Analytical methods peptides

The specification development process is a data-driven activity that requires a validated analytical method. The levels of data needed include assay precision, replicate process results (process precision), and real-time stability profiles. A statistical analysis of these data is critical in setting a realistic specification. Most often, aggregation and fragmentation degradation mechanisms are common to protein and peptide therapeutics. Therefore, the SE-HPLC method provides a critical quality parameter that would need to be controlled by a specification limit. 

Electrospray ionization mass spectrometry (ESI-MS) is an analytical method for mass determination of ionized molecules. It is a commonly used method for soft ionization of peptides and proteins in quadmpole, ion-trap, or time-of-flight mass spectrometers. The ionization is performed by application of a high voltage to a stream of liquid emitted from a capillaty. The highly charged droplets are shrunk and the resulting peptide or protein ions are sampled and separated by the mass spectrometer. 

Systematic investigations on the appearance of peptides in urine were initiated no more than ten years ago, when modem analytical methods were introduced. This relatively short period devoted to the studies on peptiduria, as well as the diversity of the methods used, has undoubtedly been the chief reason for the confusion in the state of our knowledge on this subject. Nevertheless, two important facts can now be accepted with-... 

The intrinsic inertness of the peptide bond is demonstrated by a study of the chemical hydrolysis of N-benzoyl-Gly-Phe (hippurylphenylalanine, 6.37) [67], a reference substrate for carboxypeptidase A (EC 3.4.17.1). In pH 9 borate buffer at 25°, the first-order rate constant for hydrolysis of the peptide bond ( chem) was 1-3 x 10-10 s-1, corresponding to a tm value of 168 y. This is a very slow reaction indeed, confirming the intrinsic stability of the peptide bond. Because the analytical method used was based on monitoring the released phenylalanine, no information is available on the competitive hydrolysis of the amide bond to liberate benzoic acid. 

Boss H J., Watson D.B., and Rush R.S. (1998), Peptide capillary zone electrophoresis mass spectrometry of recombinant human erythropoietin an evaluation of the analytical method, Electrophoresis 19(15), 2654—2664. 

Castagnola, M., Rossetti, D. V, Misiti, R, Cassiano, L., Giardina, B., and Messana, I. (1997). Analytical methods for peptide drugs applicable to process control. Process Control Qual. 10, 181-203. 

Finally, when RPC methods are used in preparative studies with peptides, the opportunity routinely exists for subsequent analysis of the recovered fractions by a variety of analytical methods including high-speed RP-HPLC, HP-IEX, HP-HILIC, or HP-IMAC, zonal or micellar electrokinetic high-performance capillary electrophoresis (HP-CZE and MECK-CZE), capillary electrochromatography (CEC), or capillary isotachophoresis. The combination of the RPC information, drawn from the In k versus i > plots, with the data derived from on-line spectroscopic detection thus readily provides a comprehensive opportunity to assess the purity of an isolated peptide, many of the physicochemical features of the interaction, as well as a means to optimize the resolution in the RPC separation. 

In the last 20 years, analytical methods (HPLC and CE) that are more resolutive and quantitative than TLC have been developed for the analysis and quantitation of peptides. As a result, only a limited number of applications of TLC that remain of current interest will be addressed. 

Among the analytical methods presently used for the characterization of natural and synthetic peptides and proteins, the primary value of amino acid analysis is the determination of absolute peptide and protein content in solids and solutions and the quantitation of their amino acid composition and stoichiometry. It involves two steps, i.e. complete hydrolysis of peptides and proteins, followed by photometric determination of the released amino adds. The steps are laborious and time-consuming, and there is a continuous need for improvement of the techniques to increase precision and sensitivity. 

Colistin is a linear-ring peptide antibiotic. Its main components are colistin A and colistin B. It is a member of the polymyxin family of antibiotics that is stable in dry form and in water solution. The sulfate salt of colistin, which is usually administered as feed additive, is soluble in water, slightly soluble in methanol, and practically insoluble in acetone and ether. Colistin components do not have any specific fluorophore and UV chromophore, so detection by liquid chromatography at residue levels of interest is difficult without including a suitable derivatization step in the analytical method. 

Seven years later, in a critical review on the synthesis of peptides, the following statement was made "Chemists in particular should respect the classical criteria of what constitutes synthesis of a natural product, i.e., that synthesis of a natural product has been achieved, when the physical, chemical and biological properties of the synthetic compound match those of the natural prototype. Unfortunately not a single one of the "synthetic proteins" satisfies these criteria. It is frequently argued that these criteria are not applicable to more complex situations, but lowering standards of purity is not likely to advance the field. Presently available analytical methods cannot adequately detect inhomogeneity in a high molecular peptide that is produced by stepwise synthesis. Consequently, the synthetic method must be chosen so that the product can be purified and critically evaluated by the available analytical techniques. F.M. Finn, 1976). 

Besides monitoring bulk solution qualities by conventional analytical methods, measurement of the phase transition may also be warranted. Slight differences in the nature of the formulation owing to aging, undetected by typical analytical methods, may influence the phase transition of the product formulation. For example, absorption of carbon dioxide from the air over an extended time period may cause a pH shift, consume one component of a buffering system, or promote degradation. For a peptide or protein with both a hydrophilic and hydrophobic nature, alterations to desired secondary, tertiary, or quaternary... 

Analytical methods used to identify monomers have improved significantly from those that quantify whole classes of compounds, such as amino acids, peptides, proteins, and primary amines (Undefriend et al., 1972) or carbohydrate-like compounds (Johnson and Sieburth, 1977) to ones that are molecule-specific (Table II). Most of these methods are based on combining chromatography techniques that can separate complex mixtures of molecules with highly sensitive detectors that can approach the nanomolar or picomolar range. Monomers are usually present at low concentrations, so... 

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