- Novel icIEF Fractionation Coupled with ZipChip CZE-MS for Rapid Charge Variant Characterization
- Cutting to the Chase: Enzymatic Workflows for Easy Characterization of Complex Glycoprotein Charge and Size Variants on Maurice
- Charge Characterization of Viruses and Virus Like Particles (VLPs) Using icIEF with UV Fluorescence Detection
- Characterization of Maurice CE-SDS PLUS for USP <129> Suitability
- Concentrating on AAV Impurities With Ultrasensitive Total Protein Detection on Simple Western
- Simplifying AAV Protein Analytics with Maurice
- A Single Platform for icIEF and CE-SDS Analysis of Adeno-Associated Virus (AAV) for Gene Therapy
- In-Capillary Immunoassay and Total Protein Detection for Adeno-Associated Virus (AAV) Proteins During Purification from Whole-Cell Lysate
- Detection of MISEV recommended EV Protein-Markers using Automated Western Blotting
- Determination of Antibody’s Specificity Towards Phosphorylated Protein Targets with Automated In-capillary Enzyme Treatment and Immunoassay
- Profiling Immune Cell Populations in the Tumor Microenvironment with Complementary Capillary-based and Single-cell Western Assays
- Novel Approach for Automated Sequential Immunoassay for Quantitation and Characterization of PI3K/AKT Pathway Proteins
- Multiplexed Protein and RNA Quantification on a Single Instrument Harmonizes Multi-omic Analyses of Biomarkers for Immunotherapies and Targeted Therapies in Non-Small Cell Lung Cancer
Novel icIEF Fractionation Coupled with ZipChip CZE-MS for Rapid Charge Variant Characterization
Presented by Dr. Cheng Zhou
Abstract: Therapeutic monoclonal antibodies (mAbs) make up a large portion of the rapidly growing drug market. Ensuring safety and efficacy through comprehensive understanding of these products’ critical quality attributes (CQAs), including charge heterogeneity, is a regulatory requirement. Various charge isoforms of mAbs can result from cell culture or production processes, potentially affecting the mAb structure and function. While imaged capillary isoelectric focusing (icIEF) is the preferred method for charge profiling, ion-exchange chromatography (IEC) has been the major tool for fractionation combined with characterization. However, IEC is not compatible with certain types of molecules, hydrophobic antibody drug conjugated (ADCs) for example, and icIEF typically provides higher separation resolution. Moreover, an individual charge variant obtained from IEC fractionation may not be comparable to the variant peak in the icIEF profile. Therefore, there is an unmet need for IEF-based fractionation of charge variants for characterization.
We have developed a novel icIEF fractionation solution, which involves icIEF separation and collection of charge variants. This solution enables Maurice icIEF-based peak identification followed by downstream analysis. Here we report icIEF fractionation followed by ZipChip-based mass spectrometry (MS) characterization of the NIST mAb and XMT-1535 mAb. ZipChip (CE-ESI) was utilized for mass spectrometry characterization of the fractions due to its broad sample matrix compatibility, easy sample prep, and fast mass spectrometry analysis time. Individual charge variants of each antibody were successfully collected in less than 2 hours with purity > 80% using icIEF separation conditions with or without urea. Rapid analysis using ZipChip chowed the mass spec identification of major and minor isoforms correlated well with reported mass spec data (literature and report). Urea in icIEF separation did not affect the quality of fractionation nor the mass spec result. Multiple fractionation runs of the NIST mAb suggested good reproducibility of the system. We believe this novel icIEF fractionation solution coupled with other analysis methods, such as mass spectrometer, will deliver a powerful charge variant characterization tool for biotherapeutic analytical tool kit.
Cutting to the Chase: Enzymatic Workflows for Easy Characterization of Complex Glycoprotein Charge and Size Variants on Maurice
Presented by Dr. Priyanka Sarkar
Abstract: Keeping track and close control of glycosylation in therapeutic monoclonal antibodies and fusion proteins is crucial to ensure safety and efficacy of these important classes of biotherapeutics. The removal of glycans from biotherapeutics pose a challenge when investigating the changes in physicochemical and pharmacological properties. In this work we used the highly specific enzymes SialEXO®, to enable simplified workflows for glycoprofile analysis by desialylation, OglyZOR® for specific hydrolysis of O-glycans, and FabRICATOR® for digestion of antibodies or Fc-fusion proteins. The results demonstrate simplified workflows to quantitate charge and size heterogeneity associated with the removal of glycans and digestion of antibodies, fusion proteins using capillary iso-electric focusing (cIEF) and CE-SDS. The combination of specific enzymatic sample preparation with robust cIEF and CE-SDS has potential to speed up, increased through-put and simplify routine testing of critical quality attributes when developing or manufacturing biotherapeutics.
Charge Characterization of Viruses and Virus Like Particles (VLPs) Using icIEF with UV Fluorescence Detection
Presented by Dr. Jiaqi Wu
Abstract: icIEF is well-established as the gold standard tool in the biopharmaceutical industry for protein charge characterization. Newer therapeutic modalities are coming to market, including viruses and virus like particles. A challenge associated with icIEF analysis of virus and virus like particle (VLP) samples is sample aggregation during IEF. For most sample (mAbs, proteins), adding solubilizers into the sample solution, such as urea and non-iconic surfactants, can prevent aggregation. However, intact viruses and VLPs may disassociate when these additives are used. While reducing the final sample concentration can help minimize aggregation, UV light absorbance-based detection may not possess sufficient sensitivity to analyze these samples at lower concentrations. In the presentation, we will illustrate the results of using an icIEF-UV fluorescence instrument for charge characterization of viruses and VLP samples. The UV fluorescence method requires no dye labeling and shows significantly higher sensitivity than UV absorption detection.
Characterization of Maurice CE-SDS PLUS for USP <129> Suitability
Presented by Dr. Pouya Amrollahi
Abstract: Since the first therapeutic monoclonal antibody (mAb) was commercialized in the mid-80’s, close to 100 therapeutic mAb products (accounting for around a quarter of all biotech drugs) have hit the market; making it a $125 billion industry that targets critical pathological health conditions – including but not limited to products for antitumor, antiviral, and antiplatelet therapies. From early-stage process development to batch lot release testing, the efficacy, safety, identity, stability, and purity of therapeutic mAb products throughout their shelf life are of crucial importance. Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) has become the gold standard technique for the quality-control of therapeutic mAbs and proteins due to its ease of implementation, robustness, and reproducibility, replacing the more traditional and labor-intensive technique such as SDS-PAGE gel. Successful CE-SDS method development, under both reducing and nonreducing conditions, aims to reduce assay-associated impurities, fragmentations, and aggregations.
Here, we have used the monoclonal IgG System Suitability Reference Standard developed by U.S. Pharmacopeia (USP) to assess the rigor and robustness of an optimized Maurice™ CE-SDS PLUS method compared to the recommended USP protocol provided in monograph <129>. The optimization leveraged Design of Experiments (DOE) to optimize key components in sample preparation, denaturing conditions, and sample injection. The results show that the optimized methods: (1) cause less fragmentation compared to the USP <129> method, (2) are not susceptible to sample injection variations that might differ between instruments, and (3) provide comparable data to the USP <129> monograph for mAbs.
Concentrating on AAV Impurities With Ultrasensitive Total Protein Detection on Simple Western
Presented by Dr. Chris Heger
Abstract: Purity is a critical quality attribute (CQA) that must be monitored during AAV manufacturing. Impurities in protein products can be dangerous and impact efficacy. For example, protein impurities in final drug products may lead to undesirable immune responses in patients, so detecting total protein is critical for revealing impurities in preparative protein production. Traditional methods for total protein detection rely on SDS-PAGE with dyes like Coomassie Blue, or more sensitive stains like SYPRO Ruby and silver stain. However, SDS-PAGE requires large sample volumes, a lot of hands-on time, and it is poorly reproducible. Also, the use of staining dyes often comes with a lot of waste and can require specialized imaging equipment to which not every researcher has access. Here, we present a new workflow that enables total protein detection of AAVs on the Simple Western capillary electrophoresis-based immunoassay platform with a sensitivity that exceeds SYPRO Ruby. While SYPRO Ruby requires at least 1 ng of protein for reliable detection, Simple Western can reliably detect as little as 150 pg. These findings should enable researchers who are currently using SDS-PAGE to monitor purity to apply the automated platform and sensitivity improvements enabled.
Simplifying AAV Protein Analytics with Maurice
Presented by Dr. Chris Heger
Abstract: Adeno-associated viruses (AAV) are promising vectors for the delivery of genetic material in gene therapy. During the manufacture of AAV, critical quality attributes (CQAs) like charge heterogeneity, purity, and empty/full status must be carefully monitored because they can impact the product's safety and efficacy. Imaged capillary isoelectric focusing (icIEF) and capillary electrophoresis sodium dodecyl sulfate (CE-SDS) are two powerful methods to respectively characterize and quantitate charge heterogeneity and purity, but, traditionally, two separate platforms are required to perform these analyses. Here, we used a single Maurice platform to analyze AAVs by icIEF and CE-SDS methods to ensure product stability, identity, and purity. In addition, we show that the icIEF mode on Maurice, coupled with dual wavelength detection, affords insights into the empty/full status of AAVs. Taken together, the poster will show that Maurice is a powerful fully integrated analytical tool for gene therapy development.
A Single Platform for icIEF and CE-SDS Analysis of Adeno-Associated Virus (AAV) for Gene Therapy
Presented by Dr. Chris Heger
Abstract: Adeno-associated viruses (AAV) are promising vectors for the delivery of genetic material in gene therapy. During the manufacture of AAV, critical quality attributes like charge heterogeneity and purity must be carefully monitored because they can impact the product’s safety and efficacy. Imaged capillary isoelectric focusing (icIEF) and capillary electrophoresis sodium dodecyl sulfate (CE-SDS) are two powerful methods to respectively characterize charge heterogeneity and purity, but traditionally two separate platforms are required to run these methods. Here, we used a single platform to develop icIEF and CE-SDS methods to analyze AAV2 and AAV6 serotypes to monitor product stability, identity and purity. We show that these methods could reproducibly quantify both intact (by icIEF) and denatured AAV (by icIEF and CE-SDS) samples. The CE-SDS method could separate and quantify individual AAV capsid proteins, showed robust repeatability (<5% RSD) while also detecting impurities. The icIEF method was useful for measuring both denatured and intact particles with high repeatability (<4% RSD). Interestingly, preliminary evidence suggests that icIEF can also distinguish between full (1 x 1013 GC/mL) and ‘empty’ (<1012 GC/mL) AAV capsids.
In-Capillary Immunoassay and Total Protein Detection for Adeno-Associated Virus (AAV) Proteins During Purification from Whole-Cell Lysate
Presented by Dr. Chris Heger
Abstract: During adeno-associated virus (AAV) manufacture, critical quality attributes must be monitored including the presence, identity, and purity of viral vector proteins. Traditionally, the identity and purity of these proteins is monitored by Western blot using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). However, SDS-PAGE is notoriously challenging and labor-intensive. Additionally, AAVs are limited in sample size as they are difficult to manufacture, and demand outstrips supply. Here, we have developed a method to monitor the purification of AAV2 using automated capillary electrophoresis followed by immunoassay and total protein detection directly in the capillary, eliminating the need for SDS-PAGE. AAV2 was purified from HEK293 cell lysate using affinity chromatography, and the steps of the purification process (load, flow-through, wash and elution) were monitored by the capillary-based immunoassay. VP1, VP2 and VP3 capsid proteins were resolved and identified either individually or simultaneously, depending on the AAV antibody used for detection, and the total protein assay monitored the presence of impurities. The sensitivity of this assay reduced sample size down to 3 µL of sample, corresponding to approximately 400 pg or 1x108 genomic copies loaded per well. We anticipate that this in-capillary immunoassay and total protein detection can replace traditional SDS-PAGE methods in AAV manufacturing workflows.
Detection of MISEV recommended EV Protein-Markers using Automated Western Blotting
Presented by Lisa Meyer
Abstract: The limited amount of material and the diverse methods for isolation of extracellular vesicles (EV) pose unique challenges to proper characterization of experimental EV preparations. The “Minimal Information for Studies of Extracellular Vesicles” (MISEV) guidelines recommend characterizing preparations for both trans-membrane-, cytosolic- and contaminating non-EV proteins. However, compliance with these guidelines can be a considerable effort due to lack of easy and robust analytical protocols and the time consuming and user variable nature of standard western blotting protocols. Here we present a simple method for isolation of EVs and a simple western blotting platform for automated protein separation and immunodetection of MISEV-recommended proteins. The total EVs were isolated by affinity-membrane spin columns from pre-filtered 0.5-4 mL plasma or 2-20 mL urine, respectively. Intact vesicles were eluted and the EV-depleted biofluid fraction was collected from the flow-through. A small fraction (4 μL) was analyzed by a simple western blot workflow providing automated capillary electrophoresis-based protein separation and immunodetection, characterizing each fraction for presence or absence of MISEV-recommended proteins. A range of specific antibodies were identified and the EV fractions were shown to be enriched in EV-proteins, whereas contaminating non-EV proteins were significantly reduced. Isolation of EVs was necessary to allow detection of the low abundant EV protein markers, whereas non-EV proteins were readily detectable both in the neat biofluids and in the EV-depleted flow-through. We characterized the effect of washing on the purity of EV isolates and defined the dynamic range of the workflow using titrations of input volume of both plasma and urine EV isolations. In conclusion, Simple western blotting protocols were established for quality control of isolated EVs in accordance with MISEV-guidelines. EVs isolated using affinity-membrane spin columns were shown to be enriched in EV markers and depleted for non-EV proteins.
Determination of Antibody’s Specificity Towards Phosphorylated Protein Targets with Automated In-capillary Enzyme Treatment and Immunoassay
Presented by Dr. Daryl Taketa
Abstract: Protein phosphorylation is a reversible reaction that is integral in numerous signaling cascades. Characterization of signaling cascades has been largely detected by immunoblotting with phospho-specific antibodies, which may or may not have enough specificity or affinity. Currently, a separate lysate without any phosphatase inhibitors or a separate blot is needed to determine an antibody’s specificity. Here we describe a simple assay that leverages automation and quantitation with capillary electrophoresis-based immunoassay (CEIA) to assess the specificity of these antibodies with a single lysate preparation. In this study, three lysate models are used: K562 ± TNFα treatment, 50 ng/mL phorbol myristate acetate (PMA) differentiated THP-1 ± 1 μg/mL lipopolysaccharide (LPS) treatment, and cytotoxic T lymphocytes (CTL) ± 10 ng/mL PMA and 500 ng/mL ionomycin treatment. K562 cell lysates are commercially purchased whereas THP-1 lysates are generated in-house. For CTL cells, whole blood cells from a single donor are isolated and expanded with commercially available kits. Expanded CTL cells are then stimulated with PMA and ionomycin for 15 minutes. Untreated and treated lysate samples are separated and captured to the inner lumen of the capillary wall with UV activated crosslink chemistry. Cross-linked proteins are treated with lambda phosphatase for 1 hour followed by the immunoassay to investigate the specificity of antibodies against phosphorylated protein targets respective to each activated pathway using either chemiluminescent or fluorescent detection. Preliminary data suggest phospho-specific signal decreased >90% with no significant changes to the non-specific noise. The method described here eliminates the need for multiple lysate preparations or an additional blot to assess an antibody’s specificity to a phosphorylated protein target.
Profiling Immune Cell Populations in the Tumor Microenvironment with Complementary Capillary-based and Single-cell Western Assays
Presented by Dr. Charles Haitjema
Abstract: The tumor microenvironment (TME) is a complex mixture of cancerous and non-cancerous cells, including immune cells like T-cells, macrophages, and neutrophils. The TME plays a key role in tumorigenesis and metastasis, and it has recently been recognized that it can dramatically shape a response to therapy. Thus, there is a pressing need to accurately identify and quantify the variety of cell types in any given TME. However, studying the TME presents major challenges. For example, the heterogeneity of the environment requires sensitive and high-resolution techniques to parse subpopulations of different cell types. This challenge is compounded by the severely limited sample size that can be obtained from donor tissues. To address these challenges, we use a capillary immunoassay (Wes from ProteinSimple) with small sample sizes (3 µL) to identify immune cells commonly found in the TME. We also leverage single-cell western (Milo from ProteinSimple) to uncover trends in population heterogeneity. Human peripheral blood mononuclear cells (PBMCs) were differentiated into dendritic cells (DCs) and regulatory T cells (Tregs), and natural killer (NK) cells were expanded from isolated NK cells. These samples were then analyzed by Wes and Milo. These analyses revealed the identification and characterization of cell types, at both the single-cell and population level, based on the differential expression of protein biomarkers. Specifically, Wes analysis identified mature populations by a CD56+/CD3- phenotype for NK cells, a CD209+/CD14- phenotype for DCs, and a CD25+/FoxP3+ phenotype for Tregs. Milo analysis provided further detail within these populations, for example, we observed FoxP3low and FoxP3high subpopulations in Tregs, and an unexpectedly large (81%) CD56-/CD3- subpopulation in undifferentiated PBMCs, suggesting the presence of other cell subtypes. We anticipate that the small sample size, automation, single-cell resolution, and multiplexing ability of these assays collectively will enable a more efficient and deeper characterization of the TME not possible with traditional immunoassays like western blot and flow cytometry.
Novel Approach for Automated Sequential Immunoassay for Quantitation and Characterization of PI3K/AKT Pathway Proteins
Presented by Dr. Jessica Dermody
Abstract: The P13K/Akt signaling pathway modulates growth, survival and apoptosis and this pathway is frequently modulated in human cancers contributing to resistance to radiation and chemotherapy treatments. Akt is a target for specific inhibition and recently, a number of small molecules have been developed the pharmacologic properties of known inhibitors like wortmannin and LY294002. However, Pan-Akt inhibitors can result in unanticipated side effects due to the lack of specificity for Akt isoforms 1, 2 and 3. Therefore, detection and quantitation of Akt isoforms and their downstream targets for both expression levels and phosphorylation states is crucial for therapeutic drug development. Here we demonstrate application of Replex™ to characterize the P13K/Akt signaling pathway. This approach uses sequential analysis of proteins separated and immobilized in a capillary, by performing either dual immune assays or immunoassay with total protein on the Simple Western platform Chemiluminescence detection. Assays with control and LY294002 inhibitor-treated samples were developed. Proteins were first separated based on molecular weight based on capillary electrophoresis, followed by immobilization via UV-crosslinking. Next P13K/Akt pathway targets were sequentially probed in the same capillary with total and phospho-specific antibodies to determine the phosphorylated fraction relative to the total fraction. Primary antibodies from the first immunoprobe was removed with the detection probe with >95% efficiency, as confirmed by re-probing with the same secondary antibody. Target protein loss was negligible due to covalent immobilization to the capillary wall, which was confirmed with re-probing, thus validating the quantitative data generated using this sequential approach. In addition, total protein normalization was performed in tandem with the immunoassay in the same capillary. This approach enables normalization of phosphorylation levels and/or target abundance in cell line or tissue samples, correcting for change in protein content due to treatment, loading and/or other systemic errors. These results present the utility of the RePlex™ to quickly characterize and quantify proteins involved in signaling pathways targeted during development of cancer therapies.
Multiplexed Protein and RNA Quantification on a Single Instrument Harmonizes Multi-omic Analyses of Biomarkers for Immunotherapies and Targeted Therapies in Non-Small Cell Lung Cancer
Presented by Dr. Chris Heger
Abstract: Multi-omic approaches can combine protein, DNA, and RNA analyses to elucidate diagnostic biomarkers and pathways, advancing our understanding of complex diseases. These assays, however, require different technologies and platforms to resolve the distinct physico-chemistries of protein and DNA/RNA. In contrast, single-platform quantification of proteins and nucleic acid markers offers many potential benefits, including reduced sample requirements, decreased inter-assay variability, streamlined and less error-prone workflows, and integrated results reporting. Here we demonstrate expanded capabilities of an established protein analysis system (Simple Western, ProteinSimple®) to characterize nucleic acids, and show that this system can quantify oncogenic tyrosine kinases, immune checkpoint proteins, RNA translocations, and other mRNA transcripts associated with targeted or immune-based therapies for non-small cell lung cancer (NSCLC).
- View webinar: "Single-Platform Technology for Protein and Nucleic Acid Biomarker Analysis in Non-Small Cell Lung Cancer"