USP Tailing Research Articles

Monitoring EDTA process residuals in recombinant protein manufacturing using liquid chromatography

We have developed a chromatographic method for the high sensitivity quantitation of EDTA process residuals in recombinant protein manufacturing validation studies. The reversed-phase HPLC method is based upon the detection of Cu 2+/EDTA complexes at 254 nm, and has been qualified for use on intermediates from a purification process for a recombinant protein expressed in E. coli. Quantitation of EDTA in recombinant protein process intermediates is linear in the range of 0.2 to 64 μ M with LOD/LOQ values below 2.0 μ M. The assay is suitable for use in process backgrounds containing Tris, HEPES, MES, NaCl, hexanediol, NH 4SO 4, and PEG. EDTA spike recovery values in all process samples tested were greater than 90% at the 4.0 μ M concentration. System suitability parameters for the chromatographic method were developed based upon peak area and retention time precision, column efficiency and USP tailing. Peak area precision and intermediate precision values across the linear range of the assay exhibited C.V. values less than 15% at any concentration tested in all sample backgrounds. The assay robustness was tested by transfer of the assay to a second laboratory and analyst with use of multiple process intermediate lots, reagent/column lots, and HPLC systems.

Enantioselective separation of thalidomide on an immobilized α1-acid glycoprotein chiral stationary phaseglycoprotein chiral stationary phase

An easy and rapid enantioselective separation for assay of racemic thalidomide on an immobilized α1-acid glycoprotein chiral stationary phase (GPA CSP) is described. The effects of tetrahydrofuran (THF) as organic modifier, buffer concentration to control the ionic strenth, and mobile phase pH were studied. These variations have consequences in terms of chromatographic retention (k), resolution (R s), selectivity (α), and peak asymmetry (USP tailing factor). The main condition affecting chromatographic retention was mobile phase pH. At pH 4.5, no separation of thalidomide enantiomers was achieved whereas at pH 7.9 chiral separation was optimum. Peak tailing was directly related to changes in pH and to addition of THF as mobile phase modifier. Results also indicated that the resolution factor is THF concentration-dependent, and that the separation factor (α) is the best parameter for evaluating enantioselectivity. The best mobile phase was pH 7.0, 30 mM ammonium acetate containing 0.3% THF. Under these conditions validation including linearity, recovery, and precision was performed. The suitability of this method has been successfully proved in a limited in-vivo study after intravenous administration of thalidomide to a New Zealand male rabbit.

High performance liquid chromatographic determination of phenolic compounds in seed exudates of Festuca arundinacea and F. pratense

Derivatives of benzoic and cinnamic (caffeic, protocatechuic, vanillic, ferulic, p-coumaric, p-hydroxybenzoic and syringic) acids and 3,4-dihydroxybenzaldehyde in seed exudates of Festuca arundinacea cv. “Kora” and F. pratense cv. “Otava” have been separated and quantified on a Zorbax SB C18 Rapid Resolution™ column with linear gradient elution using a mobile phase containing acetonitrile and 0.5% acetic acid. The USP tailing factors were less than 1.15 for all peaks. Strictly linear calibration curves (r > 0.9997) with limits of detection of 90–880 nM were obtained for all analytes in the concentration ranges from units or tenths up to 600 µM. RP 105 and OASIS™ HLB polymeric sorbents have been shown to be the best of the tested sorbents for the separation and preconcentration of phenolic compounds from both plant materials. The solid-phase extraction procedures have been compared with classical liquid–liquid extractions and consistent results were obtained. Copyright © 2000 John Wiley & Sons, Ltd.

An approach to the evaluation and comparison of reversed-phase high-performance liquid chromatography stationary phases

An approach to the evaluation and comparison of reversed-phase high-performance liquid chromatography stationary phases with particular emphasis on data analysis and presentation is described. Assessment is based on the peak efficiency, asymmetry (USP tailing factor) and relative retention properties shown by 24 basic compounds having a wide range of structural and physico-chemical properties. A novel approach to data normalisation and presentation is described. This overcomes the problems associated with the quality of the column packing process, as well as differences in stationary phase selectivity which in conjunction with extra column band broadening effects can make comparisons meaningless.