Technical User Guide
Aminophenylboronate A6XL
Product Code: 0355

Introduction
Aminophenylboronate affinity adsorbents are used for the purification of a diverse range ofmacromolecules which possess 1,2-diols, 1,3-diols, 1,2-hydroxy acids and 1,2-hydroxylaminegroups. These functionalities are present in glycoproteins, carbohydrates, nucleic acids(nucleosides, nucleotides and RNA’s) and polyphenols (catechols, flavonoids).
Aminophenylboronate Agarose affinity adsorbents are particularly suited to the purification of
glycoproteins, or the removal of glycoprotein and carbohydrate impurities form nonglycosylated molecules.
These adsorbents can also be used for the purification and removal of certain enzymes suchas proteases and hydrolases where the boronic acid group has affinity for the active site.
The chemically stable affinity ligand is bonded to 6% cross-linked agarose beads to produce ahighly robust adsorbent which is resistant to concentrated sodium hydroxide and suitable foruse in downstream purification process applications and incorporation into diagnostic assays.

Properties of Aminophenylboronate A6XL:
Ligand:m-Aminophenylboronic acidAdsorbent appearance:White

Mean particle size (µm)

90 μm(45-165 μm ≥95% of beads within this range)

Matrix:Binding capacity:
Recommended packing
conditions:
Recommended packing
solution:
Recommended operational
flow rates:

Highly cross-linked 6% agarose
Glycoproteins – in the range of 10-20 g/L
Carbohydrates – up to 30 g/L
Packing pressure – up to 3 bar
0.1 M NaCl solution or any process buffer(not NaOH)
Up to 500 cm/hr with column diameter less than 3 cm
Up to 200 cm/hr with column diameter greater than 5 cm

Operating pH:

pH 2 to 14 (intermittent)

pH stability:

Long term (3 months) pH 3 to 12

Chemical stability:
Cleaning/sanitization:
Storage:

PC: 0355-v2

All commonly used aqueous buffers and co-solvents
0.5 to 1.0 M NaOH
2 – 30 °C, 20% ethanol

1Column Packing
Aminophenylboronate A6XL is supplied in 20% ethanol solution. Before commencing thecolumn pack, consult the relevant manufacturer’s instructions for the selected column.
The method below describes the packing of Aminophenylboronate A6XL into axial columns:
1. Decant off the shipping preservative and prepare a 50% slurry of the adsorbent with either
0.1 M NaCl solution or equilibration buffer (packing solution).
2. 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.
3. Allow all materials to equilibrate to the temperature at which the chromatography process
is to be performed.
4. If required to obtain a fixed bed height (i.e. for larger column packs), it is recommended
to accurately determine the slurry percentage. For example; weigh a sample of thecomplete slurry, drain away the preservative and re-weigh the adsorbent. The final weightover the total weight will determine the slurry percentage.
5. 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.
6. Allow the adsorbent to settle in the column leaving a dead volume of packing solution
above the adsorbent bed.
7. 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 up to but not exceeding 3
bar (~45 psi).
8. 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.
9. 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 switch
valve 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.

PC: 0355-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 Aminophenylboronate A6XL is between
0.8 to 1.2. The recommended plate count for an acceptable pack is ≥ 2000 N/m.

PC: 0355-v2

3

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.
Aminophenylboronate A6XL 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.
•Sample treatment:
Clarify the feedstock/protein solution using an appropriate filter and adjust the pHand or conductivity of the solution if required.

•Equilibration buffer:
50mM Glycine NaOH buffer at pH 9.0 or 50mM sodium phosphate buffer at pH 8.0.

•Wash buffer:
If a wash step is required to remove any loosely bound non-target material, initially amoderate amount of salt (0.2 – 0.6 M NaCl) can be added to the equilibration buffer
to remove non-specifically bound proteins. Alternatively, the addition of up to 1.0 M
Tris to the equilibration buffer can also have the same desired effect.

•Elution buffer(s):For selective desorption of the target protein use up to 200mM sorbitol in theequilibration buffer. Other recommendations are using buffers at low pH, see below
for details:
50 mM sodium acetate buffer at pH 4.0 or 50mM Glycine HCl buffer at pH 3.0•

Cleaning-in-Place (CIP) and Sanitization:
Removal of any residual adsorbed material including micro-organisms, viruses andendotoxins can be achieved by washing the column with 0.5 M to 1.0 M NaOH.
A contact time of 1 hour will normally suffice to ensure destruction of viableorganisms, although up to 5 hours contact time may be required. No less than 5column volumes are recommended.
Once cleaning with NaOH is complete, wash with at least 5 bed volumes of
equilibration buffer until the pH and conductivity of the column eluate is equal tothat of the buffer entering the column. Complete this process prior to further use or
storage in the storage buffer.

•Storage:
Aminophenylboronate A6XL should be stored in 20% ethanol at 2-30°C.

PC: 0355-v24

Recommended Column Chromatography Conditions for
Using Aminophenylboronate A6XL
The following method is recommended (as a starting point). An initial flow rate of 100 cm/hr
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.
1. Equilibrate the column with 3 to 5 CV of equilibration buffer or until the pH/conductivity
is at baseline (Note: ensure the equilibration buffer of the column is comparable to theprotein feedstock).
2. Apply the protein feedstock onto the equilibrated column. A residence time of 3 minutes(or greater) is recommended.
3. Remove any non-bound material in the column with 5 CV of wash buffer, or until the UV
trace returns to baseline.
4. Elute the bound protein using up to 5 CV of an appropriate elution buffer.
5. If a clean-in-place is required, use 5 CV of 0.5 M NaOH.
6. Re-equilibrate column with 5 CV of equilibration buffer (until the pH of the eluent iscomparable to the equilibration buffer entering the column).
7. It is recommended to store the column in 20% ethanol at 2-30°C.

PC: 0355-v2

5

Order information
Gel Slurry
CodeDescription

Pack Size0355-00025Aminophenylboronate A6XL

25 mL

0355-00100

Aminophenylboronate A6XL

100 mL

0355-00500

Aminophenylboronate A6XL

500 mL

0355-01000

Aminophenylboronate A6XL

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 Size4444-0001

Aminophenylboronate A6XL Column kit

4 x 1 mL columns

PC: 0355-v2

6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.