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Affinity Chromatography Media
Cellufine™ Amino and Formyl

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.


Cellufine Amino > Reference > MSDS / Technical Information > Catalog No.


Cellufine Formyl > Reference > MSDS / Technical Information > Catalog No.



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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


Characteristics
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


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Pressure / Flow Characteristics


Figure1
Column: 16 x 200mm

Figure2


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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
table1


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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.


Figure3
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


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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.


Figure4
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


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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.



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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.


Figure5


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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.



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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.


Figure6


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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.



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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.
table2


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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).


Figure7
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


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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.


Figure8
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.


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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.


Option1
Option1
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.

Option2
Option2
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.
Figure9


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