Cellufine Sulfate – Cellulose Chromatography Medium for Virus Purification

Topics Covered

Introduction
Chromatography Media Product Range
Cellufine Sulfate
     Virus Purification Using Cellufine Sulfate
     Purification of blood coagulation factor
     Purification of Other Proteins
     Dynamic Binding Capacity ( DBC ) of Cellufine Sulfate
     References
About AMSBIO

Introduction

Cellufine is the liquid chromatography media for the purification of proteins, enzymes and other bio-active substances. Since it is made from spherical cellulose particles having high chemical stability, high mechanical strength and bio-compatibility, it is suitable for the production in pharmaceutical and food industry. Leaking from this matrix is much less than that from the synthetic polymer media.

Cellufine product range covers all liquid chromatography modes (Gel Filtration, Ion Exchanger, Affinity and Hydrophobic Interaction chromatography) in order to purify target molecules to requested specifications. In addition, we can provide custom made grades corresponding to user requests, such as the ion exchanger made from big beads and the affinity with a designated ligands. The production of Cellufine is guaranteed by ISO 9001 and 14000.

Chromatography Media Product Range

The chromatography media product range is given below:

Cellufine Sulfate

Cellufine Sulfate is the group specific media which can be used for purification of viruses and blood coagulation factors, and the manufacture of vaccines, etc.

Virus Purification Using Cellufine Sulfate

The conditions for virus purification are shown in Table 1 and the applications of cellufine sulfate in the concentration or purification of viral and microbial proteins, antigens and viruses are shown in Table 2.

Table 1. Typical conditions for virus purification using Cellufine Sulfate.

target Adsorption wash Elution ref.
Rabies virus 0.01M PB,pH7.2 0.01M PB,pH7.2 0.01M PB,pH7.2+1M NaCl  
Influenza Virus 0.01M PB,pH7.4 0.01M PB,pH7.2+0.2M NaCl 0.01M PB,pH7.0+1.5M NaCl  
Japanese Encephalitis Virus 0.01M PB,PH7.2 0.01M PB,pH7.2 0.01M PB,pH7.2+1M NaCl  
0.01M carbonate,pH9.0 0.01M carbonate,pH9.0 0.01M carbonate,pH9.0+0.2M NaCl 1
AssociatedViralVector 0.01M PB,pH7.4 0.01M PB,pH7.4 0.01M PB,pH7.4+1M NaCl 2
0.01M PB,PH7.2 0.01M PB,PH7.2 0.01M PB,pH7.2+1M NaCl 3

PB=Phosphate buffer

Table 2. There are many applications of Cellufine Sulfate in the concentration or purification of viral and microbial antigens, proteins and viruses.

Virus Viral/Microbial Agents
Rabies Feline Calicivirus Herpes Simplex gA and gB
Influenza Respiratory Syncytial Virus Glycoprotein Subunits
Japanese Rnchephalitis Human Herpes Simplex Hepatitis B suface Antigen
Feline Leukemia Human Measles Filamentous Hemagglutinin from B.pertusis
Feline Herpes Human Prainfluenza Loucocytosis Promoting Factor Hemagglutin

Cellufine Sulfate can perform purification of a virus efficiently. Like the typical example shown in Figure 1, purification of a virus can be performed in three steps; loading, washing, and elution. It is an excellent alternative to sucrose gradient centrifugation or other complicated operations.

Figure 1. Purification of Rabies virus from chick embryo tissue culture fluid on Cellufine Sulfate

Column : I.D.50mm- bead height 70mm
Starting/washing : 0.01M PB,pH7.2
Elution buffer : 0.01M PB,pH7.2 +1M NaCl

Purification of blood coagulation factor

Cellufine Sulfate is applicable to purification of a blood coagulation factors. Separation by Cellufine Sulfate of Prethrombin produced by the CHO cell culture is shown in Figure 1. Other blood coagulation factors purified by Cellufine Sulfate include recombinant Factor IX4) and Factor X/Xa5).

Figure 2. Purification of Recombinant Prethrombin

Sample: Culture medium 15L
Media: Cellufine Sulfate
Column: Φ90×345mm
Flow rate : 100ml/min (94cm/hr)
Adsorption and Washing buffer: 50mM Tris-HCl,0.05M NaCl,pH8.0
Elution buffer: 50mM Tris-HCl,1M NaCl,pH8.0

Purification of Other Proteins

By the affinity interactions similar to heparin, Cellufine Sulfate can purify GAG related enzyme, Growth factors, Follistatin, and Activin, etc. Please look at the list of references.

Starting/washing : 0.01M PB,pH7.2
Elution buffer : 0.01M PB,pH7.2 +1M NaCl

Dynamic Binding Capacity ( DBC ) of Cellufine Sulfate

Cellufine Sulfate has a unique performance advantage. Its DBC hardly changes with increasing flow rates.

Figure 3. Binding capacity of Lysozyme on a 1mL Cellufine Sulfate Mini-Column at various flow rates.

Sample: Lysozyme (1mg/ml)
Media: Cellufine Sulfate

Figure 4. Flow rate vs Lysozyme Binding Capacity at 10% breakthrough volume

The unique DBC character of Cellufine Sulfate is reported by S.Yamamoto , E.Miyagawa. The reference shows Dynamic Binding Capacity of γ-Globulin and Lysozyme by Cellufine Sulfate did not depend on the flow rate.

References

  1. Sugawara K, Nishiyama K, Ishikawa Y, Abe M, Sonoda K, Komatsu K, Horikawa Y, Takeda K, Honda T, Kuzuhara S, Kino Y, Mizokami H, Mi zuno K, Oka T, Honda K. Development of Vero cell-derived inactivated Japanese encephalitis vaccine Biologicals. 2002;30(4): pp 303-314 .
  2. JorisDeWit, RubenEggers, RobertEvers, EeroCastre´n, and JoostVerhaagen Long-Term Adeno-Associated Viral Vector-Mediated Expression of Truncated TrkBin the Adult Rat Facial Nucleus Results in Motor Neuron Degeneration The Journal of Neuroscience, 2006•26(5):1516–1530
  3. KENJI TAMAYOSE,YUKIHIKO HIRAI,TAKASHI SHIMADA A New Strategy for Large-Scale Preparation of High-Titer Recombinant Adeno-Associated Virus Vectors by Using Packaging Cell Lines and Sulfonated Cellulose Column Chromatography Human Gene Therapy ,1996 ;7:pp 507-513
  4. Harrison S, Adamson S, Bonam D, Brodeur S, Charlebois T, Clancy B, Costigan R, Drapeau D, Hamilton M, Hanley K, Kelley B, Knight A, Leonard M, McCarthy M, Oakes P, Sterl K, Switzer M, Walsh R, Foster W. The manufacturing process for recombinant factor IX.Semin Hematol. 1998 Apr;35(2 Suppl 2):4-10. Related Articles, Links
  5. Enjyoji, Keiichi; Miyazaki, Kaoru; Kato, Hisao Characterization of rat factors X and Xa: demonstration of factor Xa in rat plasma J. Biochem. (Tokyo) 1991;109(6):pp890-8
  6. Shuichi Yamamoto , Eiji Miyagawa Retention behavior of very large biomolecules in ion-exchange chromatography Journal of Chromatography A, 852 (1999) 25–30 Short communication

About AMSBIO

AMSBIO’s mission is to be a profitable premier provider of quality life science research reagents and services helping customers develop innovative methods, processes, products and medicines.

This is achieved by offering small and medium size manufacturers, academic groups and revenue generating biotechs a unique partnership for the global market and by providing state of the art and cost effective solutions to end users and partners.

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Date Added: May 17, 2013 | Updated: Jun 11, 2013
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