Affinity Chromatography

CellufineTMSulfate

For concentration, purification and depyrogenation of virus, viral/microbial antigens and heparin-binding proteins

Advances in vaccines and clinical diagnostics have created an increasing demand for large volumes of highly purified and concentrated virus and viral or microbial antigens. Cellufine Sulfate affinity media is a simple, rapid and effective means for concentration, purification and depyrogenation of these important products.

Cellufine Sulfate eliminates cumbersome, time-consuming and potentially unsafe classical ultra-centrifugation and density gradient methods. It can also provide a significant improvement in concentration and purity. Cellufine Sulfate can reduce or eliminate the expense, ligand leakage and reproducibility problems associated with immobilized dextran sulfate, chondroitin sulfate or heparin.Elution of the bound product is affected through simple stepwise or gradient increases in ionic strength.

Features

  • Affinity for a wide range of live, killed or disrupted viruses, viral or microbial antigens and heparin-binding proteins
  • Closed column operation assures safety and product sterility
  • Endotoxins do not bind, allowing a rapid and contaminant free depyrogenation
  • Rigid, high-strength beads
  • Autoclavable

Benefits

  • More effective than ultracentrifugation at removing contaminants from culture media and host cells
  • Avoids excessive product handling and safety concerns, particularly with viral preparations
  • Simultaneous concentration and purification improve yield, reduce processing steps, time and costs
  • Gentle binding and elution conditions provide high capacity and product yield
  • Resists compression, providing rapid flow for high-speed processing, even in large columns, making it easily scalable
  • Resistant to chemical depyrogenation with base and chemically sterilizable with formalin
Sulfate
Figure1Partial Structure of Cellufine Sulfate
Characteristics
Support Matrix Cellulose
Particle Size ca. 40 – 130 µm
Particle Shape Spherical
Gel Exclusion Limit 3kD
Activated Group Sulfate Ester
Total Sulfur >700 µg/g dry
Protein
Lysozyme :
Hepatitis B Surface Antigen :

>3 mg/ml
7 mg/ml
Environmental Resistance Resistant to 0.1M NaOH,0.1 % of 37 % Formalin
Operating Pressure <2 bar (30 psi)
Autoclavable In suspension at neutral pH; 30 min at 121 °C
Supplied Suspension in 20 % Ethanol

Flow Properties

Sulfate
Figure2Pressure / Flow Curves

The nearly rigid properties of the spherical cellulose support matrix allow outstanding flow properties, particularly in large production columns.

Column A
90 x 200 mm
Column B
350 x 200 mm

Virus, Viral/Microbial Antigens

Viruses Viral/Microbial Agents
  • Rabies*
  • Influenza*
  • Japanese Enchephalitis*
  • Feline Leukemia
  • Feline Herpes
  • Feline Calicivirus
  • Respiratory Syncytial Virus
  • Human Herpes Simplex
  • Human Measles
  • Human Parainfluenza
  • Human Coronavirus OC43
  • Herpes Simplex gA and gB
    Glycoprotein Subunits*
  • Hepatitis B Surface Antigen
  • Filamentous Hemagglutinin from B. pertussis *
  • Leucocytosis Promoting Factor Hemagglutinin*
*These applications are covered by US and foreign process patents.
Please inquire regarding details and licensing arrangements.

Table 1There are many applications of Cellufine Sulfate in the concentration or
purification of viral and microbial anifigensm proteins and viruses.

Example of purification of human coronavirus (OC43) with Cellufine Sulfate

Here is an example of purification of human coronavirus (OC43). Coronavirus has an envelope protein. So, Cellufine Sulfate is capable of adsorbing and purifying coronavirus.

Detailed technical data can be downloaded from the following.

Sulfate
Column
I.D. 5 mm x height 15 mm (0.3 ml)
Sample
Vero cell lysate including BPL-inactivated virus
Virus strain
Human corona virus(HCoV) OC43
Flow rate
0.3 ml/min (residence 1 min)
Equilibration
10 mM sodium phosphate, 150 mM sodium chloride, pH 7.4
Elution
10 mM sodium phosphate, 2 M sodium chloride, pH 7.4

Purification of Rabies Virus

The example in Figure 3 illustrates the high degree of concentration, purification and yields obtained with Cellufine Sulfate on typical viral preparations.

Sulfate
Figure3Purification of Rabies virus from chick embryo tissue culture fluid
Column
50 x 70 mm (140 ml)
Buffer
0.01M Phosphate (pH 7.2)
Eluant
1M NaCl/0.01M Phosphate (pH 7.2)
Load Eluate
Volume (ml) 4,200 50
Virus titer 32 4,096
Protein (µg/ml) 8.5 14
Yield (%) 100 152
Purification factor 79x
Concentration factor 126x

Table 2Concetration and Purification of virus with Cellufine Sulfate

Purification of Influenza Virus

Hen’s egg allantoic fluid was loaded directly onto a 33.3 mL gel bed and 94.5%virus was recovered in the eluate fraction.

Volume(ml) Virus Titer TCA-N µg/ml Recovery(%) Fold Purification
Allantoic Fluid 4200 77 337.1 100 1
Wash 6700 1 209.2 2.1 -
Eluate 170 1797 448.0 94.5 20.1

Table 3Purification of Influenza virus from hen's egg allantoic fluid

Column
50 x 170 mm
Buffer
0.01M Phosphate pH 7.4
Wash
0.01M Phosphate pH 7.2 + 0.2M NaCl
Elution
0.01M Phosphate pH 7.0 + 1.5M NaCl

Antigenic Protein Purification and Depyrogenation

Cellufine Sulfate is ideal for depyrogenating virus and other microbial extracts because it does not bind endotoxins. Figure 4 shows the purification of filamentous hemagglutinin (FHA) from the whooping cough bacterium Bordetella pertussis.

Sulfate
Figure4Purification of filamentous hemagglutinin from B. pertussis
Column
16 x 70 mm (20 ml)
Sample
800 ml B. pertussis culture fluid
(endotoxin titer > 1015 by Limulus lysate test)
Buffer
0.01M Phosphate (pH 7.6)
Eluant
1M NaCl/0.01M Phosphate (pH 7.6)
FHA Yield
94%
Purification Factor
20x
Concentration Factor
28x (30 ml product)
Endotoxin
Below standard level by Limulus lysate, rabbit pyrogen and mouse toxicity tests

Protein Purification

Cellufine Sulfate mimics the affinity of heparin or dextran sulfate for many proteins. It can function as an affinity support for selected plasma proteins, cellular growth factors and lipases. Its capacity comparable to conventional heparin gels.

Binding Proteins Non-binding Proteins
  • Antithrombin III
  • β-Lipoprotein
  • Complement C5, C6, C8
  • Complement C3 Activator
  • Trypsin
  • Trypsin Inhibitor
  • Chymotrypsinogen
  • Lysozyme
  • Urease
  • Catalase
  • Factor IX
  • Albumin
  • α-Lipoprotein
  • Complement C3, C9
  • Complement C1, C3b
  • Inactivators
  • IgG
  • Ceruloplasmin
  • α2-Macroglobulin
  • RNase
  • Bacitracin
  • Glucose Oxidase
*Binding and elution are extremely rapid and very fine separations can be generated in gradient mode.

Table 4

Purification of Partially Purified Casein Kinase II from Calf Thymus

Sulfate
Figure5
Column
10 x 20 mm
Sample
7 ml
Buffer
50mM Tris-HCl (pH 7.9)
+ 50mM MgCl2
+ 0.1mM EDTA
+ 0.1mM PMS
+ 0.5mM DTT
+ 25 % glycerol
Eluant
0.05 – 1.0M NaCl in buffer

Development of the grade

Development of the grade according to demand of a customer (inquiry)

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