Bioanalysis. change in mass around the membrane caused by the binding of the complex to the membrane results in a signal proportional to the mass of anti-PEG antibodies. The data indicate that an assay with a sensitivity of less than 1000?ng/mL for IgG is achievable. This level of sensitivity is better than current published reports on IgG anti-PEG antibody detection. strong class=”kwd-title” KEY WORDS: acoustic membrane microparticle technology, anti-peg antibodies, emerging technology, immunogenicity assays, pegylated biotherapeutics INTRODUCTION PEGylation is usually a well-documented modification used to increase therapeutic protein half-life. However, immune responses to the PEG itself have caused, in some cases, loss of product efficacy and adverse safety consequences, which highlights the importance of developing a strategy to monitor anti-PEG antibodies based on risk assessment (1). Also, the recently published FDA Guidance for Industry on Immunogenicity Assessment for Therapeutic Protein Products has recommended that for PEGylated therapeutic protein products, anti-drug antibody (ADA) assays should be able to detect both anti-protein antibodies and antibodies against the PEG moiety (2). This recommendation has proven to be a Atrasentan HCl tall order, as developing and validating assays to Mouse monoclonal to CD86.CD86 also known as B7-2,is a type I transmembrane glycoprotein and a member of the immunoglobulin superfamily of cell surface receptors.It is expressed at high levels on resting peripheral monocytes and dendritic cells and at very low density on resting B and T lymphocytes. CD86 expression is rapidly upregulated by B cell specific stimuli with peak expression at 18 to 42 hours after stimulation. CD86,along with CD80/ an important accessory molecule in T cell costimulation via it’s interaciton with CD28 and CD152/CTLA4.Since CD86 has rapid kinetics of is believed to be the major CD28 ligand expressed early in the immune is also found on malignant Hodgkin and Reed Sternberg(HRS) cells in Hodgkin’s disease detect antibodies against a PEG moiety is usually a major challenge. In a review paper by Schellekens em et al /em . (3), the authors concluded that most, if not all, assays used for detecting anti-PEG antibodies are flawed due to the lack of specificity as well as poor characterization of positive controls (3, 4). Until recently, traditional bridge immunoassay format assays have been able to detect anti-PEG IgM antibodies but have struggled to detect IgG isotype antibodies with sufficient sensitivity in human matrix (5, 6), suggesting that the type of PEG and/or protein therapeutic may play a role. In addition to previously published comments (7), our own observations during assay development have noted that high levels of IgG in a sample make detection of low affinity anti-PEG IgG antibodies difficult in a plate-based or non-plate-based assay format using anti-human IgG detection reagents. A well-characterized antibody positive control and strong assay to detect anti-PEG IgG isotype will help to understand the mechanism of induced anti-PEG response following PEGylated therapeutic protein injection in human (8, 9). In this rapid communication, we report preliminary results for detecting IgG anti-PEG antibodies using an Acoustic Membrane MicroParticle (AMMP) platform. The Acoustic Membrane MicroParticle platform is an emerging technology that utilizes a non-optical detection system to determine analyte concentration by measuring the change in the oscillating frequency of a piezoelectric membrane (10). This rapid communication describes a method in which human serum spiked with monoclonal chimeric IgG is usually diluted in buffer and incubated with paramagnetic beads coated with either PEGylated therapeutic protein or biotinylated PEG to capture anti-PEG antibodies. The complex is then detected by magnetically pulling all paramagnetic beads onto an acoustic membrane Atrasentan HCl sensor coated with Protein A. Beads that are complexed with anti-PEG antibodies remain bound to the membrane sensor through the Protein A, following removal of the magnet. The change in mass around the membrane results in a signal proportional to the mass of anti-PEG antibodies. Biotinylated PEG of various molecular weights can be coupled to streptavidin-coated Atrasentan HCl paramagnetic beads, making this technology able to detect anti-PEG antibodies against a variety of PEG molecules. The work presented here focuses on the implementation of AMMP for the detection of anti-PEG antibodies for immunogenicity assessment. MATERIALS AND METHODS Materials Commercial Reagents Biotin-PEG 20?kDa and biotin-PEG 40?kDa were purchased Atrasentan HCl from Nanocs (, New York, NY). All biotinylated PEG molecules used in this study as well as in positive control characterization were linear chain molecules with a single biotin attached at one end and a methyl cap at the other end except for the PEG (40?kDa branched) attached to BMS drug A. The following buffers were purchased from Thermo Fisher Scientific, Waltham, MA: Blocker Casein in phosphate-buffered saline (PBS) with 1% ( em w /em / em v /em ) casein (Hammarsten grade), pH 7.4; Super Block buffer in PBS with proprietary protein, pH 7.4; and Super Block buffer in Tris-buffered saline (TBS) with proprietary protein, pH 7.4. Normal human sera were purchased from Bioreclamation LLC, Westbury, NY. AMMP Type I Labeling Kit for Assay Discovery, AMMP Type II Labeling Kit for Assay Discovery, and Protein A Cartridges with ViBE Cartridge Regeneration Buffer were purchased from BioScale, Billerica, MA. Polypropylene plates (96-well) for inline incubations were purchased from BioScale. Proprietary Reagents Drug A is usually a BMS biotherapeutic with a 40?kDa PEG attached to a 12?kDa protein. Custom-made monoclonal anti-PEG antibody (PEG.2): Briefly, mice were immunized at BMS with a panel of PEGylated BMS therapeutics and hybridomas were selected that showed binding to PEG.