RESUMEN
Current source of recombinant human interleukin-11 (rhIL-11) is isolated from a fusion protein expressed by E. coli that requires additional enterokinase to remove linked protein, resulting in product heterogeneity of N-terminal sequence. Due to lack of glycosylation, rhIL-11 is suitable to be expressed by yeast cells. However, the only available yeast-derived rhIL-11 presents an obstacle in low production yield, as well as an unamiable process, such as the use of reverse-phase chromatography employing plenty of toxic organic solvents. Our findings showed that the low yield was due to self-aggregation of rhIL-11. A novel process recovering bioactive rhIL-11 from the yeast secretory medium therefore has been developed and demonstrated, involving fermentation from Pichia pastoris, followed by a two-phase extraction to precipitate rhIL-11. After renaturing, the protein of interest was purified by a two-column step, comprising a cation-exchanger, and a hydrophobic interaction chromatography in tandem at high sample loads that was facile and cost-effective in future scale-up. Identity and quality assessments confirmed the expected amino acid sequence without N-terminal heterogeneity, as well as high quality in potency and purity. Such a process provides an alternative and adequate supply of the starting material for the PEGylated rhIL-11.
Asunto(s)
Interleucina-11/genética , Pichia/genética , Línea Celular , Proliferación Celular , Cromatografía en Gel , Clonación Molecular/métodos , Fermentación , Expresión Génica , Humanos , Interleucina-11/química , Interleucina-11/aislamiento & purificación , Interleucina-11/metabolismo , Pichia/metabolismo , Agregado de Proteínas , Replegamiento Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , SolubilidadRESUMEN
A stability-indicating reversed-phase liquid chromatography (RP-LC) method was validated for the assessment of recombinant human interleukin-11 (rhIL-11), based on the ICH guidelines. The method was carried out on a Jupiter C(4) column (250 mm × 4.6 mm i.d.), maintained at 25°C. The mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) and the mobile phase B was acetonitrile with 0.1% TFA, run at a flow rate of 1 mL/min, and using a photodiode array (PDA) detection at 214 nm. Separation was obtained with a retention time of 27.6 min, and was linear over the concentration range of 1-200 µg/mL (r(2) = 0.9995). Specificity was established in degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.22% with bias lower than 1.25%. Moreover, the in vitro cytotoxicity test of the degraded products showed non-significant differences (p > 0.05). The method was applied to the assessment of rhIL-11 and related proteins in biopharmaceutical dosage forms, and the results were correlated to those of a bioassay.
Asunto(s)
Bioensayo , Cromatografía de Fase Inversa/métodos , Interleucina-11/aislamiento & purificación , Interleucina-11/farmacología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Humanos , Interleucina-11/análisis , Interleucina-11/toxicidad , Límite de Detección , Control de Calidad , Proteínas Recombinantes/análisis , Proteínas Recombinantes/toxicidadRESUMEN
Mature human interleukin-11 (HuIL-11) is a cytokine consisting of 178 amino acid residues that results from scission of the N-terminal signal peptide, consisting of 21 amino acid residaues, from the corresponding nascent polypeptide. A DNA fragment encoding a truncated HuIL-11 (trHuIL-11), with an additional 5 amino acid residues removed from the N-terminus, was cloned into vector pGEX-2T between the BamHI site and the EcoRI site. Upon transformation with Escherichia coli BL21, the construct over-produced a glutathione S-transferase (GST)-fused protein in a soluble form after IPTG induction. The fusion protein was initially fractionated with butyl-Sepharose 4 fast flow column and by affinity chromatography using a GSH-Sepharose 4B column. On-site enzymatic release with thrombin gave the target protein at 96% purity as judged by SDS-PAGE and HPLC. Expression of the interleukin as a GST-fused protein thus greatly improved downstream processing. Subsequent biological activity assay suggested that trHuIL-11 had similar activity profile to the naturally produced sample and may be a promising candidate for further development as biopharmaceutical.
Asunto(s)
Escherichia coli/genética , Interleucina-11/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proliferación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Clonación Molecular , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Hibridomas , Interleucina-11/química , Interleucina-11/genética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Plásmidos/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Análisis de Secuencia de ProteínaRESUMEN
This study first time report a method to purify the rhIL-11 which expressed by Pichia pastoris. rhIL-11 was secreted into the supernatant and collected by centrifugation. The purity of rhIL-11 reached 97% through the steps of ultrafiltration, SP Sepharose FF, Phenyl Sepharose HP and Sephadex G25. Analysis of SDS-PAGE, Western-blotting, IEF, RP-HPLC, Mass spectrometer, N and C terminus amino acid sequence and bioactivity was conducted. All the analysis results proved that the rhIL-11 expressed by Pichia pastoris was the same as Neumeg which was expressed in E. coli with fusion expression system. So it is possibly a cheaper and easier method to produce rhIL-11 for clinical use.
Asunto(s)
Interleucina-11/aislamiento & purificación , Pichia/genética , Fermentación , Humanos , Interleucina-11/farmacología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacologíaRESUMEN
OBJECTIVE: To express recombinant human interleukin-11 (rhIL-11) in methylotropic yeast Pichia pastoris. METHODS: By designing and synthesizing an artificial gene for IL-11, the expression vector pPICZ alpha-A-IL-11 was constructed and introduced into Pichia pastoris by linearized electroporation. The rhIL-11 protein was identified by ELISA and SDS-PAGE analysis. The bioactivity was analyzed by B9-11 cell line. A combination of liquid chromatography was developed to purify the rhIL-11 from ferment supernatant. RESULTS: The nucleotide sequence analysis indicated that the sequence of cloned artificial IL-11 gene accorded with that of designed; the secreted yield of rhIL-11 by yeast Pichia pastoris KM71-2424 in flask reached 60 mg/L. The biological activity of IL-11 in yeast supernatant and E. coli standard determined by B9-11 was 5.5 x 10(7) U/mg and 2.2 x 10(7) U/mg respectively. The rhIL-11 was purified to electrophoretic purity by a combination of liquid chromatography. CONCLUSION: The human IL-11 artificial gene was obtained and successfully expressed in the Pichia pastoris(KM71-2424). The biological activity of IL-11 in yeast supernatant was significantly higher than that of E. coli standard. The rhIL-11 was purified to electrophoretic purity.