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 new efficient method for isolation of phenolics from sea algae prior to their rapid resolution liquid chromatographic-tandem mass spectrometric determination.

The extraction of phenolic compounds from 4 different sea algae samples, three brown algae (Cystoseira abies-marina, C. abies-marina grinded under cryogenic conditions with liquid nitrogen, Undaria pinnatifida and Sargassum muticum) and one red algae (Chondrus crispus) via solid phase extraction using micro-elution solid-phase extraction (µ-SPE) plate method was studied. Prior to µ-SPE, 50mg of algae with 80% methanol mixture was extracted in hyphenated series by various extraction techniques, such as pressurized liquid extraction and Ika Ultra-Turrax® Tube Drive, in combination with ultrasound assisted extraction. The µ-SPE plate technique reduced the time of sample pre-treatment thanks to higher sensitivity and pre-concentration effect. Selected groups of benzoic acid derivatives (p-hydroxybenzoic, protocatechuic, gallic, vanillic, and syringic acids), hydroxybenzaldehydes (4-hydroxybenzaldehyde, and 3,4-dihydroxybenzaldehyde), and cinnamic acid derivatives (p-coumaric, caffeic, ferulic, sinapic, and chlorogenic acids) were determined using rapid resolution liquid chromatography coupled to mass spectrometry detection with negative ion electrospray ionization (RRLC-ESI-MS) using multiple reactions monitoring. LOQs of measured samples varied in the range 0.23-1.68ng/mL and LODs in the range 0.07-0.52ng/mL. The applied method allowed a simultaneous determination of phenolics (i.e. free, esters soluble in methanol, glycosides, and esters insoluble in methanol) in less than 5min (including alkaline or acidic hydrolysis of raw extracts) from sea algae extracts.

New and Evolving Techniques for the Characterization of Peptide Therapeutics.

Advances in technologies related to the design and manufacture of therapeutic peptides have enabled researchers to overcome the biological and technological challenges that have limited their application in the past. As a result, peptides of increasing complexity have become progressively important against a variety of disease targets. Developing peptide drug products brings with it unique scientific challenges consistent with the unique physicochemical properties of peptide molecules. The identification of the proper characterization tools is required in order to develop peptide formulations with the appropriate stability, manufacturability, and bioperformance characteristics. This knowledge supports the build of critical quality attributes and, ultimately, regulatory specifications. The purpose of this review article is to provide an overview of the techniques that are employed for analytical characterization of peptide drug products. The techniques covered are highlighted in the context of peptide drug product understanding and include chemical and biophysical approaches. Emphasis is placed on summarizing the recent literature experience in the field. Finally, the authors provide regulatory perspective on these characterization approaches and discuss some potential areas for further research in the field.

Recent developments in the chromatographic bioanalysis of approved kinase inhibitor drugs in oncology.

In recent years (2010-present) there has been an increase in the number of publications reporting the development, validation and use of bioanalytical methods in the rapidly expanding drug class of small molecule protein kinase inhibitors. Most reports describe the technological set-up of the methods that have allowed for drug concentration measurements from various sample types. This includes plasma, dried blood-spot, and tissue-analysis. Also method development, exploration of various techniques, as well as measurement and identification of metabolites were addressed. For the bioanalysis, a variety of sample-pretreatment methods like protein-precipitation, liquid-liquid extraction, and solid-phase extraction have been employed, all varying in complexity, cleanliness and time-consumption. Chromatographic separation, nowadays, is more focused on separating components from ion-suppressive effects, since for MS/MS detection, various components do not have to be baseline separated. For detection multiple types of detectors were used, ranging from state-of-the-art high resolution, and tandem mass spectrometry with low picogram per milliliter detection limits to the classical UV-detector with several nanograms per milliliter limits. As new bioanalytical methods have arisen that do rely on chromatographic separation, for example for high-throughput analysis, these are addressed in this review as well.

The Metabolism of Anticancer Drugs by the Liver: Current Approaches to the Drug Development Process.

The worldwide scientific community is in agreement that the activities of metabolic enzymes greatly impact the efficacies of anticancer drugs. Elucidation of the influences of these drugs on metabolism, especially that occurring in the liver, appears to be an extremely important step in the development of new anticancer drugs. Considering the continuous need to search for safe and effective chemotherapeutics, studies of the metabolism of new potent drugs are very important and should be included in the modern, innovative drug development pipeline. This article summarizes most of the current metabolic case studies involving anticancer drug development. Firstly, the impacts of diverse metabolic enzymes, particularly cytochrome P450, and the utilities of a few model in vitro enzymatic systems are described. Then, different analytical techniques, with particular emphasis on liquid chromatography- mass spectrometry detection and structural elucidation, are discussed. Finally, some computer-aided strategies for decision making in the drug design process are described. Recent advances in drug development, including microdosing, in vitro-in vivo correlation and pharmacologic audit trail, are also discussed in relation to metabolic studies.

Clinical applications of LC-MS sex steroid assays: evolution of methodologies in the 21st century.

PURPOSE OF REVIEW: The purpose of this review is to summarize why and how liquid chromatography tandem mass spectrometry (LC-MS/MS) is increasingly replacing other methodologies for the measurement of sex steroids. RECENT FINDINGS: Measurement of sex steroids, particularly testosterone and estradiol, is important for diagnosis or management of a host of conditions (e.g. disorders of puberty, hypogonadism, polycystic ovary syndrome, amenorrhea, and tumors of ovary, testes, breast and prostate). Historically, metabolites of testosterone and estradiol were measured as ketosteroids in urine using colorimetric assays that lacked sensitivity and specificity due to endogenous and exogenous interferences. Extracted competitive manual radio-immunoassays provided improved, but still imperfect, specificity, and offered increased sensitivity. As testing demand increased, they were displaced by automated immunoassays. These offered better throughput and precision, but suffered worse specificity problems. Moreover, agreement between different immunoassays has often been poor and they are all compromised by a limited dynamic measurement range. To overcome these problems, LC-MS/MS methods have been developed and validated for quantitation of sex steroids. These methods reduce interferences, provide better specificity, improve dynamic range, and reduce between-method bias. SUMMARY: Endocrine Society and Urology Society guidelines have highlighted the limitations of the immunoassays for sex steroids and have provided convincing evidence that mass spectrometric methods are preferable for measurement of sex steroid hormones. In this review, we describe LC-MS/MS methods for measurement of testosterone and estradiol.

Development and validation of a sensitive LC-MS/MS assay for the simultaneous quantification of allitinib and its two metabolites in human plasma.

Allitinib, also known as AST1306, is a novel irreversible inhibitor of the epidermal growth factor receptors 1 and 2. Allitinib is currently used in clinical trial to treat solid tumors. A previous study showed that allitinib is extensively metabolized in humans. Amide hydrolysis metabolite (M6) and 29,30-dihydrodiol allitinib (M10) are the major metabolites in circulation. To study the pharmacokinetics of allitinib and its two major metabolites in cancer patients, a rapid, sensitive and reliable LC-MS/MS method was developed and validated for the simultaneous determination of allitinib, M6 and M10 in human plasma. After simple protein precipitation, the analytes and the combined internal standards (lapatinib and NB-2, an analog of allitinib) were separated on a Zorbax Eclipase XDB C18 column (50 mm × 4.6 mm, 1.8 µm, Agilent) using a mobile phase of 5 mM ammonium acetate with 0.1% formic acid (phase A) and 50% (v/v) methanol in acetonitrile (phase B) with gradient elution. Mass spectrometric detection was conducted by atmospheric-pressure chemical ionization in positive ion multiple reaction monitoring modes using AB Sciex Triple Quad 6500 system. Linear calibration curves were obtained for the following concentration range: 0.300-200 ng/ml for allitinib; 0.030-20.0 ng/ml for M6; and 0.075-50.0 ng/ml for M10. Intra-day and inter-day accuracy and precision were within the acceptable limits of ±15% at all of the concentrations. The method was successfully applied to a preliminary clinical pharmacokinetic study following oral administration of allitinib tosylate tablets in cancer patients.

Recent advances in the direct and indirect liquid chromatographic enantioseparation of amino acids and related compounds: a review.

Amino acids are essential for life, and have many functions in metabolism. One particularly important function is to serve as the building blocks of peptides and proteins, giving rise to complex three dimensional structures through disulfide bonds or crosslinked amino acids. Peptides are frequently cyclic and contain protein as well as non-protein aminoacids in many instances. Since most of the proteinogenic α-amino acids contain a chiral carbon atom (with the exception of glycine), the stereoisomers of all these amino acids and the peptides in which they are to be found may possess differences in biological activity in living systems. The impetus for advances in chiral separation has been highest in the past decade and this still continues to be an area of high focus. The important analytical task of the separation of isomers is achieved mainly by chromatographic methods. This review surveys indirect and direct HPLC separations of biologically and pharmaceutically important enantiomers of amino acids and related compounds, with emphasis on the literature published from 2005.

The requirement for proteomics to unravel stem cell regulatory mechanisms.

Stem cells are defined by their ability to self-renew and to differentiate, the processes whereby these events are achieved is the subject of much investigation. These studies include cancer stem cell populations, where eradication of this specific population is the ultimate goal of treatment. Whilst cellular signalling events and transcription factor complex-mediated changes in gene expression have been analysed in some detail within stem cells, full systematic understanding of the events promoting self-renewal or the commitment process leading to formation of a specific cell type require a systems biology approach. This in turn demands a need for proteomic analysis of post-translational regulation of protein levels, protein interactions, protein post-translational modification (e.g. ubiquitination, methylation, acetylation, phosphorylation) to identify networks for stem cell regulation. Furthermore, the phenomenon of induced pluripotency via cellular reprogramming also can be understood optimally using combined molecular biology and proteomics approaches; here we describe current research employing proteomics and mass spectrometry to dissect stem cell regulatory mechanisms.

Polycyclic aromatic hydrocarbons in food and beverages. Analytical methods and trends.

Polycyclic aromatic hydrocarbons (PAHs) are compounds widespread in the environment, many of them showing carcinogenic effects. These compounds can reach the food chain by different ways and, therefore, the analysis of PAHs in food is a matter of concern. This article reviews the extraction methodologies together with the separation and detection techniques which are currently applied in the determination of PAHs in food and beverages. Specific extraction conditions, performance characteristics, chromatographic and detection parameters are discussed. A review of the occurrence of these compounds in the matrixes under study is also provided.

Statistical learning of peptide retention behavior in chromatographic separations: a new kernel-based approach for computational proteomics.

BACKGROUND: High-throughput peptide and protein identification technologies have benefited tremendously from strategies based on tandem mass spectrometry (MS/MS) in combination with database searching algorithms. A major problem with existing methods lies within the significant number of false positive and false negative annotations. So far, standard algorithms for protein identification do not use the information gained from separation processes usually involved in peptide analysis, such as retention time information, which are readily available from chromatographic separation of the sample. Identification can thus be improved by comparing measured retention times to predicted retention times. Current prediction models are derived from a set of measured test analytes but they usually require large amounts of training data. RESULTS: We introduce a new kernel function which can be applied in combination with support vector machines to a wide range of computational proteomics problems. We show the performance of this new approach by applying it to the prediction of peptide adsorption/elution behavior in strong anion-exchange solid-phase extraction (SAX-SPE) and ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC). Furthermore, the predicted retention times are used to improve spectrum identifications by a p-value-based filtering approach. The approach was tested on a number of different datasets and shows excellent performance while requiring only very small training sets (about 40 peptides instead of thousands). Using the retention time predictor in our retention time filter improves the fraction of correctly identified peptide mass spectra significantly. CONCLUSION: The proposed kernel function is well-suited for the prediction of chromatographic separation in computational proteomics and requires only a limited amount of training data. The performance of this new method is demonstrated by applying it to peptide retention time prediction in IP-RP-HPLC and prediction of peptide sample fractionation in SAX-SPE. Finally, we incorporate the predicted chromatographic behavior in a p-value based filter to improve peptide identifications based on liquid chromatography-tandem mass spectrometry.