Design and Validation of a Novel Generic Platform for the Production of Tetravalent IgG1-like Bispecific Antibodies.

We have designed and validated a novel generic platform for production of tetravalent IgG1-like chimeric bispecific Abs. The VH-CH1-hinge domains of mAb2 are fused through a peptidic linker to the N terminus of mAb1 H chain, and paired mutations at the CH1-CL interface mAb1 are introduced that force the correct pairing of the two different free L chains. Two different sets of these CH1-CL interface mutations, called CR3 and MUT4, were designed and tested, and prototypic bispecific Abs directed against CD5 and HLA-DR were produced (CD5xDR). Two different hinge sequences between mAb1 and mAb2 were also tested in the CD5xDR-CR3 or -MUT4 background, leading to bispecific Ab (BsAbs) with a more rigid or flexible structure. All four Abs produced bound with good specificity and affinity to CD5 and HLA-DR present either on the same target or on different cells. Indeed, the BsAbs were able to efficiently redirect killing of HLA-DR(+) leukemic cells by human CD5(+) cytokine-induced killer T cells. Finally, all BsAbs had a functional Fc, as shown by their capacity to activate human complement and NK cells and to mediate phagocytosis. CD5xDR-CR3 was chosen as the best format because it had overall the highest functional activity and was very stable in vitro in both neutral buffer and in serum. In vivo, CD5xDR-CR3 was shown to have significant therapeutic activity in a xenograft model of human leukemia.

Validation of plant dynamic model by online and laboratory measurements – a tool to predict online COD loads out of production of mechanical printing papers

COD discharges out of processes have increased in line with elevating brightness demands for mechanical pulp and papers. The share of lignin-like substances in COD discharges is on average 75%. In this thesis, a plant dynamic model was created and validated as a means to predict COD loading and discharges out of a mill. The assays were carried out in one paper mill integrate producing mechanical printing papers. The objective in the modeling of plant dynamics was to predict day averages of COD load and discharges out of mills. This means that online data, like 1) the level of large storage towers of pulp and white water 2) pulp dosages, 3) production rates and 4) internal white water flows and discharges were used to create transients into the balances of solids and white water, referred to as “plant dynamics”. A conversion coefficient was verified between TOC and COD. The conversion coefficient was used for predicting the flows from TOC to COD to the waste water treatment plant. The COD load was modeled with similar uncertainty as in reference TOC sampling. The water balance of waste water treatment was validated by the reference concentration of COD. The difference of COD predictions against references was within the same deviation of TOC-predictions. The modeled yield losses and retention values of TOC in pulping and bleaching processes and the modeled fixing of colloidal TOC to solids between the pulping plant and the aeration basin in the waste water treatment plant were similar to references presented in literature. The valid water balances of the waste water treatment plant and the reduction model of lignin-like substances produced a valid prediction of COD discharges out of the mill. A 30% increase in the release of lignin-like substances in the form of production problems was observed in pulping and bleaching processes. The same increase was observed in COD discharges out of waste water treatment. In the prediction of annual COD discharge, it was noticed that the reduction of lignin has a wide deviation from year to year and from one mill to another. This made it difficult to compare the parameters of COD discharges validated in plant dynamic simulation with another mill producing mechanical printing papers. However, a trend of moving from unbleached towards high-brightness TMP in COD discharges was valid.

Validation of an analytical methodology for the quantitative analysis of petroleum hydrocarbons in marine sediment samples

This work describes a validation of an analytical procedure for the analysis of petroleum hydrocarbons in marine sediment samples. The proposed protocol is able to measure n-alkanes and polycyclic aromatic hydrocarbons (PAH) in samples at concentrations as low as 30 ng/g, with a precision better than 15% for most of analytes. The extraction efficiency of fortified sediments varied from 65.1 to 105.6% and 59.7 to 97.8%, for n-alkanes and PAH in the ranges: C16 - C32 and fluoranthene - benzo(a)pyrene, respectively. The analytical protocol was applied to determine petroleum hydrocarbons in sediments collected from a marine coastal zone.

Validation of a simple and rapid UV spectrophotometric method for dexamethasone assay in tablets

This work reports the validation of an analytical UV spectrophotometric method to assay dexamethasone in tablets (assay and dissolution studies). The method was linear in the range between 1 and 30 µg mL-1 presenting a good correlation coefficient (r = 0.9998, n = 7). Precision and accuracy analysis showed low relative standard deviation (< 2.00%) and good percentual recoveries (95-105%). The procedure was linear, accurate, precise, and robust. The method is simple, and it has low cost. It does not use polluting reagents and can be applied in dissolution studies, being an adequate alternative to assay dexamethasone in tablets.

Development and validation of UV spectrophotometric method for determination of levofloxacin in pharmaceutical dosage forms

The objective of this research was to develop and validate an alternative analytical method for quantitative determination of levofloxacin in tablets and injection preparations. The calibration curves were linear over a concentration range from 3.0 to 8.0 μg mL-1. The relative standard deviation was below 1.0% for both formulations and average recovery was 101.42 ± 0.45% and 100.34 ± 0.85% for tablets and injection formulations, respectively. The limit of detection and limit of quantitation were 0.08 and 0.25 μg mL-1, respectively. It was concluded that the developed method is suitable for the quality control of levofloxacin in pharmaceuticals formulations.

Development and validation of a LC-method for the determination of phenols in a pharmaceutical formulation containing extracts from Stryphnodendron adstringens

A sensitive RP-HPLC method with UV detection successfully measured phenol(s) in an ointment containing 3% Stryphnodendron adstringens extract. Chromatography used acetonitrile (0.05% trifluoroacetic acid):water (0.05% trifluoroacetic acid) (v/v), flow rate 0.8 mL min-1. Quantitation was accomplished by the external-standard method. Linearity for 2.00 to 16.00 μg mL-1 (gallic acid) and 1.14 to 18.24 μg mL-1 (gallocatechin) was established. Intra- and inter-day precision levels were under 5%. LOD and LOQ were 0.231 and 0.770 μg mL-1 (gallic acid) and 0.151 and 0.504 μg mL-1 (gallocatechin), respectively. Determination of phenols was unaffected by product excipients.

Development and validation of a method for the analysis of tetracyclines in chicken-muscle by liquid chromatography-electrospray-mass spectrometry in tandem (LC-ESI-MS/MS)

A LC-ESI-MS/MS method was developed and validated according to the European Union decision 2002/657/EC, for the determination of tetracyclines (TCs) in chicken-muscle since Europe is one of the main markets for Brazilian products. Linearity of r > 0.9979, limits of quantification in the range of 7.0-35.0 ng/g, average recoveries of 89.38 - 106.27%, within-day and between-day precision were adequate for all TCs. The decision limit and the detection capability were 93.00-106.46 ng/g and 95.84-114.38 ng/g, respectively. This method is suitable for application in surveillance programmes of residues of TCs in chicken-muscle samples.

Development and validation of a high performance liquid chromatographic method for determination of cyclosporine-A from biodegradable intraocular implants

An HPLC method was developed and validated aiming to quantify the cyclosporine-A incorporated into intraocular implants, released from them; and in direct contact with the degradation products of PLGA. The separation was carried out in isocratic mode using acetonitrile/water (70:30) as mobile phase, a C18 column at 80 ºC and UV detection at 210 nm. The method provided selectivity based on resolution among peaks. It was linear over the range of 2.5-40.0 µg/mL. The quantitation and detection limits were 0.8 and 1.2 µg/mL, respectively. The recovery was 101.8% and intra-day and inter-day precision was close to 2%.

Development and validation of a stability-indicating UPLC method for rosuvastatin and its related impurities in pharmaceutical dosage forms

The present work describes a novel stability-indicating reversed-phase ultra performance liquid chromatography method for the separation and quantification of rosuvastatin (RSV) and its related impurities in the pharmaceutical dosage forms under forced degradation conditions. An unknown degradation impurity detected in the acid degradation was identified by using quadrupole time-of-flight mass spectrometry. The chromatographic separation was carried out on C-18 column (100 x 2.1 mm, 1.7 μm) using isocratic elution with methanol and 0.1% trifluoroacetic acid (50:50). The total run time was 12 min within which RSV as well as all related impurities and degradation products were separated. The developed method was validated for RSV and related impurities in pharmaceutical dosage forms.

Validation of a spectrophotometric method to estimate the adsorption on nanoemulsions of an antimalarial oligonucleotide

This study describes the validation of a spectrophotometric method to estimate oligonucleotides association with cationic nanoemulsions. Phosphodiester and phosphorothioate oligonucleotides targeting Plasmodium falciparum topoisomerase II were analyzed at 262 nm. Linear response (r > 0.998) was observed from 0.4 to 1.0 nmol/mL, the relative standard deviation values for the intra- and inter-days precision were lower than 2.6% and the recovery ranged from 98.8 to 103.6% for both oligonucleotides. The association efficiency was estimated based on an ultrafiltration/centrifugation method. Oligonucleotides recovery through 30 kDa-membranes was higher than 92%. The extent of oligonucleotides association (42 to 98%) varied with the composition of nanoemulsions

Development and validation of a method for the analysis of Ochratoxin A in roasted coffee by liquid chromatography/electrospray-mass spectrometry in Tandem (LC/ESI-MS/MS)

A method using LC/ESI-MS/MS for the quantitative analysis of Ochratoxin A in roasted coffee was described. Linearity was demonstrated (r = 0.9175). The limits of detection and quantification were 1.0 and 3.0 ng g-1, respectively. Trueness, repeatability and intermediate precision values were 89.0-108.8%; 2.4-13.7%; 12.5-17.8%, respectively. To the best of our knowledge, this is the first report in which Ochratoxin A in roasted coffee is analysed by LC/ESI-MS/MS, contributing to the field of mycotoxin analysis, and it will be used for future production of Certified Reference Material.

Development and validation of a novel stability indicating RP-UPLC method for simultaneous determination of nizatidine, methylparaben and propylparaben in oral liquid pharmaceutical formulation

A selective and accurate stability-indicating gradient reverse phase ultra performance liquid chromatographic method has been developed and validated for the simultaneous determination of nizatidine, methylparaben and propylparaben in pharmaceutical oral liquid formulation. The separation was achieved on Acquity UPLC TM HSS T3 1.8 µm column by using mobile phase containing a gradient mixture of solvent A (0.02 Mol L-1 KH2PO4, pH 7.5) and B (60:40 v/v mixture of methanol and acetonitrile) at flow rate of 0.4 mL min-1. Drug product was exposed to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. The developed method was validated as per international ICH guidelines with respect to specificity, linearity, accuracy, precision and robustness.

Development and validation of a High Performance Liquid Chromatographic method for determination of etoposide in biodegradable polymeric implants

A method using HPLC-UV was developed and validated for the determination of etoposide incorporated into polycaprolactone implants. The method was carried out in isocratic mode using a C18 column (250 x 4.6 mm; 5 µm), at 25 ºC, with acetonitrile and acetic acid 4% (70:30) as mobile phase, a flow rate of 2 mL/min, and UV detection at 285 nm. The method was linear (r² > 0.99) over the range of 5 to 65 µg/mL, precise (RSD < 5%), accurate (recovery of 98.7%), robust, selective regarding excipient of the sample, and had a quantitation limit equal to 1.76 µg/mL. The validated method can be successfully employed for routine quality control analyses.

Validation of an hplc method for the determination of 1,7-dihydroxy-2,3-methylenedioxyxanthone in extracts obtained from Polygala cyparissias A. St. Hil. & Moq. (Polygalaceae) and evaluation of the influence of its content on the gastroprotective activity

Polygala cyparissias is a plant widespread in Southern Latin America. Recently, we demonstrated the gastroprotective activity of the extract, as well as for one of the isolated metabolites-1,7-dihydroxy-2,3-methylenedioxyxanthone (MDX). In this study, a HPLC method for the quantification of MDX was validated. The HPLC method was linear (0.5-24 µg mL-1 of MDX) with good accuracy, precision and robustness. The content of MDX in the extracts from whole and different parts of the plant ranged from 0 to 5.4 mg g-1 and the gastroprotective index ranged from 72.1 to 99.1%. Thus, the method might be used for the standardization of the extracts based on the MDX marker.