The analytical demands placed on vaccines are changing. Measuring the amount of antigen in a formulation is important, but it is only part of the story; potency, structure, and biological activity all matter too.
For David Hage, Professor of Analytical Chemistry at the University of Nebraska-Lincoln, this is where high-performance immunoaffinity chromatography comes in. His group has spent decades developing affinity-based separations for complex biological, pharmaceutical, and environmental samples. Now, that expertise is being applied to vaccine analysis – with the aim of creating faster, reusable, and more automated assays for assessing vaccine antigens.
Following his presentation at SciX on the topic, we spoke with Hage about the evolving needs of vaccine characterization, the advantages of affinity-based chromatographic methods, and why bringing biological recognition into HPLC could open up new possibilities for pharmaceutical analysis.
Please could you give me a brief introduction to you and your group’s research? Is there a “red thread” that connects your research interests?
My general research interests are in the theory, design and use of new affinity-based separations and methods in high-performance liquid chromatography, capillary electrophoresis, and other systems for the study or analysis of complex analytes and samples. The general thread that connects these areas together is the use of bio-related binding agents, or “affinity ligands,” as recognition elements that can be employed in applications such as the selective capture and separation of specific targets, separation-based biosensors, and tools for the characterization and study of chemical and biochemical interactions. For instance, we have used these methods in areas such as chiral separations, chromatographic immunoassays, and multidimensional HPLC. We have also utilized these methods to study the transport of drugs and hormones in the body, the binding of antibodies with antigens or agents such as protein A and G, and the binding and transport of pharmaceuticals in the environment. We are also interested in the production of new supports, such as affinity monoliths, and immobilization schemes for bio-related agents in HPLC, and the creation of miniaturized separation platforms that use affinity ligands for areas such as personalized medicine and environmental studies.
What motivated your recent work on vaccine analysis? Why is this area particularly timely right now?
My recent work in vaccine analysis grew out of two things. First, there was our prior interest in working with antibodies and antigens in HPLC systems to create new formats for chromatographic-based immunoassays. Second, there was an observed need in vaccine analysis for not only more rapid and convenient ways of measuring vaccine antigens but also means for obtaining information related to the biological and immunological activity of these antigens. For example, new vaccines are now being continuously developed and produced to address various diseases in both humans and animals. In addition, these vaccines must meet requirements by agencies such as the U.S. Food and Drug Administration and U.S. Department of Agriculture and have data available on their safety and quality, including an analysis of their activity. In examining these trends, my co-authors and I realized that the affinity methods I had been developing for other applications provided many of the features that were being sought by manufacturers and regulators for the analysis of vaccines and their biological or immunological activity.
How do you see analytical demands for vaccines evolving?
There are now many types of vaccines, such as those based on recombinant viral proteins, inactive viral particles, and mRNA. There have been many analytical methods used to characterize the levels and properties of the agents in these vaccines. Some prior methods used for this purpose have been enzyme-linked immunosorbent assays, Western blots, reversed-phase liquid chromatography, and mass spectrometry, among others. Many of these methods can be used to provide some estimate of the total amount that is present for the main component of a vaccine. Some of these methods also give information on the structure of such components. However, information is also needed on the expected potency of the vaccine, as well as its biological and immunochemical reactivity. I expect that as vaccines continue to develop, new analytical methods that can better address each of these needs will continue to appear. I further believe that the need for more detailed information, especially on features such as changes in structure and activity, will continue in the future as analytical methods become available that can provide this information in a faster or more convenient manner.
What makes immunoaffinity chromatography especially powerful for vaccine analysis compared with more conventional approaches?
The combined use of HPLC and immunoaffinity chromatography has a number of appealing features for vaccine analysis. For instance, enzyme-linked immunosorbent assays and related standard immunoassay methods have long been the main approach for characterizing the biological/immunochemical properties of vaccine antigens. There are, however, potential limitations to these standard methods. For instance, they often involve several manual steps for sample preparation and handling. Also, they can require long incubation times between the added antibodies or related supports and reagents with antigens in the vaccine samples, with this time sometimes reaching hours or even days for low-concentration antigens. These issues can both be solved in chromatographic immunoassays by using antibodies with supports, columns, and systems designed for use with HPLC. The resulting method, known as high-performance immunoaffinity chromatography, can provide assays that now often take only minutes, provide high precision, and can be easily automated.
Another practical limitation is traditional immunoassay methods are usually designed for the antibodies, supports and other regents to be disposable. This is unfortunate because the antibodies and supports are typically the most expensive components of the assay, and their use in only single assays leads to a loss of assay reproducibility. In contrast to this, HPLC-based chromatographic immunoassays can typically be used with the same columns and set of antibodies for many samples and assay formats. For instance, the antibodies and columns used in our current study for vaccine measurements were found to be useful 450-to-500 application and elution cycles. This allowed us to use each column for many samples and even in different assay formats, such as those involving direct antigen detection or a sandwich immunoassay.
Another thing we did in our work was to create a general platform for the analysis of vaccines that used microcolumns containing immobilized streptavidin. These microcolumns were then utilized to adsorb biotin-labeled antibodies against the vaccine antigen that was to be measured. This platform had several advantages. First, we could use this with HPLC and several immunoassay formats, as I mentioned earlier, as well as with several types of detection, including absorbance or fluorescence. This capability allowed us to easily adapt this platform for the analysis of different levels of vaccine antigens. The same microcolumn platform could also be easily modified for use with other vaccine antigens or biopharmaceutical products by using the streptavidin microcolumns and different types of biotinylated antibodies or capture agents for the desired target.
Do you expect to see more in-line or at-line vaccine monitoring taking place? Do you see affinity-based chromatographic methods playing a role here?
I do see the need for more in-line and at-line monitoring of vaccines to continue. I expect this to come about as the development of additional vaccines expands and as regulatory requirements for these vaccines become more stringent and broader in scope. This type of monitoring has the potential to make vaccine creation both faster and safer by providing real-time information on these materials during their production. Affinity-based chromatographic methods like those described in our paper are a natural fit to this need as they are often directly compatible with the types of media and solutions that are used in the production of vaccines and can quickly provide information on both the levels and biological activities of the components in these products.
What are the main technical or practical barriers that still limit wider adoption of emerging techniques such as high-performance immunoaffinity chromatography in pharmaceutical settings?
It is clear from research such as our own that high-performance immunoaffinity chromatography can be a valuable tool for a pharmaceutical setting. One barrier to the wider adoption of such methods is simply the fact that many analytical and pharmaceutical chemists are not as familiar with affinity-based separation methods as they are with more routine separation techniques, such reversed-phase or ion-exchange chromatography. Another barrier often encountered with traditional immunoaffinity chromatography is that a separate antibody column is often needed for each antigen of interest. The work in this project addresses this limitation by using a general platform with a column containing immobilized streptavidin that can then be used with many types of biotinylated antibodies. Finally, there is a need for more columns that are available commercially that can easily be used by new workers in the field who do not have a great deal of experience with affinity-related methods. We are now starting to see such columns appear on the market, which should lead to a much greater use of this powerful method for the selective separation and analysis of biopharmaceuticals and other targets in complex biological samples.
What research directions is your group most excited about right now?
We are always exploring new applications and unique approaches that can be combined with the use of biological agents in affinity-based separations. One area we are quite interested in, and which is illustrated in this paper, is in the use of microscale separation platforms that employ affinity ligands. Affinity separations are well-suited to miniaturization due to the strong and selective binding of many biologically-related ligands. Advantages of using microscale systems with affinity ligands include their need for only tiny amounts of these binding agents, the fast separation times that can be obtained, and the unique formats that can now be created for the separation and analysis of chemicals and biochemicals. Examples include the capability of doing immunoassays and the analysis of solute-ligand binding with microscale systems in the minute, second or even sub-second time domain. We are also interested in creating new schemes for combining microscale affinity separations with other methods in multi-dimensional separation and analysis methods. The combination of these microscale systems with HPLC also makes it possible to create assay formats that are not feasible with standard immunoassay supports. For instance, we have shown how HPLC systems can be used with antibodies and microscale columns for new types of displacement, competitive binding, and immunometric assays for both low- and high-mass targets.
Another ongoing theme in our recent research is the development of general immobilization schemes that can be quickly adapted for use with a broad set of binding agents or with binding agents that cannot be easily immobilized by current coupling methods. Examples of novel immobilization approaches we have used for this purpose have included new forms of biospecific adsorption, as illustrated in this paper with streptavidin microcolumns and biotinylated agents, and non-covalent immobilization methods based on steric entrapment. Areas in which we have used these immobilization approaches have included studies of changes in drug binding with modified proteins during certain diseases, the screening of drug candidates against proteins or receptors of therapeutic interest, and studies of drug interactions in biological or environmental systems that may contain complex binding agents. These new methods and applications serve to further demonstrate the immense potential that affinity-based chromatographic separations hold for both the analysis and studies of chemicals and biochemicals in a variety of matrices of importance to modern analytical and bioanalytical research.
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