Technical User Guide
Anion Exchange Adsorbents
Q PURABEAD® HF
Product Code: 3452

DEAE PURABEAD® HF
Product Code: 3451

Introduction
Q PuraBead® HF is a high-performance strong anion exchange chromatography adsorbent witha quaternary ammonium group (Q).
DEAE PuraBead® HF is a high-performance weak anion exchange chromatography adsorbent
with a diethylamino ethyl group.
Both ligands are coupled to highly cross-linked near monodisperse 6% beaded agarose(PuraBead® 6HF) which has excellent flow properties. Both adsorbents are stable in up to1.0 M sodium hydroxide which allows for stringent cleaning and sanitization protocols.

Properties of anion exchange adsorbents:
Q PuraBead® HF

DEAE PuraBead® HF

Quaternary ammonium

Diethylamino ethyl

Type of ion exchanger:

Strong Anion

Weak Anion

Total ionic capacity:

40 - 60 μmol/g settled gel

110 to 160 μmol/g settled gel

Adsorbent:
Ligand:Mean particle size (µm)
Matrix:Binding capacity:
Recommended packing
conditions:
Recommended packing
solution:
Recommended operational
flow rates:
Operating pH:
Chemical stability:
Cleaning/sanitization:
Sterilization:
Storage:AIEX User Guide-v2

90 ± 10 μm
PuraBead® 6HF(Highly cross-linked 6% near monodisperse agarose)
75 - 100 mg/g of adsorbent
76 - 120 mg/g of adsorbent
(BSA)
(BSA)
Pack at a constant pressure of 1.5 bar (~ 22 psi)
0.1 M NaCl solution or equilibration buffer
Up to 600 cm/h
pH 2.0 – pH 14.0
All commonly used aqueous buffers and co-solvents
0.5 to 1.0 M NaOH
Autoclavable in 0.1 M NaCl solution at 121 °C for 30 minutes
2 – 30 °C, 20% ethanol

1Column Packing
The anion exchange adsorbents are supplied in a preservative containing 20% ethanol. Due tothe presence of ethanol, there may initially be an increased back pressure during the pack;however, this should reduce after ~ 1 column volume (CV). There is no requirement toremove the preservative prior to packing. Before commencing the column pack, consult therelevant manufacturer’s instructions for the selected column. The method below describes
the packing of Astrea’s anion exchange adsorbents into axial columns:
1. Assemble the column and remove air from the dead spaces by flushing the end piece andadaptor with packing solution (0.1 M NaCl solution or equilibration buffer) then close thecolumn outlet.
2. Allow all materials to equilibrate to the temperature at which the chromatography process
is to be performed.
3. If required to obtain a fixed bed height (i.e. for larger column packs), it is recommended
to determine the slurry percentage. For example; weigh a sample of the complete slurry,drain away the preservative and re-weigh the adsorbent. The final weight over the total
weight will determine the slurry percentage. The compression factor of this adsorbent is
1.15 to 1.20.
4. Carefully pour the adsorbent slurry into the column in a single continuous step. Pouring
the adsorbent down the side of the column helps to prevent air becoming trapped within
the adsorbent bed.
5. Allow the adsorbent to settle in the column leaving a dead volume of packing solution
above the adsorbent bed.
6. Attach the (open) top adaptor to the top of the column and adjust the adaptor to~1 cm above the bed, tighten the adaptor and attach to the workstation. Open the column
outlet and apply the desired flow to the bed (for ~ 2 CV). The recommended packing
condition (to obtain a uniform pack) is at a constant pressure of 1.5 bar (~ 22 psi). The
flow rate is dependent on column dimensions, however, will range from 700 to 1000 cm/h.
7. Once the adsorbent has packed (after ~ 2 CV), measure and mark the bed height underpacking flow and close the column outlet and stop the liquid flow through the bed.
8. Lower the top adaptor (the top adaptor must allow free flow from the workstation either
by loosening the top adaptor connection or if present switching the top valve to waste) toup to ~1 cm below the position of the marked bed height.
Note: once the flow is paused the bed may relax and rise. The seal of the top adaptor may
need to be loosened to allow the adaptor to be lowered.
9. Re-tighten the top adaptor (if loosened) and attach back to the workstation (or switchvalve back in-line). Apply the packing flow to the column again for ~ 1 CV. If a space isformed between the top of the bed and the adaptor repeat the steps above. If no space forms
the column is packed and ready to use.

AIEX User Guide-v2

2Column Efficiency Test
1. Test the column at a flow rate of 100 cm/h
2. Attach the column to an equilibrated workstation.
3. Commence flow for 1 CV (i.e. to equilibrate and obtain baseline).
4. Inject 2% to 5% CV of a 2% acetone or 2 M NaCl solution.
5. Continue flow until a UV (or conductivity) peak is observed and the trace has returned tobaseline (1 to 1.5 CV).
6. End run and determine the asymmetry factor:

7. The recommended asymmetry factor for packed anion exchange adsorbents is between 0.8
to 1.2. The recommended plate count for an acceptable pack is ≥ 2000 N/m.

AIEX User Guide-v23

Operating Instructions
Note: The following recommendations are not prescriptive and thorough investigation ofthese parameters at small-scale should be conducted to reveal the level of flexibility that canbe tolerated with the chromatography adsorbent, buffer and protein combination selected.
AIEX column kits are also available for screening experiments.
The following method is recommended (as a starting point), using an initial flow rate of
100 cm/h for the column chromatography steps is recommended. Subsequent
increases/decreases in the flow rate can be investigated to improve binding
capacity/resolution or decrease processing times.
Filter all buffers and feedstock through an appropriate filter, prior to running the column.
1. Equilibrate the column with up to 5 CV of equilibration buffer 20 mM Tris buffer, pH 7.5
Note: The equilibration buffer pH and conductivity should match that of the protein
feedstock
2. Other buffers suitable for use with Astrea’s anion exchange adsorbents to obtain optimalbinding include:
Sodium phosphate, sodium citrate and HEPES.
Note: AIEX adsorbents are designed for adsorption of negatively charged proteins (pH >pI). It is recommended to use an equilibration buffer with a pH of at least 0.5 units above
the isoelectric point (pI) of the target protein. The ionic strength of the equilibrationbuffer should also be low, with preferably no or minimal salt present.
3. Apply the clarified / filtered protein feedstock onto the equilibrated column.
Recommended residence time of 3 minutes (or greater).
4. Remove any non-bound material in the column with up to 5 CV of equilibration solution/buffer, or until the UV trace returns to baseline.
5. Elute the bound protein by increasing the conductivity of the solution with up to 5 CV ofelution buffer.
6. Any of the recommended equilibration buffers with the addition of up to 1.0 M sodium
chloride (NaCl) would be suitable.
7. For initial investigations, it is recommended to carry out a salt elution gradient (e.g. 20 CVfrom 0 to 1.0 M NaCl in equilibration buffer) to determine the appropriate elution
condition for your target biomolecule and will also identify a purification strategy (i.e.
separation of non-target proteins).
8. 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.

AIEX User Guide-v2

4

9. Re-equilibrate column with up to 5 CV of equilibration buffer (to remove sodiumhydroxide) and check pH and conductivity of the column eluate is equal to that of thebuffer entering the column before storage or re-use.
10.If the column is to be stored for future use, place the column into the storage solution(20% ethanol recommended, 0.1 M NaOH acceptable for short term storage) and store at
2 – 30 °C

AIEX User Guide-v25Order information
Gel Slurry
Code

Description

Pack Size3452-00025

Q PuraBead® HF

25 mL

3452-00100

Q PuraBead® HF

100 mL

3452-00500

Q PuraBead® HF

500 mL

3452-01000

Q PuraBead® HF1000 mL

3451-00025

DEAE PuraBead ® HF

25 mL

3451-00100

DEAE PuraBead ® HF

100 mL

3451-00500

DEAE PuraBead ® HF

500 mL

3451-01000

DEAE PuraBead ® HF

1000 mL

We also offer a range of larger pack sizes for supply of bulk resins into cGMP development andmanufacturing 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

Column Kits
CodeDescription

Pack Size

6600

Q PuraBead® HF

4 x 1 mL columns

6601

Q PuraBead® HF

4 x 5 mL columns

6602

DEAE PuraBead ® HF

4 x 1 mL columns

6603

DEAE PuraBead ® HF

4 x 5 mL columns®

1 x 1 mL Q PuraBead HF
6608

IEX selection kit (1 mL columns)®

1 x 1 mL SP PuraBead HF®

1 x 1 mL CM PuraBead HF®

1 x 1 mL DEAE PuraBead HF

6609

AIEX User Guide-v2

IEX selection kit (5 mL columns)

1 x 5 mL Q PuraBead® HF
1 x 5 mL SP PuraBead® HF
1 x 5 mL CM PuraBead® HF
1 x 5 mL DEAE PuraBead® HF6

PuraBead® - this product or portions thereof is manufactured and sold under license 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.
All other trademarks, trade names, trade dress, product names and logos appearing on thisdocument are the property of Astrea Bioseparations Ltd.

Copyright © 2020 Astrea Bioseparations Ltd.