Impact of New Columns on Drug Development

Apr 01, 2014
Volume 32, Issue 4, pg 36–41

This article will examine the development of new types of columns based on different particle types, sizes, and other physical characteristics and how they can improve the speed and efficiency of high performance liquid chromatography used to support more expansive and complicated analyses.

Liquid chromatography (LC) has always been an important analytical technique in the drug development process. For small-molecule drugs, the use of high performance liquid chromatography (HPLC) for assay and impurity analyses, analysis of process samples, support of toxicology and clinical studies, and stability monitoring methods plays a crucial role in the entire development. Many of the separations for this class of drugs are based on reversed-phase HPLC and sometimes other techniques, such as chiral analysis. For biological drugs, the use of HPLC is even more important and expands to the use of size-exclusion chromatography, ion-exchange chromatography, and other specific techniques such as affinity chromatography.

The recent Affordable Health Care Act of 2010 in the United States provided a pathway for the development of biosimilar products, similar to the generic drug pathway for traditional drugs that was established by the Hatch-Waxman Act of 1984. Although the specific requirements for approval of a biosimilar are still under development by the United States Food and Drug Administration (US FDA), it is clear from recent guidance and discussions that an increased emphasis will be placed on the analytical characterization as a part of the pathway for approval for biosimilars. A recent presentation by Kozlowski (1) highlighted the desire for an increased level of analytical and physical characterization in the development of biosimilar products.

This trend is creating an even greater need for high-efficiency and high-performance analytics, many of which are based in part on HPLC. The development of new types of columns based on different particle types, sizes, and other physical characteristics will be a key contributor to the expanded use of HPLC to support these additional analytics. This article examines a few of these new technologies and how they can improve the speed and efficiency of HPLC used to support more expansive and complicated analyses.

Monolith Columns

One of the first needs for rapid HPLC analyses is the support of process development for biological molecules. Many large proteins are produced in cell-based fermentation systems. These cell culture production systems require significant process development to optimize the input starting materials, feed rates, and frequencies, and other parameters to generate protein at a commercially viable concentration in the final product. The need to continuously and rapidly monitor protein concentration (often referred to as titer) is important to initially aid in the selection of a proper clone to be used and then as additional process development work continues.

Silica-based monolith HPLC columns were developed in the early 1990s (2) as an alternative to traditional porous HPLC columns. These columns use channels rather than pores for flow through the column and are less likely to clog with other materials in the sample, such as cell debris in a cell culture sample. In addition, they rely on convective mass transfer leading to flow rate–independent separations that can provide very rapid analysis of high-molecular-weight samples such as proteins. A specific example of this is a monolith column, which contains protein A bound to the surface of the monoliths. Protein A selectively retains IgG proteins in an affinity type separation, therefore, these columns can be used to separate the IgG (the protein of interest in the cell culture) from other proteins and cell debris made during the cell culture process.

Figure 1: Sample titer analysis of traztuzumab. Comparison of supernatants from clone (red) versus originator purified drug (blue). Samples injected into a 50 mM phosphate buffer (pH 7.4) and eluted with a 0.1 M citric acid buffer (pH 2.8). Figure adapted and provided by Maureen Joseph of Agilent Technologies and Koen Sandra of the Research Institute for Chromatography.
An example of this type of analysis is shown in Figure 1. In this case, the blue trace is a 0.5 mg/mL sample of the originator drug (traztuzumab) injected as the drug product. The red trace is a cell culture sample from one clone that was centrifuged for 5 min at 5000 g and the supernatant was directly injected onto an Agilent Biomonolith Protein A column (5.2 × 4.95 mm). The IgG protein is retained on the column while the other cell proteins and debris are eluted rapidly in less than 20 s in the initial mobile phase. A change to the mobile-phase B (citric acid buffer) elutes the IgG protein with a total analysis time of less than 2 min. Because multiple clone samples are often analyzed at once during clonal screening, this rapid analysis permits the screening of dozens or hundreds of samples quickly.

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