Cândida T. Tomaz

Hydrophobic interaction chromatography of proteins.

In this article, an overview of hydrophobic interaction chromatography (HIC) of proteins is given. After a brief description of protein hydrophobicity and hydrophobic interactions, we present the different proposed theories for the retention mechanism of proteins in HIC. Additionally, the main parameters to consider for the optimization of fractionation processes by HIC and the stationary phases available were described. Selected examples of protein fractionation by HIC are also presented.

Studies on the chromatographic fractionation of Trichoderma reesei cellulases by hydrophobic interaction.

This work reports new studies on cellulases fractionation by hydrophobic interaction chromatography. The purification procedure for the Trichoderma reesei cellulase complex consists of gel permeation chromatography on Sephadex G-25M followed by an ultrafiltration step. The concentrated enzyme solution was then fractionated on Sepharose CL-6B modified by covalent immobilization of 1,4-butanediol diglycidyl ether. The influence of the mobile phase composition on the chromatographic behaviour of the T. reesei cellulase complex was investigated. By using 13% (w/v) ammonium sulphate in eluent buffer, a selective separation of beta-glucosidase with a two-fold increase in specific activity and a recovery of 60% cellobiase activity were obtained. Other commercial hydrophobic supports (octyl- and phenyl-Sepharose) were also tested and compared under the same conditions.

Performance of a non-grafted monolithic support for purification of supercoiled plasmid DNA

The use of therapeutics based on plasmid DNA (pDNA) relies on procedures that efficiently produce and purify the supercoiled (sc) plasmid isoform. Several chromatographic methods have been applied for the sc plasmid purification, but with most of them it is not possible to obtain the required purity degree and the majority of the supports used present low capacity to bind the plasmid molecules. However, the chromatographic monolithic supports are an interesting alternative to conventional supports due to their excellent mass transfer properties and their high binding capacity for pDNA. The separation of pDNA isoforms, using short non-grafted monolithic column with CarbonylDiImidazole (CDI) functional groups, is described in the current work. The effect of different flow rates on plasmid isoforms separation was also verified. Several breakthrough experiments were designed to study the effect of different parameters such as pDNA topology and concentration as well as flow rate on the monolithic support binding capacity. One of the most striking results is related to the specific recognition of the sc isoform by this CDI monolith, without flow rate dependence. Additionally, the binding capacity has been found to be significantly higher for sc plasmid, probably because of its compact structure, being also improved when using feedstock with increased plasmid concentrations and decreased linear velocity. In fact, this new monolithic support arises as a powerful instrument on the sc pDNA purification for further clinical applications.

Successful application of monolithic innovative technology using a carbonyldiimidazole disk to purify supercoiled plasmid DNA suitable for pharmaceutical applications

The growing demand on plasmid DNA (pDNA) manufacture for therapeutic applications requires a final product with higher quality and quantity, spending the least time. Most of the current processes for pDNA production use at least one chromatographic step, which often constitutes a key-step in the purification sequence. Monolithic stationary phases are new alternatives to the conventional matrices, which offer fast separation of pDNA due to their excellent mass transfer properties and their high binding capacity for large molecules, as pDNA. However, the efficient recovery of pure pDNA focuses on a suitable balance of the feedstock, adsorbent and mobile phase properties. To satisfy the increasing demand for pharmaceutical grade plasmids, we developed a novel downstream process which overcomes the bottlenecks of common lab-scale techniques while complying with all regulatory requirements. This work reports an integrative approach using the carbonyldiimidazole monolith to efficiently purify the supercoiled (sc) pDNA active conformation from other plasmid topologies and Escherichia coli impurities present in a clarified lysate. The monolith specificity and selectivity was also assessed by performing experiments with plasmids of several sizes of 2.7, 6.05 and 7.4 kilo base pairs (kbp), verifying the applicability to purify different plasmids. Hence, the process yield of the pDNA purification step using the CDI monolith was 89%, with an extremely reduced level of impurities (endotoxins and gDNA), which was reflected in good transfection experiments of the sc plasmid DNA sample. Overall, the analytical results and transfection studies performed with the pDNA sample purified with this monolithic enabling technology, confirmed the suitability of this pDNA to be used in pharmaceutical applications.

Latex allergy: new insights to explain different sensitization profiles in different risk groups.

Background  Differences in latex allergen sensitization profiles have been described between children subjected to repetitive surgical interventions and health care workers (HCW). ‘Major’ allergens for patients with spina bifida are Hev b 1, 3 and 7, while for HCW, ‘major’ allergens are Hev b 2, 5, 6.01 and 13. The reason for these differential sensitization profiles is currently unknown.

Specific berenil–DNA interactions: An approach for separation of plasmid isoforms by pseudo-affinity chromatography

Small molecules, like some antibiotics and anticancer agents that bind DNA with high specificity, can represent a relevant alternative as ligands in affinity processes for plasmid DNA (pDNA) purification. In the current study, pDNA binding affinities of berberine, berenil, kanamycin, and neomycin were evaluated by a competitive displacement assay with ethidium bromide using a fluorimetric titration technique. The binding between pDNA and ethidium bromide was tested in different buffer conditions, varying the type and the salt concentration, and was performed in both the absence and presence of the studied compounds. The results showed that the minor groove binder berenil has the higher pDNA binding constant. Chromatographic experiments using a derivatized column with berenil as ligand showed a total retention of pDNA using 1.3M ammonium sulfate in eluent buffer. A selective separation of supercoiled and open circular isoforms was achieved by further decreasing the salt concentration to 0.6M and then to 0M. These results suggest a promising application of berenil as ligand for specific purification of pDNA supercoiled isoform by pseudo-affinity chromatography.

Quantitative analysis of five sterols in amniotic fluid by GC–MS: Application to the diagnosis of cholesterol biosynthesis defects

Cholesterol and its precursors, namely 7-dehydrocholesterol, desmosterol and lathosterol are important biochemical markers of cholesterol biosynthesis, and their quantification in body fluids is useful for the diagnosis of cholesterol biosynthesis pathway disorders. A rapid and sensitive gas chromatographic-mass spectrometric method was developed and validated for quantitative analysis of five sterols (cholesterol, 7-dehydrocholesterol, desmosterol, lathosterol and sitosterol) in amniotic fluid. The method was linear for all compounds (r(2)>0.99), and intra and inter-assay coefficients of variation were typically below 5%, and inaccuracy was within a +/-12% interval. The method was applied to 330 amniotic fluid samples, grouped by gestational age between 13 and 22 weeks of pregnancy, in order to establish reference intervals for sterols in this specimen. The obtained concentrations (mumol/L) for each sterol was as follows: 22.1758+/-4.2716 at 13 weeks and 78.5082+/-12.9041 at 22 weeks for cholesterol; 0.0039+/-0.0007 at 13 weeks and 0.1150+/-0.0212 at 22 weeks for 7-dehydrocholesterol; 0.1562+/-0.0406 at 13 weeks and 0.7691+/-0.0821 at 22 weeks for desmosterol; 0.0272+/-0.0035 at 13 weeks and 0.8551+/-0.1791 at 22 weeks for lathosterol; and 0.0404+/-0.0039 at 13 weeks and 0.2326+/-0.0386 at 22 weeks for sitosterol. The method was also applied to one pathological sample that showed decreased levels of cholesterol, and higher concentration of 7-dehydrocholesterol, which is consistent with a 7-dehydrocholesterol-reductase deficiency. Our results showed that as long as pregnancy goes on, the concentrations of cholesterol and precursors increase in amniotic fluid, which is related to the increased need for cholesterol by the fetus. The reference range of each sterol in amniotic fluid was calculated at different gestational ages and will be useful for the interpretation and validation of biochemical prenatal diagnosis of inborn errors of sterol biosynthesis.

Alternaria alternata allergens: Markers of exposure, phylogeny and risk of fungi-induced respiratory allergy

Alternaria alternata spores are considered a well-known biological contaminant and a very common potent aeroallergen source that is found in environmental samples. The most intense exposure to A. alternata allergens is likely to occur outdoors; however, Alternaria and other allergenic fungi can colonize in indoor environments and thereby increase the fungal aeroallergen exposure levels. A consequence of human exposure to fungal aeroallergens, sensitization to A. alternata, has been unequivocally associated with increased asthma severity. Among allergenic proteins described in this fungal specie, the major allergen, Alt a 1, has been reported as the main elicitor of airborne allergies in patients affected by a mold allergy and considered a marker of primary sensitization to A. alternata. Moreover, A. alternata sensitization seems to be a triggering factor in the development of poly-sensitization, most likely because of the capability of A. alternata to produce, in addition to Alt a 1, a broad and complex array of cross-reactive allergens that present homologs in several other allergenic sources. The study and understanding of A. alternata allergen information may be the key to explaining why sensitization to A. alternata is a risk factor for asthma and also why the severity of asthma is associated to this mold. Compared to other common environmental allergenic sources, such as pollens and dust mites, fungi are reported to be neglected and underestimated. The rise of the A. alternata allergy has enabled more research into the role of this fungal specie and its allergenic components in the induction of IgE-mediated respiratory diseases. Indeed, recent research on the identification and characterization of A. alternata allergens has allowed for the consideration of new perspectives in the categorization of allergenic molds, assessment of exposure and diagnosis of fungi-induced allergies.

Rapid quantification of supercoiled plasmid deoxyribonucleic acid using a monolithic ion exchanger

The demand for high-purity supercoiled plasmid DNA to be applied as a vector for new therapeutic strategies, such as gene therapy or DNA vaccination has increased in the last years. Thus, it is necessary to implement an analytical technique suitable to control the quality of the supercoiled plasmid as a pharmaceutical product during the manufacturing process. The present study describes a new methodology to quantify and monitor the purity of supercoiled plasmid DNA by using a monolithic column based on anion-exchange chromatography. This analytical method with UV detection allows the separation of the plasmid isoforms by combining a NaCl stepwise gradient. The specificity, linearity, accuracy, reproducibility and repeatability of the method have been evaluated, and the lower quantification and detection limits were also established. The validation was performed according to the guidelines, being demonstrated that the method is precise and accurate for a supercoiled plasmid concentration up to 200μg/mL. The main advantage achieved by using this monolithic column is the possibility to quantify the supercoiled plasmid in a sample containing other plasmid topologies, in a 4min experiment. This column also permits the assessment of the supercoiled plasmid DNA present in more complex samples, allowing to control its quality throughout the bioprocess. Therefore, these findings strengthen the possibility of using this monolithic column associated with a powerful analytical method to control the process development of supercoiled plasmid DNA production and purification for therapeutic applications.

Differential expression of allergens on the internal and external surfaces of latex surgical gloves

BACKGROUND Differences in latex allergen sensitization profiles have been described between children undergoing repeated surgical interventions and health care workers. The purpose of this study was to determine whether such sensitization profiles are associated with differences in the expression of latex allergen between the internal and external surfaces of surgical gloves. METHODS Extracts were obtained from whole surgical gloves as well as from their external and internal surfaces. The extracts were centrifuged, filtered, concentrated, dialyzed and lyophilized. The protein profile of the extracts was analyzed using hydrophobic interaction chromatography (HIC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblotting was performed using sera from two patients with confirmed latex allergy. Latex recombinant allergen-specific IgE in these two patients was determined using a fluorescence enzyme immunoassay (FEIA) method. Latex allergen quantification was determined on both glove surfaces using an ELISA method. RESULTS HIC and SDS-PAGE showed qualitative and quantitative differences in proteins between the internal and external glove surfaces, with the former being much richer in proteins. Immunoblotting of glove extracts using sera from two latex-allergic health workers showed differences between glove surface extracts. ELISA quantification of latex allergens demonstrated that the internal glove surface had high amounts of Hev b 5 and Hev b 6.02 whereas the external surface showed Hev b 1, Hev b 3, and Hev b 6.02. CONCLUSIONS Our results reveal substantial differences in the composition of latex allergen profiles between the internal and external surfaces of surgical latex gloves, which may suggest a relationship between latex allergen localization and sensitization routes in different risk groups.