A New Dawn for Real Time Characterization?

In my last article (1), I discussed the benefits of surface plasmon resonance (SPR). As a quick recap, SPR has been around for over 25 years and, in particular, has become a popular method for characterizing biotherapeutics and biosimilars. SPR is label-free, allows for real-time analysis and can characterize binding in terms of kinetics and affinity. In this article, I will explain in more detail some specific examples of how SPR can be used. Biopharmaceuticals are far more complex than defined chemical drugs, so analytical tools that provide in-depth understanding of how upstream and downstream processes can affect the critical quality attributes (CQAs) of a product are crucial for developing an effective bioprocess control strategy. In the early stages of biopharma development, the focus is often on kinetic analysis, but in later phases, where the manufacturing process is in place, analysis can be simplified. Here, it is important to demonstrate that the drug substance and drug product maintain binding properties, and to determine drug potency to ensure correct dosage. For this purpose, kinetic analysis can be replaced with dose response curves. By comparison with a reference batch, the relative potency can be estimated from median effective concentration values (EC50) or by parallel line analysis (PLA). The output from EC50 analysis or PLA is a concentration. However, EC50 values can also shift because of changes in binding kinetics. To establish more conclusive dose comparisons, GE Healthcare has developed Biacore software that enables direct comparison with established kinetic profiles in a way that avoids kinetic modeling and determination of rate constants. By combining the information obtained from dose response curves and sensorgram comparison, more stringent batch-to-batch comparisons can be obtained than when only using dose response curves. Data for dose response curves and sensorgram comparison can readily be obtained in a single experiment. To cover all CQAs, an array of potency assays may be required – an aspect recently discussed by researchers from Roche, who developed Biacore assays for their bispecific CrossMab (2). The assay is based on a bridging format that enables you to look at binding of the antibody to two different antigen specificities in a single sensorgram. The final readout, reported as a single response, represents the relative amount of CrossMab molecules that simultaneously bind both antigens.

Though the Biacore assay format is flexible, pitfalls can arise when applying a bridging assay (for example, a change of antigen activity upon immobilization), so the same team developed an alternative SPR-based assay that allowed individual assessment of both targets in solution (3). Comparison of data from the two assays showed that simultaneous binding can be calculated based on both individual readouts and revealed a good correlation. Hence, the SPR-based assay principles enabled “full” functional analysis of a bispecific CrossMab in only one assay.  SPR is also commonly used for biosimilar development to compare the biosimilar with the approved reference product. Indeed, SPR is even mentioned specifically in FDA biosimilar guidelines. Other techniques, including liquid chromatography-mass spectrometry and capillary electrophoresis, are often used in biosimilar development, but they only provide structural information, whereas SPR can be used for functional studies to characterize and compare target and effector function of the biosimilar to that of the reference product. Functional studies are further used to investigate whether subtle changes in higher order protein structure or glycosylation patterns impact antigen or Fc-Receptor binding. Our Biacore systems are applied in drug discovery, but also increasingly in early development and production, replacing traditional ELISA-based methods and some cell-based assays. In June 2016, we introduced our next generation of Biacore SPR systems. Biacore 8K is a highly sensitive, eight-needle parallel SPR system to boost operational efficiency throughout drug discovery, development and manufacturing. Having been involved in the development of Biacore systems for more than 30 years, it’s no surprise that I’m passionate about their potential. Users are continuously pushing the boundaries of SPR as they seek to better understand their molecules and to develop better products for patients. We are not standing by idly and I expect the technology to continue to advance in leaps and bounds. Robert Karlsson is staff scientist in the Purification and Analysis team at GE Healthcare Life Sciences.

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