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Cobetter Pultrix XQ Membrane Adsorber Solution for Efficient Plasmid Purification

2025.04.10 416


Plasmid-based mRNA vaccines, as well as viral vector technology, are at the forefront of the fight against cancer, cardiovascular disease, immunological and infectious diseases. In order to realize their full potential and reach as many patients as possible, we need to optimize the production process to achieve greater efficiency, and a key step in this process is plasmid production. One of the key steps is plasmid production. 


However, traditional production methods suffer from low plasmid yields and require complex purification protocols, so today we are introducing the Cobetter Pultrix XQ membrane adsorber (chromatography) solution for efficient plasmid purification.


Process Challenges

The target plasmid accounts for only about 3% of the total E. coli lysate, and many of the impurities are negatively charged and similar in size to the plasmid, making purification of the plasmid very difficult, and the final purified ultra-helical plasmid must comply with the purity standard set by the regulatory agency of >90%.


In addition, since plasmids are very sensitive to shear forces, which may cause topological changes during the concentration process, tangential flow ultrafiltration needs to be minimized in the process to improve the quality of the final plasmid product.


On the one hand, plasmids are much larger than proteins, which can result in low binding loads and slow mass transfer for conventional chromatographic packing materials due to their large size and inability to enter the dead-end pore structure of the packing material. On the other hand, the increased size of the plasmid can also significantly affect the differential pressure and processing time of the packed column due to feed viscosity and potential fouling.


In spite of their many drawbacks, conventional chromatographic packing processes are still used in large-scale purification processes. However, as manufacturing processes and their products have evolved, plasmid manufacturers are also considering enhancing the chromatography step with products that use convective media (i.e., membranes, monolithic columns, etc.) in order to improve productivity and increase overall throughput.


Cobetter Solutions

The Pultrix XQ membrane adsorber offers a significant improvement over traditional resins. The convective flow channels created by the great pore membrane structure, the high density of quaternary ammonium binding sites and the fast mass transfer efficiency allow the binding sites of the Pultrix XQ membrane adsorber to rapidly capture larger target molecules (e.g., plasmids) at high flow rates while achieving the target purity required for GMP production. The Pultrix XQ membrane adsorber therefore provides a significant increase in productivity (g/L/hr).


We use the XQ membrane to capture plasmids at a binding load of ~8mg/ml directly after clarifying the lysate with an appropriate amount of P3 solution (or NaCI solids), and then elute to obtain pure plasmids, which can remove >99.99% of the RNA from the lysate. Pultrix XQ membrane adsorber purified plasmid purity ≥99% (SC plasmid >90%), yield >80%, and run time per lysate capture cycle only 30 minutes (based on RT=6S). minutes per lysate capture cycle (based on RT=6S and running flow rate of 10MV/min).


In addition to performance, the easy-to-install, scalable capsule design reduces preprocessing time and footprint, allowing for operation in different production environments, eliminating the need for column loading and potency measurements, and allowing for easy, quick access. 


Overall, the increased productivity and simplicity of operation make the Pultrix  XQ Membrane Adsorber (Chromatography) an excellent solution for plasmid production.


Case Study

The starting material for the Pultrix XQ membrane adsorber capture process is E. coli lysate.


Table 1. Lysate for Salt Concentration Adjustment in Classical Lysis System


Cracking system P1:P2:P3=15:15:15


RNA binds less to anion exchange media than more strongly charged plasmids. Therefore, prior to Pultrix XQ membrane adsorber capturing, salt is added to the lysate (e.g., supplemented with P3 solution in the appropriate amount) to obtain better conductivity conditions, so that impurities such as RNA are flow-through during Pultrix XQ membrane adsorber uploading and plasmids are bound at high loads and eluted with high purity plasmids in the subsequent elution step. 


This allows direct capture of the plasmid in the lysate without the need for RNase or CaCl to precipitate the RNA. It was further found that, under appropriate salt concentration conditions, the ring-opening plasmid would flow through the lysate before the superhelical plasmid due to competition for adsorption, so that controlling the amount of sample could help to increase the overall proportion of superhelixes.


Table 2. Information on Materials, Equipment and Consumables

Table 3. Chromatography Buffer Information


Experiments were carried out using Pultrix  XQ membranes (MV:0.3 ml) according to the experimental conditions in the table below: 


Table 4. Experimental Conditions



During the sampling process, flow-through samples were taken, eluted and collected according to the peaks and then sampled to run the gel, measure the concentration, HPLC, and various impurity check items, and the results are shown below:


Table 5. Experimental Results


Experimental results show that the Pultrix XQ membrane adsorber has a one-step plasmid capture yield of 80+%, a super-helical plasmid ratio of 90+%, and impurities such as endotoxin, HCD, etc. can also be effectively controlled. 

The project has successfully achieved stable linear scale-up on Pultrix  XQ95ml with 2 cycles per batch and a total process time of less than 90min.


Comparison of Cost, Efficiency: Packing vs. Membrane Adsorber



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