Chromatographic analysis of therapeutic proteins

Abstract

Analysis of J V Therapeutic ProteinsRapid LC applications HPLC has become a principal tool for the rapid separation and characterization of many classes of pharmaceuticals, including those based on peptides and proteins.Most HPLC methods for biomolecules use an aqueous-based mobile phase; the stationary phase can vary, depending on the chromatographic mode. Aqueous solvents are used because, in general, nonaqueous solvents denature proteins by interfering with their uniquely folded structure.With the emergence of new proteinand peptide-based drugs, it has become necessary to develop rapid LC methods that can separate and characterize these substances or be used for preparative purposes without disrupting the unique three-dimensional structure upon which pharmacological activity depends. In general, these methods include, but are not limited to, size exclusion chromatography (SEC), ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), reversed-phase LC (RPLC), and affinity chromatography (AC).Each method has its strengths and weaknesses; the appropriate one is selected on the basis of the structure of the Development of new pharmaceuticals is increasingly expensive.Not only are the costs of R&D, laboratory testing, clinical trials, administration, and overhead escalating, but the maturing of the pharmaceutical industry itself is adding to the burden. Ten years ago, the rule of thumb was that development of a new drug cost about $200 million. Today that figure has doubled. A drug patent gives its owner exclusive rights to the invention for 17 years. Because it often takes as many as 12 years to bring a drug to market, it is crucial that product development proceed as efficiently as possible. Today the general rule for evaluating return on investment for pharmaceutical products is that for each day's delay in bringing the product to market, $1 million in sales is lost.Analysis and purification are essential parts of drug R&D, manufacturing, and quality assurance. Using rapid analytical methods can significantly cut develop-

The effect of the mobile phase additives on sensitivity in the analysis of peptides and proteins by high-performance liquid chromatography–electrospray mass spectrometry

“…acetonitrile). Although there are no examples of the use of HIC coupled on-line with ESI-MS, a few examples can be found for the off-line combination with MALDI [37,38].…”

“…KH 2 PO 4 , NaCl, Na 2 SO 4 , etc.) to overcome interactions between the stationary phase and the protein, as well as protein-protein interactions [37,69].…”

Elution behavior of insulin on high-performance size exclusion chromatography at neutral pH

“…Electrostatic and hydrophobic interactions are major causes of non-specific adsorption of proteins to the column material in HP-SEC [21,23,24]. Electrostatic interactions are often reduced by modifying the pH and ionic strength of the HP-SEC eluent.…”

“…In principle, HP-SEC separates analytes on the basis of their shape and hydrodynamic volume [14,22] and molecular weight determination of the analytes is possible by using appropriate protein molecular weight markers [23,24]. However, erroneous size estimations may occur due to the non-spherical nature of the selected protein standards or aggregates and protein-column interactions [25].…”

An artificial receptor for glycoproteins

“…Standardized multi-step product enrichment procedures in industry often incorporate orthogonal chromatographic techniques such as size exclusion chromatography (SEC), ion exchange, hydrophobic interaction chromatography (HIC), reverse-phase liquid chromatography and affinity chromatography (Compton and Kreilgaard, 1994;Scopes, 1996). Besides affinity techniques, all the other purification regimes are non-specific and, in fact, ion exchange, the most commonly used technique, can only be used for charged (sialylated) glycans and is inapplicable to the resolution of uncharged oligomannose-type proteins.…”