Ion Exchange
Chromatography Media

CellufineTM MAX S, Q, CM, DEAE, GS

High Flow Rate, High Binding Capacity

Cellufine™ MAX is the new, high-flow, Cellufine media. JNC’s advanced cross-linking technologies have created more robust base beads operable at high flow and pressure. Further, Cellufine MAX ion exchange (IEX) media are made using surface modification techniques that dramatically increase ligand availability, which translates to higher dynamic binding capacities. Cellufine MAX IEX media are offered in six products, including both anion and cation chemistries.

Cellufine MAX Base Resin

Cellulose, natural polysaccharide, possesses unique crystalline molecular structure differing from non-crystalline polysaccharides such as agarose. Thus Cellufine has unique pore structure as shown in the pictograph (Fig. 1). The new Cellufine MAX series offers the largest pore size of all Cellufine chromatography media. The benefit of such pore size in Cellufine MAX IEX media provides superior strength and excellent mass transfer. This is seen in the break-through curves for thyroglobulin, a very large protein (Fig. 2).

SEM analysis of Cellufine MAX base resin
Fig 1SEM analysis of Cellufine MAX base resin
Typical break-through curves for Cellufine DEAE weak anion exchange media with thyroglobulin
Fig 2Typical break-through curves for Cellufine DEAE weak anion exchange media with thyroglobulin

Partial Structure of Cellufine MAX IEX Media

Ligand structure for Cellufine MAX IEX media are described in Fig. 3. S, Q, CM and DEAE are correspondingly strong cation, strong anion, weak cation and weak anion exchangers. Two sub-types, h and r, are available for Cellufine MAX S and Q.
The differences between X-h and X-r type Cellufine MAX strong ion exchange media (X) are due to the design of the media. The X-h type is designed for higher binding capacity than the X-r type by optimizing the ligand content and dextran scaffold.

Ligand structure of Cellufine MAX IEX
Fig 3Ligand structure of Cellufine MAX IEX

Characteristics of Cellufine MAX IEX Media

The basic characteristics of Cellufine MAX IEX media are shown in Table 1. All Cellufine MAX IEX media are based on 90 μm (average) highly cross-linked cellulose beads, which are surface-modified with dextran. Cellufine MAX IEX media are designed for use in bio-pharmaceuticals purification processes.

Type MAX CM MAX S-r MAX S-h MAX DEAE MAX Q-r MAX Q-h
Matrix Cross-linked cellulose with dextran scaffold
Particle size (μm) 40 - 130
Ligand CM S S DEAE Q Q
Ion exchange capacity (meq / ml-gel) 0.09 - 0.22 0.09 - 0.21 0.10 - 0.22 0.12 - 0.22 0.10 - 0.20 0.13 - 0.22
10% DBC(mg/ml) Lysozyme 220 144 191
BSA 197 141 225
human-γ-globulin 104 131 216 108 74 135
pH stability 2 -13 2 -13 3 -14 2 -12 2 -12 2 -12
Storage 20% Ethanol

Pressure-flow Properties of Cellufine MAX IEX Media

Cellufine MAX IEX media enable high-flow operation, which is essential to efficient purification of bio-pharmaceuticals.
The figures below show pressure-flow velocity curves of Cellufine MAX IEX media in a 30 cm column with a 20 cm bed height (Fig. 4). All Cellufine MAX IEX media are operable at practical flow velocities (500 cm/h) and pressures.

Pressure-flow Properties of Cellufine MAX IEX Media
Fig 4Pressure-flow velocity curves for Cellufine MAX IEX exchange media
Column
30 cm I.D. x 20 cm L
Mobile phase
Pure Water at 24 ºC

Dynamic Binding Capacities of Cellufine MAX IEX Media

Efficient mass-transfer characteristics of Cellufine MAX IEX media translate to superior dynamic binding capacities (DBC). Figure 5 to 7 show DBC of model proteins at different residence times for Cellufine MAX IEX media. All Cellufine MAX IEX media are stable over a range of residence times.
Fig. 8 shows that Cellufine MAX S exhibits superior dynamic binding performance across a range of protein characteristics to competitive media.
These unique characteristics of Cellufine MAX IEX media make it suitable for use in up-stream as well as to down-stream steps in bio-pharmaceuticals purification.

Cellufine MAX Cation Exchange Media

Dynamic Binding Capacities of Cellufine MAX IEX Media
Fig 5Residence time vs. IgG-DBC for Cellufine MAX S
Column
5 mm ID×50 mm L
Sample
human polyclonal IgG (1 mg/ml)
Buffer
10 mM Acetate-50 mM NaCl (pH 4.3)

Cellufine MAX Anion Exchange Media

Cellufine MAX Anion Exchange Media
Fig 6Residence time vs. BSA-DBC fer Cellufine MAX Q
Column
5 mm I.D. x 100 mm L
Sample
BSA (1 mg/ml)
Buffer
50 mM Tris-HCl (pH 8.5)

Cellufine MAX Weak ion Exchange Media

Cellufine MAX Weak ion Exchange Media
Fig 7Residence time vs. DBC for Cellufine MAX CM (polyclonal IgG) and DEAE (BSA)
Column
5 mm ID x 50 mm L
Sample
human polyclonal IgG (1 mg/ml)
BSA (1 mg/ml)
Buffer
10 mM Acetate (pH 5.6) for IgG
Tris-HCl (pH 8.5) for BSA

Cellufine MAX Cation Exchange Media

Cellufine MAX Cation Exchange Media
Fig 8DBC of Cellufine MAX S and agarose base media with various model proteins (R.T. = 1 min)
Polyclonal IgG
10 mM Acetate (pH 4.3) - 50 mM NaCl
BSA
10 mM Acetate (pH 4.3) - 50 mM NaCl
Lysozyme
Tris-HCl (pH 9.5)

Model Proteins Separation Performance for Cellufine MAX IEX Media

Cellufine MAX IEX media are optimized for high adsorption and high resolution. Model protein separation with MAX S-h and MAX CM (Strong Cation vs. Weak Cation) is demonstrated in Fig. 9 and 10.

Cellufine MAX Cation Exchange Media

Model proteins separation for Cellufine MAX S-h and MAX CM
Fig 9Model proteins separation for Cellufine MAX S-h and MAX CM
Column
6.6 mm ID×50 mm L
Buffer A
10 mM phosphate buffer (pH 7)
Buffer B
10 mM phosphate (pH 7) + 1 M NaCl
(0→50 % linear gradient)
Flow rate
0.86 ml/min (residence time: 2min)
Proteins
Ribonuclease A (5 mg/ml),
Cytochrome C (2.5 mg/ml),
Lysozyme (1.5 mg/ml)
Injection volume
1.5ml

Cellufine MAX Anion Exchange Media

Model proteins separation for Cellufine MAX Q-h and MAX DEAE
Fig 10Model proteins separation for Cellufine MAX Q-h and MAX DEAE
Column
6.6 mm ID×50 mm L
Buffer A
50 mM Tris-HCl (pH 8.5)
Buffer B
50 mM Tris-HCl (pH 8.5) - 1 M NaCl
(0→75 % linear gradient)
Flow rate
0.86 ml/min (residence times 2 min)
Proteins
Transferrin (5 mg/ml),
BSA (10 mg/ml),
Pepsin (5 mg/ml)
Injection volume
1.5 ml

Chemical Stability and Cleaning-In-Place

Cellulose is well-known as a natural product having chemical and physical stability. Thus, since Cellufine is derived from cellulose, it also is stable to chemicals, caustic and acidic solutions. CIP of all Cellufine MAX IEX media can be carried out with 0.5 M NaOH solution. Used media should be stored in 20 % ethanol at 2 - 25 ºC after cleaning.

Cellufine MAX GS has been developed as a new strong cation chromatography media, with optimized ligand density. Cellufine MAX GS shows a superior performance for aggregate removal from therapeutic Mabs.

Characteristics of Cellufine MAX GS Media

The basic characteristics of Cellufine MAX GS are shown in Table 1. The base matrix for Cellufine MAX GS is 90 μm (average) highly cross-linked cellulose beads, the same as other Cellufine MAX IEX media.

Matrix Highly Cross-linked Cellulose
Particle size 40~130 μm
Ligand type -R-SO3-Na+
Ion Exchange Capacity (m mol / ml) 0.09〜0.15
Lysozyme adsorption capacity (mg / ml) ≧ 100
Polyclonal IgG 10% DBC (mg / ml) ≧ 70
Operating pressure < 0.3 MPa
pH stability pH 2 ~ 13

Pressure-Flow Properties of Cellufine MAX GS

Figure 1 shows pressure-flow velocity curves of Cellufine MAX GS in 30 cm I.D. column with 20 cm bed height. Cellufine MAX GS has excellent flow properties and is applicable for process production.

Pressure-Flow Properties of Cellufine MAX GS
Figure 1
Column
30 cm I.D. x 20 cm L
Mobile Phase
Pure water (24℃)

Model Protein Separation Performance for Cellufine MAX GS

Cellufine MAX GS is highly effective aggregates from IgG monomer by NaCl or pH gradient. Figure 2 shows the comparison of Cellufine MAX GS with S agarose media for monomer /aggregate separation of polyclonal IgG (Figure 2a) and monoclonal antibody (Figure 2b) by NaCl gradient elution. Cellufine MAX GS has been shown to be applicable an effective media for aggregate removal from Mabs.

Model Protein Separation Performance for Cellufine MAX GS
Figure 2a
Poly IgG
Thermal and acid stressed Poly IgG
Buffer
Acetate (pH5.0), 50mM →1 M NaCl Poly IgG
injection
1 ml
Poly IgG conc
2 mg/ml
Model Protein Separation Performance for Cellufine MAX GS
Figure 2b
Column
5 mm ID × 50 mm L
Buffer
Citrate Buffer (pH 5.0)
NaCl Gradient
0.2→0.5 M
Mab Injection
1 ml
Flow rate
0.66 ml/min

Dynamic Binding Capacities of Cellufine MAX GS

Efficient mass-transfer characteristics of Cellufine MAX GS translate to superior dynamic binding capacities (DBC). Figure 3 shows DBC of Poly IgG at various residence times. Cellufine MAX GS is suitable for use in down-stream steps in antibody purification.

Dynamic Binding Capacities of Cellufine MAX GS
Figure 3
Conditions
Column
φ5 mm×5 cm L
Poly_IgG Concentration
1 mg/ml
Adsorption Buffer
10 mM Acetate (pH 5.0) + 50 mM NaCl

Development of the grade

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

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