Epoxy-Activated PuraBead PC IP00062 & IP00064 User Guide-v1
Epoxy-Activated PuraBead®
Product Code: IP00062 & IP00064
INTRODUCTION
Astrea Bioseparations has over 30 years of expertise in chromatography chemistry. Our trusted PuraBead® resins are used by global pharmaceutical companies in 21 FDA-regulated processes.
PuraBead® resins are available in two different sizes, PuraBead® 6HF (90 µm) and PuraBead® Edge (65 µm), that both achieve superior uniformity, porosity, and flow properties compared to leading market products.
Epoxy-Activated PuraBead® offers a cost-effective, easy-to-use solution for custom ligand attachment. These beads provide the flexibility and efficiency you need, allowing you to tailor your process without the hassle of bead activation, giving you greater control while streamlining your workflow.
Ideal for protein purification, antibody capture, enzyme immobilization, and biomolecule separation, it offers a stable platform for attaching amino, thiol, and hydroxyl groups, creating strong bonds between ligands and resin. This versatility makes it perfect for attaching both small molecules and larger biomolecules like peptides and proteins.
Epoxy-Activated PuraBead® resin is supplied in a preservative containing 20% ethanol.
The method below describes a guide for coupling ligand to Astrea Bioseparations’
Epoxy-Activated PuraBead® .
Preparation of activated slurry
1. Determine the % slurry of the resin by agitating the resin in the bottle to form a complete slurry. Pour out volume of approximately 15 mL into an appropriate measuring cylinder. Leave to settle for a minimum of 3 hours. Calculate the % slurry:
Slurry % calculation:
2. Calculate the volume of slurry you require, a compression factor (CF) of 1.19 (1.15–1.23) is recommended:
Column volume (CV) calculation:
Where ‘r’ is the radius of the column hardware in cm and ‘BH’ is the target bed height in cm.
Slurry volume (SV) calculation:
Where ‘CV’ is the column volume calculated above, ‘CF’ is the compression factor, and ‘slurry percentage’ is the slurry as calculated above.
3. Measure out the calculated volume of slurry required.
OPERATING INSTRUCTIONS
Note: The following recommendations are not prescriptive A thorough investigation of these parameters at small-scale should be conducted to reveal the level of flexibility that can be tolerated with the activated resin, buffer, and ligand combination selected.
1. Drain the weighed-out slurry and wash the 20% EtOH preservative with water.
2. Dissolve the ligand in the coupling buffer (pre-warmed to 60°C). A medium to buffer ratio of 1:1 gives a suitable suspension for coupling
3. Mix the coupling solution to the medium in a stoppered vessel. Use a shaker water bath for 22 hours at 60°C.
4. Wash away excess ligand using coupling buffer.
5. Block any remaining active groups. Transfer the medium to 1 M NaOH. Let it stand overnight at approximately 40°C.
6. Wash the product thoroughly with 0.1 M sodium dihydrogen phosphate dihydrate buffer, pH 7.0 followed by a water wash.
INFLUENCING FACTORS
pH
The coupling reaction proceeds most efficiently in the pH range of 8 –13. Coupling to hydroxyl groups requires high pH and should therefore be performed around pH 13.
The stability of the ligand limits the maximum pH which can be used.
Coupling solution
Coupling should be performed in a boronate or phophate solution (such as 1 M potassium dihydrogen phosphate buffer solution, pH 8.0 – 8.2). Sodium hydroxide may be used for making solutions of high pH.
Tris and other buffer salts containing amino groups or other nucleophilic components should not be used since these will couple to the medium.
Organic solvents may be needed to dissolve the ligand. Dimethylformamide and dioxane may be used to up to 50% of the final mixture. The same concentration of organic solvents should be included in the coupling buffer. Always adjust the pH after dissolving the ligand, since organic solvent usually lowers pH.
Temperature
Coupling is performed at 60 ± 2°C, preferably using a shaker in a water bath. Direct heating and magnetic stirrer should be avoided. The stability of the ligand limits the maximum temperature which can be used.
Time
The time for the reaction depends largely on the pH of the coupling solution, properties of the ligand, and temperature of coupling. The coupling time decreases at higher temperatures. The efficiency of coupling is also pH and temperature dependent. 22 hours at 60°C is most often used.
Ligand concentration
A very high ligand concentration can have adverse effects on affinity chromatography. Firstly, the binding efficiency of the adsorbent may be reduced due to steric hindrance between the active sites. Secondly, substances are more strongly bound to the immobilized ligand which may result in difficult elution. Thirdly, the extent of non-specific binding increases at high ligand concentrations. As a general guideline; load 100 to 400 μmole ligand/mL drained medium (approximately 5 to 10 times concentration of active groups).
Blocking excess remaining groups
Remaining active groups on the gel should be deactivated or blocked after the coupling. Leave the medium in 0.5 M NaOH overnight at 40°C.
Washing the adsorbent
To remove excess uncoupled ligand after coupling, the medium should be thoroughly washed with coupling solution, including organic solvent if used for coupling. The medium should then be washed pH 7 buffer solution at least three times. Acetate buffer (0.1 M, pH 4) and coupling buffer (pH 8.2) each containing 0.5 M NaCl are suitable. This procedure ensures that no free ligand remains ionically bound to the immobilized ligand.
Regeneration conditions
Regeneration depend on which ligand has been coupled. Literature references and textbooks may give good guidelines.
Storage
Epoxy-Activated PuraBead® should be stored 2 – 8°C.
Coupled medium should be stored in a solution that maintains the stability of the ligand and contains a bacteriostatic agent, for example, 20% ethanol. Do not freeze.
Optimization
Optimization may be required to gain the required performance
Epoxy-Activated PuraBead
ORDER INFORMATION
Gel slurry
Code
IP00062-00050
IP00062-01000
IP00064-00050
IP00064-01000
Research use only.
Epoxy-Activated PuraBead® Edge 50 mL
Epoxy-Activated PuraBead® Edge 1000 mL
Epoxy-Activated PuraBead® 6HF 50 mL
Epoxy-Activated PuraBead® 6HF
Astrea Bioseparations also supplies larger volumes of bulk resins for cGMP development and manufacturing-scale processes.
Astrea Bioseparations can also provide column packing services. For more information on this, or any other matters please do not hesitate to contact us at sales@astrea-bio.com
