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Haptoglobin (Hp) is a positive acute phase protein, synthesized in the liver, with four N-glycosylation sites carrying mainly complex type N-glycans. Its glycosylation is altered in different types of diseases but still has not been extensively studied mainly due to analytical challenges, especially the lack of a fast, efficient, and robust high-throughput Hp isolation procedure. Here, we describe the development of a high-throughput method for Hp enrichment from human plasma, based on monolithic chromatographic support in immunoaffinity mode and downstream Hp N-glycome analysis by hydrophilic interaction ultrahigh-performance liquid chromatography with fluorescent detection (HILIC-UHPLC-FLR). Chromatographic monolithic supports in a 96-well format enable fast, efficient, and robust Hp enrichment directly from diluted plasma samples. The N-glycome analysis demonstrated that a degree of Hp deglycosylation differs depending on the conditions used for N-glycan release and on the specific glycosylation site, with Asn 241 being the most resistant to deglycosylation under tested conditions. HILIC-UHPLC-FLR analysis enables robust quantification of 28 individual chromatographic peaks, in which N-glycan compositions were determined by UHPLC coupled to electrospray ionization quadrupole time of flight mass spectrometry. The developed analytical approach enables fast evaluation of total Hp N-glycosylation and is applicable in large-scale studies., (© 2022 Wiley Periodicals LLC.)

Erythrocytosis is a blood disorder characterised by an increased red blood cell mass. The most common causes of erythrocytosis are acquired and caused by diseases and conditions that are accompanied by hypoxaemia or overproduction of erythropoietin. More rarely, erythrocytosis has a known genetic background, such as for polycythaemia vera and familial erythrocytosis. The majority of cases of polycythaemia vera are associated with acquired variants in JAK2, while familial erythrocytosis is a group of congenital disorders. Familial erythrocytosis type 1 is associated with hypersensitivity to erythropoietin (variants in EPOR), types 2-5 with defects in oxygen-sensing pathways (variants in VHL, EGLN1, EPAS1, EPO), and types 6-8 with an increased affinity of haemoglobin for oxygen (variants in HBB, HBA1, HBA2, BPGM). Due to a heterogenic genetic background, the causes of disease are not fully discovered and in more than 70% of patients the condition remains labelled idiopathic.The transfer of next-generation sequencing into clinical practice is becoming a reality enabling detection of various variants in a single rapid test. In this review, we describe the current research on erythrocytosis gene variants and the mechanisms associated with disease development, along with the currently used diagnostic tests.

Background: Erythrocytosis is a condition with an excessive number of erythrocytes, accompanied by an elevated haemoglobin and/or haematocrit value. Congenital erythrocytosis has a diverse genetic background with several genes involved in erythropoiesis. In clinical practice, nine genes are usually examined, but in approximately 70% of patients, no causative mutation can be identified. In this study, we screened 39 genes, aiming to identify potential disease-driving variants in the family with erythrocytosis of unknown cause., Patients and Methods: Two affected family members with elevated haemoglobin and/or haematocrit and negative for acquired causes and one healthy relative from the same family were selected for molecular-genetic analysis of 24 erythrocytosis and 15 hereditary haemochromatosis-associated genes with targeted NGS. The identified variants were further analysed for pathogenicity using various bioinformatic tools and review of the literature., Results: Of the 12 identified variants, two heterozygous variants, the missense variant c.471G>C (NM_022051.2) (p.(Gln157His)) in the EGLN1 gene and the intron variant c.2572-13A>G (NM_004972.3) in the JAK2 gene, were classified as low-frequency variants in European population. None of the two variants were present in a healthy family member. Variant c.2572-13A>G has potential impact on splicing by one prediction tool., Conclusion: For the first time, we included 39 genes in the erythrocytosis clinical panel and identified two potential disease-driving variants in the Slovene family studied. Based on the reported functional in vitro studies combined with our bioinformatics analysis, we suggest further functional analysis of variant in the JAK2 gene and evaluation of a cumulative effect of both variants., (© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Personalized medicine is a developing field of medicine that has gained in importance in recent decades. New diagnostic tests based on the analysis of circulating cell-free DNA (cfDNA) were developed as a tool of diagnosing different cancer types. By detecting the subpopulation of mutated DNA from cancer cells, it is possible to detect the presence of a specific tumour in early stages of the disease. Mutation analysis is performed by quantitative polymerase chain reaction (qPCR) or the next generation sequencing (NGS), however, cfDNA protocols need to be modified carefully in preanalytical, analytical, and postanalytical stages. To further improve treatment of cancer the Food and Drug Administration approved more than 20 companion diagnostic tests that combine cancer drugs with highly efficient genetic diagnostic tools. Tools detect mutations in the DNA originating from cancer cells directly through the subpopulation of cfDNA, the circular tumour DNA (ctDNA) analysis or with visualization of cells through intracellular DNA probes. A large number of ctDNA tests in clinical studies demonstrate the importance of new findings in the field of cancer diagnosis. We describe the innovations in personalized medicine: techniques for detecting ctDNA and genomic DNA (gDNA) mutations approved Food and Drug Administration companion genetic diagnostics, candidate genes for assembling the cancer NGS panels, and a brief mention of the multitude of cfDNA currently in clinical trials. Additionally, an overview of the development steps of the diagnostic tools will refresh and expand the knowledge of clinics and geneticists for research opportunities beyond the development phases., Competing Interests: Potential conflict of interest: None declared., (Croatian Society of Medical Biochemistry and Laboratory Medicine.)

Chinese hamster ovary (CHO) epithelial cells are one of the most used therapeutic medical lines for the production of different biopharmaceutical drugs. They have a high consumption rate with a fast duplication cycle that makes them an ideal biological clone. The higher accumulated amounts of toxic intracellular intermediates may lead to lower organism viability, protein productivity and manufactured biosimilar, so a careful optimal balance of medium, bioreactor operational parameters and bioprocess is needed. A precise phenomenological knowledge of metabolism's chemical transformations can predict problems that may arise during batch, semi-continuous fed batch and continuous reactor operation. For a better detailed understanding (and relations), future performance optimization and scaling, mechanistic model systems have been built. In this specific work, the main metabolic pathways in mammalian structured CHO cultures are reviewed. It starts with organic biochemical background, controlling associated phenomena and kinetics, which govern the sustaining conversion routes of biology. Then, individual turnover paths are described, overviewing standard mathematical formulations that are commonly applied in engineering. These are the core of black box modeling, which relates the substrates/products in a simplified relationship manner. Moreover, metabolic flux analysis (MFA)/flux balance analysis (FBA), that are traditionally characterizing mechanisms, are presented to a larger portion extent. Finally, similarities are discussed, illustrating the approaches for their structural design. Stated variables' equations, employed for the description of the growth in the controllable environmental conditions of a vessel, the researched reaction series of proliferating dividing CHO population, joint with the values of maximal enzymatic activity, and solutions are outlined. Processes are listed in a way so that a reader can integrate the state-of-the-art. Our particular contribution is also denoted.

Fibrinogen (FIB) is a secretory glycoprotein synthesized by hepatocytes that has a key role in blood clotting. Its glycosylation has not been studied in detail and little is known about the biological variability of FIB N-glycosylation, mainly due to the lack of fast, simple, and robust approaches to purify FIB from blood plasma samples. In recent years, customised chromatographic monoliths have been used for a variety of biological applications due to their unique characteristics. Here we describe development and optimisation of monolithic supports bearing monoclonal anti-human fibrinogen antibodies in a single column as well as in multi-well plate formats with high FIB specificity and binding capacity for fast immunoaffinity purification of FIB from human blood samples. The developed semi-high-throughput workflow has been successfully applied for FIB immunoaffinity isolation and subsequent ultra performance liquid chromatography N-glycosylation analysis in ten healthy human individuals, demonstrating the potential of monolithic supports in glycomics studies., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Metal oxide affinity chromatography has been one of the approaches for specific enrichment of phosphopeptides from complex samples, based on specific phosphopeptide adsorption forming bidentate chelates between phosphate anions and the surface of a metal oxide, such as TiO 2 , ZrO 2 , Fe 2 O 3 , and Al 2 O 3 . Due to convective mass transfer, flow-independent resolution and high dynamic binding capacity, monolith chromatographic supports have become important in studies where high resolution and selectivity are required. Here, we report the first synthesis and characterization of immobilisation of rutile TiO 2 nanoparticles onto organic monolithic chromatographic support (CIM-OH-TiO 2 ). We demonstrate the specificity of CIM-OH-TiO 2 column for enrichment of phosphopeptides by studying chromatographic separation of model phosphorylated and nonphosphorylated peptides as well as proving the phosphopeptide enrichment of digested bovine α-casein. The work described here opens the possibility for a faster, more selective enrichment of phosphopeptides from biological samples that will enable future advances in studying protein phosphorylation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Protein pegylation is a process of covalent attachment of a polyethylene glycol (PEG) group to the protein tertiary structure that can "mask" the agent from the immune system and also increases the hydrodynamic size of the agent. Usually the pegylation prolongs the protein stability in the organism due to reduced renal clearance and provides superior water solubility to hydrophobic molecules. The mono-pegylated form of protein is usually prefered for medical applications. Different conditions with different PEG reagents have to be tested to find optimal pegylation procedure with specific protein. The goal of this study was to prepare screening method for separation of random mono-pegylated protein. Cytochrome C and beta lactoglobulin were pegylated with four reagents and a complete screening of several chromatographic monoliths in ion exchange mode with different buffers was performed to optimaly separate each mono-pegylated protein. The screening method was developed that produces optimal separation of target pegylated protein on CIM monoliths. Because of short chromatographic run time, CIM monoliths are perfect candidates to test alot of parameters. The results obtained show that each protein has its own unique separation parameters (pH, ionexchange ligand, buffer type). Two biopharmaceuticals were isolated using protocol: super human leptin antagonist (SHLA) was purified from inclusion bodies and mono-pegylated super mouse leptin antagonist (SMLA) from pegylated mixture. During study it was observed that the convective interaction media (CIM) monoliths additionally discriminate between protein isoforms pegylated on different sites in 3D structure of the protein., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We investigated effect of immobilization procedure and monolith structure on chromatographic performance of methacrylate monoliths bearing affinity ligands. Monoliths of different pore size and various affinity ligands were prepared and characterized using physical and chromatographic methods. When testing protein A monoliths with different protein A ligand densities, a significant nonlinear effect of ligand density on dynamic binding capacity (DBC) for IgG was obtained and accurately described by Langmuir isotherm curve enabling estimation of protein A utilization as a function of ligand density. Maximal IgG binding capacity was found to be at least 12mg/mL exceeding theoretical monolayer adsorption value of 7.8mg/mL assuming hexagonal packing and IgG hydrodynamic diameter of 11nm. Observed discrepancy was explained by shrinkage of IgG during adsorption on protein A experimentally determined through calculated adsorbed IgG layer thickness of 5.4nm from pressure drop data. For monoliths with different pore size maximal immobilized densities of protein A as well as IgG dynamic capacity linearly correlates with monolith surface area indicating constant ligand utilization. Finally, IgGs toward different plasma proteins were immobilized via the hydrazide coupling chemistry to provide oriented immobilization. DBC was found to be flow independent and was increasing with the size of bound protein. Despite DBC was lower than IgG capacity to immobilized protein A, ligand utilization was higher., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Affinity depletion of abundant proteins such as HSA is an important stage in routine sample preparation prior to MS/MS analysis of biological samples with high range of concentrations. Due to the charge competition effects in electrospray ion source that results in discrimination of the low-abundance species, as well as limited dynamic range of MS/MS, restricted typically by three orders of magnitude, the identification of low-abundance proteins becomes a challenge unless the sample is depleted from high-concentration compounds. This dictates a need for developing efficient separation technologies allowing fast and automated protein depletion. In this study, we performed evaluation of a novel immunoaffinity-based Convective Interaction Media analytical columns (CIMac) depletion column with specificity to HSA (CIMac-αHSA). Because of the convective flow-through channels, the polymethacrylate CIMac monoliths afford flow rate independent binding capacity and resolution that results in relatively short analysis time compared with traditional chromatographic supports. Seppro IgY14 depletion kit was used as a benchmark to control the results of depletion. Bottom-up proteomic approach followed by label-free quantitation using normalized spectral indexes were employed for protein quantification in G1/G2 and cleavage/blastocyst in vitro fertilization culture media widely utilized in clinics for embryo growth in vitro. The results revealed approximately equal HSA level of 100 ± 25% in albumin-enriched fractions relative to the nondepleted samples for both CIMac-αHSA column and Seppro kit. In the albumin-free fractions concentrated 5.5-fold by volume, serum albumin was identified at the levels of 5-30% and 20-30% for the CIMac-αHSA and Seppro IgY14 spin columns, respectively., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)