Extração e purificação do biofármaco antileucêmico L-asparaginase (ASNase) utilizando sistemas aquosos bifásicos com polímeros e líquidos iônicos
Carregando...
Data
2019-03-22
Autores
Magri, Agnes
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
A L-asparaginase destaca-se como um importante biofármaco utilizado no tratamento de leucemia, além da sua utilização na indústria alimentícia. Seu alto custo de produção se deve principalmente às etapas de purificação, que correspondem em geral a mais de 70% do valor do produto final. Assim, novos processos de extração líquido-líquido, como a aplicação de Sistemas Aquosos Bifásicos (SABs), surgem como técnicas alternativas de extração/purificação mais econômica e biocompatível. Nesse sentido, este trabalho avaliou um processo alternativo para a purificação de baixa resolução da enzima L-asparaginase (ASNase) utilizando-se SABs com polímeros e sais ou líquidos iônicos (LIs). Inicialmente, foi realizado um estudo comparativo de diferentes metodologias de quantificação da atividade da ASNase comercial, a fim de compreender as interferências dos métodos em diferentes condições, e assim estabelecer um método de quantificação adequado para determinação da atividade de ASNase nos SABs. A seguir, a estabilidade da ASNase foi avaliada frente aos diferentes componentes de fases dos SABs. Dessa forma, LIs derivados de colinas e polímeros foram testados como solventes alternativos na biocatálise, e a fim de compreender o efeito do tamanho da cadeia do ânion dos LIs sob a estabilidade da enzima, foram testadas soluções aquosas contendo as colinas com os seguintes ânions: cloreto ([Ch]Cl); acetato ([Ch][Ac]); propanoato ([Ch][Pro]); butanoato ([Ch][But]) e hexanoato ([Ch][Hex]). O aumento da cadeia alquílica do ânion teve um efeito negativo na atividade enzimática devido à maior afinidade desses compostos pela proteína, portanto, [Ch]Cl e [Ch][Ac] foram selecionados como os melhores solventes alternativos, mantendo a estabilidade e aumentando a atividade enzimática da ASNase. Estes LIs foram então avaliados como agentes formadores de fase em diferentes SABs. Quanto aos polímeros, avaliou-se a estabilidade e atividade da ASNase em soluções aquosas de polietilenoglicol de massa molecular média 600 g.mol-1 (PEG 600) e polipropilenoglicol de massa molecular média 400 g.mol-1 (PPG 400), e constatou-se que a ASNase se manteve estável e ativa, podendo esses polímeros serem também utilizados como agentes formadores de fase em diferentes SABs. Dessa forma, foram avaliadas inicialmente as capacidades extrativas de sistemas polímeros-sal/LIs usando como modelo a ASNase comercial. Os SABs testados foram capazes de concentrar a ASNase comercial, com eficiências de extrações > 95%, podendo a partição da mesma ser totalmente controlada pela escolha apropriada da natureza do polímero ou sais/LIs utilizados. Por fim, os melhores SABs se mostraram plataformas promissoras para extração da ASNase a partir de lisado celular de E. coli. O conjunto de resultados mostraram que os SABs podem ser eficientemente aplicados na extração e purificação de biomoléculas complexas de interesse farmacêutico, com capacidade para serem utilizados como plataformas de integração dos processos upstream e downstream em modo contínuo e/ou semi-contínuo. Além disso, foi demonstrado que os LIs derivados de colinas podem ser utilizados como solventes alternativos na estabilização/ativação da ASNase, com grande potencial para serem utilizados em meios reacionais catalíticos ou sintéticos, para outras enzimas e moléculas, em processos industriais.
L-asparaginase (ASNase) is an important biopharmaceutical used in the treatment of leukemia, as well in industrial food processes. Its high production costs are mainly due to purification steps, in general, up to 70% of the final product value. Therefore, new liquid-liquid extraction processes, such as Aqueous Biphasic Systems (ABS), have appeared as more economical and biocompatible extraction/purification alternatives. In this work an alternative process for the purification of the enzyme L-asparaginase (ASNase) using ABS composed of polymers and salts or ionic liquids (ILs) was evaluated. In the first stage, a comparative study of different activity quantification methods of the commercial ASNase was carried out. This study intended to determine the interferences of the quantification methods and to establish the most adequate method for the quantification of ASNase activity after the extraction with different ABS. Further, the stability of the ASNase in the presence of different type and concentration of phase-forming agents, namely cholinium based-ILs ([Ch]+-ILs) and polymers, was evaluated. Thus, in order to understand the effect of the increase of anion alkyl chain length of the ILs in the enzyme stability, aqueous solutions of [Ch]+-ILs, with the following anions, were tested: chloride ([Ch]Cl), acetate ([Ch][Ac]), propanoate ([Ch][Pro]), butanoate ([Ch][But]) and hexanoate ([Ch][Hex]). The increase of the anion alkyl chain length had a negative effect on the enzymatic activity due to the affinity of these compounds to the protein structure. [Ch]Cl and [Ch][Ac] were selected as the best alternative solvents, because of their ASNase stability aptitude, as well enzymatic activity enhancing effect. The stability and activity of ASNase in aqueous solutions of polyethylene glycol of average molecular weight 600 g.mol-1 (PEG 600) and polypropylene glycol of average molecular weight 400 g.mol-1 (PPG 400) was also evaluated, in which ASNase remained stable and active, being these polymers selected as ABS phase forming agents. Therefore, the partition and extraction of commercial ASNase (used as model) using different ABS composed of polymer/salts or polymer/[Ch]+-ILs was evaluated. All ABS were able to concentrate the commercial ASNase, exhibiting extraction efficiencies > 95%. Furthermore, it was obtained a full control of the ASNase partition by the appropriate choice of the polymer nature or salts/ILs used as phase forming agents. To end, it was demonstrated the excellent aptitude of the best ABS to act as promising platforms for the extraction of ASNase from E. coli cell lysate. The set of results shown that the ABS can be efficiently applied in the extraction and purification of complex pharmaceutical biomolecules, with high capability to be used as continuous and/or semi-continuous upstream-downstream integrative platforms. Moreover, it was demonstrated that [Ch]+-ILs can be used as alternative ASNase stabilizing/activating solvents, which may be promising candidates for composing catalytic or synthetic reactive media used in industrial processes, not only for ASNase but also for other enzymes and biomolecules.
L-asparaginase (ASNase) is an important biopharmaceutical used in the treatment of leukemia, as well in industrial food processes. Its high production costs are mainly due to purification steps, in general, up to 70% of the final product value. Therefore, new liquid-liquid extraction processes, such as Aqueous Biphasic Systems (ABS), have appeared as more economical and biocompatible extraction/purification alternatives. In this work an alternative process for the purification of the enzyme L-asparaginase (ASNase) using ABS composed of polymers and salts or ionic liquids (ILs) was evaluated. In the first stage, a comparative study of different activity quantification methods of the commercial ASNase was carried out. This study intended to determine the interferences of the quantification methods and to establish the most adequate method for the quantification of ASNase activity after the extraction with different ABS. Further, the stability of the ASNase in the presence of different type and concentration of phase-forming agents, namely cholinium based-ILs ([Ch]+-ILs) and polymers, was evaluated. Thus, in order to understand the effect of the increase of anion alkyl chain length of the ILs in the enzyme stability, aqueous solutions of [Ch]+-ILs, with the following anions, were tested: chloride ([Ch]Cl), acetate ([Ch][Ac]), propanoate ([Ch][Pro]), butanoate ([Ch][But]) and hexanoate ([Ch][Hex]). The increase of the anion alkyl chain length had a negative effect on the enzymatic activity due to the affinity of these compounds to the protein structure. [Ch]Cl and [Ch][Ac] were selected as the best alternative solvents, because of their ASNase stability aptitude, as well enzymatic activity enhancing effect. The stability and activity of ASNase in aqueous solutions of polyethylene glycol of average molecular weight 600 g.mol-1 (PEG 600) and polypropylene glycol of average molecular weight 400 g.mol-1 (PPG 400) was also evaluated, in which ASNase remained stable and active, being these polymers selected as ABS phase forming agents. Therefore, the partition and extraction of commercial ASNase (used as model) using different ABS composed of polymer/salts or polymer/[Ch]+-ILs was evaluated. All ABS were able to concentrate the commercial ASNase, exhibiting extraction efficiencies > 95%. Furthermore, it was obtained a full control of the ASNase partition by the appropriate choice of the polymer nature or salts/ILs used as phase forming agents. To end, it was demonstrated the excellent aptitude of the best ABS to act as promising platforms for the extraction of ASNase from E. coli cell lysate. The set of results shown that the ABS can be efficiently applied in the extraction and purification of complex pharmaceutical biomolecules, with high capability to be used as continuous and/or semi-continuous upstream-downstream integrative platforms. Moreover, it was demonstrated that [Ch]+-ILs can be used as alternative ASNase stabilizing/activating solvents, which may be promising candidates for composing catalytic or synthetic reactive media used in industrial processes, not only for ASNase but also for other enzymes and biomolecules.
Descrição
Palavras-chave
L-asparaginase, líquidos iônicos, estabilidade, extração, purificação, sistemas aquosos bifásicos, ionic liquids, stability, extraction, purification, aqueous biphasic systems