Purification of influenza deoxyribonucleic acid-based vaccine using agmatine monolith.

Lately, researchers have made several efforts to improve vaccine production to fight highly contagious respiratory diseases like influenza. One of the most promising options for reducing the impact of this virus is DNA vaccination. However, a large quantity of highly pure plasmid DNA (pDNA) is necessary to attain this goal. The present work describes the production and purification of the plasmid NTC7482-41H-VA2HA expressing influenza virus hemagglutinin using an agmatine monolith. This ligand was chosen to purify supercoiled (sc) pDNA from complex lysates because of its versatile multimodal character. Its natural intervention in several biological systems together with its similarity with the highly studied arginine ligand allowed the development of a simpler and more specific purification process. Agmatine works under two strategies: descending ammonium sulfate gradient and ascending sodium chloride gradient. Furthermore, pH manipulation revealed an important role in pDNA isoforms selectivity. Dynamic binding capacity (DBC) experiments were performed varying different parameters and showed an increase with pDNA concentration, while high flow rate and high pH had the opposite effect. Sc pDNA was purified with high yield and was efficient with respect to cell transfection and cell viability. This monolith showed to be appropriate to purify the plasmid NTC7482-41H-VA2HA, providing a valuable tool for pDNA influenza vaccines preparation.

Alt a 15 is a new cross-reactive minor allergen of Alternaria alternata.

Alternaria alternata is one of the most common saprophytes worldwide that is clinically and epidemiologically associated with severe asthma. Therefore, the identification and characterization of all A. alternata allergens are of major clinical importance. This study describes a new cross-reactive A. alternata allergen that was officially named Alt a 15 by the official Allergen Nomenclature Subcommittee. The complete coding region for Alt a 15 was amplified using 5' and 3' rapid amplification of cDNA ends and PCR. The recombinant protein was produced in Escherichia coli as a 65-kDa fusion protein, and the protein sequence exhibits high homology with several important fungal allergens. Immunoblotting analyses revealed that IgE antibodies from A. alternata-sensitized patients (n=59) bound to rAlt a 15 with a prevalence of 10.2%. All patients who presented sIgE to rAlt a 15 were apparently poly-sensitized to A. alternata and C. lunata. The extensive cross-reactivity between A. alternata and C. lunata serine proteases was confirmed using immunoblotting inhibition assays. Overall, Alt a 15 is an important new cross-reactive allergen of A. alternata that explains some allergies to A. alternata without Alt a 1 sensitization and initial diagnostic errors for allergies to Alternaria. This molecule may improve the accuracy of the diagnosis, the understanding, and the management of IgE-mediated fungal diseases.

Development of a PCR-based tool for detecting immunologically relevant Alt a 1 and Alt a 1 homologue coding sequences.

Alt a 1 has been recognized as the most important allergen produced by the Pleosporaceae family and is a risk factor for asthma development and/or exacerbation. The aim of this study was to develop a detection tool that is highly specific for species that produced airborne Alt a 1. Based on the highly conserved internal nucleotide region of the several Alt a 1 sequences that are available in GenBank, a pair of primers (Alta1CF/Alta1CR) was designed. A set of primers used by other authors for the production of recombinant Alt a 1 (A21F/A21R) was also tested. The molecular analyses were based on the polymerase chain reaction (PCR) amplification and sequencing of the cDNA that was obtained from thirteen common fungal species. The PCR system that utilized Alta1CF/Alta1CR was highly specific, sensitive, and was able to detect an amplicon of approximately 180 bp from Alt a 1 and Alt a 1-like encoding genes from A. alternata, A. tenuissima, A. infectoria, U. botrytis, and S. botryosum. In contrast, the A21F/A21R primers were specific for the very close taxonomically related species A. alternata and A. tenuissima. Thus, this rapid, sensitive, and specific detection tool can be used to assess Alt a 1 exposure levels and to inform the implementation of the appropriate public health measures.

Aromatic ligands for plasmid deoxyribonucleic acid chromatographic analysis and purification: an overview.

The aromatic ring systems are among the most stable chemical structures known and in combination with many other chemical groups, they can originate an extraordinary variety of molecules, with interesting chemical and physical properties. Many aromatic molecules have been applied for the purification of various biomolecules, such as proteins, carbohydrates and nucleic acids. Combining aromatic chromatography with optimized production, extraction and clarification procedures, can offer a number of advantages for pharmaceutical plasmid DNA (pDNA) purification. This review focuses on pDNA chromatographic purification and analysis using aromatic ligands. The goal is to give an updated view of all existing aromatic ligands, their main characteristics, applicability and technical features of the chromatographic methods in which they have been applied. Also, a critical assessment of each method is performed as well as a comparison of the different procedures, their key features and limitations.

Negative pseudo-affinity chromatography for plasmid DNA purification using berenil as ligand.

The present study, reports the utilization of berenil as ligand in a negative pseudo-affinity chromatographic step to purify the plasmid pVAX1-LacZ from Escherichia coli clarified lysates. The chromatographic support was prepared by coupling berenil to epoxy-activated Sepharose and was qualitatively and quantitatively characterized using scanning electron microscopy, Fourier transformed infrared spectroscopy and elemental analysis. The clarified lysate was loaded onto the berenil-Sepharose support with 0.55M of ammonium sulphate in the eluent, achieving the immediate elution of plasmid DNA. The impurities tightly bound to the support, were eluted after decreasing the salt concentration to 0M. The overall process enabled the recovery of 87% of loaded plasmid DNA with a HPLC purity of ≫99% and according to FDA specifications. This method represents an alternative approach to the previous utilization of the same chromatographic pseudo-affinity support in a positive mode. It uses lower amounts of salt and one-step chromatographic procedure, resulting in smaller operating time and costs and representing an alternate procedure for plasmid DNA purification.

Dynamic binding capacity and specificity of 3,8-diamino-6-phenylphenanthridine-Sepharose support for purification of supercoiled plasmid deoxyribonucleic acid.

Affinity chromatography represents a sole technique in purification of different biomolecules. The specific recognition between affinity ligands and target biomolecules has a major role in the specificity of the process. Therefore, choosing the right ligand is a crucial step for the development of a successful purification system. This work describes the application of the DNA intercalator 3,8-diamino-6-phenylphenanthridine (DAPP) as a chromatographic affinity ligand for the specific separation and purification of supercoiled plasmid DNA (pDNA). The support was prepared by coupling DAPP onto an epoxy-activated Sepharose matrix, using mild conditions and resulting in a ligand density of 0.15mmolDAPP/g derivatized Sepharose. The characterization of DAPP-Sepharose support in terms of dynamic binding capacity was achieved by studying the effect of plasmid DNA concentration and flow rate on pDNA adsorption. The maximum capacity value of 336.75µgpDNA/mL gel was obtained at 1mL/min with a pDNA concentration of 150µg/mL. Moreover, the values did not vary significantly with the variation of flow rate. In addition, the DAPP-Sepharose showed a high affinity towards pDNA as quantified by the respective dissociation constant (Kd=2.29±0.195×10(-7)M). The support was also tested for the purification of two plasmid molecules with different sizes (pVAX1-LacZ and pCAMBIA-1303, with 6.05kbp and 12.361kbp, respectively) from clarified Escherichia coli lysate solutions. Total retention of all lysate components was achieved without any added salt to the eluent buffer. The selective elution of impurities and supercoiled pDNA was accomplished simply by the addition of small amounts of salt to the buffer solution. The yield for pCAMBIA-1303 was 65% and for pVAX1-LacZ was 94%, with all host impurity levels in accordance with the requirements established by the regulatory agencies.

Specific recognition of supercoiled plasmid DNA by affinity chromatography using the intercalator DAPP as ligand.

Small molecules that bind DNA with high specificity present a promising opportunity for application as chromatographic ligands for plasmid DNA (pDNA) purification. This research used the intercalator 3,8-diamino-6-phenylphenanthridine (DAPP) as an immobilized ligand for the specific separation of supercoiled (sc) pDNA by affinity chromatography. The results showed that the protonated DAPP-Sepharose support has a great affinity for sc pDNA isoform, separating it from the less active open circular and linear isoforms. All pDNA isoforms were retained in the column using 10mM acetate buffer pH 5. Selective elution of oc and linear isoforms was achieved with 0.22M of sodium chloride in the same buffer. Finally, increasing the concentration to 0.55M led to the elution of the sc isoform. The binding of pDNA to DAPP-Sepharose varies in function of pH, and the stability of the protonated DAPP-DNA complex decreases with increasing salt concentration.

Evaluation and comparison of commercially available latex extracts for skin prick tests.

BACKGROUND: Crude latex extracts are commonly used in skin prick tests (SPT) for the diagnosis of natural rubber latex (NRL) allergy. Nevertheless, variations in protein and allergen composition between latex extracts from different manufacturers can hamper a correct diagnosis. OBJECTIVES: To analyze the heterogeneity of proteins and allergens in latex extracts from 7 different manufacturers and to assess its relevance in the diagnosis of latex allergy. METHODS: Seven latex SPT extracts were analyzed for protein content using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The 4 major allergens Hev b 1, Hev b 3, Hev b 5, and Hev b 6.02 were also quantified using enzyme immunoassay. All commercial extracts were tested for their in vitro allergenic capacity using microarray inhibition assays and for their ability to induce biological reactivity in latex-allergic patients undergoing SPT. RESULTS: The protein content of the extracts varied widely from 8.0 microg/mL to 526.5 microg/mL. SDS-PAGE revealed broad differences in protein profiles between the extracts. Marked variability in the contents of all 4 major allergens was observed, and Hev b 3 and Hev b 5 were undetectable in some extracts. Microarray inhibition assays and SPT demonstrated relevant differences in allergenic capacity between the extracts. CONCLUSIONS: The marked heterogeneity in protein and allergen content of latex extracts from different manufacturers could explain the broad spectrum of SPT results recorded. Our findings suggest that the extracts used for the diagnosis of latex allergy should be improved and standardized.

Purification of plasmid DNA from clarified and non-clarified Escherichia coli lysates by berenil pseudo-affinity chromatography.

In this study, berenil was tested as a ligand, specifically to purify plasmids of different sizes pVAX1-LacZ (6.05 Kbp) and pCAMBIA-1303 (12.361 Kbp) from clarified Escherichia coli alkaline lysates. For this purpose, chromatographic experiments were performed using Sepharose derivatized with berenil. The results showed that both pDNA molecules are completely purified using lower amounts of salt in the eluent than those previously reported for other pseudo-affinity and hydrophobic interaction chromatography based processes. Total retention of all lysate components was achieved with 1.3M ammonium sulphate in the eluent buffer and pDNA elution was obtained by decreasing the salt concentration to 0.55 M. All impurities were eluted after decreasing the concentration to 0M. The recovery yield for pCAMBIA-1303 (45%) was lower than that obtained for pVAX1-LacZ (85%), however the larger pDNA showed a higher purity level. Purification of pVAX1-LacZ was also performed using non-clarified E. coli process streams, replacing the clarification step with a second chromatographic run on the berenil-Sepharose. Using the same binding and elution conditions as before, a pure plasmid sample was obtained with a 33% yield and with all host impurity levels in accordance with the requirements established by the regulatory agencies. These results suggest that this chromatographic method is a promising alternative to purify pDNA for therapeutic use.

Different in vivo reactivity profile in health care workers and patients with spina bifida to internal and external latex glove surface-derived allergen extracts.

BACKGROUND: Allergy to natural rubber latex is a well-recognized health problem, especially among health care workers and patients with spina bifida. Despite latex sensitization being acquired in health institutions in both health care workers and patients with spina bifida, differences in allergen sensitization profiles have been described between these two risk groups. OBJECTIVE: To investigate the in vivo reactivity of health care workers and patients with spina bifida to extracts of internal and external surfaces of latex gloves and also to specific extracts enriched in major allergens for these risk groups. METHODS: Gloves from different manufacturers were used for protein extraction, and salt precipitation and hydrophobic interaction chromatography (HIC) were applied to obtain the enriched latex extracts. The major latex allergens were quantified by an enzyme immunoassay. The extracts obtained were tested in 14 volunteers using skin prick tests (SPT). RESULTS: Latex glove extracts enriched in the hydrophobic allergens that are most often seen in patients with spina bifida were obtained by selective precipitation, whereas HIC produced extracts enriched in the hydrophilic allergens commonly found in health care workers. The health care workers had positive SPTs to glove extracts from internal surfaces and to the hydrophilic allergen-enriched extracts. By contrast, patients with spina bifida had larger skin reactions both to external glove extracts and to the extracts enriched with the hydrophobic major allergens for this risk group. Despite the protein concentration of these extracts being less than half the concentration of the commercial extract, the weal-and-flare reactions were of similar magnitude. CONCLUSION: Using novel latex extracts, our study showed a different in vivo reactivity pattern in health care workers and in patients with spina bifida to extracts of the internal and external surfaces of gloves, which suggests that sensitization may occur by different routes of exposure, and that this influences the allergen reactivity profiles of these risk groups.

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.

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.

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.

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. OBJECTIVES: To investigate latex allergen profiles on internal and external surfaces of natural rubber latex gloves. METHODS: Eighty-two samples of commonly used surgical gloves (41 glove brands) were used for analysis. Specific allergen levels of Hev b 1, 3, 5 and 6.02 on both surfaces of the gloves were quantified using an enzyme immunometric assay, a FITkit (FIT Biotech, Tampere, Finland). RESULTS: Differences in allergen levels were observed between internal and external surfaces of all glove types. Concentrations of Hev b 1 and Hev b 3 were significantly higher on external surfaces, while internal surfaces had higher allergen levels of Hev b 5 and Hev b 6.02. Analysis of surgical and examination gloves, powdered and nonpowdered gloves also showed that the content of Hev b 5 and Hev b 6.02 was significantly higher on internal surfaces while that of Hev b 1 and Hev b 3 was higher on external surfaces. CONCLUSIONS: Our study showed different allergen profiles on internal and external surfaces of natural rubber latex gloves. These results may suggest a relationship between latex allergen localization and sensitization routes in different risk groups.

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.