CEInfinite AESlytes Carrier Ampholytes

Name: Isogen Life Science B.V.
Address: Veldzigt 2a, De Meern, 3454 PW, NL
Telephone: +31 30 6880771

Carrier ampholytes are essential for isoelectric focusing (IEF) separation of proteins.

Since early 60s, carrier ampholytes were developed before the introduction of imaged capillary IEF (icIEF), the unique requirement of low UV absorbance could not be applied in icIEF application. In addition, the interaction of carrier ampholytes with protein in gel media and solution phase is different. Therefore, it is advantageous to have carrier ampholytes for icIEF with low protein interaction in solution phase for reproducible icIEF protein separation and with low UV absorbance for minimised electropherogram baseline noise.

Recently, the routine commercial brands of ampholytes have been frustrating the biopharmaceutical industry. The use of these ampholytes can cause spectral shifts which have extremely negative impacts on pI measurement and quantitation of protein charge. These artifacts include a “dip” of the baseline and “shift” of both peak and pI during icIEF separation. A “dip” is commonly observed in the acidic range when using pH 3-10 and mixed ampholytes 5-8 and 8-10.5 provided by these routine commercial brands and occurs because of variable lots and sample excipients. Similarly, a “shift” of both peak and pI results from instable lots of ampholytes and is disastrous in quality control and product release. Moreover, method optimisation usually cannot overcome the above troubles with the use of routine commercial ampholytes.

AESlytes are composed of a remarkably high percentage of effective carrier ampholytes (CAs) with small ΔpK, which provides faster focusing, more stable pH gradient and proven to be consistency between manufactured lots compared to the other commercial CAs brands of the same pH range. Thanks to our unparallel lot-to-lot stability of AESlyte CAs mitigates “Dip” occurrence and “shift” of pI and peak in icIEF separation, which guarantees consistency in QC method development.

Furthermore, AESlytes have a greater proportion of “working” CA molecules, so that sufficient resolution is maintained at lower concentrations (1-2%). Finally, AESlytes with ranges as narrow as one pH unit were created for further increased resolution

Proteins are so complex, and they react differently with CAs, depending on the functional groups of CAs. Therefore, we developed three series of AESlytes that possess different basic functional groups to minimise protein interaction. AESlytes are available in High Resolution (HR), Super High (SH) and Ultra High (UH) resolution series.

Comprehensive AESlytes
AESlytes changed icIEF' world for complex proteins
Other AESlytes Applications
Overview of other CEInfinite Solutions
Custom Made Solutions Services

Comprehensive AESlytes

We provide the most comprehensive AESlytes ampholytes with the widest pH range and the highest pI resolutions in the industry.

Please order your AESlytes ampholytes and other icIEF reagents and consumables here!

Our AESlytes and other icIEF reagents can be used on any (i)cIEF platform.

Our unique Solution for you

Lot-to-lot consistency and reduced background noise

AESlytes HR series has proven to be alternative to other commercial brands while providing better lot-to-lot consistency and minimal background interference. In the below example shows the comparison to the three lots of AESlytes, the other commercial brand show an increase in haemoglobin isomer variability (see below table).

icIEF separation of ASFC Hb to demonstration pH gradient consistency across three Lots of 3-10 HR AESlytes (bottom three) and other commercial brands 3-10 carrier ampholytes (top three).

icIEF Baseline signals of three Lots of 3-10 HR AESlytes and of other commercial brands 3-10 carrier ampholytes

Relative Standard Deviation (%RSD) of Main Haemoglobin Isomers Separated with AESlytes versus other CAs

Relative Standard Deviation (%RSD) of Main Haemoglobin Isomers Separated with AESlytes versus other CAs

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

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pH gradient linearity

The below figure demonstrates the AESlytes ampholytes (A) linearity is comparable to the other commercial 3-10 carrier ampholytes (B). For the determination of the linearity of the pH gradient, 15 pI markers between pH 3 and 10 were focused.

Linearity of pH gradient generated with 3-10 HR AESlytes (A) and other 3-10 carrier ampholytes (B)

Linearity of pH gradient generated with 3-10 HR AESlytes (A) and other 3-10 carrier ampholytes (B)

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

Extremely high pH CAs and pI Markers

Challenges such as protein drugs with high pI, for the extremely high pH CAs and pI markers, we offer in the below example the AESlytes HR 9-12 and pI Marker 11.20.

Resolution at lower concentrations

The AESlytes have an increased proportion of “working” CAs such that they sufficiently separate samples at lower concentrations than any other recommended CAs. The below example demonstrates the maintained resolution of icIEF separation of the NISTmAb reference material with AESlyte concentrations decreasing from 4% to 1%.

For direct coupling of icIEF to a mass spectrometer (MS), a lower concentration of CAs entering the MS with the sample will result in a stronger sample signal for deconvolution.

icIEF Separation of the NISTmAb Reference Material at Decreasing Concentra¬tions of AESlytes HR 8.5-9.5.

icIEF Separation of the NISTmAb Reference Material at Decreasing Concentrations of AESlytes HR 8.5-9.5.

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Increased separation resolution

Increased CA resolving power is desired, especially for more basic samples (pH 7-11). Narrow range AESlytes, with pH ranges as small as 1 pH unit used in combination with CEInfinite Preparative icIEF facilitate isolation of narrower charged isomer peaks.

The below example demonstrates the increase in resolving power of AESlytes HR 8.5-9.5 compared to a mixture of AESlytes HR 3-10 and 8-10.5 when focusing the NISTmAb reference material.

Comparison of NISTmAb separations with standard range AESlytes and new narrow range AESlytes HR 8.5-9.5

Comparison of NISTmAb separations with standard range AESlytes and new narrow range AESlytes HR 8.5-9.5

AESlytes changed icIEF' world for complex proteins

Complex protein drugs including bi-specific Ab, ADC (antibody-drug-conjugate), fusion proteins, mRNA and LNP are popular in biopharmaceutical industry due to their targeted drug delivery and selective therapeutic effects. However, their complex charge heterogeneity makes icIEF characterization rather difficult and routine commercial CAs cannot address such a challenge. AESlytes provide unmatched high-resolution capacity to ultra-highly characterise the charge variants of difficult protein drugs.

Please contact us for further information or consult with us for the right choice.

AESlytes for fusion proteins

The below figure demonstrates much better separation resolution of Etanercept protein isomers than other commercial brand CAs 3-10.

 AESlytes for fusion proteins

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

The below figure demonstrates a much better separation resolution of Fusion Protein FP-AT-1 protein isomers than other routine commercial brand CAs 2-9.

icIEF comparison of fusion protein (FP-AT-1) between AESlytes UH3-10 (top) and routine commercial ampholyte 2-9 (bottom)

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

The below figure demonstrates some more examples of Fusion Proteins - Enbrel analogues, RhVEGFR-Fc (Aflibercept analogue), RHVEGFR-FC (Conbercept analogue) and EPO-HSA

Fusion proteins with unparallel resolutions using AESlytes CAs

Fusion proteins with unparallel resolutions using AESlYtes CAs

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

AESlytes for bi-specific antibody

The below figures show the charge variants of a bi-specific Ab (BSAb-AT-1) that were not well separated using other commercial brand CAs (bottom) but could be well solved by AESlytes (top). This demonstrates the outstanding resolution of AESlytes for improving the separation of a complex Bispecific Ab.

AESlytes (top) achieved outstanding resolution compared to other commercial brand CAs (bottom) for bi-specific antibody (BSAb-AT-1)

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

AESlytes for ADCs

The below figures demonstrate the use of AESlytes HR and UH series CAs were for characterising complex ADCs with excellent resolution of protein isomers with different binding ratios.

ADC icIEF with unparallel resolutions using AESlytes RH and UH CAs

ADC icIEF with unparallel resolutions using AESlytes RH and UH CAs

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

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AESlytes in icIEF-MS

The established methodology for CEInfinite icIEF-MS is highly-sensitive and allows rapid heterogeneity characterization of monoclonal antibodies and other complex proteins including antibody-drug-conjugate (ADC) and bi-specific Abs. In addition, just by changing capillary separation cartridge, the icIEF-MS configuration can flexibly switch to a High Yield Fractionation icIEF-based fraction collection model for isolating charged variants for subsequent peptide mapping by high performance liquid chromatography (HPLC) tandem high resolution mass spectrometry (HRMS).

The use of the AESlyte CAs and permanent MC or AD coated cartridge (patent pending) for better electrospray ionization and MS signal. The AESlytes are also designed for reducing the background noise that has traditionally been an issue for icIEF-MS. The below figures demonstrates that the AESlytes has much lower MS background interference than other routine commercial CAs, meaning adequate MS sensitivity and selectivity could be obtained for trace protein characterisation.

Much lower MS background interference from AESlytes series (left) than other routine commercial brand CAs (right).

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Overview of other CEInfinite Solutions

All CEInfinite test kits, solutions are fully tested and certified. The products are manufactured under strict quality control guidelines to save you time as well as eliminating variability for a more reproducible result.

Please note that the CEInfinite AESlytes CAs and other solutions can be used for any iCIEF as well as for CIEF assays.

If you need any additional information, please do not hesitate to contact us.

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Custom Made Solution Services

We understand how complex a protein drug product can be and that the available standard icIEF reagents and/or coated cartridges do not always provide you the required high efficient separation.

Therefore, we are offering standard broader choices of icIEF reagents and special coated cartridges besides the custom made solutions for the separation of more complex protein samples. Our icIEF reagents can be used on any (i)cIEF platform. Our special coated cartridges can be used on the CEInfinite icIEF instruments.

You can choose from the following:

Custom made ampholytes; specify your required pH range or consult with us for the right choice. You can review our available standard AESlyte ampholytes. We are the only provider on the market that offers more choices than any other suppliers.
Custom made pI Markers; specify your require pI marker or review our available standard over 50 pI Markers. Our comprehensive pI Markers are suitable for UV Absorbance as well as Fluorescence detection.
Custom made coated cartridge; specify your required coating or consult with us for the right choice. We are the only provider on the market who offers the most comprehensive choices of coated cartridges for icIEF separation.

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Why Isogen Life Science?

At Isogen Life Science, we believe that long term relationships with life science professionals and suppliers are fundamental to solve life science problems. Our curiosity drives us to continually stay involved and to dig deep in dialogue, with both clients and suppliers. It’s these intensive collaborations that enable us to develop tailored, innovative products that push life science’s boundaries. Partner up with us and enjoy our quick support for troubleshooting and questions!