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

Webinar series

Join scientific experts from the National Institute of Bioprocess Research and Training ﴾NIBRT﴿ in Dublin, Ireland and Thermo Fisher Scientific to hear about the latest analytical advances for intact protein characterization.

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

In this study salt and pH gradients were evaluated for separation of charge variants of denosumab. The pH buffer gave enhanced separation of charge variants and offered faster screening possibilities.

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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Type Title Product Focus Year
Application Brief High Salt Gradient Analysis of Post-Translational Modifications- Deamidation Monitoring HPLC / UHPLC 2015
Application Note A Fast and Robust Linear pH Gradient Separation Platform for Monoclonal Antibody (mAb) Charge Variant Analysis HPLC / UHPLC 2013
Application Note A Fast and Robust Linear pH Gradient Separation Platform for Monoclonal Antibody (mAb) Charge Variant Analysis HPLC / UHPLC 2014
Application Note A Novel pH Gradient Separation Platform for Monoclonal Antibody (MAb) Charge Variant Analysis HPLC / UHPLC 2013
Application Note High-Resolution Charge Variant Analysis for Top-Selling Monoclonal Antibody Therapeutics Using a Linear pH Gradient Separation Platform Chemistries and Consumables 2015
Application Note High-Resolution Separation of Intact Monoclonal Antibody Isoforms Chemistries and Consumables 2014
Application Note High-Throughput, High-Resolution Monoclonal Antibody Analysis with Small Particle Size HPLC Columns Chemistries and Consumables 2014
Application Note Separation of Intact Monoclonal Antibody Sialylation Isoforms by pH Gradient Ion-Exchange Chromatography Chemistries and Consumables 2016
Application Note Utilizing the Native Fluorescence of Monoclonal Antibodies for the Sensitive Detection of Charge Variants HPLC / UHPLC 2016
Application Note High-Resolution Separation of a Fusion Protein HPLC / UHPLC 2016
Application Note Monoclonal Antibody Analysis on a Reversed-Phase C4 Polymer Monolith Column Chemistries and Consumables 2015
Application Note Separation of Monoclonal Antibody (mAb) Oxidation Variants on a High-Resolution HIC Column Chemistries and Consumables 2014
Application Note Separatoion of Intact Monoclonal Antibody Sialylation Isoforms by pH Gradient Ion-Exchange Chromatography Chemistries and Consumables 2014
Application Note Using the NISTmAb reference standard to demonstrate a simple approach to charge variant analysis HPLC / UHPLC 2017
Poster Automated MAb Workflow: from Harvest Cell Culture to Intact Mass Analysis of Variants Workflow 2012
Poster Note Analysis of Monoclonal Antibodies and Antibody-Drug Conjugates Using New Hydrophobic interaction Chromatography (HIC) Columns Chemistries and Consumables 2015
Poster Note A Fast and Robust Linear pH Gradient Separation Platform for Monoclonal Antibody (mAb) Charge Variant Analysis HPLC / UHPLC 2013
Poster Note Advantages of High-Resolution Separation Media for Monoclonal Antibody Analysis HPLC / UHPLC 2013
Poster Note Advantages of Small Particle High-Resolution Separation Media for Monoclonal Antibody Analysis Chemistries and Consumables 2013
Poster Note Development of a Cation-Exchange pH Gradient Separation Platform Chemistries and Consumables 2013
Poster Note High-Resolution Charge Variant Analysis for Top-Selling Monoclonal Antibody Therapeutics Using a Linear pH Gradient Separation Platform Chemistries and Consumables 2015
Poster Note Intact Mass Analysis of Monoclonal Antibody (MAb) Charge Variants Separated Using Linear pH Gradient Chemistries and Consumables 2013
Poster Note MAbPac SCX 3 and 5 µm Particle Phases for Monoclonal Antibody (MAb) Variant Analysis Chemistries and Consumables 2012
Poster Note Monoclonal Antibody Heterogeneity Characterization Using Cation-Exchange Columns Chemistries and Consumables 2011
Poster Note Small Particle Media for High Throughput, High Resolution Monoclonal Antibody Analysis Chemistries and Consumables 2014
Poster Note Novel Ways to Introduce the Traditional Salt Based Chromatography Techniques of Size Exclusion and Ion Exchange Chromatography of Biopharmaceutical Proteins Into High Resolution Mass Spectrometry Workflow 2017
Presentation New chromatographic workflows for charge variant profiling, intact mAb analysis and DAR determination Workflow 2016
Product Specifications pH Gradient Buffers Chemistries and Consumables 2013
Technical Note Development of Ultra-fast pH-Gradient Ion Exchange Chromatography for the Separation of Monoclonal Antibody Charge Variants HPLC / UHPLC 2014
White Paper Characterizing Therapeutic Monoclonal Antibodies HPLC / UHPLC 2016
Case Study Bringing Biosimilar Therapeutics To Market Faster – A CRO Case Study Chemistries and Consumables 2017

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