Eugene C. Rickard

Correlation of electrophoretic mobilities from capillary electrophoresis with physicochemical properties of proteins and peptides

Excellent correlation was observed for the electrophoretic mobilities measured by capillary zone electrophoresis versus q/MW2/3, where q is the calculated charge and MW is the molecular weight. Mobilities of a set of 33 diverse peptides from enzymatic digests and 10 intact proteins were measured for separations at pH 2.35, 8.0, and 8.15 with constant ionic strength, temperature, and viscosity. The correlation suggests that the frictional drag is proportional to the surface area of a sphere that has a volume proportional to the MW. The correlation of electrophoretic mobility with physicochemical properties will facilitate the elucidation of optimum separation strategies for protein and peptide mixtures.

Separation of antibody—antigen complexes by capillary zone electrophoresis, isoelectric focusing and high-performance size-exclusion chromatography

The separation of antibody-antigen complexes by free-solution capillary zone electrophoresis (CZE) has been demonstrated. The antigen, a monoclonal antibody specific for the antigen, and the complex were well resolved. The entire separation was achieved in less than 10 min using on-column UV detection. The pI values for the three species were estimated separately by isoelectric focusing (IEF) experiments on polyacrylamide gels. Reasonably good agreement was found between the relative migration times measured by CZE and the pI values. Both IEF and high-performance size-exclusion chromatography of the antibody-antigen mixtures confirmed the formation of the complexes observed by CZE. This study demonstrates the utility of CZE as a new and complementary technique for the characterization of antibody-antigen complexes.

Capillary zone electrophoresis of insulin and growth hormone

The separation power of capillary zone electrophoresis was examined using highly purified and well-characterized biosynthetic human insulin, growth hormone, their derivatives, and related proteins. Mixtures of proteins were chosen to illustrate practical applications of this technique. Proteins differing slightly in structure, but equivalent in net charge, were not completely separated. Degradation of insulin by dilute acid treatment was followed by capillary zone electrophoresis, native polyacrylamide gel electrophoresis, and reversed-phase liquid chromatography. Excellent correlation was observed between these techniques. Simple equipment requirements and analysis times on the order of 10 min make capillary zone electrophoresis attractive for analytical protein separations.

Influence of peroxide impurities in povidone and crospovidone on the stability of Raloxifene hydrochloride in tablets : Identification and control of an oxidative degradation product

The purpose of this study was to identify a degradation product in a tablet formulation of raloxifene hydrochloride (R-HCl), delineate the role of excipients in its formation, and develop a rational strategy for its control. The degradant was identified as an N-oxide derivative of the drug substance based upon spectroscopic characterization and chromatographic comparison to the synthetic N-oxide. To identify the factors contributing to the formation of N-oxide, binary mixtures of each excipient with R-HCl were exposed to 125°C in open containers. Raloxifene hydrochloride underwent an order of magnitude increase in conversion to the N-oxide in the presence of two excipients, povidone and crospovidone, as compared with its conversion in the presence of other excipients. To confirm a hypothesis that peroxide impurities in these two excipients contributed to the oxidation of the drug substance, tablet lots were spiked with quantities of H2O2 equivalent to 200, 400, 600, and 800 ppm peroxide over the intrinsic levels present in povidone and crospovidone. A strong correlation was observed between the total peroxide level and the quantity of the N-oxide formed upon accelerated storage. From these experiments a rational limit test for peroxide content in povidone and crospovidone was adopted as part of a control strategy to limit formation of the degradation product.

Capillary zone electrophoresis of peptide fragments from trypsin digestion of biosynthetic human growth hormone

Capillary zone electrophoresis (CZE) was applied to the separation of the 19 peptide fragments produced by enzymatic digestion of human growth hormone (hGH). The fragments of hGH produced by trypsin digestion under non-reducing conditions were identified in the electropherogram. Almost all of the fragments were resolved by CZE in less than 15 min. There is a marked difference in selectivity between reversed-phase high-performance liquid chromatography (RP-HPLC) and CZE. CZE is demonstrated to be a powerful complement to RP-HPLC for routine identification of hGH using trypsin digests.

Optimization of a capillary electrophoresis method to determine the chiral purity of a drug

Abstract Capillary electrophoresis (CE) using hydroxypropyl-β-cyclodextrin (HP-β-CD) in the separation buffer was investigated to determine the overall chiral purity of a drug containing a single stereogenic center. The effects of primary factors —pH, buffer components, buffer concentration, cyclodextrin concentration and sample amount (concentration and injection volume) — on the resolution of the enantiomers were investigated. Secondary factors such as the HP-β -CD source, lot and degree of substitution that were expected to affect the robustness of the assay were investigated also. The linearity, quantitation limit for the trace enantiomer and the precision of the measurements were determined. This study shows that understanding and optimizing the assay conditions leads to a chiral CE separation that is comparable to that obtained by chiral HPLC. However, chiral CE separations achieved with buffer additives have the advantages of shorter run times, higher numbers of theoretical plates (greater resolution), smaller amounts of chiral additive (less cost) and greater ruggedness (separation virtually independent of column properties unlike HPLC).

Method optimization in capillary zone electrophoretic analysis of hGH tryptic digest fragments

The mobile phase composition was optimized for the separation of tryptic digest fragments of human growth hormone by capillary zone electrophoresis. The effect of pH (pH 2.4, 6.1, 8.1 and 10.4) was evaluated since pH determines the relative charge of species, the prime contributor to selectivity; pH 8.1 was selected for the optimization studies. Tricine (buffer), sodium chloride (ionic strength adjustor), and morpholine (mobile phase additive) concentrations were systematically varied a pH 8.1. All three exhibited major effects on the electroosmotic flow velocity and current, and minor effects on selectivity. Tricine was the most crucial for good resolution, although addition of morpholine helped to resolve closely eluting species. The optimum separation conditions were found to be pH 8.1 with 0.1 M tricine, 0.02 M morpholine and no salt.

Role of capillary electrophoresis methods in the drug development process

In the past several years, capillary electrophoresis (CE) has generated considerable interest from pharmaceutical companies for control of both the chiral and achiral purity of bulk drugs and drug products. This paper evaluates the use of CE as: (1) a technique complementary to HPLC for the determination of peak homogeneity of a drug, (2) for determination of chiral purity, and (3) for determination of achiral purity. It would be greatly advantageous if CE could be used to determine both the chiral and achiral purity in a single assay. This investigation compares the results obtained for the separation of the enantiomers of duloxetine using several neutral cyclodextrins to those obtained using anionic cyclodextrins (sulfobutyl ether derivatives) as chiral selectors added to the separation buffer. In addition, it reports chiral separations obtained by using neutral cyclodextrins in a sulfonic acid-coated capillary column, which give a negatively charged capillary surface and electro-osmotic flow even in low pH buffers. The possible mechanism of separation is discussed.

Solution stability of the monoclonal antibody-vinca alkaloid conjugate, KS1/4-DAVLB

A 3-month solution stability study at 5°C of the monoclonal antibody–vinca alkaloid conjugate KS1/4-DAVLB indicated that phosphate-buffered saline solutions at pH 4.5–5.5 had little tendency to lose vinca by hydrolysis, improved vinca stability, showed acceptable physical stability, and formed minimal amounts of soluble aggregates compared to solutions at pH 6.5–7.4. Hydrolysis and aggregation with concomitant loss of stability were accelerated at 30°C throughout the pH range investigated. As determined by ELISA, the binding properties of KS1/4-DAVLB to tumor antigens were not affected by pH or temperature. A formulation suitable for initial clinical trials in cancer patients is described.

Chromatographic detection limits in pharmaceutical method development

Abstract A procedure for the determination of chromatographic method detection limits that incorporates the variabilities associated with sample preparation, measurement, and calculations is described. Theoretical considerations lead to an equation for the calculation of a method detection limit that evaluates the precision of the method near the detection limit and incorporates the number of sample and standard replicates used for a determination. Guidelines for the experimental determination of the method detection limit and for subsequent method procedure limitations were developed from the assumptions employed. This procedure is general and can be applied to chromatographic and non-chromatographic techniques. Several examples which demonstrate the effectiveness of this procedure for chromatographic methods are given. Method detection limits determined in this way should provide consistency for methods developed for pharmaceutical applications.