CHROMATOGRAPHY MPC Ceramic Hydroxyfluoroapatite

Transcription

1 CHROMATOGRAPHY MPC Ceramic Hydroxyfluoroapatite High physical and chemical stability Unmatched selectivity Clearance of impurities and aggregates in a single step Rapid and simple column packing Separation of Impurities in a Single Step The newest addition to Bio-Rad s line of ceramic apatite chromatography media is MPC ceramic hydroxyfluoroapatite. MPC is a secondgeneration CHT ceramic hydroxyapatite mixedmode chromatography media, a composite of hydroxyapatite and fluoroapatite prepared by chemically substituting 2% of the hydroxyl groups of hydroxyapatite nanocrystals with a fluorine reagent. Its chemical formula is Ca 1 (PO 4 ) 6 (OH) 1. (F).. The partial fluorination confers a more chemically stable form of the matrix during ph excursions that are inherent to buffer exchanges. MPC has unique separation properties, unmatched selectivity, and resolution similar to CHT, including binding capacity (Tables 1 and 2), protein separations (Figure 1), and clearance (Figures 2 and 3, Table 3). MPC is 4 µm and is directly comparable to CHT Type I, 4 µm (Tables 1 and 2). Mechanism of Action and Standard Chromatography MPC interacts with biomolecules by multiple modes. Cation exchange occurs when negatively charged phosphate groups interact with protein amino groups. Much stronger coordination complexes can form between carboxyl clusters, phosphoryl moieties, or both on biomolecules and the calcium sites on MPC via metal affinity. Repulsion effects and the geometric charge distribution on MPC provide unique selectivity. Typically, acidic, basic, and neutral proteins are bound to MPC using low ionic strength phosphate buffer. Elution is accomplished through the use of a sodium chloride or phosphate gradient of increasing strength or via similar step elutions. Regeneration of the support with phosphate buffers at neutral ph is followed by sanitization with up to 2 N NaOH. Regardless of greater chemical stability, we recommend implementing ph mitigation steps, for example, the surface neutralization system (SNS). A pilot-scale study with MPC using SNS achieved 12 cycles. For more detailed information, refer to the CHT user s guide at Table 1. Specifications. CHT Type I, 4 µm Functional groups Ca 2+, PO 4, OH Ca 2+, PO 4, OH, F Observed dynamic binding capacity lysozyme (Lys) 2 mg Lys/g CHT MPC 2 mg Lys/g MPC Nominal pore diameter 6 8 Å 6 8 Å Maximum backpressure 1 bar (1, psi) 1 bar (1, psi) Nominal mean particle size 4 ± 4 μm 4 ± 4 μm Tap settled density* (g/ml tap settled bed) * Under ideal conditions..63 g/ml.72 g/ml

2 Table 2. Characteristics. CHT Type I, 4 µm Observed dynamic binding capacity IgG 2 6 mg IgG/ml CHT* 2 mg IgG/ml MPC** Typical linear flow rate range 1, cm/hr 1, cm/hr ph stability ph ph Base stability At least 1 year in.1 N NaOH At least 1 year in.1 N NaOH Regeneration.4. M sodium phosphate, ph 7 7., is generally sufficient. If higher concentrations are needed, use potassium phosphate MPC.4. M sodium phosphate, ph 7 7., is generally sufficient. If higher concentrations are needed, use potassium phosphate Autoclavability (bulk) 121 C, 2 min in phosphate buffer, ph C, 2 min in phosphate buffer, ph 7 Sanitization 1 2 N NaOH 1 2 N NaOH Recommended column storage.1 N NaOH.1 N NaOH * 4 μm particles, 3 cm/hr, mm sodium phosphate, ph 6.. ** 4 μm particles, 3 cm/hr, mm sodium phosphate, 2 mm NaCl, ph Ovalbumin Myoglobulin a-chymotrypsinogen A Cytochrome C Fig. 1. Separation of protein standards. MPC separates the standard protein mixture similarly to CHT Type I, 4 µm. Sample: 6 mg of ovalbumin, 3 mg of myoglobulin, 3 mg of a-chymotrypsinogen A, and 3 mg of cytochrome C in 1. ml 1 mm sodium phosphate (NaPi), ph 6.8. CHT column:. x 1.3 cm, packed bed volume 2 ml; MPC column:. x 1.4 cm, packed bed volume 2 ml. Protocol: 1. ml/min; 4 mm NaPi, ph 6.8, column volumes (CV); 1 mm NaPi, ph 6.8, CV; load volume µl; linear gradient elution to 7% 4 mm NaPi, ph 6.8, over CV; strip 4 mm NaPi, ph 6.8, CV; sanitization 1 N NaOH, CV. CHT ( ); MPC ( ). 1 Fig. 2a. MAb-S size exclusion chromatography (SEC) profile of starting material. SEC profile shows the higher molecular weight impurities (dimers/aggregates) and monomers in the starting material (material applied to the second step (Figure 2b) using CHT or MPC). Sample: 2.9 mg/ml mab-s in 1 mm NaPi, ph 7. Column: 3 x 7.8 mm, Bio-Sil SEC 2 HPLC. Protocol: 1 ml/min; equilibration in 1 ml of.1 M NaPi,. M NaCl,.2% azide, ph 7; injection volume.1 ml; elution in ml of.1 M NaPi,. M NaCl,.2% azide, ph Fig. 2b. MAb-S purification profile. Elution profile shows separation of the monomer from higher molecular weight impurities. Sample: 7.26 mg mab-s/ml packed bed in ml 1 mm NaPi, ph 7. Column:. x 1 cm, packed bed volume 2.1 ml of UNOsphere SUPrA rprotein A media. Protocol:. ml/min; 1 mm NaPi, ph 7, column volumes (CV); load volume ml; 1 mm NaPi, ph 7, CV; linear gradient elution to 1% 1 mm NaPi, 1 M NaCl, ph 7, over 4 CV; strip 1 mm NaPi, 1 M NaCl, ph 7, CV; sanitization 1 N NaOH, CV. CHT ( ); MPC ( ). 1 Fig. 2c. SEC profile of pooled monomer fractions. The SEC profile of the pooled fractions confirms aggregate clearance from the monomer. Column: 3 x 7.8 mm, Bio-Sil SEC 2 HPLC. Protocol: 1 ml/min; 1 mm NaPi, mm NaCl,.2% azide, ph 7, 1 ml; load volume.1 ml; elution 1 mm NaPi, mm NaCl,.2% azide, ph 7, ml. CHT ( ); MPC ( ). 213 Bio-Rad Laboratories, Inc. Bulletin 6432

3 Fig. 3a. MAb-G SEC profile of starting material. SEC profile shows the higher molecular weight impurities (dimers/aggregates) and monomers in the starting material (material applied to the second step (Figure 3b) using CHT or MPC). Sample: 4.41 mg/ml mab-s in 1 mm NaPi, ph 7. Column: 3 x 7.8 mm, Bio-Sil SEC 2 HPLC. Protocol: 1 ml/min; equilibration in 1 ml of.1 M NaPi,. M NaCl,.2% azide, ph 7; injection volume.1 ml; elution in ml of.1 M NaPi,. M NaCl,.2% azide, ph Fig. 3b. MAb-G purification profile. Elution profile shows separation of the monomer from higher molecular weight impurities. Protocol as described in Figure 2b. CHT ( ); MPC ( ) Fig. 3c. SEC profile of pooled monomer fractions. The SEC profile of the pooled fractions confirms aggregate clearance from the monomer. Protocol as described in Figure 2c. CHT ( ); MPC ( ). Table 3. Quantified data from all assays. All pooled fractions and mab starting material purified on MPC and CHT were analyzed for host cell protein (HCP ELISA kit, Cygnus Technologies), for dsdna (PicoGreen dsdna quantitation kit, Invitrogen Corporation), for protein A leachables (Protein A ELISA kit, Cygnus Technologies), and for aggregate clearance by SEC (Bio-Sil SEC 2, Bio-Rad Laboratories, Inc). Sample MAb-S IgG Concentration, mg/ml Protein A, ppm HCP, ppm DNA, ppm Dimer/Aggregate, % Protein A eluate CHT pool.4 <.6 <2 2.6 <.3 MPC pool.46 <. <2 2.8 <.3 MAb-G Protein A eluate < CHT pool.91 <.3 <1 1.4 <.3 MPC pool.77 <.3 <1 1.4 < Bio-Rad Laboratories, Inc. Bulletin 6432

4 BSA pl = 4.9 Apo ferritin pl =.. Carboxypeptidase pl = Fetuin pl = 3.3 Pepsin pl = Fig. 4. Elution profiles of five acidic proteins. CHT and MPC produce similar elution characteristics for the five acidic proteins studied. Samples: porcine carboxypeptidase B (CBP), pi 4.6.2, pepsin pi 2.9, fetuin, pi 3.3, BSA, pi 4.9, and apoferritin, pi.., each prepared to a final concentration of 2 3 mg/ml in water. Column:.7 x 2.6 cm, packed bed volume 1 ml, executed using a BioLogic DuoFlow system and software. Protocol: 3 cm/hr; load volume 1 µl; linear gradient elution to 1%.2 M HEPES,.1 M NaPi, ph 6., to.2 M HEPES,.4 M NaPi, ph 6., over 2 CV. CHT ( ); MPC ( )..8 1, 2 Phosphate group 3 Adenosine (1) AMP (2) 1 ADP (3) 2 ATP (4) A ph Fig.. Nucleic acids separation profile. Elution profile shows separation of ADP and ATP. Sample: 1 mg each of adenosine, AMP, ADP, and ATP mixture dissolved in 1 ml of 1 mm NaPi, ph 6.8, filtered with.22 μm membrane. Column:.4 x 1 cm. Protocol: 1 ml/min; load volume.3 ml; wash 1 mm NaPi, ph 6.8, 2 CV; linear gradient elution to 7% 4 mm NaPi, ph 6.8, over 12 CV; strip 4 mm NaPi, ph 6.8, 4 CV. CHT ( ); MPC ( ). 213 Bio-Rad Laboratories, Inc. Bulletin 6432

5 Storage MPC ceramic hydroxyfluoroapatite should be stored in.1 N NaOH at room temperature. In dry powder form, MPC ceramic hydroxyfluoroapatite should be stored in a secured, closed container at room temperature. Technical Assistance For more detailed information on process step development, use recommended steps as described in the CHT Applications Guide ( Regulatory support file is available upon request. Bio-Rad Laboratories is an ISO 91 registered corporation. For additional information and technical assistance, contact your local Bio-Rad office. In the USA and Canada, call 1-8-4BIORAD. You can also send an to lsg_techserv_us@bio-rad.com. Visit us on the Web at for more information on Bio-Rad s complete line of process chromatography supports. Ordering Information Catalog # Description MPC Ceramic Hydroxyfluoroapatite, Type I 8-2 MPC Ceramic Hydroxyfluoroapatite, 4 µm, Type I, 1 g 7-2 MPC Ceramic Hydroxyfluoroapatite, 4 µm, Type I, 1 g 7-21 MPC Ceramic Hydroxyfluoroapatite, 4 µm, Type I, 1 kg 7-2 MPC Ceramic Hydroxyfluoroapatite, 4 µm, Type I, kg Foresight Columns Foresight MPC Type I Column, 4 μm, 1 ml Foresight MPC Type I Column, 4 μm, ml Foresight Plates* Foresight MPC Type I Plates, 4 μm, 2 μl Foresight RoboColumn Units** Foresight MPC Type I RoboColumn Units, 4 μm, 2 μl Foresight MPC Type I RoboColumn Units, 4 μm, 6 μl * Package size: 2 x 96-well plates ** Package size: one row of eight columns Catalog # Description CHT Ceramic Hydroxyapatite, Type II 8-22 CHT Ceramic Hydroxyapatite, 2 μm, Type II, 1 g 7-2 CHT Ceramic Hydroxyapatite, 2 μm, Type II, 1 g 7-21 CHT Ceramic Hydroxyapatite, 2 μm, Type II, 1 kg 7-2 CHT Ceramic Hydroxyapatite, 2 μm, Type II, kg 8-42 CHT Ceramic Hydroxyapatite, 4 μm, Type II, 1 g 7-4 CHT Ceramic Hydroxyapatite, 4 μm, Type II, 1 g 7-41 CHT Ceramic Hydroxyapatite, 4 μm, Type II, 1 kg 7-4 CHT Ceramic Hydroxyapatite, 4 μm, Type II, kg 8-82 CHT Ceramic Hydroxyapatite, 8 μm, Type II, 1 g 7-8 CHT Ceramic Hydroxyapatite, 8 μm, Type II, 1 g 7-81 CHT Ceramic Hydroxyapatite, 8 μm, Type II, 1 kg 7-8 CHT Ceramic Hydroxyapatite, 8 μm, Type II, kg Foresight Columns Foresight CHT Type I Column, 4 μm, 1 ml Foresight CHT Type I Column, 4 μm, ml Foresight CHT Type II Column, 4 μm, 1 ml Foresight CHT Type II Column, 4 μm, ml Foresight Plates* Foresight CHT Type I Plates, 4 μm, 2 μl Foresight CHT Type II Plates, 4 μm, 2 μl Foresight RoboColumn Units** Foresight CHT Type I RoboColumn Units, 4 μm, 2 μl Foresight CHT Type I RoboColumn Units, 4 μm, 6 μl Foresight CHT Type II RoboColumn Units, 4 μm, 2 μl Foresight CHT Type II RoboColumn Units, 4 μm, 6 μl * Package size: 2 x 96-well plates ** Package size: one row of eight columns For More Information Request or download bulletins 686 and 667 PicoGreen is a trademark of Invitrogen Corporation. Related Items CHT Ceramic Hydroxyapatite, Type I 8-2 CHT Ceramic Hydroxyapatite, 2 μm, Type I, 1 g 7-2 CHT Ceramic Hydroxyapatite, 2 μm, Type I, 1 g 7-21 CHT Ceramic Hydroxyapatite, 2 μm, Type I, 1 kg 7-2 CHT Ceramic Hydroxyapatite, 2 μm, Type I, kg 8-4 CHT Ceramic Hydroxyapatite, 4 μm, Type I, 1 g 7-4 CHT Ceramic Hydroxyapatite, 4 μm, Type I, 1 g 7-41 CHT Ceramic Hydroxyapatite, 4 μm, Type I, 1 kg 7-4 CHT Ceramic Hydroxyapatite, 4 μm, Type I, kg 8-8 CHT Ceramic Hydroxyapatite, 8 μm, Type I, 1 g 7-8 CHT Ceramic Hydroxyapatite, 8 μm, Type I, 1 g 7-81 CHT Ceramic Hydroxyapatite, 8 μm, Type I, 1 kg 7-8 CHT Ceramic Hydroxyapatite, 8 μm, Type I, kg 213 Bio-Rad Laboratories, Inc. Bulletin 6432

6 Bio-Rad Laboratories, Inc. Life Science Group Web site USA Australia Austria Belgium Brazil Canada China Czech Republic Denmark Finland France Germany Greece Hong Kong Hungary India Israel Italy Japan Korea Mexico The Netherlands New Zealand Norway Poland Portugal Russia Singapore South Africa Spain Sweden Switzerland Taiwan Thailand United Kingdom Bulletin 6432 Rev B US/EG Sig 1212