HIC Selection Kit 5 mL | 6615

HIC Selection Kit 5 mL Column Product Code: 6615 Includes: 6611, 6613, 6635, 6637

INTRODUCTION Properties of the 5 mL Hydrophobic Interaction (HIC) columns: COLUMN:

5 mL axial flow column (ID: 1.6 cm diameter x 2.5 cm bed height)

COLUMN MATERIAL:

LIGAND:

Polypropylene Phenyl: Aromatic ring

MEAN PARTICLE SIZE (µm)

Butyl: Aliphatic 4 carbon chain

Hexyl: Aliphatic 6 carbon chain

90 ± 10 Cross-linked 6% near-monodisperse agarose (PuraBead® 6HL)

MATRIX: MAXIMUM RECOMMENDED OPERATING PRESSURE (DELTA COLUMN PRESSURE):

Not exceeding 5 bar (~72 psi) *

RECOMMENDED OPERATIONAL FLOW RATE:

Up to 5 mL/min

OPERATING PH:

pH 2.0 to pH 14.0

PH STABILITY:

Long term: pH 3.0 – pH 12.0

CHEMICAL STABILITY:

All commonly used aqueous buffers and solutions

CLEANING/SANITIZATION: STORAGE:

* Delta pressure across the column (not the system pressure) ** To minimize the risk of the columns drying out.

HIC Selection Kit 1 mL Column PC6615 User Guide-v2

Octyl: Aliphatic 8 carbon chain

0.5 to 1.0 M NaOH, 25 °C 2 – 8°C**, 20% ethanol

INTRODUCTION Hydrophobic interaction chromatography (HIC) adsorbents are designed for intermediate purification steps for the purification of mildly hydrophobic biomolecules.

Astrea Bioseparations manufactures a range of four HIC adsorbents: • • • •

Phenyl PuraBead® HF (aromatic phenyl ring) Butyl PuraBead® HF (4 carbon aliphatic chain) Hexyl PuraBead® HF (6 carbon aliphatic chain) Octyl PuraBead® HF (8 carbon aliphatic chain)

All HIC adsorbent are coupled to highly cross-linked near monodisperse 6% beaded agarose (PuraBead® 6HF) which has excellent flow properties. These HIC adsorbents are stable in up to 1.0 M sodium hydroxide which allows for stringent cleaning and sanitization protocols.

HIC Selection Kit 1 mL Column PC6615 User Guide-v2

OPERATING INSTRUCTIONS 1. The preferred option is to use the HIC 5 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. Filter all buffers and feedstock through an appropriate filter, prior to running the column.

3. Protein adsorption: The binding of protein to a HIC adsorbent is influenced by: a) The ionic strength of the equilibration buffer. The salts that cause salting out (i.e. ammonium sulphate) promote the binding of the proteins to HIC adsorbents. b) The binding of proteins to a HIC adsorbent decreases when temperature decreases.

To ensure that target molecules bind to a HIC adsorbent, the most common strategy is to apply the sample at high salt concentration in a buffered solution, pH range from pH 4.0 to pH 9.0 and high salt concentrations. The addition of a salting out solution should be added to the protein solution/sample yet ensuring not to precipitate the target protein.

The recommended salt solution is ammonium sulphate (up to a final concentration of 2.0 M ammonium sulphate).

The ordering of cations and anions to their respective salting out ability is called the Hoffmeister Series.

The cations are arranged as follows: NH4+> K+> Na+ >Li+ >Mg2+ >Ca2 + where ammonium has the highest ability to precipitate other protein-based solutes.

The order for anions is: SO4 2-> H2PO4-> H3CCOO-> Cl-> NO3Adjust the pH of the feedstock/solution and clarify the feedstock/protein solution using a 0.45 μm filter if required.

4. Allow the column, buffers and sample to reach the operational temperature.

5. Remove the inlet (Top) stopper of the 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.

6. Snap off the outlet (Bottom) plug of the column and attach to the workstation as in step 4. Note: The reverse of the bottom plug is threaded and can be used as a stopper.

HIC Selection Kit 1 mL Column PC6615 User Guide-v2

Column Inlet (TOP)

Column Outlet (BOTTOM)

7. Apply the protein solution/sample onto the column at a flow rate of 2.5 mL/min (2-minute residence time).

8. Remove any non-bound material in the column with up to 5 CV of equilibration buffer, or until the UV trace returns to baseline.

9. Elute the bound material by decreasing the salt concentration either step wise (e.g. 5 CV) or for initial investigations, a linear negative salt gradient (20 CV) from high to no salt is recommended.

The ionic strength of the elution buffer should be low, with preferably no or minimal salt present, e.g. 20 mM sodium phosphate, pH 6.0.

For tightly bound proteins (i.e. strong hydrophobic) increasing the pH can improve recovery. Additionally, for proteins that are tightly bound to the adsorbent, a positive gradient of polarity-reducing organic solvents (i.e. ethylene glycol, glycerol, ethanol, propan-2-ol at concentrations of up to 50%) can be used.

10.If a CIP is required, use up to 5 CV of 0.5 to 1.0 M NaOH. A contact time of 1 hour will normally suffice to ensure destruction of viable organisms, although up to 5 hours contact time may be required. No less than 5 column volumes are recommended.

11.Re-equilibrate column with up to 5 CV of equilibration buffer (to remove sodium hydroxide) and check pH and conductivity of the column eluate is equal to that of the buffer entering the column before storage or re-use.

12.If the column is to be stored for future use, place the column into the storage solution and store at 2 – 8 °C (to minimize the risk of the columns drying out).

Note: The columns cannot be opened or re-packed.

HIC Selection Kit 1 mL Column PC6615 User Guide-v2

ORDER INFORMATION Prepacked Columns Code

Description

Pack Size

6610

Phenyl PuraBead® HF

4 x 1 mL

6611

Phenyl PuraBead® HF

4 x 5 mL

6612

Octyl PuraBead® HF

4 x 1 mL

6613

Octyl PuraBead® HF

4 x 5 mL

6614

HIC Selection Kit (1mL columns)

1x 1mL Phenyl PuraBead® HF 1x 1mL Octyl PuraBead® HF

6615

HIC Selection Kit (5mL columns)

1x 5mL Phenyl PuraBead® HF 1x 5mL Octyl PuraBead® HF

6634

Butyl PuraBead® HF

4 x 1 mL

6635

Butyl PuraBead® HF

4 x 5 mL

6636

Hexyl PuraBead® HF

4 x 1 mL

6637

Hexyl PuraBead® HF

4 x 5 mL

We also offer a range of pack sizes for supply of loose resins. For more information on this or any other supply related matters please contact us on sales@astrea-bio.com

HIC Selection Kit 1 mL Column PC6615 User Guide-v2

Issue Date: 31 May 2022 CCR Number: CCR-1729 Author Name: R Dodd QA Reviewer Name: R Hawkins 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 on this document are the property of Astrea UK Services Ltd. Copyright © 2022 Astrea Bioseparations Ltd. All rights reserved.