Content/Potency Assessment of Botulinum Neurotoxin Type-A by Validated Liquid Chromatography Methods and Bioassays.

Botulinum neurotoxin type-A (BoNTA) is one of the seven different serotypes (A to G) produced by Clostridium botulinum. A stability-indicating size-exclusion chromatography (SEC) method was developed and validated, and the specificity was confirmed by forced degradation study, interference of the excipients, and peaks purity. The method was applied to assess the content and high-molecular-weight (HMW) forms of BoNTA in biopharmaceutical products, and the results were compared with those of the LD50 mouse bioassay, the T-47D cell culture assay, and the reversed-phase chromatography (RPC) method, giving mean values of 0.71% higher, 0.36% lower, and 0.87% higher, respectively. Aggregated forms showed significant effects on cytotoxicity, as well as a decrease in the bioactivity (p < 0.05). The employment of the proposed method in conjunction with the optimized analytical technologies for the analysis of the intact and altered forms of the biotechnology-derived medicines, in the correlation studies, enabled the demonstration of the capability of each one of the methods and allowed for great improvements, thereby assuring their safe and effective use.

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

Evaluation of recombinant human parathyroid hormone by CZE method and its correlation with in vitro bioassay and LC methods.

A stability-indicating capillary zone electrophoresis (CZE) method was validated to assess the content/potency of the recombinant human parathyroid hormone (rhPTH 1-34), using ranitidine as internal standard (IS). A fused-silica capillary, (i.d. of 50µm; effective length of 40cm) was used at 25°C; the applied voltage was 20kV. The background electrolyte solution consisted of 50mmolL-1 sodium dihydrogen phosphate solution at pH 3.0. Injections were performed using a pressure mode at 50 mbar for 45s, with detection by photodiode array (PDA) detector set at 200nm. Separation was obtained with a migration time of 5.3min, and was linear over the concentration range of 0.25-250µgmL-1 (r2 =0.9992). Specificity and stability-indicating capability were established in degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.28% with bias lower than 0.85%. Analyses of the same batches showed mean differences of the estimated content/potencies of 0.61%, 1.31% higher and 0.86% lower as compared to the validated reversed-phase and size exclusion liquid chromatography methods, and to the UMR-106 cell culture bioassay, respectively, with non-significant differences (p>0.05). Degraded forms were also subjected to the in vitro cytotoxicity test. The results obtained showed the capabilities of each one of the methods, and constitute an alternative strategy to monitor stability, improve the quality control and ensure the batch-to-batch consistency of bulk and finished biotechnology-derived medicine.

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).