UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE ENGENHARIA QUÍMICA
ANDRÉ DELANO DOMINGOS CAVALCANTI
ANÁLISE DO DESEMPENHO DE PROCESSOS DE SEPARAÇÃO E PURIFICAÇÃO DO ÁCIDO HIALURÔNICO PRODUZIDO POR FERMENTAÇÃO
ANALYSIS OF THE PERFORMANCE OF SEPARATION AND PURIFICATION PROCESSES OF HYALURONIC ACID PRODUCED BY FERMENTATION
Campinas 2019
ANDRÉ DELANO DOMINGOS CAVALCANTI
ANALYSIS OF THE PERFORMANCE OF SEPARATION AND PURIFICATION PROCESSES OF HYALURONIC ACID PRODUCED BY FERMENTATION
ANÁLISE DO DESEMPENHO DE PROCESSOS DE SEPARAÇÃO E PURIFICAÇÃO DO ÁCIDO HIALURÔNICO PRODUZIDO POR FERMENTAÇÃO
Tese de Doutorado apresentada à Faculdade de Engenharia Química da Universidade de Campinas como parte dos requisitos exigidos para obtenção do título de Doutor em Engenharia Química.
Thesis presented to the School of Chemical Engineering of the University of Campinas in partial fulfilment of the requirements for the degree of Doctor in Chemical Engineering
Supervisora/Orientadora: Profª. Drª. Maria Helena Andrade Santana
Este trabalho corresponde à versão final da dissertação/tese defendida pelo aluno André Delano Domingos Cavalcanti e orientada pela profa. Maria Helena Andrade Santana
Campinas 2019
Folha de Aprovação da Defesa de Tese de Doutorado defendida por André Delano Domingos Cavalcanti aprovada em 25 de fevereiro de 2019 pela banca examinadora constituída pelos seguintes doutores:
Profa. Dra. Maria Helena Andrade Santana FEQ / UNICAMP
Dr. Reinaldo Gaspar Bastos Universidade Federal de São Carlos
Dra. Gisella Maria Zanin UEM
Profa. Dra. Ana Silvia Prata Soares FEA/UNICAMP
Dr. Adriano Rodrigues Azzoni Escola Politécnica da USP
ATA da Defesa com as respectivas assinaturas dos membros encontra-se no SIGA/Sistema de Fluxo de Dissertação/Tese e na Secretaria do Programa da Unidade.
Agradecimentos
Antes de mais nada, agradeço a Deus, nosso Pai e Provedor. Sem Ele, nada disso seria possível, começando pela chegada em Campinas até a conclusão dessa jornada.
Agradeço imensamente aos meus pais queridos, Marcelo e Valéria, por me ensinarem, educarem e fortalecerem ao longo dessa caminhada, além de serem grande fonte de inspiração. Gostaria de agradecer também ao meu irmão Alexandre, meu melhor amigo e com quem eu sempre pude e posso contar. Importante lembrar de minhas avós Emília e Tereza, que são as matriarcas e pilares da família, bem como aos meus falecidos avôs Manuel e Heleno, que olham lá de cima pela nossa família.
Gostaria de agradecer a Carolina, minha namorada, amiga e confidente, mulher que amo e que esteve comigo em boa parte dessa caminhada, sempre me escutando e me aconselhando muitas vezes com visões complementares as minhas, ajudando no meu crescimento. Agradeço também a família Lima que me acolheu em Campinas, em especial dona Graça e a Marcila (Má).
À professora Maria Helena, que sempre acreditou em mim e nunca me deixou acomodar nem me conformar com menos, se o mais é possível. Agradeço muito a paciência e os ensinamentos da professora, que me ajudaram no crescimento pessoal e profissional.
Aos meus amigos de laboratório Carla, Daniel, Fabiane e Andréa pelas conversas e companhia ao longo desses anos. Um agradecimento especial à Bruna, cuja parceria sob a orientação da prof. Maria Helena rendeu meu primeiro artigo publicado. E um agradecimento ainda maior aos meus companheiros de experimentos, histórias e risadas, Rhelvis (Papai), Gilson e Alex, que ajudaram com que os momentos no laboratório fossem ainda mais agradáveis.
A minhas amigas e trigêmeas, Fernanda e Renata, sem as quais eu nem em Campinas teria chegado. Com certeza uma grande amizade que se formou ao longo desses anos.
Por fim, a todos os demais amigos que fiz em Campinas (em especial pessoal da Cleide’s House, Lucas e Gi) e a todos os amigos que ficaram em Maringá, mas que sempre me recebem quando eu regresso (Fernando, Luiz Fernando, Gabriel, Juliano, Miguel, Bruno, entre outros). Com certeza cada palavra de vocês me influenciou positivamente de alguma forma.
E finalmente, agradeço ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) pelo apoio financeiro ao longo desses 4 anos.
RESUMO
O ácido hialurônico (AH) é um polieletrólito natural, presente amplamente na natureza em todos os tecidos humanos e animais (como pele, fluido sinovial e humor vítreo do olho) e também em bactérias, especialmente nos Streptococci. O AH é um produto de alto valor agregado, de grande interesse das indústrias de cosméticos e médica. A grande maioria dos trabalhos na literatura apenas apresentam as condições operacionais e o resultado final da purificação do AH, sem a demonstração de quaisquer estudos sobre as especificações das operações utilizadas. O aprofundamento do conhecimento sobre os fenômenos envolvidos nas etapas de um processo de purificação permite localizar condições operacionais de melhor desempenho, e otimização do processo global.
Neste trabalho foi estudada a purificação do AH produzido por Streptococcus zooepidemicus em meio composto somente por glicose e peptona de soja. O foco principal foram os estudos da precipitação e posteriormente o processo integrado com a adsorção como etapa seguinte, visando-se estabelecer uma sequência de duas etapas para obtenção de alto nível de pureza do AH. Tendo a precipitação como principal operação nos processos de purificação do AH, a performance da operação e o comportamento do AH de acordo com a variação de parâmetros operacionais são melhor compreendidos com o presente trabalho, além da alta pureza atingida com um processo simples.
Os parâmetros operacionais estudados na precipitação do AH com etanol e isopropanol foram: proporção solvente:caldo, pH e presença de cloreto de sódio (NaCl). A massa molar, tamanhos de partícula, potencial zeta e tensão superficial foram caracterizados, de forma a entender o comportamento da operação. Os melhores resultados para a precipitação com etanol foram obtidos na proporção de 2:1 etanol:caldo (v/v). Na ausência de NaCl, foram obtidas pureza de 55,0±0,2% e recuperação de 85,0±0,7% a pH 4, enquanto na presença de NaCl, pureza de 59,0±3,0% e recuperação de 89,0±5,0% foram obtidas a pH 3, enquanto que a pH 7 foram obtidas pureza de 59,0±0,9% e recuperação de 82,0±4,0%. Para a precipitação com IPA, os melhores resultados foram novamente obtidos na proporção solvente:caldo de 2:1 (v/v). No pH 3, foi obtida pureza de 79,0±1,8% e recuperação de 76,0±6,6%, enquanto no pH 7 foi obtida 74,0±2,7% de pureza e 72,0±7,5% de recuperação. Esses resultados foram diretamente relacionados com as variáveis operacionais e propriedades físico-químicas, além das condições iniciais do caldo, em especial as concentrações de AH e proteína. Por fim, o processo integrado apresentou pureza de 90,2±0,7% e recuperação global de 51,8±5,0% em apenas duas etapas, com manutenção da integridade da alta massa molar. Esses resultados são relevantes para obtenção de AH de alta pureza produzido por fermentação em processo de somente duas etapas.
O entendimento do comportamento do AH nas etapas estudadas possibilita estudos posteriores de otimização e/ou adição de etapa complementar para que o grau médico de pureza para o AH injetável (> 99%) seja atingido.
ABSTRACT
Hyaluronic acid (HA) is a natural polyelectrolyte, ubiquitous in human and animal tissues (skin, synovial fluid and eye’s vitreous humor), and also in bacteria, specially at Streptococci. HA is a valuable material, with interest from cosmetics and medical industries. Even though there were successful results in various purification sequences, most of these works have not explored how the parameters and conditions of each operation would influence on its performance. A deeper knowledge about the involved phenomena allows to find improved operational conditions, leading to the optimization of the global process.
In the present work, the main goal was to study the purification of HA produced by Streptococcus zooepidemicus in a medium containing only glucose and soy peptone. The study focuses on precipitation studies, with later integration with the adsorption operation as a second step, aiming to establish a simple sequence to obtain a highly purified HA. As precipitation is the main operation in the HA purification processes, the operational performance and HA behavior according to the parameters are better comprehended in this work, added to the high purity obtained in a simple process.
Precipitation with etanol and isopropanol (IPA) was studied according to operational parameters, solvent:broth proportion, pH and sodium chloride (NaCl) presence. The molar mass, particle size, zeta potential and surface tension were characterized, in order to understand the operational behaviour. The best results of ethanol precipitation were obtained at the 2:1 ethanol:broth proportion (v/v). Without sodium chloride (NaCl) at pH 4, a purity of 55.0±0.2% and a recovery of 85.0±0.7% were observed. Meanwhile, in NaCl presence, purities of 59.0±3.0% and 59.0±0.9% and recoveries of 89.0±5.0% and 82.0±4.0% were obtained in pH 3 and 7, respectively. With regard to isopropanol (IPA) precipitation, the better results were also obtained at the 2:1 proportion (v/v). At pH 3, a purity of 79.0±1.8% and recovery of 76.0±6.6% were obtained, while pH 7 presented a purity of 74.0±2.7% and recovery of 72.0±7.5%. These results were directly linked to the operational parameters and the physicochemical properties, and also with the initial broth conditions, specially HA and protein concentrations. At last, the precipitation-adsorption process showed a purity of 90.2±0.7% and a global recovery of 51.8±5.0% in only two steps, keeping HA high molar mass integrity. These results are relevant to obtain a highly purified HA from fermentation in only two steps. The understanding of HA behaviour in the studied operations allows later studies of optimization and/or adding a complementary step, aiming to reach the required purity to injectable HA (> 99%).
2. Fundamentação Teórica - Performance das Operações Principais das Sequências de
Purificação do Ácido Hialurônico (Artigo Revisão) ... 12
3. Resultados e Discussão ... 46
3.1. Recuperação e pureza do Bio-Ácido Hialurônico de alta massa molar via estratégias de precipitação moduladas por pH e cloreto de sódio ... 46
3.2. Propriedades estruturais e de superfície controlam a recuperação e pureza do Bio-Ácido Hialurônico na precipitação com isopropanol ... 71
3.3. Purificação do Bio-Ácido Hialurônico de alta massa molar em duas etapas: precipitação com isopropanol e adsorção em carvão ativado ... 92
4. Conclusões ... 109
4.1. Geral ... 109
4.2. Específicas ... 109
5. Sugestões para trabalhos futuros ... 110
1. Introdução
O ácido hialurônico (AH) é um polissacarídeo composto de unidades alternadas de ácido D-glicurônico e N-acetil-D-glicosamina, unidas por ligações β-(13) e β-(14), sendo um polieletrólito natural e um dos glicosaminoglicanos presentes na natureza (Kakehi et. al, 2003). É considerado um hidrogel, isto é, tem redes de ligação que absorvem grandes quantidades de água, causando modificações macroscópicas nas suas dimensões (Kazakov, 2012). Sua presença no fluido sinovial é o principal responsável pela alta viscosidade e viscoelasticidade do mesmo (Ogston e Stanier, 1950 e 1952; Silpananta et al., 1968 e 1969). O AH confere lubrificação às articulações e tem função na absorção de choques sofridos. Além disso, o AH tem um importante papel na embriogênese, transdução de sinais e mobilidade (Kogan et. al, 2007).
O mercado global do AH foi avaliado em US$ 7,2 bilhões em 2016 e espera -se que alcance um valor de US$ 15,4 bilhões até 2025, graças ao aumento do interesse da população a respeito de produtos na áreas da saúde e estética (Grand View Research, 2016). Por muito tempo, as únicas fontes de AH eram animais, tais como a crista de galo ou o humor vítreo bovino, das quais o AH era extraído. Entretanto, a obtenção de uma alta pureza do AH animal é mais dispendiosa, devido a formação de complexos entre AH e proteoglicanos, presentes no tecido animal. (Armstrong e Johns, 1997; Jagadeeswara Reddy et al., 2013). Com isso, o AH produzido por fermentação microbiana, intitulado de Bio-AH (AH de fonte biotecnológica), passou a receber atenção especial, até por evitar infecções virais e o uso de solventes tóxicos que podem ser usados na extração do AH de tecido animal (Chong et al., 2005; Lapcík et al., 1998). Os meios de cultura utilizados tem preponderantemente a glicose como fonte de carbono (Armstrong e Johns, 1997; Ogrodowski et al., 2005), enquanto que a principal fonte de nitrogênio varia com mais frequência, entre fontes animais como o BHI, extrato de leveduras e outras fontes complexas (Benedini e Santana, 2013; Rangaswamy e Jain, 2008; Ellwood et al., 1995). Ainda não há produção de AH em larga escala no Brasil, o que destaca a carência de maior entendimento sobre a produção e purificação do produto no país.
Trabalhos na literatura apresentaram a obtenção do AH altamente purificado a partir de diferentes operações formando diversas sequências de purificação, compostas principalmente por precipitação, filtração e adsorção (Rangaswamy e Jain, 2008; Jagadeeswara Reddy et al., 2013; Murado et al., 2012). A precipitação é comum à ampla maioria desses processos, graças a sua alta efetividade e menor custo, se comparada com outras operações. A grande maioria dos
trabalhos na literatura apenas apresentam as condições operacionais e o resultado final da purificação do AH, sem a demonstração de quaisquer estudos que justifiquem as especificações utilizadas na sequência de purificação, somado a falta da demonstração de purezas intermediárias, mostrando o impacto de cada operação dentro dos processos. Condições operacionais que possibilitem um melhor desempenho, sobretudo nas operações iniciais, beneficiaria as etapas subsequentes e o processo de purificação como um todo.
O objetivo deste trabalho foi estudar a recuperação e purificação do AH obtido por fermentação submersa produzido por Streptococcus zooepidemicus em meio composto somente por glicose e peptona de soja. A sequência de purificação consiste no uso da precipitação com solvente orgânico seguida de adsorção em carvão ativado, visando a obtenção de AH com alto nível de pureza. Nosso grupo de pesquisa tem trabalhado no processo integrado de produção, purificação e aplicação do AH, com enfoque na medicina regenerativa. As elevadas purezas necessárias nas diferentes aplicações do AH, sobretudo na área médica, somadas a falta de maiores informações das operações de purificação do AH, configuram a importância do presente estudo.
Esta tese é composta de quatro artigos: o primeiro traz uma revisão bibliográfica sobre a purificação do AH presente na literatura de 1970 até 2019, explorando as diferentes fontes de obtenção do AH, bem como processos que detalham ou não os resultados obtidos após cada operação, com enfoque nas principais operações utilizadas. O segundo e o terceiro artigos apresentam os estudos da precipitação do AH com etanol e isopropanol (IPA), respectivamente. Por fim, o quarto artigo contempla o processo de purificação composto por precipitação e adsorção em carvão ativado. O presente trabalho contribui com o melhor entendimento da precipitação, principal operação de purificação do AH, em termos de performance e comportamento do polissacarídeo, além da obtenção de alta pureza com um processo simples de apenas duas etapas.
2. Fundamentação
Teórica
-
Performance
das
Operações Principais das Sequências de Purificação
do Ácido Hialurônico (Artigo Revisão)
Performance of the Main Downstream Operations on
Hyaluronic Acid Purification
André Delano Domingos Cavalcanti
1, Bruna Alice Gomes de Melo
1, Bruno
Armenio Moreira Ferreira
1, Maria Helena Andrade Santana
1*
1Development of Biotechnological/Microbial Processes Laboratory, School of Chemical Engineering, University
of Campinas, 13083-852, Campinas, São Paulo, Brazil *Corresponding Author
e-mail address: lena@feq.unicamp.br
Abstract
Hyaluronic acid (HA), or hyaluronan, is the name given to a natural polyelectrolyte, ubiquitous in human tissues. Exogenous HA has been a valuable material, due to its wide range of medical applications such as in osteoarthritis treatment, ophthalmic surgery, adhesion prevention after surgery and wound healing, besides cosmetic applications. However, to ensure the physicochemical and biological properties, a purity of 99% is a primary requirement, enabling clinical applications. To achieve this goal, various downstream operations have been used, aiming HA concentration, separation and purification. Precipitation with organic solvents has been a common operation in the majority of processes in combination with other downstream operations such as quaternary salts precipitation, filtration, adsorption and ion exchange. This work presents an updated review of HA purification emphasizing the performance of the main downstream operations to achieve high purity HA, in the period from 1970 to 2019. This work lead to the conclusion that there is a lack of studies of the operational conditions used and absence of intermediate purities during the processes, in the majority of the published works.
1. Introduction
Hyaluronic acid (HA) or “hyaluronan”, term attributed by Balázs et al. in 1986 [1], is a polysaccharide with hydrogel properties, composed of alternating units of D-glucuronic acid and N-acetylglucosamine, linked by β-(13) and β-(14) bonds. It is a natural polyelectrolyte and one of the many glycosaminoglycans (GAGs) in mammalian connective tissues, such as skin, synovial fluid and eye’s vitreous humor [2]. HA functions in human promote elasticity of skin and cartilage, joints lubrication, and shocks absorption [3-6]. It has also an important role in embryogenesis, signal transduction and cell mobility [7].
The importance of HA biological functions raised great interest in medical and cosmetics applications, making exogenous HA a valuable product. Nowadays, HA is widely used in orthopedics [8-13], in plastic, intraocular [14-16], wound healing [7, 17-20], abdominal surgeries [21-26] and regenerative therapies [27-31]. Other uses of HA include controlled release of drugs [32-36], implants [37-40] and anti-aging filling products [41-45].
Firstly isolated from the viscous bovine humor by Karl Meyer and John Palmer [46], HA was extracted from rooster combs in subsequent years [47-50]. Nowadays, the HA for medical applications is mainly produced by microbial fermentation using Streptococci bacteria [51-60], especially Streptococcus zooepidemicus [61-65].
From the context of application, HA degree of purity has proved to be determining on successful clinical results. Balázs reported that the use of a non-purified HA caused inflammation in intraocular surgeries and other applications [66]. Because of the molecular structure, HA retains contaminants in its highly hydrated and negatively charged structure when in physiological pH. From this date, separation and purification processes involving various downstream operations have been studied using HA from different sources. The main studied downstream operations in the downstream process for HA are: precipitation [53, 56, 67-72];
filtration [53,73-77]; adsorption [59,72,78-82]; electrophoresis [75, 83-85]; and ion-exchange, which is a refined method but also more expensive, and therefore, less described in the literature [59, 86-90]. The combination of the different operations for HA purification may lead to an optimized process in terms of effectiveness and efficiency for removal of impurities [37,53,56,75,78,79,91].
In this review we examined the role of the main downstream operations used in HA purification processes described in patents and articles from 1970 to 2017. Initially the structural properties of HA were described. The performance of the downstream operations were analyzed in terms of yield, purification factor and purity degree of the final product, individually or from an entire process, according to the provided information. Finally, we highlighted promising sequences of downstream operations concerning HA purification processes for medical applications.
2. Structural properties of HA
The HA molecular structure is shown in Figure 1A. HA is a heteropolymer composed by repeated units of glucuronic acid and N-acetylglucosamine with molar mass ranging from 103 to 107 Da. In physiologic conditions, HA is a highly hydrated polyanionic structure. The ionic strength of the medium directly affects its behavior in solution. In water, HA presents a pKa, of about 2.9 [92,93] derived from its carboxyl group [94]. The pH influence is notorious in HA structure in aqueous solutions. HA behaves as a random coil in a wide range of pH (Figure 1B).
(A)
(B)
Fig. 1. A) – Hyaluronic acid molecular structure composed by glucuronic acid and N-acetylglucosamine linked alternately by glycosidic bonds β-(13) and β-(14); B) Models of hyaluronan behaviour in solution. In dilute solution, hyaluronan behaves as a stiffened random coil. In concentrated solutions,
stiffened random coils show entanglement as a physical crosslinking [96].
At high concentrations, HA self-associate, due to hydrogen bonding, electrostatic repulsion, and hydrophobic interactions between chains, forming entanglements [95]. However, the volume occupied by the monomers decreases hindering the molecules movement, what confers to HA high viscoelasticity and hydrogel properties [96].
HA secondary and tertiary structures, which include double helixes and an extensive ramification network [97], are mediated by hydrophobic and hydrophilic interactions among HA monomers [98]. Therefore, HA has the properties of a highly hydrophilic material simultaneously with hydrophobic parts and lipid characteristics, being considered an amphiphilic compound.
By the mentioned self-associations, with the addition of steric interactions, high molecular weight HA at high concentration in solution is able to generate entangled molecular networks, with different properties from isolated hyaluronan molecules. With a rapid and short-duration flow, the entanglement can be maintained, with the exhibition of elastic properties. Meanwhile, a long-duration slow fluid flow is able to separate partially and align the molecules, exhibiting viscous properties [99].
3. Production and Obtainment of HA
As mentioned previously, HA was obtained predominantly from animal sources in a first moment, in which rooster combs [70, 100] and bovine vitreous humor [101, 102] were the major options. The process consisted in HA extraction from the animal tissues by the use of solvents, with later precipitations with organic solvents and washing as final treatments of the extraction. The extraction solvents, which were earlier composed by mixtures of chloroform and water [70], were replaced by mixtures of water and organic solvents like ethanol or acetone [87, 103]. After the extraction process, the solutions containing HA were submitted to later treatments aiming bacteria removal and further separation of the protein chains linked to HA structure, with the use of bactericides and proteolytic enzymes, respectively.
However, with disadvantages presented by animal HA, like the protein chains linked directly to the molecule, higher amounts of nucleic acids and viral contamination risks [104],
the HA production by fermentation became gradually the main option, as the process is reported in many works [51-53]. The most part of HA fermentations presented the Streptococcus bacteria in the production, in which Streptococcus zooepidemicus was predominant [61-63]. Through the fermentations, there were substantial variations in the culture medium, which were initially composed by animal sources like BHI and sheep blood [64,65], and replaced by microbial sources, highlighting yeast extract [56,119] and even vegetal derivatives like the soy peptone [51, 105]. Even though the different culture medium compositions, the fermented broths presented high concentrations of HA, varying from 1 to 7 g/L [53,56].
4. Downstream operations for HA recovery and purification
Highly purified HA has been a challenge to researchers involved in purification area. Tables 1 and 2 shows intermediate purities of downstream operations used for purification of HA from fermentation process and fish eyeball, in which the purities are expressed in HA or in protein percentage, respectively. Moreover, the operations are discussed regarding the obtained purity, reported in terms of HA or remaining proteins.
As observed, there are not many processes which detail the purities after each operation. Besides, many of the detailed processes are composed by a single operation subdivided in steps, like diafiltration cycles or subsequent microfiltration and ultrafiltration. However, the impact of each operation inside the process was better noticed in the detailed, multi-operation sequences [56,73]. Precipitation presented higher increases in purity, even though it figured in different moments among the processes, with different HA sources.
Table 1
Hyaluronic acid sources and purity into each downstream operations (expressed in HA percentage) into purification sequences. The arrow represents the sequence order.
HA Source Downstream Operations Purity (%) Reference Fermentation Process (Streptococcus zooepidemicus) Precipitation (ethanol; 1.5:1, v/v; four consecutive steps) 87% [106] Fermentation Process (Streptococcus zooepidemicus) Filtration (Tangential, UF, 100 kDa, diafiltration) 90% [74] Fish Eyeball Filtration (300 kDa; diafiltration) + Protein Electrodeposition 18% [75] Precipitation with Ethanol (1.5:1, v/v) 88% Table 2
Hyaluronic acid sources and purity into each downstream operations (expressed in protein percentage) into purification sequences. The arrow represents the sequence order.
HA Source Downstream Operations Purity (%) Reference
Fermentation Process (Streptococcus zooepidemicus) Precipitation (2-propanol, 1:1 v/v) 14.1% (protein) [56] Adsorption (Silica Gel,
2% w/v)
4.5% (protein) Filtration + Adsorption
(Charcoal filter assembly, 0.45 μm) 0.6% (protein) Filtration (Diafiltration, 5x, 50 kDa) 0.06% (protein) Fermentation Process (Streptococcus zooepidemicus) Filtration (Tangential, MF and UF, 100 and 300
kDa, diafiltration)
4.1. Precipitation
Proteins are the main source of impurities in the process of HA production processes. Precipitation by organic solvents or quaternary salts has been widely used. In general, precipitation figures as the first steps in the HA purification processes, due to its effectiveness in removing most part of proteins and other contaminants.
4.1.1. Precipitation by Organic Solvents
Organic solvents promote dehydration and reduce the dielectric constant of the medium causing an increase in electrostatic interactions, leading to intra and intermolecular aggregation [107]. McDowall [108] emphasized the organic solvent precipitation as one of the most used operations for protein removal by using solvents such as ethanol, methanol, acetone, propanol, etc. The use of ethanol and propanol are preferable, due to the inherent toxicity of methanol and acetone.
Regarding HA purification, Roden et al. [109] reported the partition of proteins between the structure of HA and the aqueous medium favoring co-precipitation. Figure 2 illustrates the effects of the organic solvents on HA precipitation in the presence of proteins.
Ethanol is the most used solvent for HA precipitation, due to its lower cost compared to other alcohols and ketones. Proportions of 1:1 to 3:1 v/v ethanol: broth have been used as an initial step of HA precipitation from Streptococcus zooepidemicus fermentation [55,58,59,78,79,106,110-113]. Murado et al. [75] also studied the influence of ethanol proportion, on HA precipitation from fish eyeball, ranging the ethanol: extract from 0.5 to 2:1. According to their results, ethanol proportions of 1.5:1 or higher yielded an optimal recovery
(A)
(B)
Fig. 2. The effects caused by the organic solvents on HA precipitation : (A) HA dehydration (B)
electrostatic interactions with proteins due to decreasing of the dielectric constant.
of HA (>80%). Most works involving HA precipitation by ethanol use 1.5:1 up to 3:1 proportions.
The use of 2-propanol (or isopropanol) is also common in HA precipitation [59,78,110,114-117]. Rangaswamy and Jain [56] precipitated HA from a fermented broth produced by Streptococcus zooepidemicus using a 1:1 v/v proportion, obtaining a protein/HA
ratio of 16.5%. Jagadeeswara Reddy and Karunakaran [53] also used isopropanol at a 3:1 v/v proportion to precipitate microbial HA, but they do not specify intermediate purities.
Nimrod et al. [59] investigated the possibility of consecutive precipitations instead of a single one. Three consecutive precipitations were performed, with different
sequences involving isopropanol, ethanol or acetone to precipitate HA from a Streptococcus zooepidemicus fermentation broth. The final purities ranged from 87-91%. Sousa et al. [106] conducted four consecutive precipitations with ethanol (1.5:1, v/v) achieving a reduction of 87% of the initial protein content of a fermentation broth.
4.1.2. Precipitations with salts
4.1.2.1. Quaternary salts
An alternative to the organic solvents is the use of quaternary salts to precipitate HA. The salts form complexes with HA, scavenging it from the medium. A further separation of the HA-salt complex is required, which can be accomplished by using a highly ionic solution or an organic solvent. Cleland and Sherblom [67] used the cetylpiridinium chloride (CPC) precipitation as a step in the HA purification from bovine nasal septum cartilage, obtaining a final protein content of 8%. Swann [100] used CPC precipitation to purify and clarify HA solutions extracted from rooster combs, obtaining protein contents around 3-7%.
Precipitation of HA with cetyltrimethylammonium bromide (CTAB) from Streptococcus pyogenes fermentation broth in consecutive steps of CTAB and ethanol precipitations, yielded a final protein content 0.03 to 0.3% depending on the method of analysis [58]. Lago et al. [69] also precipitated HA with CTAB achieving a final purity of 99% by combining the CTAB
precipitation with organic solvent precipitation. As known, CTAB is also used for HA quantification [118-120].
4.1.2.2. Salts as Adjuvant
Murado et al. [75] studied the sodium chloride (NaCl) effect on protein removal by salting out in HA precipitation by ethanol from fish eyeball extracts. NaCl concentrations varied from 0.5 to 2 mol.L-1. The best results were obtained for NaCl concentrations equal or above 1.5 mol.L-1. The calcium and potassium salts were used as adjuvants for HA filtration before NaCl treatment followed by ethanol precipitation at 1:1 and 2:1 (v/v) ethanol:extract. The HA purity from this sequence was 90%. [121].
Changes in shape and density were observed by Won et al. [79] in HA precipitates. At low concentration of salt, the HA precipitates were in the form of fibers, while when at high concentration they formed particles, resulting in a more homogeneous precipitate with reduced volume.
4.2. Filtration
Filtration operation is based on retaining particles according to their size on a porous membrane. The fouling phenomenon is the main limitation of conventional filtration in which pore obstruction may occur along time, increasing both flux resistance and pressure drop. The tangential filtration avoids this limitation with the feed flow tangent to the membrane instead of the perpendicular flow as shown in Figure 3.
Fig. 3. Illustrative scheme of A) conventional filtration and B) tangential filtration.
The diafiltration mode operates by continuous solvent recirculation through the membrane, promoting better purification. Filter aids such as diatomaceous earth, are efficient due to their high porosity that improves the filtration rate.
The use of filtration in HA purification processes is widely reported to impurities removal, as well as cells separation in the specific case of fermented HA (53,58,114,122-124). Zhou et al. [73] studied HA purification from Streptococcus zooepidemicus by using consecutive tangential microfiltration (MF) and ultrafiltration (UF). Polyvinylidene fluoride membranes with cut-offs of 0.2 and 0.45 μm (MF) and 100 kDa and 300 kDa (UF) achieved better results with a purification factor of 1000 (protein content of 0.07%, approximately) and HA recovery of 77%. From mass balances, Oueslati et al. [74] proposed a predictive equation for HA yield, purity and process productivity from diafiltration. Its predictions were validated by tangential UF using a 100 kDa cut-off membrane to purify HA from Streptococcus zooepidemicus fermentation. The results showed that 90% HA purity was achieved with seven diavolumes.
In general, filtration is associated with other steps for HA purification from fermented broth. Choi et al. [125] initially combined centrifugation and filtration with diatomaceous earth to assure cells removal, followed by tangential filtration in diafiltration mode. The HA recovery was 60% with removal of most proteins. Rangaswamy and Jain [56] used UF (50 kDa cut-off membrane) in diafiltration mode as the final step of HA purification. The protein content was 0.06% after the whole process. Jagadeeswara Reddy and Karunakaran [53] used precipitation, MF and UF in diafiltration mode and adsorption followed by another precipitation step, obtaining HA with 99% purity.
Murado et al. [75] also used UF (300 kDa membrane) in diafiltration mode, simultaneously to a protein electrodeposition operation as the first step for purifying HA from fish eyeball.. The conjugate operations yielded HA with 18% purity only.
4.3. Adsorption
Adsorption is based on the capability of retention of compounds on the surface of porous solids (Figure 4). For HA purification adsorption is normally used in batch mode after precipitation and/or filtration [59,72,78,81,126,127]. Activated charcoal is, by far, the most used adsorbent [79,122,125] due to a low cost and high availability. However, the use of resins and silica gel are also common, as well as the use of different adsorbents in the same process.
Won et al. [79] compared the effect of different concentrations of charcoal and gamma alumina, alone or in combinations, for HA produced by Streptococcus sp. The best results were obtained with 3% of charcoal and 1.5% of gamma alumina that yielded HA with 0.8 μg.mL-1 protein content. Protein concentrations of 1.7 μg.mL-1 and 1.9 μg.mL-1 were achieved with the individual use of 3% of charcoal and 2% of gamma alumina, respectively. Hemant et al. [91]
Fig. 4. Illustrative scheme of adsorption operation on the surface of porous solids.
used adsorption steps with bentonite and activated charcoal as adsorbents at 10 and 30 g.L-1 respectively.
Yang and Lee [72] constructed isotherms based on the hyaluronic acid adsorption on chitosan-magnetite particles, as the adsorption was performed at different HA concentrations and a fixed amount of adsorbent, at three different pH (6, 7 and 8). The adsorption was more effective at pH 6, once the adsorbent and HA presented opposite charges. In the subsequent step, HA was directly adsorbed from fermented broth, resulting in a lower recovery due to electrostatic interactions among HA and medium components. However, after a pre-purification with ethanol precipitation (3:1, v/v), the adsorption at pH 6 was successful in terms of purity, with no detected proteins. Despite the expressive purification, HA presented a very low recovery (8%) after elution at pH 8.
4.3.1. Ion Exchange
The ion-exchange operation explores the interactions between a highly charged resin and opposite-charged compounds into solution, which are selectively adsorbed as illustrated in Figure 5. Usually ion exchange separation is used in the end of downstream sequences, Romeo and Lorenzi [87] used a Dowex M-15 resin for HA purification from rooster comb. Nebinger [86] compared the performance of different types of resins to separate sodium hyaluronate oligosaccharides. The best results were obtained with the Dowex and DEAE-Sephacel resins, as reported in previous works [128-130]. Hemant et al. [91] used cation-exchange chromatography, to remove metallic impurities from fermented HA, varying 10 to 100 g resin/L (Indion 225H).
Fig. 5. Representation of the ion-exchange chromatography in cationic resin for HA adsorption.
Besides purification, ion exchange is reported to be used in fractioning for HA characterization. Rosa et al. [48] used a Mono-Q column to fractionate glycosaminoglycans (GAGs) derived from rooster combs, in which HA corresponded to 90% of total GAGs. Lago et al. [69] characterized HA from umbilical cord through, by using a Mono Q HR 5/5 Column after a purification sequence involving precipitations with CTAB, ethanol and acetone.
4.4. Electrophoresis
Electrophoresis is a widely used method for protein separation and identification, in which the efficiency depends on the used gel, size and density of target molecules, as well as its purity (Figure 6). The use of electrophoresis is not usual for HA purification, due to the low capacity for protein removal compared to other operations.
Fig. 6. Illustration of HA separation by electrophoresis
Murado et al. [75] used a combined electrophoresis-diafiltration for HA recovery. Previously diafiltered, HA extract was submitted to electrophoresis aiming protein electrodeposition. A HA purity of approximately 18% was obtained after the conjugate operation. Another alternative is HA deposition, as explored by Hutterer and Jorgenson [83]. Capillary electrophoresis was evaluated with commercial HA, comparing the separation efficiencies of two different voltages (15 and 95 kV). The latter voltage favored separation, increasing by tenfold the HA deposition. The authors also observed that the peak resolution decreased with increasing of HA molecular weight.
4.5. HA Purification Processes
Tables 3 and 4 summarize the whole purification processes concerning one or more of the discussed operations. The results are expressed in HA purity and remaining proteins, respectively. Figure 7 presents a flow chart illustrating the main possibilities of the purification processes into a 3-step sequence. As mentioned before, precipitation figures in most purification sequences, due to its accessibility, simplicity and effectiveness. The highest purities were achieved in the downstream processes applied to microbial HA, in which filtration and adsorption are also frequent. Animal sources requires more refined downstream sequences for HA purification. The use of operations such as enzymatic digestion by proteases and ionic exchange operations can be justified by the presence of proteoglycans linked to HA.
Table 3
Purification Processes of HA and their Main Operations (purity expressed in HA percentage)
HA Source Downstream processes Purity Reference
Streptococus
zooepidemicus
Precipitation: 2-propanol (3:1 v/v); Filtration: 2 filtration steps; ultrafiltration in diafiltration mode; Adsorption:
Charcoal (1-2% w/v);
Precipitation: Ethanol (1.5 to 3:1 v/v); Filtration; Adsorption: Aromatic Resin and Active Carbon (3% w/v
each)
Precipitation: Organic Solvents (1-3:1 v/v); Adsorption: Bentonite (1% w/v) and Activated Carbon (3% w/v); Ion
Exchange Chromatography
Precipitation: Isopropanol, Ethanol or Acetone (1-1.5% v/v) and Cetylpiridinium Chloride (10% solution); Adsorption:
Activated Charcoal (0.1% w/v) 99.2% 99% 99.3% 87-92% [53] [78] [91] [59]
Rooster comb Precipitation: Ethanol (1-2:1 v/v); Enzymatic Treatment: Protease
90% [121]
Human Umbilical Cord
Residues
Precipitation: 1% solution of CTAB (300-600 mL to 1 kg of sample); Precipitation (Ethanol 75% and Acetone); Ion
Exchange Chromatography (characterization)
Table 4
Purification Processes of HA and their Main Operations (purity expressed in protein percentage
Microorganism Downstream processes Purity Reference
Streptococus
zooepidemicus
Precipitation: Ethanol (1:1 v/v); Filtration (Diafiltration Mode); Adsorption: Charcoal (2% w/v) and
Gamma-Alumina (1% w/v) 0.8% [79] Streptococcus pyogenes
Precipitation: Ethanol (3:1 v/v) and CTAB 0.32%; Tangential Filtration (Diafiltration Mode)
0.16% [58]
Streptococcus equi
Precipitation: Isopropyl Alcohol (2:1 v/v); Filtration 0.2% [110]
Rooster comb Precipitation: Ethanol (3:1 v/v); Filtration (Diafiltration Mode); Enzymatic Treatment: Papain, Pepsin, Trypsin or
PRONASE® (0.2 g enzyme/300 g dry powder); Ion Exchange Resin
Precipitation: Ethanol (3:1 v/v); Filtration; Enzymatic Treatment: PRONASE® 0.2% (protein) 0,05% (protein) [127] [132] Bovine Nasal Septum Cartillage
Precipitation: CPC and Methanol (3-4:1 v/v); Enzymatic Treatment: Papain
8% (protein) [67]
Bovine Vitreous Body
Precipitation: Trichloroacetic Acid 5%; Adsorption: CH-Sepharose 4B
0.1% (protein)
[133]
Synovial Fluid Filtration; Enzymatic Treatment: Trypsin 0.5% (protein)
[134]
5. Conclusions and future perspectives
Reports from the specific literature presented the achievement of a highly purified HA, with different downstream sequences, reaching the medical requirements in most of them. Precipitation is common at the beginning of most downstream process, due to its effectivity, ease of operation and cost benefit. Filtration can also be highlighted, specially in diafiltration mode. Adsorption and ion-exchange chromatography are used at the final of the sequences to reach highly purified HA.
Purification of microbial HA yielded similar or even higher purities using less refined operations compared to HA purification from animal sources. Although the various processes described in the literature, still there is a lack of information about the performance of the individual operations as evaluated by their specific parameters. Future studies may comprise the development of representative mathematical models for process optimization, as well as upstream strategies in order to reduce downstream operation sequences. Moreover, the influence of the operations on the HA structure and molar mass distribution must be investigated concerning its biological activity.
Acknowledgements
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico [CNPq - Projeto nº:142480/2014-2], Fundação de Amparo à Pesquisa do Estado de São Paulo [FAPESP – Project no: 2015/23134-8] and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
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