Ionic liquids (ILs), as non-molecular type solvents, possess excellent physical-chemical properties, which make them useful in important separation applications in gas chromatography, liquid chromatography, and capillary electrophoresis. Among a plethora of potential uses of ionic liquids in separation science, capillary electrophoresis can utilize its resolution-enhancing effect in the analysis of proteins and carbohydrates, via the formation of intermolecular interactions, e.g., hydrophobic, hydrogen bonding, or electrostatic. ILs and polymeric ionic liquids (PIL) also represent an excellent choice as background electrolyte (BGE) additives for capillary coatings in CE, which is especially important in protein analysis. Another interesting utilization of ILs is the fabrication of monoliths for capillary electrochromatography in which instance the mechanism of retention is based on ion exclusion interactions. Carbohydrates can also be readily analyzed by CE with the help of ionic liquids without the need for an extra derivatization step. One of the future perspectives on the use of ILs is their utilization in the recently emerging biopharmaceutical industry exploiting the increased resolution of proteins and carbohydrates, two of the important components of glycoprotein therapeutics. In this paper, we address the so-far not-reviewed ionic liquid-mediated analysis of proteins and carbohydrates by capillary electrophoresis-based techniques also addressing their impact on the separation mechanism.

This review summarizes the fundamental principles, basic methodologies, strength and weaknesses of capillary gel electrophoresis of proteins by providing both a short historical overview and highlighting new developments and applications in biopharmaceutical, biomedical as well as food and agriculture fields. The subsets of the method including native capillary gel electrophoresis, SDS capillary gel electrophoresis, capillary gel isoelectric focusing, capillary gel isotachophoresis and capillary affinity gel electrophoresis of proteins are all critically reviewed. Relevant protein labeling techniques are also addressed.

Capillary electrophoresis (CE) handles complex samples with excellent resolution, but has poor sensitivity because of the small detection volume and suffers from destacking of high-conductivity samples; therefore, sample preparation is often required for matrix cleanup and analyte enrichment. This review discusses operationally-simple sample preparation schemes which are easily adoptable by researchers using commercial CE instruments, wherein various solid-phase microextraction (SPME) and liquid-phase microextraction (LPME) techniques are ‘in-line’ coupled (i.e., integrated) to the separation capillary without modifying the commercial CE instrument with sophisticated interfaces. In-line coupling refers to scenarios where the prepared sample is injected directly into the separation capillary. Then, sample loss is reduced and the enrichment factors can be significantly increased as the acceptor volume can be reduced to the nanoliter level. Cases involving subsequent on-line preconcentration are also discussed. In-line SPME-CE and LPME-CE are powerful tools for analyzing various samples, especially trace substances in complex biological matrices.

Capillary electrophoresis-related microseparation techniques include ultrathin-layer gel electrophoresis and microchip gel electrophoresis. Since this latter can be a subject of another book, in this chapter we only discuss the former one. Analysis of DNA molecules was accomplished by ultrathin-layer gel electrophoresis in early sequencing applications and fragment analysis. Ultrathin gels were also utilized in multilane separation of sodium dodecyl sulfate–protein complexes, but showed their real potential in two-dimensional gel electrophoresis.

Micropollutants are persistent and hazardous materials in low concentrations (ng L −1 –μg L −1 ), including substances such as pharmaceuticals, personal care products and industrial chemicals. The advancement of analytical chemistry has allowed for the detection of micropollutants; however, an efficient and economical treatment solution is yet to be installed. Fungal laccase has been a successful biocatalyst of these compounds. However, large-scale application of free enzyme is currently not feasible for removing water-borne micropollutants, partly due to relatively rapid loss in enzyme stability. In this paper, three types of cyclodextrin, α, β and γCD, were chosen to immobilise the laccase under various conditions with the aim to improve the stability of the enzyme. Laccase activity was chosen as a response parameter, and laccase-cyclodextrin binding was evaluated by Fourier-transform infrared spectroscopy (FTIR). Results showed an optimum using α-cyclodextrin immobilisation. At that level, α-cyclodextrin increased the half-life of laccase and slightly improved its activity in all tested pH by physically bonding to laccase. By protecting the enzyme structure, activity was maintained under a range of circumstances (acidic conditions, from 10 to 50 °C). Under room temperature and at pH 5, α-cyclodextrin-laccase nanocomposite had a better removal efficiency of diclofenac compared to free laccase of the same concentration. Graphical abstract

In order to explain the different catalytic activity in hydrogenation of two new intermetallic Ni/Ga clusters, [Ga-7](NiCp*)(6) (1A) and [NiGa6](NiCp*)(6) (1B) (Cp* = C5Me5), investigations of structure-function relationship have been performed based on Raman and infrared (IR) spectroscopy and theoretical (density functional theory [DFT] and normal coordinate) calculations. Full interpretation of the Raman, far-IR, and mid-IR spectra of these dark colored solids has been proposed. Based on the overview of metal-Cp* complexes, all the 14 characteristic Cp*(-) skeletal fundamental modes have been identified. By comparison of the Ni-Cp* stretching and tilting external modes (350-380 cm(-1)), their force constants, and bond lengths, cluster 1B exhibited slightly stronger metal-ligand bonding. Vibrations of Ga-7 and NiGa6 cluster cores showed that the stretching wavenumbers and force constants of Ni-Ga (100-350 cm(-1)) and Ga-Ga (60-250 cm(-1)) bonds are higher for cluster 1B, in agreement with the shorter averaged experimental and calculated bond lengths of Ga-Ga bonds (2.873 and 2823 angstrom for clusters 1A and 1B, respectively). Cluster hydrogenation experiments with H-2 and D-2 showed strong Ni-H and N-D stretching features (at 1750 and 1260 cm(-1), respectively), and at the same time, characteristic bands of self-hydrogenated Cp*H and remained nondegraded clusters have been detected. The extent of H-D exchange in cluster deuteration was obtained about 1.5 times more effective with cluster 1B than cluster 1A. The stronger hydrogen or deuterium uptake by cluster 1B and the more intensive self-hydrogenation of Cp* clearly support the higher hydrogenation activity of cluster 1B compared with that of 1A.

The market segment of new biological drugs (monoclonal antibodies, fusion proteins, antibody-drug conjugates, and new modality protein therapeutics) is rapidly growing, especially after the patent expiration of the original biologics, initiating the emergence of biosimilars. N-glycosylation of therapeutic proteins has high importance on their stability, safety, immunogenicity, efficacy, and serum half-life. Therefore, Nglycosylation is considered to be one of the critical quality attributes. Consequently, it should be rigorously monitored during the development, manufacturing, and release of glycoprotein biologicals. In this review, first, the regulatory considerations for biosimilars are shortly summarized, followed by conferring the analytical techniques needed for monitoring and characterization of the N-glycosylation of biological drugs. Particular respect is paid to liquid phase separation techniques with high sensitivity and highresolution detection methods, including laser-induced fluorescence and mass spectrometry.

Capillary electrochromatography (CEC) is a powerful hybrid separation technique that combines capillary electrophoresis and capillary chromatography, capable to address the analytical challenges of proteomics and glycomics. The focus of this paper is to review the recent developments in capillary electrochromatography of proteins and carbohydrates. The different column types applied in capillary electrochromatography such as packed bed, open tubular and monoliths are conferred in detail with respective separation examples. A comprehensive comparison is also given listing the mostly utilized coating methods, stationary phase materials and column preparation methods. The choice of porogenic solvent combinations for monolithic column fabrication is thoroughly discussed, paying close attention to the fine tuning options for the separation driving electroosmotic flow. Application examples of CEC in process analytical technology for the biopharmaceutical and biomarker discovery in the biomedical fields are also given.

Abstract Capillary electrochromatography (CEC) is a powerful hybrid separation technique that combines capillary electrophoresis and capillary chromatography, capable to address the analytical challenges of proteomics and glycomics. The focus of this paper is to review the recent developments in capillary electrochromatography of proteins and carbohydrates. The different column types applied in capillary electrochromatography such as packed bed, open tubular and monoliths are conferred in detail with respective separation examples. A comprehensive comparison is also given listing the mostly utilized coating methods, stationary phase materials and column preparation methods. The choice of porogenic solvent combinations for monolithic column fabrication is thoroughly discussed, paying close attention to the fine tuning options for the separation driving electroosmotic flow. Application examples of CEC in process analytical technology for the biopharmaceutical and biomarker discovery in the biomedical fields are also given.

Lung adenocarcinoma is one of the leading causes of mortality among cancer patients worldwide and Chronic Obstructive Pulmonary Disease (COPD) is also high in death statistics. In addition, patients with Chronic Obstructive Pulmonary Disease (COPD) have a high risk of developing primary lung cancer. Prevention, risk estimation and a non-invasive diagnostics are essential to decrease COPD and lung cancer mortality. Therefore, better and more accurate molecular diagnostic markers (biomarkers) are needed for the early differential diagnosis of these lung diseases to help clinicians make better therapeutic decisions. This review focuses on recently discovered adenocarcinoma and COPD biomarkers at the proteome and glycome level. In the first part, the protein markers are summarized, while the second part is focused on glycan markers. Their use to differentiate between chronic inflammation (COPD) and malignant (adenocarcinoma) diseases is discussed in detail.

Absztrakt: A krónikus obstruktív tüdőbetegség (COPD) világszerte előkelő helyet foglal el a morbiditási és mortalitási statisztikákban. A COPD megelőzhető és kezelhető betegség, kialakulásáért döntően a dohányzás tehető felelőssé. A prevenció kulcsfontosságú, de korlátozottan kivitelezhető, így a rizikó meghatározása és a korai noninvazív diagnosztika révén lehetne tovább csökkenteni a COPD miatti halálozást. A fejlődő diagnosztikus technikák ellenére az optimális szűrővizsgálat felfedezése még várat magára. Kellően szenzitív és specifikus biomarkerek felfedezése megfelelő eszközt adhat a klinikus kezébe a betegség korai diagnosztizálásához, a differenciáldiagnosztikához, a fenotipizáláshoz és a prognózis becsléséhez. Közleményünkben a COPD vonatkozásában a közelmúltban felfedezett potenciális fehérje és glikán biomarkereket foglaljuk össze. Orv Hetil. 2020; 161(4): 123–128.

Chronic obstructive pulmonary disease (COPD) is worldwide a significant representative of morbidity and mortality statistics. COPD is a preventable and treatable disease and smoking is the main risk factor of disease development. Prevention is crucial, but it has its limitations, so risk estimation and early non-invasive diagnostics are essential to decrease COPD mortality. Although diagnostic techniques are evolving, the perfect screening tool is lacking. Discovery of properly sensitive and specific biomarkers is important. They could be effective diagnostic, differential diagnostic, phenotyping and prognostic tools to clinicians. The manuscript is focusing on recently discovered potential protein and glycan biomarkers for COPD. Orv Hetil. 2020; 161(4): 123-128.

Adeno-associated virus (AAV) is one of the most promising viral gene delivery vectors with long-term gene expression and disease correction, featuring high efficiency and excellent safety in human clinical trials. During the production of AAV vectors, there are several quality control (QC) parameters that should be rigorously monitored to comply with clinical safety and efficacy. This review gives a short summary of the most frequently used AVV production and purification methods, focusing on the analytical techniques applied to determine the full/empty capsid ratio and the integrity of the encapsidated therapeutic DNA of the products.

Vibrational spectroscopic study of crystalline copper hexacyanoferrate complexes of composition K4Cu6II [Fe-II(CN)(6)](4)nH(2)O (1) and Cu-6(II)[Fe-III(CN)(6)](4)nH(2)O (2) with -Cu-N equivalent to C-Fe- bridging structures have been performed. The cubic Fmm (O-h(5)) unit-cells contain ideally 4 Fe and 4 Cu ions which were calculated by periodic density functional theory (DFT) (using the Gaussian09 C.01 software package) for ideal lattice compositions of K8Cu4II[Fe-II(CN)(6)](4) (1a), K4Cu4II[Fe-III(CN)(6)](4) (2a) and with lattice water molecules KCu4II[Fe-III(CN)(6)](3)6H(2)O (3a). Systematically, non-linear Cu-N equivalent to C structure was fitted with Cu-N equivalent to C bond angles about 155 degrees for complexes 1a, 2a, and 3a. Practically, all optically active internal modes of Fe(CN6)(n-) moieties resulted from factor group analysis as 4A(1g) + 6E(g) + 4F(1g) + 10F(1u) were experimentally observed and assigned. Some low-frequency translatory and librational modes were also interpreted. Vibrational bands were assigned to cis- and trans-Cu(NC)(4)(OH2) complexes which are formed in the lattice holes of both complexes. Vibrational spectra and force constants of a great number of transition metal hexacyano complexes of compositions K-4[M-II(CN)(6)], K-3[M-III(CN)(6)], CsLi2[M-III(CN)(6)] and Prussian blue analogues have been reexamined and recalculated. Internal and external modes of 6 different lattice water species (coordinated, hydrogen bonded, or zeolitic type) have been interpreted for complex 2 using results of periodic DFT calculation of model complex 3a.

The recent expiration of several protein therapeutics opened the door for biosimilar development. Biosimilars are biologic medical products that are similar but not identical copies of already-authorized protein therapeutics. Critical quality attributes (CQA), such as post-translational modifications of recombinant biotherapeutics, are important for the clinical efficacy and safety of both the innovative biologics and their biosimilar counterparts. Here, we summarize biosimilarity CQAs, considering the regulatory guidelines and the statistical aspects (e.g., biosimilarity index) and then discuss glycosylation as one of the important attributes of biosimilarity. Finally, we introduced the 'Glycosimilarity Index', which is based on the averaged biosimilarity criterion.

The carbohydrate moieties on the polypeptide chains in most glycoprotein based biotherapeutics and their biosimilars play essential roles in such major mechanisms of actions as antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, anti-inflammatory functions and serum clearance. In addition, alteration in glycosylation may influence the safety and efficacy of the product. Glycosylation, therefore, is considered as one of the important critical quality attributes of glycoprotein biotherapeutics, and consequently for their biosimilar counterparts. Thus, the carbohydrate moieties of such biopharmaceuticals (both innovator and biosimilar products) should be closely scrutinized during all stages of the manufacturing process. In this paper we introduce a rapid, capillary gel electrophoresis based process to quantitatively assess the glycosylation aspect of biosimilarity (referred to as glycosimilarity) between the innovator and a biosimilar version of etanercept (Enbrel® and Benepali®, respectively), based on their N-linked carbohydrate profiles. Differences in sialylated, core fucosylated, galactosylated and high mannose glycans were all quantified. Since the mechanism of action of etanercept is TNFα binding, only mannosylation was deemed as critical quality attribute for glycosimilarity assessment due to its influence on serum half-life.