Ion Exchange Chromatography Media. Cellufine IEX. Technical Data Sheet

Transcription

1 Ion Exchange Chromatography Media IEX Technical Data Sheet 2-1, Otemachi 2-Chome Chiyoda-ku, Tokyo JAPAN Phone / Fax cellufine@jnc-corp.co.jp web: http: //

2 Technical DATA Sheet Ion Exchange Media What is? is porous and spherical chromatography medium manufactured from cellulose. has been approved in many bio-pharmaceutical GMP production processes since Cellulose is well-known as natural product having unique crystalline molecular structure which brings to have chemicals stability and mechanical strength. Owing to these properties, has an excellent column packing characteristics and can be packed at high flow rates due to high mechanical strength of produced particles. IEX Media is available with six different IEX media as shown in Table 1 and their ligand structure are described in Fig. 1. IEX 5 series media in Table 1 is used as conventional typed media. The pore property (pore size) of media influences on the performance of chromatography medium. Fig. 2 shows the relationship Kav profile on each base resin of IEX media. JNC offers 3 different weak anion media having different from pore property described in the next. A 2, A 5, A 8 Q 5 C 5 S 5 C 2 H 5 OCH 2 CH 2 N + H Cl - C 2 H 5 CH 3 RCH 2 N + CH 3 Cl - CH 3 O OCH 2 CO - Na + O OCH 2 CH 2 CH 2 CH 2 SO - Na + O Fig. 1. Ligand structure of IEX media Kav E+4 1.E+5 1.E+6 1.E+7 M.W. [proteins] A-2 Base resin m Base resin A-8m Base resin Fig. 2. Kav profile on each base resin of anion exchange chromatography Characteristics of IEX Media The basic characteristics of IEX media are shown in Table 1. All IEX media are based on 9 μm (average) cross-linked cellulose beads. Figure 3 shows particle-size distributions of which is a standard media as IEX 5 series. freaquency (%) particle size(μm) Fig. 3. Particle-size distributions of 5 type (, Q-5, C-5 and S-5) is a standard medium in IEX medium. The media have good flow property for industrial. These are resins with relatively large pore size which is enough effective for IgG. A-2 has the smallest pore size in IEC media. As the adsorption and desorption of protein is occurred on the surface of A-2 resins, a typical breakthrough curve in the media is very sharp as shown in Fig. 4. Thus A-2 is used as flow through mode with taking advantage of its unique characteristic in adsorption. A-8 has large pore size which makes huge protein such as thyroglobulin (MW=66kDa) stay inside. While A-8 media is poor flow property as in Fig. 5, dynamic binding capacity of the medium is 通過分積算 (%) 2

3 excellent as in Fig. 6. Especially the medium is suitable for the purification of large protein. Q-5 is manufactured from by attaching tri-methyl ammonium ligand. One of characteristics in the medium is to be able to use under high salt condition as in Fig.9. Pressure-flow Properties of IEX Media Figure 5 shows pressure-flow velocity curves of IEX media in a 2.2 cm column with a 2 cm bed height. Figure 6 also shows pressure-flow velocity curves of media in a 3 cm column with a 2 cm bed height. IEX media enable highflow operation, which is essential to efficient purification of bio-pharmaceuticals. Table 1. Characteristics of IEX media A 2 A 5 A 8 Q 5 C 5 S 5 Base Matrix Cross linked cellulose Particle size (μm) 4 13 Ion exchange type Weak Strong (DEAE) (QA) Weak (CM) Strong (S) Ion Exchange (meq/ml gel) A-2 Q-5 A-8 Pressure [Mpa] A-2 A-8 Q-5 C Linear velocity [cm/h] Fig.4. Typical break through curves of BSA for anion IEX media Column: 5 mm ID 5 mm L Flow rate: 15 cm/h Sample: 1 mg/ ml Buffer: 5 mm Tris-HCl (ph 8.5) for A-2, A5 and A-8 5 mm Tris-HCl (ph 8.)+ 5 mm NaCl for Q-5 Fig 5. Pressure-flow velocity curves of IEX media (2.2 cm I.D. x 2 cm L), Mobile phase with pure water at 24, 3

4 .5 2 Lysozyme Pressure [MPa] A5 2.2 cm ID A5 3 cm ID % DBC (mg/ml) C 5 S 5 Agar type CM media IgG Agar type S media Linear velocity [cm/h] Fig 6. Pressure-flow velocity curves of media (2.2 cm I.D. x 2 cm L and 3 cm I.D. x 2 cm L ), Mobile phase with pure water at 24, Dynamic Binding Capacities of IEX Media IEX media have efficient mass-transfer characteristics. Especially they exhibit superior dynamic binding performance for large molecular proteins such as immunoglobulin in comparison with competitive medium as shown in Fig. 7 and Fig.8. These unique characteristics of IEX media make it suitable for use in up-stream as well as to downstream steps in bio-pharmaceuticals purification. BSA 1%DBC (mg/ml) Fig. 8. DBC with model proteins in C-5 weak cation IEX media and in competitive medium Column: 5 mm ID 5 mm L Flow rate: 15cm/h Sample: 1 mg/ml Buffer: 1 mm Acetate (ph 5.3) for IgG 5 mm Tris-HCl (ph 8.5) for Lysozyme BSA mm NaCl 2 mm NaCl 5 mm NaCl 1 mm NaCl 1%DBC mg/ml γ-globulin A-2 A-8 Competitive agar Fig. 9. DBC of BSA at different NaCl concentration for Q-5 IEX medium Column: 5 mm ID 5 mm L Flow rate: 15 cm/h Sample: 1 mg/ml Buffer: 5 mm Tris-HCl (ph 8.5) + NaCl Fig. 7. DBC with model proteins in weak anion IEX media and in competitive media Model Proteins Separation Performance for Column: 5 mm ID 5 mm L IEX Media Flow rate: 15 cm/h IEX media are optimized for high adsorption Sample: 1 mg/ml and high resolution. Model protein separations with Buffer: 5 mm Tris-HCl (ph 9.5) for IgG IEX media are demonstrated in Fig. 1 to mm Tris-HCl (ph 8.5) for BSA 4

5 Agar type DEAE media A-8 A-2 Chemical Stability and Cleaning-In-Place Cellulose is well-known as natural products having chemical and physical stability. Thus, since is derived from cellulose, it also is stable to chemicals, caustic and acidic solutions. CIP of all IEX media can be carried out with.5 M NaOH solution. Used media should be stored in 2 % ethanol at 2-25 after cleaning. Fig. 1 Model proteins separation for IEX media and competitive agar. Column: 6.6 mm ID 5 mm L Buffer A: 5 mm Tris-HCl (ph 8.5) Buffer B: 5 mm Tris-HCl (ph 8.5) + 1 M NaCl ( 75 % linear gradient) Flow rate:.86 ml/min (residence time 2 min) Proteins: Transferrin (5 mg/ml), SA (1 mg/ml), Pepsin (5 mg/ml) Injection volume: 1.5 ml Q-5 Fig. 11 shows that Model protein separation with and Q-5. Column: 6.6 mm ID 5 mm L Buffer A: 5 mm Tris-HCl (ph 8.5) Buffer B: 5 mm Tris-HCl (ph 8.5) + 1 M NaCl ( 75 % linear gradient) Flow rate:.86 ml/min (residence time 2 min) Proteins: Transferrin (5 mg/ml), BSA (1 mg/ml), Pepsin (5 mg/ml) Injection volume: 1.5 ml 5

6 Ordering Information Product Name A-2 A-8 Pack Size Catalogue No. Product Name Pack Size Q C-5 S-5 Catalogue No Technical Support contacts (North & South America, Europe) JNC America Incorporated 555 Theodore Fremd Avenue, Suite C-26 Rye, NY 158 USA TEL: FAX: (Asia & Others) JNC Corporation Life Chemicals Sales Dept. 2-1, Otemachi 2-Chome, Chiyoda-ku Tokyo Japan Tel: Fax: Please Send Purchase Orders to: (North America & Europe) JNC America Incorporated 555 Theodore Fremd Avenue, Suite C-26 Rye, NY 158 USA TEL: FAX: (Asia & Others) JNC Corporation Life Chemicals Sales Dept. 2-1, Otemachi 2-Chome, Chiyoda-ku Tokyo Japan Tel: Fax: Please visit our web site for more information http: // 6