iCE technical library

icIEF analysis of Adeno-Associated Virus (AAV) proteins for Gene Therapy App Note

In this Application Note, learn how Maurice CE-SDS outperforms SDS-PAGE for protein purity analysis.

In this application note, we’ll hone in on the 21 CFR Part 11 tools integrated into Compass for iCE for batch execution and data processing, plus audit trails and electronic signatures.

In this application note, we demonstrate data equivalency across iCE instruments by running multiple molecules across all three systems. Data equivalency between iCE280 and iCE3 systems using Alcott and PrinCE autosamplers has been demonstrated before, so we focused on comparing system quantitation and reproducibility using absorbance mode on iCE280-PrinCE, iCE3-PrinCE, and Maurice systems.

Most post-translational and degradation events affect the biological activity of therapeutic proteins, making charge heterogeneity analysis a critical quality attribute for molecule characterization. Both iCE280 and iCE3 use protein absorption at 280 nm to monitor charge heterogeneity, and now Maurice adds native fluorescence detection to greatly increase cIEF capabilities.

Maurice's native fluorescence detection for cIEF works by measuring the fluorescence emission of tryptophan's aromatic group. It's labelfree so you're not wasting time optimizing protein labeling or dealing with the background noise when label unconjugates from your protein. Baselines are significantly cleaner and less sensitive to ampholyte interference, giving you more options when optimizing your pH gradient.

You'll also get 3-5X more sensitivity compared to UV absorption. That means you can nix concentrating or desalting your samples, decreasing your sample preparation time. And, because proteins tend to aggregate less at lower concentrations, you'll be able to reduce or even remove urea completely in some of your methods.

iCE3 is the next-generation iCE280 and features a number of technical improvements for better system fluidics, systemto-
system reproducibility, and improved 21 CFR Part 11 options. All improvements are designed for direct transfer of
methods from iCE280. As a direct replacement for the iCE280 system it was a requirement that iCE3 and iCE280 have
equivalent applications performance. This document demonstrates system performance of iCE3 in comparison to iCE280
for all system configurations. The comparability experiments were performed using the following instruments and assays.

This guidance defines the requirements for GMP compliant electronic records and signatures including procedural controls such as training and standard operating procedures as well as software technical controls to maintain data security.

Three major usability improvements are now available for the iCE3 system. The new HT Cartridge improves resolution and run times by eliminating the need for methyl cellulose, saving up to 5 minutes per run when compared to the original FC cIEF Cartridge. Redesigned locking electrode arm hardware also reduces evaporation and minimizes cathodic drift, and updated software features allow automated pI calibration and data export.

Now you can run your Maurice platform directly from the Empower® software

iCE consumables make operating your system fast and
easy � and get you to more consistent data at the same
time. Our kits help you develop methods and run system
qualifications, and come with all the reagents you need
to simplify day-to-day operation. They�re also designed
to work with our FC Cartridges (P/N 101700, 101701),
which need methyl cellulose in both the rinse and sample
solutions to reduce sample interactions with the capillary.
GMP/GLP needs? All consumables are tested and validated
to meet them!

Analysis with the iCE3 system just got faster! The new HT Cartridge reduces run times by 30% on the iCE3 with comparable results to the FC Cartridge. Its all-new, proprietary capillary coating eliminates the need for methyl cellulose in sample mixtures, which speeds sample loading and cuts focusing time - so runs that used to take 15 minutes are now done in 10. The HT Cartridge was developed and validated for the iCE3 and optimized for use with the iCE3 HT Upgrade.

Over 100 publications, presentations, and posters can't be wrong. Customers across sites, countries and continents rely on iCE™ technology to solve their toughest challenges and meet regulatory requirements.

We've listed a select number of publications for you, but if you visit
www.proteinsimple.com/cIEF_forum/ you'll find plenty more.

The iCE3 with Alcott 720NV Autosampler offers accurate injection combined with simple start up and maintenance. On-board sample preparation eliminates protein stability concerns by mixing ampholytes, pI markers and methylcellulose just prior to injection. Multiple tray options include 48 standard sample vials and 96-well plate compatibility.

The best designed biologics manufacturing processes are efficient and reproducible. Streamlining the many steps in process development translates to less time required to get your product to market. We’ll help you test all your samples during development to save you time and money downstream. We’ll also help you confirm the integrity of your final products.

In this eBook, we examine technological advances in protein analysis that are at the forefront of immuno-oncology research and therapeutic development.

Robust and fast analytical techniques are critical for achieving commercial success of therapeutic biologics and achieving it first. Speed up the development process with iCE and MFI from ProteinSimple. These highly sensitive technologies will streamline each step in the biologics characterization and manufacturing process so that you can win the race to FDA approval.

Prof. Dr. Hermann Wätzig discusses the benefits of capillary electrophoresis and the automated solutions in the modern laboratory

We want to keep you updated on all of our advances in protein discovery and analysis, so here is our quarterly newsletter. It's never been easier to see what we're up to because each page covers a product platform and, if you want to attend an event, all you have to do is click on it to register. From new application notes and publications to product updates, we've got you covered.

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.

Here, we provide a protocol for analyzing fluorescent ADC mimics using Maurice. Our findings demonstrate that a fluorescent conjugate can be imaged independently of the protein signal, and the dye and antibody peaks can be assigned by overlaying the absorption and fluorescence profiles.

Analysis of Rituximab by Maurice

Analysis of Golimumab by Maurice

Analysis of Bevacizumab by Maurice

Charge heterogeneity is a critical quality attribute in the development of ADCs and must be carefully monitored throughout the development pipeline. For this purpose, ProteinSimple offers iCE3™ and Maurice™ systems, which are powerful tools to analyze the charge heterogeneity of your ADCs.

Quick Start Guide to use Waters™ Empower® 3 Chromatography Data Software with Maurice.

Maurice cIEF Fluorescence Calibration Standard 5.5 mL

This technical note introduces SimpleSol, a novel and alternative reagent to urea to solubilize proteins for icIEF analysis on iCE and Maurice systems.

In this method development guide, we’ll go over how to quickly and easily develop and optimize a CE-SDS method on Maurice® or Maurice S. We’ll run through all the main parameters to adjust when optimizing assay performance, and how to start running your samples.

The CE-SDS Application Kit is a one-stop shop for all your size application development needs! It includes the CE-SDS cartridges, all sample preparation reagents and consumables that'll let you tackle any monoclonal antibody. This Application Guide will help you every step of the way.

If you add the IgG Standard, which is produced with a known quantity of non-glycosylated heavy chain, you can test resolution and quantitation suitability to make sure your Maurice is ready to go.

Review CE-SDS technology and associated failure rates in the context of QC testing and explore considerations for workflow and recovery implementation.