Analytical techniques for biopharmaceutical development by Roberto Rodríguez-Díaz, Tim Wehr, Stephen F. Tuck

By Roberto Rodríguez-Díaz, Tim Wehr, Stephen F. Tuck

This reference assists scientists within the choice and alertness of analytical thoughts for biopharmaceutical id, formula, and assessment-providing a helpful survey of exploratory tools usually applied in the course of the preclinical, medical, and advertisement levels of improvement.

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The surface silanols on silica can serve as sites for covalent attachment of ligands. A major limitation of silica as a matrix for chromatography of peptides and proteins is the potential for interaction of basic amino acid side chains with residual silanol groups on the silica surface. These can participate in secondary retention mechanisms through hydrogen bonding or ion exchange. The presence of highly active silanol sites can cause peak tailing. If the characteristics of the underlying silica change from one batch of column packing to another, columns may exhibit variations in peak symmetry and selectivity.

Surfactants will also bind to the reversed-phase surface and can permanently change the behavior of the column. Therefore, the use of surfactants is usually reserved for applications where they are required for solubilization of the Copyright © 2005 by Marcel Dekker. 44 Wehr protein and to achieve satisfactory recovery. For example, RPLC methods for integral membrane proteins often employ ionic, zwitterionic, or nonionic surfactants as additives. Retention–Solvent Strength Relationships The relationship between retention and solvent strength in reversed-phase chromatography is described by the linear solvent strength model:19,20 Log k = log kw – SΦ (1) where kw is the capacity factor (k) in water as eluent, Φ is the volume fraction of organic solvent, and S is a constant.

However, chromatography of peptides and proteins is generally carried out using gradient elution with organic modifier concentrations rarely exceeding 60 to 80%. Under these conditions, the alkyl chains probably exist largely in a self-associated state such that the effective hydrophobicity of the different alkyl chains is similar. 13 Phenyl and cyano phases are likely to exhibit different selectivities than the straight-chain alkyl phases and therefore are often selected when C8 or C18 columns fail to provide satisfactory resolution.

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