| | A Single Platform for icIEF and CE-SDS Analysis of Adeno-Associated Virus (AAV) for Gene Therapy | [概要表示] |
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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. |
| | Computer-aided Assay Development for Charge Heterogeneity Analysis by iCE | [概要表示] |
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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. |
| | High Throughput IEF Analysis Using Hands Free cIEF | [概要表示] |
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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.
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| | Faster and Easier Charge Heterogeneity Analysis with the iCE 3 | [概要表示] |
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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.
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| | Optimizing Parameters for Hands Free cIEF | [概要表示] |
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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.
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.
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| | Capillary Electrophoresis for Upstream and Downstream Biopharmaceutical Development
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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.
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| | iCE3 GMP Ready Out of the Box, Guide to iCE3 Software's Key 21 CFR Part 11 Technical Controls (WCBP 2013) | [概要表示] |
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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.
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| | An Even Faster iCE Method (CE Pharm 2012) | [概要表示] |
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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. |
| | iCE3 and iCE280 Comparability (CE Pharm 2012) | [概要表示] |
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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. |
| | Optimizing Hands Free Capillary Isoelectric Focusing (cIEF) (CE Pharm 2012) | [概要表示] |
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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. |
| | Capillary Isoelectric Focusing (cIEF) Analysis of a Monoclonal Antibody under a Denatured and Reducing Condition (CE Pharm 2011) | [概要表示] |
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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. |
| | Enhancing Peak Pattern Stability and Reproducibility in Capillary Isoelectric Focusing (cIEF) by Plugging Capillary Column During Focusing (CE Pharm 2011) | [概要表示] |
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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. |
| | Improved System to System Reproducibility with the iCE3 IEF Analyzer (WCBP 2012) | [概要表示] |
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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. |
| | Carrier Ampholytes in Capillary Isoelectric Focusing, Which One Do You Use? (WCBP 2012) | [概要表示] |
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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. |
| | Hands-Free Capillary Isoelectric Focusing (cIEF) (WCBP 2012) | [概要表示] |
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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. |