AldeActive PuraBead®
Product Code: IP00075 & IP00076
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 bead sizes, PuraBead® 6HF (90 µm) and PuraBead® Edge (65 µm), that both achieve superior uniformity, porosity, and flow properties compared to leading market products.
AldeActive 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.
An ideal choice for applications requiring protein or antibody immobilization, aldehyde-activated beads provide an excellent platform for attaching primary amine groups through the formation of stable Schiff base linkages. They are particularly effective when specific interactions between ligands and target molecules are crucial, such as the capture of proteins, peptides, and nucleic acids.
Properties of AldeActive PuraBead® 6HF:
ACTIVE GROUP: Aldehyde group
ACTIVE GROUP DENSITY: ≥14 μmol/g settled
MEAN PARTICLE SIZE (µm): 90 ± 10 µm
MATRIX:
PuraBead® 6HF (Highly cross-linked 6% nearmonodisperse agarose)
CHEMICAL STABILITY: pH 3.0 to pH 14.0
STORAGE: 2 –30°C, 20% ethanol
Properties of AldeActive PuraBead® Edge:
ACTIVE GROUP: Aldehyde group
ACTIVE GROUP DENSITY: ≥14 μmol/g settled gel
MEAN PARTICLE SIZE (µm): 65 ± 10 µm
MATRIX:
PuraBead® Edge (Highly cross-linked 6% nearmonodisperse agarose)
CHEMICAL STABILITY: pH 3.0 to pH 14.0
STORAGE: 2 –30°C, 20% ethanol
LIGAND COUPLING
AldeActive PuraBead® resin is supplied in a preservative containing 20% ethanol.
The method below describes a guide for coupling ligand to Astrea Bioseparations’
AldeActive 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 out the 20% EtOH preservative with water.
2. Dissolve the ligand in the coupling buffer (pre-warmed to an appropriate temperature, typically 20 – 60°C dependent on ligand stability). A medium to buffer ratio of 1:1 gives a suitable suspension for coupling reactions.
3. Mix the coupling solution to the medium in a stoppered vessel. Use a shaker water bath for up to 22 hours at the appropriate temperature (20 – 60°C dependent on ligand stability)
4. Monitor ligand coupling reaction progress, if on-line testing method is available.
5. Wash away excess ligand using coupling buffer.
6. Block any remaining active groups.
7. Wash the product thoroughly with an appropriate buffer (Example: 10 washes using 0.1 M sodium dihydrogen phosphate dihydrate buffer, pH 7.0) followed by a water wash.
INFLUENCING FACTORS
pH
The stability of the ligand limits the maximum pH which can be used.
Coupling solution
Coupling should be performed in a buffer solution (such as 1 M potassium dihydrogen phosphate buffer solution).
The coupling process involves the functionalized aldehyde groups on the agarose beads, reacting with primary amines (-NH₂) on biomolecules (e.g., lysine residues in proteins) to form Schiff bases (imine bonds, R-CH=NR’), and the subsequent reduction using a reducing agent to form a stable secondary amine (-CH₂-NH-R’), ensuring permanent immobilization.
Temperature
Coupling can be performed at 20 – 60°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 could decrease at higher temperatures. The efficiency of coupling is also pH and temperature dependent. 16–22 hours at 20–60°C is most often used.
Ligand concentration
A very high ligand concentration can influence 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.
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 could then be washed pH 7 buffer solution at least three times, if required. Acetate buffer (0.1 M, pH 4) and coupling buffer (pH 8.2) each containing 0.5 M NaCl can also be suitable. This washing procedure ensures that no free ligand remains ionically bound to the immobilized ligand.
Regeneration conditions
Regeneration depends on which ligand has been coupled. Literature references and textbooks may give good guidelines.
Storage
AldeActive PuraBead® should be stored 2 - 30°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.
ORDER INFORMATION
Gel slurry
IP00076-00050
IP00076-01000
IP00075-00050
IP00075-01000
Research use only.
PuraBead® Edge
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
