ion exchange chromatography

Summary

Ion exchange chromatography is a chromatographic method in which ion exchange genes (CM, SP, Q, DEAE, etc.) are bonded to certain inert carriers (cellulose, crosslinked dextran, crosslinked agarose, etc.), which are used as stationary phases, and the samples are separated according to the degree of interaction with the ion exchange groups on the stationary phases based on the differences in the charges they carry. Ion exchange chromatography has been widely used for the purification of proteins, peptides, oligonucleotides, viruses, phages, polysaccharides, and so on.

Operation method

ion exchange chromatography

Principle

Ion exchange chromatography is a chromatographic method in which ion exchange genes (CM, SP, Q, DEAE, etc.) are bonded to certain inert carriers (cellulose, crosslinked dextran, crosslinked agarose, etc.), which are used as stationary phases, and the samples are separated according to the degree of interaction with the ion exchange groups on the stationary phases based on the differences in the charges they carry. Ion exchange chromatography has been widely used for the purification of proteins, peptides, oligonucleotides, viruses, phages, polysaccharides, and so on. The ion exchanger is bonded to some ion exchange groups on the carrier, and the ion exchange groups release free ions through ionization in aqueous solution, which can be positively or negatively charged, and can be replaced by other ions with the same charge in the solution and have a strong binding force to the ion exchanger. The magnitude of the binding force is affected not only by the force between the ion and the ion exchanger, but also mainly by the ion concentration, the force in this process is electrostatic attraction, and the principle of ion exchange is electrostatic interaction. The retention time of proteins on ion exchange chromatography depends on the amount of electrostatic charge carried by proteins under the corresponding chromatographic conditions, and the amount of electrostatic charge carried by proteins is determined by the pI of the protein molecule and the pH of the solution environment in which it is located. Under acidic conditions, proteins are positively charged; under alkaline conditions, proteins are negatively charged. On the cation exchange column, only the proteins with pI greater than the pH of the mobile phase are retained on the column, while the proteins with pI less than or equal to the pH of the mobile phase are not retained, and even if their pI values differ from each other, they are all flushed out as solvent peaks at the same time directly. The order of the retained proteins is that those with small pI peak first and those with large pI peak later. The opposite is true for anion-exchange columns. The process of ion exchange chromatography is divided into four stages: equilibrium-adsorption-desorption-regeneration, in which adsorption and desorption are the main stages, and Q-Sepharose FF and SP-Sepharose FF, which are applied in the process of purifying TNF, are used as examples.

Materials and Instruments

TNF
Tris-HCl EDTA PB buffer Ammonium sulfate Dialysis solution Sodium chloride
Dialysis Bags Centrifuge Centrifuge Tubes Pipette Guns Tips Anion Exchange Matrix Q-Sepharose FF Protein Detector Cation Columns SP-Sepharose FF Test Tubes

Move

I. Preparation of reagents
1. Prepare pH 8.5 20 mmol/L Tris-HCl solution containing 1 mmol/L EDTA (or its reserve solution, just dilute it at the time of use) for use after filtration.
2. Prepare PB buffer pH 7.5 20 mmol/L, filter and use.II. Operational steps
1. Pre-treatment of samples
2. Equilibration of samples
The supernatant obtained by precipitation and centrifugation of 50% saturated ammonium sulfate in experiment I was placed in a dialysis bag in 20-40 times volume of buffer pH 8.5 20 mmol/L Tris-HCl, 1 mmol/L EDTA, stirred and dialyzed at 4°C for 24 h, with dialysate changed 3-4 times in the middle, and then centrifuged at 12 000 rpm at 4°C for 15 min, and the supernatant was collected to determine the protein concentration, and the volume was recorded.
3. Equilibration and sampling on anionic column Q-Sepharose FF
The column was loaded with anion-exchange matrix Q-Sepharose FF with a column volume of 5.4×20 cm and equilibrated with pH 8.5 20 mmol/L Tris-HCl 1 mmol/L EDTA buffer flow-washing the chromatographic column, and adjusted the range of the protein detector and the sensitivity of the recorder. The collected supernatant was upsampled at a flow rate of 8 ml/min. Equilibrate the slow liquid rinse until the effluent absorbance value returns to baseline. Collect across the peaks, measure the volume and measure the protein content.
4. Elution
Elution solution A was pH 8.5 20 mmol/L Tris-HCl, 1 mmol/L EDTA buffer, and elution solution B was a linear gradient elution of solution A containing a final concentration of 1 mol/L NaCl (according to the purification process set by the purification conditions), and the volume of the amount of each elution peak was collected, and the concentration of the proteins was measured, and the peaks where TNF was located were subjected to SDS-PAGE or to activity identification.
5. The active peak was dialyzed with pH 7.5 20 mmol/L PB for 24 h with 3 intermediate changes. Centrifugation was performed and the supernatant was taken and volume measured for protein concentration.
6. Equilibration and sampling on cationic column SP-Sepharose FF
Take the cation exchange matrix SP-Sepharose FF loaded column, column volume 2.5 × 10 cm, wash the equilibrium chromatography column with pH 7.5 20 mmol/L PB buffer flow, adjust the range of the protein detector and the sensitivity of the recorder, the peak solution of TNF after dialysis and centrifugation was up-sampled at the flow rate of 4 ml/min, washed with equilibrium solution to the baseline, collected across the peaks, measured volume , and measure the protein concentration.
7. Elution
The cation chromatography column SP-Sepharose FF was subjected to a linear gradient elution with eluent A of pH 7.5 20 mmol/L PB and eluent B of solution A containing 1 mol/L NaCl, and the elution peaks were collected, the volume was measured, and the protein concentration was measured and subjected to SDS-PAGE or activity identification.
8. Post-processing
The purity and activity of TNF are identified, and TNF products with certain potency are made by freeze-drying with excipients according to the requirements.

Common Problems

I. Selection of ion exchangers

1. Selection of formulations

Before deciding the class of ion exchanger to be selected, it is necessary to understand the pH range in which the biological macromolecule under study maintains its biological activity and solubility, and then observe the charge of the macromolecule according to its isoelectric point as well as its electrophoretic behavior within the above pH range, and then select the appropriate ion exchanger accordingly. The specific method is as follows; when electrophoresis is carried out under the pH conditions of the mobile phase, proteins swimming toward the anode can be adsorbed by the anion exchanger, therefore, anion-exchange chromatography is selected; conversely, proteins swimming toward the cathode can be adsorbed by the cation exchanger, and cation-exchange chromatography should be selected.

Usually, weak ion exchangers are used to separate proteins with high electrostatic forces and strong ion exchangers are used to separate proteins with low electrostatic forces.

2. Selection of particle size

The size of the particle size of the ion exchanger has little effect on the adsorption amount, mainly on the resolution and flow rate. Columns packed with coarse particles are not compact, with large particle gaps, which can easily cause zone diffusion; due to large particles, coarse particles of the same adsorbed amount occupy a large volume of the column bed, which broadens the peaks formed. These will lead to a decrease in chromatographic resolution. However, the flow rate of large particles is fast, the separation speed is fast, so that the purification time is shortened. Fine particle packing can be filled into a dense adsorption bed with high resolution, but with slow flow rate and high column pressure. We can choose according to the needs of their own work, if you need to shorten the separation time in order to maintain the biological activity of the components to be separated, in the preparation of large quantities of genetically engineered products, you can choose coarse particles. If we want to get high resolution, we can use ultra-fine particles. Usually we use fine or medium coarse particles in our laboratory work.

3. Buffer selection

In ion exchange chromatography, it is more critical to select a suitable buffer system to maintain an accurate mobile phase pH. The following requirements should be met when selecting a buffer:

(1) Do not destroy the structure of protein and do not affect the activity of protein.

(2) It is non-toxic and harmless to human body.

(3) Large buffer capacity, the smaller the ion concentration, the better under the premise of sufficient buffering capacity in the selected pH range.

(4) Apply anionic buffer when using cation exchange and cationic buffer when using anion exchange to avoid unnecessary ion exchange process.

(5) Do not interfere with the identification of the isolate.

At present, the common buffer ions used in cation exchange chromatography are alkylamine, aminoethanolamine, ethylenediamine, imidazole, Tris, pyridine, etc., of which Tris is the most commonly used; anion exchange chromatography commonly used buffer ions are acetate, barbiturate, citrate, glycine, phosphate, etc., of which acetate and phosphate are the most commonly used.

In practice, we decide what buffer system to use according to different mobile phase pH conditions. For example, we use ammonium formate buffer when the mobile phase is pH 3-5; we use sodium acetate or potassium acetate buffer when the mobile phase is pH 4-6; we use sodium phosphate or potassium phosphate buffer when the mobile phase is pH 6-8. In addition, Tris-hydrochloric acid buffer in the pH range of 7-9, Tris-phosphoric acid buffer in the pH range of 6-9, we often choose.

4. Selection of elution method

There are three types of elution methods in ion exchange chromatography: one is to change the buffer pH to change the protein from adsorption state to desorption state. For example, in anion-exchange chromatography, the negatively charged proteins adsorbed on the column are positively charged by lowering the pH of the mobile phase, so as to achieve desorption, and in cation-exchange chromatography, the desorption is achieved by elevating the pH of the mobile phase; secondly, the ionic strength of the buffer is increased, so as to replace the molecules that are strongly adsorbed from the ion-exchanger; and thirdly, the pH of the buffer and the ionic strength are changed at the same time. Either method can be performed by both phase elution and gradient elution.

Stage elution is performed stepwise with several buffers of different pH or several buffers of different salt concentrations. That is, the pH and ionic strength changes are discontinuous. In gradient elution, on the other hand, the elution capacity of the buffer is continuously increased, and the latter is widely used because of its better resolution.

A gradient mixer is required to create a linear gradient in classical chromatography, and the gradient mixer consists of two containers resting on the same level, one containing the starting buffer and the other containing a buffer with a higher ionic strength or a different pH. The two are connected by tubing to maintain hydrostatic equilibrium of the solutions. As the buffer flows into the column from the first container, the buffer from the second container enters the first container and automatically tops up, creating a gradient of buffer.

Nowadays, many low and medium pressure chromatography instruments (e.g., some products from Waters, Pharmacia, etc.) and high performance liquid chromatography can be used to automate the preparation of mobile phases with computer-controlled dual pumps, and it is easy to obtain linear or non-linear, continuous or non-continuous gradient modes to meet the different separation needs.

The usual requirements for a linear gradient are:

(1) The total volume of the eluent should be large enough, generally the gradient should be at least four times the bed volume, to give good resolution of the individual peaks of the separation;

(2) The upper limit of the gradient should be strong enough to allow even the most tightly adsorbed substances to be eluted from the column;

(3) The slope of the gradient should not be too large to ensure that the peaks can be separated, but it should not be too small so that the peaks are too broad or even form a trailing tail; it is generally believed that the larger the gradient volume and the slower the gradient change, the better the resolution.

Experimental operation

1. Pretreatment of packing material

2. Column loading

Ion exchange chromatography differs from gel chromatography in that the columns are usually shorter. For general work, a column of 10-40 cm in length is usually sufficient. The diameter of the column is chosen according to the amount of substance to be analyzed. For very complex mixtures and fine analyses, long, thin columns are used for better resolution; for preliminary separations, thicker columns with higher capacity can be used. The loaded columns are well equilibrated with the starting buffer and the samples are taken.

3. Sampling

The ion exchange column must be fully equilibrated before sampling. The sample volume depends on the exchange capacity of the column, usually 70-80% of the exchange capacity, and the sample volume can be unlimited. Typically, low concentration samples can be sampled at several to several hundred times the bed volume. This is extremely favorable to the more dilute samples, can play a dual role of separation and concentration at the same time, can be said to be two in one. However, when used for analysis, in order to improve the resolution, the volume of the sample volume is appropriately reduced. The ionic strength and pH of the sample solution used for ion exchange chromatographic separation must be consistent with the starting buffer (i.e., mobile phase A liquid); if not, buffer conversion should be performed, which can be accomplished by dialysis or gel chromatography.

4. Elution, sample collection

The sample is added and washed with a sufficient amount of starting buffer to remove non-adsorbed material, followed by elution. The elution can be done in different ways, with the gradient controlled by a controller or a gradient mixer. The effluent sample fractions are detected in real time by a UV detector (detection wavelength typically 280 nm) and collected by a segmented collector or manually.

5. Regeneration

After the sample has been eluted it is regenerated to remove some of the proteins that are still bound to the column and have not been completely washed off, usually by eluting 1 more column volume with 100% mobile phase B solution.

6. Equilibration

The regenerated column should be re-equilibrated for re-sampling. The equilibrium is usually performed by washing the column with at least four column volumes of 100% Solution A (i.e. 0% Solution B).

7. Preservation

When the column is not in use, it should be washed with a strong eluent to remove impurities bound to the column, then washed thoroughly with distilled water and stored with bacteriostatic agents.

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