Products & Services

Cellufine™ Chromatography Media

Cellulose Beads Chromatography Media

The Cellufine™ chromatography media is based on spherical cellulose beads, which exhibit high chemical stability, high mechanical strength, and inherent bio-compatibility. Cellufine™ chromatography products are appropriate for all chromatographic techniques. Gel Filtration, Ion Exchange, Affinity, and Hydrophobic Interaction chromatography media are available for preparation of a broad range of target molecules.

Applications

Chromatography Media Product Range

Typical Product Range
TypeApplicationsSpecification
Ion Exchange
Cellufine™ IEX
For initial separation and final polishing of proteins, peptides and enzymes by electrostatic interaction Ion exchange resins (DEAE, QA, CM, S)
MAX IEX (High performance Ion exchange)
Graft polymer modification
Cellufine™ MAX GS
Mix mode
Cellufine™ MAX IB
For high performance impurities removal from target molecules Strong cation exchange on Graft chain
Mix mode Anion exchange resin
Affinity
Cellufine™ Sulfate
Cellufine™ ET Clean
Cellufine™ Chlete, etc.
For high specificity separation of a wide range of molecules by specific interaction Heparin mimic for virus & heparin binding proteins
Endotoxin removal
IMAC metal binding molecules
Molecules with diol & EPO
Nucleic acid related molecules
Hydrophobic Interaction
Cellufine™ Butyl
Cellufine™ Phenyl
For purification of proteins and enzymes by hydrophobic interaction Butyl
Phenyl
Gel filtration
Cellufine™ GCL-2000HF
Cellufine™ GH-25
For purification of bio-molecules and proteins by molecular size.
For salt and solvent removal and buffer exchange
MW 50 - 3000 kD
3 kD gel exclusion limit
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Ion Exchange

Chemical Structures of Cellufine™ IEX Series Ligands

▶Strong Anion Exchange (Q) Weak Anion Exchange (DEAE)
Cellufine™ MAX Q-h
Cellufine™ MAX Q-r
Cellufine™ Q-500
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Cellufine™ MAX DEAE
Cellufine™ A-200
Cellufine™ A-500
Cellufine™ A-800
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Strong Cation Exchange (S) Weak Cation Exchange (CM)
Cellufine™ MAX S-h
Cellufine™ MAX S-r
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Cellufine™ MAX CM
Cellufine™ C-500
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Superior Binding Capacity under High Flow Rates

Cellufine™ MAX IEX series media demonstrate higher DBC compared to other agarose or acrylic media, even under high flow rate conditions.

Figure 4
BSA DBC at 10% Breakthrough for Various Flow Rates

▶Strong Anion Media ▶Weak Anion Media
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BSA concentration: 1 mg/mL
Column: 5 mm I.D. x 100 mm
Buffer: 50 mM Tris-HCl (pH 8.5)

BSA concentration: 1 mg/mL
Column: 5 mm I.D. x 50 mm
Buffer: 50 mM Tris-HCl (pH 8.5)

IgG DBC at 10% Breakthrough for Various Flow Rates

▶Strong Cation Media ▶Weak Cation Media
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IgG concentration: 1 mg/mL (polyclonal, human)
Column: 5 mm I.D. x 100 mm
Buffer: 10 mM Na-acetate, 50 mM NaCl (pH 4.3)

IgG concentration: 1 mg/mL (polyclonal, human)
Column: 5 mm I.D. x 50 mm
Buffer: 10 mM Na-acetate (pH 5.6)

High Pressure Resistance Enables Efficient Purification

Cellufine™ MAX IEX chromatography media perform effectively under high pressure conditions, enabling efficient purification of biopharmaceuticals.
The figure on the right shows pressure flow curves of Cellufine™ MAX IEX series media used in large columns. All Cellufine™ MAX IEX media can be operated at high pressures.
Pressure Flow Curves of Cellufine™ MAX IEX Chromatography Media
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Column: 300 mm I.D. x 200 mm
Mobile phase: pure water (20 ºC)

Excellent Adsorption & Resolution

Cellufine™ MAX IEX chromatography media are optimized for high adsorption and resolution. Figures below show the separation of model proteins. Good separation profiles are observed.

Separation of Model Proteins by Cellufine™ MAX Q-h and Cellufine™ MAX DEAE Separation of Model Proteins by Cellufine™ MAX S-h and Cellufine™ MAX CM
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Column: 6.6 mm I.D. x 50 mm
Equilibration buffer (A): 50 mM Tris-HCl (pH 8.5)
Elution buffer (B): 1 M NaCl in (A)
Elution: (B) 0% → (B) 75%, linear gradient
Flow rate: 0.86 mL/min (residence time 2 min)
Sample loaded: 1.5 mL

Transferrin (5 mg/mL)
BSA (10 mg/mL)
Pepsin (5 mg/mL)

Column: 6.6 mm I.D. x 50 mm
Equilibration buffer (A): 10 mM phosphate (pH 7)
Elution buffer (B): 1 M NaCl in (A)
Elution: (B) 0% → (B) 50%, linear gradient
Flow rate: 0.86 mL/min (residence time 2 min)
Sample loaded: 1.5 mL

Ribonuclease A (5 mg/mL)
Cytochrome C (2.5 mg/mL)
Lysozyme (1.5 mg/mL)

High Salt Tolerance

Cellufine™ MAX IEX chromatography media maintain high binding capacity over a range of salt concentrations. This characteristic enables the omission of buffer exchange steps prior to the use of Cellufine™ chromatography media.

BSA DBC at 10% Breakthrough for Various NaCl Concentrations IgG DBC at 10% Breakthrough for Various NaCl Concentrations
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BSA concentration: 1 mg/mL
Column: 5 mm I.D. x 100 mm
Buffer: NaCl in 50 mM Tris-HCl (pH 8.5)
Flow rate: 300 cm/h (residence time 2 min)

IgG concentration: 1 mg/mL (polyclonal, human)
Column: 5 mm I.D. x 100 mm
Buffer: NaCl in 10 mM Na-acetate (pH 4.3)
Flow rate: 300 cm/h (residence time 2 min)

Optimization of Binding Conditions

The binding capacity of ion exchange chromatography media depends on ionic strength and pH. Separation conditions need to be optimized depending on the characteristics of the target proteins.
Figures below shows that Cellufine™ MAX S-h media has a higher IgG binding capacity than Agarose S type media with 10 mM NaCl at all pH levels tested. Even with 100 mM NaCl, Cellufine™ MAX S-h media demonstrates higher IgG DBC at pH 4.3.

IgG DBC at 10% Breakthrough for Various Salt Concentrations and pH Levels

▶Cellufine™ MAX S-h ▶Agarose S Type Media
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IgG concentration: 1 mg/mL (polyclonal, human)
Column: 5 mm I.D. x 100 mm
Buffer: NaCl in 10 mM Na-acetate
Flow rate: 300 cm/h (residence time 2 min)

IgG concentration: 1 mg/mL (polyclonal, human)
Column: 5 mm I.D. x 100 mm
Buffer: NaCl in 10 mM Na-acetate
Flow rate: 300 cm/h (residence time 2 min)

Characteristics & Specifications

Cellufine™ MAX IEX Series

ProductMAX Q-rMAX Q-hMAX S-rMAX S-hMAX DEAEMAX CM
Ligand Q Q S S DEAE CM
Ion exchange type Strong anion Strong cation Weak anion Weak cation
Support matrix Highly cross-linked cellulose with dextran scaffold
Particle size 40-130 µm
Ion exchange capacity 0.10-0.20 mEq/mL 0.13-0.22 mEq/mL 0.09-0.21 mEq/mL 0.10-0.22 mEq/mL 0.12-0.25 mEq/mL 0.09-0.22 mEq/mL
Adsorption capacityLysozyme - - ≥110 mg/mL ≥140 mg/mL - ≥140 mg/mL
BSA ≥120 mg/mL ≥190 mg/mL - - ≥120 mg/mL -
Chemical stability Stable in commonly used aqueous buffers and
cleaning solutions (NaOH up to 0.5 M)
pH stability 2-12 2-12 2-13 3-14 2-12 2-13
Operating pressure <0.3 MPa (3 bar, 44 psi)
Supplied as Suspension in 20% ethanol

Cellufine™ IEX Series

ProductQ-500A-200A-500A-800C-500
Ligand Q DEAE DEAE DEAE CM
Ion exchange type Strong anion Weak anion Weak cation
Support matrix Cross-linked cellulose
Particle size 40-130 µm
Degree of swelling 5-9
mL/g-dry wt
6-9
mL/g-dry wt
8-10
mL/g-dry wt
12-16
mL/g-dry wt
9-11
mL/g-dry wt
Ion exchange capacity 1.2-1.9
mEq/g-dry wt
0.8-1.1
mEq/g-dry wt
1.1-1.4
mEq/g-dry wt
0.6-1.0
mEq/g-dry wt
0.9-1.2
mEq/g-dry wt
Adsorption capacity Lysozyme - - - - ≥70 mg/mL
BSA ≥10 mg/mL ≥80 mg/mL ≥60 mg/mL ≥45 mg/mL -
Exclusion limit (protein) Approx.
500 kDa
Approx.
30 kDa
Approx.
500 kDa
Approx.
1,000 kDa
Approx.
500 kDa
Chemical stability Stable in commonly used aqueous buffers and
cleaning solutions (NaOH up to 0.5 M)
pH stability 1-13
Operating pressure <0.2 MPa (2 bar, 29 psi)
Supplied as Suspension in 20% ethanol

Catalog Numbers

► Cellufine™ MAX IEX Series

ProductQuantityCatalog Number
Cellufine™ MAX Q-r 1 mL x 5 (mini column) 20500-51
5 mL x 5 (mini column) 20500-55
100 mL 20500
500 mL 20501
5 L 20502
10 L 20503
Cellufine™ MAX Q-h 1 mL x 5 (mini column) 20600-51
5 mL x 5 (mini column) 20600-55
100 mL 20600
500 mL 20601
5 L 20602
10 L 20603
Cellufine™ MAX S-r 1 mL x 5 (mini column) 20300-51
5 mL x 5 (mini column) 20300-55
100 mL 20300
500 mL 20301
5 L 20302
10 L 20303
Cellufine™ MAX S-h 1 mL x 5 (mini column) 20400-51
5 mL x 5 (mini column) 20400-55
100 mL 20400
500 mL 20401
5 L 20402
10 L 20403
Cellufine™ MAX DEAE 1 mL x 5 (mini column) 21000-51
5 mL x 5 (mini column) 21000-55
100 mL 21000
500 mL 21001
5 L 21002
10 L 21003
Cellufine™ MAX CM 1 mL x 5 (mini column) 20900-51
5 mL x 5 (mini column) 20900-55
100 mL 20900
500 mL 20901
5 L 20902
10 L 20903

► Cellufine™ IEX Series

ProductQuantityCatalog Number
Cellufine™ Q-500 1 mL x 5 (mini column) 19907-51
5 mL x 5 (mini column) 19907-55
100 mL 675 982 327
500 mL 19907
5 L 19908
10 L 675 982 335
Cellufine™ A-200 1 mL x 5 (mini column) 19611-51
100 mL 676 980 327
500 mL 19611
5 L 19612
10 L 676 980 335
Cellufine™ A-500 1 mL x 5 (mini column) 19805-51
5 mL x 5 (mini column) 19805-55
100 mL 675 980 327
500 mL 19805
5 L 19806
10 L 675 980 335
Cellufine™ A-800 1 mL x 5 (mini column) 19865-51
5 mL x 5 (mini column) 19865-55
100 mL 673 980 327
500 mL 19800
5 L 19801
10 L 673 980 335
Cellufine™ C-500 1 mL x 5 (mini column) 19800-51
5 mL x 5 (mini column) 19800-55
100 mL 675 983 327
500 mL 19865
5 L 19866
10 L 675 983 335

Graft Polymer Modification

MAX GS
An ion exchange chromatography medium for
monoclonal antibody aggregate removal

Aggregate formation is a common issue during monoclonal antibody (mAb) manufacturing processes, including cultivation, Protein A column elution, low-pH viral inactivation, and ultrafiltration/diafiltration. These mAb aggregates can decrease pharmacological activity and induce overactive immune responses such as anaphylactic shock, and thus need to be removed.

Cellufine™ MAX GS is a strong cation exchanger designed to remove mAb aggregates.

The Cellufine™ MAX GS matrix comprises highly cross-linked cellulose. Surface modification with graft chains containing ion-exchange groups creates a specialized structure that offers a unique mechanism for separating antibody monomers and aggregates. Pore diameters are also optimized to deliver high adsorption capacity.

Cellufine™ MAX GS provides a valuable tool for tackling aggregate removal.

Separation with Cellufine™ MAX GS vs. S agarose media for polyclonal IgG aggregates

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Poly IgG: thermal and acid stressed poly IgG
Buffer A: 10 mM acetate (pH 5.0) + 50 mM NaCl
Buffer B: 10 mM acetate (pH 5.0) + 1 M NaCl
Dynamic binding capacity of Cellufine™ MAX GS vs. S agarose media

Merits

・Efficient separation of antibody monomers and aggregates
・High adsorption capacity
・Low pressure drop
・Excellent compatibility with large-scale column chromatography
・High stability during repeated alkali washing
・Simple regeneration and depyrogenation
・Low non-specific adsorption

Dynamic binding capacity of Cellufine™ MAX GS vs. S agarose media

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Column: 5mm ID x 50mm L
Polyclonal antibody: 1mg/mL in buffer A
Buffer A: 10mM acetate (pH 5.0) + 50 mM NaCl

Contour plot of DBC with Cellufine™ MAX S-h (upper) and Cellufine™ MAX GS (lower)

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Characteristics of Dynamic Binding Capacity (DBC) with Cellufine™ CEX

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▶Graft modified Cellufine™ MAX GS showed to be less susceptible to pH condition in comparison with dextran modified Cellufine™ MAX S-h.

Load condition

mAb: momoclonal antibody (5 mg/mL)
Actual bed volume: 0.59 mL (3 cm bed height)

▶Graft modified Cellufine™ MAX GS showed to be less susceptible to pH condition in comparison with dextran modified Cellufine™ MAX S-h.

MAb aggregate separation using Cellufine™ MAX GS
(Gradient elution with low sample loading)

Distinct MAb monomer and dimer peaks are obtained with gradient elution.

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Buffer A: 10 mM acetate (pH 4.5) + 50 mM NaCl

Buffer B: 10 mM acetate (pH 4.5) + 0.5 M NaCl

Gradient: 75 CV

Injection: acid-treated mAb1

Catalog Numbers

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Mixed mode resin, Cellufine™ MAX IB

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▶Benefit
Salt tolerance
Superior impurity removal
Less susceptible to buffer especially multivalent buffer
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Affinity

Sulfate

Cellufine™ Sulfate chromatography media is an affinity media for the concentration, purification and depyrogenation of virus, viral/microbial antigens and heparin-binding proteins.
Unlike heparin chromatography, the ligand of Cellufine™ Sulfate media does not have an animal origin, and safety concerns are minimized.

Structure of Cellufine™ Sulfate

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Virus & Protein Binding by Non-animal Derived Affinity Ligand

Similar to conventional heparin chromatography, Cellufine™ Sulfate media can be used for purification of various types of viruses. In the case of the influenza virus (H7N7) purification study shown below, high degrees of concentration and yield are achieved, while proteins and DNA are effectively removed.
As Cellufine™ Sulfate media, unlike heparin chromatography, does not use animal-derived ligands safety concerns are minimized.

Purification of Influenza Virus

Virus strain: Egg-derived A/duck/Hokkaido/vac-2/2004 (H7N7)
Column: Cellufine™ Sulfate, 70 mL (I.D. 22 mm x H 185 mm)
Flow rate: 100 cm/h
Equilibration buffer (A): 0.01 M phosphate (pH 7.4)
Washing buffer: 0.19 M NaCl in (A)
Elution buffer: 3 M NaCl in (A)

Notes:
1. HA-titer: Virus titer was estimated by hemagglutination assay.
2. A280 readings during parts of the load and wash stages of the run exceeded the detection limit and are shown as a dashed line.

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Superior Chemical Stability

Cellufine™ Sulfate media is more stable in acid and alkali conditions than is heparin agarose chromatography media. The graph on the right shows that Cellufine™ Sulfate media maintains its adsorption capacity of lysozyme over 40 days even with the use of 0.2 M HCl or 0.5 M NaOH, while heparin agarose media shows a sharp drop in adsorption capacity with the use of 0.01 M HCl or 0.5 M NaOH.

Chemical Stability of Cellufine™ Sulfate and Heparin Agarose Media

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Linear Flow Rate under High Pressure Operation

Cellufine™ Sulfate media can be operated under high flow rate conditions. The graph on the right shows that the flow rate of Cellufine™ Sulfate media is linear up to about 250 cm/h. Even under high pressure operation, compression of the bed height is less than 15%.
This data was obtained from a study using three different lots of Cellufine™ Sulfate media.

Flow Rate Curve of Cellufine™ Sulfate

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Column: 9 cm I.D. x 38 cm bed height
Mobile phase: pure water

Characteristics & Specifications

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Catalog Numbers

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for removal of endotoxins during Downstream Processing

The Cellufine™ ET clean is poly(ε-lysine) immobilized Cellufine™ (cellulose spherical beads). The beads bind and remove endotoxin from your sample solution. The poly(ε-lysine) is a microbial poly (amino acid) that consist of 30-35 lysine residues produced by Streptomyces albulus. The poly(ε-lysine) ligand and cellulose beads are products of JNC Corporation.

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The adsorption of endotoxin was measured as follows: The beads were washed and equilibrated with 0.02 M phosphate buffer (pH 7.0) – 0.05 M NaCl. The removal of endotoxin was determined using a batchwise method with 0.3 mL of wet beads and 2 mL of a protein solution (1 mg/mL) containing natural endotoxin. The suspension was shaken for 2 hr at 25 ℃. The endotoxin content was measured after filtrating through a 0.8 μm cellulose acetate filter to remove the beads from the suspension.

Molecular weight Exclusion

Cellufine™ ET Clean-S: 2 kDa
Cellufine™ ET Clean-L: 2000 kDa

Features
•Low ligand leakage due to stable coupling chemistry and cellulose base bead,
•High endotoxin loading capacity,
•Salt tolerant removal of endotoxin up to 0.4M NaCl,
•ET clean-L is compatible with high MWt. proteins due to its large pore size.
•Affinity ligand is an FDA approved food additive,
•Endotoxin levels in protein samples reduced by >99% in 1 hr,
•Simple cartridge based syringe protocol,
•Scale able into columns up to 30 cm in diameter.

Ordering Information

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Cellufine™ Chelate Media

Cellufine™ Chelate media is designed for use in immobilized metal chelate affinity chromatography of proteins and peptides. The media comprises cross-linked cellulose to which iminodiacetic acid (IDA) is immobilized. Its rigid structure allows operating under high flow rates, which gives rapid processing times. When exposed to metal salts, IDA readily complexes with the cation. The resulting metal chelate moiety interacts (primarily) with the surface accessible residues, such as histidine tag.

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Hydrophobic Interaction

Hydrophobic Interaction Chromatography Media
Cellufine™ MAX Butyl,
Cellufine™ MAX Phenyl

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Gel filtration

Gel Filtration Chromatography Media
Cellufine™ GCL-2000HF

Gel Filtration, or size exclusion chromatography, has proven to be a simple and effective purification method for proteins and other biomolecules. However, the traditionally slow flow rates and limiting resolving power of most gel filtration media have restricted its usefulness. These problems, inherent to the packings, existed not only at the laboratory preparative scale but especially at process scale. The limited throughput of gel filtration columns has often created a significant process bottleneck. Cellufine™ GCL media overcome these limitations. The mechanical strength of the spherical cellulose matrix allows high flow rates, even in large industrial columns.

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Range of Fractionalization
This media offer an extraordinarily broad selection of fractionation ranges: from the unique Cellufine™ GCL-2000 and Cellufine™ GCL-2000HF for very high molecular weight protein complexes. The following table shows the exclusion limit for the media.

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This media offer an extraordinarily broad selection of fractionation ranges: from the unique Cellufine™ GCL-2000 and Cellufine™ GCL-2000HF for very high molecular weight protein complexes. The following table shows the exclusion limit for the media.

Gel Filtration Chromatography Media
Cellufine™ GH-25

For rapid protein desalting buffer exchange and removal of alcohol and detergents

Cellufine™ GH-25 desalting media is based on porous, spherical, highly crosslinked cellulose particles. The sharp 3kD exclusion limit allows proteins to pass through the column in the void volume while retarding smaller molecular weight solutes in the internal pores. Outstanding mechanical strength allows operation at high flow rates even in large diameter process scale columns, thus minimizing run times.

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Applications
► Desalting before lyophilization or concentration
► Buffer exchanges
► Removal of alcohol or other organic solvents
► Removal of aromatic compounds (e.g., phenol) in purification of nucleic acids
► Removal of detergents used to solubilize proteins (e.g. Triton® X-100, SDS)
► Permits use with all commonly used solvents and buffers without shrinkage or swelling
► Removal of chaotropic agents, (e.g. urea, guanidine)

High Speed Desaltings

Although the rigidity of Cellufine™ GH-25 makes it ideally suited to large scale column use, its ability to operate at very high flow rates enables the use of smaller columns running multiple cycles giving similar throughput to lower flow rates on larger columns. Unlike conventional chromatography, the performance (as measured by sample load, salt removal, and dilution) of desalting chromatography can actually improve as the flow rate increases, due to decreased sample dilution at high volumetric loads.

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