Optimizing Plasmid DNA Purification with Monoliths at AstraZeneca

With the introduction of Version 2 process, AstraZeneca has achieved a 5-fold increase in pDNA binding capacity.

Plasmid DNA (pDNA) is the backbone of most cell and gene therapy production workflows. As a key raw material, its quality significantly impacts downstream processes. The increasing demand for highly purified pDNA at the gram scale necessitates controllable, robust, and scalable processes. This blog demonstrates how Justina Martinkiene’s team at AstraZeneca incorporated monolithic columns into the pDNA purification process, significantly enhancing the binding capacity in the pDNA capture step, all while preserving product quality and step recovery.

Meet the Expert: Justina Martinkiene

The goal was to develop a process that is:

• Scalable
• Suitable for different sizes of plasmids
• Delivers required pDNA attributes despite initial cell lysate quality

To develop the process, AstraZeneca enabled three analytical tools for pDNA quality and purity monitoring. For routine testing, they use the AEX HPLC method with CIMac pDNA monolithic columns. Their analytical team employs capillary gel electrophoresis (CGE) as lot release methods. Sometimes agarose gel electrophoresis (AGE) is also used for pDNA monitoring.

1. Bottlenecks in the pDNA Purification Process Version 1

The first version of their purification process successfully purified a range of high-quality plasmids at Tox scale, achieving 30-60% purification recoveries and delivering 3 to 10 grams of pDNA per 100 L bioreactor. However, due to the low binding capacity in the capture step, the process required many cycles and multiple manufacturing slots to process a full 100 L bioreactor.

As they began working with complex constructs for novel biopharmaceutical products, the new pDNA constructs became larger and had more impurities, as well as concatemers in the starting lysate material. This complexity necessitated the development of a new capture method.

2. Development of an Improved Platform for Downstream Processing | pDNA Purification Process Version 2

To improve the pDNA capture, AstraZeneca began by evaluating various chromatography resins, media, and membranes. In the first round of media evaluation tests with a crude E. coli lysate, only monoliths showed very high yield but needed better purity. Conditioning with NaCl improved yields and purity, particularly for monoliths, leading to the selection of CIMmultus DEAE (6 µm) for further development.

Subsequently, they replaced NaCl with CaCl₂ for conditioning optimization, as NaCl was not effectively removing impurities. After developing the purification process plan, they confirmed that the AEX monolith step (CIMmultus DEAE 6 μm) was effective across various constructs and scalable (proven working across 1 mL to 800 mL). Optimization work across the purification process enabled the monolith columns to handle different size pDNA constructs (4 and 12 kB) and deliver high purification yields while maintaining high pDNA purity.

Conclusions

Comparing both purification processes, the improved process significantly increased the capture step capacity, which was previously a bottleneck, and improved productivity and process recovery. The only drawback of the improved process is that it takes a day longer to complete, extending from 5 to 6 days of work, which does not have a significant impact.

The new version 2 process achieved a 5-fold increase in pDNA binding capacity, significantly reducing batch processing times and buffer consumption, making it more sustainable. An 11- to 30-times increase in productivity was observed. The average pDNA capture step recovery increased from 72% (process version 1) to 100% (improved process version 2).

Watch how AstraZeneca enables CIMmultus DEAE columns at scale for Plasmid DNA capture.