Dajana Gašo-Sokač

Use of proteomics for validation of the isolation process of clotting factor IX from human plasma

The use of proteomic techniques in the monitoring of different production steps of plasma-derived clotting factor IX (pd F IX) was demonstrated. The first step, solid-phase extraction with a weak anion-exchange resin, fractionates the bulk of human serum albumin (HSA), immunoglobulin G, and other non-binding proteins from F IX. The proteins that strongly bind to the anion-exchange resin are eluted by higher salt concentrations. In the second step, anion-exchange chromatography, residual HSA, some proteases and other contaminating proteins are separated. In the last chromatographic step, affinity chromatography with immobilized heparin, the majority of the residual impurities are removed. However, some contaminating proteins still remain in the eluate from the affinity column. The next step in the production process, virus filtration, is also an efficient step for the removal of residual impurities, mainly high molecular weight proteins, such as vitronectin and inter-alpha inhibitor proteins. In each production step, the active component, pd F IX and contaminating proteins are monitored by biochemical and immunochemical methods and by LC-MS/MS and their removal documented. Our methodology is very helpful for further process optimization, rapid identification of target proteins with relatively low abundance, and for the design of subsequent steps for their removal or purification.

The role of proteomics in plasma fractionation and quality control of plasma-derived therapeutic proteins

Human plasma is still an important row material for isolation of therapeutic proteins. Human serum albumin (HSA) was the first therapeutic protein isolated from human plasma, and plasma-derived (pd) HSA concentrates are still being used for patient treatment1, 2. Preparations of human immunoglobulins for intravenous use (IVIG) are currently the driving force for usage of human plasma in the fractionation industry1. Von Willebrand factor (VWF), protease inhibitors such as alpha-1 antitrypsin (A1-AT) and antithrombin III (AT III) and clotting factors and inhibitors are further important therapeutic proteins that are isolated form human plasma. Their concentration in this biological fluid is up to six orders of magnitude lower than HSA and IgG. Consequently, the manufacturing process for isolation of these proteins with economically sufficient yield and in form of safe and active concentrates can be a true challenge2,3. Virological safety of human plasma and human plasma-derived therapeutic products is still a primary concern, and high safety standards have been implemented to make these products virologically safe2–4. Although all plasma-derived therapeutic preparations that are on the market belong to so-called “well-characterized biologicals” 5 they still contain relatively high amount of foreign proteins. In contradiction to this name, most impurities are poorly characterized6. After a relatively slow start, proteomics has finally progressed into different branches of transfusion medicine1,7,8. As shown in figure 1, proteomic technologies can be used as an efficient tool for process development and characterization of human plasma-derived therapeutic proteins. As shown in this figure, proteomics can be used for process validation and quality control of these therapeutic proteins. It has been demonstrated that proteomics also offer fast and efficient ways for the identification of potentially harmful impurities6. After their identification, the time for process optimization towards their removal can be significantly shortened6, 9. For quality control of final products, proteomics can be used for characterization of the active component, detection of impurities and determination of batch-to-batch variations10–12. Figure 1 Application of proteomics for validation of the production process of a therapeutic protein concentrate from human plasma and for final product characterization. Human serum albumin Human serum albumin (HSA) is present in human plasma at about 35 mg/mL, and it is the most abundant protein in this biological fluid. Essentially all therapeutic pd HSA preparations are manufactured form human plasma after cryoprecipitation by ethanol fractionation2. These concentrates contain between 95 and 98% HSA, and it is well known that they contain some plasma proteins as impurities. To get highly pure HSA, e.g. for measurement of its potential biological activity, additional purification steps are necessary13. Fortis et al. 10 analyzed the 98% pure, injectable HSA solution, and after direct analysis by SDS-PAGE followed by mass spectrometry of electrophoretically separated proteins, only a single impurity, haptoglobin, was identified. However, after enrichment of trace proteins by use of hexapeptide library beads, 13 additional plasma proteins were identified as impurities in this preparation. The list of these impurities is given in table I. As shown in this table, no potentially harmful proteins such as proteases, clotting factors and inhibitors were identified, and this preparation can be considered as biologically safe. However, this work demonstrates, that proteomic investigation gives a lot of additional information about the concentrate composition, and can be very useful, especially if rare side reactions occur after use of such therapeutics. Consequently, further investigations, especially comparison between the products of different producers and batch-to-batch variations for single products are still necessary. Table I Proteins identified from injectable HSA concentrate after treatment with peptide ligand library. Modified from Reference10 with permission IVIG Clotting factor VIII used to be the driving force for human plasma usage at the end of the last century. Now, IVIG concentrates are the leading products of the plasma fractionation industry. Recent reviews about the production and quality control of these concentrates deal mostly the aspects of yield and virus safety14, 15. Quality controls that are performed for release of IVIG batches are determination of subclass composition and contents of some potentially harmful proteins that can use adverse reactions (e.g. IgA and IgM), endotoxins and chemical (e.g. residues of viral inactivation treatments) impurities15. Recently, Buchacher et al.16 investigated the anticomplementary activity (ACA) of IVIG concentrates caused by high concentration of large size polymers in some concentrates. They could demonstrate that conditions under which polymers are formed have an influence on the ACA outcome, but also other impurities related to the starting material and unforeseen changes of process condition might affect the ACA and lead to batch-to-batch variations. Further detection and characterization of these impurities, e.g. by above discussed hexapeptide library beads10 could give additional information about their identity and possible side effects. In an earlier paper, Page et al.17 demonstrate that IgG fragmentation that can cause impaired efficacy of IVIG concentrates can be caused by contamination with serum proteases such as plasmin and kallikrein. However, proteomic investigations of different IVIG preparations and batch-to-batch comparison to show potential variations of the concentrates that are on the market are still outstanding and have to be performed.

Separation of proteins from human plasma by sample displacement chromatography in hydrophobic interaction mode

Sample displacement chromatography (SDC) in reversed‐phase and ion‐exchange modes was introduced approximately 20 years ago. This method was first used for the preparative purification of peptides and proteins. Recently, SDC in ion‐exchange mode was also successfully used for enrichment of low‐abundance proteins from human plasma. In this paper, the use of SDC for the separation of plasma proteins in hydrophobic interaction mode is demonstrated. By use of two or more columns coupled in series during sample application, and subsequent elution of detached columns in parallel, additional separation of bound proteins was achieved. Further low‐abundance, physiologically active proteins could be highly enriched and detected by ESI‐MS/MS.

Therapeutic plasma proteins – application of proteomics in process optimization, validation, and analysis of the final product

An overview is given on the application of proteomic technology in the monitoring of different steps during the production of therapeutic proteins from human plasma. Recent advances in this technology enable the use of proteomics as an advantageous tool for the validation of already existing processes, the development and fine tuning of new production steps, the characterization and quality control of final products, the detection of both harmful impurities and modifications of the therapeutic protein and the auditing of batch‐to‐batch variations. Further, use of proteomics for preclinical testing of new products, which can be either recombinant or plasma‐derived, is also discussed.

Volatile Organic Compounds from Centaurium erythraea Rafn (Croatia) and the Antimicrobial Potential of Its Essential Oil

GC and MS were used for the analysis of Croatian Centaurium erythraea Rafn essential oil (obtained by hydrodistillation) and headspace (applying headspace solid-phase microextraction). The headspace contained numerous monoterpene hydrocarbons (the major ones were terpinene-4-ol, methone, p-cymene, γ-terpinene and limonene). Oxygenated monoterpenes were present in the headspace and oil, while 1,8-cineole, bornyl acetate and verbenone were present only in the headspace. High headspace percentages of toluene and naphthalene were found, followed by hemimellitene. Lot of similarities were observed with Serbian C.erythraea oil [neophytadiene (1.4%), thymol (2.6%), carvacrol (6.1%) and hexadecanoic acid (5.7%)], but different features were also noted such as the presence of menthol, menthone and phytone. The oil fractionation enabled identification of other minor compounds not found in total oil such as norisoprenoides, alk-1-enes or chromolaenin. The essential oil showed antimicrobial potential on Escherichia coli, Salmonella enteritidis, Staphylococcus aureus and Bacillus cereus. On the other hand, no antibacterial activity of the oil was observed on Pseudomonas fluorescens and Lysteria monocytogenes.

Foodomics and Food Safety: Where We Are

The power of foodomics as a discipline that is now broadly used for quality assurance of food products and adulteration identification, as well as for determining the safety of food, is presented. Concerning sample preparation and application, maintenance of highly sophisticated instruments for both high-performance and high-throughput techniques, and analysis and data interpretation, special attention has to be paid to the development of skilled analysts. The obtained data shall be integrated under a strong bioinformatics environment. Modern mass spectrometry is an extremely powerful analytical tool since it can provide direct qualitative and quantitative information about a molecule of interest from only a minute amount of sample. Quality of this information is influenced by the sample preparation procedure, the type of mass spectrometer used and the analysts skills. Technical advances are bringing new instruments of increased sensitivity, resolution and speed to the market. Other methods presented here give additional information and can be used as complementary tools to mass spectrometry or for validation of obtained results. Genomics and transcriptomics, as well as affinity-based methods, still have a broad use in food analysis. Serious drawbacks of some of them, especially the affinity-based methods, are the cross-reactivity between similar molecules and the influence of complex food matrices. However, these techniques can be used for pre-screening in order to reduce the large number of samples. Great progress has been made in the application of bioinformatics in foodomics. These developments enabled processing of large amounts of generated data for both identification and quantification, and for corresponding modeling.

Proteomic analysis of food borne pathogens following the mode of action of the disinfectants based on pyridoxal oxime derivatives

A comprehensive proteomic analysis of food borne pathogens after treatment with disinfectants based on ammonium salts of pyridinium oxime was performed. Changes in proteomes of the Gram-positive bacterium Bacillus subtilis and the Gram-negative one, Escherichia coli, were evaluated. Up and down-regulated proteins in these bacteria after growth under the inhibition with four different disinfectants based on chloride and bromide salts of pyridinium oxime were identified and their cellular localizations and functions were determined by gene ontology searching. Proteome changes presented here demonstrate different mechanisms of action of these disinfectants. In the Gram-positive food pathogen Bacillus subtilis, the inhibitory substances seem to act mainly at the cell surface and cause significant alterations of membrane and cell surface proteins. On the other hand, intracellular proteins were more affected in the Gram-negative pathogen Escherichia coli. This research is a contribution to the investigation of the virulence and pathogenicity of food borne bacteria and their survival under stress conditions, and can also lead the way for further development of new inhibitors of microbial growth and studies of mechanism of their actions.

An Efficient Synthesis of Pyridoxal Oxime Derivatives under Microwave Irradiation

Quaternary salts of pyridoxal oxime have been synthesized by the quaternization of pyridoxal oxime with substituted phenacyl bromides using microwave heating. Microwave-assisted rapid synthesis was done both in solvent (acetone) and under solvent-free conditions. Good to excellent yields (58%–94%) were obtained in acetone in very short reaction times (3–5 min) as well as in the solvent-free procedure (42%–78%) in very short reaction times (7–10 min) too. Effective metodologies for the preparation of pyridoxal oxime quaternary salts, having the advantagies of being eco-friendly, easy to handle, and performed in shorter reactions time are presented. The structure of compound 7, in which a 4-fluorophenacyl moiety is bonded to the pyridinium ring nitrogen atom, was unequivocally confirmed by the single-crystal X-ray diffraction method.

Data set of proteomic analysis of food borne pathogens after treatment with the disinfectants based on pyridoxal oxime derivatives

Food borne pathogens, namely the Gram-positive bacterium Bacillus subtilis and the Gram-negative bacterium Escherichia coli, were grown under the inhibition with four different disinfectants based on chloride and bromide salts of pyridinium oxime. Bacterial samples were subjected to the sequential extraction of proteins and the in-solution tryptic digestion of obtained extracts was performed prior to the identification of proteins with LC-ESI-MS/MS. Proteomic analysis identified up- and down-regulated proteins in these bacteria after treatment with each compound. The tables with differently expressed proteins are presented with this article.

Occurence of pharmaceuticals in surface water

Pharmaceuticals constitute a large group of human and veterinary medicinal organic compounds which have long been used throughout the world. According to their therapeutic activity they are classified in several groups: antibiotics, analgesics/antipyretic, CNS (Central nervous system) drugs, cardiovascular drugs, endocrinology treatments, diagnostic aidadsorbable organic halogen compounds. Pharmaceuticals are designed to have a physiological effect on humans and animals in trace concentrations. Pharmaceuticals end up in soil, surface waters and eventually in ground water, which can be used as a source of drinking water, after their excretion (in unmetabolized form or as active metabolites) from humans or animals via urine or faeces. The possible fates of pharmaceuticals once they get into the aquatic environment are mainly three: (i) ultimately they are mineralized to carbon dioxide and water, (ii) the compound does not degrade readily because it is lipophilic and is partially retained in the sedimentation sludge and (iii) the compound metabolizes to a more hydrophilic molecule, passes through the wastewater treatment plant and ends up in receiving waters (which are surface waters, mainly rivers). These compounds exhibit the highest persistence in the environment. In recent years, and in particular after the use of the advanced measurement technologies, many pharmaceuticals have been identified worldwide and detected at ng/L levels (trace concentrations) in the aquatic environment, and are considered as an emerging environmental problem due to their continuous input and persistence in the aquatic ecosystem even at low concentrations.