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

Abstract

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.

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

“…CHO–K1 cell line (Chinese hamster ovary, ATCC number CCL‐61) and NCTC Clone 929 cell line (mammalian fibroblasts, ATCC number CCL‐1) were used to assess cytotoxicity by mean of measuring the neutral red uptake (NRU) by the cells after exposure to degraded forms of STK, and the absorbance was measured at 540 nm, following the procedure described elsewhere .…”

Quantitation of certolizumab pegol by validated liquid chromatography methods

“…One of the most challenging problems in pharmaceutical monoclonal antibody formulations is protein aggregation, which is directly associated with the quality of the final product, wherein high-molecularweight aggregates form and can induce immunogenicity (Hernández-Jiménez et al, 2018). Size exclusion liquid chromatography (SEC) is commonly used and acknowledged as the best analytical methodology for the quantitation of the native protein and to monitor potential aggregation because it allows for characterization with minimal impact on the conformational structure (Stamm et al, 2013;Shahbazi et al, 2017;Perobelli et al, 2018).…”

Evaluation of botulinum toxin type A by bioassays and a validated reversed-phase liquid chromatography method

“…In vitro cytotoxicity test. The in vitro cytotoxicity method was performed as described elsewhere, 25 based on a neutral red uptake (NRU) assay, with the exposure of NCTC Clone 929 cell line (mammalian broblasts, ATCC number CCL-1) to degraded samples, the pH of the samples was adjusted to 7.0, and positive and diluents control were included in the assay together with R-BoNTA solution. The NRU assay was performed on 96-well microplates, maintained at 37 C in a CO 2 incubator for 24 h, with a cell suspension density of approximately 2 Â 10 5 cells per mL.…”