Mixed-Mode Chromatography in Downstream Process Development

Salt-tolerant adsorption and unique selectivity are the major advantages of mixed-mode materials over single-mode resins.
Mar 02, 2010

Mixed-mode chromatography materials contain ligands of multimodal functionality that allow protein adsorption by a combination of ionic interactions, hydrogen bonds, and/or hydrophobic interactions. Complex mixtures like fermentation supernatants or cell lysates can be applied directly at relatively high conductivity, and elution is usually achieved by electrostatic charge repulsion. We used mixed-mode materials for capturing and intermediate purification of several recombinant therapeutic proteins from various expression systems like yeast, Escherichia coli, and mammalian cells. Product-related impurities as well as process-related impurities from fermentation media were efficiently removed while the desired product was bound with high selectivity. Because these purification protocols can be scaled up easily to production scale, mixed-mode materials are being considered as potential elements of a general purification platform for recombinant therapeutic proteins produced in various expression systems.

(Bayer Schering Pharma)
The impurity profile of a starting material for downstream processing depends mainly on the conditions of the fermentation process, such as the expression system, medium composition, fermentation regimen, time point of harvest, or shear forces during isolation. This leads to a variety of process- and product-related impurities which must be removed by an efficient, orthogonal, and robust purification process. Modern downstream processes often consist of only two or three separation steps, and usually avoid conditioning steps like ultrafiltration/diafiltration (UF/DF) for buffer exchange to reduce the total number of process steps. However, this strategy significantly increases the risk that contaminants are not completely removed during the purification process, especially if complex microbial feedstocks or E. coli lysates are used as starting materials. In these cases, single-mode chromatography materials like ion exchange or hydrophobic resins often fail, in particular if the feedstock contains large amounts of complex colored impurities like melanoidins in combination with a relatively high conductivity of 15–30 mS/cm. Mixed-mode resins may help to overcome these problems because multimodal ligands offer excellent selectivity in combination with a salt-tolerant adsorption of the target protein.

The ligands of mixed-mode materials typically contain a combination of multiple binding modes like ion exchange, hydrogen bonding, and hydrophobic interactions.1 The target protein itself is also a multimodal molecule. This situation results in a variety of protein–ligand interactions and often leads to unique selectivity, which in some cases includes pseudo-affinity. Furthermore, binding of the desired protein often is achieved at the conductivity of the feedstock without further dilution or addition of lyotropic salts. That feature makes mixed-mode resins an excellent choice for direct capture steps.

Commercially available mixed-mode materials are based on several principles. Resins containing hydrocarbyl amine ligands (e.g., PPA Hypercel, HEA Hypercel, Pall Corporation) allow binding at neutral or slightly basic pH values by a combination of hydrophobic and electrostatic forces. Elution usually is achieved by electrostatic charge repulsion when the pH value is lowered below the isoelectric point (pI) of the target protein and the pKa of the ligand.2 Another ligand, 4-mercapto-ethyl-pyridine (MEP Hypercel, Pall), is based on a similar principle, but hydrophobic interaction is achieved by an aromatic residue and the sulphur atom facilitates binding of immunoglobulins by thiophilic interaction.3–6 Another group of mixed-mode materials (Capto MMC and Capto adhere, GE Healthcare) contains ligands with hydrogen bonding groups and aromatic residues in the proximity of ionic groups, which leads to the salt-tolerant adsorption of proteins at different conductivities.7,8 However, in contrast to electrostatic charge repulsion, which is driven mainly by changes in the pH value, the elution of proteins from Capto MMC usually requires an increase in the pH value and salt concentration. It also should be noted that many other chromatography materials that have been around for decades, like affinity resins with dye ligands, hydroxyapatite, and some ion-exchange resins, such as Amberlite CG 50 (Rohm & Haas) or Lewatit CNP 105 (Lanxess) often show a unique specificity that is based mainly on a multimodal protein–ligand interaction.1,3

Table 1. The use of mixed-mode materials in the downstream processes of various therapeutic proteins
Mixed-mode materials were used in our laboratories to purify a variety of different therapeutic proteins, like cytokine muteins, protease inhibitors, and antibody fragments (Table 1). Although these resins are particularly useful for capture steps because of their selectivity and salt tolerance, they can be used for intermediate purification or polishing as well. In the following sections, we describe two case studies in which mixed-mode materials showed significant advantages compared with single-mode resins for removing a product-derived impurity containing a post-translational modification, and for purifying a therapeutic protein with challenging physicochemical properties.

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