- Introduction
- Advantages of Endotrap®
- General specifications of EndoTrap®
- Priciple of EndoTrap®
- Application of EndoTrap®
- EndoTrap®
Introduction
Removal of endotoxin is one of the most difficult downstream processes during protein purification. Many commercially available products are unable to remove endotoxin satisfactorily, or require time consuming incubation steps. In many cases, complete endotoxin removal is only achieved with massive substrate loss.
Reduction or removal of endotoxin to less than 1 ng/mg (10 EU/mg) is a very difficult task. By using selective sorbents, endotoxin removal from proteins has clear limits. Only methods with highest endotoxin removal capacity combined with excellent recovery rates of the targets substance are reasonable and acceptable. To meet exactly these most challenging requirements, Hyglos GmbH has developed an endotoxin trap: EndoTrap®.
EndoTrap® is an affinity matrix for the efficient removal of bacterial endotoxins from solutions. EndoTrap® can be employed in both batch and chromatography mode. Non specific binding of proteins to EndoTrap® is extremely low, delivering a mass yield which typically exceeds 95%. The EndoTrap® system can be reused at least three times (in general 10 times) without loss of endotoxin removal efficiency!
Advantages of EndoTrap®
- Highly Specific and efficient LPS removal from complex mixtures
- Quick and easy to use in column (gravity flow) or batch mode
- Upscaling on fully automated liquid-chromatographic systems possible
- Average sample/protein recovery > 95%
- Broad pH tolerance and broad pI range of target proteins (5-9)
- Compatible with commonly used buffers and high salt conditions
- Reusable at least 10 times (EndoTrap® HD)
- Not Polymyxin B based, non toxic
- Regulatory Support File (EndoTrap® HD) and toxicological data available on request
General specifications of EndoTrap®
|
Ligand: |
EndoTrap® blue or EndoTrap red respectively |
|
Binding capacity: |
2.000.000 EU/ml resin ( 5,000,000 EU/ml for EndoTrap ® HD) (each cleaning step theoretically yields a two log reduction of LPS) |
| Dissociation constant for LPS: |
KD = 5 x 10-8 M |
|
Support matrix: |
Highly cross-linked 4% agarose, spherical beads |
|
Void volume: |
0.3 to 0.5 ml |
| Mean particle size: |
90 µm |
| Max. flow rate: |
0.2-1 ml/min |
| Max. pressure: |
3 bar, 43 psi, 0.3 MPa (when using automated systems) |
| Temperature stability: |
At 2-8°C in regeneration buffer (RB blue or RB red) |
|
Storage: |
supplemented with 0.02% sodium azide. Do not freeze ! |
|
Shelf life: |
EndoTrap is stable until the stated expiry date when stored correctly. |
Principle of EndoTrap®
 |
1. Endotoxin-contaminated proteins
and aqueous solutions are applied
2. Endotoxin is captured, proteins
elute
3. Regeneration of ligand by using
regeneration buffer
|
 |
Elution profile and Application of EndoTrap columns
LPS-free protein elutes immediately after the column void volume. Endotoxin remains bound until eluted by a purge with regeneration buffer. Columns are now ready to be reused. |
 |
Efficient endotoxin removal 1 ml Bovine Serum Albumine (BSA) in EndoTrap equilibration buffer (1 mg/ml, yellow bars) was spiked with 2500 EU endotoxin (E. coli, green bars) and applied onto a 1-ml EndoTrap column. |
 |
1-ml prepacked ready-to-use EndoTrap columns |
Application of EndoTrap®
| Pharmacology |
immune modulation e.g. T cell or B cell stimulation immune suppression e.g. dendritic cells apoptosis e.g. primary endothelial cells pro-inflammatory responses TLR (toll-like receptors) |
|
Animal Models |
proliferation assays vaccine e.g. injection into mouse and monkey immune stimulation e.g. HIV envelope Sepsis |
| Cell Culture |
Process development, down stream process |
EndoTrap®:
highly specific endotoxin removal and excellent sample recovery without incubation
| Efficiency | Average LPS removal rates of > 95% per cycle |
| Best sample recovery | Average sample recovery of > 90% per cycle |
| Reliability |
Endotoxin removal widely independent from -pH -ionic strength - temperature ( |
| Regeneration | The EndoTrap® system can be reused in general 10 times |
| Endotoxin variability | Endotoxin removal from broad range of bacteria strains |
| Individuality | also economical batch procedure possible |