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

Recombinant human interferon beta 1b (rhIFNß-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 rhIFNß-1b, contributing to improve the quality control and to ensure the efficacy of the biotherapeutic

Produção da proteína recombinante Interferon-ß1b na plataforma se síntese proteica livre de célula / Cell-free protein synthesis of recombinant Interferon-ß1b

Biofármacos e produtos imunobiológicos representam uma parcela significativa no mercado farmacêutico global. Cerca de 400 milhões de pessoas em todo o mundo dependem desses produtos e, muitas vezes, necessitarão delas para o resto de suas vidas. Até o momento, a tecnologia convencional para síntese dessas proteínas recombinantes é a expressão baseada em células. Porém, este sistema está frequentemente associado a problemas como a degradação da proteína-alvo devido à presença de nucleases e proteases, agregação com formação de corpos de inclusão e perda de molde de DNA. A plataforma de síntese proteica livre de células (do inglês Cell-Free Protein Synthesis ou CFPS) tem surgido como uma alternativa à expressão baseada em célula, permitindo a síntese de diversas proteínas, inclusive as de difícil expressão, na forma solúvel, em elevado rendimento e com capacidade de escalonamento. Este estudo buscou sintetizar a proteína interferon-ß1b humano recombinante (rhINF-ß1b) no sistema CFPS. Através de um trabalho de bioinformática, otimizou-se a sequência nucleotídica da proteína para expressão em lisado de E. coli e construiu-se um vetor de expressão linear, contendo promotor T7, RBS, região codificadora e um terminador T7. O sistema CFPS aceita molde de DNA no formato linear, que possui a vantagem de agilidade na preparação, sem a necessidade das etapas de clonagem, transformação, cultivo, extração e purificação do DNA. Também foi testado a expressão em DNA plasmidial, em que o gene do IFN foi clonado em vetor TOPO-TA Cloning. Para comparar os níveis de expressão foi utilizado esses mesmos vetores no sistema baseado em célula, com cepas de BL21(DE3) e Rosetta(DE3). Apesar da proteína de interesse não ter sido sintetizada com sucesso no sistema CFPS, foram dados alguns passos importantes para atingir este objetivo. No futuro, uma das prioridades é padronizar um sistema livre de células do laboratório capaz de fornecer um elevado rendimento para síntese de proteínas

Biopharmaceutical and immunobiological products represent a growing share of the global pharmaceutical market. Around 400 million people worldwide are dependent of such proteins and, often, they are going to need for the rest of their lives. So far, the most employed biopharmaceutical production technology is cell-based expression. However, this system is usually associated with problems such as degradation of proteins due to the presence of endogenous nucleases or proteases, aggregation with inclusion bodies formation and loss of DNA template. Cell-Free Protein Synthesis (CFPS) platform has emerged as an alternative to cell-based expression, allowing synthesis of many types of proteins, including difficult-to-express proteins, proteins in soluble form, in high yield and possibility to scale up or down the process. This work sought to synthesize recombinant human interferon-ß1b (rhINF-ß1b) in CFPS system. Through a bioinformatics study, a nucleotide sequence of the target protein was optimized for expression in E. coli lysate and a linear DNA template was designed, containing a T7 promoter, a RBS, a coding sequence and a T7 terminator. The CFPS system accepts linear template DNA, which has the advantage of agility in the preparation, without the need for the steps of cloning, transformation, cultivation, extraction and purification of DNA template. Expression in plasmid DNA was also tested, wherein the IFN gene was cloned into TOPO-TA Cloning vector. To compare expression levels, the same vectors were used in the cell-based system with BL21 (DE3) and Rosetta (DE3) strains. Although the protein of interest was not successfully synthesized in the CFPS system, some important steps were taken to achieve this goal. In the future, one of the priorities is to standardize the lysate preparation for a high throughput protein production