This product is an agarose-based chromatography medium designed for endotoxin removal, with Polymyxin B as the ligand. It maintains high resolution and gravity flow rates.
Polymyxin B specifically binds to lipopolysaccharides (LPS) from Gram-negative bacteria, enabling efficient endotoxin removal under mild conditions with high recovery rates. After purification with this endotoxin removal resin, endotoxin levels in protein solutions can be reduced to 0.1–0.25 EU/mL. The resin is compatible with buffers of various pH levels and ionic strengths, making it suitable for endotoxin removal in biopharmaceuticals, protein purification, and other biological products.
Aladdin Endotoxin Removal Resin is stored in 20% ethanol, with a settled gel to storage solution ratio of 1:1. The product specification refers to the actual volume of the settled gel.
Parameter
| Value / Description
| Matrix
| 4% highly cross-linked agarose
| Ligand
| Polymyxin B, ~2 mg/mL
| Particle Size Range ①
| 30~70 μm
| Average Particle Size
| ~50 μm
| | Endotoxin Binding Capacity④ | ~2000000 EU/mL
| Recommended Operating Flow Rate ②
| 30~150 cm/h
| Maximum Flow Rate & Pressure ③
| 300 cm/h, 0.3 MPa
| Operating pH Range
| 3–10 (recommended), 3–12 (short-term)
| Chemical Stability
| Stable in common aqueous buffers, 300 mM imidazole, 10 mM DTT, etc.
| Storage Conditions
| 20% ethanol, 2–8°C
| Shelf Life
| 3 years |
|
Notes:
① Over 90% of the beads fall within this size range.
② This flow rate range ensures sufficient contact time for effective binding.
③ Maximum tested flow rate at 10 cm column height.
④The determination of endotoxin-binding capacity (~2000000 EU/mL) requires that: The original sample's endotoxin concentration reaches a certain detection threshold; The binding efficiency is positively correlated with the endotoxin content. Refer to the table below for relevant reference data.
Protocol
1. Column Packing
The following procedure describes column packing when connected to a chromatography system:
(1) Equilibrate all materials to the operating temperature. Degas liquids if possible.
(2) Resin Quantity Calculation:
Settled resin volume = Column volume × Compression factor (1.20 for this resin).
(3) Resin Preparation: Resuspend the resin slurry thoroughly. Measure the required volume, remove storage solution by filtration, and wash 3 times with ~3 resin volumes of purified water.
(4) Packing Slurry Preparation: Transfer resin to a suitable container and add packing solution to achieve a 50–75% (v/v) slurry. Mix well before use.
(5) Pre-packing Setup: Fill the bottom of the clean column with packing solution to remove air from the bottom frit and outlet. Retain a small amount of liquid, tighten the bottom end fitting, and ensure the column is vertical.
(6) Packing: Pour the well-mixed slurry into the column in one slow, continuous motion (use a packing reservoir if needed). After adding all resin, fill the reservoir with packing solution, attach the top lid, and connect the column to the system.
(7) Compression: Allow the resin to settle naturally (or use a low flow rate of 50–150 cm/h). Apply a high flow rate (~150 cm/h; ensure pressure ≤0.3 MPa) until the interface is stable. Mark the stable height, stop the pump, and lower the adapter to the position corresponding to the compression factor (1.20). Tighten the adapter and equilibrate the column at a high flow rate.
Packing Condition
| Endotoxin Removal Resin
| Compression Factor
| 1.20
| Packing Flow Rate
| ~150 cm/h |
|
2. Column Efficiency Testing
After packing, assess column efficiency using HETP (Height Equivalent to a Theoretical Plate) and As (Asymmetry Factor) with acetone or NaCl as tracers:
Tracer
| 1.0% Acetone
| 0.8~1.0 M NaCl
| Sample Volume
| 1.0% CV
| 1.0% CV
| Mobile Phase
| Pure Water
| 0.4 M NaCl
| Flow Rate
| 30 cm/h
| 30 cm/h
| Detector
| UV-280 nm
| Conductivity |
|
Calculate HETP, N, and As using:
HETP = L / N
N = 5.54 × (Vʀ / Wₕ)²
As = a / b
HETP should be <3× the average particle diameter (HETP/D₅₀ < 3), and As should be 0.8–1.5.
3. Regeneration
Regenerate the resin before first use and after each use to minimize endotoxin levels:
Use 1% Triton X-114 (at 2–8°C) or 1% DOC (sodium deoxycholate).
Wash with 5–10 CV of regeneration solution, followed by 5–10 CV of endotoxin-free buffer.
4. Equilibration and Loading
Use endotoxin-free water, buffers, and consumables to avoid contamination.
Recommended Buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 7.0–7.4. Adjust pH to 7–8 and NaCl concentration to 150–500 mM to reduce non-specific adsorption and electrostatic interactions.
Equilibrate the column with 5–10 CV of buffer at the operating flow rate until baseline (conductivity/pH) stabilizes.
Load sample at a resin-to-sample volume ratio of 1:1 to 1:100. Larger sample volumes may improve recovery. Collect the flow-through containing the target protein.
Repeat loading 1–3 times if needed.
5. Storage
Store in 20% ethanol at 2–8°C. Do not freeze.
ZJ25F1230387