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.

Reversed-phase and size-exclusion liquid chromatography methods were validated for the assessment of streptokinase. The reversed-phase method was carried out on a Jupiter C column (250 mm × 4.6 mm id) maintained at 25°C. The mobile phase consisted of 50 mM sodium sulfate solution pH 7.0 and methanol (90:10, v/v), run isocratically at a flow rate of 0.8 mL/min. The size-exclusion method was carried out on a Protein KW 802.5 column (300 mm × 8.0 mm id), at 25°C. The mobile phase consisted of 40 mM sodium acetate solution pH 7.0, run isocratically at a flow rate of 1.0 mL/min. Retention times were 19.3 min, and 14.1 min, and calibration curves were linear over the concentration range of 0.25-250 μg/mL (25.75-25 750 IU/mL) (r = 0.9997) and 5-80 μg/mL (515-8240 IU/mL) (r = 0.9996), respectively, for reversed-phase and size exclusion, with detection at 220 and 204 nm. Chromatographic methods were employed in conjunction with the in vitro bioassay for the content/potency assessment of Streptokinase, contributing to improve the quality control and ensure the efficacy of the biotherapeutic.

Recombinant human interferon beta 1b (rhIFNβ−1b) is clinically used to treat multiple sclerosis. A reversed−phase liquid chromatography (RP−LC) method was carried out on a Jupiter C 4 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 (r 2 = 0.9998) and 0.50−200 µg/mL (r 2 = 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 rhIFNβ−1b, contributing to improve the quality control and to ensure the efficacy of the biotherapeutic.

Background: Botulinum neurotoxins (BoNTs) are among the most potent toxins known and are also used for therapeutic and aesthetic applications. Objective: An alternative in vitro cell culture bioassay based on the induction of apoptosis on T−47D breast cancer cells, after exposure to BoNTA, was developed and validated. Methods: The T-47D cells (ATCC HTB−133) were seeded at a density of 3 × 105 cells mL−1, and the bioassay was performed with doses of BoNTA, between 3 and 81 U mL−1. The responses were assessed using 10 µL of Alamar Blue®. The absorbances were read at 570 and 600 nm. Results: The results were compared with those of the in vivo LD50 mouse bioassay, showing a non-significant 1.08% higher, mean difference of the estimated potencies (p>0.05). Besides, the biopharmaceutics is analyzed by the size exclusion and reversed-phase liquid chromatography methods, showing a significant correlation with values 1.15% higher and 0.85% lower, respectively, related to the cell culture bioassay. Conclusion: It is concluded that the validated T−47D cell culture assay represents an advancement toward the establishment of an alternative approach for the potency assessment, in the context of the 3 Rs. Besides, the employment of chromatographic methods in conjunction with the bioassays contributes to assessing the quality attributes of the biopharmaceutical formulations of BoNTA.