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A new method for the purification of adenovirus serotype 5 (Ad5) by two-column, size-exclusion simulated countercurrent chromatography (SEC) has been published in the Journal of Chromatography A.
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A new method for the purification of adenovirus serotype 5 (Ad5) by two-column, size-exclusion simulated countercurrent chromatography (SEC) has been published in the Journal of Chromatography A.1 The new method could potentially improve productivity sixfold compared to conventional batch systems.
Ad5 is a viral vector that has been developed by the biopharmaceutical industry as a potential delivery route for vaccines and gene therapy. Coauthor Paulo Mota told The Column: “We started this study because the vaccine industry is becoming very interested in process intensification. Specifically, there is now a clear need to decrease the cost per dose of new vaccines and viral vectors in order to be able to access developing country markets where the cost matters. Adenoviruses are considered one of the most suitable platforms for the production of viral vaccines and gene therapy vectors.”
One of the most costly steps in the manufacture of recombinant adenoviral vectors (as with any biopharmaceutical manufacturing process) is downstream processing to clear impurities and unwanted compounds. SEC is one standard technique performed by the biopharmaceutical industry to cleanup batches of adenoviruses, but suffers from disadvantages such as low productivity and product dilution.
To tackle these issues the team swapped single-column batch SEC for simulated moving bed (SMB) SEC using two columns switching from a batch process to a continuous process. The system had an open-loop configuration and only required two high performance liquid chromatography (HPLC) pumps. Mota said: “Calibration with acetone, blue dextran, and standard proteins was performed with each separate column and with the columns placed in series to determine the average values of porosity, number of theoretical plates, and retention factors for each key component. These data were the basis for computer-aided design of the cycle.” It was found that virus yield was increased from 57% for the batch system to 86% for the two-column SEC as contaminated Ad5 was recycled back through the system.
When asked for advice for those chromatographers wanting to test the new method, Mota said: “In multicolumn processes it is very important to first understand the batch chromatogram. We advise to perform several experiments by varying the initial feed concentration or volume and to perform the elution step at different flow rates to assess its impact on band broadening. The flow rate can have a great impact on the performance of the SEC step, especially when the objective function is the maximization of the feed throughput. Therefore, this parameter should be properly assessed before defining the cycle parameters and steps.” They also added that it is important to identify the product and the two closest eluting impurities because this will allow the establishment of modelling and cycle design. - B.D.
1. P. Nestola, R.J.S. Silva, C. Peixoto, P.M. Alves, M.J.T. Carrondo, and J.P.B. Mota, Journal of Chromatography A1347, 111–121 (2014).
This article is from The Column. The full issue can be found here