Current possibilities of liquid chromatography for the characterization of antibody-drug conjugates.

Antibody Drug Conjugates (ADCs) are innovative biopharmaceuticals gaining increasing attention over the last two decades. The concept of ADCs lead to new therapy approaches in numerous oncological indications as well in infectious diseases. Currently, around 60 CECs are in clinical trials indicating the expanding importance of this class of protein therapeutics. ADCs show unprecedented intrinsic heterogeneity and address new quality attributes which have to be assessed. Liquid chromatography is one of the most frequently used analytical method for the characterization of ADCs. This review summarizes recent results in the chromatographic characterization of ADCs and supposed to provide a general overview on the possibilities and limitations of current approaches for the evaluation of drug load distribution, determination of average drug to antibody ratio (DARav), and for the analysis of process/storage related impurities. Hydrophobic interaction chromatography (HIC), reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC) and multidimensional separations are discussed focusing on the analysis of marketed ADCs. Fundamentals and aspects of method development are illustrated with applications for each technique. Future perspectives in hydrophilic interaction chromatography (HILIC), HIC, SEC and ion exchange chromatography (IEX) are also discussed.

Development of RP UPLC-TOF/MS, stability indicating method for omeprazole and its related substances by applying two level factorial design; and identification and synthesis of non-pharmacopoeial impurities.

A new UPLC-TOF/MS compatible, reverse phase-stability indicating method was developed for determination of Omeprazole (OMP) and its related substances in pharmaceutical dosage forms by implementing Design of Experiment (DoE) i.e. two level full factorial Design (2(3)+3 center points=11 experiments) to understand the Critical Method Parameters (CMP) and its relation with Critical Method Attribute (CMA); to ensure robustness of the method. The separation of eleven specified impurities including conversion product of OMP related compound F (13) and G (14) i.e. Impurity-I (1), OMP related compound-I (11) and OMP 4-chloro analog (12) was achieved in a single method on Acquity BEH shield RP18 100 × 2.1 mm, 1.7 µm column, with inlet filter (0.2 µm) using gradient elution and detector wavelength at 305 nm and validated in accordance with ICH guidelines and found to be accurate, precise, reproducible, robust and specific. The drug was found to degrade extensively in heat, humidity and acidic conditions and forms unknown degradation products during stability studies. The same method was used for LC-MS analysis to identify m/z and fragmentation of maximum unknown impurities (Non-Pharmacopoeial) i.e. Impurity-I (1), Impurity-III (3), Impurity-V (5) and Impurity-VIII (9) formed during stability studies. Based on the results, degradation pathway for the drug has been proposed and synthesis of identified impurities i.e. impurities (Impurity-I (1), Impurity-III (3), Impurity-V (5) and Impurity-VIII (9)) are discussed in detail to ensure in-depth understanding of OMP and its related impurities and optimum performance during lifetime of the product.

Development and validation of a hydrophilic interaction chromatography method coupled with a charged aerosol detector for quantitative analysis of nonchromophoric α-hydroxyamines, organic impurities of metoprolol.

The European Pharmacopeia (EP) metoprolol impurities M and N are polar, nonchromophoric α-hydroxyamines, which are poorly retained in a conventional reversed-phase chromatographic system and are invisible for UV detection. Impurities M and N are currently analyzed by TLC methods in the EP as specified impurities and in the United States Pharmacopeia-National Formulary (USP-NF) as unspecified impurities. In order to modernize the USP monographs of metoprolol drug substances and related drug products, a hydrophilic interaction chromatography (HILIC) method coupled with a charged aerosol detector (CAD) was explored for the analysis of the two impurities. A comprehensive column screening that covers a variety of HILIC stationary phases (underivatized silica, amide, diol, amino, zwitterionic, polysuccinimide, cyclodextrin, and mixed-mode) and optimization of HPLC conditions led to the identification of a Halo Penta HILIC column (4.6 × 150 mm, 5 µm) and a mobile phase comprising 85% acetonitrile and 15% ammonium formate buffer (100 mM, pH 3.2). Efficient separations of metoprolol, succinic acid, and EP metoprolol impurities M and N were achieved within a short time frame (<8 min). The HILIC-CAD method was subsequently validated per USP validation guidelines with respect to specificity, robustness, linearity, accuracy, and precision, and could be incorporated into the current USP-NF monographs to replace the outdated TLC methods. Furthermore, the developed method was successfully applied to determine organic impurities in metoprolol drug substance (metoprolol succinate) and drug products (metoprolol tartrate injection and metoprolol succinate extended release tablets).

Current trends in separation of plasmid DNA vaccines: a review.

Plasmid DNA (pDNA)-based vaccines offer more rapid avenues for development and production if compared to those of conventional virus-based vaccines. They do not rely on time- or labour-intensive cell culture processes and allow greater flexibility in shipping and storage. Stimulating antibodies and cell-mediated components of the immune system are considered as some of the major advantages associated with the use of pDNA vaccines. This review summarizes the current trends in the purification of pDNA vaccines for practical and analytical applications. Special attention is paid to chromatographic techniques aimed at reducing the steps of final purification, post primary isolation and intermediate recovery, in order to reduce the number of steps necessary to reach a purified end product from the crude plasmid.

New trends in reversed-phase liquid chromatographic separations of therapeutic peptides and proteins: theory and applications.

In the pharmaceutical field, there is considerable interest in the use of peptides and proteins for therapeutic purposes. There are various ways to characterize such complex samples, but during the last few years, a significant number of technological developments have been brought to the field of RPLC and RPLC-MS. Thus, the present review focuses first on the basics of RPLC for peptides and proteins, including the inherent problems, some possible solutions and some directions for developing a new RPLC method that is dedicated to biomolecules. Then the latest advances in RPLC, such as wide-pore core-shell particles, fully porous sub-2 µm particles, organic monoliths, porous layer open tubular columns and elevated temperature, are described and critically discussed in terms of both kinetic efficiency and selectivity. Numerous applications with real samples are presented that confirm the relevance of these different strategies. Finally, one of the key advantages of RPLC for peptides and proteins over other historical approaches is its inherent compatibility with MS using both MALDI and ESI sources.

Development and validation of a RP-HPLC method for the quantification of sparfloxacin in pharmaceutical dosage forms

Un modo rápido y sensible de cromatografía líquida en fase reversa de alta resolución ha sido desarrollado para el análisis cuantitativo de parfloxacino en preparaciones farmacéuticas. El método fue validado de acuerdo a las normas de precisión, especificidad y linealidad aportadas por la FDA, ICH y USP. El método fue desarrollado utilizando la fase móvil compuesta por solución acuosa de ácido acético al 1% y acetonitrilo en la proporción 71:29% (v/v) bajo una velocidad de flujo de 0.7 ml/min sobre una columna de sílica c-8 a temperatura ambiente. La recuperación fue de más del 97% para cada muestra adicionada de parfloxacino lo que demuestra la precisión del protocolo. La precisión intradía e interdiario fue inferior al límite máximo permitido (RSD ≤ 2.0), de acuerdo a la FDA. El método mostró una respuesta linear con un coeficiente de correlación del 0.998. Por lo tanto, el método fue preciso, reproducible, sensible y rápido, por lo que puede ser utilizado para el análisis de formulaciones de sparfloxacino (AU)

Trends in nonpolar polymer-based monolithic columns for reversed-phase capillary electrochromatography.

This review article is concerned with describing the various strategies that have been introduced for the preparation of nonpolar polymer-based monolithic columns for RP-CEC. First, the various traditional ways of generating the EOF that involved the introduction of fixed charges on the surface of the monoliths are reviewed. This is followed by a description of the development of neutral monoliths as the most promising monoliths for the separation of a wide range of neutral and charged species at a relatively moderate to strong EOF in the absence of electrostatic attraction or repulsion.

Development and validation of a stability-indicating RP-HPLC method for assay of betamethasone and estimation of its related compounds.

Betamethasone (9alpha-fluoro-16beta-methylprednisolone) is one of the members of the corticosteriod family of active pharmaceutical ingredient (API), which is widely used as an anti-inflammatory agent and also as a starting material to manufacture various esters of betamethasone. A stability-indicating reverse-phase high performance liquid chromatography (RP-HPLC) method has been developed and validated which can separate and accurately quantitate low levels of 26 betamethasone related compounds. The stability-indicating capability of the method was demonstrated through adequate separation of all potential betamethasone related compounds from betamethasone and also from each other that are present in aged and stress degraded betamethasone stability samples. Chromatographic separation of betamethasone and its related compounds was achieved by using a gradient elution at a flow rate of 1.0mL/min on a ACE 3 C18 column (150mmx4.6mm, 3microm particle size, 100A pore size) at 40 degrees C. Mobile phase A of the gradient was 0.1% methanesulfonic acid in aqueous solution and mobile phase B was a mixture of tert-butanol and 1,4-dioxane (7:93, v/v). UV detection at 254nm was employed to monitor the analytes. For betamethasone 21-aldehyde, the QL and DL were 0.02% and 0.01% respectively. For betamethasone and the rest of the betamethasone related compounds, the QL and DL were 0.05% and 0.02%. The precision of betamethasone assay is 0.6% and the accuracy of betamethasone assay ranged from 98.1% to 99.9%.