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Novel HPLC assays allow for better biopharmaceutical development

Meet the expert: Xia, Analytical Assay Scientist.

HPLC has been widely used for decades in the analysis of small molecules and is gaining momentum as an important tool for analyzing biotechnology products. For instance, HPLC has been successfully introduced for the analysis of monoclonal antibody-based products and is more and more used for large particle products like vaccines. Which brings us the possibility of setting up orthogonal methods for product characterization instead of classical biochemical assays, such as ELISA or SRID. One of the major advantages of using HPLC over an ELISA or SRID is that the method is not dependent on antibodies which are often not commercially available. In contrast, standard HPLC columns can generally be applied for the development of quantitative assays. Furthermore, HPLC methods have higher precision and lower detection limits than biochemical assays. Especially in the field of process development, precision of testing is essential to allow optimization of the process for maximum product yield.

Case study: cation exchange (CEX) assay for poliovirus quantification

For the quantification of polioviruses, traditionally an infectivity assay or a D-antigen ELISA is used. The cell-based infectivity assay has low precision, while the ELISA assay needs different antibodies for each strain of poliovirus. Parallel quantification of the different virus strains would be a preferred characteristic of a quantitative assay, as a polio vaccine typically contains 3 strains (trivalent vaccine). Additionally, the relatively low precision of the D-antigen ELISA makes it necessary to perform 16 ELISA runs per polio strain to assure the quality of the result is sufficient to be used for release of a Drug Product. Also in formulation development, the ability to have rapid and precise results for the three strains in one run is highly valuable, as for each condition that is tested a quantification test is needed.

To tackle the above challenges with the D-antigen ELISA assay for poliovirus quantification we developed an HPLC-based poliovirus quantification assay. We reasoned that a cation exchange (CEX) chromatography based HPLC method, capable of analyzing large molecules or particles based on their differences in surface charge would provide a starting point for such an assay. CEX separates ionic molecules based on their affinity with the cation exchanger in the column. The decision to explore a cation exchange (CEX) chromatography approach was fueled by the fact that the three polio virus strains have slightly different capsid proteins. Further analysis indeed showed small variations in acidity meaning that the isoelectric points of the capsid proteins from the strains differ.

Based on our extensive experience with chromatography, we predicted that using either a pH or salt gradient would not result in sufficient separation between the peaks of the three strains. Subsequent laboratory testing proved our hypothesis. We therefore decided to create a cunning combination of a salt and pH gradient to ensure elution of the three strains separately. This new method resulted in a complete separation and determination of each of the three virus strains, delivering very precise quantitative and qualitative results in one go. This novel CEX assay allows testing of the drug product 3 times more cost-effective with a significantly lower invalid rate and a significantly lower detection limit per strain compared to the D-antigen ELISA. Additionally, when using the CEX assay for in process samples, cross contaminations of the other virus strains can be detected, which would not have been detected with the ELISA.

This example is a clear demonstration of our ability at Batavia Biosciences to think out of the box and to deliver relevant, fit for purpose, robust assays. We believe that there are countless possibilities for the development of novel assays, be it biochemical, analytical or biomolecular in nature. It is always very rewarding to see our creation making a difference in the development of a novel product.

As a company dedicated to help bringing biopharmaceuticals to the market at higher speed, with reduced costs, and with a higher success rate, Batavia Biosciences has vast experience in developing product specific assays. With our team of experienced researchers, using the newest technologies and techniques, we are well equipped to take on any challenge associated with assay development.

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