BACKGROUND: The efficient, controlled and robust purification of conjugates from PEGylation has a growing demand in the biopharmaceutical's market. In general, the yield and purity reached through the conventional chromatographic modes are not particularly high or efficient. Affinity chromatography has so far scarcely been explored. The present work introduces the purification of mono-PEGylated lysozyme from a PEGylation reaction by heparin affinity chromatography (HAC) for the first time in a single step. Response surface methodology (RSM), particularly a Box-Behnken design (BBD) was employed to optimize the separation. RESULTS: Protein adsorption of PEGylated and native lysozyme on Heparin Sepharose 6 Fast Flow resin was described byLangmuir isotherms, showing a relatively low affinity for the PEGylated proteins. From the experimental design, optimal elution conditions in a linear gradient of sodium chloride (NaCl) for the three response variables (yield, purity and productivity) were: gradient length of 13 column volumes (CVs), flow at 0.8 mL min −1 and protein load of 1 mg mL −1 . Based on this optimization, a step gradient procedure was designed that achieved the purification of mono-PEGylated lysozyme with approximately 100% yield and purity in comparison with 92.7% and 99.7% with the linear gradient. Productivity was c. 0.048 ± 0.001 mg mL −1 min −1 using 0.05 mol L −1 NaCl for its elution. CONCLUSIONS: Mono-PEGylated lysozyme was completely separated from a PEGylation mixture with high yield and purity using HAC for first time. Applying response surface methodology (RSM), adequate conditions for more than one requirement were found as well as optimal conditions for a linear gradient of NaCl. Based on this optimization a step gradient procedure was designed that achieved the purification of mono-PEGylated lysozyme in one step with advantages respect to time, resolution, yield and purity compared with other chromatographic modes such as hydrophobic interaction chromatography (HIC) and cation exchange chromatography (CEX).
The aim of this research was to identify, extract and isolate pristimerin in leaves, stems and roots of the Mexican plant Mortonia greggii (Celastraceae). The principal objective was to determine the best laboratory experimental conditions for the extraction and isolation of this powerful natural anticancer agent from the root tissue. Six experimental factors in solid-liquid pristimerin extraction were analyzed: solvent systems, number of extractions, ratio of plant weight (g)/solvent volume (mL) used, time of extraction, temperature and agitation. A mathematical model was generated for pristimerin purity and yield. Ethanol, first extraction, 0.5 ratio of plant weight/solvent volume (g/mL), 0.5 h, 200 rpm and 49.7°C were optimal conditions for the extraction of this phytochemical. The degree of purification of pristimerin root extract was studied by size-exclusion chromatography (SEC) using Sephadex LH-20 reaching fractions with purification indexes (PI) greater than 2 and recoveries of 28.3%. When fractions with purification indices higher than 1 and less than 2 were accumulated, the recovery of pristimerin increased by about 73.6%. By combining the optimum extracts and SEC purification protocols, an enriched fraction containing 245.6 mg pristimerin was obtained from 100 g of root bark, representing about 14.4%, w/w, pristimerin from the total solids presented in the fraction.
Plant tissue culture provides an alternative approach to improve the quality of soybean (Glycine max (L.) Merrill) cultivars. This study was undertaken to analyze the susceptibility of Mexican soybean for direct shoot regeneration and to determine the critical factors that affect in vitro performance. Our hypothesis was that Mexican soybean is suitable for in vitro regeneration using a cotyledonary node as explant. The effects of the seed disinfection procedure, soaking pretreatment before germination, soybean variety, as well as the culture medium composition of the shoot induction medium, were evaluated by two split-plot statistical designs. According to the statistical analysis, the seed disinfection procedure, the soaking pretreatment before germination, and the soybean genotype were the factors that brought about a significant effect (p£0.01), while the hormones composition of the shoot induction medium did not have a significant effect. The best response for multiple shoot formation was observed using a chlorine gas seed disinfection method, 3% hydrogen peroxide soaking pretreatment, and Huasteca-100, Nainari and Suaqui soybean genotypes. A robust protocol was developed, and under these selected conditions, it is possible to obtain more than 10 shoots per explant. Well-developed plantlets were obtained after 60 d of in vitro culture.
The process of brewing is a complex one, in which several biological and chemical reactions occur that involve many variables and their interactions. This pilot study is an attempt to understand and to control the chemical and biological nature of the process of 'beer cooking'. Through data collection and analysis the measurement system was initially evaluated and improved to allow the assessment of the stability of the analysed response variable: wort's F (F is a fictitious name for this variable due to confidentiality). Next, a deeper analysis was carried out to characterize, improve and control the behaviour of this factor by means of confidence intervals and several regression analyses. The way to control F is by adding a certain amount of element X according to a previously empirically developed table. After the analyses, this table was questioned and a new one was developed. This study is the outcome of the willingness of a group of people in this company to incorporate into its traditional and, at some stages, artisan way of producing beer, the utilization of statistical techniques for analysing and improving its processes and products.SPC, brewing process, quality improvement,
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