Buffer replacement solution during protein purification

Buffer replacement during protein purification

To ensure that the proteins are in the same buffer system as those used in the downstream purification process.That is, the pH value and salt concentration of the environment where the protein is located should be consistent with the purification process, in order to optimize the purification process. Increase the purification multiple, increase the recovery rate, reduce the process cost and so on.

1.Buffer replacement during protein purification

When the buffer environment where the protein is located is not sufficient for subsequent purification, buffer replacement is required.

1. The secreted and expressed protein has more components in the culture medium in the environment where the protein is located, which will affect the subsequent process. At this time, it is necessary to replace the buffer. For example, when the protein secreted and expressed by CHO cells is purified with Ni Focurose 6FF (IDA / IMAC), Due to the complex composition of the medium, which affects the combination of its protein and the nickel column or affects the performance of the nickel column, buffer replacement is required at this time.

2. After ion-exchange chromatography and partial affinity chromatography, such as heparin affinity chromatography and metal chelation affinity chromatography, proteins usually need to remove the high concentration of salt in the eluent of the previous step before continuing the next ion chromatography. Desalting is necessary, and the process of desalting is also a process of buffer replacement.

3. After the protein is subjected to ion exchange chromatography and partial affinity chromatography, the salt in the buffer system must usually be reduced to a certain extent before storage, and buffer replacement is also required at this time.

4. After the inclusion body protein is dissolved with denaturing agent, the denaturing agent should be removed in the process of renaturation, which also involves the replacement of buffer.

2.Common methods of buffer replacement

The common methods of buffer replacement mainly include membrane method (dialysis and ultrafiltration) and gel filtration chromatography.In addition, in some cases, dilution can also be used to achieve the replacement of buffer solution. For example, in the process of denaturing agent after inclusion body dissolution, denaturing agent is usually removed by dilution to achieve the purpose of buffer replacement and denaturing agent is removed to complete protein renaturation.

1. Replacement of buffer by dialysis

Dialysis uses a semi-permeable membrane to wrap the protein solution and immerse it in a dialysis buffer. Because the buffer concentration is lower than the concentration inside the membrane, substance exchange occurs on the semi-permeable membrane; however, the protein cannot pass through the semi-permeable membrane, only Small molecules such as salt ions can pass through; therefore, the salt impurities in the protein solution are exchanged to the outside through the semi-permeable membrane.

The larger the volume of dialysis buffer, the greater the driving force of small molecule diffusion.We generally recommend a buffer to sample volume ratio of 100:1, the best is 500:1.When the diffusion rate decreases and the solution on both sides of the membrane tends to equilibrium, the diffusion driving force and dialysis rate of small molecules can be maintained by replacing the buffer.We generally recommend replacing the buffer 2 to 3 times within 12 to 24 hours. The specific arrangement is as follows:

First buffer replacement: 2-3 hours after dialysis begins

Second buffer replacement: 4-5 hours after dialysis begins

Last buffer replacement: before dialysis overnight

Due to the long cycle and the need for a large amount of replacement fluid, the application of dialysis replacement buffer is greatly limited. It is seldom used in industry and is mainly still used in the laboratory. In the laboratory, it is gradually replaced by ultrafiltration tube/ultrafiltration cup and gel filtration chromatography.

2. Gel filtration chromatography

Focudex G-25 gel filtration media is widely used in the desalination and buffer replacement of biological macromolecules. Because of its mild process, it is widely used in biopharmaceutical processes, especially in centrifugal exchange, desalination and buffer replacement after affinity The application is universal.

3. Ultrafiltration

Ultrafiltration method is a membrane filtration method, which takes porous film as the separation medium, and relies on the pressure difference between the two sides of the film as the driving force to separate substances with different molecular weights in the solution, thus playing the role of desalination, concentration, purification and other methods.It has the advantages of no phase conversion, no heating, less energy consumption, mild operating conditions, no need to add chemical reagents, no damage to the heat sensitive drugs and so on.

The ultrafiltration method is relatively mature from the ultrafiltration tube for micro-processing in the laboratory, the ultrafiltration cup for small-volume processing, and the industrial ultrafiltration system technology.

Ultrafiltration is divided into direct pressure ultrafiltration and tangential flow filtration. At present, the industry is mainly tangential flow filtration, which is widely used in desalination and buffer replacement.