iCE technical library

このポスターではMaurice CE-SDSの分子量範囲（10～270 kDa）をどのように広げ、小さいタンパク質や巨大タンパク質を分析することができるのかを示します。

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.

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.

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.

CRISPR/Cas9 has become a revolutionary tool for precise genome editing in a wide variety of prokaryotes and eukaryotes, and multiple Cas9 variants have been engineered to further broaden its functionality. Purity, heterogeneity and stability are critical properties in the biophysical characterization, chemical modification and structural investigation of Cas9 and its variants. At the forefront of techniques to monitor these properties are capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and capillary isoelectric focusing (cIEF). Maurice™ from ProteinSimple enables both CESDS and imaged cIEF (icIEF) in a single unit, with several key advantages over other CE-SDS and cIEF systems, such as ease-of-use, high data quality, and speed.

In this poster we show that this reagent, SimpleSol can
effectively solubilize proteins for icIEF but is a significantly more stable agent than urea, eliminating the need for analysts to prepare urea fresh every time SimpleSol is also stable when pre mixed with methylcellulose as opposed to urea and is compatible with absorbance and native fluorescence detection on Maurice icIEF In addition we show that SimpleSol had less of an impact on the acidic portion of the pH gradient formed during icIEF compared to urea, resulting in more stable pI values of protein peaks relative to using urea.

Most post-translational and degradation events affect the biological activity of therapeutic proteins, making charge heterogeneity analysis a critical quality attribute when assessing molecule efficacy. Regulatory agencies require characterization of charge variants as well as monitoring them throughout the product development and manufacturing process. The iCE platform reproducibly delivers this data in less than 10 minutes per injection. A simple workflow adds the ability to quickly develop platform methods giving you the gold standard for protein charge heterogeneity characterization.

In this poster, we introduce the new member of the iCE family, Maurice. Maurice delivers the same high quality data as the iCE3 system and also adds the Native Fluorescence (NF) detection mode for higher sensitivity. NF detection gives you 4x higher sensitivity compared to absorbance, potentially decreasing the need to desalt samples or add urea to prevent molecule aggregation. Additionally, reduced ampholyte background in the fluorescence detection mode gives you more options when it comes to improving profile resolution. Resulting separations had baseline resolution with %CV < 2% for peak with higher than10% composition over 100 injections. Data is automatically analyzed for you at the end of the run using Compass for iCE.

Sample preparation for cIEF analysis requires protein formulations to be modified with carrier ampholytes, pI markers, and other additives prior to injection. This can pose challenges, as some proteins are chemically unstable when exposed to highly basic environments, undergoing degradation reactions when stored under these conditions for extended periods. The onboard sample preparation feature of the new iCE3 IEF Analyzer can elevate preparative artifacts such as protein degradation by facilitating just-in-time (JIT) sample preparation. The automated sample preparation capability of the iCE 3 also has added benefit of reducing operator to operator variability. This feature can be implemented with either a 96 well plate or a 48 position standard vial tray. In this poster we demonstrate the results of automated sample preparation of 96 samples in a 96 well plate and compare them to the results using manual sample preparation.

cIEF analysis requires the protein sample to be pre-mixed with carrier ampholytes, pI markers, and other additives. Proteins can be sensitive to highly basic environments and can experience degradation when exposed to these conditions for extended periods. The iCE3 automates sample preparation minimizing protein sample degradation in IEF buffers and operator to operator variability. The sample is prepared immediately prior to injection limiting sample exposure to cIEF buffers and preventing degradation.
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The development of a robust and repeatable automated sample preparation assay requires optimizing sample mixing parameters. During optimization, several factors need to be considered, such as the shape of the sample vial, the number of mixing strokes, air bubbles and sample dilution by the mixing action.

Electronic data authenticity and integrity are an integral part of GMP manufacturing. The FDA guidance 21 CFR Part 11 defines the characteristics required for GMP compliant electronic records and signatures.
It is important to note that 21 CFR Part 11 compliance specifies additional procedural controls (i.e. notification, training, SOPs, and administration) to be put in place by the user in addition to the technical controls that the software provides. iCE Software contains the following 21 CFR Part 11 technical controls:

• User Log-In Function limits system access to authorized individuals
• Electronic Signature is required throughout run execution, processing and exporting
• A secure, computer generated, time stamped audit trail records the date and time of operator entries and actions that create, modify, or delete electronic records
• The software provides accurate and complete copies of records in both printed and electronic format. Note: All iCE software designs are and will remain backwards compatible
• Uses operational system and network domain features to ensure data authenticity and integrity are maintained The software uses file string encryption and applies the industry standard checksum algorithm to verify data integrity. In addition, in QC-function, operational restrictions strengthen GMP compliant batch execution. For example, once started, a batch may not be modified - the batch must be stopped/aborted, then renamed and restarted. The batch may not be paused and modified.

When methods and instruments are implemented in Quality Control, it is critical that any replacement instrument provide equivalent data with minimal or no changes to the method. The iCE280 system has been adopted by most major biopharmaceutical companies worldwide for charge heterogeneity analysis. The iCE3 is the next-generation iCE280 and the first update to the iCE280 in 15 years. The iCE3 features a host of technical improvements, all designed for direct transfer of methods from the iCE280. As a direct replacement for the iCE280, the iCE3 must have equivalent applications performance. In this poster, we demonstrate direct method transfer and system equivalency using the following instruments and assays.

Capillary isoelectric focusing (cIEF) is the best tool for protein charge heterogeneity characterization. Common sources of charge heterogeneity for monoclonal antibodies include heavy chain C-terminal lysine heterogeneity, deamidation, and sialyation associated with the glycosylation sites on the antibodies. Under the denatured and reduced condition, disulfide bonds in the antibodies can be reduced and the monoclonal antibodies are broken into heavy chains and light chains. The heavy chain and light chain are expected to have different pI values, thus, they can be separated from each other by IEF. In this way, the contributions of the heavy chain and light chain to the entire antibodies' charge heterogeneity can be observed by IEF analysis.

In this presentation, monoclonal antibodies are analyzed by cIEF under a 8 M urea (denatured) and DTT condition. In the example shown in the presentation, the heavy chain and light chains are well separated and the charge heterogeneity of both is observed.

During capillary isoelectric focusing (cIEF), proteins are focused at their isoelectric point. When performing IEF in a capillary, two forces within the capillary can destroy the IEF process within the capillary column: electroosmotic flow (EOF) and hydrodynamic flow. For reproducible cIEF, both of these forces must be eliminated. EOF is suppressed by neutral coatings on the inner wall of the capillary. Elimination of hydrodynamic flow is dependent on the system design. Traditional capillary electrophoresis instruments minimize hydrodynamic flow by leveling the capillary inlet and outlet vials. In imaged cIEF, specifically the iCE280, elimination of hydrodynamic flow depends on the autosampler. Small differences in hydrodynamic flow can result in differences in reported pI values from system to system. Although the difference is relatively small, it may create issues during method transfer between labs.

In this poster, we describe how the new iCE3 IEF Analyzer simplifies method transfer and minimizes system-to-system variability caused by hydrodynamic flow.

The well-characterized biopharmaceutical requires an assessment of charge heterogeneity. Techniques like IEF gels, ion exchange chromatography, and traditional capillary IEF all have benefits, but each one has its own set of challenges. Imaged cIEF (ProteinSimple's iCE IEF Analyzer) combines the best of these three worlds by providing rapid analysis, platform methods, and simple method development.

For cIEF analysis, samples are pre-mixed with carrier ampholytes, pI markers, and other additives. Although iCE IEF Analyzer offers rapid analysis and high throughput, some proteins can still experience degradation when exposed to these conditions for extended periods. On-Board Sample Preparation with the new iCE3 IEF Analyzer solves this problem. The system prepares the sample immediately prior to injection, limiting sample exposure to cIEF buffers and preventing degradation. As an additional benefit, automated sample preparation eliminates tedious pipetting. Simply load your sample and go.

This poster presents the application of On-Board Sample Preparation to the analysis of proteins.