Ricardo Bizogne Souto

Stability-indicating capillary zone electrophoresis method for the assessment of recombinant human granulocyte-macrophage colony-stimulating factor and its correlation with reversed-phase liquid chromatography method and bioassay

A stability-indicating capillary zone electrophoresis (CZE) method was validated for the analysis of granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using leuprorelin acetate (LA), as internal standard (IS). A fused-silica capillary (75 μm i.d.; effective length, 72 cm) was used at 25 °C; the applied voltage was 12 kV. The background electrolyte solution consisted of 50mM di-sodium hydrogen phosphate solution at pH 8.8. Injections were performed using a pressure mode at 50 mbar for 9s, with detection by photodiode array detector set at 200 nm. Specificity and stability-indicating capability were established in degradation studies, which also showed that there was no interference of the excipients. The method was linear over the concentration range of 2.5-200 μg mL(-1) (r(2)=0.9995) and the limit of detection (LOD) and limit of quantitation (LOQ) were 0.79 μg mL(-1) and 2.5 μg mL(-1), respectively. The accuracy was 99.14% with bias lower than 1.40%. The method was applied to the quantitative analysis of biopharmaceutical formulations, and the results were correlated to those of a validated reversed-phase LC method (RP-LC), and an in vitro bioassay, showing non-significant differences (p>0.05).

Simultaneous Determination of Nimesulide and Valdecoxib by Micellar Electrokinetic Capillary Chromatography Method

Abstract A micellar electrokinetic capillary chromatography (MEKC) method for the analysis of nimesulide and valdecoxib, using celecoxib as internal standard, has been developed and validated. The MEKC method was carried out on a fused silica capillary (50 µm I.D., effective length 56 cm). The background electrolyte consisted of 35 mM borate buffer and 35 mM of anionic detergent SDS (pH 9.75)/acetonitrile (95:5, V/V). The capillary temperature was maintained at 35°C, the applied voltage was 30 kV, and the injection was performed using the pressure mode at 50 mbar for 5 s, with detection at 234 nm using a photodiode array detector. Method validation investigated parameters such as the linearity (r2=0.9999), range, precision, accuracy, robustness, and specificity, giving results within the acceptable range. The detection limit calculated for nimesulide and valdecoxib was 0.25 and 0.86 µg mL−1, respectively, and the quantitation limit evaluated experimentally was 2 µg mL−1 for both the compounds. The proposed method was successfully applied for the quality control analysis of pharmaceutical products and the results compared to the liquid chromatography method, showing non‐significant difference (P>0.05).

Assessment of recombinant human parathyroid hormone: correlation of LC methods with bioassays

Reversed-phase liquid chromatography (RP-LC) and size exclusion liquid chromatography (SE-LC) methods were validated for the assessment of recombinant human parathyroid hormone (rhPTH 1-34). The gradient RP-LC method was carried out on a Zorbax 300 SB C(18) column (150 mm × 4.6 mm i.d.), maintained at 40 °C. The mobile phase A consisted of 0.1 M sodium sulphate buffer, pH 2.3, and the mobile phase B was acetonitrile. The SE-LC method was carried out on a BioSep-SEC-S 2000 column (300 mm × 7.8 mm i.d.), maintained at 25 °C. The mobile phase consisted of 0.1 M phosphoric acid buffer, pH 2.5, run isocratically at a flow rate of 0.7 mL min(-1). Chromatographic separation was obtained with retention times of 12.2 min, and 13.2 min, and was linear over the concentration range of 1-250 μg mL(-1) (r(2) = 0.9997) and 2-300 μg mL(-1) (r(2) = 0.9993), respectively, for RP-LC and SE-LC, with photodiode array (PDA) detection at 214 nm. Specificity was established in degradation studies, which also showed that there was no interference of the excipients. Equally, the accuracy was 100.49% and 100.22%, with bias lower than 1.12% and 0.81% respectively. Moreover, the in vitro cytotoxicity test of related proteins and higher molecular weight forms showed significant differences (p < 0.05). Chromatographic methods were applied for the content/potency assessment of rhPTH and related proteins in biopharmaceutical injectable dosage forms, and the results were correlated with those of in vitro and in vivo bioassays. It is concluded that the employment of the methods in conjunction allows a great improvement in monitoring stability, contributing to evaluate alternatives which improve the quality control and thereby assure the therapeutic efficacy of the biotechnology-derived medicine.

Validation of an RP-LC Method for the Determination of Interferon-α2a in Pharmaceutical Formulations

Abstract A reversed phase liquid chromatography (RP-LC) method was validated for the determination of interferon-α2a in pharmaceutical formulations. The RP-LC method was carried out on a Jupiter C4 column (250 mm × 4.6 mm I.D.), maintained at room temperature. The mobile phase A consisted of 0.1% TFA and the mobile phase B was acetonitrile with 0.1% TFA, run at a flow rate of 1 mL/min, and using photodiode array (PDA) detection at 214 nm. The chromatographic separation was obtained with the retention time of 32.6 min, and was linear in the concentration range of 0.5–50 MIU/mL (r 2 = 0.9999). The specificity was proven through degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.84% with bias lower than 1.87%. The limits of detection and quantitation were 0.19 and 0.5 MIU/mL, respectively. Moreover, method validation demonstrated acceptable results for precision and robustness. The proposed method was applied for the analysis of the interferon-α2a and their related proteins in parenteral dosage forms, contributing to establishing alternatives to improve the quality control assuring the therapeutic efficacy of the biological medicine.

Stability-indicating capillary zone electrophoresis method for the assessment of recombinant human interleukin-11 and its correlation with reversed-phase liquid chromatography and biossay.

A stability-indicating capillary zone electrophoresis (CZE) method was validated for the analysis of recombinant human interleukin-11(rhIL-11) using rupatadine fumarate, as internal standard (IS). A fused-silica capillary, (50 µm i.d.; effective length, 40 cm) was used at 25°C; the applied voltage was 20 kV. The background electrolyte solution consisted of 50 mmol L(-1) sodium dihydrogen phosphate solution at pH 3.0. Injections were performed using a pressure mode at 50 mbar for 45 s, with detection by photodiode array detector set at 196 nm. Specificity and stability-indicating capability were established in degradation studies, which also showed that there was no interference of the excipients. The method was linear over the concentration range of 1.0-300 µg mL(-1) (r(2)=0.9992) and the limit of detection (LOD) and limit of quantitation (LOQ) were 0.2 µg mL(-1) and 1.0 µg mL(-1), respectively. The accuracy was 100.4% with bias lower than 1.1%. Moreover, the in vitro cytotoxicity test of the degraded products showed significant differences (p<0.05). The method was applied for the content/potency assessment of rhIL-11 in biopharmaceutical formulations, and the results were correlated to those of a validated reversed-phase LC method (RP-LC) and an TF-1 cell culture assay, showing non-significant differences (p>0.05). In addition the CZE and RP-LC methods were applied for the analysis of rhIL-11 in human plasma. Therefore, the proposed alternative method can be applied to monitor stability, to assure the batch-to-batch consistency and quality of the bulk and finished biotechnology-derived medicine.

Assessment of recombinant human erythropoietin by alternative bioassay and its correlation with liquid chromatography methods

Biotechnology-derived erythropoietin is a sialoglycoprotein, which stimulates erythropoiesis, and it is clinically used for the treatment of anaemia related to chronic renal failure. N-Acetylneuraminic acid content was quantified by a reversed-phase liquid chromatography method with fluorescence detection giving values higher than 108.74 ng μg−1. An alternative in vitro TF-1 cell proliferation bioassay was studied showing a lower mean difference of the estimated potency of 2.67%, compared to the normocythaemic mice bioassay, with non-significant differences (p > 0.05). Biopharmaceutical products were also analyzed by validated reversed-phase and size-exclusion liquid chromatography methods and compared to the in vivo bioassay, showing lower mean differences of the content/potencies of 2.11 and 1.21%, respectively. Higher molecular mass forms and deamidated/sulphoxide forms showed mean bioactivities reduced to about 10%, for both. The TF-1 cell culture assay in conjunction with the determination of sialic acids represents an advance that can be correlated with the normocythaemic mice bioassay and the physicochemical methods, allowing for the establishment of alternative methods, which can be applied to monitor the stability, quality control, and thereby ensuring the therapeutic efficacy of the biological medicine.

Granulocyte-macrophage colony stimulating factor: Evaluation of biopharmaceutical formulations by stability-indicating RP-LC method and bioassay.

The granulocyte-macrophage colony stimulating factor (GM-CSF) is a cytokine that regulates the proliferation and differentiation of hematopoietic cells and activates granulocytes and macrophages. A reversed-phase liquid chromatography (RP-LC) method was validated for the assessing of the stability of non-glycosylated recombinant rhGM-CSF (Molgramostim) in biopharmaceutical formulations. The RP-LC method was carried out on a Jupiter C(4) column (250 mm × 4.6 mm i.d.), maintained at 45 °C. The mobile phase A consisted of 0.1% TFA and the mobile phase B was acetonitrile with 0.1% TFA in acetonitrile, run at a flow rate of 1 mL/min, and using photodiode array (PDA) detection at 214 nm. Chromatographic separation was obtained with a retention time of 29.2 min, and was linear over the concentration range of 2-300 μg/mL (r(2) = 0.9992). Specificity was established in degradation studies. Moreover, the in vitro cytotoxicity test of the degraded products showed significant differences (p < 0.05). The method was applied to the assessment of rhGM-CSF and related proteins in biopharmaceutical dosage forms, and the results were correlated to those of a bioassay. It is concluded that the employment of RP-LC in conjunction with current methods allows a great improvement in monitoring stability, quality control and thereby assures the therapeutic efficacy.

Biological potency evaluation and physicochemical characterization of unfractionated heparins

Unfractionated heparins are used clinically as anticoagulants. The biological potency of thirteen samples of raw material and pharmaceutical formulations were assessed utilizing the 5th International Standard of heparin using the sheep plasma coagulation inhibition assay, activated partial thromboplastin time, anti-factor Xa assay, and anti-factor IIa assay, resulting in mean potencies of 101.15%, 96.15%, 98.15% and 99.37%, respectively. The samples were also evaluated by the protamine neutralization test giving results within the range of 92 - 138 IU/mg. The anti-factor IIa assay was performed showing reproducibility and significant correlation with the pharmacopoeial assays, thus demonstrating it to be a feasible alternative to the sheep plasma coagulation inhibition assay. Moreover, an analysis by nuclear magnetic resonance and capillary electrophoresis showed some peaks attributable to oversulfated chondroitin sulfate. The results show that batch-to-batch variations and the quality of samples contributed to improvements in the quality control of pharmaceutical products and to assure the safe use and clinical efficacy of this biological medicine.

Validation of a stability-indicating micellar electrokinetic capillary method for the assessment of febuxostat and its correlation with the reversed-phase LC method

A stability-indicating micellar electrokinetic capillary chromatography (MEKC) method was validated for the analysis of febuxostat in pharmaceutical formulations, using lisinopril as the internal standard (IS). A fused-silica capillary (50 μm i.d.; effective length, 40 cm) was maintained at 30 °C, and the applied voltage was 20 kV. The background electrolyte solution consisted of 15 mmol L−1 sodium tetraborate buffer and 25 mmol L−1 sodium dodecyl sulphate solution at pH 10. Injections were performed using a pressure mode at 50 mbar for 45 s, with detection using a photodiode array detector set at 216 nm. Specificity and stability-indicating capability were established in degradation studies, which also showed that there was no interference of the excipients. The method was linear over the concentration range of 0.10–50 μg mL−1 (r2 = 0.9993) and the limit of detection (LOD) and limit of quantitation (LOQ) were 0.05 μg mL−1 and 0.10 μg mL−1, respectively. The accuracy was 99.89% with a relative error lower than 1.04%. The proposed method was applied to the quantitative analysis of febuxostat in tablet dosage forms and in human plasma, and the results were correlated with those of a validated reversed-phase (RP-LC) method, in attempts to improve quality control of pharmaceutical products.