SCIEX

Roxana McCloskey

Charge variant analysis is a fundamental tool for quality control of biopharmaceutics, providing critical insights into the structural heterogeneity of products. This type of analysis is essential because any alteration in the structure of a biopharmaceutical can affect the quality, efficacy, and safety of the final product, which can lead to clinical impacts such as unintended side effects. Charge variants are primarily induced through inherent cellular processes, such as post-translational modifications (PTMs) and genetic mutations. PTMs—which include glycosylation, deamidation, and glycation—change the protein's structure, while genetic mutations introduced during cell line development can cause changes in the protein sequence.

The key to charge variant analysis is its ability to identify and manage the charge profile of biopharmaceuticals early in upstream process development to avoid carryover into the downstream phase, where they cannot be removed. As such, charge variant analysis is critical for establishing a valid release method and, consequently, ensuring the quality and consistency of biopharmaceutical products.

Overcoming Challenges Related to Comprehensive Charge Variant Analysis

Imaged capillary isoelectric focusing (IEF) is frequently used for charge heterogeneity analysis and provides a snapshot of the proteoforms present. However, mass identification of those proteoforms is typically a manual and inherently time-consuming process, often ranging from one to two months. So often, this type of in-depth identification of a charged variant is pushed to a later stage of development. However, it is crucial to understand a protein's personality and identify steps in the manufacturing process that could introduce undesirable PTMs into the final drug products. Having this information early in the drug development process provides more data points to make decisions on a drug candidate's developability. 

To overcome longstanding time challenges associated with charge variant analysis and identification, the industry has been pioneering novel approaches and developing systems that merge icIEF with other analytical instruments to provide unified workflows for a more comprehensive analysis of charge variants. Integrating mass spectrometry (MS) with icIEF enables researchers to identify what the charge proteoforms are and to monitor critical quality attributes of a particular protein therapeutic.

A Novel Approach for Streamlined Analysis and Deeper Insight

Combining icIEF and MS provides multidimensional information. icIEF allows for real-time separation of protein isoforms with improved resolution and the ability to distinguish closely related variants. The integration of MS can enable accurate identification of product attributes—such as deamidation, glycosylation, and glycation—accelerating the identification process and reducing labor associated with multiple assays.

The direct integration of icIEF and MS streamlines comprehensive charge variant analysis, enabling the identification of PTMs at the intact protein level and accelerating the timelines associated with drug development. SCIEX has integrated these core analytical methods for charge variant analysis and identification with the Intabio ZT system, which couples icIEF separation and UV detection with high-resolution mass spectrometry on the ZenoTOF 7600 system. This technology streamlines the separation, quantitation, and identification of individual charge variants. A process that typically takes weeks and requires manual sample manipulation, fraction collection, and multiple pieces of equipment can be performed on a single platform within minutes. This integrated approach provides multidimensional data to enable researchers to make informed decisions about a protein therapeutic’s potential for development.

This was the case for Kristen Nields, senior scientist at Johnson & Johnson, who applies the Intabio ZT system to analyze the charge profiles of proteins in upstream process development. During a recent podcast, Nields shared how the system can integrate seamlessly into existing workflows while quickly and accurately identifying charge variants in real-time—enabling a detailed analysis of the peaks in charge variant profiles and providing insights that were difficult and time-consuming to obtain in the past.

Ensuring Safer Products with Continuous Improvement

Over the past 30 years, we have witnessed significant progress toward overcoming bottlenecks related to drug development. Continuous improvements have provided more detailed and accessible data to equip scientists with the data to make decisions. Technologies such as the Intabio ZT system that integrate key analytical functions enable workflows to be streamlined to ensure safe biopharmaceutical products can get to market faster.