Charge Variant Analysis Information

Get structural insight into protein charge variants

The analysis of charged variants is a regulatory requirement for bio-therapeutic proteins. These large heterogeneous molecules can be subject to a variety of enzymatic post-translational modifications during manufacture, such as glycosylation and lysine truncation. In addition, chemical modifications can occur during purification and storage such as oxidation or deamination. Ion exchange charged variant analysis is a high-resolution technique that has proved very useful in the analysis of such variants. Protein charge variant profiling is mandated in regulations such as ICH Q6B .


Protein charge variant analysis by ion exchange chromatography

Protein charge variants can be separated using physicochemical fractionation—based on charge characteristics of antibodies or other proteins. Historically, protein separation has been performed using ion exchange chromatography with salt gradients. A key challenge of salt-based gradients is that a unique gradient needs to be developed for each individual target protein molecule. Moreover, poor reproducibility of salt-based gradients meant that method replication and robustness was often poor and method development was time consuming.

In 2009 Genentech first published the use of pH gradients for charge variant separations instead of salt gradients. The advantages of pH-based gradients include a global applicability of the method to any monoclonal antibodies (mAb), greatly simplifying the method development. It was found that mAbs with isoelectric points in the range of around 7–9 could be well separated using cation exchange columns operated in a pH-based elution mode. Moreover, the approach was more generic and could easily be used to separate the different charge variants in a range of antibodies using a single method, that could also be significantly shorter using pH gradients (30 min.) rather than conventional ionic strength salt gradients (90 min.).

New columns and instrument technologies have shown the potential to decrease the run times even further:
shorter small particle size columns combined with linear pH gradients can bring this analysis into the domain of a true UHPLC application.

A difficulty with this technique is how to produce a truly linear pH gradient. Several buffers have to be employed to cover the whole of the pH range at concentrations so that the buffering capacities of each buffer match each other. The column itself will act as a buffer against any pH changes, so careful selection of a high resolution/low capacity column is required. The difficulty in producing a linear pH gradient with buffer cocktails can be seen clearly from the image below, where a clear curved gradient is produced in response to a programmed linear gradient. This can be overcome through the use of commercially available gradient buffers which, in combination with the correct column, produces a linear gradient. This enables method optimization to be achieved easily and logically and delivers robustness and consistency between operators and laboratories.


Are there alternative techniques?

Isoelectric focusing using capillary electrophoresis (CE) has also been used as a fast global method for charged variant analysis. Whilst this method has shown promise, it is prone to long equilibration times and operator inconsistency. HPLC or UHPLC pH gradients are now faster and more reproducible than CE alternatives. HPLC or UHPLC also have global applicability and familiarity in all biopharmaceutical laboratories. Moreover, HPLC or UHPLC allows the fractionation of any new variant peak detected, so that it can be positively identified with further analysis.

For more information on the products and technologies for protein charge variant analysis please visit our Charge Variant products page.


Featured charge variant profiling learning content

Video

Take just 5 minutes to learn how patented pH gradient buffer kits enable you to easily produce linear pH gradients for fast and robust therapeutic protein separations.

Application

This study shows that pH gradient–based strong cation exchange chromatography on the ProPac SCX-10 column can provide excellent resolution for MAb charge variants. The variants were partially identified as sialylation variants. Compared to the routine IEF method, this method is more convenient and straightforward for protein quality control.


Protein charge variant profiling literature library

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TypeTitleProduct FocusYear
Application BriefHigh Salt Gradient Analysis of Post-Translational Modifications- Deamidation MonitoringHPLC / UHPLC2015
Application NoteA Fast and Robust Linear pH Gradient Separation Platform for Monoclonal Antibody (mAb) Charge Variant AnalysisHPLC / UHPLC2013
Application NoteA Fast and Robust Linear pH Gradient Separation Platform for Monoclonal Antibody (mAb) Charge Variant AnalysisHPLC / UHPLC2014
Application NoteA Novel pH Gradient Separation Platform for Monoclonal Antibody (MAb) Charge Variant AnalysisHPLC / UHPLC2013
Application NoteHigh-Resolution Charge Variant Analysis for Top-Selling Monoclonal Antibody Therapeutics Using a Linear pH Gradient Separation PlatformChemistries and Consumables2015
Application NoteHigh-Resolution Separation of Intact Monoclonal Antibody IsoformsChemistries and Consumables2014
Application NoteHigh-Throughput, High-Resolution Monoclonal Antibody Analysis with Small Particle Size HPLC ColumnsChemistries and Consumables2014
Application NoteSeparation of Intact Monoclonal Antibody Sialylation Isoforms by pH Gradient Ion-Exchange ChromatographyChemistries and Consumables2016
Application NoteUtilizing the Native Fluorescence of Monoclonal Antibodies for the Sensitive Detection of Charge VariantsHPLC / UHPLC2016
Application NoteHigh-Resolution Separation of a Fusion ProteinHPLC / UHPLC2016
Application NoteMonoclonal Antibody Analysis on a Reversed-Phase C4 Polymer Monolith ColumnChemistries and Consumables2015
Application NoteSeparation of Monoclonal Antibody (mAb) Oxidation Variants on a High-Resolution HIC ColumnChemistries and Consumables2014
Application NoteSeparatoion of Intact Monoclonal Antibody Sialylation Isoforms by pH Gradient Ion-Exchange ChromatographyChemistries and Consumables2014
Application NoteUsing the NISTmAb reference standard to demonstrate a simple approach to charge variant analysisHPLC / UHPLC2017
Application NoteEvaluation and Application of Salf- and pH-Based Ion-Exchange Chromatography Gradients for Analysis of Therapeutic mAbsHPLC / UHPLC2018
Application NoteSimple Charge Variant Profile Comparison of an Innovator mAb and a Biosimilar CandidateHPLC / UHPLC2018
PosterAutomated MAb Workflow: from Harvest Cell Culture to Intact Mass Analysis of VariantsWorkflow2012
Poster NoteAnalysis of Monoclonal Antibodies and Antibody-Drug Conjugates Using New Hydrophobic interaction Chromatography (HIC) ColumnsChemistries and Consumables2015
Poster NoteA Fast and Robust Linear pH Gradient Separation Platform for Monoclonal Antibody (mAb) Charge Variant AnalysisHPLC / UHPLC2013
Poster NoteAdvantages of High-Resolution Separation Media for Monoclonal Antibody AnalysisHPLC / UHPLC2013
Poster NoteAdvantages of Small Particle High-Resolution Separation Media for Monoclonal Antibody AnalysisChemistries and Consumables2013
Poster NoteDevelopment of a Cation-Exchange pH Gradient Separation PlatformChemistries and Consumables2013
Poster NoteHigh-Resolution Charge Variant Analysis for Top-Selling Monoclonal Antibody Therapeutics Using a Linear pH Gradient Separation PlatformChemistries and Consumables2015
Poster NoteIntact Mass Analysis of Monoclonal Antibody (MAb) Charge Variants Separated Using Linear pH GradientChemistries and Consumables2013
Poster NoteMAbPac SCX 3 and 5 µm Particle Phases for Monoclonal Antibody (MAb) Variant AnalysisChemistries and Consumables2012
Poster NoteMonoclonal Antibody Heterogeneity Characterization Using Cation-Exchange ColumnsChemistries and Consumables2011
Poster NoteSmall Particle Media for High Throughput, High Resolution Monoclonal Antibody AnalysisChemistries and Consumables2014
Poster NoteNovel Ways to Introduce the Traditional Salt Based Chromatography Techniques of Size Exclusion and Ion Exchange Chromatography of Biopharmaceutical Proteins Into High Resolution Mass SpectrometryWorkflow2017
PresentationNew chromatographic workflows for charge variant profiling, intact mAb analysis and DAR determinationWorkflow2016
Product SpecificationspH Gradient BuffersChemistries and Consumables2013
Technical NoteDevelopment of Ultra-fast pH-Gradient Ion Exchange Chromatography for the Separation of Monoclonal Antibody Charge VariantsHPLC / UHPLC2014
White PaperCharacterizing Therapeutic Monoclonal AntibodiesHPLC / UHPLC2016
Case StudyBringing Biosimilar Therapeutics To Market Faster – A CRO Case StudyChemistries and Consumables2017

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