The Role of Elution Gradient Shape in the Separation of Protein Therapeutics

Sep 01, 2014
Volume 32, Issue 9, pg 736–740

In this article, we discuss the role of the gradient in protein separations by reversed-phase and ion-exchange high performance liquid chromatography (HPLC). To illustrate the key points, we show data from two products. Granulocyte colony stimulating factor (GCSF) is a microbial protein that is expressed in E. coli. For this molecule, reversed-phase HPLC is examined for separation of the product-related variants. The other molecule is a biosimilar monoclonal antibody product and, in this case, ion-exchange HPLC is explored as a tool for analysis of the acidic, main, and basic variant species.

The number of therapeutic protein products available for use has radically increased in recent years. They include a wide variety of molecules such as recombinant human cytokines (for example, α and β interferon), cellular growth factors (such as granulocyte-macrophage colony-stimulating factor [GM-CSF]), hormones (such as glucagon), neuromuscular antagonists (for example, botulinum toxin), blood products (such as clotting factor VIII), and monoclonal antibodies (mAbs) (1). For protein therapeutics to be effective, they must be synthesized in their biologically active forms, with proper folding and post-translational modifications (2). However, these products are known to be associated with a variety of heterogeneities because of modifications such as glycosylation, deamidation, oxidation, and disulfide bond formation, which occur as a consequence of events during protein expression, purification, and storage (3). In view of these heterogeneities, thorough characterization using multiple orthogonal techniques is necessary for receiving regulatory approval for product commercialization. Of the many tools that are used, high performance liquid chromatography (HPLC) is the primary workhorse for analysis of biopharmaceutical proteins (4,5). The significant advantages that HPLC offers include high reproducibility, high sample throughput because of autosampling capabilities, high separation resolution, easy quantitation, high precision, and high robustness (6).

HPLC can further be classified into normal-phase, reversed-phase, ion-exchange, and gel filtration (size-exclusion) chromatography. Each mode is based on a different underlying mechanism and together make HPLC a powerful tool in the analytical arsenal. Since typical HPLC applications involve separation of product from product-related variants and impurities that have very similar physicochemical properties as compared to the product, the elution strategy has a significant impact on the quality of separation. The most commonly used strategies are isocratic elution and gradient elution. The latter technique can be implemented in linear, segmented, convex, and concave shapes (7). Elution is isocratic when the eluent strength is kept constant throughout the separation. Gradient elution implies that the mobile-phase composition will be varied during sample separation as per the chosen trajectory. Linear gradients are generally preferred because they are easy to create and are relatively robust. However, nonlinear gradients offer several distinct advantages, including reduced separation time, improved sample resolution, and higher detection sensitivity (8). In this installment, we primarily focus on reversed-phase HPLC and ion-exchange HPLC.

Separation by reversed-phase HPLC is achieved because of the interactions between the hydrophobic ligands covalently attached to the adsorbent and the hydrophobic patches of the species in the feed. Loading conditions are chosen such that the product binds strongly to the adsorbent. Thereafter elution is performed by using organic solvents such as acetonitrile, ethanol, or methanol. The molecules are eluted in the order of increasing hydrophobicity. More hydrophobic species are retained strongly and hence are eluted later, while the less hydrophobic species are eluted earlier. Several systematic approaches for reversed-phase HPLC method development have been described in the past (9–14). In most cases, an attempt is made to optimize sample retention (values of the retention factor, k), column efficiency (plate number, N), and selectivity (separation factor, α). Major emphasis is usually given to the optimization of selectivity, often using a preselected series of experiments plus a computer program for predicting retention (k and α) as a function of one or more experimental variables. About four decades ago, the gradient elution was merely used for the prediction of isocratic behavior because the gradient run covers all binary compositions of possible interest to isocratic separation (14,15). In the past, favored method development strategies have been based on varying experimental conditions that are believed to have the largest effect on α; for example, solvent type, solvent strength (%B), and column type for neutral samples, or pH and ion-pair-reagent concentration for ionic samples. The use of gradient elution with temperature and gradient steepness as variable parameters for the optimization of selectivity and separation have been used as an approach for method development (13).

Ion-exchange HPLC is another popular, nondenaturing analytical method that is used for the separation of species based on their charge (16). Separation in this case is achieved by either changing the pH of the mobile phase or increasing the salt content in the mobile phase. Either of these alterations change the charge on the species and thus affect the interaction between the species and the stationary phase. The resolution of peaks is generally based on the differential retention of the protein on the column (17). There are several studies published on this topic that focus on the importance of development and validation of charge heterogeneity analysis of protein therapeutics using an ion-exchange method (18–24). Several approaches have been published in literature on the types of elution to resolve the charge variants of mAb, including salt gradient, pH gradient, and salt hybrid gradient. All of these types of gradients have advantages and disadvantages associated with them. The most popular of these is the formation of a salt gradient. In a salt gradient, apart from the routinely optimized parameters (column, pH, conductivity, temperature, and the type of salt), the optimization of the shape of the elution gradient can have a significant impact on the resolution (25).

In this installment, we discuss the role of the gradient in separation by reversed-phase and ion-exchange HPLC. To illustrate the key points, we show data from two products. Granulocyte colony stimulating factor (GCSF) is a microbial protein that is expressed in E. coli. For this molecule, reversed-phase HPLC is examined for separation of the product-related variants (oxidized, main, and reduced species) in view of the difference in the hydrophobicity of these variants (24). The other molecule is a biosimilar monoclonal antibody product and in this case ion-exchange HPLC is explored as a tool for analysis of the charged variants (acidic, main, and basic species) because of the difference in the charges on these variants.

Materials and Methods

Protein Samples

The two therapeutic protein samples used in this study were recombinant human GCSF (E. coli derived) and an IgG1 mAb (CHO cell culture derived). Both were donated to us by major domestic biotech manufacturers.

Instrumentation and Columns

An Agilent 1200 series HPLC unit was used, consisting of a quaternary pump with degasser, an autosampler with a cooling unit, and a variable-wavelength detector.

Two ion-exchange columns were used in this study: a MAbPac SCX-10 strong-cation-exchange column and a 250 mm × 4.6 mm, 10-µm d p MAbPac WCX-10 weak-cation-exchange column. Both columns were purchased from Dionex (now part of Thermo Fisher Scientific).

Three reversed-phase columns were used: a 100 mm × 4.6 mm Chromolith High Resolution RP-18 column from Merck, a 250 mm × 4.6 mm, 5-µm d p C4 column from Phenomenex, and a 250 mm × 4.6 mm, 3.5-µm d p X-bridge BEH300 C4 column from Waters.


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