BESTCHROM DEAE Bestarose FF Weak Anion Exchange Resin Instruction Manual

BESTCHROM DEAE Bestarose FF Weak Anion Exchange Resin

Introduction

Ion exchange chromatography(IEC)is a very effective method for the separation and purification of biomolecule. The method mainly relies on the interaction between positive and negative charges, and uses the charge properties and differences of different biological molecules under specific conditions to separate them. It has the characteristics of high load, good resolution, controllable condition and easy scale-up. It has been widely used in medicine, chemical industry, metallurgy, food and other fields.

IEC resin is composed of three parts: (1)Cross-linked agarose matrix, that has the characteristics of porous, hydrophilic and good chemical stability; (2)Functional groups fixed to the matrix, which are electrically charged groups, determine the properties of the IEC resin; (3)An ion (called an equilibrium ion) that has an opposite charge to the functional group and can be reversibly bound to the functional group.

DEAE Bestarose FF(Fast Flow) is a weak anion exchange resin formed by coupling diethylaminoethyl to high-flow agarose microspheres.

Technical characteristics

GuHCl,8M Urea, 70% ethanol,30% Isopropyl alcohol, 20% ethanol, 2%Benzyl alcohol

pH stability| 2~14(CIP), 2~12 (Working)
Temperature tolerance| 2~40℃, Can’t freeze, Can tolerate 121℃ high pressure sterilization

(50mM PB , pH7)

Storage| 2~30℃，20%ethanol
Recommend flow rate| 60~300cm/h
Avoid| Avoid contact with oxidizing agents, cationic detergents.

Avoid long time exposures (1 week, 20℃) to pH <4.

+Particle size is normally distributed, and particles within this range account for more than 95% of the total.
++The flow rate is linear flow rate of BXK50/30 column, column bed height 15cm, 20℃ 0.1 MPa pressure. Pressure flow rate is the relationship between pressure and flow rate at a particular pressure, and the pressure given should not be regarded as the maximum tolerated pressure of the resin. Pressure flow rate is shown in Fig 1
+++1M NaOH and 1M HAc only be used for cleaning

Method of chromatographic

Column packing

Note: It is best to equilibrate the resin slurry to room temperature before column packing.

• According the column volume to calculate the amount of resin.

Resin volume=column volume×1.15 (Compression factor=1.15) According to the volume of the settlement resin required, the suspended slurry of the resin required is calculated by the follow:

Required resin slurry1 volume = Settlement resin volume ÷ Resin slurry 1 concentration.The original concentration of resin slurry 1 is shown in the follow table

1. It refers to the original packaging resin slurry sold by Bestchrom.

Note: For non-original packaging, customer can calculate the required volume according to the actual concentration of resin slurry

• Washing the resin: Suspend the resin by shaking and pour into a funnel, remove the liquid, and wash 3 times with about 3mL distilled water/mL resin. Use a glass stick or stirrer to stir each time you add the distilled water, in order to better clean the storage solution.

• Prepare the packing slurry: Move the washed resin from the funnel into a beaker or other appropriate container, and add distilled water to obtain a 50%~75% slurry concentration, stir well and set aside for use.

• Take a cleaned BXK column (BXK series columns with diameters ranging from 1cm to 30cm can satisfy different scale chromatography applications). Take BXK16/20 for example, purge the bubbles trapped at the end-piece net by draining some distilled water through the column outlet. Leave about 1cm water at the bottom of the column and close the bottom outlet. Adjust the column so that it is perpendicular to the ground.

• Slowly pour the slurry into the column at one time (use a packing reservoir if necessary). Do not bring any air bubbles into the column.
  Packing reservoir: Empty glasstube with same diameter as the BXK column.

• Fill the remainder of the column with packing solution. Connect the packing reservoir to the chromatography system, open the flow rate, drain the bubbles in the hose, close the flow rate, and tighten the top cover of the packing reservoir.

• After pouring, stir well again with Stirrer, and then wash the resin particles on the inner wall of the column from top to bottom with the packing solution, and let the resin settle naturally until there is about 1cm of clarifying solution on the suspension. Mount the adapter and connect the adapter to the chromatography system or peristaltic pump. Lower the adapter to descend to contact with the clarifying solution and tighten the sealing ring after it is fully immersed in the  clarifying solution. With the outlet of the top piece is opened, slowly move the adapter down until all bubbles are drained.
  Note: This operation is only applicable to BXK 100 and above columns.Flushing the inner wall reduces the resin particles sticking between the seal ring and the column wall, avoiding the risk of leakage.

• When the bed height is 10cm, the flow rate can be set to 300cm/h. Open the bottom outlet, start the pump and run the setting flow rate until the bed is stability. Mark the bed height.

• Remove the packing reservoir (if any), lower the adapter to about 0.5cm above the resin surface, set the flow rate at 480cm/h,and continue to press the column using the above flow rate until the bed is completely consolidated, marking the consolidated bed height.

• Stop the pump, open the outlet of the top piece, close the outlet of the bottom piece, loosen the seal ring slightly, press the adapter to about 0.3cm below the marked position, tighten the seal ring, close the outlet, and complete the column packing

Evaluation of Packing

• Test the column efficiency to check the quality of packing. The tests are required after the column packing, during the column working life and when the separation and purification performance is deteriorated. The method of the expressing the efficiency of a packed column is in terms of the height equivalent to a theoretical plate(HETP) and the asymmetry factor(As).
• Acetone or Na Cl can be used as sample for the testing. Sample solution and eluent buffer can be prepared according to the following table.

Acetone method  Na Cl method

• Method for measuring HETP and As:

According the UV curve or the conductivity curve to calculate the column efficiency(HETP), and the asymmetry (As):

HETP=L/N
N=5.54(VR/Wh)
2
Note: VR = retention volume
Wh = half-peak width
L = column height
N = the number of theoretical plates
(The units of VR and Wh should be the same)
As=b/a

Note:

a= 1st half peak width at 10% of peak height
b= 2nd half peak width at 10% of peak height

• Evaluation the column packing
  As a guideline, if the value of HETP is less than 3 times the average particle size(d50) of the resin and the As is between 0.8~1.8, it is very efficient. The unsatisfactory results should be analyzed and the column should be repacked .

Chromatographic method

• Buffer selection: Buffer salts whose buffer groups do not act on the resin should be selected. The buffer solution with low salt (less than 5mS/cm) and high pH (usually 1 pH unit higher than the isoelectric point of the target) should be adopted to facilitate the combination of substances. Meanwhile, the stability of samples in the buffer solution should be considered. Elution buffers are usually made by adding a high concentration of salt (e.g. 1M NaCl) or low pH elution to balance buffer.
• Flow rate: According the column bed high to use the flow rate 60~300cm/h，the higher column bed high and lower flow rate.
• Sample preparation: In order to prevent blocking of the column, the sample needs to be filtered by microporous membrane of 0.45μm before loading, the pH and conductivity of the sample are adjusted to be consistent with the equilibration buffer (the pH and conductivity of the sample can be adjusted by dilution, ultrafiltration, and desalination with Bested G-25).
• Equilibration: Washing the column with equilibration buffer until the pH and conductivity of the column outlet buffer are basically the same as the equilibration buffer, which usually needs 3-5CV.
• Sampling: The loading volume is determined according to the substance content in the sample and the binding load of DEAE Best arose FF.
• Rinse: Wash the column with equilibration buffer until the UV absorption value is reduced to an appropriate value.
• Elution: Linear gradient or step-gradient can be used to increase the elution strength in the elution buffer, eluting substances with different binding strength from the chromatography column, collecting different components and detecting the location of the target.
• Regeneration: Flush the column with a high concentration of salt (eg: 2M NaCl).
• Rebalancing: After rinsing with equilibration buffer, the second sample can be loaded and repeated.

Application

Application of DEAE Bestarose FF in a certain protein drug.

Column: BXK 16/20 DEAE Best arose FF
Equilibration: 20mM Tris-H Cl pH 8.0
Elution: ①20mM Tris-H Cl 0.1M Na Cl pH 8.0 ②20mM Tris-H Cl 0.3M Na Cl pH 8.0 ③20mM Tris-H Cl 1M Na Cl pH 8.0
Sample: Protein solution

Order：
Lane 1: Sample
Lane 2~4: Flow through
Lane 5: Peak1
Lane 6: Peak2
Lane 7: Peak3
Lane 8: Peak4
Lane 9: Peak5

Cleaning-in-place(CIP)

With the increasing use of chromatography resin, the accumulation of contaminants on the chromatography column is also increasing. Cleaning-in- place can prevent the accumulation of contaminants and maintain a stable working state. Determine the frequency of CIP according to the degree of contamination of the resin (if the contamination is serious, CIP should be carried out after each use to ensure repeatability of the results).

The recommended CIP for different types of impurities and contaminants are as follows:

• 2~3CV of 2M NaCl was used to wash out the proteins with relatively tight binding.
  Removal of strong hydrophobic proteins and precipitating proteins: Clean with 1M NaOH of 2~3CV first, then rinse immediately with 5~10CV pure water.

• Removal of lipoproteins and lipids: Clean with 70% ethanol or 30% isopropanol by volume of 5~10CV first, then rinse with pure water by volume of 5~10CV.

• The above two cleaning conditions can also be combined for cleaning, namely 30% isopropanol solution containing 1M NaOH.

Note: 70% ethanol or 30% isopropanol should be degassed before use. In the CIP process, the flow rate can be chosen as 30~60cm/h. Reverse flushing can be used when the blockage is serious.

Sterilization

Since the 20% ethanol or 2% benzyl alcohol preservation solution does not have sterilization and dehydrogenation, it is recommended that DEAE Best arose FF can be treated with 1M Nao for more than 0.5-1h to reduce the risk of microbial contamination before and during use. It can also be autoclaved at 121℃ (50mM PB, pH7) for 30min.

Storage

DEAE Best arose FF is supplied in 20% ethanol or 2% benzyl alcohol. It should be stored in 20% ethanol and sealed at 2-30℃ after use, in order to prevent ethanol volatilization and microbial growth, it is recommended to replace 20% ethanol every 3 months.

Disposal and Recycling

DEAE Best arose FF is very difficult to degrade in nature, incineration is recommended to protect the environment.

Order information

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www.bestchrom.com

References

• 博格隆（上海）生物技术有限公司

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