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Cellufine Amino en 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.

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

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

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

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.

Figure5

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.

Figure6

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.

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

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.


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.

 

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