the BLI platform. The ForteBio Octet allowed for rapid ranking of hybridoma clones, determination of relative affinities during the cloning process and as a reliable method to monitor antibody production during scale-up. The capture of stable Ltx on the biosensor provided us the opportunity to screen for orientations of multiple mAbs in pursuit of a highly sensitive rapid immunodiagnostic.

Keith Luhrs, Allergan, Inc.
Presented at the May 2013 ForteBio User Meeting, San Diego, CA
In early-stage biologics programs it is often necessary to identify tool reagents for use in proof-of-concept animal studies, sometimes even for multiple species. For species outside of the most commonly used small rodents, sourcing target materials can be a significant challenge. By combining off-the-shelf cell-free translation systems, co-translational site-specific biotinylation, and the Octet BioLayer Interferometry platform, we have developed a technique to rapidly screen potential tool biologics, drug candidates, and variants for binding to recombinant target protein from multiple species. In many cases functional activity can be assessed by competitive binding to the target cognate ligand or receptor. Analyses can be performed using the crude expression reaction following buffer exchange without further purification or quantitation of the expressed target material. Additionally, this technique can be broadly applied to analyses of other protein variant libraries, allowing rapid qualitative screening without purification.

Paul Rogers, Imperial College
Presented at the April 2013 ForteBio User Meeting, London, UK
Investigations into the design of an effective vaccine regimen to stimulate protective immunity to HIV have been ongoing for the last 30 years.

In order for a vaccine to successfully prevent infection following exposure to HIV it will likely need to generate effective antigen-specific antibodies in both plasma and the mucosa. These antibodies should be directed against conserved viral antigens, have high affinity and will preferably be neutralising, that is preventing viral infection of target cells.

During vaccine experiments, responses including the titre, affinity and class of antibodies produced in experimental models are evaluated over time to evaluate the effectiveness of the regimen. As a grant-funded academic group, involved in preclinical and clinical trials, a compact, robust, adaptable and cost-effective system to investigate the interaction of vaccine-elicited antibodies with antigens and with cell-surface receptors is highly desirable. Here we present on the use of the ForteBio's BLItz system and how it has been employed during these investigations.

Our experiences of using different biosensor surface chemistries in two separate projects are presented; namely the analysis of whole serum binding to immobilised antigen following DNA vaccination and the association of antibody-bound HIV virions with immobilised cell-surface receptors.

Svend Kjaer, Ph.D., London Research Institute, Cancer UK
Presented at the April 2013 ForteBio User Meeting, London, UK
The regulation of the Rnd family (1-3) of small GTPases has remained a puzzle for cell biologist, since it is not regulated by the classical GTP/GDP switch. Here we present, how the adaptor protein 14-3-3 interacts with Rnd3 using a previously undescribed mode of interaction, where it recognizes a phosphoserine residue and C-terminal farnesylation moiety. By use of biotinylated peptides with different modifications, we evaluated the impact of different lipid length modification on the kinetic profiles as well as overall affinity of the interaction.

Phil Buckle, ForteBio Pall Europe
Presented at the April 2013 ForteBio User Meeting, London, UK
The Octet and Blitz platforms offer a significant advantage over flow based systems in that they able to measure binding from complex, crude media without the possibility of blockages and carry-over. In this presentation I will describe how, using off-the-shelf Biosensors, kinetic constants and off-rate ranking data can be obtained directly without prior sample clean-up.

Yasmina Abdiche, Research Fellow, Rinat-Pfizer, Inc.
Presented at the April 2013 ForteBio User Meeting, South San Francisco
The Octet is a versatile platform for characterizing protein/protein interactions and is suitable for both a multi-project and multi-user research environment. Here, we highlight how the Octet can be used in the discovery of therapeutic antibodies. A breadth of applications is presented, including the kinetic screening of crude antibodies, lot-to-lot kinetic comparisons of purified antibodies, blocking of ligand/receptor interactions, epitope binning, and epitope mapping.

Ryan Case, Senior Scientist, Therapeutic Discovery; Biologics, Amgen, Inc.
Presented at the April 2013 ForteBio User Meeting, South San Francisco
Accurate kinetic profiles of protein-protein interactions are desired and it is important that disruptive processes such as nonspecific binding and baseline drift are minimized. Optimization of both target tip loading and buffer blocking components can reduce observed nonspecific binding, stabilize baselines and lead to accurate kinetic profiles. A case study is presented in which this assay development was used for accurate kinetic and affinity characterization in a screen against multiple targets.

Andy Stephen, Acting Director, Protein Chemistry Laboratory, SAIC-Frederick, Inc.
Presented at the April 2013 ForteBio User Meeting, South San Francisco
The Optim 1000 (Pall Corp.) measures the temperature dependent changes in the emission spectra of as little as 2mg of a target protein. From this information the onset of aggregation and melting temperatures of the protein can be determined. This is essential information in protein formulations and also in development of protein biophysical measurements. The Optim 1000 has been used to screen suitable buffer conditions to improve the solubility and stability of our proteins of interest. In addition ligand-induced changes in the melting temperature of a protein have been measured to gain insight into the protein-ligand interaction. This approach has been used to characterize the interactions between an annealing protein and complementary oligonucleotides and transcription factor binding to a phosphorylated peptide.

Sydney Zaremba, Sr. Associate Scientist, Analytical Science, Boehringer Ingelheim. Presented at Antibody Development 2012. This presentation describes the successful development and validation of two Octet assays for a Fab molecule; a titer assay and an activity assay for in-process and lot release samples.

Philip Buckle, Applications Manager Europe, Pall ForteBio, 2012 Molecular interactions play a myriad of important roles in drug discovery and development. In this webinar, we discuss kinetics analysis on the Octet® and BLItz® Systems. We answer fundamental questions, including: What processes apply to each part of the binding curve? What do the rate constants actually mean? How do rate constants affect the shapes of curves? How can non-ideal behavior affect the results?

Brian Bullard,Ph.D., R&D Scientist, KPL, 2012

Mark Fisher, Ph.D., University of Kansas Medical Center, 2012 Monitoring protein folding events is central to the majority of protein based experiment. A high-throughput assay to detect protein folding has been developed using chaperone proteins and ForteBio's Octet instrument, which operates in standard microtiter plates in a Dip and Read format. Examples of the approach are discussed, including detection of small molecules that correct folding for a cystic fibrosis protein and detection of a conformational change of a component of anthrax toxin.

Lioudmila Campbell, Ph.D., Pfizer, Inc., 2012 CovX-Bodies are bioconjugates that combine the therapeutic potential of peptides with beneficial clinical properties of antibodies. A CovX-Body consists of a humanized monoclonal antibody scaffold and a therapeutic pharmacophore that is covalently, site-specifically conjugated to the antibody via a proprietary linker. Novel therapeutic peptides are identified through phage display and high-throughput screening and further optimized using chemical and genetic mutagenesis strategies. The Octet RED96 system is used for screening peptides from phage display, characterization of peptide binding kinetics, and epitope mapping studies.

Judy Shih, Ph.D., Pharmacokinetics and Drug Metabolism, Amgen, Octet System User Workshop - NBC, 2012 Quality assay reagent is the key for ligand binding assays, particularly for assay specificity and sensitivity. Assay reagent screening, characterization and selection process has been well established. ELISA has been widely and routinely used. Complementary tools include Biocore and cell based assays. This presentation demonstrates that the Octet platform is another complementary tool with high throughput and increased efficiency. Two case studies address assay reagent screening, paring, and bridging assessment. The third case study describes how the Octet platform facilitated assay troubleshooting. Furthermore, the platform has been implemented for reagent biotinylation assessment. In summary, the Octet platform is an efficient tool for ligand binding assays.

Subhashchandra Naik et al., University of Kansas Medical Center, 2012 The authors have developed a high-throughput platform using the GroEL chaperonin in conjunction with label free protein interaction techniques such as SPR and Bio-layer interferometry (BLI) to detect general protein unfolding and stabilization. They demonstrate that the nucleotide free form of Cystic Fibrosis Nucleotide Binding Domain 1 (CFTR NBD1) undergoes partial unfolding reactions that can be detected with GroEL when the chaperonin is either immobilized or free in solution.

Brian Bullard, Ph.D. et al., KPL Inc., 2012 Detection of foodborne pathogens is the primary means of preventing contaminated foodstuffs from entering the market. While traditional 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 bacteria strains while avoiding time consuming enrichment steps.

Jiamin Yu, Senior Scientist, Diadexus, at the 2nd Annual Workshop on Label-Free Assays for Bioprocessing Ligand Binding and Antibody Characterization, Boston MA, August 2011 Identification of constructs suitable for the recombinant protein production pipeline is a bottleneck for structural genomics efforts. A novel high-throughput approach, Binding Rate Screen, that can alleviate this bottleneck by screening crude lysates has been developed using the hexahistidine (His(6)) tag as a reporter. The constructs with the highest binding rates also exhibit high expression of soluble monomeric protein. Constructs expressing variations of the target protein can be prioritized on a time scale of minutes, providing a times savings of 10-100 fold.

Amy Bass, Research Associate, Takeda Pharmaceuticals, at the 2nd Annual Workshop on Label-Free Assays for Bioprocessing Ligand Binding and Antibody Characterization, Boston MA, August 2011 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 platform 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.

Oren Beske, Vice President, Aragen Biosciences at the 2nd Annual Workshop on Label-Free Assays for Bioprocessing Ligand Binding and Antibody Characterization, Boston MA, August 2011 This case study presents an example of a protein therapeutic expressed in mammalian cells that lost significant bioactivity when adapted to an industrial CHO production platform. Further investigation using various bioassays revealed that the reduction in specific activity was due to a fraction of the protein being expressed by the cells in an inactive form. Alternative purification processes were developed to identify and remove the inactive protein species in the preparation. Using the ForteBio Octet platform, an in vitro binding assay was rapidly developed and a kinetic analysis of the interaction revealed that the inactive form of the product possessed a faster dissociation constant from the target, resulting in a reduced affinity constant. With the source of the inactivity understood, a cell culture process was developed to increase specific activity of the expressed protein and identify clones producing product with high specific activity. Both the bioactivity assay and the ForteBio kinetic assay were used to track product quality during process development.

Mark Dysinger, Senior Scientist, Pfizer Global R&D, at the ForteBio Second Annual Ligand Binding Assay workshop at the AAPS National Biotechnology Conference, May 2011 Here we present a strategy and results for the development and qualification of an Octet platform assay for the quantitation of a humanized antibody therapeutic with reagents previously used in a validated ELISA. Octet system qualification and ELISA validation results are compared, and samples analyzed by the ELISA in support of a toxicokinetic (TK) study are analyzed via an Octet instrument. From these results we draw conclusions about the practical quantitative use of BLI in both non-GLP and GLP environments.

Dan Schuessler, Analytical Scientist, GlaxoSmithKline, at the ForteBio Second Annual Ligand Binding Assay workshopat the AAPS National Biotechnology Conference, May 2011 The quantitation of CHO Host Cell Proteins in monoclonal antibody therapeutics is commonly performed using a laborious sandwich ELISA assay taking approximately 5.5 hours to test one 96-well assay plate. With minimal method optimization, a CHO HCP quantitation assay was developed using ForteBio Streptavidin biosensors and a Metal DAB precipitating substrate that can be completed in approximately 2 hours. Subsequently, this method was fully automated using a Hamilton liquid handling robot integrated with the Octet QK384 instrument allowing a scientist to run up to three 96 well assay plates in a single run without any user involvement after the initial setup.

Ryan Case, Ph.D., Amgen, 2011 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.

Henry R. Maun, Ph.D., Genentech, Inc., 2011 Dysregulation of Sonic hedgehog (Shh) signaling has been implicated in cancer, where inhibition shows therapeutic benefit. The Shh-neutralizing antibody 5E1 and receptor Hedgehog-interacting protein (Hhip) are potent inhibitors of this pathway. We solved crystal structures of 5E1 Fab and Hhip, each complexed with Shh, which revealed a common binding site. These interactions depend on Zn2+ and Ca2+ being bound to Shh and mutations in these sites are linked to the genetic disease BDA1. Octet RED platform kinetic values matched closely with data produced in Isothermal Titration Calorimetry (ITC) experiments.

Daniel Eustace, Pfizer Inc., at IBC Analytical Technologies for Biotherapeutic Development, March 17, 2011 The concentration of recombinant human Factor IX (rFIX) in bioreactor harvest samples manufactured at Pfizer is monitored using an ELISA. 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 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.

Yasmina Noubia Abdiche, Ph.D., Rinat-Pfizer, South San Francisco, CA, Sep 21 2010 Dr. Yasmina Abdiche of Rinat/Pfizer presented a seminar at GTCBio conference in September 2010 on the efficient use of an Octet 384 system for epitope binning monoclonal antibodies. She compared the Octet platform to SPR technology to highlight the advantages of the Octet instrument for this application.

A tutorial on the use of Octet systems for protein titer determination in bioprocessing applications. The role of Octet QK systems in the protein expression workflow at Pfenex is described in this June 15, 2010 issue of GEN magazine.

Jian Li, Centocor R&D, presented at AAPS NBC, 2010 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 the Octet QK system and mock ADA samples we demonstrated that this method offered advantages over ELISA and ECLIA methods. We then investigated the capability of the Octet RED platform 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 biolayer interferometry platform offers much promise for the development of drug tolerant ADA immunoassays. Experimental results of our study will be presented.

The Pseudomonas-based Pfenex Expression Technology^TM has proven to be a robust and cost effective platform for the production of numerous classes of therapeutic proteins, including various types of antibody derivatives. High cell densities in the fermentor along with high specific protein yields result in high target protein titers. Very high cell densities are also achievable even in small scale growth (96-well plates). An extensive tool box of unique host strains has also been developed. These strains have phenotypes selected to impact the amount of target protein produced and its solubility and activity. Coupled to an automated sample preparation platform, parallel through-put biolayer interferometry (BLI) allows hundreds of different expression strategies and host cell combinations to be rapidly tested for both quantity and functionality.

Arnout Gerritsen, 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.

John Wang, Ph.D., at SBS and CHI's DDC, 2010 Dr. Wang presented on the use of the Octet RED384 system for higher throughput screening of small molecule libraries and fragment libraries. Validation data from Roche is included along with results from an internal Maybridge library screen.

Yasmina Noubia Abdiche, Ph.D., Rinat-Pfizer, South San Francisco, CA, 2010 Dr. Yasmina Abdiche 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 platform quickly discriminated between tight, medium and weak binders and picked up some weak binders missed by ELISA. For epitope binning, the Octet QK384 system 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.

Shirley Li, et al., Hoffman LaRoche, poster at SBS in Lille, France, 2009 Dr Shirley Li describing the first use of the ForteBio Octet RED platform and its label-free biolayer interferometry technology for successfully completing a fragment library screening campaign for a protein target. The unique features of the system allowed for rapid screening against immobilized target, minimal protein degradation, and the sensitivity to detect the binding of small molecules in the 200 MW range.

Yasmina Noubia Abdiche, Ph.D., Rinat-Pfizer, South San Francisco, CA, 2009 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 the Octet instruments matched those from SPR and that solution affinities from the Octet instruments also matched those from SPR.

Brian Miller, Senior Scientist, Antibody Engineering, Biogen Idec, presented at PEGS, Boston MA, April 2009 Dr. Brian Miller from Biogen Idec presented a seminar on the use of the Octet system at Biogen for protein quantitation, off-rate screening of antibodies in crude samples, monoclonal antibody cross-blocking, epitope scanning and affinity ranking of purified antibodies.

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

Frank Podlaski, Roche, Nutley, NJ, at the SBS conference, Dresden, Germany, June 2008 Dr. Podlaski talks about the extensive testing his laboratory did to evaluate the use of the Octet RED instrument for small molecule kinetic analysis. He finds that the Octet RED platform affords many specific advantages including ease-of-use, fast assay development, ability to identify aggregating and reactive compounds, and the ability to assay complex or insoluble analytes.

Keith A. Davis, Ph.D., Scientist, Global Biologics, Pfizer Inc, at IBC Antibody Production, San Diego, CA, 2008 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.

Sarah Koob, Wyeth, at BioProcess International in Boston, MA, 2007 The Octet QK instrument was successfully used for quick turnaround Protein A-based titer analysis of diluted, high concentration, in-process cell culture samples. The Octet QK system performed successfully for primary titer screening of 720 cell lines with same day turnaround of result. Strong linear correlation between Octet platform assay results, and HPLC and BioVeris results. The Octet QK system offers considerable time savings over both Pro A HPLC and BioVeris for quantitative titer analyses.

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

Poster presentation by Raven Biotechnologies @ WilBio Waterside conference in Chicago, IL - 2006

The Pioneer and Pioneer FE Next Generation SPR systems by ForteBio employ an innovative gradient injection technology based on Taylor dispersion called OneStep. A OneStep injection produces a sigmoidal concentration gradient of analyte from a single sample vial by dispersing the sample through a buffer-filled capillary line that flows into the SPR flow cell. From this single injection, kinetic analysis can be performed to accurately determine the kinetic rate constants and affinity (K_D) of the interaction between analyte in solution and immobilized ligand. In contrast, traditional SPR methods require a dilution series of analyte be prepared that spans two orders of magnitude from top to bottom.

The Ready BLI Detection kit for Residual Protein A (PN 18-5128) is intended for the detection and quantitation of recombinant Protein A or other Protein A constructs such as MabSelect SuRe™. It has been developed as a simpler, faster alternative to ELISA method with reduced hands-on time for customers who require a sensitive and robust assay for measuring small amounts of leached Protein A in antibody or Fc-fusion protein samples. This kit is for research and manufacturing use only and is not intended for diagnostic use in humans or animals. 

Anti-Human Fab-CH1 2nd Generation (FAB2G) biosensors from Pall ForteBio come pre-immobilized with a high affinity ligand that is specific for the CH1 region of Human IgG. In conjunction with the Octet^® and BLItz^® systems, FAB2G biosensors provide a rapid and label-free solution for analysis of Fab fragments or full-length human IgG for lead identification and optimization, cell line screening, process development, and QC applications. The high specificity of the anti-human CH1 ligand enables direct analysis of IgG or Fab analytes directly from complex samples such as cell culture supernatants, partially purified samples, and cell lysates, offering a time-saving alternative to traditional analytical methods.

The DYKDDDDK polypeptide tag (the  FLAG® tag) is fused to recombinant proteins to facilitate their detection and purification. Pall ForteBio’s High Precision Streptavidin (SAX) biosensors are designed for customization by direct immobilization of biotin-labeled proteins for both kinetic characterization and quantitation measurements on Octet® or BLItz® systems. By immobilizing a biotinylated anti-FLAG antibody onto SAX biosensors, FLAG-tagged proteins can be easily captured from crude or purified samples for detection, quantitation, or affinity measurement to an interacting protein.

HIS1K biosensors are disposable fiber optic tips for use with Pall ForteBio’s Octet and BLItz instruments. The biosensor tip surface is factory-coated with the monoclonal Penta-His antibody from Qiagen, enabling specific, high affinity capture and detection of His-tagged proteins. When the biosensor is dipped into a sample, the binding of protein molecules to the tip alters the interference pattern of light reflected from the biosensor surface to a detector, allowing molecular association and dissociation events to be monitored in real time with Octet instruments. Since the rate of binding is proportional to concentration, His-tagged protein samples can be readily quantified against a standard curve. Kinetic analysis can also be performed with HIS1K biosensors to determine affinity of a His-tagged protein to interacting biomolecular analytes. The surface enables capture and analysis directly from complex mixtures and can be used as an alternative to chemical protocols such as EDC/NHS and biotinylation.

Host cell proteins (HCPs) are contaminants found in biopharmaceuticals expressed in bacterial, yeast or mammalian production cell lines. Among protein expression cell lines, Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for industrial production of recombinant protein therapeutics. However, manufacturing and production processes of biopharmaceuticals often leave behind contaminating HCPs from CHO cells. Such residual HCPs carry substantial risk of decreasing efficacy of the drug and causing adverse immunogenic reactions in patients. Hence, the detection of residual host cell protein contaminants and methods that reduce them to the lowest acceptable levels have become critical aspects of drug safety and qualification.

Streptavidin-coated surfaces are widely used as a simple and straightforward method of molecular immobilization. Utilized with Bio-Layer Interferometry (BLI), Streptavidin biosensors enable quick and easy modification and customization of the biosensor with any biotintagged molecule for quantitative and kinetic measurements. The new High Precision Streptavidin Biosensor (SAX) was specifically developed and qualified for applications in downstream drug discovery and regulated environments that have more stringent assay precision requirements — and is QC-tested at Pall ForteBio to meet our precision-controlled coefficient of variation (CV) specification of <4%.

The ability to provide quick checks at-line for titer provides valuable insight into process variability and opportunities for increased control over them.  This technical note demonstrates the utility of the BLItz system in evaluating monoclonal antibody recovery post-filtration

The polyhistidine-tag (HIS-tag) is a common peptide tag fused to recombinant proteins during cloning. Many tools have been developed that enable this tag to be used for detection and purification of tagged proteins. The Anti-HIS (HIS2) Biosensor provides a rapid, label-free method for quantitation of HIS-tagged proteins on Octet® and BLItz® systems. This biosensor comes pre-immobilized with the next-generation high-affinity, high-specificity anti-HIS antibody from MBS (Maine Biotechnology Services), and is ready to use for detection and quantitation of HIS-tagged proteins.

QIAGEN's Tris-NTA is charged with nickel (Ni²⁺) and pre-immobilized onto the biosensor and will bind specifically to a HIS-tag attached to recombinant proteins. This binding is monitored in real time using an Octet^© or BLItz^© system and can be compared to binding a known calibrator to determine concentration. With appropriate dilution, it is possible to measure analytes in complex matrix conditions. The concentration is calculated based on the binding kinetics of the HIS-tagged protein, therefore best results are achieved when the protein used for calibration and the unknown samples are identical. The use of Ni-NTA also requires that the samples and buffers be free from EDTA, imidazole, or other similar chelating agents.

QIAGEN's Tris-NTA is charged with nickel (Ni²⁺) and pre-immobilized onto the biosensor and will bind specifically to a HIS-tag attached to recombinant proteins. The Ni2+ captures HIS-tagged analytes for both quantitation and kinetic applications. The binding of the target analyte to the immobilized HIS fusion proteins alters the interference pattern of light reflected from the biosensor surface, allowing molecular association and dissociation events to be monitored in real time using the Octet^® or BLItz^® instrument platforms. The surface is well suited for capture and analysis directly from complex mixtures as an alternative to chemical protocols such as EDC/NHS and biotinylation.

Glutathione-S-transferase (GST) is commonly fused to recombinant proteins as a means of facilitating detection, purification and increasing 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 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.

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.

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

The Amine Reactive 2nd Generation (AR2G) Reagent Kit is intended for use with the AR2G biosensor to enable covalent immobilization of a protein, peptide or other primary amine containing biomolecule onto the AR2G biosensor surface via a stable amide bond. Immobilization of proteins is achieved through standard EDC-catalyzed amide bond formation to create a covalent bond between a reactive amine on the protein and the carboxy-terminated biosensor surface. The kit contains sufficient reagents to perform 1000 standard immobilization reactions.

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.

Anti-Mouse IgG Fc Capture (AMC) biosensors enable kinetic
characterization of macromolecular interactions between mouse Fc-containing proteins and target analytes (Figure 1). 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 AMC biosensors extremely useful in high-throughput applications.

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.

Details use of Protein L biosensors for quantitation of antibodies and antibody fragments.

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.

This technical note describes a rapid assay method for quantitation and subtype identification of human and mouse IgG. The antibodies can be quantified in buffer, serum-free media or lysates, using the ForteBio Anti-Human IgG Fc or Anti-Murine Fv biosensors. Using the same biosensors, the subtype of the antibody can be identified using subtype-specific secondary antibodies. The flexible microplate-based format of the Octet platform allows up to 48 samples to be analyzed on an Octet QK or Octet RED system and up to 96 samples on an Octet QK384 or Octet RED384 system in 1 hour.

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 Pall ForteBio’s Octet 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. These protocols 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.

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.

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

This technical note outlines a general batch mode procedure for immobilizing a biotinylated ligand onto Streptavidin biosensors in the biosensor tray assembly on the benchtop. The goal is to develop a procedure that creates a binding surface having a maximum and reproducible response. The protocol outlines determining the optimal concentration and time for immobilization on-line and then the transfer of these parameters to off-line immobilization.

Traditional methods for analyzing the kinetics of biomolecular interactions such as ELISA are often cumbersome and may require rigorous sample preparation. In addition, ELISA may not provide the information required for the confident understanding of a drug molecule’s MOA. In contrast the 96-well sample plate based Octet® family of instruments (Figure 1) such as the Octet RED96e system provide researchers with an easy to use real time label free system that can detect the interactions of a diverse range of biomolecules; from small molecules to proteins to mammalian cells. The Octet platform offers an advanced fluidics-free approach with a wide variety of off-the-shelf Dip and Read™ biosensors for rapid binding kinetics and quantitation analysis, enabling direct detections of not only purified biomolecules, but even those in complex media such as cell culture supernatants and lysates. The 96-channel Octet HTX system performs quantitation of 96 samples in as little as 2 minutes, and kinetic screening of 384 samples in 15 minutes. Analysis can be done using a single channel or up to 96 channels, enabling more flexibility in sample throughput when needs change. In addition, the Octet systems have been developed to operate reliably in a regulated environment. Fortebio offers 21 CFR Part 11 software and a full line of GxP products and services as part of the GxP Package. To demonstrate the performance of the BLI technology, applications in protein-protein and protein-small molecules interactions will be described.

Assessing clonality is key to the establishment of a cell line, and evidence of monoclonality is required by regulatory agencies to get a biopharmaceutical drug to the marketplace. The traditional and most accepted approach involves the visualization of microplate wells using transmitted white light (brightfield) on day 0 to confirm the presence of a single cell. However, the definitive identification of a single cell on the initial day of cell seeding is not without challenge, as cellular debris and well artifacts can be easily mistaken for cells. Here, we demonstrate an optimized workflow using the fluorescence reagent, calcein AM, in conjunction with a fluorescence-capable CSI that shows similar viability to label-free conditions while simultaneously providing high assurance of clonality. In this workflow, we determine a concentration of calcein AM that is ideal for detection of single cells on a fluorescence-capable CloneSelect™ Imager while minimizing cytotoxic effects on clonal outgrowth. We describe guidelines for establishing an optimal concentration of viability dye for different cell types.

Cell line development is a crucial step in upstream processes for generating biopharmaceutical molecules like monoclonal antibodies. Traditional workflows in cell line development have major drawbacks such as inefficient single-cell isolation, reduced cell viability, and limited evidence of monoclonality. We present an approach to address these challenges by utilizing a microfluidics-based singlecell isolation technology and a high-throughput imaging system. The CloneSelect™Single-Cell Printer™ offers documented proof of clonality and provides efficient and fast single-cell seeding combined with excellent cell viability and zero risk of cross-contamination. Meanwhile, the CloneSelect™ Imager allows for fast and objective confirmation of monoclonality as well as the growth characteristics of single cell-derived clones. The two systems can be used in concert to increase throughput with the confidence of clonality and objective analysis of clones for downstream steps in the cell line development workflow.

The Octet^® platform that utilizes Bio-Layer Interferometry (BLI) technology is an alternative and convenient analytical method that is easy to use and can be customized through the use of the highly reproducible High Precision Streptavidin biosensors (SAX) to quantify any recombinant protein. In this work, we give the example of its use in the accurate quantification of five therapeutic recombinant proteins: Insulin, Factor VIII, Factor IX, Granulocyte Colony Stimulating Factor (GCSF) and Erythropoietin (EPO) in different cell culture matrix.

Fast, accurate determination of vaccine titre during influenza vaccine manufacture is important in understanding process performance and correctly scaling each process step. Traditionally Single Radial Immunodiffusion (SRID) assays have been used as the 'gold standard' but the assay requires very skilled operators to obtain reproducible results and is relatively low throughput. ELISAs have also been used to determine titre but have lower precision and dynamic range. BLI combines the high throughput characteristics of a 96-well plate based ELISA assay in conjunction with improvements in accuracy and repeatability derived from a simpler direct measurement of mass transfer on binding.

Pall ForteBio LLC has teamed up with Cygnus Technologies to jointly develop an Anti-CHO HCP Detection Kit for quantitation of residual host cell proteins. While Pall ForteBio LLC Octet systems are known industry-wide for easy and rapid high-throughput protein analysis, Cygnus 3G HCP ELISA kits are known for their broad HCP recognition and excellent sensitivity. The Pall ForteBio LLC-Cygnus Anti-CHO HCP assay kits bring scientists the best of both worlds. Assay performance using sample data sets are described as evidence of assay robustness, sensitivity, and precision.

The new Octet HTX instrument monitors up to 96 biosensors simultaneously. The high-throughput read-head, in conjunction with new data analysis software features, opens up new applications such as epitope binning and allows higher throughput for standard applications such as quantitation. The flexibility to perform demanding applications such as small molecule kinetics is addressed by a high-sensitivity 16 sensor mode within the same instrument. Additionally, the ability to use both modes of acquisition in a single experiment provides a straightforward workflow for immobilization of capture protein onto a full tray of 96 biosensors, followed by high sensitivity quantitation or kinetic experiments without user intervention.

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.

This presentation was made by Dr. Sriram Kumaraswamy, at the CHI Bioprocessing Summit in August 2010. The anti-penta HIS biosensors have been developed as a simple, one-step, Dip and Read method on the Octet label-free detection platform for the quantitation and detection of His-tagged proteins. The platform is especially suited for high-throughput expression screening and the optimization of protein expression systems where sample purification needs to be avoided. The assay utilizes the Octet optical biosensing platform and involves direct, label-free detection and quantitation of his-tagged proteins that bind to highly specific penta-HIS antibody pre-immobilized on Octet biosensors. The anti-penta HIS biosensors complement other pre-fabricated and custom biosensors on the Octet platform to provide a comprehensive set of tools for use in the entire bioprocess workflow from cell line development to production monitoring and quality analysis.

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 poster was presented by ForteBio at the Peptalk conference in San Diego, January 11 to 15, 2010.
The poster describes the use of a specific anti-penta HIS antibody pre-immobilized on an Octet biosensor to capture and quantify HIS-tag proteins in a label-free method that does not require any secondary detection antibody. Using this method, we tested proteins with molecular weights ranging from 13 kDa up to 43 kDa and determined the assay dynamic range to be about 0.25 to 200 micrograms per milliliter in both purified form and spiked into spent CHO cell media. This assay provides a very rapid and easy method for quantitation of HIS-tag proteins in both purified and crude media.

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 microtiter plates without the use of microfluidics, and generates binding profiles, responses, and kinetic constants that correlate with SPR including k_on, k_off , and K_D.

Presented by John Proctor at BPI 2009 in Raleigh, NC Detection of residual Protein A in product purified using a Protein A, or similar column matrix such as MabSelect SuRe™, is a critical quality control step in antibody therapeutic development and production. During the IgG purification process, Protein A can leach from the purification column and co-elute with the IgG. This contaminant in the therapeutic mixture can cause adverse reactions in patients and thus must be detected and minimized. The Octet family of instruments utilizes a unique label-free dip and read detection technology to quantify biomolecules in solution. ForteBio has developed a new kit for use on this system to detect and quantify residual Protein A. The new Dip and Read™ Residual Protein A Detection Kit contains biosensors and all the reagents required to quantitate Protein A and MabSelect SuRe. The data presented here demonstrates that the Octet system can accurately and precisely detect 100 pg/mL Protein A or MabSelect SuRe in bioprocess samples.

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.

We report a label-free method for determining kinetic constants for the binding of small molecule inhibitors (<500 Daltons) to carbonic anhydrase, including rate constants and affinity constants. These parameters are derived from data obtained using ForteBio's Octet RED System, a label-free, 8-channel system that is compatible with 96-well microplates.

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 protein-protein and protein-small molecules will be described.

Project timelines and workflow are adversely impacted by the amount of assay development required for each protein when using traditional flow-cell based biosensors. Several factors contribute to delays in assay development, such as limited throughput, sample constraints due to fluidics and parallel processing of multiple conditions or epitopes. Data for rapid assay development using Amine Reactive Biosensors on the Octet System will be presented covering a variety of conditions for optimization, shortened worklfow analysis and parallel rrocessing of various concentrations or molecule types.

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 specificity and affinity, 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 off-rate using Streptavidin biosensors in the kinetics mode. Presented at the Society of Biomolecular Screening in Seattle, WA, 2006

Quantitation of antibodies using a Protein A biosensor on the Octet. Replace ELISA and HPLC in your lab and increase productivity by many fold.

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

Efficient and cost-effective workflow for antibody - Octet QK generates quantitative or kinetic results in real time, using an integrated system that is user-friendly for training, set-up, assay time and process development. The system provides: K_D, K_obs, K_a, K_d, minimal interference from media, compatibility with crude lysates, affinity ranking for 96 samples in 1 hour and automation bioprocessing.

The GlyS Kit lets you perform high-throughput glycan screening of crude and purified samples on Octet systems. No sample purification or digestion is required, preparation time is reduced by up to 3 hours. Screen for samples with desired sialylation levels – high, medium or low - and combine titer data with sialic acid data for more informed decisions.

ForteBio Octet Data Acquisition and Data Analysis software is rapidly evolving to better address the growing applications and needs of our customers. These comparison tables provide a general guide to the features included in various versions of the software. The latest release, Version 11, is available for use on all Octet systems.

Recent enhancements to our Octet CFR v11.0 software and the ForteBio FB Server Monitor Module, along with software validation package support enable all Octet instruments to be integrated seamlessly into GxP regulated environments.

Regulations and global standards, including US FDA 21 CFR Part 11, EU GMP Annex 15 and USP <1058>, require documented verification that your instruments are delivered, installed and  routinely calibrated and functioning according to their operational specifications. Our trained and certified scientists and engineers at Pall ForteBio are here to help you with a full line of qualification products and services to help you meet your initial and ongoing system qualification requirements.

We offer a selection of surface chemistries for use on the Pioneer and Pioneer FE platforms — from amine reactive surfaces with varying capacities to pre-immobilized capture chemistries. Our high quality SPR biosensors enable researchers to optimize for a diverse array of ligand types and applications, ensuring generation of high quality data from interaction analysis studies.

Feature comparison for v8.x, v9.x and v10.x.

The new Octet© RED96e system detects a diverse range of biomolecules from small molecules to proteins to mammalian cells. The Octet platform offers an advanced fluidics-free approach with a wide variety of off-the-shelf Dip and Read biosensors for rapid binding kinetics and quantitation analysis. The system utilizes Pall ForteBio's Bio-Layer Interferometry (BLI) technology, enabling direct detection of specific proteins and other biomolecules — even in complex mixtures like cell culture supernatants and lysates. The 8-channel Octet RED96e system performs quantitation of 96 samples in 32 minutes, and kinetic screening of 64 samples in 1.5 hours. Analysis can be done using a single channel or up to eight channels, enabling more flexibility in sample throughput when needs change. An optional microplate evaporation cover minimizes losses in sample volume, allowing full post-analysis sample recovery even after a 12 hour experiment.

Octet® software provides an intuitive and easy to use interface for data acquisition and analysis on all Octet instruments, enabling label-free kinetic, affinity, epitope binning, activity, concentration and screening applications. Note: Octet Data Analysis HT software is not available in the Octet CFR software suite installer. To upgrade your software to v10.0, please complete the software download form or contact your local sales representative.

The Anti-Human IgG Fc Capture (AHC) biosensors enable kinetic characterization of macromolecular interactions between human Fc-containing proteins and target analytes. Immobilization of human Fc-containing proteins is achieved through a factory immobilized anti-human Fc-specific antibody whose high-affinity for the human Fc domain provides the stable baseline required for demanding kinetics applications. Cost-effective regeneration of the biosensors and the ability to directly immobilize human Fc-containing proteins from crude matrices make the AHC Biosensor extremely useful in high-throughput applications.

Protein A binds tightly to the Fc region of IgG and is commonly used to purify IgG from crude protein mixtures such as clarified bioreactor cell cultures. During the purification process, Protein A can leach from chromatography matrix and co-elute with IgG drug substance. Protein A can cause adverse reactions in patients, therefore detecting and minimizing Protein A contaminant is a critical quality control step in antibody therapeutic development and production. 

The FAB2G Biosensor is qualified for both quantitation and kinetic applications. It enables researchers to quickly and easily detect and quantitate human Faband IgG, or to capture them for affinity measurements with other analytes. Combined with the ease of use provided by the BLItz® platform or the high throughput provided by the Octet® systems, the Anti-Human Fab-CH1 Biosensor greatly accelerates laboratory workflows and reduces time to results. In addition, the BLItz platform’s low (4 μL) sample volume enables measuring precious samples with the Anti- Human Fab-CH1 Biosensor. The biosensor can be easily regenerated using a 10 mM glycine solution at pH 1.7.

Octet software provides an intuitive and easy to use interface for data acquisition and analysis on all Octet instruments, enabling label-free kinetic, affinity, epitope binning, activity, concentration and screening applications. 

Feature comparison for v7.x, v8.x and v9.x.

Feature comparison for v7.x, v8.x and v9.x.

The Anti-Penta-HIS Dip and Read Biosensor is qualified for both kinetic and quantitation applications. It enables users to quickly and easily detect His-tagged recombinant proteins for quantitation measurements, or to capture them for affinity measurements with interacting analytes. Together with the BLItz system’s ease of use or the Octet platform’s throughput, HIS1K biosensors greatly accelerate laboratory workflows and reduce time to results. The BLItz system further enables measurement of precious samples with sample volume requirements as low as 4 µL. The HIS1K Biosensor can be regenerated for kinetic applications, providing a cost-effective solution for generating replicate data for ligand-analyte pairs, or for analyzing multiple analytes.

The Octet K2 2-channel system brings you unprecedented access to exquisite biomolecular interactions data. Priced for start-up labs, low volume users are no longer bound by the trade-off between cost and performance when choosing a label-free assay system. The Octet K2 system detects protein-protein and protein-small molecule interactions, with molecules down to 150 daltons in size.

Octet service contracts are not just for repair services. They help keep your Octet system in top condition, and provide you priority access to our global network of Technical Support, Field Applications, Product Support, and Field Service Engineers. We are dedicated to helping you succeed, and to providing rapid solutions to your problems.

Compare features in Versions 6, 7 and 8 of Octet Data Acquisition and Data Analysis Software.

Pall ForteBio LLC and Cygnus Technologies have jointly developed the Anti-CHO HCP Detection Kit for quantitation of residual HCPs. The Octet platform’s rapid high-throughput protein analysis combined with the broad HCP recognition and sensitivity of the industry-standard Cygnus 3G anti-CHO HCP antibody bring scientists the best of both worlds for HCP analysis. 

Streptavidin-coated surfaces are widely used as a simple and straightforward method of molecular immobilization. Utilized with Bio-Layer Interferometry (BLI), Streptavidin biosensors enable quick and easy modification and customization of the biosensor with any biotin-tagged molecule for quantitative and kinetic measurements. The new High Precision Streptavidin Biosensor (SAX) was specifically developed and qualified for applications in downstream drug discovery and regulated environments that have more stringent assay precision requirements. SAX biosensors are QC-tested at Pall ForteBio to meet our precision-controlled coefficient of variation (CV) specification of <4%.

Each type of Octet Biosensor is designed to analyze specific biomolecular interactions. A range of surface chemistries is available. Pall ForteBio biosensors are coated with a proprietary biocompatible matrix that is uniform and non-denaturing with minimal non-specific binding. The tips are derivatized and ready for use in a diverse set of biomolecular applications. Biosensors can also be customized for unique applications. Multiple layers can be bound and detected, opening a vast array of approaches to detecting and characterizing biological interactions.  To date, over 2.5 million biosensor assays have been run on Octet systems, attesting to the high reproducibility, accuracy, precision and robustness of the platform. Octet biosensors are manufactured in a ISO 9001:2008 certified facility.

The BLItz system is enabling label-free detection and analysis across protein manufacturing, purification, and processing workflows.  To support customer requirements in regulated environments, Pall ForteBio offers additional tools to qualify BLItz systems and to protect the integrity of acquired data.

The Octet HTX instrument monitors up to 96 biosensors simultaneously, enabling label-free detection for protein quantitation and kinetic characterization at unmatched speed.  The system's ability to read 8, 16, 32, 48 or 96 wells in parallel lets you tailor your assay design to maximize analytical throughput or sensitivity.

The polyhistidine tag, commonly known as HIS-tag, is fused to recombinant proteins as a means of facilitating detection and purification. The Anti-HIS (HIS2) Biosensor consists of high affinity, high specificity Anti-HIS antibody from Maine Biotechnology Services (MBS) pre-immobilized on a ForteBio fiber optic biosensor. In conjunction with the Octet&reg; and BLItz&reg; systems, the Anti-HIS (HIS2) Biosensor provides a rapid and label-free method for HIS-tagged protein quantitation. The high specificity of the antibody-based biosensor enables the direct quantitation of HIS-tagged proteins in crude lysates, column eluents, cell lysates and cell culture supernatants, serving as an alternative to traditional time-consuming analytical methods such as HPLC and ELISA.

ForteBio’s Aminopropylsilane (APS) biosensors, in conjunction with the Octet system, are designed for hydrophobic immobilization of most proteins used in assaying protein:protein interactions. Using Aminopropylsilane biosensors, the Octet system supports applications from kinetics screening to full kinetics characterization.

ForteBio’s Dip and Read Anti-Murine (AMQ) IgG biosensors, in conjunction with the Octet system, are designed for monitoring antibody concentrations in mouse or rat sera and cell culture supernatants. Using Anti-Murine IgG biosensors, the Octet system supports applications from assay development, clonal selection, and cell culture screening to concentration monitoring during development and manufacturing.

The Ni-NTA biosensor is pre-immobilized with novel nickel-charged Tris-NTA from QIAGEN, and enables an easy and rapid method of quantitation and kinetic characterization of HIS-tagged biomolecules. The strong binding of Ni2+ to the HIS-tag makes the Ni-NTA Biosensor particularly suitable for stable capturing of HIS-tagged proteins for affinity measurements.

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.

The AR2G biosensor is a newly engineered biocompatible surface matrix that delivers enhanced performance for kinetic assays compared to the original Amine Reactive (AR) biosensor for most proteins. AR2G routinely achieves greater signal intensity than the AR biosensor during both the loading (immobilization of ligand) and association (detection of analyte) steps of an assay while delivering the same reliable kinetic values

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.

Pall 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 protein-small molecule interactions. The Octet RED96 system 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.

Accurate antibody quantitation is critical to selecting cell lines for developing and optimizing antibody production. Traditional methods for measuring antibody concentration include HPLC, ELISA and densitometry—all of which have long analysis times, lack of specificity, and precision. Using Dip and Read Protein A (ProA) biosensors with the Octet system streamlines a variety of bioprocessing applications by providing precise results which require minimal sample handling and give rapid turnaround of results.

Dip and Read Protein G (ProG) biosensors provide a rapid and direct method for quantifying mammalian IgGs from buffer, media or other complex matrices. Protein G is pre-immobilized onto the biosensors, and binds to rodent and many other mammalian IgGs with higher affinity than Protein A, but does not bind to IgM, IgD or IgA. In combination with the Octet system, Protein G biosensors can streamline bioprocessing applications by providing precise results with minimal sample handling and turnaround times as fast as two minutes. The biosensors can be regenerated multiple times, providing a cost-effective and time-saving assay format.

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 Octet QKe system has been designed to be an enhanced performance version of the Octet QK instrument. Three acquisition speed settings to customize assay performance and a higher performance spectrometer combine to provide greater sensitivity and greater dynamic range for quantitation and kinetic analysis. In addition, biosensor re-racking ability provides enhanced flexibility and operational cost savings for running assays.

The 384TW microplate is a black, polypropylene, 384-well tilted-bottom plate can be used on the Octet RED384, QK384 and HTX instruments, both of which accommodate the 384-well plate format. The 384TW 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.

Overview of ForteBio's Super Streptavidin biosensors.

The Sidekick station is an accessory to Pall ForteBio's Octet family of real-time label-free biomolecular interaction analysis instruments. The instrument 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 station, freeing the Octet instrument for other users. The Sidekick station 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 Anti-Human IgG Fc Capture (AHC) biosensor is designed for kinetic characterization of macromolecular interactions between proteins containing an Fc region and a target analyte. This biosensor enables the immobilization of human IgG or other protein containing a human Fc region using a high-affinity anti-human Fc-specific antibody. The biosensor surface has been designed to provide stable interactions and can be regenerated, making it particularly suitable for high-throughput kinetics applications. AHC biosensors can be used with many sample types, including purified proteins and cell culture supernatants.

Biosensor Mount Cleaning Trays from Pall ForteBio are intended for periodic, regular, automated cleaning of biosensor mounts on Octet HTX, RED384, and QK384 instruments.

The evaporation cover minimizes sample evaporation by only exposing one column on the sample plate at a time. Biosensors will dip into the open sample well column, and once the read is complete, the cover will return to its original position. The use of the evaporation cover can extend the experimental run time to up to 12 hours, with minimal sample evaporation, allowing for maximum sample recovery after concluding an assay.