As quality by design (QbD) has become increasingly adopted in biopharmaceutical manufacturing operations, the need for rapid analytical techniques for the analysis of proteins has also risen. These methods must be robust, easy-to-use, and applicable during product development and on through commercialization. Susan Darling, director of product marketing with ProteinSimple spoke with Cynthia Challener, editor of the Pharmaceutical Sciences, Manufacturing & Marketplace Report, about imaged capillary isoelectric focusing (icIEF), a new capillary-based method for protein charge variant analysis that can dramatically reduce analysis times.
Rapid, robust, and easy-to-use
Pharmaceutical Sciences, Manufacturing and Marketplace Report: What are the key attributes of a good protein analysis system used in pharma manufacturing?
Darling (ProteinSimple): The ideal protein analysis system is rapid, robust, and reproducible and provides critical information on product quality that is easily transferred across the entire pharmaceutical process. It should allow biopharmaceutical companies to accelerate product development.
A good example relates to the analysis of charge variants of therapeutic proteins, because this property is critical for the characterization and monitoring of the quality attributes of proteins. Different types of charge variants include deamidation, formation of N-terminal pyroglutamate, aggregation, isomerization, sialylated glycans, antibody fragmentation, and glycation at the lysine residues. In some cases, such changes affect binding, biological activity, patient safety, and shelf life.
Pharmaceutical Sciences, Manufacturing and Marketplace Report: What performance requirements are needed to ensure effective and efficient analysis throughout the pharmaceutical development process and during commercial manufacture?
Darling (ProteinSimple): Methods for charge heterogeneity need to detect small changes reproducibly, but also have to be automated and easy to use. Newer capillary-based methods, such as imaged capillary isoelectric focusing (icIEF), are becoming increasingly adopted across the biopharmaceutical industry because they provide high resolution separation of charge variants with minimal development and start-up times.
Pharmaceutical Sciences, Manufacturing and Marketplace Report: How does icIEF work and how is it different from conventional separation/analysis techniques for protein charge variants?
Darling (ProteinSimple): The icIEF method involves imaged capillary isoelectric focusing, or isoelectric focusing performed in a capillary. Protein isoforms are separated based on their isoelectric points in an ampholytic pH gradient and detected by a whole column UV absorption detector. The process takes 15 minutes start to finish and combines the resolution of a traditional gel IEF system with the advantages of quantitation and automation found in standard column-based systems.
Pharmaceutical Sciences, Manufacturing and Marketplace Report: In addition to high resolution, what other advantages does icIEF provide?
Darling (ProteinSimple): An additional benefit of capillary-based techniques such as icIEF is that these methods are not product specific. Given the large pipeline of pharmaceuticals, the ability to use generic methods reduces the time required for analytical method development. A technique that has these benefits can be implemented across the entire pharmaceutical process, from cell culture development and optimization to commercial quality control (QC) release and stability activities, resulting in an accelerated product development process.
High throughput capability
Pharmaceutical Sciences, Manufacturing and Marketplace Report: What recent advances in protein analysis technology have been made and what need(s) in the marketplace led to their development?
Darling (ProteinSimple): Biopharmaceutical manufacturing is evolving from a traditional approach to a QbD strategy. QbD requires determination and measurement of key quality attributes during manufacturing development. As a result, significantly more samples must be run during process development to set the design space of QbD. High throughput methods are therefore very important for the implementation of a QbD strategy.
For example, Simon Briggs, a senior scientist on the in-process analytics team at UCB Pharma, has shown that a process characterization study at his company may generate more than 1000 samples for all of the upstream and downstream processes that must be evaluated. A traditional tool for charge heterogeneity analysis, such as ion-exchange chromatography, which has an average run time of 70 minutes, would require 50 days to analyze all of these samples. Fortunately, advances in high throughput techniques such as icIEF for the determination of charge heterogeneity have recently been made. At UCB, according to Briggs, by switching to an icIEF method with a 15 minute run time, UCB was able to reduce the analysis time for process characterization studies to 7 days.More information on the work at UCB Pharma can be found in a Separations Now webinar presented by Briggs entitled “Charge Variant Analysis for High Throughput Process Development,” which can be found at: http://www.separationsnow.com/details/webinar/13d161c91da/Charge-variant-analysis-for-high-throughput-process-development.html.
Protein complexity still a challenge
Pharmaceutical Sciences, Manufacturing and Marketplace Report: What are some of the current issues/limitations that still remain that must be addressed to improve protein analysis?
Darling (ProteinSimple): To keep up with the throughput demands of QbD and protein therapeutic development, analysis methods still need to be faster and more sensitive, and ideally, simpler to use. However, proteins aren’t simple. They are complex and highly individual, and post-translational modifications like glycosylation can result in a large number of isoforms. In addition, protein therapeutics are constantly evolving to include fusion proteins, antibody drug conjugates, and bispecific antibodies. Developing protein analysis systems that work robustly with all of these types of molecules and those new ones that haven’t even been developed yet is a major challenge.