Separation of isobaric phosphorothioate oligonucleotides in capillary electrophoresis: study of the influence of cationic cyclodextrins on chemo and stereoselectivity

Phosphorothioate (PS) oligonucleotides have drawn more and more attention lately due to their great therapeutic potential. The presence of one (or several) phosphorothioate moiety (ies) improves pharmacokinetic properties but at the same time creates an additional chiral center for each phosphorothioate linkage, and thus diastereomers. It is therefore important to develop analytical strategies to monitor individual species to enable more in-depth investigations. In this study, a PVA coated capillary with a background electrolyte made of 100 mM phosphoric acid adjusted to pH 3.0 with triethanolamine was used. A design of experimental approach provides the optimal conditions for the separation of the eight isobaric diastereomers bearing one phosphorothioate linkage (Mix 1), and the separation of the 12 isobaric diastereomers of a mixture made of oligonucleotides with two phosphorothioate linkages (Mix 2). Remarkably, full separation in Mix 1 could be achieved using a combination of a cationic cyclodextrin (2-hydroxy-3-N,N,N-trimethylamino) propyl-γ-CD chloride, and an anionic cyclodextrin (carboxymethyl-β-cyclodextrin sodium salt), while a second cationic cyclodextrin (2-hydroxy-3-N,N,N-trimethylamino) propyl-β-CD chloride) was required for Mix 2, providing additional selectivity

Comparison of Three Complementary Analytical Techniques for the Evaluation of the Biosimilar Comparability of a Monoclonal Antibody and an Fc-Fusion Protein.

peer reviewedIn this work, a monoclonal antibody, adalimumab, and an Fc-fusion protein, etanercept, were studied and compared to one of their biosimilars. Samples submitted to stress conditions (agitation and high temperature) were used for method development. The developed methods were also applied to samples reduced by beta-mercaptoethanol to evaluate their capability to distinguish the expected species. Capillary gel electrophoresis (CGE), reversed-phase liquid chromatography (RPLC), and size-exclusion chromatography (SEC) methods coupled with UV detection were used to analyze the biopharmaceuticals. Their complementarity was investigated. For further molecular weight determination, SEC-multi angle light scattering and RPLC-quadrupole time-of-flight were occasionally used. For adalimumab, a larger amount of fragments and aggregates was observed in the biosimilar compared with the reference product. For etanercept, more related species were found in the reference product. Those three separation techniques showed good complementarity. Indeed, RPLC enabled the separation of hydrophilic and hydrophobic degradation products. CGE provided good selectivity for several adalimumab fragments, and SEC was useful for the analysis of aggregates and certain fragments that cannot be separated by the other approaches. Moreover, those formulations were submitted to mild stress conditions (30°C, 300 rpm for 4 h) that mimic shipping conditions. No additional peak was found under these conditions for the two studied biopharmaceuticals

Validation of an LC-MS/MS Method Using Solid-Phase Extraction for the Quantification of 1-84 Parathyroid Hormone: Toward a Candidate Reference Measurement Procedure.

peer reviewed[en] BACKGROUND: Parathyroid hormone (PTH) measurement is important for patients with disorders of calcium metabolism, including those needing bone-turnover monitoring due to chronic kidney disease-mineral bone disorder. There are currently 2 generations of PTH immunoassays on the market, both having cross-reactivity issues and lacking standardization. Therefore, we developed an LC-MS/MS higher-order method for PTH analysis. METHODS: The method was calibrated against the international standard for 1-84 PTH (WHO 95/646). Antibody-free sample preparation with the addition of an isotope-labeled internal standard was performed by solid-phase extraction. Extracts were analyzed by LC-MS/MS. EDTA-K2 plasma was used throughout the development and validation. Bias and uncertainty sources were tested according to ISO 15193. Clinical Laboratory Standards Institute guidelines and reference measurement procedures were consulted for the design of the validation. Patient samples and external quality controls were compared between LC-MS/MS and 2 third-generation immunoassays. RESULTS: The method was validated for 1-84 PTH from 5.7 to 872.6 pg/mL. The interassay imprecision was between 1.2% and 3.9%, and the accuracy ranged from 96.2% to 103.2%. The measurement uncertainty was <5.6%. The comparison between LC-MS/MS and the immunoassays showed a proportional bias but moderate to substantial correlation between methods. CONCLUSIONS: This LC-MS/MS method, which is independent of antibodies, is suitable for a wide range of PTH concentrations. The obtained analytical performance specifications demonstrate that development of a reference measurement procedure will be possible once a higher order reference standard is available

Chemotherapy of Ovarian Cancer: State of the Art

peer reviewedEpithelial ovarian cancer is frequently diagnosed at advanced-stage disease and chemotherapy is nearly always required. Optimally debulked patients may need adjuvant chemotherapy while, most of the time this chemotherapy will be given to those with advanced-stage disease. Also relapses will be treated differently whether they occur early or late in the course of the disease. This paper reviews medical treatment modalities according to stage based on published data. Maintenance and consolidation treatments are also discussed. Finally a brief insight into new therapeutic tools is also given

Enantioseparations in nonaqueous capillary electrophoresis using charged cyclodextrins.

The enantioseparation of acidic and basic compounds can be successfully achieved in nonaqueous capillary electrophoresis using single-isomer charged beta-cyclodextrin (beta-CD) derivatives of opposite charge to that of the analytes. This chapter describes how to separate the enantiomers of three basic substances selected as model compounds, i.e., alprenolol, bupranolol, and terbutaline, using the negatively charged heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-CD. The enantiomers of three acidic drugs (tiaprofenic acid, suprofen, and flurbiprofen) are resolved using a monosubstituted amino beta-CD derivative, namely, 6-monodeoxy-6-mono(3-hydroxy)propylamino-beta-CD

Application of dual cyclodextrin systems in capillary electrophoresis enantioseparations.

The enantioseparation of acidic and neutral compounds can be successfully achieved in capillary electrophoresis (CE) using dual cyclodextrin (CD) systems. This chapter describes how to separate the enantiomers of acidic or neutral substances using dual CD systems made up of a negatively charged CD derivative, i.e., sulfobutyl-beta-CD (SB-beta-CD) or carboxymethyl-beta-CD (CM-beta-CD), in combination with a neutral one, namely, heptakis(2,3,6-tri-O-methyl)-beta-CD (TM-beta-CD). An acidic compound (carprofen) and a weakly acidic drug (pentobarbital) were selected as model compounds

Stability of 90 mg/ml cefuroxime sodium solution for administration by continuous infusion

peer reviewedContinuous infusions of beta-lactam antibiotics increase pharmacokinetic/pharmacodynamic target attainment. However, this way of administration brings about some practical issues such as stability. This study aims to determine the stability of a 90 mg/mL cefuroxime sodium solution. Cefuroxime sodium was reconstituted and mixed with 50-mL 0.9% saline to produce 90 mg/mL solution in polypropylene syringes which were stored at 4 degrees C, 25 degrees C and 40 degrees C. Cefuroxime sodium concentration was determined periodically over 14 days using a stability-indicating high-performance liquid chromatographic method with ultra-violet detection. The loss in concentration was less than 10% after 2 days of storage at 25 degrees C and less than 5% after 14 days of storage at 4 degrees C. The concentration fell below 60% after 1 day at 40 degrees C. Solutions darken in appearance with time and heat. A 90 mg/mL cefuroxime sodium solution stored in polypropylene syringes is stable for 2 days at 25 degrees C and for at least 14 days at 4 degrees C

Effects of background electrolyte composition and addition of selectors on separation selectivity in nonaqueous capillary electrophoresis

This review gives a survey of the approaches employed to obtain, enhance and tune selectivity in nonaqueous capillary electrophoresis (NACE). Recent developments in NACE are described and the effects of background electrolyte composition and addition of selectors on separation selectivity are discussed. The use of one organic solvent, a mixture of several organic solvents or the use of additives to tune separation selectivity in NACE is presented and a list of relevant applications is included