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Cellufine MAX S-r, S-h and Q-r, Q-h Ion Exchange Chromatography Media

Ion Exchange Chromatography Media
Cellufine MAX S, Q, CM, DEAE

High Flow Rate, High Binding Capacity

Cellufine MAX is the new, high-flow, Cellufine media. 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).

Fig 1.　SEM analysis of Cellufine MAX base resin	Fig 2.　Typical 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.

Fig 3.　Ligand 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.

Characteristics
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	0.12 - 0.24	0.13 - 0.25	0.14 - 0.25	0.13 - 0.20	0.17 - 0.24
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.

Cellufine MAX Cation Exchange Media

Cellufine MAX Anion Exchange Media

Fig 4.　Pressure-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

Cellufine MAX Anion Exchange Media

Fig. 5 Residence 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)

Fig 6.　Residence 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 Cation Exchange Media

Fig. 7 Residence 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

Fig. 8 DBC 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

Cellufine MAX Anion Exchange Media

Fig. 9　Model 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

Fig. 10　Model 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 S-r