Downstream processing of virus-like particles with aqueous two-phase systems: Applications and challenges

The advancement of recombinant virus-like particle-based vaccines has attracted global attention owing to substantially safety and high efficacy in provoking a protective immunity against various chronic and infectious diseases in humans and animals. A robust, low-cost, and scalability separation and purification technology is of utmost importance in the downstream processing of recombinant virus-like particles to produce affordable and safe vaccines. Being a relatively simple, environmentally friendly, and efficient biomolecules recovery approach, aqueous two-phase systems have received great attention from researchers worldwide. This review aims to highlight the challenges and outlook in addition to the current applications of aqueous two-phase systems in downstream processing of virus-like particles. The efforts will confidently reinforce scholars’ knowledge and fill in the valuable research gap in the aspect of concerning recombinant virus-like particle-based vaccines development, particularly related to the virus-like particles downstream production processes.     

聚乙二醇双水相萃取光度法测定锌

A novel method of extraction spectrophotometric determination of Zn~(2+) in aqueous two-phase system formed by polyethylene glycol-2000,sodium sulfate and zincon has been established.Amounts of PEG solution,zincon solution,sodium sulfate added and the acidity of the system were optimized.The interference effects of various coexistent ions and the method to remove the interference were investigated.The Zn-zincon complex that had been formed in the presence of H_2BO_3-Na_2B_4O_7 buffer solution at pH 7.4 was extracted into the upper PEG-rich phase,and the absorbance of the extracted complex in the upper PEG-rich phase was measured at 630 nm.The apparent molar absorptivity was 7.3 × 10~4 L / (mol·cm).Beer's law was obeyed over the range of 0～2.0 μg / mL Zn~(2+).The analytical results obtained for Zn~(2+) in water samples were in good agreement with values measured by atomic absorption spectrometry.     

PEG/(NH4)2SO4双水相体系萃取甘草中的有效成分

甘草的主要有效成份甘草酸具有消炎解毒、抗氧自由基、调节机体免疫力等作用,目前已被广泛应用于食品、饮料、化妆品、医药、卷烟等行业。工业上广泛应用的是水提法,该法操作容易、设备简单、溶剂价廉,但提取效率不高,易造成资源浪费。双水相萃取技术(Two-aqueous phase extraction,简称．ATPS),是近年出现的新型生物化工分离技术。由于双水相萃取分离过程条件温和、可调节因素多、易于放大和操作,不存在有机溶剂残留问题,特别适用于生物物质及天然资源中有效成分的分离和提纯。     

Hg (II) extraction in a PEG-based aqueous two-phase system in the presence of halide ions. I. Liquid phase analysis

The partition behaviour of Hg (II) was studied in an aqueous polyethylene glycol (PEG) — (NH₄)₂SO₄ two-phase system as a function of halide, halide concentration, and pH. For a system prepared by mixing equal volumes of 40 % (w/w) PEG (1550) with 40 % (w/w) (NH₄)₂SO₄, Hg(II) remains almost exclusively in the salt-rich phase. The addition of NaX (X = Cl⁻, Br⁻, I⁻) enhances Hg (II) partition into the PEG-rich phase due to the formation of halide complexes. The efficiency of halide extractants increases in the order: Cl⁻ < Br⁻ < I⁻. Mercury extraction is improved at lower halide ion concentration by higher stock salt solution acidity. From the distribution coefficients determined as a function of halide ion concentration, the extracted species were identified. The Hg (II) extractability is determined by the type and stability of the Hg (II) halide species, and depends on the stock salt solution acidity. The observed behaviour is discussed and a possible extraction mechanism is proposed.     

聚乙二醇-硫酸铵双水相萃取次甲基蓝的研究

利用分光光度法研究了次甲基蓝在聚乙二醇-硫酸铵双水相体系的萃取行为,探索了质量配比(mPEc/m(NH4)2SO4)、温度和次甲基蓝的浓度对双水相及次甲基蓝萃取率(Y)的影响.实验表明:(1)在一定的温度和浓度下,随着mPEC/m(NH4)2SO4的减小,分配系数K( cup/clow)增加,萃取率Y略有减小;(2)在一定的质量配比和浓度下,分配系数K(cup/clow)随温度的升高而增大,但萃取率Y却基本保持不变;(3)在一定的质量配比和温度下,随着次甲基蓝浓度的增加,分配系数K(cup/clow)增大;萃取率Y略有增加.在mPEG/m( NH4) 2SO4为1.50∶1,c次甲基蓝=8.915×10-5mol/L、t=30℃的实验条件下,K(cup/clow)=22.14,萃取率(y)可达95.43％.     

两次双水相萃取结合高效液相色谱法选择性分离唾液蛋白质的初步研究（英文）

双水相萃取是一种新型的液-液萃取技术,具有方法简单,易操作,成本低,易放大,条件温和,可保持蛋白质活性等明显优势,特别适用于生物样品的前处理和组分分离。本文建立了15% PEG-4000/8% NaH₂PO₄双水相体系,通过两次双水相萃取结合高效液相色谱法（HPLC）分离了唾液中的多种蛋白质。经过双水相萃取,对上、下两相中的蛋白质进行色谱的梯度洗脱分析。50 min内蛋白质的色谱峰可分为10组,根据其在上、下两相的分配规律还可划分为6个组分区。结果表明,两次双水相萃取结合HPLC可以实现唾液中的蛋白质的选择性分离。该法为复杂生物样品中的蛋白质多维度、选择性分离和分析提供了新的思路。     

Purification of plasmid DNA vectors by aqueous two-phase extraction and hydrophobic interaction chromatography

The current study explores the possibility of using a polyethyleneglycol(PEG)-ammonium sulphate aqueous two-phase system (ATPS) as an early step in a process for the purification of a model 6.1 kbp plasmid DNA (pDNA) vector. Neutralised alkaline lysates were fed directly to ATPS. Conditions were selected to direct pDNA towards the salt-rich bottom phase, so that this stream could be subsequently processed by hydrophobic interaction chromatography (HIC). Screening of the best conditions for ATPS extraction was performed using three PEG molecular weights (300, 400 and 600) and varying the tie-line length, phase volume ratio and lysate load. For a 20% (w/w) lysate load, the best results were obtained with PEG 600 using the shortest tie-line (38.16%, w/w). By further manipulating the system composition along this tie-line in order to obtain a top/bottom phase volume ratio of 9.3 (35%, w/w PEG 600, 6%, w/w NH4)2 SO4), it was possible to recover 100% of pDNA in the bottom phase with a three-fold increase in concentration. Further increase in the lysate load up to 40% (w/w) with this system resulted in a eight-fold increase in pDNA concentration, but with a yield loss of 15%. The ATPS extraction was integrated with HIC and the overall process compared with a previously defined process that uses sequential precipitations with iso-propanol and ammonium sulphate prior to HIC. Although the final yield is lower in the ATPS-based process the purity grade of the final pDNA product is higher. This shows that it is possible to substitute the time-consuming two-step precipitation procedure by a simple ATPS extraction.     

Partition behavior and partial purification of hexokinase in aqueous two-phase polyethylene glycol/citrate systems

This study dealt with the partition behavior and partial purification of hexokinase (HK) from baker’s yeast by liquid-liquid extraction using aqueous two-phase polyethylene glycol (PEG)/citrate systems. First, we investigated the effect of agitation type (vortex and 8 rpm rotation) on the stability of the system, and then the effects of sodium citrate concentration, PEG concentration, and molar mass of PEG on the partition coefficient of this enzyme by using a 2⁵ factorial experimental design. The results of this factorial experiment showed the possibility of a partial purification of HK by using two extraction steps, since the enzyme preferentially migrated to the top phase and the total proteins (mainly contaminants) remained in the bottom phase. The purification factor (Pur _TOP) of the enzyme in the top phase was 1.87, and the partition coefficient of the total proteins (K _Prot ) was 0.47.     

Influence of magnetic field on aqueous two-phase extraction of horse ferritin in the polyethylene glycol/hydroxyethyl starch system

The presented experiments show the model of expectation of equine spleen ferritin extraction in a new aqueous two-phase system which was formed by mixing polyethylene glycol (PEG) and hydroxyethyl starch (HES). The tendency of the protein to migrate in the analyzed systems was dependent on the concentrations of HES and PEG as well as PEG molecular weight. The highest concentration of ferritin in the top phase (rich in PEG) was recorded in the system composed of 6% PEG 3000 and 3% HES. The obtained concentration was 0.88 mg mL⁻¹. The lowest concentration was 0.42 mg mL⁻¹ in the system composed of 5% PEG 6000 and 1% HES.     

Effective extraction of tylosin and spiramycin from fermentation broth using thermo-responsive ethylene oxide/propylene oxide aqueous two-phase systems

Recyclable aqueous two-phase systems with thermo-responsive phase-forming materials have been employed to separate macromolecules; however, these systems have achieved very limited separation efficiency for small molecules, such as antibiotics. In this study, aqueous two-phase systems composed of the ethylene oxide/propylene oxide copolymer and water were developed to extract alkaline antibiotics from the fermentation broth. In the aqueous two-phase systems with an ethylene oxide ratio of 20 and propylene oxide ratio of 80, the partition coefficients of tylosin and spiramycin reached 16.87 and 20.39, respectively, while the extraction recoveries were 70.67 and 86.70%, respectively. Coupled with mechanism analysis, we demonstrated the feasibility of extracting alkaline antibiotics using this aqueous two-phase system, especially for 16-membered macrolide antibiotics. The molecular dynamic simulation was employed to visualize the process of dual-phase formation and the partition behavior of antibiotics in an aqueous two-phase system. The dynamic simulation revealed the binding energy between the antibiotic and ethylene oxide/propylene oxide copolymers, which provides a simple indicator for screening suitable antibiotics in aqueous two-phase systems. Our recyclable aqueous two-phase systems provide a robust approach for the extraction of 16-membered macrolide antibiotics with ease of operation and high recovery rates, which is appropriate for large-scale extraction in the fermentation industry.     

Liquid-liquid equilibrium of aqueous two-phase systems composed of poly(ethylene oxide) 1500 and different electrolytes ((NH4)2SO4, ZnSO4 and K2HPO4): Experimental and correlation

Phase diagrams of aqueous two-phase systems composed of PEO1500 + salt (di-potassium phosphate + potassium hydroxide or ammonium sulfate or zinc sulfate) + water were determined at (283.15, 298.15, and 313.15) K. All systems produce a large two-phase region; however the influence of temperature on the binodal position seems to be very small. By analyzing the effects of ammonium sulfate or zinc sulfate, it was observed that zinc was more effective in promoting phase separation than ammonium. The consistency of the tie-line data was ascertained by applying the Othmer-Tobias correlation. In this paper, aqueous two-phase systems data for nine ternary systems are correlated by using the NRTL model and UNIFAC for the activity coefficient. The results are very satisfactory, with root mean square deviations between experimental and calculated compositions as low as 0.99 and 1.21%, respectively. However the NRTL model better represents the systems in study, when compared with UNIFAC.     

On the influence of some strong electrolytes on the partitioning of acetic acid to aqueous/organic two-phase systems in the presence of tri-n-octylamine: Part II: Toluene as organic solvent

Water-insoluble amines (dissolved in an organic solvent/organic solvent mixture) are often used for the extractive recovery of carboxylic acids from aqueous phases. The basic design of the extraction process requires a thermodynamic framework that should be able to describe the liquid–liquid phase equilibrium not only in the phase forming systems (water + carboxylic acid + organic solvent + reactive extractant), but also when the aqueous feed phase contains additionally small amounts of strong electrolytes. Even small amounts of strong electrolytes might considerably reduce the recovery rate. In part I of this series such a model was presented and discussed for methyl isobutyl ketone as organic solvent and tri-n-octylamine (TnOA) as the chemical extractant. The present part II is to demonstrate that the procedures/methods described for methyl isobutyl ketone as organic solvent can be applied also for other organic solvents. By way of example, here toluene is that organic solvent. New experimental results are reported for the influence of sodium chloride, sodium nitrate, sodium sulfate, sodium acetate and hydrochloric acid on the partitioning of acetic acid to coexisting aqueous/organic liquid phases of the system (water + toluene + tri-n-octylamine) at 25 °C. An extension/adaptation of the previously published thermodynamic framework is successfully applied to describe/predict the new experimental liquid–liquid phase equilibrium data.