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IEX Chromatography Leading the Charge in Charge Variant Analysis

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In a recent Q&A, Matthew Lauber, a consulting scientist at Waters told us about the importance of accurately characterizing biotherapeutic proteins and discussed the most popular techniques.

Regulatory bodies require charge variants – acidic or basic variants which indicate the level of chemical stability of a product – to be characterized.

Ion exchange chromatography (IEX) was highlighted as having advantages over other methods, as it enables the collection of separated species for additional analyses. These analyses may include structure-function characterization and tests to confirm biological significance, i.e. if binding to the therapeutic target is preserved.

To follow-up, Matthew explains how the biopharmaceutical industry has benefited from advances in chromatography technology, with a particular focus on ion exchange chromatography.

Abbreviations: HPLC: high performance liquid chromatography; IEX: ion-exchange chromatography; LC: liquid chromatography; mAbs: monoclonal antibodies; QC: quality control. 

Michele Wilson (MW): How have advances in chromatography affected modern biopharmaceutical development? 

Matthew Lauber (ML):
Rapid charge variant separations are now possible in modern biopharmaceutical development workflows due to recent advancements in chromatography, such as columns and associated reagents. A unique benefit is the flexibility to use advanced IEX columns across high performance liquid chromatography (HPLC), ultra-high performance liquid chromatography (UHPLC) and UltraPerformance liquid chromatography (UPLC), which means there is no need to change columns, and there is the chance to generate comparable high resolution mAb variant separations across different systems.

To help ensure batch-to-batch consistency and column-to-column performance in transferred validated methods, select columns are now specifically QC-tested with mAb charge variant standards. From the initial discovery stage through to manufacturing, successful mAb characterization is driven by combining advanced column technology with optimized pH buffer concentrates and mAb charge variant standards, as well as biocompatible LC systems. With the versatility of advanced IEX technology, it is possible to select pH, salt, or pH/salt gradients, and extend column life, without sacrificing separation performance.

MW: What kind of cost and time efficiences can be made by using innovative analytical approaches?

Due to minor compositional/structural differences in the various protein-based drugs being tested, method development for the LC-based determination of biotherapeutic protein charge heterogeneity can be a very time-consuming process. Fortunately, the time needed to evaluate gradients of increasing pH, increasing ionic strength, or simultaneous increases in both pH and ionic strength is minimized due to advances in instrumentation design, as well as informatics and method development software. Increased productivity and improved workflow efficiencies can be achieved by making effective use of automatable method development capabilities and the use of appropriate software packages for taking on a quality-by-design approach during method development.

MW: What’s on the horizon for this area of chromatography? 

Despite the recent innovations in biopharmaceutical characterization, it remains very laborious to fingerprint the molecular details of a biologic. However, obtaining sufficient biotherapeutic charge variant information to satisfy international regulatory agencies is becoming easier. This is thanks to continuous innovations in IEX chromatography technology, such as superior component resolution, column-to-column reproducibility, and enhanced column lifetimes. Advanced column technology, combined with robust HPLC, UHPLC, and UPLC instrumentation, is now driving reliable identification and quantification of mAb charge variants in biopharmaceutical discovery and development, and monitoring throughout the manufacturing process.

MW: Are there any new innovations that could help improve the technique for users?

ML: Stationary phases are being re-examined and more purposefully built for the separation of mAb-based therapeutics. The polymerization reactions that are the basis of the materials are being optimized by DoE (design of experiments) approaches. Moreover, it is becoming a norm for them to be quality-controlled with a mAb separation, so that nuances in the manufacturing process can be more tightly monitored for their impact on separation quality.  Mobile phase systems that are rigorously developed for broad applicability to diverse types of mAbs have also been developed. In turn, simple to implement, ready-to-use methods are available to give the right answer even from a new analyst’s very first run with the technology. Direct coupling of ion exchange to mass spectrometry is also proving to be very promising. With the right stationary phase, it is becoming possible to employ volatile mobile phase systems and to begin making direct identification of charge variants.

MW: How do such innovations in analytical technology address the challenges in IEX chromatography?

They reduce method development time, increase sample throughput, and enhance method robustness. 

MW: Can you give examples of how new technological advances have made positive changes to the way things are done?

ML: When assay technology takes a step forward, the biopharmaceutical industry is able to accelerate their research, development, and manufacture of biotherapeutics. More data can be generated with greater reliability and confidence. In turn, there is a cost savings to be had when aiming to develop a biosimilar or a specific therapeutic wherein a certain physicochemical property is being targeted. Additionally, during development, there is greater opportunity to pinpoint product-related impurities and to understand degradation paths of potential concern to safety or efficacy.

Matthew Waters was speaking to Michele Wilson, Science Writer for Technology Networks.