Technical User Guide
Insulin Adsorbent
Product Code: 3160

Introduction
Insulin Adsorbent is a high-performance synthetic ligand affinity chromatography adsorbent
for the capture and purification of insulin and insulin analogues from a variety of sources.
The Insulin Adsorbent ligand is coupled to highly cross-linked near monodisperse 6% beaded
agarose (PuraBead® 6HF) which has excellent flow properties. The adsorbent is stable in up to
0.5 M sodium hydroxide which allows for stringent cleaning and sanitization protocols.

Properties of Insulin Adsorbent:
Ligand:Synthetic derivative structure

Function:

For the purification of Insulin and Insulin analogues

Adsorbent appearance:

Brown

Mean particle size (µm)

90 ± 10 μm

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

Highly cross-linked 6% near monodisperse agarose(PuraBead® 6HF)
Up to 25 g/L of Adsorbent(dependent on target protein and feedstock)
At a constant pressure of 1.5 bar (~ 22 psi)
0.1 M NaCl solution (saline)
Up to 600 cm/h

Operating pH:

pH 2.5 to pH 8.0

pH Stability:

Long term (3 months) pH 3.0 to pH 13.0

Chemical stability:

Stable in all commonly used buffers and solutions

Cleaning / sanitization:

0.5 – 1.0 M NaOH

Recommended storage
condition†:

Store in the dark, 20% ethanol, 2 – 30 °C** Please do not store the Insulin Adsorbent in buffers of extreme pH or elevated temperature

PC: 3160-v3

1

Column Packing
Insulin Adsorbent is supplied in 20% ethanol solution. Due to the presence of ethanol, there
may initially be an increased back pressure however, this should reduce after ~1 column
volume (CV) during the pack. Before commencing the column pack, consult the relevant
manufacturer’s instructions for the selected column. The method below describes the packingof Insulin Adsorbent 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 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.
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 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 of 1.5 bar (~ 22 psi).
7. Once the adsorbent has packed (after ~ 2 CV), measure and mark the bed height underpacking flow, 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 (do not push
the top adaptor further into the adsorbent bed).
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.
Note: it is recommended that either before first use or after prolonged storage in thepreservative solution, the packed column is washed with 30% iso-propanol/0.2 M NaOH (2CV) to dislodge loosely bound agarose chains and attached ligand which may arise from thevery low-level hydrolysis of the agarose polymer chains.

PC: 3160-v32

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. Insulin Adsorbent 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: 3160-v3

3

Operating Instructions
Note: the following recommendations are not prescriptive and thorough investigation of theseparameters at small-scale recommended to reveal the level of flexibility that can be
tolerated with the chromatography adsorbent, buffer and protein combination selected.
Insulin Adsorbent 1 mL Column Kits (code: 4444-00001) are available for scouting
experiments.
The following instructions are recommended (as a starting point) for the purification ofalbumin-fusion proteins. Filter all buffers and feedstock through an appropriate filter, prior
to running the column.
An initial flow rate of 100 cm/h for all 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 up to 5 column volumes (CV) of equilibration buffer.
Recommended equilibration buffer: 0.2 M sodium acetate buffer, pH 5.5.
2. Apply filtered sample onto the column at a flow rate of 100 cm/h. A minimum residence
time of 3 minutes is recommended.
3. 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.
4. If required, use an appropriate wash strategy to remove non-specifically bound material
prior to elution.
Note: Wash buffers can be used containing from 0.01 to 0.4 M sodium acetate (from pH 5.0
to pH 5.6) to improve target protein purity or recovery if required.
5. Elute the bound protein using 5 CV of elution buffer. It is recommended to use an acidic
solution of 0.1 M acetic acid (increasing the molarity up to 1.0 M of the acetic acid can
improve target protein recovery)
Note: 20% ethanol, 1.0 M acetic acid can be used as a strip post elution to remove any
tightly bound target and non-target material if required.
6. If a clean-in-place is required, use up to 5 CV 0.5 M NaOH. Removal of any residualadsorbed material including micro-organisms, viruses and endotoxins can be achieved bywashing the column with 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 berequired. No less than 5 column volumes are recommended.
7. Re-equilibrate the column with 5 CV of equilibration solution/buffer (to remove the CIP
solution) and check pH and conductivity before re-use.
8. For long term storage, it is recommended to store Insulin adsorbent in the dark, in 20%
ethanol at 2 – 30 °C.

PC: 3160-v3

4

Order information
Gel Slurry
Code

Description

Pack Size

3160-00025

Insulin Adsorbent

25 mL

3160-00100

Insulin Adsorbent

100 mL3160-00500

Insulin Adsorbent

500 mL3160-01000

Insulin Adsorbent

1000 mL

4444-00001

Insulin Adsorbent 1 mL Column Kit

4 x 1mL columns

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

PC: 3160-v3

5

All trademarks, trade names, trade dress, product names and logos appearing in thisTechnical User Guide are the property of Astrea Bioseparations Ltd.
PuraBead® - this product or portions thereof is manufactured and sold under licence fromGE 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.
Copyright © 2020 Astrea Bioseparations Ltd.