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Engineering in Life Sciences



Wiley Online Library : Engineering in Life Sciences



Published: 2017-11-01T00:00:00-05:00

 



Cell-free production of pore forming toxins: Functional analysis of thermostable direct hemolysin from Vibrio parahaemolyticus

2017-11-09T05:20:26.645652-05:00

The pore forming characteristic of TDH1 and TDH2 variants of thermostable direct hemolysin (TDH), a major toxin involved in the pathogenesis of Vibrio parahaemolyticus, was studied on a planar lipid bilayer painted over individual picoliter cavities containing microelectrodes assembled in a multiarray. Both proteins formed pores upon insertion into the lipid bilayer which was shown as a shift in the conductance from the baseline current. TDH2 protein was able to produce stable currents and the currents were influenced by external factors like concentration, type of salt and voltage. The pore currents were influenced and showed a detectable response in the presence of polymers which makes them suitable for biotechnology applications. This article is protected by copyright. All rights reserved



Protein adsorption onto Monoliths – A surface energetics study

2017-11-08T02:50:23.32356-05:00

This part of work was done to explore the basic understanding of the adsorption chromatography by determining the interaction of selected model proteins (n = 5) to monolithic chromatographic materials, with varying densities of butyl and phenyl ligands. Surface energetics approach was applied to study the interaction behavior. The physicochemical properties of the proteins and monolithic chromatographic materials were explored by contact angle and zeta potential values. These values were used to study protein to monolith interaction under various operating conditions. Surface energetics approach allowed the calculation of interaction energy as a function of distance, i.e. energy minimum values. Calculations were performed at various conditions to analyze the effect of major operating parameters on the interaction strength. The interaction strength exposed the hydrophobic nature of the monoliths which increases with increasing ligand density. Further, interaction energy of proteins were higher with monolith with butyl ligand compared to monolith with phenyl ligand. For instance, lactoferrin interaction to monoliths with butyl represents more interaction, i.e. 24.38 kT as compared to monoliths with phenyl i.e. 23.28 kT, keeping lambda as 0.2 nm and salt concentration as 100 mM of ammonium sulphate. Hence, more energy and time will be consumed for elution of proteins immobilized to monoliths with butyl. Similarly, the effect of solid surface for proteins immobilization, effect of ligand density and effect of lambda showed some interesting insights on the interaction behavior. The knowledge generated from the present work will help in the basic understanding as well as development of an efficient, low cost downstream processing design and may mimic the real chromatographic experiments. This article is protected by copyright. All rights reserved



Influence of cavitation and high shear stress on HSA aggregation behavior

2017-11-08T01:50:34.556988-05:00

The influence of high shear rates on proteins is not fully understood, and the influence of cavitation has not yet been evaluated. The effect of cavitation bubble collapse-derived hydroxyl radicals on the aggregation behavior of human serum albumin (HSA) was investigated. Radicals were generated by pumping through a micro-orifice, ultra-sonication, or chemically by Fenton's reaction. The amount of radicals produced by the two mechanical methods (0.12 and 11.25 nmol L−1 min−1) was not enough to change the protein integrity. In contrast, Fenton's reaction resulted in 382 nmol L−1 min−1 of radicals, inducing protein aggregation. However, the micro-orifice promoted the formation of soluble dimeric HSA aggregates. A validated computational fluid dynamic model of the orifice revealed a maximum and average shear rate on the order of 108 s−1 and 1.2 × 106 s−1, respectively. Although these values are among the highest ever reported in the literature, dimer formation did not occur when we used the same flow rate but suppressed cavitation. Therefore, aggregation is most likely caused by the increased surface area due to cavitation-mediated bubble growth, not by hydroxyl radical release or shear stress as often reported. This article is protected by copyright. All rights reserved



A modified 384-well-device for versatile use in 3D cancer cell (co-)cultivation and screening for investigations of tumor biology in vitro

2017-11-03T10:50:23.381709-05:00

Pancreatic cancer exhibits a worst prognosis owed to an aggressive tumor progression i.a. driven by chemoresistance or tumor-stroma-interactions. The identification of candidate genes, which promote this progression, can lead to new therapeutic targets and might improve patient's outcome. The identification of these candidates in a plethora of genes requires suitable screening protocols. The aim of the present study was to establish a universally usable device which ensures versatile cultivation, screening and handling protocols of cancer cells with the 3D spheroid model, an approved model to study tumor biology. By surface modification and alternative handling of a commercial 384-well plate, a modified device enabling a) 3D cultivation either by liquid overlay or by a modified hanging drop method for b) screening of substances as well as for tumor-stroma-interactions c) either with manual or automated handling was established. The here presented preliminary results of cell line dependent dose-response-relations and a stromal-induced spheroid-formation of the pancreatic cancer cells demonstrate the proof-of-principle of the versatile functionality of this device. By adapting the protocols to automation, a higher reproducibility and the ability for high-throughput analyses were ensured. This article is protected by copyright. All rights reserved



Potential application of Bacillus subtilis SPB1 biosurfactants in laundry detergent formulations: Compatibility study with detergent ingredients and washing performance

2017-11-17T11:15:29.791545-05:00

Surfactants play a very important role in laundry and household cleaning products ingredients. In this research, the application of lipopeptide biosurfactants, produced by Bacillus subtilis SPB1, in the formulation of a washing powder was investigated. The SPB1 biosurfactant was mixed with sodium tripolyphosphate as a builder and sodium sulfate as filler. The efficiency of the formulated detergent composition with different washing conditions to remove a stain from cotton fabric was examined. The results showed that the formulated detergent was effective in oil removal, with optimal washing conditions of pH, temperature, striate and time of washing system of 7, 65°C, 1000 RPM and 60 min, respectively. A comparative study of different detergent compositions (biosurfactant-based detergent, combined biosurfactant-commercial detergent, and a commercial detergent) for the removal of oil and tea stains, proved that the bio-scouring was more effective (>75%) in terms of the stain removal than the commercial powders (<60%). Moreover, the results demonstrated that the biosurfactant acts additively with a commercial detergent and enhances their performance from 33 to 45% in removing oil stain and from 57 to 64% in removing tea stain. As a conclusion, in addition to the low toxicity and the high biodegradability of the microbial biosurfactants, the results of this study have shown that the future use of this lipopeptide biosurfactant as laundry detergent additive is highly promising.



Promotion of phenolic compounds production in Salvia miltiorrhiza hairy roots by six strains of rhizosphere bacteria

2017-11-16T09:35:30.805538-05:00

Salvia miltiorrhiza Bunge is an important herb for the treatment of cerebrovascular and cardiovascular diseases with bioactive compounds (phenolic acids and tanshinones). Abundant studies showed that tanshinones could be stimulated by biotic and abiotic stresses, but limited information is available on biosynthesis of phenolic acids promoted by biotic stresses. The aim of the present work was to isolate and identify rhizosphere bacteria which stimulated phenolic compound in Salvia miltiorrhiza hairy roots and investigated the internal mechanism, providing a potential means to enhance content of pharmaceuticals in S. miltiorrhiza. The results showed that six bacteria, namely, HYR1, HYR26, SCR22, 14DSR23, DS6, and LNHR13, belonging to the genus Pseudomonas and Pantoea, significantly promoted the growth and content of major phenolic acids, RA and SAB. Bacteria LNHR13 was the most effective one, with the contents of RA and SAB reaching ∼2.5-fold (30.1 mg/g DW) and ∼2.3-fold (48.3 mg/g DW) as those of the control, respectively. Phytohormones and polysaccharides produced by bacteria showed potential responsibility for the growth and biosynthesis of secondary metabolites of S. miltiorrhiza. Meanwhile, we found that the more abundant the types and contents of phytohormones, the stronger their stimulating effect on the content of salvianolic acids.



Removal of dimethylphenols and ammonium in laboratory-scale horizontal subsurface flow constructed wetlands

2017-11-02T02:36:33.180639-05:00

Phenolic compounds in industrial wastewaters are toxic pollutants and pose a threat to public health and ecosystems. More recently, focus is being directed toward combining the treatment of these compounds with a cost-effective and environmentally sound technology. The removal efficiency of dimethylphenol and ammonium nitrogen was studied, for the first time, in three different laboratory-scale horizontal subsurface flow constructed wetlands planted with Juncus effusus. Two of the wetlands used were filled with gravel. One of these was planted and the second left without vegetation. The third wetland was a hydroponic system. It was found that the removal efficiencies of dimethylphenol was dependent on the inflow loading of the contaminant and was higher in the planted systems. Both planted systems yielded 99% removal efficiency up to loads of 240 mg/d, compared to only 73% for the unplanted constructed wetland. Factors and processes such as redox dynamics, methanogenesis, reduction of ammonium and low nitrate and nitrite concentrations imply simultaneous aerobic and anaerobic dimethylphenol transformations. A significant surplus of organic carbon was detected in the planted wetlands, which may originate from intermediates of the dimethylphenol transformation processes and/or organic plant root exudates. The present study demonstrates that horizontal subsurface flow constructed wetlands are a promising alternative system for the treatment of effluents contaminated with dimethylphenol isomers.



Shear treatment of starter culture medium improves separation behavior of Streptococcus thermophilus cells

2017-11-02T02:35:48.578673-05:00

A central step in the production of starter cultures is the separation of the cells from the fermentation medium, which is usually achieved by disk centrifuges. In case of microorganisms which produce exopolysaccharides (e.g., various strains of lactic acid bacteria), the properties of the respective exopolysaccharides may interfere with this separation step. By using six strains of Streptococcus thermophilus the hypothesis was tested that a shear treatment of the fermented culture medium improves subsequent cell separation markedly. Depending on the type of exopolysaccharides (freely present in the medium, or as capsules around the cells) an energy input of up to 2.5 kJ/mL generated with an Ultra-Turrax affected cell chain length of the strains and viscosity of fermentation medium differently. For bacteria producing capsular exopolysaccharides, space- and time-resolved centrifugation experiments revealed an increase of sedimentation velocity after shear treatment. In general, viability of the microorganisms, detected by flow cytometry measurements and fermentation experiments, was not affected by the shearing procedure. The results therefore indicate that strain-targeted shearing is helpful to improve the separability of cells from the fermented media.



In-line monitoring of amino acids in mammalian cell cultures using raman spectroscopy and multivariate chemometrics models

2017-10-26T03:51:55.237348-05:00

The application of PAT for in-line monitoring of biopharmaceutical manufacturing operations has a central role in developing more robust and consistent processes. Various spectroscopic techniques have been applied for collecting real-time data from cell culture processes. Among these, Raman spectroscopy has been shown to have advantages over other spectroscopic techniques, especially in aqueous culture solutions. Measurements of several process parameters such as glucose, lactate, glutamine, glutamate, ammonium, osmolality and VCD using Raman-based chemometrics models have been reported in literature. The application of Raman spectroscopy, coupled with calibration models for amino acid measurement in cell cultures, has been assessed. The developed models cover four amino acids important for cell growth and production: tyrosine, tryptophan, phenylalanine and methionine. The chemometrics models based on Raman spectroscopy data demonstrate the significant potential for the quantification of tyrosine, tryptophan and phenylalanine. The model for methionine would have to be further refined to improve quantification.



Selectively screen the antibacterial peptide from the hydrolysates of highland barley

2017-10-26T03:51:25.438582-05:00

Highland barley is one of the most important industrial crops in Tibetan plateau. Previous research indicated that highland barley has many medical functions. In this work, the antibacterial abilities of highland barley were investigated. The protein solutions hydrolyzed by trypsin for 4 h exhibited the highest antibacterial activity. An antibacterial peptide, barleycin, was screened and purified by magnetic liposome extraction combining with the protein profiles of reversed-phase high-performance liquid chromatography (RP-HPLC). Structure, characterization, and safety evaluation of barleycin were further investigated. Amino acids sequence was determined as Lys-Ile-Ile-Ile-Pro-Pro-Leu-Phe-His by N-sequencing. Circular dichroism spectra indicated the a-helix conformation of barleycin. The activity spectrum included Bacillus subtilis, Staphylcoccus aureus, Listeria innocua and Escherichia coli and the MICs were from 4 to 16 μg/mL. Safety evaluations with cytotoxicity and hemolytic suggested this antibacterial peptide could be considered as safe at MICs. Finally, mode of action of barleycin on sensitive cells was primarily studied. The results suggested the damage of cell membrane.



Conversion of glucose-xylose mixtures to pyruvate using a consortium of metabolically engineered Escherichia coli

2017-10-16T08:55:52.325551-05:00

Two strains of Escherichia coli were engineered to accumulate pyruvic acid from two sugars found in lignocellulosic hydrolysates by knockouts in the aceE, ppsA, poxB, and ldhA genes. Additionally, since glucose and xylose are typically consumed sequentially due to carbon catabolite repression in E. coli, one strain (MEC590) was engineered to grow only on glucose while a second strain (MEC589) grew only on xylose. On a single substrate, each strain generated pyruvate at a yield of about 0.60 g/g in both continuous culture and batch culture. In a glucose-xylose mixture under continuous culture, a consortium of both strains maintained a pyruvate yield greater than 0.60 g/g when three different concentrations of glucose and xylose were sequentially fed into the system. In a fed-batch process, both sugars in a glucose-xylose mixture were consumed simultaneously to accumulate 39 g/L pyruvate in less than 24 h at a yield of 0.59 g/g.



Optimization of cell culture-derived influenza A virus particles purification using sulfated cellulose membrane adsorbers

2017-10-16T08:55:40.443482-05:00

Downstream processing remains one of the biggest challenges in manufacturing of biologicals and vaccines. This work focuses on a Design of Experiments approach to understand factors influencing the performance of sulfated cellulose membrane adsorbers for the chromatographic purification of a cell culture-derived H1N1 influenza virus strain (A/Puerto Rico/8/34). Membranes with a medium ligand density together with low conductivity and a high virus titer in the feed stream resulted in optimum virus yields and low protein and DNA content in the product fraction. Flow rate and salt concentration in the buffer used for elution were of secondary importance while membrane permeability had no significant impact on separation performance. A virus loss of 2.1% in the flow through, a yield of 57.4% together with a contamination level of 5.1 pgDNA HAU−1 and 1.2 ngprot HAU−1 were experimentally confirmed for the optimal operating point predicted. The critical process parameters identified and their optimal settings should support the optimization of sulfated cellulose membrane adsorbers based purification trains for other influenza virus strains, streamlining cell culture-derived vaccine manufacturing.



Comparison of different solvents for extraction of polyhydroxybutyrate from Cupriavidus necator

2017-10-02T09:00:31.219027-05:00

Polyhydroxybutyrate (PHBs) have attracted much attention due to their biodegradability and biocompatibility properties. The main drawback to the commercial production of them is their high cost. The recovery of PHB from bacterial cytoplasm significantly increases total processing costs. Efficient, economical, and environment-friendly extraction of PHB from cells is required for its industrial production. In the present study, several nonhalogenated organic solvents (ethylene carbonate, dimethyl sulfoxide, dimethyl formamide, hexane, propanol, methanol, and acetic acid) were examined for their efficacy regarding recovery at different temperatures from culture broth containing Cupriavidus necator cells. The highest recovery percentage (98.6%) and product purity (up to 98%) were seen to be those of ethylene carbonate-assisted extraction at 150°C within 60 min of incubation time. Average molecular weight of the recovered PHB (1.3 × 106) was not significantly affected by the extraction solvent and conditions. The melting point of PHB extracted using ethylene carbonate was measured to be 176.2°C with an enthalpy of fusion of 16.8% and the corresponding degree of crystallinity of 59.2%. NMR and GC analyses confirmed that the extracted biopolymer was PHB. The presented strategy can help researchers to reduce the cost to obtain the final product.



Parametric studies on droplet generation reproducibility for applications with biological relevant fluids

2017-09-27T01:55:53.224374-05:00

Although the great potential of droplet based microfluidic technologies for routine applications in industry and academia has been successfully demonstrated over the past years, its inherent potential is not fully exploited till now. Especially regarding to the droplet generation reproducibility and stability, two pivotally important parameters for successful applications, there is still a need for improvement. This is even more considerable when droplets are created to investigate tissue fragments or cell cultures (e.g. suspended cells or 3D cell cultures) over days or even weeks. In this study we present microfluidic chips composed of a plasma coated polymer, which allow surfactants-free, highly reproducible and stable droplet generation from fluids like cell culture media. We demonstrate how different microfluidic designs and different flow rates (and flow rate ratios) affect the reproducibility of the droplet generation process and display the applicability for a wide variety of bio(techno)logically relevant media.



Advanced monitoring and control of pharmaceutical production processes with Pichia pastoris by using Raman spectroscopy and multivariate calibration methods

2017-09-25T03:16:47.44123-05:00

This contribution includes an investigation of the applicability of Raman spectroscopy as a PAT analyzer in cyclic production processes of a potential Malaria vaccine with Pichia pastoris. In a feasibility study, Partial Least Squares Regression (PLSR) models were created off-line for cell density and concentrations of glycerol, methanol, ammonia and total secreted protein. Relative cross validation errors RMSEcvrel range from 2.87% (glycerol) to 11.0% (ammonia). In the following, on-line bioprocess monitoring was tested for cell density and glycerol concentration. By using the nonlinear Support Vector Regression (SVR) method instead of PLSR, the error RMSEPrel for cell density was reduced from 5.01 to 2.94%. The high potential of Raman spectroscopy in combination with multivariate calibration methods was demonstrated by the implementation of a closed loop control for glycerol concentration using PLSR. The strong nonlinear behavior of exponentially increasing control disturbances was met with a feed-forward control and adaptive correction of control parameters. In general the control procedure works very well for low cell densities. Unfortunately, PLSR models for glycerol concentration are strongly influenced by a correlation with the cell density. This leads to a failure in substrate prediction, which in turn prevents substrate control at cell densities above 16 g/L.



Ultrasensitive SPR detection of miRNA-93 using antibody-enhanced and enzymatic signal amplification

2017-09-25T03:16:06.356898-05:00

MiRNAs are endogenous noncoding RNA molecules. They play important gene-regulatory roles by binding to the mRNA of target genes thereby leading to either transcript degradation or translational repression. In virtually all diseases, distinct alterations of miRNA expression profiles have been found thus suggesting miRNAs as interesting biomarkers. Here, we present an SPR biosensor that utilizes disposable, injection-molded sensor chip/microfluidic hybrids combined with a lateral imaging optical system for parallel analysis of three one-dimensional spot arrays to detect miRNA-93. To increase the sensitivity of the biosensor we used two different amplification strategies. By adding an RNA-DNA-hybrid antibody for primary signal amplification, a limit of detection of 10 pmol/L was achieved. Based on that method we demonstrate the detection of miRNA-93 in total RNA lysate from HEK-293 cells. Utilizing an enzymatic signal amplification with Poly(A) polymerase, the sensitivity could be increased even further leading to a limit of detection of 1 fmol/L.



Protein micropatterns printed on glass: Novel tools for protein-ligand binding assays in live cells

2017-09-15T13:12:06.121094-05:00

Micrometer-sized patterns of proteins on glass or silica surfaces are in widespread use as protein arrays for probing with ligands or recombinant proteins. More recently, they have been used to capture the surface proteins of mammalian cells seeded onto them, and to arrange these surface proteins into pattern structures. Binding of small molecule ligands or of other proteins, transmembrane or intracellular, to these captured surface proteins can then be quantified. However, reproducible production of protein micropatterns on surfaces can be technically difficult. In this review, we outline the wide potential and the current practical uses of printed protein micropatterns in a historical overview, and we detail some potential pitfalls and difficulties from our own experience, as well as ways to circumvent them.



Anionic lipopeptides from Bacillus mojavensis I4 as effective antihypertensive agents: Production, characterization, and identification

2017-09-11T01:51:32.825404-05:00

A new isolated Bacillus mojavensis strain I4 was found as producer of biosurfactants by different screening methods, such as parafilm M test, hemolytic activity, oil displacement test, emulsification index, surface tension, and lipase production assay. Enhanced biosurfactants production was obtained using glucose and glutamic acid as carbon and nitrogen sources, respectively. The optimal production of the biosurfactants was obtained by using a C/N ratio of 17, pH of 7.0, and temperature of 37°C. The surface tension was reduced to 29 mN/m and the emulsification index E24 of 62% was achieved after 72 h of culture. The purified biosurfactants showed stability with regard to surface tension reduction and emulsification in a wide range of temperatures (4–120°C), pH (4–10), and salinity (2–12% of NaCl). The thin-layer chromatography showed that the produced biosurfactants were lipopeptides. The biosurfactants were characterized as a group of anionic lipopeptides with zeta potential measurement. Chromatographic characterization using HPLC revealed that I4 lipopeptides contained numerous isoforms and surfactin was the major component. Moreover, the I4 lipopeptides showed interesting angiotensin-converting enzyme-inhibitory activity.



A soft computing tool for species classification and prediction of glucomannan content in Amorphophallus genus

2017-09-11T01:45:44.337363-05:00

The proposed work aims at designing a classification system for automatic identification of A. muelleri species, grown as a potential cash crop in many Asian countries, from the DNA fingerprints of Amorphophallus genus. Four sets of 48 DNA fingerprints belonging to 37 species of the Amorphophallus genus, developed with the help of four different primers are considered for the experiment, with an objective to identify only the fingerprints of the species of interest. A second experimental setup deals with the automatic classification of species containing high amounts of glucomannan from the same set of DNA fingerprints of the Amorphophallus genus. For each set of 48 DNA fingerprints generated with a specific primer, the DNA fingerprints are preprocessed to extract a 42 dimensional feature vector which is used to generate a k-Nearest Neighbor based classifier based on the Leave One Out Cross Validation protocol. Final classification based on outputs from individual classifiers constructed with respect to the four different primers is performed according to a n-star consensus strategy. The n-star consensus predicts species A. muelleri with cent per cent accuracy while it predicts species containing glucomannan with a more modest accuracy of 81.25%.



Measurement of heat transfer coefficients in stirred single-use bioreactors by the decay of hydrogen peroxide

2017-08-21T09:05:31.558343-05:00

Single-use bioreactors are barely described by means of their heat transfer characteristics, although some of their properties might affect this process. Steady-state methods that use external heat sources enable precise investigations. One option, commonly present in stirred, stainless steel tanks, is to use adjustable electrical heaters. An alternative are exothermic chemical reactions that offer a higher flexibility and scalability. Here, the catalytic decay of hydrogen peroxide was considered a possible reaction, because of the high reaction enthalpy of –98.2 kJ/mole and its uncritical reaction products. To establish the reaction, a proper catalyst needed to be determined upfront. Three candidates were screened: catalase, iron(III)-nitrate and manganese(IV)-oxide. Whilst catalase showed strong inactivation kinetic and general instability and iron(III)-nitrate solution has a pH of 2, it was decided to use manganese(IV)-oxide for the bioreactor studies. First, a comparison between electrical and chemical power input in a benchtop glass bioreactor of 3.5 L showed good agreement. Afterwards the method was transferred to a 50 L stirred single-use bioreactor. The deviation in the final results was acceptable. The heat transfer coefficient for the electrical method was 242 W/m2/K, while the value achieved with the chemical differed by less than 5%. Finally, experiments were carried out in a 200 L single-use bioreactor proving the applicability of the chemical power input at technical relevant scales.



Micro free-flow isoelectric focusing with integrated optical pH sensors

2017-08-02T09:55:57.270876-05:00

Recently, a new observation method for monitoring of pH gradients in microfluidic free-flow electrophoresis has emerged. It is based on the use of chip-integrated fluorescent or luminescent micro sensor layers. These are able to monitor pH gradients in miniaturized separations in real time and spatially resolved; this is particularly useful in isoelectric focusing. Here these multifunctional microdevices that feature continuous separation, monitoring, and in some instances other functionalities, are reviewed. The employed microfabrication procedures to produce these devices are discussed and the different pH sensor matrices that were integrated and their applications in the separation of different types of biomolecules. The procedures for obtaining spatially resolved information about the separated molecules and the pH at the same time and different detection modalities to achieve this such as deep UV fluorescence as well as time-resolved referenced pH sensing and the integration of a precolumn labeling step into these platforms are also highlighted.



Progress in enzyme inhibition based detection of pesticides

2017-08-02T09:55:27.527987-05:00

The previous few decades have seen the development of biosensors and their use in monitoring of pesticides in food and environmental samples. Although inhibition-based biosensors have been subject of several recent research works, their performance characteristics greatly depend on the type of immobilization and the presence of interfering compounds in the samples. Moreover, sensitivity, detection limits, and rapidity of the response are few of the other major features that need to be investigated further if they are to become operationally user-friendly. This review will highlight research carried out in the past on biosensors that are based on enzyme inhibition for determination of organophosphorus compounds and carbamate pesticides.



Stability of polymersomes with focus on their use as nanoreactors

2017-08-01T01:26:21.319156-05:00

The increased membrane stability of polymersomes compared to their liposomal counterparts is one of their most important advantages. Due to this benefit, polymer vesicles are intended to be used not only as carrier systems for drug delivery purposes but also as nanoreactors for biotechnological applications. Within this work, the stability of polymersomes made of the triblock copolymer poly(2-methyloxazoline)15-poly(dimethylsiloxane)68-poly(2-methyloxazoline)15 (PMOXA15-PDMS68-PMOXA15) toward mechanical stress, typically prevailing in stirred-tank reactors being the most often used reactor type in the biotechnological industry, was characterized. Dynamic light scattering and turbidity measurements showed that stirrer rotation causing a maximum local energy dissipation of up to 1.23 W/kg−1 did not result in any loss of vesicle quality or quantity. Nevertheless, most probably due to local membrane defects, 6.6% release of the previously encapsulated model dye calcein was recognized at 25°C within 48 h. Moreover, increased temperature, leading to decreased membrane viscosity and increased membrane fluidity, respectively, led to a higher molecule leakage. Besides, the stability of polymersomes in two-phase systems was investigated. Although alkanes and ionic liquids were shown not to lead to complete vesicle damage, no efficient calcein retention was achieved in either case.



Experimental characterization and simulation of amino acid and peptide interactions with inorganic materials

2017-07-26T01:20:28.355368-05:00

Inspired by nature, many applications and new materials benefit from the interplay of inorganic materials and biomolecules. A fundamental understanding of complex organic–inorganic interactions would improve the controlled production of nanomaterials and biosensors to the development of biocompatible implants for the human body. Although widely exploited in applications, the interaction of amino acids and peptides with most inorganic surfaces is not fully understood. To date, precisely characterizing complex surfaces of inorganic materials and analyzing surface–biomolecule interactions remain challenging both experimentally and computationally. This article reviews several approaches to characterizing biomolecule–surface interactions and illustrates the advantages and disadvantages of the methods presented. First, we explain how the adsorption mechanism of amino acids/peptides to inorganic surfaces can be determined and how thermodynamic and kinetic process constants can be obtained. Second, we demonstrate how this data can be used to develop models for peptide–surface interactions. The understanding and simulation of such interactions constitute a basis for developing molecules with high affinity binding domains in proteins for bioprocess engineering and future biomedical technologies.



A microfluidic device for the delivery of enzymes into cells by liposome fusion

2017-01-13T14:15:28.445212-05:00

Liposomes are versatile carriers of drugs or biomolecules and are ideally suited to transport molecules into cells. However, mechanistic studies to understand and improve the fusion of liposomes with cell membranes and endosomes are difficult. Here, we report a method that allows for stable coimmobilization of liposomes and living cells, thereby bringing the membranes into close contact, which is essential for membrane fusion. The small unilamellar liposomes are tethered to the surface by a linker so that no modification of the liposome membrane for cell binding is required. The cells are positioned above the liposomes by posts that are integrated into the microfluidic device, and a pH drop induces the fusion of the cell-liposome membranes. Both membrane fusion and release of molecules into the cytosol are visualized by fluorescence dequenching assays. Furthermore, we proved the efficient delivery of the enzyme β-galactosidase into the cells when a fusogenic liposome composition was used. The device could be used for fusion studies but is also a versatile means for cell transfection.



Cover Picture: Engineering in Life Sciences 11'17

2017-11-03T09:13:38.149629-05:00




Issue Information

2017-11-03T09:13:40.709068-05:00







Downstream process development strategies for effective bioprocesses: Trends, progress, and combinatorial approaches

2016-05-13T01:30:53.093383-05:00

The biopharmaceutical industry is at a turning point moving toward a more customized and patient-oriented medicine (precision medicine). Straightforward routines such as the antibody platform process are extended to production processes for a new portfolio of molecules. As a consequence, individual and tailored productions require generic approaches for a fast and dedicated purification process development. In this article, different effective strategies in biopharmaceutical purification process development are reviewed that can analogously be used for the new generation of antibodies. Conventional approaches based on heuristics and high-throughput process development are discussed and compared to modern technologies such as multivariate calibration and mechanistic modeling tools. Such approaches constitute a good foundation for fast and effective process development for new products and processes, but their full potential becomes obvious in a correlated combination. Thus, different combinatorial approaches are presented, which might become future directions in the biopharmaceutical industry.



A scalable software framework for data integration in bioprocess development

2016-09-05T02:35:59.866263-05:00

Effectiveness in lab workflows—despite progresses made in automation and lab informatics—is often hindered by insufficient integration of devices and data. The iLAB software framework, a middleware connecting and integrating devices and data, provides a plugin architecture that can be adapted to individual lab environments. Integration of devices is preferably based on standardized data and communication protocols. In addition to device integration, process data and result data from different sources (e.g. readers) are converted to a standard format and administered by a powerful database for further processing. In this paper, the use of iLAB in a bioprocess development application is described. Process parameters and measured values of two high-throughput bioreactor systems (96-well plates and 10 mL reactor vessels) are collected in the iLAB database. Data from screening experiments and offline data are visualized, analyzed, and compared. A filter algorithm allows searching for matching parameters in experiments as well as the comparison of correlated datasets, independently from the used bioreactor system. The database model enables the consolidation of data, the transition from data to information, and a solid base for management decisions on enterprise level.



Design of experiments-based high-throughput strategy for development and optimization of efficient cell disruption protocols

2016-10-05T01:25:51.730997-05:00

Efficient and reproducible cell lysis is a crucial step during downstream processing of intracellular products. The composition of an optimal lysis buffer should be chosen depending on the organism, its growth status, the applied detection methods, and even the target molecule. Especially for high-throughput applications, where sample volumes are limited, the adaptation of a lysis buffer to the specific campaign is an urgent need. Here, we present a general design of experiments-based strategy suitable for eight constituents and demonstrate the strength of this approach by the development of an efficient lysis buffer for Gram-negative bacteria, which is applicable in a high-throughput format in a short time. The concentrations of four lysis-inducing chemical agents EDTA, lysozyme, Triton X-100, and polymyxin B were optimized for maximal soluble protein concentration and ß-galactosidase activity in a 96-well format on a Microlab Star liquid handling platform under design of experiments methodology. The resulting lysis buffer showed the same performance as a commercially available lysis buffer. The developed protocol resulted in an optimized buffer within only three runs. The established procedure can be easily applied to adapt the lysis buffer to other strains and target molecules.



Intensified design of experiments for upstream bioreactors

2016-10-14T03:41:02.361386-05:00

Statistical Design of Experiments (DoE) is a widely adopted methodology in upstream bioprocess development (and generally across industries) to obtain experimental data from which the impact of independent variables (factors) on the process response can be inferred. In this work, a method is proposed that reduces the total number of experiments suggested by a traditional DoE. The method allows the evaluation of several DoE combinations to be compressed into a reduced number of experiments, which is referred to as intensified Design of Experiments (iDoE). In this paper, the iDoE is used to develop a dynamic hybrid model (consisting of differential equations and a feedforward artificial neural network) for data generated from a simulated Escherichia coli fermentation. For the case study presented, the results suggest that the total number of experiments could be reduced by about 40% when compared to traditional DoE. An additional benefit is the simultaneous development of an appropriate dynamic model which can be used in both, process optimization and control studies.



Fast-track development of a lactase production process with Kluyveromyces lactis by a progressive parameter-control workflow

2016-11-21T09:30:31.109627-05:00

The time-to-market challenge is key to success for consumer goods affiliated industries. In recent years, the dairy industry faces a fast and constantly growing demand for enzymatically produced lactose-free milk products, mainly driven by emerging markets in South America and Asia. In order to take advantage of this opportunity, we developed a fermentation process for lactase (β-galactosidase) from Kluyveromyces lactis within short time. Here, we describe the process of stepwise increasing the level of control over relevant process parameters during scale-up that established a highly efficient and stable production system. Process development started with evolutionary engineering to generate catabolite-derepressed variants of the K. lactis wild-type strain. A high-throughput screening mimicking fed-batch cultivation identified a constitutive lactase overproducer with 260-fold improved activity of 4.4 U per milligram dry cell weight when cultivated in glucose minimal medium. During scale-up, process control was progressively increased up to the level of conventional, fully controlled fed-batch cultivations by simulating glucose feed, applying pH- and dissolved oxygen tension (DOT)-sensor technology to small scale, and by the use of a milliliter stirred tank bioreactor. Additionally, process development was assisted by design-of-experiments optimization of the growth medium employing the response surface methodology.



Online bioprocess data generation, analysis, and optimization for parallel fed-batch fermentations in milliliter scale

2016-11-14T03:41:17.666861-05:00

Bioprocess development, optimization, and control in mini-bioreactor systems require information about essential process parameters, high data densities, and the ability to dynamically change process conditions. We present an integration approach combining a parallel mini-bioreactor system integrated into a liquid handling station (LHS) with a second LHS for offline analytics. Non-invasive sensors measure pH and DO online. Offline samples are collected every 20 min and acetate, glucose, and OD620 subsequently analyzed offline. All data are automatically collected, analyzed, formalized, and used for process control and optimization. Fed-batch conditions are realized via a slow enzymatic glucose release system. The integration approach was successfully used to apply an online experimental re-design method to eight Escherichia coli fed-batch cultivations. The method utilizes generated data to select the following experimental actions online in order to reach the optimization goal of estimating E. coli fed-batch model parameters with as high accuracy as possible. Optimal experimental designs were re-calculated online based on the experimental data and implemented by introducing pulses via the LHS to the running fermentations. The LHS control allows for various implementations of advanced control and optimization strategies in milliliter scale.



Rapid process synthesis supported by a unified modular software framework

2017-02-08T02:28:27.988792-05:00

Although known to be very powerful, the widespread application of model-based techniques is still significantly hampered in the area of bio-processes. Reasons for this situation can be found along the whole chain to set up and implement such approaches. In a time-consuming step, models are typically hand-crafted. Whether alternatives of better models exist to actually fulfill the final goals is undocumented, most often even unknown. In a next step, model-based process control methods are hand-coded in an error-prone procedure. For many of these methods given in the literature, only simulation studies are shown, leaving the interested reader with the unanswered question whether the implementation of a specific method in a real process is viable. As the potentially time-consuming implementation of such a method presents a risk for a rapid process development, promising candidates may be overlooked. To remediate this unsatisfactory situation, a combination of theoretical methods and information technology is proposed here. By an exemplarily realized software tool, it is shown how such an environment helps to promote model-based optimization, supervision, and control of bio-processes and allows for an inexpensive test of new ideas as well in real-life experiments. The contribution concentrates on an overview of a possible software architecture with respect to necessary methods and a meaningful information strategy, highlighting some of the more crucial building blocks. Experimental results exploiting parts of the proposed methods are given for a yeast strain synthesizing a product of industrial interest.



Detection of growth rate-dependent product formation in miniaturized parallel fed-batch cultivations

2017-08-29T11:16:10.345196-05:00

Saccharomyces cerevisiae is a popular expression system for recombinant proteins. In most cases, production processes are performed as carbon-limited fed-batch cultures to avoid aerobic ethanol formation. Especially for constitutive expression systems, the specific product formation rate depends on the specific growth rate. The development of optimal feeding strategies strongly depends on laboratory-scale cultivations, which are time and resource consuming, especially when continuous experiments are carried out. It is therefore beneficial for accelerated process development to look at alternatives. In this study, S. cerevisiae AH22 secreting a heterologous endo-polygalacturonase (EPG) was characterized in microwell plates with an enzyme-based fed-batch medium. Through variation of the glucose release rate, different growth profiles were established and the impact on EPG secretion was analyzed. Product formation rates of 200–400 U (gx h)−1 were determined. As a reference, bioreactor experiments using the change-stat cultivation technique were performed. The growth-dependent product formation was analyzed over dilution rates of D = 0.01–0.35 with smooth change of D at a rate of 0.003 h−2. EPG production was found to be comparable with a qp of 400 U (gx h)−1 at D = 0.27 h−1. The presented results indicate that parallel miniaturized fed-batch cultures can be applied to determine product formation profiles of putative production strains. With further automation and parallelization of the concept, strain characterization can be performed in shorter time.