The Advantages of Implementing icIEF in the Lab
"Imaged cIEF, also known as icIEF, is a platform analytical technology and offers not only high throughput with a quick turn-around of ~10-min per sample, but also quantitation capability for charge variants, while maintaining the highest resolution of IEF technology."
- Xiaoping He, Sr. Principal Scientist, Pfizer (retired)
What drove your decision to bring imaged cIEF technology into your laboratory?
As one of the critical quality attributes (CQA) for biotherapeutic drug product development, protein charge heterogeneity had traditionally been determined with slab-gel isoelectric focusing (IEF) or HPLC Ion Exchange (IEX) methods until the birth of imaged cIEF or iCE technology. The slab-gel IEF technique is known to be tediously time-consuming and only semi-quantitative. By contrast, IEX is quantitative, but often requires product-specific separation method development which could take weeks or months to achieve. Besides, the HPLC based IEX resolution for charge species could be compromised and/or complicated by a bimodal HPLC separation nature.
Imaged cIEF technology overcomes the limitations of both slab-gel IEF and IEX mentioned above. Imaged cIEF, also known as icIEF, is a platform analytical technology and offers not only high throughput with a quick turn-around of ~10-min per sample, but also quantitation capability for charge variants, while maintaining the highest resolution of IEF technology.
Did icIEF with iCE replace other technologies that were being used in the lab, and why?
Yes, we completely replaced the slab-gel methods in the lab, and IEX (AEX or CEX) methods wherever possible for the reasons stated above.
What were the timelines for bringing in each generation of iCE instruments (iCE280, iCE3 and Maurice)?
Imaged cIEF technology overcomes the limitations of both slab-gel IEF and IEX mentioned above. Imaged cIEF, also known as icIEF, is a platform analytical technology and offers not only high throughput with a quick turn-around of ~10-min per sample, but also quantitation capability for charge variants, while maintaining the highest resolution of IEF technology.
iCE instruments Timeline Details iCE280 2004 Purchased the first iCE280 in 2002, developed platform methods in 2004, qualified and validated both platform and product specific iCE methods for GMP support by 2005 iCE3 2013 Purchased iCE3 instruments and qualified iCE3 to replace iCE280 for GMP testing Maurice 2017 - 2017: purchased Maurice systems after onsite demo
- 2017-2022: conducted bridging study and started testing support of early-stage projects in non-GMP labs
- 2022-2023: completed a multi-lab and multi-analyst equivalency test and confirmed the equivalency between iCE3 and Maurice instruments for charge variant determination using a same iCE method
- 2023: added Maurice to iCE methods for GMP testing
MauriceFlex 2023 Method development for charge variant fraction collection for subsequent mass spec characterization What were the major improvements you experienced between each instrument?
iCE instruments Major improvements iCE280 Able to quantitate charge variants, much quicker turn-around time compared to the previously stable lab technology iCE3 Simpler mechanics of switch valves and tubing connection, no need to use a syringe pump for sample transfer. Therefore, more user friendly, especially for new analysts, resulting in decreased human error and overall assay failure. Maurice - Able to run both cIEF and CE-SDS assays
- Maurice with Compass software is overall amazingly easy to set up a run
- Empower Driver for direct data acquisition
- For cIEF, native fluorescence detection for high sensitivity
- Equivalent to iCE3 for charge analysis and can use the same iCE method
MauriceFlex Has all the features listed above for Maurice, plus fractionation capability for separated charge variants collection, which can be used for subsequent characterization by MS. What are some of the key advantages of the Maurice and MauriceFlex instruments compared to the iCE3 system?
- Very intuitive and easy setup both hardware and software, extremely user friendly for running assay and system maintenance
- Direct data acquisition bridges the compliance gap of data integrity
- Native fluorescence features increased detection sensitivity 3-20 folds, making it possible for low abundance sample analysis
- No new method development necessary for existing iCE3 users, simply adopt the iCE3 method onto Maurice when using UV 280 detection
- Capability of icIEF charge and CE-SDS size analyses with one instrument
- Does everything Maurice can, as listed above
- Allows fraction collection of separated charge species for subsequent analysis including MS characterization for intact protein structural identification, peptide mapping for more detailed study on post-translational modification (PTM) analysis, and potentially potency assay for degraded charge species of stability samples to gain insight on charge-related CQAs.
How long did it take you to complete your bridging study between the iCE3 and Maurice?
We took the time to plan the experiments aiming to cover as much the research portfolio as possible. It spanned about a year for the DOE and sample preparation, and three months for lab experiment execution, data analyses and reports.
Is there any specific advice or resources you would recommend to other iCE3 Users who are transitioning to Maurice?
- Do custom appropriated bridging study using representative iCE methods on iCE3 and Maurice instruments
- prepare a set of samples representing the customer’s portfolio
- include multiple analysts of various levels of iCE experience, if possible
- involve DoE design to simplify experiment execution and data analysis integrity and validity
- If any significant discrepancy between iCE3 and Maurice occurs in a well-designed and executed bridging study, investigate the iCE test method to make sure that the separation conditions are optimal, e.g., sample prep composition of ampholyte range, strength, brands, detergent or other additive usage, focus time, etc.
- Some particularly valuable resources I would like to recommend
- Bio-Techne website and tech support
- CASSS CE Pharm conference
- Peers in the Pharma industry
- iCE3 and Maurice equivalency evaluation reports and presentations from Pfizer and Janssen, which might be accessed via Bio-Techne Tech Seminars at CE Pharm 2024, WCBP 2025, CE Pharm 2025 to come.
- Do custom appropriated bridging study using representative iCE methods on iCE3 and Maurice instruments