Integrated separation process for isolation and purification of biosuccinic acid

Biotechnologically produced succinic acid has the potential to displace maleic acid and its uses. Therefore, it is of high interest for the chemical, pharmaceutical, and food industry.In addition to optimized production strains and fermentation processes, an efficient separation of succinic acid from the aqueous fermentation broth is indispensable to compete with the current petrochemical production of succinic acid. Isolation and purification of succinic acid from an Escherichia coli fermentation broth were studied with two amine-based reactive extraction systems: (i) trihexylamine in 1-octanol and (ii) diisooctylamine and dihexylamine in a mixture of 1-octanol and 1-hexanol. Back extraction of succinic acid from the organic phase was carried out using an aqueous trimethylamine solution. The trimethylammonium succinate generated after back extraction was split with an evaporation-based crystallization.The focus was on process integration, for example, reuse of the applied amines for extraction and back extraction. It was shown that the maximum trimethylamine concentration for back extraction should not exceed the stoichiometric amount (2 mol trimethylamine/mol the succinic acid in the organic phase) to ensure maximal extraction yields with the reused organic phase in subsequent extractions. Moreover, mixer-settler extraction and back extraction of succinic acid were scaled up from the milliliter- to the liter-scale making use of liquid–liquid centrifuges. The overall yield was 83.5% of the succinic acid from thefermentation supernatant. The final purity of the succinic acid crystals was 99.5%. Organic phase and amines can easily be recycled and reused.     

An integrated process for mammalian cell perfusion cultivation and product purification using a dynamic filter

In the present work, a dynamic filter was employed to develop an integrated perfusion/purification process. A recombinant CHO cell line producing a human anti-HIV IgG was employed in the experiments. In the first part of this work, the dynamic filter was fitted with conventional microfiltration membranes and tested as a new external cell retention device for perfusion cultivations. The filter was connected to a running perfusion bioreactor and operated for approximately 400 h at an average cell concentration of 10 million cells mL(-)(1), whereby cell viability remained above 90% and no problems of sterility were experienced. In the second part of this work, the dynamic filter was employed to simultaneously carry out cell separation and product purification, using membrane adsorbers containing Protein A affinity ligands. An automated system was built, which integrated the features of an automated perfusion bioreactor and of a liquid chromatography system. The IgG was continuously adsorbed onto the affinity membranes and was periodically recovered through elution cycles. After connection of the filter, the system was operated for approximately 300 h, whereby three elution cycles were carried out. No progressive increase in transmembrane pressure was observed, indicating no membrane fouling problems, and the IgG was recovered practically free of contaminants in a 14-fold concentrated form, indicating that the integrated, one-step perfusion/purification process developed during this work is a promising alternative for the production of biologicals derived from mammalian cells.     

Smart process development: Application of machine-learning and integrated process modeling for inclusion body purification processes

The development of a biopharmaceutical production process usually occurs sequentially, and tedious optimization of each individual unit operation is very time-consuming. Here, the conditions established as optimal for one-step serve as input for the following step. Yet, this strategy does not consider potential interactions between a priori distant process steps and therefore cannot guarantee for optimal overall process performance. To overcome these limitations, we established a smart approach to develop and utilize integrated process models using machine learning techniques and genetic algorithms. We evaluated the application of the data-driven models to explore potential efficiency increases and compared them to a conventional development approach for one of our development products. First, we developed a data-driven integrated process model using gradient boosting machines and Gaussian processes as machine learning techniques and a genetic algorithm as recommendation engine for two downstream unit operations, namely solubilization and refolding. Through projection of the results into our large-scale facility, we predicted a twofold increase in productivity. Second, we extended the model to a three-step model by including the capture chromatography. Here, depending on the selected baseline-process chosen for comparison, we obtained between 50% and 100% increase in productivity. These data show the successful application of machine learning techniques and optimization algorithms for downstream process development. Finally, our results highlight the importance of considering integrated process models for the whole process chain, including all unit operations.     

An integrated microfluidic system for bovine DNA purification and digital PCR detection

In this paper, we described an integrated modularized microfluidic system that contained two distinct functional modules, one for nucleic acids (NA) extraction and the other for digital PCR (dPCR), allowing for detecting the bovine DNA in ovine tissue.     

An interactive biomolecule graphics system

This paper describes an interactive biological macromolecule graphic system on the VAX-11/750 + Model75 image-processing system. It can display the structure of types of macromolecules on the color monitor, and various user-defined options are available. The display model can be either a stick-and-ball model or a space-filling model. The user can easily display windows, rotate and shift interactively. For the space-filling model, a light source can be adjusted to produce a satisfactory three-dimensional effect. A fast algorithm for shading processing is proposed based on the use of spatial correlation information.     

Chromatographic control of the process of rifamycin B isolation and purification]

A method of chromatography in a thin layer of Silica Cel No. 2 was developed for rifamicin B, rifamicin complex and some admixtures. Extracts of rifamicin B in an organic solvent, aqueous buffer reextracts and dry preparations of rifamicin B were analyzed with the above method. A half-quantitative chromatographic procedure for estimation of the main admixture in dry preparations was proposed.     

An improved procedure for the isolation and purification of protoplasts from carrot suspension culture

A procedure is reported for the rapid and highly reproducible isolation of protoplasts from carrot suspension culture. The method utilizes Onozuka R 10 cellulase which has been purified by chromatography on Sephadex G75. Protoplast isolation, using this procedure, is quantitative and complete within 1 to 1.5 h. Intact protoplasts were separated from broken ones and other cellular debris by application of a polyethylene glycol/dextran two-phase system. The protoplasts isolated in this manner lack any detectable cell wall and are greater than 95% viable when assayed using fluorescein diacetate. It is concluded that such protoplasts are highly suitable for biochemical studies.Abbreviation PCM protoplast culture medium     

An integrated process for the production and biotransformation of penicillin.

The extraction of Penicillin G (PG) from the filtered cultivation medium of Penicillium chrysogenum and its conversion into 6-amino penicillanic acid (6-APA) and phenyl acetic acid (PhA) at pH 8 was performed in a 10 l kühni extractor during the production by means of penicillin-G-amidase immobilized in a liquid membrane carrier system (LM). 6-APA was enriched in LM, and the PhA returned to the cultivation medium. After electrocoalescence of LM, the 6-APA was converted into ampicillin with the same enzyme at pH 6, while the liquid membrane phase and enzyme were recycled and reused.     

Different types of aqueous two‐phase systems for biomolecule and bioparticle extraction and purification

Upstream improvements have led to significant advances in the productivity of biomolecules and bioparticles. Today, downstream processes are the bottleneck in the production of some biopharmaceuticals, a change from previous years. Current purification platforms will reach their physical limits at some point, indicating the need for new approaches. This article reviews an alternative method to extract and purify biomolecules/bioparticles named aqueous two‐phase system (ATPS). Biocompatibility and readiness to scale up are some of the ATPS characteristics. We also discuss some of ATPS applications in the biotechnology field. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1343–1353, 2013     

Improved methodologies for the isolation and purification of tropoelastin

Increased yields of purified tropoelastin were obtained from lathyritic chicks which had been raised on normal commercial starting feed for 1 week followed by 8 days on commercial starting feed supplemented with 0.1% aminoacetonitrile·HCl and 0.05% ?-aminocaproic acid. The increased age of chicks prior to lathyrogen administration plus the addition of the trypsin-like enzyme inhibitor, ?-aminocaproic acid, to the diet resulted in a 110% increase in the yield of tropoelastin. In addition, a cation-exchange system was developed for the fractionation of tropoelastin and removal of minor acidic protein contaminants.     

Chromatographic techniques for the isolation and purification of lipoproteins

Various modes of chromatography are available for lipoprotein separation. Gel permeation and affinity chromatography are used for preparative purposes and to separate lipoproteins according to size and apolipoprotein content, respectively. Development of rigid supports for gel permeation has led to large improvements in speed and resolution. Reversed-phase high-performance liquid chromatography (HPLC) of apolipoproteins offers the best performance in terms of speed and resolution of structural variants. Due to its high speed and superior resolving power, the recently developed technique of capillary electrophoresis should emerge as an important method for lipoprotein analysis.     

An integrated process for the treatment of CETP wastewater using coagulation, anaerobic and aerobic process

The aim of this study was to treat the wastewater collected from equalization tank of Common Effluent Treatment Plant (CETP), which was a mixture of waste coming from 525 small-scale industries manufacturing textile and dyestuff intermediate, pigments and pharmaceuticals. Initially a pretreatment using ferric chloride and lime was carried out to increase the biodegradability (BOD(5)/COD) of the effluent, which showed color removal of 74% and COD reduction of 75% at a concentration of 10 and 4 g/L. respectively. The biological treatment system using anaerobic fixed film reactor was investigated as secondary treatment. A mixture of bacterial consortium DMAB and cowdung slurry was used for the formation of biofilm. The effect of hydraulic retention time (HRT) and organic loading rate (OLR) on the efficiency of treatment of anaerobic reactor was analysed. Subsequent aerobic treatment after anaerobic step using aerobic culture Pseudomonas aeroginosa helped in further removal of COD and color. Formation of aromatic amines during anaerobic treatment was mineralized by sequential aerobic treatment.     

An integrated electrocoagulation-phytoremediation process for the treatment of mixed industrial wastewater

The elimination of organic contaminants in highly complex wastewater was tested using a combination of the techniques: electrocoagulation with aluminum electrodes and phytoremediation with Myriophyllum aquaticum. Under optimal operating conditions at a pH of 8 and a current density of 45.45 A m(-2), the electrochemical method produces partial elimination of contaminants, which was improved using phytoremediation as a polishing technique. The combined treatment reduced chemical oxygen demand (COD) by 91%, color by 97% and turbidity by 98%. Initial and final values of contaminants in wastewaters were monitored using UV-vis spectrometry and cyclic voltammetry. Finally, the morphology and the elemental composition of the biomass were characterized with using scanning electron microscopy (SEM) and energy dispersion spectroscopy (EDS). The presence of Al in the roots of plants in the system indicates that the aluminum present in the test solution could be absorbed.     

An impurity characterization based approach for the rapid development of integrated downstream purification processes

In this study, we describe a new approach for the characterization of process‐related impurities along with an in silico tool to generate orthogonal, integrated downstream purification processes for biological products. A one‐time characterization of process‐related impurities from product expression in Pichia pastoris was first carried out using linear salt and pH gradients on a library of multimodal, salt‐tolerant, and hydrophobic charge induction chromatographic resins. The Reversed‐phase ultra‐performance liquid chromatography (UPLC) analysis of the fractions from these gradients was then used to generate large data sets of impurity profiles. A retention database of the biological product was also generated using the same linear salt and pH gradients on these resins, without fraction collection. The resulting two data sets were then analyzed using an in silico tool, which incorporated integrated manufacturing constraints to generate and rank potential three‐step purification sequences based on their predicted purification performance as well as whole‐process “orthogonality” for impurity removal. Highly ranked sequences were further examined to identify templates for process development. The efficacy of this approach was successfully demonstrated for the rapid development of robust integrated processes for human growth hormone and granulocyte‐colony stimulating factor.     

An integrated treatment system for coffee processing wastewater using anaerobic and aerobic process

The experiment was conducted to develop an integrated treatment system for coffee processing wastewater (CPWW) through the combination of biomethanation with aeration and wetland plants treatment. The biomethanation was carried out at different hydraulic retention times (HRTs) using upflow anaerobic hybrid reactor (UAHR) and 18 h of HRT was found to be optimum. The maximum biochemical oxygen demand (BOD), chemical oxygen demand (COD) and total solids (TS) reduction were 66.0%, 61.0% and 58.0%, respectively with organic loading rate of 9.55 kg m^?3 day^?1. The reduction of pollution load of the wastewater by microbial action augmented by aeration resulted in the reduction of electrical conductivity (EC), BOD, COD, and total solids (TS). Continuous aeration of wastewater resulted in maximum reduction of BOD (74.6%), COD (68.6%) and TS (49.3%). The wetland plant, Typha latifolia reduced 85.4% and 78.0% of BOD and COD, respectively in biomethanated cum aerated CPWW.     

Intensified process for the purification of an enzyme from inclusion bodies using integrated expanded bed adsorption and refolding

This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations in an intensified process used to recover purified and biologically active proteins from inclusion bodies expressed in E. coli. Delta(5)-3-Ketosteroid isomerase with a C-terminal hexahistidine tag was expressed as inclusion bodies in the cytoplasm of E. coli. Chemical extraction was used to disrupt the host cells and simultaneously solubilize the inclusion bodies, after which EBA utilizing immobilized metal affinity interactions was used to purify the polyhistidine-tagged protein. Adsorptive refolding was then initiated in the column by changing the denaturant concentration in the feed stream from 8 to 0 M urea. Three strategies were tested for performing the refolding step in the EBA column: (i) the denaturant was removed using a step change in feed-buffer composition, (ii) the denaturant was gradually removed using a gradient change in feed-buffer composition, and (iii) the liquid flow direction through the column was reversed and adsorptive refolding performed in the packed bed. Buoyancy-induced mixing disrupted the operation of the expanded bed when adsorptive refolding was performed using either a step change or a rapid gradient change in feed-buffer composition. A shallow gradient reduction in denaturant concentration of the feed stream over 30 min maintained the stability of the expanded bed during adsorptive refolding. In a separate experiment, buoyancy-induced mixing was completely avoided by performing refolding in a settled bed, which achieved comparable yields to refolding in an expanded bed but required a slightly more complex process. A total of 10% of the available KSI-(His(6)) was recovered as biologically active and purified protein using the described purification and refolding process, and the yield was further increased to 19% by performing a second iteration of the on-column refolding operation. This process should be applicable for other polyhistidine tagged proteins and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution.     

Composite fluorine polymer-containing sorbents for isolation and purification of biopolymers

Composite fluoropolymer-containing sorbents based on porous silicas were synthesized for the isolation and purification of biopolymers under nondenaturing conditions. Examples of the application of these sorbents in the separation of various mixtures of peptides and proteins and purification of nucleic acids from various sources (plasmid DNA and DNA from nucleated human blood cells) using the cartridge, column, and batch (sorption in a stirred volume) methods are presented. It was shown that the sorbents can be used in laboratory practice because they are selective to nucleic acids (DNA and RNA) and proteins. These materials combine the mechanical properties of the inorganic matrix with the specific sorption properties of the polymer phase and exhibit enhanced stability to alkaline hydrolysis. Alternative methods of preparing sorbents containing polytetrafluoroethylene, polytrifluorostyrene, and polyfluorobutadiene are described. By the example of polyfluorobutadiene-containing sorbents, a completely new method for obtaining fluorinated polymer phases was developed: the polymer phase was preliminarily formed on the surface of porous disperse carriers and was fluorinated with xenon difluoride.