Next Generation Zirconia-Based Antibody Purification Media Dr. Clayton McNeff, Dwight Stoll, Danielle Hawker (ZirChrom), Dr. Andy Clausen (Merck) Dr. Peter W. Carr and Dr. Anuradha Subramanian (U of MN) 1-866-STABLE-1 www.zirchrom.com 2003 ZirChrom Separations, Inc.
Outline Supports for Bio-Chromatography Modification of zirconia for biochromatography Small scale Mab purification Fast Mab purifcation Comparison to Protein G Binding Capacity Studies Conclusions
Current Supports for Bio-Chromatography Soft-Gels: Hard-Gels: 1. Cellulose 7. Silica 2. Agarose 8. Hydroxy-apatite 3. Sepharose 9. Zirconia 4. Polyacrylamide 5. Polyamide 6. Polystyrene
Particle Sizes Available Data for Rhinophase -AB Both nonporous and porous analytical (3, 5 micron) and preparative (10, 25 micron) particles are available. 1.7 micron NPZ 3 micron 25 micron
Modification: Chelating Ligand H 2 O 3 P CH 2 CH 2 CH 2 CH 2 PO 3 H 2 N N H 2 O 3 P CH 2 CH 2 PO 3 H 2 Ethylenediamine-N,N -tetra(methylenephosphonic) acid = EDTPA Reflux particles in EDTPA solution Forms multiple bonds Blocks Lewis acid/base interactions
Nitrogen Porosimetry of P-EDTA Modified ZrO 2 1.2 Bare Unmodified ZrO 2 dv/dlogd (cm 3 /g) 1.0 0.8 0.6 0.4 Reflux 4 hrs in P-EDTA Reflux 8 hrs in P-EDTA Reflux 16 hrs in P-EDTA 0.2 0.0 10 100 1000 No pore blockage No loss of surface area Pore Diameter (A)
1 2 Gradient Chromatography on 3 Rhinophase -AB 4 5 6 7 1 BSA 2 Myoglobin 3 Ribonuclease B 4 Ribonuclease A 5 Chymotrypsin 6 Lysozyme 7 Cytochrome c 0 5 10 15 20 Time (min) Protein mixture. Mobile phase: 4 mm P-EDTA, 20 mm MES, ph 5.5; linear gradient from 0 to 1 M Na 2 SO 4 in 30 min. Flow Rate 1 ml/min. UV detection at 280 nm.
Small-Scale Purification of Mab IgG 2a Contaminated with BSA mau 140 120 100 80 VWD1 A, Wavelength=280 nm (NIH22399\IGGBSA01.D) 2.682 Area: 5329.82 BSA Note: BSA is not retained under sample loading conditions. 60 40 20 12.678 Area: 2602.62 MAB 0-20 0.05 M NaCl 0.5 M NaCl 1.0 M NaCl 0 5 10 15 20 25 30 35min LC Conditions: 100 ul injection of BSA (6.0 mg/ml) contaminated MAB (1.0 mg/ml) eluted by salt step gradient. Mobile phase: 20 mm MES, 4 mm EDTPA, 0.05 M-to-1.0 M NaCl ph=5.5. Flow rate: 2.0 ml/min. Temperature: 30 o C. Detection: 280 nm.
A New Ultrafast Preparative Purification Method Using Rhinophase -AB Due to zirconia s very high mechanical strength, Mab purifications can be performed at high mobile phase linear velocities. A simple vacuum filtration apparatus can be use to achieve very high flow rates through a packed bed (90 ml/min). This approach is not possible using soft affinity gels such as Protein A and Protein G media.
Ultrafast Preparative Purification of IgG 1 Using Rhinophase -AB UV Absorbance at 280nm 2.5 2 1.5 1 0.5 Step A (Load) Step B (Wash) Step C (Elution) Fraction 5 Total Analysis Time = 15 minutes This is 100 times faster than a Protein G purification of the same scale! 0 1 2 3 4 5 6 7 8 9 10 11 Fraction Number Step A = 20 mm MES buffer, 4 mm EDTPA, 50 mm NaCl @ ph 4.0, Step B = 20 mm MES buffer, 4 mm EDTPA, 50 mm NaCl @ ph 4.0, Step C = 20 mm MES buffer, 4 mm EDTPA, 2.0 M NaCl @ ph 4.0. Flow Rate = 60 ml/min, Injection size = 31.6 ml serum-free cell culture supernatant diluted 4-times with loading buffer, (3.98 mg of Mab), Amount of Rhinophase -AB in tube = 10 grams.
ELISA Plate Comparison of Protein G and the Ultrafast Purification on Rhinophase -AB OD 280 nm Protein Protein G Rhinophase -AB Offered Purified IgG Fraction 5 Fraction 5 15.6 0.0531 0.0502 94.5% 31.3 0.0946 0.0992 104.9% 62.5 0.1676 0.1892 112.9% 125 0.3176 0.3632 114.4% 250 0.5596 0.6362 113.7% 500 1.0166 1.1507 113.2% 1000 1.8151 1.8632 102.6% Average % 108.0% An ELISA plate analysis using the same amount of Mab from Protein G and Rhinophase -AB purifications showed an increased signal for the Rhinophase -AB purified Mab.
Mab Purity Comparison from Semi-Preparative Run Rhinophase -AB Purified Protein G Purified Purified Mab was equally pure using Protein G and Rhinophase -AB. Silver-stained, SDS-PAGE gel Comparing IgG 1 purified by Protein G (row 1 from right) and Rhinophase -AB (row 3 from right). Electrophoresis was run under reducing conditions. Sample loading at 1 ug per lane. All other lanes are standards.
The Dynamic Capacity of ZirChrom Mab Media was Determined as the Amount of higg captured per ml of Media. Effect of Mab (higg) Concentration on Dynamic Bead Capacity (Q a ) of ZirChrom Mab Media Q a, mg/ml beads 12 10 8 6 4 2 0 0.0 5.0 10.0 15.0 C, mg/ml Linear Velocity: 12 cm/min Isotherm shape Indicates Langmuir-type Adsorption Conditions: Pure higg in Loading Buffer (20mM MES, 4mM EDTPA, 50mM NaCl, ph5.5) was loaded on a 5x4.6mm i.d. ZirChrom Mab Media column at a linear velocity of 12.0cm/min and detected at 280nm. The injection was continued until the absorbance of the effluent reached 80% of the inlet concentration. The column was then washed with Loading Buffer until it had reached baseline. The Adsorbed higg was then eluted with Elution Buffer (20mM MES, 4mM EDTPA, 1.0M NaCl, ph5.5).
Effect of Linear Velocity on the Dynamic Q max of ZirChrom Mab Media Qmax, mg HIgG/ ml media 25 20 15 10 5 0 Qmax = 20.0mg/cc 13.9mg/cc 11.5 mg/cc 0 5 10 15 Linear Velocity, cm/min Typical value on Protein A = 2-4 mg/ml Conditions: Pure higg in Loading Buffer (20mM MES, 4mM EDTPA, 50mM NaCl, ph5.5) was loaded on a 5x4.6mm i.d. ZirChrom Mab Media column at a linear velocities of 3.0, 6.0, and 12.0 cc/min and detected at 280nm. The injection was continued until the absorbance of the effluent reached 80% of the inlet concentration. The column was then washed with Loading Buffer until it had reached baseline. The adsorbed higg was then eluted with Elution Buffer (20mM MES, 4mM EDTPA, 1.0M NaCl, ph5.5).
Conclusions Rhinophase -AB provides a widely applicable alternative to currently used Protein A and Protein G antibody purification media. Typical yields of Mab purifications greater than 95%, with purity levels equal to or greater than affinity gel-type media. Due to Rhinophase -AB s excellent mechanical stability, purifications can be performed 100-fold faster with equivalent results. Rhinophase -AB is chemically durable over the entire ph range, which allows for cleaning and depyrogenation. Rhinophase -AB has affinity for a wide range of immunoprotein classes and subclasses including monoclonal and polyclonal IgG, IgA, and IgM. Rhinophase -AB can be packed into large preparative scale columns and operated at high mobile phase linear velocities for fast large scale purifications of immunoproteins. Acknowledgement: National Institutes of Health