Data Sheet
Agilent Bio-Monolith Protein A,
rProtein A, and Protein G Columns
Characteristics and shipping conditions
The information in this data sheet is provided to ensure proper product care and
maximum product lifetime.
Agilent Bio-Monolith Protein A (p/n 5069-3639)
Catalog Number
Immobilized Ligand
Agilent Bio-Monolith Protein A
Agilent Bio-Monolith rProtein A
Agilent Bio-Monolith Protein G
Support Matrix
Column Dimensions
Dynamic Binding Capacity
Agilent Bio-Monolith Protein A
Agilent Bio-Monolith rProtein A
Agilent Bio-Monolith Protein G
Maximum Loading Capacity
Working Flow Rates
Maximum Allowed Pressure Over
the Column
Temperature Stability
Recommended pH*
Shipping Solvent
* At high pH, ligands can slowly degrade, therefore, only use pH higher than 11 for regeneration, cleaning-in-place,
and sanitization procedures.
Agilent Bio-Monolith rProtein A (p/n 5190-6903)
Agilent Bio-Monolith Protein G (p/n 5190-6900)
Immunoaffinity Protein A from Staphylococcus aureusis
Immunoaffinity r-Protein A from Escherichia coli
Immunoaffinity r-Protein G from Escherichia coli
Poly(glycidyl methacrylate-co-ethylene dimethacrylate) highly porous monolith
Diameter 5.2 mm, length 4.95 mm, bed volume (CV) 0.10 mL
>8 mg hIgG/mL wet support
>5 mg hIgG/mL wet support
>9 mg hIgG/mL wet support
(Conditions: hIgG 0.5 mg/mL, PBS buffer, pH 7.4, flow rate 1 mL/min)
0.4 to 0.5 mg
Recommended: 0.2 to 2 mL/min (1 to 10 cm/min, 2 to 20 CV/min)
Maximum: 3 mL/min (15 cm/min, 30 CV/min)
150 bar (15 MPa, 2,100 psi)
WARNING: Do not exceed the maximum allowed pressure, as this might
seriously damage your column.
Working 4 to 40 °C Storage 4 to 8 °C
WARNING: Avoid prolonged use at elevatedtemperatures.
Working range: 2 to 11
Cleaning in place: 2 to 13
20 mM Tris, pH 7.4 and 20 % ethanol
Agilent Bio-Monolith Protein A, rProteinA,
Flat end
direction
Pointed end
and Protein G columns are analytical
affinity columns designed for fast
separation, isolation, and quantitation
of antibodies in fermentation (cell
culture supernatants and lysates). The
columns are compatible with HPLC and
preparative LC systems. The monolithic
structure enables high reproduciblity
and long lifetimes for separation and
quantitation of immunoglobulin G (IgG)
at high speeds.
Preparing the column
before first use
Connecting the column
The flow direction is indicated in
Figure1. The flat end is the inlet, and the
pointed end is the outlet.
Flow
Figure 1. Flow direction through the flat end of
the column.
1. Remove both end caps.
2. Connect the flat end* to the nut of the
capillary, which is already connected
to the HPLC system.
3. Tighten the fitting as instructed by
themanufacturer.
4. Flow liquid into the column.
5. Connect outlet (pointed end) to
thedetector.
* Long to extra long fittings and ferrules
(p/n 5065-9967) should be used.
Column equilibration
1. The columns should be washed
with at least 2 mL (20 CV) of a 0.1M
buffer such as sodium phosphate
buffer or Tris buffer containing 1 M
NaCl, pH 7.0 to 8.0 at a flow rate of
0.5 to 1.0 mL/min.
2. Wash the columns with at least 2 mL
(20 CV) of eluting buffer (see eluting
buffers below).
3. Equilibrate the column with 30 CV of
the binding buffer (see binding and
washing buffers below) at a flow rate
of 0.5 to 1.0 mL/min.
4. The column is ready for injection.
Binding and washing buffers
Bio-Monolith Protein A, rProtein A, and
Protein G columns are compatible with
many buffers for binding and washing.
The most common ones are:
1. Sodium phosphate buffer and
Trisbuffer, 25 to 100 mM. We
recommend using 50 mM as the
starting concentration.
2. Binding and washing buffers with
pH 6 to 9. We recommend starting
with pH 7.4. This pH seems to be
the optimal for the columns to
absorb/bind most antibodies.
3. Room temperature or 24 to 25°C
is recommended for optimal
performance and extended column
lifetime. Higher temperature can be
also used, but might reduce column
lifetime.
4. The columns are compatible with
neutral salts such as sodium chloride
(NaCl) or potassium chloride (KCl).
Salt concentrations of 0.1 to 0.2
M can improve the peak shape
and recovery in some cases for
somesamples.
Note: Filtration of buffers through a 0.22
or 0.45-µm membrane is recommended
to reduce buffer impurities that build up
on the frits inside the column. This will
prevent column blockage.
Note: Binding/washing buffers should be
freshly made.
Eluting buffers
Bio-Monolith Protein A, rProtein A, and
Protein G are compatible with many
low pH buffers. The buffers commonly
used are citric, glycine, HCl, and acetate
acid. Table 1 provides details for these
commonly used buffers.
Table 1. Some common eluting buffers for use
with AgilentBio-Monolith Protein A, rProtein A, and
Protein G columns.
Column Buffer Conc. pH
Agilent
Bio-Monolith
Protein A and
rProtein A
Agilent
Bio-Monolith
Protein G
Citric acid 0.1 M 2.5 to 3.0
Glycine 0.1 M 2.5 to 3.0
Acetic acid 5 to 20 %
Citric acid 0.1 M 2.0 to 2.5
Glycine 0.1 M 2.0 to 2.5
Acetic acid 5 to 20 %
Note: Commonly, elution buffers for
affinity columns have a refractive index
(RI) very different from binding/washing
buffers; therefore, baseline noise and
an artifact peak could appear when the
eluents start flowing. This peak could
interfere with the quantitation of low
concentration samples. To minimize
this effect, high-quality chemicals are
recommended to be used and blank
runs should be included to establish the
artifact peak. Blank runs can be used for
baseline subtraction if desired.
Note: HCl has a lower RI compared to
other eluents. If a low concentration
sample is used and baseline noise and
artifact peaks are of concern, HCl can be
used as an eluent.
2
Sample preparation
Bio-Monolith Protein A, rProtein A, and
Protein G columns are compatible with
many buffers. However, some minor
preparation is required before injection
to optimize column performance and
extend column lifetimes.
Centrifuge or filter samples to remove
host cell debris and particulates from
the supernatant or lysate, to prevent
blockage of the columns.
Optimization/
re-optimization
Please keep in mind that we provide
some general guidelines on how to use
the columns. Optimization of methods
might be needed for some samples.
Methods from existing protocols with
other affinity columns might not work
with Agilent columns due to differences
in chemistry between columns and
differences between the antibody
sources. Therefore, method optimization
might be required.
Optimization methods include sample
preparation, buffer compositions, buffer
pH, binding time, washing time, and
eluting time.
Taking care of the column
Cleaning/washing the columns
between injections
It is a good practice to monitor column
performance and include regular column
cleaning cycles.
Cleaning-in-Place (CIP) protocol
1. Wash the column with 1 to 2 mL
(10to 20 CV) 0.1 M NaOH.
Note: Reverse the flow direction
and use flow rates between 0.2 to
0.5mL/min
2. Wash the column with 1 to 2 mL
(10to 20 CV) deionized (DI) water at
the working flow rate.
3. Wash the column with 1 to 2 mL
(10to 20 CV) concentrated buffer
(for example, 0.1 to 0.5 M phosphate
buffer, pH 7.4) to quickly restore the
appropriate pH.
4. Equilibrate with at least 5 mL (50 CV)
of binding phase buffer at the working
flow rate.
If the impurities are highly hydrophobic
or lipidic, and are not easily removed
from the column, 2-propanol (up to 30%)
or guanidine hydrochloride (up to 3 M)
can be used to remove these impurities.
After using these alternative cleaning
solutions, follow steps 1 through 4.
WARNING: When you wash the column
with these cleaning solutions, always
decrease the flow rate on the column to
avoid generation of high pressures that
might exceed the maximum allowed
pressure over the column.
Short-term storage
For storage overnight or for a few days,
the columns can be flushed with binding
buffer, disconnected from the instrument,
capped, and stored at 4 to 8 °C. Columns
should be equilibrated before the first
injection after short-term storage.
Long-term storage
If the column will not be in use for more
than 2 days, it should be washed with
at least 1 mL (10 CV) of DI water and
afterwards flushed with at least 2 mL
(20 CV) of 20 % ethanol with 20 mM
Tris buffer, pH 7.4 at a flow rate of 0.2 to
0.5mL/min. It should then be sealed with
column end stops and stored at 4 to 8 °C
(39 to 46 °F).
www.agilent.com/chem
DE44230.0856712963
This information is subject to change without notice.
© Agilent Technologies, Inc. 2015, 2021
Printed in the USA, February 4, 2021
5991-6040EN
Loading...