Written by expert: Gert, Associate Director Assays
HPLC assays have been widely used to analyze small molecules for decades. Now, HPLC is gaining momentum as an important tool for analysis of biopharmaceutical products. For example, after successful introduction to analyze monoclonal antibodies, HPLC is increasingly used for large particle products like vaccines. The use of HPLC allows 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. The needed antibodies for those assays 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. A polio vaccine typically contains 3 strains (trivalent vaccine). Therefore, parallel quantification of the different virus strains would be a preferred characteristic of a quantitative assay. Additionally, the relatively low precision of the D-antigen ELISA makes it necessary to perform 16 ELISA runs per polio strain. These 16 runs are needed to assure the quality of the result is sufficient for release of the Drug Product. Also, in formulation development, the ability to have rapid and precise results for the three strains in one run is highly valuable, because for each condition that is tested, a quantification test is needed.
Developing the assay
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.
Using a salt and pH gradient
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. It delivered 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 deliver relevant, fit for purpose, robust assays. We believe that there are countless possibilities for 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.
We are dedicated to help bring 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.