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

Francielle Santos Da Silva,Douglas Franco Cardoso,Valquiria Guedes Perlin,Maurício Elesbão Walter,Guilherme Zanini Calegari,Sérgio Luiz Dalmora,Rafaela Ferreira Perobelli,Bruna Xavier

doi:10.1590/s2175-97902019000218328

Francielle Santos Da Silva, Douglas Franco Cardoso + Show 6 more

Open Access

https://doi.org/10.1590/s2175-97902019000218328

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Abstract

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 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 rhIFNβ-1b, contributing to improve the quality control and to ensure the efficacy of the biotherapeutic.

Highlights

Interferons (IFNs) are natural proteins produced by immune system cells that have antiviral, antiproliferative and immunomodulatory properties
Sodium phosphate dibasic, sodium chloride, acetonitrile, trifluoroacetic acid (TFA), human serum albumin (HSA) and mannitol were supplied by Merck (Darmstadt, Germany)
For the reversed−phase liquid chromatography (RP−Liquid chromatography (LC)), TFA in water and TFA in acetonitrile resulted in higher sensitivity related to phosphate buffer, and lower retention time compared to sodium phosphate buffer

Summary

Introduction

Interferons (IFNs) are natural proteins produced by immune system cells that have antiviral, antiproliferative and immunomodulatory properties. Interferon betas represent the first class of disease modifying therapies (DMTs) for multiple sclerosis (MS) and have contributed considerably to the understanding of the immunomodulatory mechanisms. Human interferon beta (hIFN-β) is a hydrophobic glycoprotein that contains 166 amino acids produced by fibroblasts. Advances in the recombinant DNA technology facilitate the expression of hIFN-β resulting in a large-scale production of biopharmaceutical formulations. Recombinant human interferon beta 1b (rhIFNβ-1b) is engineered as a nonglycosylated protein in Escherichia coli (E. coli) with a serine residue instead of a cysteine at amino acid position 17 and lacks the methionine at the N-terminus. The substitution at position 17 was made to eliminate the free sulfhydryl of cysteine to obtain a product that is more stable upon storage. It is currently being used worldwide to treat MS in a large number of patients (Mark et al, 1984; Dendrou, Fugger, Friese, 2015; EMEA, 2017)

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