With the technology development of advanced biotherapeutics, plasmid DNA (pDNA) is becoming an increasingly important vector for DNA vaccines and gene therapies, and the demand for high quality upstream plasmid DNA products is increasing, too. Plasmid DNA is widely produced by cultivation of recombinant E. coli. Obtaining plasmid products from E. coli cultures needs to be separated from genomic DNA in a complex lysis and purification procedure. The starting material, which is typically clarified lysate from alkaline lysis after neutralization.
Lysis and Flocculation
First, for this most used alkaline lysis process for plasmid DNA recovery, many deformed proteins and host DNA are combined by hydrophobic interaction to form flocculent precipitate when adding neutralization solution, while the target plasmids are dissolved in the supernatant, making it difficult to achieve a good solid-liquid separation.
Second, in order to further reduce the content of soluble impurities, flocculants such as CaCl₂, NH₄HCO₃ or other kind solids often be used to further precipitate impurities. However, the introduction of flocculants poses an greater challenge for clarification steps and requires larger scale multi-stage filtration systems for scale-up production.
For the above clarification process, the industry often uses centrifugal, filter bags, multi-stage filtration process, and these processes often meet the following disadvantage:
- Higher system hardware cost, larger floor space requirement and complicated operation steps.
- Exposed operation for consumables replacing, unfriendly to environment or personnel.
- Lower throughput capacity, higher first clarification turbidity, generally requires a second clarification steps.
- Poor scale-up linearity performance, more often used for lab and small-scale production.
Depth Filtration in Clarification
Cobetter depth filtration products shows very good performance even without the centrifugation step or adding filtration aids. While maintaining of good throughput, high quality filtrate can be obtained with one-step filtration.
This low-cost and small-space filtration systems can meet the clarification needs of large-scale lysates production, greatly increased motivation of the application of disposable filtration consumables, solved a series of issues of high solid content lysate clarification during plasmid scale-up production.
At the same time, its super-high clarification performance can also significantly reduce the pressure and cost for downstream purification process.
This depth filtration products solve existing issues such as poor clarification effect, solid-liquid separation difficulties and amplification difficulties. Having received good feedback from our customers, we recommend the following specifications for different scales of plasmid clarification.