Primary Recovery Step Research Articles

Recovery of a Bacteriocin-Like Inhibitory Substance from Lactobacillus bulgaricus FTDC 1211 Using Polyethylene-Glycol Impregnated Amberlite XAD-4 Resins System.

Lactobacillus bulgaricus is a LAB strain which is capable of producing bacteriocin substances to inhibit Staphylococcus aureus. The aim of this study was to purify a bacteriocin-like inhibitory substance (BLIS) produced by L. bulgaricus FTDC 1211 using an aqueous impregnated resins system consisting of polyethylene-glycol (PEG) impregnated on Amberlite XAD4. Important parameters influencing on purification of BLIS, such as the molecular weight and concentration of PEG, the concentration and pH of sodium citrate and the concentration of sodium chloride, were optimized using a response surface methodology. Under optimum conditions of 11% (w/w) of PEG 4000 impregnated Amberlite XAD4 resins and 2% (w/w) of sodium citrate at pH 6, the maximum purification factor (3.26) and recovery yield (82.69% ± 0.06) were obtained. These results demonstrate that AIRS could be used as an alternate purification system in the primary recovery step.

Economic and environmental analysis of PHAs production process

Following the growing demand to improve both economic and environmental performance of PHAs production, the research focused on the evaluation of the economic and environmental performance of PHAs production process via aqueous two-phase extraction (ATPE). Thus, the process analysis of two processes with different PHAs purification and recovery strategies (which are with and without thermoseparating ATPE as primary purification step) was performed. Using the basis of 9000 tons PHAs production per year and 7920 operating hours, the process with thermoseparating ATPE as primary purification step standout in terms of both economic and environmental performance. PHA production cost of 5.77 US$/kg with a payback period of fewer than 4 years and ROI of 25.2% was achieved. The results showed that most of operating cost is contributed by facility-dependent cost and raw material cost, while the main contributor to raw material cost is carbon source. The insight from sensitivity analysis has demonstrated that the economic performance is sensitive to the fluctuation in surfactant cost. This proved that utilizing thermoseparating ATPE as primary recovery step not only helps to reduce chemical consumption, it also minimizes downstream equipment cost and wastewater treatment cost.

Design of aqueous two-phase systems for purification of hyaluronic acid produced by metabolically engineered Lactococcus lactis.

Hyaluronic acid has a wide range of biomedical applications and its commercial value is highly dependent on its purity and molecular weight. This study highlights the utility of aqueous two-phase separation as a primary recovery step for hyaluronic acid and for removal of major protein impurities from fermentation broths. Metabolically engineered cultures of a lactate dehydrogenase mutant strain of Lactococcus lactis (L. lactis NZ9020) were used to produce high-molecular-weight hyaluronic acid. The cell-free fermentation broth was partially purified using a polyethylene glycol/potassium phosphate system, resulting in nearly 100% recovery of hyaluronic acid in the salt-rich bottom phase in all the aqueous two-phase separation experiments. These experiments were optimized for maximum removal of protein impurities in the polyethylene glycol rich top phase. The removal of protein impurities resulted in substantial reduction of membrane fouling in the subsequent diafiltration process, carried out with a 300 kDa polyether sulfone membrane. This step resulted in considerable purification of hyaluronic acid, without any loss in recovery and molecular weight. Diafiltration was followed by an adsorption step to remove minor impurities and achieve nearly 100% purity. The final hyaluronic acid product was characterized by Fourier-transform IR and NMR spectroscopy, confirming its purity.

Differential response in downstream processing of CHO cells grown under mild hypothermic conditions

The manufacture of complex therapeutic proteins using mammalian cells is well established, with several strategies developed to improve productivity. The application of sustained mild hypothermic conditions during culture has been associated with increases in product titer and improved product quality. However, despite associated cell physiological effects, very few studies have investigated the impact on downstream processing (DSP). Characterization of cells grown under mild hypothermic conditions demonstrated that the stationary phase was prolonged by delaying the onset of apoptosis. This enabled cells to maintain viability for extended periods and increase volumetric productivity from 0.74 to 1.02 g L−1. However, host cell proteins, measured by ELISA, increased by ∼50%, attributed to the extended time course and higher peak and harvest cell densities. The individual components making up this impurity, as determined by SELDI-TOF MS and 2D-PAGE, were shown to be largely comparable. Under mild hypothermic conditions, cells were less shear sensitive than those maintained at 37°C, enhancing the preliminary primary recovery step. Adaptive changes in membrane fluidity were further investigated by adopting a pronounced temperature shift immediately prior to primary recovery and the improvement observed suggests that such a strategy may be implementable when shear sensitivity is of concern. Early and late apoptotic cells were particularly susceptible to shear, at either temperature, even under the lowest shear rate investigated. These findings demonstrate the importance of considering the impact of cell culture strategies and cell physiology on DSP, by implementing a range of experimental methods for process characterization. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:688–696, 2013

Expanded bed adsorption as a primary recovery step for the isolation of the insulin precursor MI3 process development and scale up

Expanded bed adsorption (EBA) was evaluated for the isolation of the human insulin precursor MI3, expressed and secreted by the yeast Saccharomyces cerevisiae. The isoelectric point of the insulin precursor (pH 5.3) makes cation exchange a prime candidate for direct adsorption. In order to find a suitable window of operation for the process the adsorption equilibrium was analysed in a wide range of operating conditions (pH and conductivity) and for three different stationary phases. The same array of operating conditions was examined with regard to stable fluidisation of the adsorbents in S. cerevisiae suspensions. Interactions of the yeast with the fluidised stationary phase were investigated by a pulse response technique and the hydrodynamics of the fluidised bed under process conditions by residence time distribution analysis. The case study demonstrates that by parallel examination of product binding and fluidisation quality a window of operation can be found. Analysis of the binding kinetics by breakthrough experiments and modelling led to the definition of a set of operating conditions, which yield a compromise between optimal use of the equilibrium capacity provided by the adsorbent and high throughput required for an industrial separation. After initial experiments on the bench scale the protocol was transferred successfully to pilot scale demonstrating the design of a reliable operation.

Evaluation of different primary recovery methods forE. coli-derived recombinant human growth hormone and compatibility with further down-stream purification

Due to advances in fermentation technology, it is now possible to obtain fermentation broth with over 30% solids. The high solid content makes the clarification step difficult, especially at large scale. The primary protein recovery step is challenging due to the heterogeneous solution of soluble and insoluble material. In this study, we compare different primary recovery routes and the compatibility with the initial capture chromatography step. The primary recovery routes studied are standard clarification by centrifugation and extraction in aqueous two-phase systems. The compatibility of the feed streams from the different primary recovery steps with the first chromatography step is addressed. An anion-exchange column was used as the first capture column in the purification process. The aqueous two-phase system was composed of a random copolymer of ethylene oxide and propylene oxide (EOPO) in combination with a waxy starch. The target protein in this study was human growth hormone (hGH) produced in recombinant Escherichia coli. The purity of hGH in the top phase after aqueous two-phase extraction was found to be significantly higher than in clarified homogenate supernatant and increased as the EOPO polymer concentration in the aqueous two-phase system increased. Stability of the supernatant and EOPO top phases and hGH were determined by turbidity measurements and LC-MS assay. All of the feed-streams from the primary recovery steps were compatible with the anion-exchange chromatography step; however, the capacity of the resin was strongly dependent on the purity of the load. Different process aspects, e.g., resin capacity, viscosity, purification, and yield of hGH and scalability are compared.

A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I–hydrophobin I

Extraction systems for hydrophobically tagged proteins have been developed based on phase separation in aqueous solutions of non-ionic detergents and polymers. The systems have earlier only been applied for separation of membrane proteins. Here, we examine the partitioning and purification of the amphiphilic fusion protein endoglucanase I core–hydrophobin I (EGI core–HFBI) from culture filtrate originating from a Trichoderma reesei fermentation. The micelle extraction system was formed by mixing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Reppal PES100. The detergent/polymer aqueous two-phase systems resulted in both better separation characteristics and increased robustness compared to cloud point extraction in a Triton X-114/water system. Separation and robustness were characterized for the parameters: temperature, protein and salt additions. In the Triton X-114/Reppal PES100 detergent/polymer system EGI core–HFBI strongly partitioned into the micelle-rich phase with a partition coefficient ( K) of 15 and was separated from hydrophilic proteins, which preferably partitioned to the polymer phase. After the primary recovery step, EGI core–HFBI was quantitatively back-extracted ( K EGIcore–HFBI=150, yield=99%) into a water phase. In this second step, ethylene oxide–propylene oxide (EOPO) copolymers were added to the micelle-rich phase and temperature-induced phase separation at 55°C was performed. Total recovery of EGI core–HFBI after the two separation steps was 90% with a volume reduction of six times. For thermolabile proteins, the back-extraction temperature could be decreased to room temperature by using a hydrophobically modified EOPO copolymer, with slightly lower yield. The addition of thermoseparating co-polymer is a novel approach to remove detergent and effectively releases the fusion protein EGI core–HFBI into a water phase.

Separation optimization for the recovery of phenyl ethyl alcohol.

Phenyl ethyl alcohol is a compound that occurs naturally in flower petals and in many common beverages, such as beer. Desire for the floral, rose-like notes imparted by phenyl ethyl alcohol has created a unique niche for this chemical in flavor and fragrance industries. Phenyl ethyl alcohol can be produced by Saccharomyces cerevisiae via bioconversion. Often this method of production results in extremely low yields, thus placing a great deal of importance on recovery and purification of the valuable metabolite. To determine the best method for recovering the chemical, a primary recovery step and a secondary recovery step were developed. The primary recovery step consisted of comparing dead-end filtration with crossflow ultrafiltration. Crossflow ultrafiltration was ultimately selected to filter the fermentation broth because of its high flow rates and low affinity for the product. The secondary recovery step consisted of a comparison of liquid- liquid extraction and hydrophobic resin recovery. The hydrophobic resin was selected because of its higher rate of recovery and a higher purity than the liquid-liquid extraction, the current practice of Brown-Forman.

Fluidized bed adsorption as a primary recovery step in protein purification.

Fluidized bed adsorption has been introduced as an integrative technology combining clarification, concentration, and initial purification in a single step. In the paper presented here, the use of fluidized adsorbents in the primary recovery of proteins starting from unclarified broths is reviewed. First the principle of fluidizing adsorbent particles is discussed, subsequently possible experimental procedures for whole broth adsorption are demonstrated. The system parameters governing the performance of the sorption process in a fluidized bed are discussed in the second part of the paper and considerations on how operating parameters and process design influence the limiting steps are provided. Finally, examples for the successful operation of whole broth adsorption employing fluidized adsorbents are shown and conditions are defined under which this technology may be an alternative to traditional protein purification methods.