CEInfinite icIEF Instruments

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CEInfinite iCIEF Instruments

Fast and in-depth characterization under a variety of different conditions including formulation and stability studies.

Our CEInfinite is a next generation cIEF system based on a Whole Column Time-resolved Imaging Detection (WCTID). It is standard equipped with a high sensitivity CMOS imaging sensor and powerful deep UV LED light source that gives an increased scan rate over 100-fold compared to any other (i)cIEF system on the market.

The icIEF-Analytical simplifies the protein charge analysis and provides the highest resolution separation. The icIEF fractionation and icIEF-MS modes allow you to characterize the post-translational modifications (PTMs), antibody-drug ratio (DAR), (im)purity, and chemical degradation underlying each protein charge isomer. The fractionated fractions can be used for your formulation and stability studies, such as peptide mapping, amino acid sequencing & composition, amino- & carboxy-terminal amino acids determination, disulfide bridge assessment, glycosylation & glycan characterization, binding studies (SPR, ELISA,..), potency studies and other biological assays

 

accelerating biologics development

Accelerating biologics development 

One icIEF workflow for your fast and in-depth product characterization

Scan rates are over 100 times faster compared to any other imaging time-resolved cIEF systems, which enables the study of:

  • Fast dynamic processes more accurately. 
  • Empowering Taylor dispersion rapid measurement of protein hydrodynamic radius.
  • Protein interaction studies.
  • Preparative time-resolved icIEF.
  • Online (native) real-time icIEF-MS.

Analytical profiling, Method Transfer - Existing CIEF Methods

Existing cIEF methods from most icIEF instruments can be transferred to CEInfinite Analytical instrument with no additional method development using the traditional cIEF FC coated capillaries.

three different kinds of USP samplesThree different kinds of USP samples

The combination of the CEInfinite high sensitive detection, the use of other CEInfinite High Performance Imaging CIEF Cartridges (Patent Pending) and the AESlyte ampholytes the resolution and separation can be significantly improved.

Analytical profiling, Method Transfer - Existing CIEF Methods 

Your Advantages – the use of CEInfinite Instrument

icIEF is the industry golden standard for protein charge analysis. The CEInfinite icIEF instrument overcomes low analytical throughput and difficult method development associated with traditional cIEF, gel electrophoresis and other available icIEF instruments:

  • User-friendly interface, reliable operation control, highly efficient data processing, choose from CEInsight, Chromeleon or Empower (coming soon) Platform.
  • Fast and simplify the protein charge variant analysis.
  • Provide you consistency, repeatability and building platform methods.
  • AESlyte ampholytes and special coated cartridges for complex proteins characterization (such as mAb, ADC, Fusion Proteins, Vaccines, mRNA, LNP and many more).
  • Easy method transfers and the utmost flexibility in method development.
  • One icIEF workflow for your fast and in-depth protein related product characterization (analytical profiling, fractionation, and online icIEF-MS).
  • Fast identification of PTMs, antibody-drug ratio (DAR), (im)purity and/or mass confirmation (online icIEF-MS).
  • Fractionated fractions are suitable for your formulation and stability studies, such as peptide mapping, amino acid sequencing, & composition, amino- & carboxy-terminal amino acids determination, disulfide bridge assessment, glycosylation & glycan characterization, binding studies (SPR, ELISA,..), potency studies and other biological assays.
  • High performance CMOS imaging sensor warrants overall system compactness, energy saving and robustness
  • Full automation for fast injection and wash cycles.
  • CE and FCC certified to guarantee your safe operation.

 

Whole Column Time-resolved Imaging Detection (WCTID) Technology - High Resolution Separation

The WCTID capillary electrophoresis (CE) systems have revolutionised protein charge related heterogeneity analysis with capillary isoelectric focusing (cIEF). 

  • Faster method development
  • Higher detection sensitivity
  • Better reliability
  • Higher analytical throughput

The CEInfinite icIEF is your total solution for high resolution cIEF assays. The CEInfinite icIEF instrument is standard equipped with a high sensitivity CMOS imaging sensor and powerful deep UV LED light source.

We recommend to use them in combination with the CEInfinite High Performance Imaging cIEF Cartridges (Patent Pending) and the CEInfinite AESlytes CAs, which are specially developed and synthesised for fast and reproducible high resolution cIEF assays. 

CEInfinite icIEF High Resolution - High Yield Fractionation

The charge variants are often analysed with ion exchange chromatography or a related technique capable of fractionation. However, it can take months to develop a proper method on these platforms, and there is no guarantee that they can match the resolution of icIEF. With the rise of ADCs, fusion proteins, and more, this problem will only become more pronounced.

Our Unique Solutions for you

The CEInfinite Preparative icIEF instrument provides you an unique, reliable and high resolution protein charged based separation for biologics. Utilising the custom-designed WCID cIEF-Prep cartridges (patent pending) delivers milligram-scale yields within a week.

Characterisation of individual peaks is essential for comprehending the nature of product isomers and protein degradation pathway. Therefore it is  desirable to perform peak fractionation or direct coupling to mass spectrometer for fast structure and other chemical degradation underlying characterisation of the separated protein isomer.

Proprietary cartridge - US Patent 10935519

Watch the video

Fast and High Reproducibility

Pressure mobilization is used instead of chemical mobilization to avoid instabilities related to Joule heating and pH gradient disruption. A typical single run is < 45 minutes with excellent reliability.

Avoid unstabilities. A typical single run is < 45 minutes with excellent reliability

                                  Analytical cartridge - 100 µm                                     icIEF-Prep cartridge - 320 µm         

 

High Resolution         

A transfer capillary with a reduced diameter and specialized coatings helps minimize dispersion during transit. A reversed polarity is also possible to improve the purity of the acidic fractions.

Improve the purity of the acidic fractions.

Automation and High Throughput

The target regions for fractionation are manually selected during the first run. Each subsequent run is then automated based on this condition, thereby enabling more than 25 reproducible runs per day with minimal user intervention.

Automation and high throughput

An estimation of the fraction purity, recovery, and quantity calculated based on the a starting concentration of 2.5 mg/mL

Orthogonal Characterization

The CEInfinite Preparative icIEF instrument can be used in combination with other analytical techniques, such as SEC-MS, to obtain a more in-depth and unique fingerprint.

High Resolution Time-resolved Imaging cIEF Fractionation – AFSC

The below work show a high resolution time-resolved imaging cIEF fractionation, fraction collection and fraction cIEF of haemoglobin control AFSC, demonstrating the speed and efficiency of the CEInfinite Preparative icIEF instrument.

High Resolution Time-resolved Imaging cIEF Fractionation – AFSC

Please contact us for further information on the materials, methods, and data analysis.

Characterizing charge heterogeneity of therapeutic antibody by icIEF-Prep and icIEF-MS

Fractionation and online mass spectrometry based on imaged capillary isoelectric focusing (icIEF) for characterizing charge heterogeneity of therapeutic antibody

Learn more about the researchers at AES, how icIEF fractionation and online icIEF-MS were developed for charge variant analysis after analytical icIEF-UV profiling. The established hybrid platform was successfully utilized for in-depth charge variant characterization of a therapeutic antibody. The fractions of a main protein component with its two major charge variants were collected for following LC-MS analysis at intact protein level.

therapeutic antibody by icIEF-Prep and icIEF-MS

The icIEF-MS coupling was performed for rapid identification of protein heterogeneity. The whole workflows of icIEF-based fractionation and MS online detection for protein heterogeneity are straight forward, reliable and accurate, which can provide comprehensive and cutting-edge technologies for protein drug quality control (QC) monitoring, online MS detection for rapid fingerprinting intact proteins and HPLC-MS in-depth characterization of collected fractions.

Read other researchers' work:

CEInfinite icIEF-MS

The direct coupling of icIEF to a Mass Spectrometer (MS) helps you to streamline the characterization process for biologics significantly. Characterisation of individual peaks is essential for comprehending the nature of product isomers and protein degradation pathway. Therefore it is desirable for direct coupling to MS for fast structure and other chemical degradation underlying characterisation of the separated protein isomer.

Or to perform peak fractionation for peptide mapping, amino acid sequencing & composition, amino- & carboxy-terminal amino acids determination, disulfide bridge assessment, glycosylation & glycan characterization, binding studies (SPR, ELISA,..), potency studies and other biological assays.

Our Unique Solutions for you

The CEInfinite icIEF-MS and Preparative icIEF instruments utilising the custom-designed WCID cIEF-MS cartridges (patent pending) for fast identification of intact charge variants. It allows you for real-time observation of the separation focusing (icIEF) as well as the subsequent mobilization process to isolate the charge isomer peaks.

  • High Resolution and Reliability

The pressure mobilization and transfer capillary, along with a carefully designed icIEF-MS interface, have enabled robust performance. No need to add a chemical mobilization agent to the sample matrix.

During the pressure mobilisation, an electric field keeps the samples in the separation capillary focused. The separation capillary has a larger Inner Diameter (ID) and the transfer capillary has a smaller ID. Hence, this difference greatly minimizes the remixing of separated protein isomers as they travel away from the cartridge.

High Resolution and Reliability

  • High Sensitivity - use of Critical Reagents

Use of the AESlyte carrier ampholytes and permanent MC or AD coated cartridge for better electrospray ionization and MS signal. AESlytes are also designed for reducing the background noise that has traditionally been an issue for icIEF-MS. Formamide can be used to replace urea which poses the same concern as MC.

aeslytes

  • MS Instrument Compatibility

The platform works with various nano source MS brands, including Thermo Fisher, Waters, Bruker, Agilent and more. For high resolution and sensitivity use the Thermo Scientific Q Exactive Plus in combination with our nanospray emitter.

MS instrument compatibility

A) The transfer capillary from CEInfinite is coupled to the B) Thermo EASY-Spray Ion Source coupling using our nanospray emitter. C) This interface connects directly to the Thermo Q Exactive Plus mass spectrometer with the entire setup in approximately 5 minutes.

Thanks to the long and small transfer capillary, the CEInfinite Preparative icIEF and icIEF-MS instruments allows also to spot the separated protein elution to a MALDI target plate.

  • Seamless Method Transfer: Switching between analytical icIEF, fractionation, and icIEF-MS simply by exchanging the cartridge and limited changes to the method. As a result, this instrument provides you a highly comprehensive and flexible charge variant characterization strategy for your biologics.
  • Makeup Solution: Acetonitrile and formic acid can be added to solution prior to MS for an improved electrospray ionization process. It is also possible to use less denaturing conditions for more sensitive molecules such as ADCs.
  • Fast identification of PTMs

The CEInfinite icIEF-MS mode enables the identification of several PTMs and chemical degradation in <45 minutes

A)icIEF profile (mAb) with 2% 8.5-10 UR AESlyte®+ 10% formamide which is mobilized towards the MS to produce a B) Total Ion Electropherogram (TIE) that displays the corresponding icIEF peaks [4]. C) Deconvoluted MS data demonstrates the identification of chemical degradation and PTMs including glycosylation, lysine residues, and deamidation.

Read other researchers' work:

Next Generation Biotherapeutics

ADCs, fusion proteins, bispecific antibodies, lipid nanoparticles (LNPs), and adeno-associated viruses (AAVs) all offer potential as the next generation of life-saving medicine. However, these modalities present unique challenges for charge variant characterization by icIEF and IEX due to a number of factors including increased glycosylation, conjugation, and hydrophobicity. Consequently, emerging tools for icIEF method optimization, fractionation, direct MS coupling seem poised to play a more prominent role in biotherapeutic development.  The below figure demonstrate how our CEInfinite icIEF Solutions can provide in-depth characterization for a highly heterogenous recombinant protein that was developed as a vaccine for SARS-CoV2.

nextgeneration biotherapeutics icief

A collaborative study demonstrating icIEF characterization of the recombinant protein vaccine which was extremely heterogeneous due to PTMs including N-glycans, O-glycans, deamidation, and oxidation . A) The icIEF profile and B) the corresponding TIE by MS illustrating at least 18 different charge isomers. C) The deconvoluted MS data for the first and second peaks highlighting D) extensive glycosylation as shown in the table.

We are pleased to announce our CEInfinite Chromeleon Driver integration of the CEInfinite icIEF and icIEF-MS.

It allows you to work in a well control laboratory environment and streamlining workflow while meeting 21 CFR Part 11 compliance.

CHAMALEON CEINFINITE AND IICIEF MS

Besides iCIEF-orbitrap HRMS using nano-ESI (Easy Spray), we have developed the nano-Emitter. It has been developed for high-efficient characterization of protein drug heterogeneity, which achievement has been published in Analytical Biochemistry*

*Imaged capillary isoelectric focusing tandem high-resolution mass spectrometry using nano electrospray ionization for protein heterogeneity characterization, Analytical Biochemistry, 2023, 680, 115312  

Have a look at our publications!

Customer reviews

Customer review on CEInfinite icIEF 

 

CEInfinite icIEF instrument enabled us to verify easily and quickly the behavior of the pI markers we have proposed.

The operation with the instrument is easy and the time for the separation in the order of minutes, while the same focusing task performed in the classical CE capillary equipment with pressure mobilization took hours.

 

 

Customer review on CEInfinite icIEF

"The CEInfinite enables direct coupling of icIEF to mass spectrometry, which is a powerful tool for the characterization of proteoforms on the intact level.

The obtained high resolution mass spectra are of excellent quality - For me a surprisingly high quality even for intact mAbs although we use generic separation methods (standard ampholytes)."

Prof. Dr. Christian Neusüß

Hochschule Aalen, Germany

 

Customer review on CEInfinite icIEF-MS

"icIEF plays a critical for monitoring charge variants during biopharmaceuticals. The CEInfinite allows us to get a massa readout for the product variants detected by icIEF, and this is a great help for understanding the clinical nature of the charge variants. 

When combining the CEInfinite with high resolution Orbitrap MS, detailed insights into product variants and product stability can be achieved. In short, icIEF MS during the CEInfinite is a very powerful characterisation tool in biopharmaceutical development."

Dan Bach Kristensen (PhD, Principal Scientist)

Symphogen A/S, Denmark 

Watch Webinar - Using direct CIEF-MS and PREP-CIEF

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