Optimizing Growth Conditions of Kluyveromyces marxianus for Mannan Production as a Bioemulsifier

 Background and objective: Mannan which is a linear glycoprotein with β-1,4 links carrying mannose units bind to proteins, includes natural amphiphiles and serves as a bioemulsifier. The aim of this study was optimization of growth and purification of Kluyveromyces marxianus for mannan production, which can use as a natural bioemulisifier.Material and methods: In this study, mannan production by Kluyveromyces marxianus was assessed using combinational method of fractional factorial design and response surface methodology optimization. Process variables include concentration of carbon source (15, 30, 45 g l-1) of glucose, and glycerol and methanol at 0, 2.5 and 5 gl-1), nitrogen source (yeast extract and peptone 4, 6 and 8 gl-1), as well as fermentation time (48, 96 and 144 h), pH (4, 6, 8) and agitation speed (150, 200 and 250 rpm).Results and conclusion: Results showed that four variables of carbon and nitrogen source concentrations, as well as fermentation time and pH included the greatest effects on mannan production. Optimization of the affecting factors using response surface methodology demonstrated appropriate conditions of mannan production by Kluyveromyces marxianus as 55.15 g l-1 of glucose, 9.35 g l-1 of yeast extract, pH of 4.99 and fermentation time of 168 h, which led to a mannan yield of 245.98 mg (100 ml)-1culture media.Conflict of interest: The authors declare no conflict of interest

A PRACTICAL RISK-BASED APPROACH TO ASSESS VIAL'S DIMENSIONS DEVIATIONS EFFECT ON THE ASEPTIC FILLING PROCESSING, ACCORDING TO ICH Q9 GUIDELINE

Objective: Qualitative risk assessment process is a new topic in the pharmaceutical industries. The main outcome of the risk assessment implementation is to help the manufacturers for a better decision-making, in a case that a quality problem arises. According to the ISO documents;  vials used in the pharmaceutical industry have a special dimension specification and Quality Control analytical results should prove that the vial samples are in the defined range. Nevertheless, the value of these tests is not the same as defined ISO specifications;  and this may have minor and/or significant impact on the final product quality.Methods: The purpose of this qualitative study was to rank the results of the vial dimention tests based on quality risk assessment. Consequently, these rankings can help to decide whether the dimension deviation from quality specification of vials is acceptable and what will be the impact of accepting the risk on the final product safety and finally how to decrease the risk.For this purpose, we consider the final product contamination could be one of the main indicators for the quality as the contamination from packaging materials in particular are more important when aseptic processing run.Results: Dimensions that are directly associated with opening the vial containing d2, d3, d4 and h4 that they affect rubber sealing and capping. Other dimensions like h1, h2, h3 and d1 affect rubber sealing and capping indirectly. Therefore, these two groups of deviations have a very high probability of contamination.Â

C-Terminal Domain Deletion Enhances the Protective Activity of cpa/cpb Loaded Solid Lipid Nanoparticles against Leishmania major in BALB/c Mice

Cutaneous leishmaniasis (CL) is the most common form of leishmaniasis with an annual incidence of approximately 2 million cases and is endemic in 88 countries, including Iran. CL's continued spread, along with rather ineffectual treatments and drug-resistant variants emergence has increased the need for advanced preventive strategies. We studied Type II cysteine proteinase (CPA) and Type I (CPB) with its C-terminal extension (CTE) as cocktail DNA vaccine against murine and canine leishmaniasis. However, adjuvants' success in enhancing immune responses to selected antigens led us to refocus our vaccine development programs. Herein, we discuss cationic solid lipid nanoparticles' (cSLN) ability to improve vaccine-induced protective efficacy against CL and subsequent lesion size and parasite load reduction in BALB/c mice. For this work, we evaluated five different conventional as well as novel parasite detection techniques, i.e., footpad imaging, footpad flowcytometry and lymph node flowcytometry for disease progression assessments. Vaccination with cSLN-cpa/cpb-CTE formulation showed highest parasite inhibition at 3-month post vaccination. Immunized mice showed reduced IL-5 level and significant IFN-ã increase, compared to control groups. We think our study represents a potential future and a major step forward in vaccine development against leishmaniasis

Production of Brucella abortus Antiserum in Goats and its Comparison with Conventional Rabbit Antiserum: Production of Brucella abortus antiserum in goats

Brucellosis, caused by Brucella species, is common among humans and animals, and is one of the most common infectious diseases in Iran. Several assays are available to detect brucellosis, but serological tests may be the only method used in many laboratories. In Iran, different kits are produced in the Pasteur Institute based on agglutination, such as Rose Bengal and 2-mercaptoethanol (2-ME). The positive antiserum control used in these kits is produced in rabbits. The purposeof this study was to produce the antiserum in goats and to compare the titer, quality, and quantity with the antiserum produced in rabbits. The goat immunization was performed by intramuscular injection. Seven days after the last injection, sera were collected. The produced antibody was used in slide and tube agglutination tests with different antigens. The results indicated that the antiserum, produced by the goats had a high quality and quantity. Slide agglutination test showed positive results at 1/6400 dilution with goat antiserum (4+) and 1/1600 dilution with rabbit antiserum (1+). The application of the goats is a better and more appropriate choice, in terms of both cost and quantity, when a high concentration of serum is required. In addition, one goat can provide a higher amount of antiserum compared to several rabbits. HIGHLIGHTS This study presents a robust and time-saving method for the production of an efficient antiserum. Goat is a better and more appropriate host when higher amount of serum is required. The amount of anti-serum produced in goat is approximately 8 times greater than that of rabbit

Determining buffer conditions for downstream processing of VLP-based recombinant hepatitis B surface antigen using multimodal resins in bind-elute and flow-through purification modes

Abstract The difficulties in purification of VLP-based recombinant hepatitis B surface antigen (rHBsAg) are mainly emerged from inefficient semi-purification step plus proteins physicochemical properties and these issues make the downstream processing (DSP) very lengthy and expensive. In this study, optimization of rHBsAg (recombinantly-expressed in Pichia pastoris) DSP was performed using selection of buffering conditions in the semi-purification step. In the semi-purification optimization step, up to 73% of the protein impurities were eliminated and the utmost increase in rHBsAg purity (ca. 3.6-fold) was achieved using 20 mM sodium acetate, pH 4.5. By using rHBsAg binding and nonbinding situations obtained from the response surface plot in design of experiments (DOE), additional bind-elute and flow-through purification mode experiments were conducted and rHBsAg with high purity (near 100%) and recovery (> 83%) was achieved. Following assessment of critical quality attributes (i.e., purity, particle size distribution, host cell DNA, host cell protein, secondary structures, specific activity and relative potency), it was indicated that the characteristics of rHBsAg purified by the new DSP were similar or superior to the ones obtained from conventional DSP. The purification performance of the resin was constantly retained (97–100%) and no significant resin damage took place after 10 adsorption–elution–cleaning cycles. The new DSP developed for production of rHBsAg in this study can substitute the conventional one with granting satisfactory target protein quality, long-lasting resin efficacy, shorter and less expensive process. This process may be also employable for purification of both non-VLP- and VLP- based target proteins expressed in the yeast

cSLN formulations and their characteristics.

<p>Nanoparticles were formulated from cetyl palmitate, cholesterol and DOTAP hydrochloride. Results represent mean±SD of three independent SLN preparations.</p

Flowcytometry analysis of the footpads.

<p>The bars represent the average percentage of GFP positive footpad cells ± SD of three mice per each group GFP fluorescence expression was significantly lower in the footpads of group 3 (*<i>p</i><0.05).</p