Benefits of a Revised Approach to Anion Exchange Flow-Through Polish Chromatography - A high-performance anion exchange resin performs well compared with membranes. In addition, the resin offers great

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Benefits of a Revised Approach to Anion Exchange Flow-Through Polish Chromatography
A high-performance anion exchange resin performs well compared with membranes. In addition, the resin offers greater flexibility and cost savings.


BioPharm International Supplements
Volume 24, Issue 4, pp. s16-s20

Demonstration of performance using a scale-down model


Table I: Scale down approach for AEX chromatography resin and membrane adsorber comparison.
An alternative and beneficial approach to AEX FT is the use of a short, 5-cm length, packed bed format with a faster operating flow rate. This format enables volumetric throughput capability that is similar to the membrane format, and increases flexibility when designing a purification scheme. The scale-down model used to compare the performance of the different AEX products/formats is summarized in Table I. We evaluated five commonly used AEX products: three resins and two membrane adsorbers for dynamic binding capacity of DNA and protein using bovine serum albumin (BSA) to mimic common contaminants, such as host cell proteins, removed during a FT step. The resin target operating flow rate was the maximum flow rate defined per the manufacturer's operating instructions. The membrane target operating flow rate was based on the common industry design space of 10 membrane volumes (MV) per minute. Although POROS chromatography resins can be operated at 2000 cm/h or faster, 1000 cm/h was evaluated as the upper limit for this evaluation. A 5 cmL POROS column can be operated at 1000 cm/h with a low pressure drop allowing for the use of high operating flow rates in conventional low pressure chromatography columns and systems (see Figure 1).

Materials and methods


Table II: DNA and BSA dynamic binding capacities of five AEX products.
DNA Dynamic Binding Capacity: The AEX product was pre-charged with 20 mM sodium phosphate, 1 M NaCl, pH 7.0 followed by an equilibration with 20 mM sodium phosphate, 50 mM NaCl, pH 7.0 (7.8 mS/cm). Each column/membrane was loaded with 2 mg/mL Herring Sperm DNA (Sigma D3159) in equilibration buffer (titrated to pH 7.0 with 0.2 M sodium phosphate dibasic, anhydrous) with a final conductivity of 8.8 mS/cm per the flow rates listed in Table I. Binding capacities at 5% (C5) and 50% (C50) breakthrough were determined based on UV absorbance.

BSA Dynamic Binding Capacity: The AEX product was pre-charged with 20 mM Tris, 1 M NaCl, pH 8.0 followed by an equilibration with 20 mM Tris, pH 8.0 (1.1 mS/cm). Each column/membrane was loaded with 10 mg/mL BSA (Sigma A7906, pI 4.7-5.3, MW 66 kDa) in equilibration buffer with a final conductivity of <2 mS/cm per the flow rates listed in Table I. C5 and C50 breakthrough were determined based on UV absorbance.

POROS HQ Viral Clearance: Polyclonal human IgG (Sigma G4386, MW:155–160 kDa; pI: ~6.9) was used for the model process. The salt concentrations evaluated and the corresponding conductivity values are summarized in Table III and Figure 2. The column format was 0.46 cmD x 5 cmL, 0.83 mL or 0.46 cmD x 20 cmL, 3.3 mL. Viral clearance was assessed at 1000 cm/hr at room temperature for the 25 mM, 50 mM and 150 mM runs and 300 cm/hr for the 100 mM NaCl run. The studies were all run at pH 7.0 using 20 mM bis-tris propane for buffering. The column was loaded with 5 mg/mL IgG with a 5% xenotropic murine leukemia virus (xMuLV, retrovirus, enveloped, ssRNA, 80-120 nm) or murine minute virus (MVM, parvovirus, non-enveloped, ssDNA, 18-26 nm). Spike and column FT samples were taken to determine viral clearance.


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