GE 71-5000-15 AD User Manual
GE Healthcare
Instructions 71-5000-15 AD Pre-activated media
CNBr-activated
Sepharose 4 Fast Flow
Introduction
The preparation and use of affinity chromatography media by coupling biospecific
ligands to CNBr-activated matrices is a widely used, successful and well-documented
technique.
CNBr-activated Sepharose
combines the advantages of CNBr coupling with the high flow and stability
characteristics of Sepharose 4 Fast Flow. In our experience, the CNBr coupling
technique has a well-proven track record for the purification of therapeutic
proteins. This, plus the performance of the matrix at large scale, makes the use of
CNBr-activated Sepharose 4 Fast Flow particularly attractive for manufacturing
applications in the biopharmaceutical industry. Fur thermore, the medium is a
member of the BioProcess
for production scale chromatography. All BioProcess media are produced with
validated methods and are tested to meet manufacturing requirements. Secure
ordering and delivery routines give a reliable supply of media for production
scale. Regulatory Support Files (RSF) are available to assist process validation and
submissions to regulatory authorities.
To ensure best performance and trouble-free operation, please read these
instructions before using CNBr-activated Sepharose 4 Fast Flow.
TM
4 Fast Flow is a pre-activated affinity matrix that
TM
media. BioProcess media are developed and suppor ted
Table of contents
1. Product description 2
2. Coupling 3
3. Column packing guidelines 5
4. Evaluation of packing 7
5. Cleaning, Sanitization and Storage 10
6. Ordering information 11
1. Product description
CNBr-activated Sepharose 4 Fast Flow is a bead-formed, highly crosslinked pre-activated matrix produced by reacting Sepharose 4 Fast Flow
with cyanogen bromide (CNBr). This coupling makes the medium more
rigid which in turn improves the pressure/flow characteristics. Proteins
and other molecules containing primary amino groups can be coupled
directly to the pre-activated medium. Multi-point at tachment of proteins
provides the immobilized product with good chemical stability. The resulting
affinity medium can isolate a specific substance from a complex mix ture,
often achieving very high yield and purity in a single step. Many references
demonstrate that binding affinity is frequently well maintained after CNBr
coupling.
A typical application of pre-activated affinity media like CNBr-activated
Sepharose 4 Fast Flow is based on antigen-antibody reactions with
immobilized monoclonal antibodies as ligands. In such cases, purification
factors of 2 000–20 000 can be obtained.
Table 1 summarizes the main characteristics of CNBr-activated Sepharose 4
Fast Flow.
p. 2
Table 1 . Medium characteristics.
Mean particle size 90 μm
Particle size range 45–165 μm
Bead structure Highly cross-linked 4% agarose, spherical
Linear flowrate
Base matrix 150 –250 cm/h, 0.1 MPa (1 bar) , XK 50/60 column, bed height 25 cm
Swelling factor 4–5 ml drained medium/g
Coupling capacity 13–26 mg α-chymotrypsinogen/ml
pH stability*
long term 3–11
short term (CIP) 3–11
* Refers to s tability of co upling betwe en ligand abd b ase matrix. L igands can be l ess stable.
Sepharose 4 Fast Flow matrix
Sepharose 4 Fast Flow is a highly cross-linked agarose matrix. In its preactivated CNBr form, it offers much improved performance when compared
with the well established CNBr-activated Sepharose 4B. The Sepharose 4
Fast Flow matrix has higher rigidity and can thus be run at high flow rates
(see Table 1).
The higher mechanical strength of the cross-linked matrix makes it wellsuited for use in large columns. Scaling up a purification developed on CNBractivated Sepharose 4 Fast Flow is therefore simple and more predictable.
The coupled product is stable at low pH, which is often required for elution
from some immunoadsorbents.
For applications that require operation at high pH, note that the amide bond
formed when using the companion product NHS-activated Sepharose 4
Fast Flow is stable up to pH 13 for normal use.
2. Coupling
CNBr-activated Sepharose 4 Fast Flow is supplied freeze-dried in the
presence of additives. These additives need to be washed away at low
pH (pH-2–3) before coupling the desired ligand. The use of low pH ( pH2–3)
preser ves the activity of the reactive groups, which otherwise hydrolyse at
high pH.
p. 3
In order to retain maximum binding capacity of CNBr-activated Sepharose 4
Fast Flow prior to coupling the ligand, use cold (0 –4 °C) solutions. The
time interval between washing and coupling must be minimised; therefore
preparations of all required solutions prior to coupling is recommended.
1. Prepare the coupling solution, i.e. dissolve the ligand to be coupled in
a suitable coupling buffer, e.g. 0.1 M NaHCO
NaCl. For good coupling efficiency avoid unnecessarily dilute solutions
pH 8.3 containing 0.5 M
3
(Recommended ratio of volumes, coupling solution/medium is 0.5:1). The
coupling pH depends on the ligand. Normally pH in the range 7–9 is used.
ppm sugar
400
300
200
100
0
0
10 20 30 40 50 60
A-method
B-method
A-method: Add portions of 4
column volumes cold 1 mM HCl,
stir and immediately remove the
liquid.
B-method: Add portions of 1
column volume cold 1 mM HCl,
stir for approx. 5 min and then
remove the liquid.
Gel vol.
Fig 1. The content of sugar in the filtrate after washing with different
medium volumes of cold 1 mM HCl.
2. CNBr-activated Sepharose 4 Fast Flow is supplied freeze-dried with
sugar additives and is washed initially with 10–15 medium volumes of
cold 1 mM HCl, see Fig 1. Use small wash por tions (e.g. 1 medium volume)
and let the mixture equilibrate a few minutes during each washing step.
After washing, determine the exact medium volume obtained using e.g.
centrifugation or PD-10 column (the medium volume may vary between
experiments).
3. Mix the washed medium and coupling solution. Adjust pH to the desired
value. To obtain good reproducibility it is wise to adjust total reaction
volume to a fixed value with coupling buffer.
4. Coupling is normally very fast. At room temperature the reaction is
usually completed after 2–4 hours. If coupling is per formed at 4 °C, it can
be performed overnight. It may be practical to follow the reaction using
UV-absorbance measurements.
p. 4
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