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.
- 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
|MW Exclusion Limit||4,000kD|
|Standard Particle Size||125 - 210µm|
|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||Formyl 0.01 % 2, 2-thio-bis (pyridine-1-oxide)|
|Support||Active Group||Spacer Length(atoms)||Density (μmol/ml)|
|Formyl||Aldehyde||8||15 - 20|
Pressure / Flow Characteristics
- 16 x 200mm
|Activated Support||Immobilized Molecule||Target Molecule|
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.
- 1200 liters semi-purified HBs Ag-positive human plasma
- 140 x 780mm (12 liters) Cellufine Formyl Horse Anti-HBs Ag
- 0.1M NaCl, 0.2M phosphate
- (pH 7) wash volume 200 liters
- 0.2M glycine/HCl (pH 3)
- Flow Rate
- 20cm/hr loading/washing 26cm/hr elution
- Product Volume
- 14 liters (85 x concentration)
- 87 %
- Single Step
- 149 x
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.
- 66ml RCA1 (30mg/ml protein)
- 0.9 x 9mm (0.6ml) Cellufine Formyl Con A
- 0.1M NaCl
- 0.2M Phosphate (pH 7.2)
- 0.2M methyl-alpha-D-mannoside
- Flow Rate
FUNCTIONAL SELECTION OF ACTIVATED SUPPORTS
The availability of formyl functional groups allows great flexibility in selecting media for optimal reaction conditions (pH, temperature, activating agents, reactant concentrations, etc.). Cellufine formyl 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.
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.
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.|
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.
- 24ml precipitated rabbit antiserum
- 14 x 34mm Cellufine Formyl BSA (5.2ml)
- Starting/Wash Buffer
- 0.05M Phosphate (pH 7.4)/0.5M NaCl
- 0.2M Glycine/HCl (pH 2.25)
- Flow Rate
- 27mg antibody
- Single Step Purification
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.