With the demand for growing plasmid quantities of recent years in response to the more widespread application of plasmid DNA in vaccination and gene therapy, plasmid manufacturing needs to become more efficient and boost productivity. Most of the existing large-scale processes are based on traditional chromatographic methods exploiting their comparatively high resolution. Anion exchange chromatography (AEC) is one of the most commonly used techniques and has been implemented for capture or intermediate purification/polishing. But its efficiency has been described as much dependent on sample composition according to its pretreatment and origin. We investigated AEC with the aim to utilise its full potential for plasmid production. Initial work targeted increase in binding capacity. We examined the influence of RNA and buffer conditions of the starting material on plasmid binding, screening a broad range of Fractogel® EMD and Eshmuno® tentacle anion exchange resins in batch assay. Severe interference from RNA was confirmed, when original lysate was used. Major capacity differences were observed between resins, and up to fivefold capacity increase was achieved by change of salt type or pH value. The uniqueness of DMAE and DEAE ligands was proven. Prototype resins indicated the performance advancements of future innovative products. Extending our study to dynamic conditions, we conducted capture runs with selected resins and the Natrix® Q chromatography membrane in direct comparision. Clarified lysates after different conditionings were used as feeds. Elimination of RNA interference was identified as key for powerful AEC operation, which could be achieved by different approaches. Applying direct supplementation of the original lysate with salt - our method of choice due to its feasibility and very low extra effort - , plasmid DNA was obtained with high purities and yields of 80 to 95 % and with the highest binding capacity of 10 g/L delivered by the chromatography membrane.

Enhanced capture of plasmid DNA using anion exchange chromatography

ACS Fall 2021

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