Endotoxin or lipopolysaccharides (LPS) (Figure 1) are highly toxic components of the cell wall of Gram-negative bacteria and are often present in significant amounts in bacterial cell culture expression systems such as E. coli.
FIGURE 1 Schematic representation of Gram-negative bacterial endotoxin (LPS).
A number of methods have been adopted for the removal of endotoxin based on adsorption, in particular ion-exchange chromatography.
Although downstream processing can significantly reduce endotoxin levels in the product, efficient and cost-effective removal of residual endotoxin from biopharmaceutical preparations remains a challenge.
This technical poster addresses the issues of efficient removal of endotoxin from biological preparations. Specific reference will be made to a new synthetic ligand affinity adsorbent, EtoxiClear™, which exhibits high affinity for endotoxin, low protein binding and can be depyrogenated using sodium hydroxide.
The bi-dentate ligand, attached to Prometic Bioseparations Ltd (PBL’s) proprietary base matrix – PuraBead®, binds in a spatially selective and optimal manner to the LPS molecule with a binding capacity for endotoxin in excess of 1,000,000 EU/mL of adsorbent in a flow through chromatography mode (5 mL disposable EtoxiClear™ column, loading at 120 cm/hr - 5 minute residence time).
A number of biomolecules with different isoelectric points have been used to demonstrate efficient protein recovery and clearance of residual endotoxin across the pH range. Protein recoveries in excess of 95% are achievable with endotoxin clearance to below 0.1 EU/mg protein.
EtoxiClear™ has a high dynamic binding capacity for endotoxin and can operate in acidic to neutral conditions (pH 4.0 to pH 8.0) without a reduction in endotoxin clearance and typically maintaining >90% recovery of various proteins (up to 5 mg/mL concentration).
Performance & Scalability:
EtoxiClear™ is available in a range of disposable column sizes including 5 mL (figure 1.) & 50 mL and can be readily packed into an Evolve™ Bioprocess column. A standard bed height of 10 cm across the product range is recommended to provide effective process scalability .
Figure 1. 5mL Disposable Column.
The disposable EtoxiClear™ columns are designed for use in either process development applications or final polishing steps used during cGMP manufacturing of biological molecules, as well as the efficient removal of endotoxin from a range of buffers. The columns demonstrate excellent performance with comparable endotoxin clearance and protein recoveries across the range.
IgG protein solutions (~5 mg/mL), containing similar starting concentrations of endotoxin, were loaded onto the range of disposable EtoxiClear™ columns, and both endotoxin clearance and protein recoveries were determined (Table 1).
TABLE 1 Performance data for endotoxin clearance from both the 5 mL column and 100 mm diameter Evolve™ Process Column (780 mL) packed with EtoxiClear™.
EtoxiClear™ was compared against two commercially available competitor endotoxin removal products (IEX and affinity based membranes), using the manufacturers instructions. HSA protein solutions (10 mg/mL), containing similar starting concentrations of endotoxin, were loaded onto each product at a flow rate of 1 mL/min and both endotoxin clearance and protein recoveries were determined (Table 2).
TABLE 2 Comparison of EtoxiClear™ versus other commercially available endotoxin removal products.
Varying endotoxin concentration:
IgG protein solutions (~5 mg/mL) containing a range of endotoxin starting concentrations (from low to high) were loaded onto EtoxiClear™, and both endotoxin clearance and protein recoveries were determined (Table 3).
TABLE 3: Protein recovery and endotoxin clearance results.
Results show that the EtoxiClear™ adsorbent gives excellent endotoxin clearance (~0.1 EU/mg protein) and high protein recoveries (up to 100%), for protein solutions containing a range of endotoxin starting concentrations.
Endotoxin Removal from buffers:
A range of buffers, commonly used in cell culture applications, containing low starting concentrations of endotoxin (~20 EU/mL) were loaded onto EtoxiClear™ and endotoxin clearance was determined (Table 4).
*equal to or below limit of detection BSS – balanced salt solution
TABLE 4: Endotoxin clearance results for various buffers
Results show that the EtoxiClear™ adsorbent gives excellent endotoxin clearance (≤0.05 EU/mL) from a range of buffers.
Endotoxin Removal – low protein binding:
EtoxiClear™ has low protein binding and a wide range of proteins can be processed independent of their iso-electric point achieving high protein recoveries. Figure 3 indicates that typically >90% recovery is achieved for various model proteins spanning the pI spectrum.
FIGURE 3: Typical protein recoveries at neutral pH.
Endotoxin Removal – purified antibody fragment:
EtoxiClear™ was used to remove residual endotoxin from an antibody fragment from an E. coli lysate partially purified using Fabsorbent™ F1P HF as a capture step (Table 5). Clarified cell lysate was loaded onto Fabsorbent™ F1P HF and the F(ab’)2 fragment eluted at pH 5.0. The resulting elution fraction was loaded directly onto EtoxiClear™.
TABLE 5: Endotoxin levels determined using a chromogenic endotoxin assay kit.
Fabsorbent™ F1P HF produced a high purity antibody fragment, with a >3.0 log reduction of endotoxin achieved using EtoxiClear™.
Endotoxin Removal – chromogenic (LAL) assays:
There are many commercially available endotoxin detection tests/kits to determine endotoxin clearance from protein solutions. However, if a chromogenic based test is used, it is recommended to include Glucashield® buffer to render the reagent insensitive to (1→3)-β-D-glucan interference which may be present in the sample.
HSA (9.1 mg/mL) and IgG (9.6 mg/mL) protein solutions, containing endotoxin, were loaded (1 mL) onto EtoxiClear™ at 1 mL/min. The flow through fractions were collected and the samples analysed using a chromogenic based test (with and without Glucashield® buffer) and by Lonza Bioscience by LAL Kinetic chromogenic assay (Table 6).
TABLE 6: Endotoxin clearance, from both HSA and IgG protein solutions, determined using a chromogenic endotoxin assay kit (± Glucashield® buffer) and by Lonza Bioscience in their endotoxin testing laboratory.
Inhibition of the β-D-glucan interference allows for more sensitive and more accurate determination of endotoxin removal comparable to the analysis performed externally at the Lonza Bioscience laboratory.
- EtoxiClear™ provides excellent endotoxin removal from a wide range of proteins across the pI spectrum, with recoveries that can be in excess of >95%, in a range of conditions from acidic to neutral pH.
- EtoxiClear™ has a high capacity for endotoxin, in excess of 1,000,000 EU/mL of adsorbent, and is a available in a range of disposable columns (5 mL, 50 mL and 500 mL) with a standard bed height to enable process scalability.
- EtoxiClear™ shows superior endotoxin clearance and protein recovery in comparison to other commercially available endotoxin removal products.
- EtoxiClear™ gives excellent endotoxin clearance (~0.1 EU/mg protein) and high protein recoveries (up to 100%) for protein solutions containing a range of endotoxin starting concentrations.
- EtoxiClear™ shows excellent endotoxin clearance (≤0.05 EU/mL) from a range of buffers commonly used cell culture applications.
- EtoxiClear™ provided a >3.0 log reduction of endotoxin following the capture and partial purification of an antibody fragment by Fabsorbent™ F1P HF.
- The introduction of Glucashield® buffer to remove interference by β-D-glucans improved the sensitivity of the chromogenic assay and provided a more accurate determination of endotoxin clearance.
For further information please e-mail: firstname.lastname@example.org.
All trademarks, trade names, trade dress, product names and logos appearing on this website are the property of Prometic Bioseparations Ltd.
PuraBead® - this product or portions thereof is manufactured and sold under licence from GE Healthcare under US patents 6841097 and 7341202 and any corresponding patents for life sciences research and the purification/separation of naturally occurring or recombinant plasma derived proteins.
Glucashield® is a registered trademark of Associates of Cape Cod.
Copyright © 2017 Prometic Bioseparations Ltd.
About the AuthorFollow on Linkedin More Content by Buzz Lobbezoo