Acoustic Membrane Microparticle Research Articles

Antibodies against polyethylene glycol in human blood: A literature review

Polyethylene glycol (PEG) conjugation, i.e. PEGylation, is a successful strategy to improve the pharmacokinetics and pharmacodynamics of biopharmaceuticals. In the past few decades, PEGylation technology has developed tremendously, and >15 PEGylated therapeutics have been brought to market, with more in development. However, the widely accepted assumption that PEG would have no antigenicity or immunogenicity is increasingly challenged with popularization of PEGylation technique. Although PEGylation indeed reduces the immunogenicities of the modified molecules, and even appears to completely eliminate their immunogenicities, yet emerging clinical evidence of anti-PEG antibodies (including both pre-existing and PEGylated therapeutics-treatment induced anti-PEG antibodies) have been attracted more and more attention. Anti-PEG antibodies were detected in not only patients treated with PEGylated therapeutics but also PEGylated drugs treatment-naïve individuals with a prevalence from <1% to 72%. In patients, the existing anti-PEG antibodies may attenuate therapeutic efficacy of PEGylated drugs and increase adverse effects. Although there is no golden standard avenue, several types of methods, including passive hemagglutination, Western Blot, enzyme linked immunosorbent assay, flow cytometry, Meso Scale Discovery technology, Acoustic Membrane Microparticle assay, and surface plasmon resonace technique, were established and used to screen, confirm and quantitatively detect anti-PEG antibodies. Herein, we focused on reviewing the prevalence of anti-PEG antibodies in healthy and PEGylated therapeutics-treated patients, and highlighting the detection methods for pre-screening and quantitative detection of anti-PEG antibodies.

Generic Anti-PEG Antibody Assay on ProterixBio's (Formerly BioScale) ViBE Platform Shows Poor Reproducibility.

PEGylation is a modification commonly used to increase the half-life of therapeutic proteins. The strategy for immunogenicity testing of these compounds should include methods to detect both anti-protein and anti-PEG antibodies. We previously reported a method for the detection of anti-PEG antibodies using ProterixBio's (formerly BioScale) acoustic membrane microparticle (AMMP) technology. Our initial method development work showed the assay was capable of detecting antibodies in human serum with a sensitivity of 1μg/mL with good reproducibility (CV < 7%). Since the publication of this initial paper, additional experimentation was performed in an effort to validate the assay for support of clinical sample analysis. This additional data indicate that the method has high variability (CV% > 20) and is unsuitable to support clinical sample analysis.

Development of a Generic Anti-PEG Antibody Assay Using BioScale's Acoustic Membrane MicroParticle Technology.

Immunogenicity testing for PEGylated biotherapeutics should include methods to detect both anti-protein and anti-PEG antibodies (anti-PEG). Although some methods have been published for the detection of anti-PEG antibodies, the information is incomplete and, in some cases, reagents used (such as Tween-20) are known to interfere with detection. This rapid communication describes the use of BioScale's Acoustic Membrane MicroParticle (AMMP®) technology using the ViBE® Workstation to measure anti-PEG antibodies in human serum samples. Briefly, a sample spiked with monoclonal human IgG anti-PEG antibody is diluted in buffer and incubated with paramagnetic beads coated with linear chain mPEG to capture anti-PEG antibodies. The complex is then captured on an acoustic membrane coated with Protein A. The change in mass on 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 1000ng/mL for IgG is achievable. This level of sensitivity is better than current published reports on IgG anti-PEG antibody detection.

Evaluation of acoustic membrane microparticle (AMMP) technology for a sensitive ligand binding assay to support pharmacokinetic determinations of a biotherapeutic.

Achieving the required sensitivity can be a challenge in the development of ligand binding assays for pharmacokinetic (PK) determinations of biotherapeutics. To address this need, BioScale's Acoustic Membrane Microparticle (AMMP) technology was evaluated for the quantification of a PEGylated domain antibody (dAb) biotherapeutic. Previous uses of this technology had shown utility in biomarker and process development applications and this is the first application, to our knowledge, for PK determinations. In this evaluation, AMMP was capable of delivering a sensitivity of 0.750 ng/mL, which surpasses the sensitivity requirements for the majority of assays to support PK determinations. This evaluation demonstrates that this emerging technology has the ability to produce the required sensitivity, reproducibility, and selectivity needed to meet the industry's standards for PK analysis.

Measurement of downstream kinase activity modulation as indicator of epidermal growth factor receptor inhibitor efficacy

The acoustic membrane micro particle (AMMP) technology has been used to quantify single analytes out of multiple sample types. In this study the technology is used to reveal molecular interactions of components of kinase pathways. Specifically, the downstream kinase activity of the EGFR receptor in the presence or absence of EGFR inhibitors is investigated. These experiments substantiate that EGFR stimulation predominantly activates the MEK/ERK pathway. The EGFR inhibitors tested had varying effectiveness at preventing phosphorylation at the EGFR or downstream kinase activity. These experiments reveal the use of the AMMP technology for observing multiple signaling pathways in a single experiment.

Competitive, immunometric assay for fusion protein quantification: Protein production prioritization

Effective drug discovery demands the availability of microgram to gram quantities of high-quality protein encoded by novel transcripts. Protein expression vectors designed for large-scale protein production often include one or more specific tags to such transcripts, to simplify the purification of the targeted protein. Optimization of the complex expression and purification process requires the evaluation of multiple expression candidate clones to identify a production-suitable construct in terms of quality and final protein yield. Efficiency of the entire expression screening process is typically assessed by direct visualization of the banding patterns from whole-cell lysates on SDS–PAGE gels, by direct staining and/or immunoblotting, using antibodies against the tag or the protein of interest. These techniques, generally run under denaturing conditions, have proven to be only marginally predictive of the purification yield and authentic folding for native proteins. Small-scale, multiparallel affinity purification followed by SDS–PAGE analysis is more predictive for expression screening; however, this approach is labor intensive and time consuming. Here we describe the development of an alternative expression efficiency assessment technique, designed to evaluate the accessibility of affinity tags expressed with the desired fusion proteins, using acoustic membrane microparticle assay technology on the ViBE protein analysis workstation.

Thrombin detection using a piezoelectric aptamer-linked immunosorbent assay

The development of diagnostic assays using highly targeted specific aptamers with existing detection platforms has been an endeavor with few opportunities until now. Many current commercially available diagnostic platforms make use of detection systems employing capture agents composed of modified antigen-specific antibodies coupled with a variety of detection modalities, including radioimmunoassays, fluorescence-based detection assays, electro/chemiluminescence assays, and immunoradiometric assays. In the studies presented here, a novel frequency-modulating technology from BioScale called Acoustic Membrane MicroParticle (AMMP) detection was used to demonstrate a sensitive and reproducible method of incorporating aptamers as capture and detection agents. The method provides a robust and rapid detection of thrombin in human serum while also eliminating the labor-intensive efforts of Western blot analysis and is not affected by the interfering substances found in serum that often affect optical-based detection systems. In addition, we have demonstrated, for the first time, the adaptation of the AMMP platform to exploit aptamers against a clinically relevant target. The AMMP platform is an ideal medium for using aptamers in commercial assay development for application in a clinical setting.

Abstract 5170: Novel discovery of P38 activation: Expression in A431 cells in in vitro and in vivo models of cancer, measured by a novel assay for p-P38 .

Abstract The p38 enzyme, also known as mitogen activated protein kinase or MAPK (pT180/pY182), is responsive to cellular stress such as osmotic shock, heat shock, and irradiation, and when activated, modulates the cell cycle, activation of DNA repair, regulation of protein translation and initiation of immune response. The p38 pathway consists of a cascade of protein kinases which ultimately leads to its phosphorylation on specific tyrosine and threonine residues. Growth factors play an important role in the activation of p38 MAPK. The binding of EGF or other specific growth factors to external receptors activate its kinase activity. The activation of p38 MAP kinase can directly influence gene transcription. A growing number of transcription factors are known to be direct targets of p38, thus its implication in many forms of cancer. In the A431 human epidermoid carcinoma cell line, proliferation and migration responses to extracellular stimuli require activation of distinct pathways. In this study we describe the use of the BioScale ViBE workstation and AMMP (acoustic membrane microparticle) assay technology to study the expression of p-p38 in lysates of A431 epidermoid carcinoma cells with or without stimulation of surface receptors for epidermal growth factor, compared to xenograft tumors of the same cell line. The AMMP p-p38 assay is robust and reproducible, with CVs less than 6% for within day and between day measurements. The assay demonstrates little interference from cell or tumor lysate matrices. Results from the p-p38 AMMP assays demonstrate that, in a group of ten A431 tumors, all were positive for the activated analyte. There was heterogeneity in the expression of the p-p38 in the tumors but all gave a positive assay response. Recombinant p-p38 was used as a positive control in each experiment. The results of the phospho-p38 AMMP assay were compared to western blots for the detection of p- p38 in EGF stimulated A431 cell lysates and untreated tumor lysates. The western blots were confirmation of presence or absence of the AMMP measured analyte in the samples. p-p38 was detected by western blot with similar intensity for equal protein loading of each sample type. The elucidation of p-p38 in A431 tumors in untreated nu/nu mice was unexpected as the EGF receptor was not activated based on AMMP results for P-EGFR derived from the same tumor sample set. This would indicate that the P38 was activated through a pathway other than the EGFR pathway. The observation of similar signals between the p-p38 levels detected between EGF stimulated A431 cells grown in vitro and in vivo tumors was also unexpected, again pointing to different pathways of activation. These results demonstrate that the BioScale AMMP assay for detection of p-p38 can be a powerful tool for the study of cancer, apoptosis or cell signalling, giving sensitive and reproducible results without interference from matrix components. Citation Format: Lee Anne Beausang, Kristen Leong, W. Matthew Dickerson, Ashley Saab, Edward M. Alderman. Novel discovery of P38 activation: Expression in A431 cells in in vitro and in vivo models of cancer, measured by a novel assay for p-P38 . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5170. doi:10.1158/1538-7445.AM2013-5170

Abstract 3208: Development of applications for acoustic membrane microparticle assay technology for translational medicine.

Abstract The detection and study of proteins and their interactions utilizing small samples of cells or tissues is an ongoing problem in cancer research. Recent advances in the early detection of many cancers have resulted in smaller sized tumors for biopsy and evaluation. This has created a need for sensitive and robust assays for pathway related proteins. One mission for the Antibody and Protein Purification Unit (APPU) of the National Cancer Institute has been to evaluate emerging technologies and create new applications for these technologies. Using Acoustic Membrane MicroParticle (AMMP) assays on the ViBE Workstation (Bioscale, Lexington, MA) for solution phase immunoassays, the APPU has developed several applications which support the great utility and breadth of use for this technology in translational research. AMMP assays employ a homogenous, sandwich assay format with paired antibodies, one coupled to a magnetic bead and the second tagged for capture on the sensor, and the analyte forming a complex which interacts with a vibrating membrane sensor. The assay measures the concentration of the analyte by a resonant frequency shift of the membrane caused by the protein interaction – without matrix interference or signal scatter seen in optical techniques. A highly sensitive assay for Prolactin showed no serum interference, which is a common problem in optical immunoassays for circulating peptide hormones. An assay for the important marker C-Met required less than 125 nanograms of cell lysate protein for detection and quantitation. An AMMP assay was further able to quantitate C-Met on the surface of Hep2 cells using an antibody capture method. In an application for detection and quantitation of a member of the MAPK kinase family, an ultrasensitive assay for the detection of MEK1 was developed as well as a highly specific solution phase assay for pMEK1 (S218/S222) as shown by cognate phosphopeptide inhibition. And finally in an application which we believe to be truly unique, a solution heterodimer assay for the ERK1/MEK1 interaction was shown to be susceptible to inhibitor treatment. In conclusion, the AMMP technology was able to perform a wide range of applications required by translational research studies. The assays showed superior lower limit of detection and were reproducible, only requiring nanogram quantities of lysate proteins. The ability to measure protein/protein interactions in solution phase is especially useful for drug development and evaluation of drug effects in targeted therapies. Citation Format: Christopher McAndrew, Christopher Heger, Ashley Saab, W Matthew Dickerson, Paul K. Goldsmith. Development of applications for acoustic membrane microparticle assay technology for translational medicine. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3208. doi:10.1158/1538-7445.AM2013-3208

Abstract 5527: A panel of in vitro kinase assays developed using a novel detection technology to predict and monitor EGFR inhibitor efficacy.

Abstract The efficacy of kinase inhibitors, a highly studied class of drugs used to treat many forms of cancer, remains difficult to predict or tailor to individual specific carcinomas. Monitoring multiple biomarkers in the course of drug development may lead to more successful therapeutic design and patient treatment regimens. Here we discuss the generation and use of assays to measure a panel of kinase biomarkers using Acoustic Membrane Microparticle (AMMP) technology on the VIBE workstation to efficiently monitor both on- and off-target effects of kinase inhibitors directed against the EGFR family of tyrosine kinase receptors. These results may be used to better predict the roots of drug resistance. Two cell lines were selected for use in this study on the basis of EGFR expression. The epidermoid carcinoma A431 cell line is a high expressor of the endothelial growth factor class of receptors and, therefore, subject to EGFR inhibition. This line was used to evaluate ‘on-target’ effects of the selected EGFR inhibitors. In contrast, the MCF-7 breast cancer cell line, a low expressor of EGFR class of receptors, should be much less susceptible to EGFR inhibition, and was used to better understand the ‘off-target’ effects of the EGFR inhibitors. Cells were preincubated at reported IC50 concentrations of EGFR inhibitors gefitinib and neratinib, then stimulated by EGF, lysed, and assessed for cell surface receptor (EGFR and HER3) and cytosolic kinase (MEK, ERK, p38, JNK and AKT) activity. As expected, EGF stimulated a large phosphorylation event of EGFR, HER3, MEK and ERK in the A431 cell line, with little observable change for the MCF-7 cell line. A significant increase in the phosphorylation of p38 but little change in JNK was seen in both lines. The phosphorylation of EGFR and HER3 in the A431 cells appeared unaffected by gefitinib, but completely inhibited by neratinib. Unexpectedly, no downstream inhibition of EGF-stimulated MEK or ERK phosphorylation was observed in the A431 cell line for either inhibitor. In contrast, EGF-induced p38 phosphorylation was significantly inhibited in both the A431 and the MCF-7 cell lines. Here we have constructed a panel of assays that may be used to better predict biological activity, not only at the target site of a kinase drug, but also downstream of that site. This panel demonstrated that EGF was still able to activate both MEK and ERK despite failing to phosphorylate either the EGFR or HER3 receptors, revealing that EGF may be acting through other receptors. Consequently, blocking of this class of receptors may only be partially effective in preventing EGF-induced activity. Further, preventing the phosphorylation of the EGF class of receptors may not be sufficient to completely inhibit the ligand induced phosphorylation of the Mitogen Activated Kinase pathway. Citation Format: W. Matthew Dickerson, Lee Anne Beausang, Ashley Saab, Kristen Leong, Edward Alderman. A panel of in vitro kinase assays developed using a novel detection technology to predict and monitor EGFR inhibitor efficacy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5527. doi:10.1158/1538-7445.AM2013-5527

Abstract 234: Snapshot of MAP kinase and related signal transduction pathways from biopsied cells using a novel non-optical assay technology - a proof of concept study

Abstract Aberrant activity of the MAP and PI3 kinases is implicated in many forms of cancer. The ability to gather quantifiable information on the activation-state of these kinases from limited in-vitro and in-vivo tumor samples would accelerate drug development and ultimately, the treatment of cancer. Here non-optical, AMMP (acoustic membrane microparticle) technology is used to quantitate the activity state of multiple kinases including EGFR, c-RAF, MEK, ERK, AKT, p38 and JNK in a single assay plate. Lysates from multiple unstimulated tumor cell lines were compared with those from the same cell lines specifically stimulated with ligands to several well-known surface receptors for expressed changes in their phosphorylation states. The data show that the AMMP assay technology can be used to monitor the MAP Kinase pathway activation from EGFR stimulation through ERK phosphorylation following EGF stimulation. Additionally, the activation states of PI3 kinase, p38 and JNK were determined from the same lysate samples. Subsequently, assays were developed to monitor kinase dimerization (e.g., MEK-ERK) by rearranging the pairing of antibodies from the kinase activity assays described above. Using fewer than 2000 cells per assessment, multiple kinases were measured, in their native states, in a single assay using AMMP technology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 234. doi:1538-7445.AM2012-234

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays. An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass. To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate. Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays. An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass. To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate. Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.