Analysis of streptokinase by validated liquid chromatography methods and correlation with an in vitro bioassay

Abstract

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.

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

“…The in vitro neutral red uptake assay was performed as described elsewhere [36], with exposure of the cell lines NCTC Clone 929 (ATCC CCL−1) and CHO-K1 (ATCC CCL−61) to intact and degraded products. The cells’ viability was measured by a color reaction, and recorded their absorbance at 540 nm.…”

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

“…Currently, analytical methodologies based on biological and physicochemical properties have proven to be particularly useful for the analysis of therapeutic proteins and have also been applied to correlation studies (Qian et al, 2008;Skrlin et al, 2010;Cardoso et al, 2017). Reversed-phase liquid chromatography (RP-LC) and sizeexclusion liquid chromatography (SE-LC) methods offer a high level of accuracy and sensitivity for the analysis of closely related protein variants, degradation products, and high-molecular-weight (HMW) substances with reduced or absent activity and altered immunogenicity (Fekete et al, 2014;Moussa et al, 2016).…”