Technical User Guide
p-Aminobenzamidine Agarose 6XL
Product Code: 0320

Introduction
p-Aminobenzamidine Agarose 6XL is an affinity adsorbent for the purification ofserine proteases and esterases (i.e. trypsin, thrombin, kallikrein, urokinase, acetylcholinesterase and alkaline phosphatase). p-Aminobenzamidine Agarose 6XL can beused in the separation of closely related proteases, for example, trypsin fromchymotrypsin, or the removal of trace protease contamination from a final product.
p-Aminobenzamidine Agarose 6XL is immobilised onto a 6% cross-linked agarose via a stable
spacer arm to provide a robust adsorbent. This product is manufactured in a controlledenvironment in accordance with an ISO 9001 quality system and is designed for use in cGMP
manufacturing.

Properties of p-Aminobenzamidine Agarose 6XL:
Appearance:
Ligand:White to off-whitepara-Aminobenzamidine

Particle size:

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

Base Matrix:

Cross-linked 6% agarose (Agarose 6XL)

Dynamic Binding capacity:

25 - 40 mg of Trypsin/mL of adsorbent

Recommended packing conditions:

0.1 M NaCl solution(at a constant pressure of up to 3 bar)

Recommended operational flow rates:
Chemical Stability:

Up to 200 cm/h (up to 1 bar)
All commonly used aqueous buffers and solutions within theoperating pH

Operating pH:

pH 2 to 12

pH stability:

The guanidine moiety of the Aminobenzamidine ligand can degrade
if exposed to pH values higher than pH 12.0, therefore DO NOT
use NaOH (alkali conditions) for cleaning

Cleaning:

8 M Urea
Low (pH 2.0) / High (pH 12.0) wash cycles
Low (0 M) / High (1 M) NaCl wash cycles

Recommended storage condition*:

2 – 8 °C, 24% ethanol / 76% 0.1 NaCl (v/v) solution* For short term storage and shipment (up to 1 month), pABA A6XL can be kept at ambient conditions (up to 25 °C) without any
negative impact to adsorbent performance or ligand leachates

PC: 0320-v3

1Column Packing
p-Aminobenzamidine Agarose 6XL is supplied in 24% ethanol / 0.1 M NaCl (v/v)
solution. Before commencing the column pack, consult the relevant manufacturer’s
instructions for the selected column. The method below describes the packing of pAminobenzamidine Agarose 6XL 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) then close the column 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 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.
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 until a dead volume of packing solution above
the adsorbent has formed (~ 2 cm).
6. Attach the (open) top adaptor to the top of the column and adjust the adaptor to justabove the bed, tighten the adaptor and attach to the workstation. Open the column outlet
and apply the desired flow to the bed. The recommended packing conditions (to obtain auniform pack) is at a constant pressure up to but not exceeding 3 bar (~ 45 psi).
Note: The flow rate will be dependent on column dimensions, however, will range from
200 to 400 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 by loosening the top adaptor seal (the top adaptor must allow freeflow from the workstation either by loosening the top adaptor connection or if present
switching the top valve to waste) to the position of the marked bed height.
Note: Once the flow is paused the bed may relax and rise.
9. Re-tighten the top adaptor (if loosened) and attach back to the workstation (or switch
valve back in-line). Open the bottom outlet and apply the packing flow to the column
again for 1 CV. If a space is formed between the top of the bed and the adaptor repeat thesteps above. If no space forms the column is packed and ready to use.

PC: 0320-v3

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. p-Aminobenzamidine Agarose 6XL is an affinity adsorbent, therefore an asymmetry factor
for an acceptable pack is between 0.8 to 1.6. The recommended plate count for an
acceptable pack is ≥ 2000 N/m.

PC: 0320-v33

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.
p-Aminobenzamidine Agarose 6XL 1 mL and 5 mL Column Kits (6618 and 6619) are available
for high throughput process optimization and scouting experiments.
The preferred option is to use p-Aminobenzamidine Agarose 6XL with a liquid chromatographysystem or automated workstation. Note: the adsorbents can also be operated manually using
a peristaltic pump or even a syringe.
An initial flow rate of 50 cm/h for the column chromatography steps is recommended.
Subsequent changes to the flow rate can be investigated to improve binding capacity /resolution or decrease processing times.
1. Equilibrate the column with 5 column volumes (CV) of equilibration buffer (e.g. 100 mM
Sodium acetate, 10 mM calcium chloride, pH 5.5). Allow the column buffers and sample toreach the operational temperature.
2. Apply the clarified feedstock / sample onto the column. A minimum residence time of ≥ 3
minutes is recommended.
3. Remove any non-bound material in the column with 5 CV of equilibration buffer, or until
the UV trace returns to baseline.
4. To remove any non-specifically bound material, p-Aminobenzamidine Agarose 6XL can bewashed with up to 5 CV of 100 mM Sodium acetate, 10 mM calcium chloride, 900 mM
sodium chloride, pH 5.5
5. Elute the bound enzyme/material with up to 5CV using 50 mM glycine-HCl, pH 2.2.
6. Depending on the nature of the feed and the extent of column fouling, conditions for CIP
will require optimization depending on which of the recommended solutions is employed.
If a Clean-in-Place (CIP) or sanitization is required, the following solutions arerecommended for CIP of the column:i.

8.0 M Urea.

ii.

Low/high pH (i.e. pH 2.0/pH 12.0) wash cycles.

iii.

Low/high salt (i.e. 0 to 1.0 M NaCl) wash cycles.

The adsorbent can also be cleaned using 20% ethanol/1.0 M acetic acid
Note: Do NOT use sodium hydroxide (NaOH) as the ligand is not stable in alkali conditions(> pH 12.0).
7. Post CIP, wash the column with at least 5 CV of equilibration buffer to remove the CIP
solution and check pH and conductivity of the column eluate is equal to that of the buffer
entering the column before re-use or storage.
8. If required at a later date, store the column into the storage solution at 2 – 8 ºC. The
recommended preservative is 24% ethanol / 76% 0.1 M NaCl.

PC: 0320-v34

Order information
Gel Slurry
CodeDescription

Pack Size

0320-00025

p-Aminobenzamidine Agarose 6XL

25 mL

0320-00100

p-Aminobenzamidine Agarose 6XL

100 mL

0320-00500

p-Aminobenzamidine Agarose 6XL

500 mL

0320-01000

p-Aminobenzamidine Agarose 6XL1000 mL

Please visit our webshop at https://www.astreabioseparations.com/product-category/serineproteases-purification-removal/. We also offer a range of larger pack sizes for supply of bulkresins into cGMP development and manufacturing scale processes.

Column Kits
CodeDescription

Pack Size

6618

p-Aminobenzamidine Agarose 6XL 1 mL HT Column Kit

4 x 1 mL columns

6619

p-Aminobenzamidine Agarose 6XL 5 mL HT Column Kit

4 x 5 mL columns

Astrea can also supply larger sized pre-packed columns for process development or smallscale manufacture. For more information on this or any other supply related matters please
do not hesitate to contact us on sales@astrea-bio.com

PC: 0320-v35

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