Quantitation of certolizumab pegol by validated liquid chromatography methods

Abstract Certolizumab pegol (CZP) is a Fab' fragment of the humanized antibody with anti-TNF-α activity that is indicated as therapy for Crohn's disease and rheumatoid arthritis. Using a BioSep-SEC-S3000 column (300 x 4.6 mm i.d., 5 µm particle size), a size exclusion liquid chromatography (SEC) method was developed. Mobile phase A consisted of 100 mM monobasic sodium phosphate and 200 mM sodium chloride (pH 7.0), while mobile phase B was ethanol (95:5, v/v), and the analysis was performed using a diode array detector (DAD) set to 214 nm and a flow rate of 0.5 ml min-1. In addition, a reversed-phase liquid chromatography (RP-LC) method based on gradient elution was developed on a Zorbax 300 SB C18 column (150 mm x 4.6 mm i.d., 3.5 µm particle size) kept at 80 °C. Mobile phase A was 0.1% (v/v) TFA in ultrapure water, and mobile phase B was a mixture of propanol, acetonitrile, ultrapure water and TFA (70 + 20 + 9.9 + 0.1, v/v) operated at a flow rate of 1.0 ml min-1, and DAD was applied at 214 nm. CZP elution was achieved with retention times of 5.6 min and 9.0 min for SEC and RP-LC, respectively.

Anti-inflammatory activity and identification of the Verbena litoralis Kunth crude extract constituents

Verbena litoralis is a plant popularly known as "gervãozinho-do-campo" in Portuguese. It is traditionally used for stomach, liver and gallbladder problems, and as an anti-inflammatory and anthelmintic. The goal of this study was to determine the chemical composition of the crude extract obtained from the aerial parts of V. litoralis by Ultra High Performance Liquid chromatography coupled with Mass Spectrometry in Tandem (UHPLC-MS/MS); assess the anti-inflammatory activity of ethyl acetate fraction and of crude extract; and verify liver, kidney and pancreas damage. In this study, the chemical composition of the extract was identified via UHPLC/MS/MS, assessing the anti-inflammatory activity of the crude extract and the acetate fraction in an induction model of the granulomatous tissue, as well as given liver, kidney and pancreas damage markers. Chlorogenic acid, luteolin, caffeic acid, apigenin, p-coumaric acid, vanillic acid, ferulic acid and quercetin were quantified in the extract. After the seven-day treatment, the granuloma of the animals treated with the plant extract and fraction presented values very close to the positive control (nimesulide). The V. litoralis crude extract and ethyl acetate fraction show anti-inflammatory activity similar to the nimesulide without evidence of liver, kidney and pancreas damage, which attributes the plant's pharmacological action to the flavonoids found.

Evaluation of recombinant human interferon beta 1b by liquid chromatography methods and bioassay

Recombinant human interferon beta 1b (rhIFNß-1b) is clinically used to treat multiple sclerosis. A reversed-phase liquid chromatography (RP-LC) method was carried out on a Jupiter C4 column (250 mm × 4.6 mm i.d.). The mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) in water, and the mobile phase B was acetonitrile with 0.1% TFA run at a flow rate of 1.0 mL/min. A size exclusion liquid chromatography (SE-LC) method was carried out on a BioSep-SEC-S 2000 column (300 mm × 7.8 mm i.d.). The mobile phase consisted of 1 mM monobasic potassium phosphate, 8 mM sodium phosphate dibasic and 200 mM sodium chloride buffer pH 7.4, run isocratically at a flow rate of 0.8 mL/min. Retention times were 31.87 and 17.78 min, and calibration curves were linear over the concentration range of 1-200 µg/mL (r2 = 0.9998) and 0.50-200 µg/mL (r2 = 0.9999), respectively, for RP-LC and SE-LC, with detection at 214 nm. Liquid chromatography (LC) methods were validated and employed in conjunction with the in vitro bioassay to assess the content/potency of rhIFNß-1b, contributing to improve the quality control and to ensure the efficacy of the biotherapeutic

Atividade anti-inflamatória e avaliação da toxicidade do extrato hidroetanólico de Morus alba (Moraceae) / Anti-inflammatory activity and biochemical parameters of the hydroethanolic leaf extract of Morus alba (Moraceae)

No presente trabalho, avaliou-se a atividade anti-inflamatória do extrato hidroetanólico das folhas de Morus alba, através do modelo de indução de tecido granulomatoso e analisaram-se os efeitos toxicológicos sobre o fígado pela dosagem de AST e ALT e sobre o rim, pela dosagem de creatinina. O extrato hidroetanólico das folhas de M. alba foi administrado oralmente, três vezes ao dia, durante 6 dias. A nimesulida (5 mg/kg/dia) foi utilizada como controle positivo e o propilenoglicol 20% como controle negativo. Após o tratamento, foi avaliada a formação do granuloma e realizada a dosagem de AST, ALT e creatinina em todos os grupos. Os animais tratados com o extrato de M. alba apresentaram inibição do processo inflamatório de 20,24 ± 6,94%, enquanto que os tratados com controle positivo apresentaram 21,42 ± 6,52%. O extrato hidroetanólico de M. alba demonstrou atividade anti-inflamatória semelhante à nimesulida com ausência de indícios de hepatotoxicidade e nefrotoxicidade.

In this study, the anti-inflammatory activity of a hydroethanolic extract of leaves of Morus alba (white mulberry) was assessed in a model of granulomatous tissue induction and the toxicological effects on the liver were analyzed by assaying AST and ALT, and those on the kidney by determining creatinine. The hydroethanolic leaf extract was administered orally three times a day six days. Nimesulide (5 mg /kg/day) was used as a positive control and 20% propylene glycol as a negative control. After the 6-day treatment, granuloma formation was assessed and the levels of AST, ALT and creatinine assayed in all groups. Animals treated with the extract showed inflammation inhibition of 20.24 ± 6.94%, while those treated with positive control showed 21.42 ± 6.52%. The hydroethanolic extract of M. alba thus exhibited anti-inflammatory activity similar to nimesulide, with an absence of hepatotoxicity or nephrotoxicity.

Atividade Antimicrobiana e Citotoxicidade do extrato bruto obtido de Morus Alba L. (Moraceae) / Antimicrobial Activity and Cytotoxicity of crude extract obtained of Morus Alba L. (Moraceae)

O presente trabalho avaliou a atividade antimicrobiana in vitro do extrato hidroetanólico e frações hexânica, clorofórmica, acetato de etila e butanólica das folhas de Morus alba L. A concentração inibitória mínima (CIM) foi determinada frente à Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Aspergillus fumigatus e Prothoteca zophii. As frações que apresentaram melhores respostas para a atividade antimicrobiana foram acetato de etila e clorofórmica com CIM de 256 ?g/mL. Não foi possível detectar atividade antimicrobiana para Aspergillus fumigatus em nenhuma das concentrações testadas. A citotoxidade do extrato hidroetanólico foi avaliada através de culturas de células de ovário de hamster chinês (CHO) e células do tecido conectivo de camundongo (NCTC) clone 929, determinando o índice de citotoxidade (IC50). O IC50 foi de 0,34 mg/mL para as células CHO e 3,24 mg/mL para as células NCTC 929. De modo geral, as frações acetato de etila e clorofórmica das folhas de M. alba L. apresentaram moderada atividade antimicrobiana e o extrato bruto demonstrou ação citotóxica in vitro frente as células CHO e NCTC 929.

This study evaluated the in vitro antimicrobial activity of hydroethanolic extract and hexane, chloroform, ethyl acetate and butanol fractions from Morus alba L. leaves. The minimum inhibitory concentration (MIC) was determined using Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Aspergillus fumigatus and Prothoteca zophii. The fractions that better responded the antimicrobial activity were ethyl acetate and chloroform with CIM of 256 ?g/mL. It was unable to detect antimicrobial activity for Aspergillus fumigatus in the tested concentrations. The cytotoxicity of the hydroethanolic extract was evaluated by cell cultures of chinese hamster ovary (CHO) and connective tissue of mouse clone 929 (NCTC), determining the level of cytotoxicity (IC50). The IC50 obtained was 0.34 mg/mL for CHO and 3.24 mg/ml for NCTC 929 cells. In general, ethyl acetate and chloroform fractions from M. alba L. leaves showed moderate antimicrobial activity and its extract presented in vitro cytotoxicity against CHO and NCTC 929 cells.

Avaliação de pirogênios em produtos de uso veterinário pelos testes da hipertermia em coelhos e do lisado de amebócitos do Limulus / Pyrogens in veterinary products by the rabbit pyrogen test and the Limulus amoebocyte lysate test

Realizou-se a avaliação de pirogênios em produtos veterinários de uso parenteral, pelo método da hipertermia em coelhos, calculando-se, para o teste das amostras, doses com concentrações de três a sete vezes superiores à terapêutica. Preconizou-se como resposta positiva o aumento de temperatura de 0,6°C. Utilizou-se também o ensaio do lisado de amebócitos do Limulus (LAL) por geleificação, semiquantitativo, executando o teste de interferentes, validando o procedimento e estabelecendo a máxima diluição válida para a análise de cada produto. Paralelamente, efetuou-se avaliação comparativa de amostras com o método do LAL cromogênico, quantitativo, demonstrando correlação e reprodutibilidade dos resultados. Avaliaram-se vinte e oito produtos de diferentes classes farmacológicas, observando-se que dois não cumpriram as especificações, sendo reprovados. Sugere-se que as especificações estudadas sejam adotadas, contribuindo para aprimorar o controle de contaminantes, garantindo a qualidade e a segurança dos produtos veterinários.

The rabbit pyrogen test was used to evaluate veterinary products, suggesting the temperature rise of 0.6°C as the ending point for the positive results. The test doses were calculated based on the therapeutic dose increased from three to seven times. The semi-quantitative Limulus amoebocyte lysate (LAL) gel clot test was performed and compared to the LAL spectrophotometric chromogenic, quantitative assay. The comparative evaluation of the samples showed correlation and reproducibility of the results. The interference test was carried out, the procedure validated and the maximum valid dilution established for the analysis of the products without Pharmacopoeial specifications. Two of the twenty-eight products of different pharmacological groups evaluated didn't meet the requirements and were reproved. The specifications investigated are suggested to be used for the purity evaluation of the veterinary parenteral products, as a contribution to assure the quality and safety of the products.

Análise físico-química de hormônio de crescimento humano recombinante em diferentes etapas do processo de produção / Physico-chemical analysis of recombinant human growth hormone in different steps of the production process

Padronizou-se metodologia de cromatografia líquida de alta eficiência em fase reversa (RP-HPLC) isocrática para determinação de hormônio de crescimento humano recombinante (rec-hGH) em extratos periplásmicos de E. coli. O procedimento possibilitou uma rápida análise inicial da qualidade e quantidade de hGH secretado no espaço periplásmico, imediatamente após, ou mesmo durante a fermentação. Considerando que a RP-HPLC não identifica isômeros de massa, estes foram determinados através da análise paralela dos extratos periplásmicos por cromatografia líquida de alta eficiência por exclusão molecular (HPSEC) e radioimunoensaio (RIE) das frações eluídas. O método permitiu obter em 24 h, do início do processo de fermentação, uma avaliação da atividade, identidade, rendimento e contaminantes relacionados ao hGH. Entre esses incluem-se os sulfóxidos e desamidados, dímero e formas de alta massa molecular. As metodologias padronizadas foram empregadas em diferentes etapas da produção de rec-hGH. O processo de fermentação de E. coli transformada foi otimizado, escolhendo-se o melhor tempo de ativação, que propiciou maior nível de secreção de hGH e a menor quantidade de formas alternadas. Analisou-se uma das primeiras fases do processo de purificação, confirmando-se a ausência de perdas significativas em formas poliméricas. A liofilização foi aprimorada, possibilitando a preparação e estudo de diferentes padrões. Todos esses estudos acrescentaram importantes conhecimentos para o controle de qualidade do produto farmacêutico injetável. que foi realizado de acordo com as recomendações das Farmacopéias Européia e Brasileira

Dosagem biológica do antiveneno botrópico / Biological evaluation of the bothropic antivenin

The determination of the LD50 of bothropic reference venom and the evaluation of the respective antivenin potency were standardized by intraperitoneal inoculation in mice. The computer program was developed for the calculations of the LD50 and the potency(ED50) by probit analysis. The final results were consistent and reproducibles. The weight obtained in independent assays was between 374 and 1762. The precision index was < 0,10