Optimized upstream processing and high-productivity cell culture increase not only target protein titers, but also impurity and contaminant concentrations to be removed from large volumes of feedstock. Simultaneously, biopharmaceutical drug production is increasingly driven by manufacturing cost reduction. These facts together increase the pressure on downstream processing and create an urgent need for more productive and streamlined chromatography operations. Key parameters to consider for enhanced process economics in chromatography are higher protein binding capacities at high flow rates (to reduce batch processing duration) and improved sorbent selectivities (to reduce the number of column chromatography steps and decrease purification cost).

Ion-exchange (IEX) chromatography is one of the most broadly used techniques for protein purification. Protein binding during ion exchange is driven by a combination of factors including primarily

• characteristics of the functional group and its density at the surface of the base matrix

• pH and conductivity of the mobile phase that drives electrostatic interactions between proteins and functional groups

• the nature of the base matrix.

PRODUCT FOCUS: PROTEINS

PROCESS FOCUS: DOWNSTREAM PROCESSING

WHO SHOULD READ: PROCESS DEVELOPMENT, ANALYTICAL

KEYWORDS: ANION-EXCHANGE, CATION-EXCHANGE, PH, GRADIENT ELUTION, SELECTIVITY, DYNAMIC BINDING CAPA#CITY, CONDUCTIVITY

LEVEL: INTERMEDIATE

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Many developments occurred in the past decade to enable manufacture of new mechanically stable base matrices and more capacitive IEX sorbents. This progress resulted mainly in enhanced capacities at high process flow rates or shorter residence times (RTs), but resin selectivity has not been substantially modified or usually taken into account. In the past few years, scientists have tried to characterize more precisely the factors that affect the selectivity of various IEX chromatography sorbents.

Wu and Walters showed that different ligand densities could alter the selectivity and order of protein elution for different ion exchangers (1). Harinarayan et al. demonstrated the existence of an exclusion mechanism on ion exchangers that leads to an optimal dynamic binding capacity (DBC) for intermediate pH and conductivity conditions even if optimum conditions for protein binding are expected to be at low conductivity and around the pH value at which the protein is more charged (2). Additionally, Hardin et al. measured that resins with large pore sizes — or smaller pores but low ligand density — could reduce the impact of the exclusion mechanism (3). Altogether, these data suggest that the selectivity of a given ion exchanger is ruled by a complex combination of parameters.

The new Q and S HyperCel IEX chromatography sorbents from Pall Life Sciences are designed to provide a very high protein binding capacity even at low RT, along with a new selectivity and a differentiated salt sensitivity. Low to moderate density of quaternary amine (Q, 99–138 µeq/mL) and sulfonic acid (S, 59–84 µeq/mL) groups are immobilized on a robust and scalable cross-linked cellulose HyperCel matrix that confers low nonspecific binding with chemical stability and excellent mechanical properties.

Here, we characterized the differentiated selectivity of both those sorbents. We first evaluated the DBC for model proteins in various pH, conductivity, and RT conditions compared with other commercially available sorbents: rigid agarose Q and S and polymeric Q and S. We also separated a mixture of model proteins to further characterize the specific selectivity of Q and S HyperCel sorbents. Finally, we performed a real-case application using Q HyperCel sorbent as a capture step for purification of a recombinant green fluorescent protein (rGFP) from an Escherichia coli lysate, and we compared that performance to two other anion exchangers: rigid agarose Q and Q Ceramic HyperD F sorbents.

Materials and Methods

Chemicals and Equipment Used: Analytical-grade reagents came from Sigma Aldrich (www.sigmaaldrich.com) except for bovine serum albumin (BSA), which was provided by Millipore (www. millipore.com). Professor Xavier Santarelli of Ecole Nationale Supérieure de Technologie des Biomolécules de Bordeaux (ENSTBB) in France kindly provided clarified E. coli lysate containing recombinant greenfluorescent protein (rGFP).

The rigid S agaroseorbents came from GE Healthcare (www.gehealthcare. com). Polymeric sorbents came from Tosoh Bioscience (www.tosohbioscience.com). All HyperCel and HyperD sorbents came from Pall Life Sciences (www.pall. com). Each sorbent was packed according to manufacturer instructions into 0.5-cm diameter columns (1 mL with 5.0-cm bed height) from Kronlab (www.ymc-europe.com/ymceurope/ products/preparativeLC/LC_ IntroForKronlab.html). We used an ÄKTAexplorer 100 system from GE Healthcare (www.gelifesciences.com) for all chromatographic runs.

For sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), we used NuPAGE 4–12% Bis-Tris gels from Invitrogen (www. invitrogen.com) stained with Coomassie SimplyBlue (also from Invitrogen). Total protein content was measured with a bicinchoninic acid (BCA) protein assay from Thermo Scientific Pierce (www. piercenet.com).

Comparison of Anion-Exchange Sorbents Used As a Capture Step for rGFP Purification from E. Coli Lysate: We screened different anion exchangers (Q HyperCel, rigid agarose Q, and Q Ceramic HyperD F) for their selectivity using linear salt-gradient elution. Before equilibration in 50-mM Tris-HCl at pH 8.0, a 100-µL sample of crude E. coli lysate was loaded. After an equilibration buffer wash, elution was performed using salt gradient over 20 column volumes (CV) using 50 mM Tris-HCl at pH 8.0 with 0.5 M NaCl before a final strip with 50 mM Tris-HCl at pH 8.0 with 1.0 M NaCl. The flow rate applied was 1 mL/min.

We transferred the initial salt gradient elution to a four-step elution according to the conductivity at rGFP elution. The sorbent was equilibrated with 50 mM Tris-HCl at pH 8.0, then an 8-mL sample of E. coli lysate was loaded onto the column at 0.5 mL/min (2 min RT). After an equilibration buffer wash, elution was carried out in steps using 50 mM Tris-HCl at pH 8.0 with 0.1, 0.2, 0.5, and 1.0 M NaCl.

We evaluated the DBC for GFP of the sorbents tested at 10% breakthrough by loading E. coli lysate onto the columns until complete saturation. Samples were diluted tenfold in equilibration buffer to delay GFP breakthrough so that saturation was clearly observed. We noted the maximum absorbance value and calculated DBC with the volume loaded until flow-through absorbance was at least 10% of this maximum.