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Activated
Supports for Immobilization of Antibodies, Antigens,
Affinity Ligands and Enzymes |
|
The growth of process-scale affinity
chromatography has created the need for a new generation of support
matrix materials and coupling chemistries suited for the industrial
environment. Classical agarose based supports perform poorly at
the large-scale for several reasons. They provide poor flow properties
in large columns. The widely used cyanogen bromide coupling chemistry
has well-documented problems with bond stability and non-specific
adsorption. Additionally, even with more modern chemistries, agarose
can shed polysaccharide chains, giving rise to significant ligand
leakage under mild operating conditions.
Cellufine activated supports provide state-of-the-art laboratory
performance at the process-scale without difficulty. The products
are based on rigid spherical cellulose beads specially optimized
for affinity chromatography to provide very large pore size and
high ligand capacity together with high flow rates in large columns.
The cellulose backbone offers very low non-specific adsorption
without the ligand leakage problems of agarose. |
Features
• High flow rates in laboratory and process columns for high
throughput
• Low ligand leakage due to exceptionally stable coupling
chemistry and support matrix
• Excellent mechanical, chemical and environmental resistance
• High ligand loading capacity
• Compatible with high molecular weight ligands and target
proteins due to pore size equivalency with 4 % cross-linked agarose
media
• Unreacted formyl groups easily converted during reduction
to neutral hydroxyls for low non-specific adsorption
• Built in hydrophilic spacer arms for maximum ligand accessibility
and low non-specific adsorption
• No media damage or fines generation with extended mixing
to allow use of simple coupling apparatus
• Ligand coupling occurs under mild conditions in short reaction
times
• Thermal stability of media allows high temperature reactions
• Long shelf-life of unreacted media |
|
|
| Substrate |
Crosslinked cellulose |
| MW
Exclusion Limit |
4,000kD |
| Standard
Particle Size |
125 - 210µm |
| Particle
Shape |
Spherical |
| Density |
0.7g/ml wet |
| Shrinkage
/ Swelling |
Will not shrink or swell substantially under changes
in pH or ionic strength
|
| Chemical
Resistance |
Can be used with
any salts, non-ionic detergents, organic solvents.
Resistant to 0.1M HCl and 0.5M NaOH. (Note: coupled
ligand may not be stable under these conditions) |
| Mechanical
Resistance |
Will withstand peristaltic
pumping and extended mixing |
| Autoclavable |
121 °C for 30
minutes at pH 7 |
| Saturation
Capacity |
Up to 40mg protein/ml
depending upon protein and conditions |
| Operating
Pressure |
< 1 bar (15 psi) |
| Supplied |
Amino and Carboxyl
20 % EtOH
Formyl 0.01 % 2, 2-thio-bis
(pyridine-1-oxide) |
|
| Support |
Active
Group |
Spacer
Length
(atoms) |
Density
(mol/ml) |
| Formyl |
Aldehyde |
8 |
15 - 20 |
| Amino |
Primary
Amine |
3 |
15 - 20 |
|
Pressure / Flow Characteristics
|
 |
Applications
Activated
Support |
Immobilized
Molecule |
Target
Molecule |
Cellufine
Formyl |
Antibodies
Antigens
Protein A, G
Lectins
Cytokines
Enzymes |
Antigens
Antibodies
Antibodies
Carbohydrates Glycoproteins
Receptors
Substrate/Product |
Cellufine
Amino |
Carboxyl-Containing
Proteins and
Small Ligands
Reducing Sugars
Heparin |
General Proteins
Lectins, Receptors
Blood Proteins Growth Factors, Viral Antigens |
|
|
Antigen Purification
Figure 3 illustrates the use of Cellufine Formyl coupled with
an antibody for largescale antigen purification. In this application
the Cellufine affinity column provides a significant concentration
and purification of antigen at high yield. The subject column
has been used for over 30 months to process over 3,000 liters
of starting plasma with no significant degradation in performance.
To produce the gel, 45 liters of horse serum containing anti-HBs
Ag antibody were first concentrated and purified by ammonium
sulfate precipitation and dialysed into 0.2M phosphate (pH 7)
with 0.1 M NaCl. The resulting antibody serum was added to 12
liters of Cellufine Formyl and reacted together with 80 grams
of NaCNBH3 at 4 to 8 °C for 24 hours. The antibody
gel was then washed with buffer and packed into the column.
The process stream consisted of human plasma positive for HBs
Ag which had been previously purified by freeze-thawing, centrifugation,
ammonium sulfate precipitation and gel filtration chromatography.
| Sample: |
1200 liters semi-purified HBs Ag-positive
human plasma |
| Column: |
140 x 780mm (12 liters) Cellufine Formyl
Horse Anti-HBs Ag |
| Starting/Wash |
0.1M NaCl, 0.2M phosphate |
| Buffer: |
(pH 7) wash volume 200 liters |
| Eluent: |
0.2M glycine/HCl (pH 3) |
| Flow Rate: |
20cm/hr loading/washing 26cm/hr elution |
| Product Volume: |
14 liters (85 x concentration) |
| Yield: |
87 % |
| Single Step: |
|
| Purification: |
149 x |
|
RCA, Purification
Cellufine Formyl can be used to immobilize lectins for glycoprotein
purification, as shown in Figure 4. Con A (50mg) was immobilized
on 0.5g (wet) of Cellufine Formyl by reacting at
4 °C overnight in 1ml of 0.1M acetate (pH 6.4) containing
1mM MgCl2, 1mM MnCl2 and 1mM CaCl2
under the presence of methyl-alpha-D-mannoside and NaCNBH3.
After washing with water, the gel was suspended at 4 °C overnight
in 2ml of 1 % glutaraldehyde with NaCNBH3. After a
second water wash the gel was suspended for one hour at room temperature
in 2ml of 1M Tris/HCl (pH 7.4) and rewashed.
| Sample: |
66ml RCA1 (30mg/ml protein) |
| Column: |
0.9 x 9mm (0.6ml) Cellufine Formyl Con A |
| Starting/Wash |
0.1M NaCl |
| Buffer: |
0.2M Phosphate (pH 7.2) |
| Eluent: |
0.2M methyl-alpha-D-mannoside |
| Flow Rate: |
12cm/hr |
|
FUNCTIONAL SELECTION OF ACTIVATED SUPPORTS
The two types of Cellufine activated support media are Cellufine
Formyl, and Amino. The availability of two functional
groups allows great flexibility in selecting media for optimal
reaction conditions (pH, temperature, activating agents, reactant
concentrations, etc.). Each is a high-stability, functional packing
optimized for an application group. Control of reaction chemistry
and ligand density is straightforward. |
A GENERAL SUPPORT FOR PROTEINS
Cellufine Formyl
The aldehyde active group on Cellufine Formyl packings reacts
with primary amine groups on the ligand to form a Schiff’s
base complex (see Figure 5). A mild reducing agent is used to
convert the Schiff’s base to a highly stable linkage. Table
2 illustrates a general ligand coupling protocol.
|
Reducing Agents
Cellufine Formyl requires a reducing agent for formation of a
highly stable linkage. A number of reducing agents are available
for good results in virtually any application. The agent should
be selected to produce a reasonable reaction rate and yet not
be so strong as to damage the protein ligand (such as by reduction
of disulfide bonds) or as to reduce the aldehyde groups. Sodium
borohydride (NaBH4), sodium cyanoborohydride (NaCNBH3)
and a newer, non-toxic reducing agent, trimethylamine borane ((CH3)3NBH3)
are successful agents, depending upon the particular requirements.
For any agent, the quantity required is typically less than 10
milligrams per gram of wet gel. |
INTERMEDIATES FOR ADVANCED CHEMISTRY
Cellufine Amino
Cellufine Amino and Carboxyl are convenient intermediates for
use in coupling reactions requiring greater chemical sophistication
than possible with Cellufine Formyl. Each support can be easily
used with carbodiimide reagents to form a media suitable for coupling
the opposite functionality (see Figure 6). Other reaction chemistries
can also be used, such as carbodiimide in conjunction with Cellufine
Carboxyl to form an active ester group (e. g., N-hydroxysuccinimide).
The excellent mechanical and chemical stability of the Cellufine
support matrix allows effective performance over a wide range
of chemistries.
|
VERSATILE COUPLING
Optimization of ligand coupling chemistry is often critical to
the success of an affinity separation. Cellufine Formyl allows
a broad range of coupling conditions to be used to maximize both
coupling efficiency and yield of active protein. Cellufine Amino
and Carboxyl give the user a wide range of further options for
custom chemistry in specialty applications. |
Coupling with Formyl
The reaction rate of Cellufine Formyl is rapid enough to be practical,
yet slow enough to be extremely gentle to most proteins. It also
allows for a fine measure of control. The rate may be controlled
effectively with temperature to achieve maximum protein stability.
The pH of effective couplings ranges between 3 and 10.
The coupling efficiency (the ratio between amount coupled and
amount offered) and total ligand density can be varied and optimized
quite easily through changes in coupling ligand concentration,
pH and temperature. A standard set of conditions will work well
for most cases, but optimization over a broad range can be used
to improve process economics for specific applications.
| 1 |
Wash media with water and
filter. Slurry media coupling buffer containing ligand. |
| 2 |
Stir or shake one-half
to two hours. |
| 3 |
Add reducing agent. |
| 4 |
Stir or shake 6 to 10 hours. |
| 5 |
Wash with 0.2M Tris/HCl
(pH 7) or 1M ethanolamine in buffer with reducing agent
to quench residual aldehydes. Stir or shake 3 to 5 hours. |
| 6 |
Wash with chromatography
elution buffer and then starting buffer. |
| 7 |
Pack and run column. |
|
Coupling with Amino
The flexibility of these media is illustrated in the use of Cellufine
Amino for the immobilization of heparin (Figure 9) and for coupling
of reduced sugars.
Amino supports have often been used to couple reducing sugars
directly through the aldehyde functionality. A major problem with
agarose-based supports, however, has been the lengthy reaction
time (often weeks) required. The thermal stability of Cellufine
media allows much faster reactions at high temperatures (Figure
7).
Reaction Conditions:
2g (wet) of Cellufine Amino was added to 0.2g of maltose
and 70mg NaCNBH3
in 2ml 0.2M Phosphate (pH 7) / 0.1M NaCl |
|
Antibody Purification
Optimization of ligand coupling to activated gels always involves
a trade-off between efficiency of uptake (the fraction of ligand
offered in the reaction that is actually coupled) and the final
ligand loading (mg of ligand coupled per ml of gel). When purified
ligand is readily available, a high loading gel can be produced
at the cost of low coupling efficiency. In the more common case,
however, purified ligand is quite precious, and good coupling
efficiency is highly desirable, even at the expense of low loading.
Low ligand density may also improve binding specificity in some
cases.
The lack of competing hydrolysis reaction in the aldehyde chemistry
of Cellufine Formyl makes fine control of the loading and efficiency
quite straightforward. In this example, high purity bovine serum
albumin is used as an antigen for the purification of rabbit anti-BSA
antibody. The coupling reaction was designed to give very high
efficiency (98 %) and relatively low ligand density. |
| Sample: |
24ml precipitated rabbit antiserum |
| Column: |
14 x 34mm Cellufine Formyl BSA (5.2ml) |
| Starting/Wash: |
0.05M Phosphate (pH 7.4) |
| Buffer: |
/0.5M NaCl |
| Eluent: |
0.2M Glycine/HCl (pH 2.25) |
| Flow Rate: |
27cm/hr |
| Yield: |
27mg antibody |
| Single Step |
|
| Purification: |
20 x |
Cellufine Formyl BSA was prepared by washing 5g (wet) of media
with 0.1M phosphate (pH 7.1), adding 5ml of 4mg/ml BSA and stirring
for 12 hours at 25 °C. After washing with buffer, the media
was suspended in 5ml of buffer containing 0.4M ethanola-mine.
After stirring for 4 hours at 25 °C the media was washed with
buffer. The BSA coupled was about 3.0 mg/ml media. |
Heparin Immobilization
The complex carbohydrate heparin can be coupled in two ways: either
through the side chain carboxyl groups by a carbodiimide reaction;
or directly through the aldehyde group located on the terminal
sugar of the molecule. Selection will depend on performance requirements.
Coupling through the carboxyl groups is faster and produces a
higher loading but the terminal aldehyde reaction normally results
in higher biological activity.
 |
 |
| 400mg of heparin in 8ml water (adjusted
to pH 4.5 with HCl) was mixed with 5g (wet) Cellufine
Amino and 300mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide.
After shaking overnight at 4 °C, the washed gel
coupled 25mg heparin/ml. |
 |
 |
| 2g of heparin in 75ml of 0.2M Phosphate
(pH 7)/ 0.1M NaCl was mixed with 50g (wet)Cellufine
Amino. After adding 0.2g NaCNBH3, the mixture
was stirred at 60 °C for two days. The washed
gel coupled 1mg heparin/ml. |
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|
| Packing |
Quantity |
Catalogue
No. |
| Cellufine
Formyl |
50
ml
0.5 L
5 L |
19853
19854
19855 |
| Cellufine
Amino |
50
ml
0.5 L
5 L |
19856
19857
19858 |
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