High performance liquid chromatography (HPLC) of peptides and proteins

Abstract

The introduction of high performance liquid chromatography (HPLC) to the analysis of peptides and proteins some 25 yr ago revolutionized the biological sciences by enabling the rapid and sensitive analysis of peptide and protein structure in a way that was inconceivable 30 yr ago. Today, HPLC in its various modes has become the pivotal technique in the characterization of peptides and proteins and has therefore played a critical role in the development of peptide and protein-based pharmaceuticals. The extraordinary success of HPLC can be attributed to a number of factors. These include 1) the excellent resolution that can be achieved under a wide range of chromatographic conditions for very closely related molecules as well as structurally quite distinct molecules; 2) the experimental ease with which chromatographic selectivity can be manipulated through changes in mobile phase characteristics; 3) the generally high recoveries and hence high productivity and 4) the excellent reproducibility of repetitive separations carried out over a long period of time, which is due partly to the stability of the sorbent materials under a wide range of mobile phase conditions. HPLC is extremely versatile for the isolation of peptides and proteins from a wide variety of synthetic or biological sources. The complexity of the mixture to be chromatographed will depend on the nature of the source and the degree of preliminary clean-up that can be performed. In the case of synthetic peptides, RPC is generally employed both for the initial analysis and the final large scale purification. The isolation of proteins from a biological cocktail however, often requires a combination of techniques to produce a homogenous sample. HPLC techniques are then introduced at the later stages following initial precipitation, clarification and preliminary separations using soft gel. Purification protocols therefore need to be tailored to the specific target molecule. Reversed phase chromatography (RPC) is by far the most commonly used mode of separation for peptides, although ion-exchange (IEC) and size exclusion (SEC) chromatography also find application. The three dimensional structure of proteins can be sensitive to the often harsh conditions employed in RPC, and as a consequence, RPC is employed less for the isolation of proteins where it is important to recover the protein in a biologically active form. IEC, SEC affinity chromatography are therefore the most commonly used modes for proteins, but RPC and hydrophobic interaction (HIC) chromatography are also employed. In addition, each mode of chromatography can be operated at different level of loading from capillary formats to large scale process systems. An appreciation of the factors that control the resolution of peptides and proteins in interactive modes of chromatography can assist in the development and manipulation of separation protocols to obtain the desired separation. The capacity factor kof a solute can be expressed in terms of the retention time tr, through the relationship k= (tr - to ) / to (1) where t0 is the retention time of a nonretained solute. The practical significance of kcan be related to the selectivity parameter ?, defined as the ratio of the capacity factors of two adjacent peaks as follows a k?i / k?j (2) which allows the definition of a chromatographic elution window in which retention times can be manipulated to maximize the separation of components within a mixture. The optimization of high resolution separations of peptides and proteins involves the separation of sample components through manipulation of both retention times and solute peak shape. The second factor that is involved in defining the quality of a separation is therefore the peak width t. The degree of peak broadening is directly related to the efficiency of the column and can be expressed in terms of the number of theoretical plates, N, as follows N = (tr)2/ ?r 2 (3) N can also be expressed in terms of the reduced plate height equivalent h, the column length L and the particle diameter of the stationary phase material dp, as N = hL / dp (4) The resolution, Rs, between two components of a mixture therefore depends on both selectivity and bandwidth according to Rs 1/ 4 N(a ?1)[1/(1k?)] (5) This equation describes the relationship between the quality of a separation and the relative retention, selectivity and the bandwidth and also provides the formal basis upon which resolution can be manipulated to achieve a particular level of separation. Thus, when faced with an unsatisfactory separation, the aim is to improve resolution by one of three possible strategies: the first is to increase , the second is to vary κwithin a defined range normally 1 < κ< 10, or third to increase N, for example, by using very small particles in narrow bore columns. The challenge facing the scientist who wishes to analyze and/or purify their peptide or protein sample is the selection of the initial separation conditions and subsequent optimization of the appropriate experimental parameters. This chapter provides an overview of the different techniques used for the analysis and purification of peptides and proteins and the experimental options available to achieve a high resolution separation of a peptide or protein mixture. The interested reader is also referred to a number of publications that provide a comprehensive theoretical and practical overview of this topic (1-6). © 2008 Humana Press.