iCE technical library

Powering Up Maurice with Waters™ Empower® Software

Recent advances in vector engineering, delivery and safety have placed viral vector-based therapy at the forefront of gene therapy, with adeno-associated virus (AAV) being one of the most actively investigated. To support the rise of AAV vectors in the clinic, technological solutions that afford robust quality control assays are essential for implementing Good Manufacturing Practice (GMP), meeting regulatory requirements and ensuring the clinical quality, safety and consistency

In this application note, we’ll show you how Maurice data compares against SCIEX PA 800 systems for reduced and non-reduced CE-SDS separation of a reference monoclonal antibody from the National Institute of Standards and Technology (NIST).

Maurice’s CE-SDS application delivers speed, automation, reproducibility, and high-resolution data. The CE-SDS PLUS system preserves features and adds enhanced sample stability and data consistency.

In this application note, we compare Maurice™ against PerkinElmer’s LabChip® GXII Touch, a chip-based electrophoretic separation system. Under reduced and non-reduced conditions, we evaluate CE-SDS separation using a reference mAb from the National Institute of Standards and Technology (NIST). Maurice and LabChip are assessed for their performance on linearity, sensitivity, precision, reproducibility and resolution, with the technological approach, workflow and data quality outlined for easy comparison.

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.

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.

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 10^13 GC/mL) and ‘empty’ (<10^12 GC/mL) AAV capsids.

Monoclonal antibodies (mAbs) are routinely used as therapeutic and diagnostic products when treating a wide variety of diseases. Due to the importance of these molecules, regulatory agencies have guidelines describing methods to assess product purity necessary for lot release and characterization. CE-SDS is one of the most common techniques used to monitor purity and heterogeneity.

iCE3 has been the go-to method for analyzing charge heterogeneity for mAbs. In this poster, we introduce Maurice, the latest member of the iCE platform family, who now gives you high resolution CE-SDS IgG data on top of the exceptional cIEF data you've come to expect.

Maurice's CE-SDS application gives you baseline resolution of reduced non-glycosylated and glycosylated IgG heavy chain in just 25 minutes with % Area RSDs less than 4%. A simplified workflow provides unparalleled ease-of-use. Once samples and reagents are prepared, it takes less than 10 minutes to install the cartridge and start your batch. And at the end of your batch, easy clean-up and automatic data analysis by Compass for iCE software lets you quickly start your next batch.

Analytical techniques like isoelectric focusing have become indispensable tools in evaluating biologic preparations. The resulting surge in charge isoform analysis has led to major advances in instrumentation, such as Imaged Capillary Electrophoresis iCE�. To maximize the benefits from improved instrumentation requires development of robust assays. Initially implemented in biopharmaceutical manufacturing, Quality by Design (QbD) has the potential to transform assay development. Key to QbD is comprehensively gauging the effects of process inputs on critical to quality (CTQ) attributes of the output. To this end, the Design of Experiments (DOE) methodology has proven itself to be a highly efficient tool in modeling the relationship between input and output.
The contents of this poster demonstrate the successful implementation of DOE tools in the iCE3 assay development process. Utilizing a Central Composite Design (CCD) strategy, a response surface of the relationship between ampholyte composition and focusing time on peak resolution was generated. Experimental validation of this model at optimal operational settings indicated a high level of accuracy with error between the predicted and experimentally derived values ranging between -0.68 and 8.06 percent.

Three major usability improvements are now available for the iCE3 system. A new HT cIEF Cartridge increases resolution while reducing run times by eliminating the need for methyl cellulose. A redesigned electrode arm assembly minimizes cathodic drift allowing robust analysis of 100 samples in a batch. The pI calibration and data export processes have been combined into a single automated procedure through the development of enhanced software features. These updates offer greater speed and improved ease of use. In this poster we present the results of these improvements on a model iCE method for analysis of a basic IgG1 mAb. The new HT cartridge reduces analysis time by five minutes, while still providing a highly resolved peak profile comparable to the original FC cartridge. An intermediate precision study demonstrated a %CV of less than 10% for peak all major peak clusters (>5% percent composition).

iCE3 Separations
Separations were performed on an iCE3 system
equipped with either a PrinCE Next MicroInjector or an
Alcott 720 NV Autosampler.

In order to bring an antibody or other biologic drug to market, each step of the process needs to be carefully monitored. Charge heterogeneity and apparent molecular weight (MW) via SDS-PAGE are two of the most commonly assessed parameters. Ideally, similar methods should be used from the beginning of development (low expression, complex matrix) to late in production and QC (high concentration, pure material). Capillary techniques, such as iCE and CE-SDS, are currently used heavily in downstream product development but are best suited to purified and higher concentration samples. Here, we present the Simple Western that combines in one novel instrument, Peggy, capillary electrophoresis with an immunoassay to provide highly reproducible and fully automated analysis of monoclonal antibodies. This sensitive technology measures either size or charge in complex samples and provides critical charge heterogeneity, size, and product titer information without the need for sample purification. Data will be presented demonstrating the application of the Simple Western technique and the iCE technology for the analysis of monoclonal antibodies against VEGF and the ability of the two techniques to provide consistency of data across the whole range of product development. In addition we show examples for the unique capability of this technology to assess affinity information for these anti VEGF antibodies to different charge isoforms of VEGF.

Platform methods, high throughput and ease of use have made the iCE system the gold standard for protein charge heterogeneity characterization for biopharmaceuticals. At 15-18 minutes per sample, iCE methods are fast and simple. However, biopharmaceutical companies are always looking for higher throughput. In this poster, we investigate a new rapid iCE method that utilizes a new column coating, eliminating the need for high viscosity polymer additives. By eliminating the viscous polymer additives, the column rinsing and sample injection cycles can be reduced from 2.5 minutes to 25 seconds. The required focusing time can also be reduced by 2-3 minutes for a high resolution monoclonal antibody platform method. The peak pattern and resolution of the new column coating is compared to the FC coating in the current iCE cartridge. The new column coating provides high resolution while increasing sample throughput to 10 minute per sample.

In cIEF analysis, samples are pre-mixed with carrier ampholytes, pI markers, and additives. Although imaged cIEF (iCE3 IEF Analyzer) offers rapid analysis and high throughput, proteins can still experience degradation when exposed to carrier ampholytes and additives for extended periods. Automated 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.

There are several important method parameters such as Mixing Rate, Mixing Strokes and Mixing Depth that require optimization. This poster describes an optimization strategy for the on-board sample preparation and provides relevant examples.

In isoelectric focusing (IEF), at the end of the focusing process, all components in a sample are focused and stop at their pI points. In order to perform IEF in a capillary column (cIEF), two forces within the column that interfere with the focusing process have to be eliminated: electroosmotic flow (EOF) and hydrodynamic flow. In commercial cIEF instruments, the EOF is substantially reduced by column coatings and the hydrodynamic flow is eliminated by placing both ends of the column at the same level during the IEF process.

In ProteinSimple's iCE280 IEF Analyzer, the hydrodynamic flow within the separation column is eliminated by using a specially designed, constant fluid level waste vial at the outlet of the column and a balancing vial at the column inlet in the autosampler that has the same fluid level as the waste vial. This design constantly provides equal fluid levels at both ends of the column regardless of the waste volume dumped into the waste vial.

However, some high concentration additives could generate an unbalancing force within the column during IEF for some unknown reasons. One example is high concentration urea. When these high concentration additives are used, the peak pattern sometimes is pushed back or forth within the separation column during the IEF process, making the peak pattern unstable. This can reduce separation resolution and reproducibility.

We found that this problem could be solved by plugging one end of the column during the IEF process with a micro switch valve. The new design will be adapted in ProteinSimple's new model iCE instrument.

In this presentation, we will compare the peak pattern stability of a monoclonal antibody on the iCE of the new design and the existing design when 8 M urea is used as the additive.

Carrier ampholytes are the single most important component of a capillary IEF separation. Unfortunately, all carrier ampholytes are not created equal. While carrier ampholytes are available from a number of manufacturers, they have significant differences. Understanding those differences accelerates method development.

Today, three brands of carrier ampholytes are commercially available: Pharmalyte by GE, Servalyt by Serva and Biolyte by Bio-Rad. While pH gradients from all three are quite linear, they do differ in baseline noise and peak resolution for some proteins. For example, Servalyts have higher resolutions for fusion proteins and proteins with heavy sialyation.

In this poster, we will discuss the effects of the carrier ampholytes on peak pattern, baseline noise and resolution.

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

A difference between CE-SDS and SDS-PAGE is that discrepancies in apparent molecular weight (MW) have been observed between the two methods. While CE-SDS and SDS-PAGE are typically not intended for high-resolution MW determination, many of their applications require a reasonable estimate of apparent MW. This Technical Note highlights relevant peer-reviewed publications that address these factors for comparing MW between CE-SDS and SDS-PAGE.

ProteinSimple iCE CFR software is used for system control, data acquisition and collection of critical audit information. The files used by the iCE CFR software are created using a file format unique to the software. The file types created by the iCE CFR software have the extensions ICF, CAL, DAT, BCH, IEF and ITF. This document provides file type descriptions and file locations for iCE CFR software V3 and V4. Recommendations for data backup are also provided.

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.