Fast protein HPLC

Table n. Repetitive Analytical Cycles for Fast Protein HPLC... 

Hsieh et al. (2003) and Zeng et al. (2003a), used direction injection to quantitate multiple drug discovery compounds simultaneously with good robustness. In one application, the flow rate of the HPLC gradient was programmed. The flow rate was first increased (4 to 8 mL/min) for fast protein clean-up, then decreased (1.2 mL/min) for better sensitivity. The column maintained similar efficiency after several hundred injections. 

High-performance liquid chromatography (HPLC) and fast protein liquid chromatography (FPLC) rely on the same separation principles as the traditional chromatography columns, but tend to be much faster because of high flow rates that are possible due to the uniform bead size and the mechanical strength of the beads. See also Chapter 4, section 1.2.2. 

There are several problems requiring careful attention. Lysozyme has a tendency to form complexes with many substances [e.g., alkyl sulfates, fatty acids, aliphatic alcohols (Smith and Stocker, 1949), cephalins (Brusca and Patrono, 1960), and other proteins]. Of particular importance is its tendency to form complexes with transferrins [e.g., ovotrans-ferrin (Ehrenpreis and Warner, 1956)]. These interactions lead to difficulties in the isolation of lysozyme. Some recent workers have used fast protein liquid chromatography (FPLC) and high-performance liquid chromatography (HPLC) (e.g., Ekstrand and Bjorck, 1986). The resolution in these procedures may not always be satisfactory, and in HPLC pressure and solvent effects must be monitored carefully if the product is to be suitable for conformation and activity studies. 

A variant of HPLC is FPLC (fast protein liquid chromatography) (Sheehan 1996), a form of chromatography in which the apparatus and column material is designed specifically with protein analysis and purification in mind. However, this does not imply that conventional HPLC with the right columns is unsuitable for protein chromatography. 

Figure 7.26. HPLC chromatogram showing the performance of a pellicular column (Zorbax Poroshell 300-SB C4, 50 x 4.6mm, 5pm) in fast protein separation. Inset shows the structure of Zorbax Poroshell 300-SB particles. Diagram courtesy of Agilent Technologies.

The following sections will describe some of the various methods of liquid chromatography suitable for separation and analysis of biological (macro)molecules. Such systems often use high pressures and rapid flow rates, and are sometimes loosely described as HPLC (high performance liquid chromatography) or FPLC (fast protein liquid chromatography). 

For discovery PK assays, the most common sample preparation procedure is protein precipitation161720 24 because it is fast, easy to automate, and requires no method development. While protein precipitation typically will not provide as clean a sample as will alternative procedures, it is sufficient for most discovery PK samples that use HPLC/MS/MS for the analytical step.21101... 

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