EtoxiClear™ 50 mL Column | 4251-00050, AS025100-PC3250-G

EtoxiClear® 50 mL Column Product Code: 4251-00050, AS025100-PC3250-G

INTRODUCTION Endotoxin are lipopolysaccharides (LPS) derived from the cell membrane of Gramnegative bacteria and are often found as impurities in final pharmaceutical products.

Endotoxin elicit a variety of pathophysiological effects when administered systemically and the removal of residual endotoxin to below the threshold level for recombinant proteins produced in E. coli continues to be a major challenge for the biopharmaceutical industry.

The maximum permitted level of endotoxin for intravenous products is 5 EU (endotoxin units) per kg adult per hour, where 1 EU is equivalent to approximately 0.1 – 0.2 ng of endotoxin.

The removal of endotoxin is more challenging when associated with large protein molecules.

While several methods adapted for specific products have been described for the removal of endotoxin from proteins, the methods based on selective adsorption are the most effective for the removal of LPS from final product.

EtoxiClear® provides cost effective and efficient endotoxin removal, using a high-performance synthetic ligand affinity chromatography adsorbent, providing superior endotoxin removal from processing solutions and final products.

The adsorbent has a high dynamic binding capacity (>1 x 10 6 EU/mL packed adsorbent) and low protein binding, with typical recoveries of > 90%, which allows for up to 4 log clearance of the residual endotoxin present in the sample.

The affinity ligand is covalently coupled to 6% cross-linked, near monodisperse agarose beads (PuraBead® 6XL) to provide a sodium hydroxide stable adsorbent with excellent selectivity for endotoxin.

The pre-packed EtoxiClear® columns are constructed using biocompatible materials. The prepacked columns have a 10-cm bed height and have been specifically designed to be cost effective, robust, and to provide a platform for fast and accurate process development and polishing steps for the removal of endotoxin.

Pre-packed EtoxiClear® columns are available in two formats: Evolve® R for research use and Evolve® S for sanitary applications.

EtoxiClear® 50 mL Column User Guide–v3

Properties of the 50 mL EtoxiClear® column COLUMN: COLUMN MATERIAL: LIGAND: MEAN PARTICLE SIZE: MATRIX:

50 mL axial flow column (2.5 cm diameter x 10 cm bed height) Polypropylene Synthetic chemical ligand (proprietary) 100 ± 10 μm Cross-linked 6% near-monodisperse agarose PuraBead® 6XL

DYNAMIC ENDOTOXIN BINDING CAPACITY:

> 1,000,000 EU/mL of adsorbent*

MAXIMUM RECOMMENDED OPERATING PRESSURE (DELTA COLUMN PRESSURE):

Not exceeding 0.5 bar (~7 psi)**

RECOMMENDED OPERATIONAL FLOW RATE:

10 mL/min (120 cm/h)

OPERATING PH:

pH 4.0 to pH 8.0

PH STABILITY:

Long term: pH 3.0 – pH 12.0

CHEMICAL STABILITY: CLEANING/SANITIZATION: STORAGE:

All commonly used aqueous buffers and solutions 0.5 to 1.0 M NaOH, 25 °C 2 – 30 °C, 20% ethanol : 80% 0.1 M NaCl (v/v)

* Loading at 120 cm/h, 5-minute residence time (EtoxiClear® pre-packed column) ** Delta column pressure

EtoxiClear® 50 mL Column User Guide–v3

Important Notes 1. It is good practice to clarify / filter all protein samples and chromatography buffers prior to use.

2. Removal of residual endotoxin from the final protein product solution may require process optimization to identify optimal buffer conditions.

3. EtoxiClear® can be operated across a wide pH range (pH 4.0 – pH 8.0) for endotoxin binding applications.

4. It is recommended that the endotoxin removal is carried out using aseptic techniques and for fraction collection use pyrogen free tubes/containers.

5. Avoid freezing the final protein solution prior to endotoxin removal. Freeze-thawing increases the formation of endotoxin micelles which can be more difficult to remove.

6. Below the pH of the isoelectric point (pI) of the target protein the net charge on the protein is positive and polyanionic (negatively charged) endotoxin molecules will interact strongly with the protein molecule. Consequently, the effective removal of endotoxin from the final product could be impaired at pH values below the pI of the target protein.

7. Although EtoxiClear® has low non-specific binding, it does have an anionic functional group which may act as a very weak ion exchanger. This could lead to a slight binding of target protein which can be minimized by increasing the ionic strength of the buffer by the addition of salt (up to 0.3 M NaCl).

8. Do not use ammonium or Tris based buffers. These buffers have shown to reduce the efficiency of endotoxin clearance with EtoxiClear®. Other buffers to avoid include MES and HEPES. 9. Increasing the concentration of the buffer salt (i.e. from 10 mM to 100 mM sodium phosphate) can improve the target protein recovery without having a detrimental effect on endotoxin clearance.

10.If required, endotoxin clearance can be optimized by decreasing the flow rate (increasing residence time) used during sample loading.

11.There are many commercially available endotoxin detection tests/kits available, however, if a chromogenic based (LAL) test is used (e.g. Associates of Cape Cod) it is recommended to include Glucashield® buffer. This renders the reagent insensitive to (1→3)-β-D-glucan interference, which may be present in the sample, by effectively blocking the Factor G pathway of the endotoxin clotting cascade.

EtoxiClear® 50 mL Column User Guide–v3

COLUMN OPERATION 1. The preferred option is to use the EtoxiClear® 50 mL column with a liquid chromatography system or automated workstation. Note: the column can also be operated manually using a peristaltic pump or even a syringe.

2. Recommended equilibration buffers are PBS or 50 mM sodium phosphate, 0.1 M NaCl, pH 7.2. Other equilibration buffers are as follows (including 0.1 M NaCl): • sodium acetate • sodium citrate 3. Allow the EtoxiClear® 50 mL column, buffers and sample to reach the operational temperature.

4. Remove the inlet (top) stopper of the EtoxiClear® 50 mL column and attach directly to the liquid chromatography system using a standard 10-32 threaded male for 1/16’’ OD tubing connector, ensuring that the tubing connecting the workstation to the column is primed with equilibration buffer. Once top connections are made, remove the outlet (bottom) stopper and connect to system.

Column Inlet (TOP)

Column Outlet (BOTTOM)

Note: We recommend that EtoxiClear® 50 mL Evolve® R columns are treated with a depyrogenation step prior to use.

5. To depyrogenate, flush the liquid chromatography system and the EtoxiClear ® 50 mL column at 60 cm/h (5 mL/min) with 0.5 M NaOH then stop the flow and hold in 0.5 M NaOH for ≥ 16 hours (overnight) at room temperature.

Note: This step is not applicable for EtoxiClear® 50 mL Evolve® S columns, as these are provided pre-depyrogenated for direct implementation into sanitary applications. The columns are supplied in 20% ethanol : 80% 0.1 M NaCl; sufficient equilibration prior to use is necessary as per Step 6 onwards.

6. Flush the NaOH out of the system and column using depyrogenated equilibration buffer at 60 cm/h (5 mL/min) until the effluent pH is equivalent to the equilibration buffer entering the column.

7. Re-equilibrate column with at least 5 CV of equilibration buffer or until the pH of the eluent is comparable to the equilibration buffer entering the column.

EtoxiClear® 50 mL Column User Guide–v3

8. Apply the protein solution/sample onto the EtoxiClear ® 50 mL column at the recommended operational flow rate of 120 cm/h (10 mL/min to obtain a recommended 5minute residence time) and collect the flow through in endotoxin free collection vessels.

9. If required, wash the EtoxiClear® 50 mL column with equilibration buffer at 120 cm/h (10 mL/min) to flush out the remaining protein solution with at least 1 CV or until a UV baseline is achieved.

The EtoxiClear® 50 mL column is designed to be a disposable column, and so endotoxin removal from the column, clean-in-place (CIP) and re-use protocols have not been developed or validated. Post-application it is recommended to discard the EtoxiClear® 50 mL column appropriately.

Note: Astrea Bioseparations does not assume responsibility for the removal of endotoxin from the EtoxiClear® 50 mL column, or integrity of the column (column performance and pack) post single use application.

EtoxiClear® 50 mL Column User Guide–v3

ORDER INFORMATION Pre-Packed Columns Astrea Bioseparations offers a range of pre-packed columns for research use, process development and small-scale production, as well as pre-sanitised columns for direct implementation into sanitary workflows.

Code

Description

4251-00050

EtoxiClear® 50 mL Evolve®R Column (research use only)

AS025100-PC3250-G

EtoxiClear® 50 mL Evolve®S Column

We also offer a range of larger pack sizes for supply of bulk resins into process development and manufacturing scale processes. For more information on this or any other supply related matters please do not hesitate to contact us on sales@astrea-bio.com

EtoxiClear® 50 mL Column User Guide–v3

Issue Date: 13 Dec 2022 CCR Number: N/A – trademarks updated Author Name: T Hayward QA Reviewer Name: R Hawkins Evolve® is a registered trademark of Delta Precision Ltd. Evolve® is a registered trademark in the EU, UK and US. This product is covered by or for use under one or more patents: www.astreabioseparations.com/patents All other trademarks, trade names, trade dress, product names and logos appearing in this document are the property of Astrea UK Services Ltd. Copyright © 2022 Astrea Bioseparations Ltd. All rights reserved.