Screening and optimization of process parameters of SARS-CoV-2 monoclonal antibody F61 in affinity chromatography stage using design of experiment / 中国生物制品学杂志

@#Objective To select and optimize the process parameters of SARS-CoV-2 F61 affinity chromatography by the screening and optimization experiment of Design of Experiment(DoE),in order to obtain the optimal process conditions.Methods Eight process parameters that may affect the experimental response results in affinity chromatography were selected and their level ranges were determined.DoE screening test was used to perform 8 factor 2 level screening tests on the selected process parameters.The response values were detected and the mathematical model was fitted by statistical software.Three key process parameters significantly affecting the key quality attributes were obtained by Pareto diagram analysis(P < 0.05).Then DoE response surface method(RSM)was selected to optimize the key process parameters.First,the full factorial experiment design was completed,the response results were detected to fit the mathematical model,and the bending term P value was analyzed to judge the range of significant factors in the optimal range(bending P < 0.05),then according to the sequential complement of the central composite face-centered design(CCF)experiment,through the detection of the response results to fit the response surface model,the range of optimal conditions was obtained,and the stability of the optimal parameters was finally verified by repeated experiments.Results In the screening experiment,it was found that the significant factors affecting F61 in the affinity chromatography stage were elution buffer pH,elution buffer salt concentration,leaching buffer salt concentration and equilibrium buffer salt concentration.The optimal conditions of key process parameters in affinity chromatography were obtained by CCF.When the pH of elution buffer was 3.2,the elution buffer salt concentration was 0.07 mol/L NaCl,and the leaching buffer salt concentration was 0.31 mol/L NaCl,the yield of F61 reached 95.25%,the residual amount of host cell protein(HCP)was 97.33 ppm,and the monomer purity of the sample was98.51% at the affinity chromatography stage.Conclusion Different types of DoE methods were used to screen and optimize the process parameters of F61 affinity chromatography stage,and the optimal process conditions were obtained,which lays a foundation for the esta-blishment of F61 purification process.

Development and verification of a reporter gene determination assay for ADCP potency of IgG2 anti-EGFR monoclonal antibody based on DOE / 中国生物制品学杂志

@#Objective To develop and verify a reporter gene assay for the determination of antibody dependent cellular phagocytosis(ADCP)potency of Ig G2 monoclonal antibody(m Ab)against epidermal growth factor receptor(EGFR)by combining Design of Experiment(DOE)and one factor at a time(OFAT).Methods The Jurkat/NFAT-Re/FcγRⅡa stably transformed cell line was used as effector cells,while the A431 cell line as the target cells.The JMP software was used to optimize the seven key factors in the experiment by combining DOE and OFAT analysis,while the ratio of upper and lower asymptotes(D/A)was used as the statistic,and the reporter gene method was developed to evaluate the ADCP potency of Ig G2 anti-EGFR m Ab.The method was verified according to the general chapter<9401>of Chinese Pharmaco-poeia（Ⅲ/Ⅳvolume,2020 edition)and used to determine the biological potency of Ig G2 anti-EGFR m Ab injection.Results After three rounds of experiments,the reporter gene method to evaluate the ADCP potency of Ig G2 anti-EGFR m Ab was developed.The method showed a dose-response relationship and was consistent with the four-parameter regression equa-tion y=(A-D）/[1+(x/C)~B]+D.The range of seven key conditions was determined:the density of effector cells was(1.25-3.75）×10~4 cells/well,the density ratio of effector cells to target cells was 1.0-2.0,the incubation time of target cells was 20-40 min,the incubation time of administration was 15-30 min,the total time was 5.5-6.5 h,and the color time was 5-30 min with luciferase detection system(Bright-Glo)as the color agent.The method had good specificity.Six independent tests were run for the five potency levels,with the correlation coefficient r of 0.994 5 and the linear regression equation slope of 1.02.The relative potency of five potency levels respectively was(62.15±1.38)%,(78.53±2.82)%,(99.12±3.95)%,(123.27±4.59)%and(155.22±7.04)%,the range of relative biases was-2.9%-0.2%,and the range of generalized cross-validation(GCV)was 2.2%-4.6%.The method had good linearity,relative accuracy and precision in the range of 64%-156%.The mean value of the potency of IgG2 anti-EGFR m Ab in three tests was(101.5±2.8)%.Conclusion The reporter gene assay developed in this study can be used to evaluate the ADCP potency of IgG2 anti-EGFR mAb

DEVELOPMENT OF ORODISPERSIBLE TABLETS OF LORATADINE CONTAINING AN AMORPHOUS SOLID DISPERSION OF THE DRUG IN SOLUPLUS® USING DESIGN OF EXPERIMENTS

Objective: The objective of the present work was to develop an orodispersible tablet of loratadine, an orally active, non-sedating anti-histaminic, belonging to BCS Class II. The drug was prepared as a solid dispersion using Soluplus® as carrier and formulated into an optimal tablet using Design of Experiments.Methods: Solid dispersions of loratadine with varying ratios of Soluplus® were prepared by solvent evaporation and subjected to solubility study in simulated salivary fluid. Selected composition was characterized by differential scanning calorimetry and X-ray diffraction and formulated into an orodispersible tablet by direct compression after addition of suitable excipients. DOE based on a full factorial design was used to optimize the product using a trial version of JMP software, so as to obtain a tablet with low friability, rapid disintegration and maximal drug dissolution within 5 min. The optimized tablet was prepared and evaluated for several attributes, including in vivo disintegration and palatability.Results: A solid dispersion prepared with a 1: 4 ratio of loratadine: Soluplus® was found to show a 130-fold increase in drug solubility in the simulated salivary fluid. X-ray diffraction revealed loratadine in amorphous form. The exercise using DOE for optimization of the orodispersible tablet formula served to balance the proportion of crospovidone as super disintegrant and PVP as dry binder and yielded a formulation with good mechanical strength, rapid in vitro disintegration (39 sec) and dissolution of 93.78% of the drug within 5 min. When evaluated in vivo, the tablets were found to disintegrate in about 60 secs and were reported to be palatable.Conclusion: A patient-friendly dosage form containing a highly soluble form of loratadine was prepared and could be of potential benefit in offering quick relief from allergic conditions.

Application of Design of Experiment in development of purification process of recombinant human growth hormone-Fc fusion protein / 中国生物制品学杂志

@#ObjectiveTo optimize the condition for anion exchange chromatography in purification process of recombinant human growth hormone-Fc(rhGH-Fc)fusion protein by Design of Experiment(DOE)so as to decrease the content of host cell protein(HCP)in bulk of protein.MethodsA complete factorial experimental design with four factors at two levels was established by the DOE in Minitab 19 software.The condition(flow rate,sample load,pH value of buffer and salt concentration of eluent)for anion exchange chromatography in purification process of rhGH-Fc was optimized by DOE to find out the operating space.ResultsThe experimental results were predicted accurately by the established DOE model.The sample load,pH value of buffer,salt concentration of eluent as well as the interaction of pH value of buffer and salt concentration of eluent showed significant influence on the HCP content in the harvest.The optimal sample load,flow rate as well as pH value and salt concentration of eluent were 15 mg/mL,120 cm/h,7.0 ～ 8.0 and 0.1 mol/L respectively.The HCP contents in eluents under the optimal condition were less than 400 ng/mg,which met the requirements for verification within the range of results in determined operating space.ConclusionThe condition for removal of HCP by anion exchange chromatography was successfully optimized by DOE,which provided a reference for further application of DOE in the biopharmaceutical field.

Successful Uterine Detorsion and its Therapeutic Management in a Doe

The present case describes successful uterine detorsion and its therapeutic management in a non-descript goat.

Successful C-section of a Non-descriptive Goat to Relieve from Dystocia

Present case study was associated with successful management of a pregnant doe from dystocia by caesarean section. A 1.5 years old pregnant non-descriptive doe was admitted in the TVCC, C.V.A.Sc., Pantnagar, Uttarakhand with a history of complete gestation period and prolonged labor (more than 12 hours). There was protrusion of both fetal forelimbs from the vaginal opening. Gynaecological examination revealed that the fetus was in Nape presentation. The case was diagnosed as dystocia due to abnormal fetal disposition. The animal was subjected to caesarean operation. The operation was performed under light sedation with 0.1 ml of xylazine and regional inverted L- block was also done with 2% Lignocaine. An oblique incision was given at left flank and recovery of one dead male fetus occurred. Post-operative treatment was done with Inj. Amoxicillin-sulbactum @10 mg/kg intramuscularly, Inj. Tribivet® 5 ml intramuscularly, Inj. Chlorpheniramine maleate @0.5 mg/kg intramuscularly, Meloxicum@0.5 mg/kg intramuscularly and NS 250 ml for five days. Local antiseptic dressing and fly repellant spray was advised for every alternative day. The skin suture was removed on 10th day post-surgery.

Recent research progress in key technologies of Chinese materia medica manufacturing process quality control / 中草药

The development of Chinese materia medica (CMM) has risen to the level of national strategy. Under the new situation that the pharmaceutical industry implements the "Made in China 2025" strategy, quality control of the production process of CMM is one of the key areas in which the CMM industry needs to accelerate its breakthrough. The key common issues in process design, analysis and detection, process modeling, and manufacturing equipment and other aspects in the field of quality control of CMM production processes was analyzed in the paper. The progress in the three aspects of process understanding, real-time analysis method development and process control strategy establishment in the quality control system of CMM production process was reviewed. Combined with the author's corporate research practices, this paper introduces the application progress of key technologies such as quality by design (QbD), process analytical technology (PAT), experimental design (DOE), and multivariate statistical analysis in the above three research directions, and analyzes the difficulties problems in practical industrial application. The application prospect is prospected. The purpose of this article is to provide reference for CMM enterprises to apply and improve the quality control technology in the production process.

Development of a Novel Optimization and Validation Method Based on DOE for the ADCC Potency of Anti-PD-1/PD-L1 Monoclonal Antibodies Using Reporter Genes / 中国药学杂志

OBJECTIVE: To develop a novel optimization and validation method based on design of experiment(DOE) for the antibody-dependent cell-mediated cytotoxicity (ADCC) potency of anti-programmed cell death 1 and anti-programmed cell death-ligand 1 (PD-1/PD-L1) monoclonal antibodies using reporter genes. METHODS: Jurkat-hFcγRIIIa-NFAT transgenic cell line was used as effector cells, 293FT-PD-1 cell line and CHO-PD-L1 cell line were used as target cells, respectively. The ADCC potency for anti-PD-1/PD-L1 monoclonal antibodies was detected with Luciferase detection system (BrightGloTM Luciferase Assay system), then the method was optimized and validated based on DOE. RESULTS: The anti-PD-1/PD-L1 monoclonal antibodies showed a dose-response relationship and the determination result complied with the following four-parameter equation: y=(A-D)/+D. The method was optimized and the testing parameters were determined as follows: the working concentration of anti-PD-1 monoclonal antibody was 10 000 ng•mL-1 to 4.833 ng•mL-1 and that of anti-PD-L1 was 2 000 ng•mL-1 to 0.488 ng•mL-1, the ratio of effector cells and target cells for anti-PD-1/PD-L1 monoclonal antibodies were 6:1 and 3:1, and the induction time for both of these antibodies was 20 h. The method possessed good specificity. The recovery rate test samples in the four different dilution groups were determined for 3 times, and the results showed that the relative potencies of anti-PD-1 monoclonal antibody were (51.74±2.22)%, (77.12±3.14)%, (118.71±2.83)% and (156.20±12.99)%, and the recoveries of which were (103.49±4.44)%, (102.83±4.19)%, (94.96±2.26)% and (104.14±8.66)%, respectively. While as for anti-PD-L1 monoclonal antibody, the relative potencies were (54.32±4.75)%, (75.24±4.25)%, (127.40±2.43)%, (156.82±3.27)% and the recoveries were (108.64±9.51)%, (100.33±5.67)%, (101.92±1.94)% and (104.55±2.18)%, respectively. The RSDs of the above results were all less than 10%. CONCLUSION: A novel optimization and validation method based on DOE for detecting ADCC potency of anti-PD-1/PD-L1 mAb is successfully developed. This detecting method based on reporter gene shows high specificity, good reproducibility and high accuracy, and might be used in the evaluation of ADCC potency of anti-PD-1/PD-L1 mAb.

Production of L-asparaginase of pharmaceutical nterest from yeasts isolated from the Antarctic continent / Produção de L-asparaginase de interesse farmacêutico a partir de leveduras isoladas do continente Antártico

The L-asparaginase (ASNase) obtained from yeasts species has been poorly studied and a new yeast ASNase could be an alternative to minimize the side effect in the treatment of lymphoblastic leukemia. The Antarctic ecosystems have a great potential to obtain novel enzymes produced from psychrophilic and psychrotolerant microorganisms. Yeasts isolated from samples collected in the Antarctic Peninsula by the PROANTAR expedition team were tested for the production of ASNase and L-glutaminase (GLNase). From this screening, the strain Leucosporidium scottii L115 presented the highest ASNase activity (6.24 U g-1 of dried cell weight (dcw)) with a combination of low GLNase activity (0.41 U g-1 dcw). The ASNase belonging to L. scottii L115 (LsASNase) was purified 227 fold with a specific activity of 137.01 U mg-1 at 37 ºC, and with 0.93 U mg-1 for GLNase. Moreover, the maximum activity was observed at pH 7.5 at 55 ºC. The enzyme is a multimer presenting a single band of 54.5 kDa of molecular weight in reduced conditions and 462 kDa by size exclusion chromatography. The LsASNase is a glycosylated enzyme that presented a band lower at 25 kDa when was treated with PGNase F. The enzymatic kinetic reveals an allosteric regulation of the enzyme and the kinetic parameters were determined at 37º C, pH 7.0 as K0.5 = 233 µM, kcat = 54.7 s-1 and nH = 1.52 demonstrating a positive cooperativity by the enzyme and the substrate. The ASNase production by L. scottii L115 was improved by applying DoE for the culture medium development. The PB and CDD designs were used to optimize the ASNase production providing the nutrient values of 6.15 g L-1 of proline, 28.34 g L-1 sucrose, and 15.61 g L-1 of glycerol for a maximal production. The synthetic medium containing the optimized quantities was added with the salts: KCl, 0.52 g L-1; MgSO4.7H2O, 0.52 g L-1; CuNO3.3H2O, 0.001 g L-1; ZnSO4.7H2O, 0.001 g L-1; FeSO4.7H2O, 0.001 g L-1.The optimized medium produces a 23.75 ULh-1 of ASNase in shake flask culture. Furthermore, L. scottii is characterized as an oleaginous yeast that accumulates lipids with a suitable fatty acid profile. The production of ASNase and lipids were scaled up in the 1 L bioreactor to evaluate the initial cell concentration, carbon source, and oxygen transfer rate (kLa).The experiments were performed at 15ºC in the bioreactor BIOSTAT®Q plus (Sartorius Stedim, Germany) in batch mode, using 0.5 L of the optimized medium culture in phosphate buffer 50 mM pH 7.0. The initial cell concentration was evaluated at 1%, 3%, and 5% (v/v). Sucrose and glycerol were tested alone to examine if the combination of both is mandatory to produce ASNase. All these assays were carried in duplicate. The kLa was assessed through a CCD design in the range of 1.42 - 123.0 h-1. The performance in bioreactor showed the productivity of 36.95 ULh-1of ASNase under the optimized conditions (growth temperature 15º C, X0: 5 g L-1, pH 7.0, 48 h, kLa 89-92 h-1). The cultivation of L. scottii L115 at 15ºC in sucrose and glycerol as carbon sources generate an interesting lipid profile, where it presents monounsaturated and polyunsaturated lipids

A L-asparaginase (ASNase) obtida a partir de espécies de leveduras tem sido pouco estudada e uma nova ASNase de levedura pode ser uma alternativa para minimizar os efeitos adversos no tratamento da leucemia linfoblástica. Os ecossistemas Antárticos têm um grande potencial para obter novas enzimas produzidas a partir de microorganismos psicrofílicos e psicotrolerantes. As leveduras isoladas de amostras coletadas na Península Antártica pela equipe de expedição do PROANTAR foram testadas para a produção de ASNase e L-glutaminase (GLNase). A partir desta triagem, a cepa Leucosporidium scottii L115 apresentou a maior atividade de ASNase (6,24 U g-1 dcw) com uma combinação de baixa atividade de GLNase (0,41 U g-1 dcw). A ASNase pertencente a L. scottii L115 (LsASNase) foi purificada 227 vezes com uma atividade específica de 137,01 U mg-1 a 37 ºC e com 0,93 U mg-1 de GLNase. A atividade máxima foi observada a pH 7,5 a 55 ºC. A enzima é um multímero que apresenta uma banda única de 54,5 kDa de peso molecular em condições redutoras e 462 kDa por cromatografia de exclusão molecular. A LsASNase é uma enzima glicosilada que apresentou uma banda menor a 25 kDa quando tratada com PGNase F. A cinética enzimática revela uma regulação alostérica da enzima e os parâmetros cinéticos foram determinados a 37º C, pH 7,0 como K0,5 = 233 µM, kcat = 54,7 s-1 e nH = 1,52 demonstrando uma cooperatividade positiva pela enzima e o substrato. A produção de ASNase por L. scottii L115 foi melhorada aplicando DoE para o desenvolvimento do meio de cultura. Os desenhos experimentais de PB e CDD forma usados para otimizar a produção de ASNase e forneceram os valores de nutrientes de 6,15 gL-1 de prolina, 28,34 gL-1 de sacarose e 15,61 gL-1 de glicerol para uma produção máxima. O meio sintético contendo as quantidades otimizadas foi adicionado com os sais: : KCl, 0.52 g L-1; MgSO4.7H2O, 0.52 g L-1; CuNO3.3H2O, 0.001 g L-1; ZnSO4.7H2O, 0.001 g L-1; FeSO4.7H2O, 0.001 g L-1.O meio otimizado produz 23.75 ULh-1 de ASNase em cultivo em frasco agitado. Além disso, L. scottii é caracterizada como uma levedura oleaginosa que acumula lipídios com um perfil adequado de ácidos graxos. A produção de ASNase e lipídios foi ampliada no biorreator de 1 L para avaliar a concentração celular inicial, fonte de carbono e taxa de transferência de oxigênio (kLa). Os experimentos foram realizados a 15ºC no biorreator BIOSTAT®Q plus (Sartorius Stedim) em modo batelada, utilizando 0,5 L da cultura de meio otimizado em tampão fosfato 50 mM pH 7,0. A concentração celular inicial foi avaliada em 1%, 3% e 5% (v / v). Sacarose e glicerol foram testados isoladamente para examinar se a combinação de ambos é obrigatória para produzir ASNase. Todos esses ensaios foram realizados em duplicado. O kLa foi avaliado através de um planejamento CCD na faixa de 1,42-123,0 h-1. O desempenho no biorreator mostrou a produtividade de 36,95 ULh-1 de ASNase sob condições otimizadas (temperatura de crescimento 15º C, X0: 5 g L-1, pH 7,0, 48 h, kLa 89-92 h-1). O cultivo de L. scottii L115 a 15ºC em sacarose e glicerol como fontes de carbono gera um perfil lipídico interessante, onde apresenta lipídios monoinsaturados e poliinsaturados

Design of Experiments (DoE) applied to Pharmaceutical and Analytical Quality by Design (QbD)

According to Quality by Design (QbD) concept, quality should be built into product/method during pharmaceutical/analytical development. Usually, there are many input factors that may affect quality of product and methods. Recently, Design of Experiments (DoE) have been widely used to understand the effects of multidimensional and interactions of input factors on the output responses of pharmaceutical products and analytical methods. This paper provides theoretical and practical considerations for implementation of Design of Experiments (DoE) in pharmaceutical and/or analytical Quality by Design (QbD). This review illustrates the principles and applications of the most common screening designs, such as two-level full factorial, fractionate factorial, and Plackett-Burman designs; and optimization designs, such as three-level full factorial, central composite designs (CCD), and Box-Behnken designs. In addition, the main aspects related to multiple regression model adjustment were discussed, including the analysis of variance (ANOVA), regression significance, residuals analysis, determination coefficients (R2, R2-adj, and R2-pred), and lack-of-fit of regression model. Therefore, DoE was presented in detail since it is the main component of pharmaceutical and analytical QbD.

Desenvolvimento e otimização de protetores solares empregando os conceitos de qualidade por design (QbD) e tecnologia analí­tica de processos (PAT) / Development and Optimization of sunscreen applying Quality by Design (QbD) and Process Analytical Technology (PAT), 2018. (Master Degree)

Os protetores solares (PS) são os grandes responsáveis pela proteção da pele quando exposta à radiação solar, por isso a importância sanitária de se otimizar o desenvolvimento deste cosmético tipo II e monitorar para que seja eficaz em seu propósito. O principal objetivo deste trabalho é aplicar os conceitos de Qualidade por Design (QbD), ferramentas estatísticas de desenho experimental (DoE - Design of Experiments) e o conceito de tecnologia analítica de processo (PAT - Process Analytical Technology) para desenvolver uma formulação e processo produtivo de um PS de modo a modernizar os processos da indústria cosmética, fazendo as análises durante o processo e eliminando o controle de qualidade final. Trata-se de um sistema de desenvolvimento sistematizado, onde se executa as ferramentas de QbD para avaliar os dados obtidos ao longo da fase experimental. Para a fase experimental, empregou-se o desenho fatorial e desenho do compósito central (CCD - Central Composite Design) como ferramenta estatística, para a execução do planejamento de experimentos (DoE - Design of Experiments). As respostas foram analisadas através da metodologia de superfície resposta (RSM - Response Surface Methodology). Tais ferramentas são fundamentais para a determinação do desenho de concepção (design space), para se obter o PS com as melhores características físico-químicas e de processo dentro do escopo delineado. Para o desenvolvimento da metodologia de análise in line, optou-se pela utilização da espectrometria UV, utilizando-se ferramentas como análise de regressão dos mínimos quadrados (PLS) devido a praticidade em transforma-la em uma ferramenta PAT, para isto, a quimiometria foi empregada para modelar sistemas que são desconhecidos e complexos, como um PS, e trazendo respostas diretas como a aprovação do produto antes de ser embalado, por exemplo. A abordagem apresentada baseia-se na construção da qualidade ao longo do desenvolvimento e otimização de PS e torna possível o monitoramento da qualidade em tempo real

The sunscreens are great responsible for the skin protection when it is exposed to direct sunlight, so it means a great importance of health to optimize the development of type II cosmetic and monitor for it to be effective in its purpose. The objective of this work is to apply the concepts of Quality by Design and statistical tools of experimental design (DoE - Design of experiments), as well as applying the process analytical technology (PAT - Process Analytical Technology) concept for formulation and manufacturing process development of a topical sunscreen being able to modernize the cosmetic industry processing, including real time analyses and eliminating quarantine step, which waits analysis approval performed by the quality assurance, and then release the product for sale. As it is a systematic development, where critical quality attributes and risk assessment were performed to evaluate over obtained data. During experimental phase, the factorial design was used as a statistical tool for design of experiments implementation, and the responses were analyzed by response surface methodology (RSM - Response Surface Methodology). This mapping is critical to determination of the product design (design space), i.e. get sunscreen with the best physical and chemical characteristics and processing within the outlined scope. For in line methodology development, UV spectrometry was opted to be used due to less effort in sample preparation and due to great easiness to turn it into a PAT tool. For this, chemometrics was used, which brings together chemical and statistical elements to obtain three main elements: empirical modeling, multivariate modeling and chemical data, making it able to model systems that are unknown and complex, as a sunscreen, getting direct answers as product release approval before being packed, for example. The presented approach was based on the construction of quality throughout the sunscreen development and optimization making possible the real time quality monitoring

Optimisaton techniques: a futuristic approach for formulating and processing of pharmaceuticals.

Designing and formulating an ideal pharmaceutical product is a very tedious job for a formulator as it comprises of multiple objectives. The traditional method followed for years is not only expensive and time consuming but it also needs lot of effort to be put in and in spite of that at times it proves to be unfavourable and unpredictable too.The recent approach to optimise i.e. to achieve the best combination of product and process characteristics under the given conditions is by using Design of Experiment (DoE).Design of Experiment (DoE) is an organised approach to determine the relationship between the factors (Xs) affecting a process and the output of that process (Ys).The recent optimisation methodologies include various approaches that come under the 2 main categories namely, simultaneous optimisation and sequential optimisation. Nowadays there are various software available to carry out the optimisation of pharmaceutical products.

Analytical quality-by-design approach for sample treatment of BSA-containing solutions / 药物分析学报

The sample preparation of samples containing bovine serum albumin (BSA), e.g., as used in transdermal Franz diffusion cell (FDC) solutions, was evaluated using an analytical quality-by-design (QbD) approach. Traditional precipitation of BSA by adding an equal volume of organic solvent, often successfully used with conventional HPLC-PDA, was found insufficiently robust when novel fused-core HPLC and/or UPLC-MS methods were used. In this study, three factors (acetonitrile (%), formic acid (%) and boiling time (min)) were included in the experimental design to determine an optimal and more suitable sample treatment of BSA-containing FDC solutions. Using a QbD and Derringer desirability (D) approach, combining BSA loss, dilution factor and variability, we constructed an optimal working space with the edge of failure defined as Do0.9. The design space is modelled and is confirmed to have an ACN range of 8373%and FA content of 170.25%.

Batch production and media optimization of anti-leukemic L-asparaginase from Pseudomonas fluorescens by Taguchi DOE methodology.

L-Asparaginase isolated from bacterial sources has been shown to possess antileukemic characteristic, mainly against the acute lymphoblastic leukemia. Large number of bacterial strains have been reported which can produce it. The production level of the enzyme by the bacterial sources is very low and hence the factors have to be distinguished which effect the growth of cells and production of enzyme directly or indirectly, in order to decrease the cost of treatment. The production of L-Asparaginase was optimized in classical fermentation process by the use of Taguchi DOE methodology. L-18 array was selected for the purpose of media optimization. The five factors at three levels were considered for the optimization. L-Asparaginase production was significantly (p<0.05) affected by the interaction of two factors present in the culture broth. The factors interaction viz. energy source-nitrogen source, energy source-phosphate source etc. having pronounced effect on the production while individually they have the minimum impact on L-Asparaginase production. So the interactions of different parameters were studied and they were used to formulate the optimized condition for the production of LAsparaginase. After the validation of result by performing the experiment under optimized condition there was about 28.48% increase in the production of the enzyme.

DoE Approach: A Stability Indicating RP-HPLC Method for Simultaneous Estimation of Methylparaben, Mometasone furoate and Eberconazole nitrate in Topical Formulations.

A rapid and sensitive stability indicating RP-HPLC method is developed for the simultaneous estimation of Methylparaben, Mometasone Furoate and Eberconazole Nitrate in topical formulations. Chromatographic separation was achieved on Waters Xterra C18 (150 × 4.6 mm, 5μm) using a mobile phase constituted of water and methanol (35:65, v/v) at a flow rate of 1.50 mL/min and column temperature of 30˚C. All three components were measured with uv detection at 235 nm. Force degradation study was conducted to determine Methylparaben, Mometasone Furoate and Eberconazole Nitrate in the presence of degradants and excipients peaks. Developed method was validated for method precision, specificity, linearity, accuracy, robustness and solution stability as per ICH guidelines. Method is showing linearity in the range of 0.25-188, 0.50-75 and 2.0- 750 μg/mL for Methylparaben, Mometasone Furoate and Eberconazole Nitrate respectively. The method was proved to be robust by conducting DOE study. The method is suitable for stability studies, routine analysis and quality control of topical formulations containing these components, either alone or in combination.

Histometric parameters of uterus and vagina in breeder female rabbits on the day after weaning according to the ordinal number of parturitions / Parámetros histométricos en útero y vagina de conejas reproductoras en el día posterior al destete según el número ordinal de partos

The objective of this study was to establish in New Zealand female rabbits, the effect of ordinal number of parturitions on some histological parameters on the day after weaning. Tissue fragments of uterus and vagina were obtained from females of first, second and third parturitions and were processed imbedding them in paraffin in order to do histological cuts. In eight microscope fields captured by a camera connected to an optic microscope, the lumen and glandular endometrial epithelium height, as well as the thickness of vaginal and myometrium epithelium thickness were measured. From the results that were obtained it was concluded that there are significant differences (p<0.05) in histological parameters evaluated in uterus and vagina between does of different parturitions, which indicates that the number of parturitions has an effect on histometric characteristics of genital tract organs in breeder female rabbits.

El objetivo del presente estudio fue determinar en conejas Nueva Zelanda el efecto del número ordinal de partos sobre algunos parámetros histológicos, en el día posterior al destete. A partir de hembras de primero, segundo y tercer parto se obtuvieron fragmentos de tejido uterino y vaginal los que se procesaron mediante la técnica de inclusión en parafina para efectuar cortes histológicos. En ocho campos microscópicos capturados con una cámara conectada a un microscopio óptico se midió la altura del epitelio luminal y glandular del endometrio; el grosor del epitelio de la vagina y del miometrio. A partir de los resultados obtenidos se concluye que existen diferencias significativas (p<0,05) en los parámetros histológicos evaluados en útero y vagina entre las conejas de distintos partos, lo que indica que el número de partos tiene un efecto sobre las características histométricas de los órganos del tracto genital de la coneja.

Macroscopic morphometric analysis of genital organs of rabbit does with differing parturitions at 24 hours post-weaning / Análisis morfométrico macroscópico de órganos genitales de conejas con diferentes partos a las 24 horas post-destete

The purpose of this study was to analyze the macroscopic morphometry of the ovaries, uterus and vagina in rabbit does in the first, second and third parturition at 24 hours post-weaning in order to determine if there are differences between parturitions. Weaning of the litter was performed at 30 days post-partum and 24 hours later the does were euthanized. Right and left ovaries, uterine horns-cervix and vagina were removed, and the length of each one was measured. Significant differences were found in the average of the right ovary length between the first group with respect to second and third parturition group (P 0.05 and P 0.01). Also, left ovary length was different between the first and second with respect to third parturition group (P <0.001). Average total ovary length increased significantly as the number of parturitions increased in second and third parturition groups (P 0.01 and P0.001). The weight of the left and right ovaries was higher in does in their second and third parturition when compared to those in their first (P 0.5; P 0.01). The average total ovary weight increased significantly as the number of parturitions in second and third parturition groups (P0.001). The length of the left and right uterine horn-cervix of the rabbits after second and third parturition decreased when compared to that of females in their first parturition (P 0.05; P0.001). The average total uterine horns-cervix length decreased significantly as the number of parturitions in second and third parturition groups (P0.001). Furthermore, vagina length in females in their third parturition was greater than that of first and second parturition (P 0.01).The color of the vulva of the majority of females in their first parturition (3/4) was pale pink while. In contrast, the majority of females in their second parturition (3/4) had their vulva intensely red. All of the females (4/4) in the third parturition group had their vulva intensely red...

El propósito de este estudio fue analizar la morfometría macroscópica de los ovarios, útero y vagina en conejas de primero, segundo y tercer parto a las 24 horas post-destete con el propósito de determinar si existen diferencias entre los partos. El destete de la camada se realizó a los 30 días después del parto y 24 horas después fueron eutanasiadas. De cada animal se removieron los ovarios derecho e izquierdo, cuernos-cuello uterinos derecho e izquierdo y la vagina y se midió la longitud de cada uno de los órganos genitales. Se encontraron diferencias significativas en la longitud del ovario derecho entre el grupo de primer parto con respecto al de segundo y tercer parto (P 0,05 y P 0,01). Además, la longitud media de ovario izquierdo fue significativamente diferente entre el grupo de primero y segundo parto con respecto al de tercer parto (P <0,001). La longitud total de los ovarios aumentó con el número de partos y fue mayor en los grupos de segundo y tercer parto (P 0,01 y P 0,001). El peso de los ovarios derecho e izquierdo fue mayor en el grupo de segundo y tercer parto al compararlo con el de primero parto (P 0,5; P 0,01). Por otra parte, el peso total de los ovarios aumentó en los grupos de segundo y tercer parto (P 0,001). La longitud del cuerno-cuello uterino izquierdo y derecho de los grupos de segundo y tercer parto disminuyó al compararlo con el de las hembras de primer parto (P 0,05; P 0,001). La longitud total del cuerno-cuello uterino izquierdo y derecho disminuyó significativamente en las hembras de segundo y tercer parto (P 0,001). La longitud de la vagina de hembras de tercer parto fue mayor que la de primero y segundo parto (P 0,01). El color de la vulva de la mayoría de las hembras de primer parto (3/4) fue rosa pálido. En contraste, la mayoría de las hembras en su segundo parto (3/4) presentaron vulva de color rojo intenso. Todas las hembras del grupo de tercer parto (4/4) presentaron vulva de color rojo intenso...

Development of ethosomes with taguchi robust design-based studies for transdermal delivery of alfuzosin hydrochloride.

In the present investigation efficiency of ethosomes as novel lipid carriers for transdermal delivery of Alfuzosin Hydrochloride (AH) has been evaluated. Taguchi robust design method was used for optimization of ethosomal formulations. Phospholipid type, concentration of phospholipid and concentration of ethanol was selected as independent variables and their effect on the dependent variables (entrapment efficiency and flux) was studied. Ethosomal formulation (EA8) with soya phosphatidylcholine (3%) and ethanol 20% were optimized. Vesicles were spherical, unilamellar with smooth surface. The optimized formulation exhibited vesicle size (6.85 ± 1.35μm), zeta potential (-8.14 ± 0.62mv), entrapment efficiency (91.86 ± 3.25%), flux (27.42 ± 0.04μg/cm2/hr), lag time (0.26±0.20hr) and skin deposition (298.01 ± 15.4μg/g). Transdermal flux was enhanced by 6.92 times over drug solution. Vesicle skin interaction studies showed fatty change in the dermis. The formulations were stable at 4°C for 120 days. Results suggested ethosomes as efficient carriers for AH transdermal delivery.

Applying design of experiments (DOE) to flavonoid extraction from Passiflora alata and P. edulis

The Passifloraceae family is extensively used in native Brazilian folk medicine to treat a wide variety of diseases. The problem of flavonoid extraction from Passiflora was treated by application of design of experiments (DOE), as an experiment with mixture including one categorical process variable. The components of the binary mixture were: ethanol (component A) and water (component B); the categorical process variable: extraction method (factor C) was varied at two levels: (+1) maceration and (-1) percolation. ANOVA suggested a cubic model for P. edulis extraction and a quadratic model for P. alata.These results indicate that the proportion of components A and B in the mixture is the main factor involved in significantly increasing flavonoid extraction. In regard to the extraction methods, no important differences were observed, which indicates that these two traditional extraction methods could be effectively used to extract flavonoids from both medicinal plants. The evaluation of antioxidant activity of the extract by ORAC method showed that P. edulis displays twice as much antioxidant activity as P. alata. Considering that maceration is a simple, rapid and environmentally friendly extraction method, in this study, the optimized conditions for flavonoid extraction from these Passiflora species is maceration with 75% ethanol for P. edulis and 50% ethanol for P. alata.

Functionality advancement of poorly soluble biovariable anti hypertensive Drug by sophisticated SD-FBP technology as per enhanced QbD.

Lacidipine (LCDP) is a dihydropyridine derivative categorized as an Anti-hypertensive Ca+2 channel blocker belonging to BCS class IV drug with low solubility and low permeability which presents a challenge to the formulation scientists. The development of a solid dispersion by solvent evaporation is a practically viable method to enhance dissolution of LCDP from oral dosage form. Solvent evaporation by Fluidized Bed Process (FBP) was the method of choice for SD as it improves wettability with simultaneous increase in porosity of granules resulting enhanced surface area producing higher dissolution rate and bioavailability of poorly water-soluble drug. Thus, the main object of the present invention is to provide stable pharmaceutical dosage form of LCDP with desired dissolution rate i.e. at least 80% drug release within 45 minutes, without use of disintegrant(s) and/or surfactant(s) or without micronization of the active ingredient per se. One more object of this invention is to provide a sophisticated robust process for the preparation of said pharmaceutical dosage form by Quality by Design (QbD) concept focusing on thorough understanding of the product and process by which it is developed and manufactured along with a knowledge of the risks involved in manufacturing by IRMA & FMEA study of the product with process and how best to mitigate those risks by developing design space with DoE & MVDA with outlined control strategy.