Agilent Protein A, rProtein A, Protein G User Manual

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 elevatedtemperatures.

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, rProteinA,

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

Figure1. 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

themanufacturer.

4. Flow liquid into the column.

5. Connect outlet (pointed end) to

thedetector.

* 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 0.1M

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
Trisbuffer, 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

somesamples.

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 AgilentBio-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