László Hajba

Continuous-flow biochemical reactors: Biocatalysis, bioconversion, and bioanalytical applications utilizing immobilized microfluidic enzyme reactors

The utilization of continuous-flow biochemical reactors, including biocatalysis, biotransformation, and biochemical interaction based flow-analytical systems, and enzyme reactors are recently the focus of attention to produce fine biochemicals and also show great potential in bioanalytical applications. Continuous-flow biochemical processes implemented in microstructured reactors enable short development time to production scale utilizing enzymatic processes to efficiently fulfill the current needs of the fine biochemical and pharmaceutical industry. Immobilization of the enzymes is preferable because it usually enhances their stability, and in some instances, immobilized enzymes can even be reused multiple times. In this review on the continuous-flow biochemical reactors, first the enzyme immobilization strategies will be briefly discussed followed by summarizing the recent developments in the field of immobilized enzyme microflow reactors for biocatalysis, bioconversion and bioanalytical purposes.

A fully automated linear polyacrylamide coating and regeneration method for capillary electrophoresis of proteins

Surface modification of the inner capillary wall in CE of proteins is frequently required to alter EOF and to prevent protein adsorption. Manual protocols for such coating techniques are cumbersome. In this paper, an automated covalent linear polyacrylamide coating and regeneration process is described to support long‐term stability of fused‐silica capillaries for protein analysis. The stability of the resulting capillary coatings was evaluated by a large number of separations using a three‐protein test mixture in pH 6 and 3 buffer systems. The results were compared to that obtained with the use of bare fused‐silica capillaries. If necessary, the fully automated capillary coating process was easily applied to regenerate the capillary to extend its useful life‐time.

Liquid phase separation methods for N-glycosylation analysis of glycoproteins of biomedical and biopharmaceutical interest. A critical review

Comprehensive carbohydrate analysis of glycoproteins from human biological samples and biotherapeutics are important from diagnostic and therapeutic points of view. This review summarizes the current state-of-the-art liquid phase separation techniques used in N-glycosylation analysis. The different liquid chromatographic techniques and capillary electrophoresis methods are critically discussed in detail. Miniaturization of these methods is also important to increase throughput and decrease analysis time. The sample preparation and labeling methods for asparagine linked oligosaccharides are also addressed.

Tilted pillar array fabrication by the combination of proton beam writing and soft lithography for microfluidic cell capture: Part 1 Design and feasibility

Design, fabrication, integration, and feasibility test results of a novel microfluidic cell capture device is presented, exploiting the advantages of proton beam writing to make lithographic irradiations under multiple target tilting angles and UV lithography to easily reproduce large area structures. A cell capture device is demonstrated with a unique doubly tilted micropillar array design for cell manipulation in microfluidic applications. Tilting the pillars increased their functional surface, therefore, enhanced fluidic interaction when special bioaffinity coating was used, and improved fluid dynamic behavior regarding cell culture injection. The proposed microstructures were capable to support adequate distribution of body fluids, such as blood, spinal fluid, etc., between the inlet and outlet of the microfluidic sample reservoirs, offering advanced cell capture capability on the functionalized surfaces. The hydrodynamic characteristics of the microfluidic systems were tested with yeast cells (similar size as red blood cells) for efficient capture.

Computational Fluid Dynamics-Based Design of a Microfabricated Cell Capture Device

A microfluidic cell capture device was designed, fabricated, evaluated by numerical simulations and validated experimentally. The cell capture device was designed with a minimal footprint compartment comprising internal micropillars with the goal to obtain a compact, integrated bioanalytical system. The design of the device was accomplished by computational fluid dynamics (CFD) simulations. Various microdevice designs were rapidly prototyped in poly-dimethylsiloxane using conventional soft lithograpy technique applying micropatterned SU-8 epoxy based negative photoresist as moulding replica. The numerically modeled flow characteristics of the cell capture device were experimentally validated by tracing and microscopic recording the flow trajectories using yeast cells. Finally, we give some perspectives on how CFD modeling can be used in the early stage of microfluidics-based cell capture device development.

Vibrational spectroscopic and theoretical studies of urea derivatives with biochemical interest: N,N’-dimethylurea, N,N,N’,N’-tetramethylurea and N,N’-dimethylpropyleneurea

Abstract Mid-infrared, far-infrared, and Raman vibrational spectroscopic studies were combined with density functional theory (DFT) calculations and normal coordinate force field analyses for N,N′-dimethylurea (DMU), N,N,N′,N′-tetramethylurea (TMU), and N,N′-dimethylpropyleneurea (DMPU: IUPAC name 1,3-dimethyltetrahydropyrimidin-2(1H)-one). The equilibrium molecular geometry of DMU (all three conformers), TMU, and DMPU and the frequencies, intensities, and depolarization ratios of their fundamental infrared (IR) and Raman vibrational transitions were obtained by DFT calculations. The vibrational spectra were fully analyzed by normal coordinate methods as well. A starting force field for DMPU was obtained by adapting corresponding force constants for DMU and TMU, resulting after refinements in the stretching force constants C=O (7.69, 7.30, 7.68 N·cm−1), C–N (5.16, 5.55, 5.05 N·cm−1), and C-Me (5.93, 4.00, 4.22 N·cm−1) for DMU, TMU, and DMPU, respectively. The dominating conformer of liquid DMU was identified as trans-trans, strong intermolecular hydrogen bonding was verified in solid DMU, and weak dipole–dipole association was found in liquid TMU and in DMPU. Special attention was paid to analyzing the methyl group frequencies, which revealed deviations from local C3v symmetry. A linear correlation was found between the CH stretching force constants and the inverse of the CH bond lengths (1/r 2). The averaged NH stretching frequencies of gaseous, dissolved, and solid urea and of DMU, with variations for hydrogen bonding of different strength, are linearly correlated to the NH stretching force constants. Characteristic skeletal vibrations were assigned for a broad variety of urea derivatives and also for pyrimidine derivatives, which all contain the N2C=O entity. The very strong IR bands of C=O stretching (1,676 ± 40 cm−1) and asymmetric CN2 stretching (1,478 ± 60 cm−1), and the very intense Raman feature of symmetric CN2 stretching or ring breathing (757 ± 80 cm−1), can be recognized as fingerprint bands also for the pyrimidine derivatives cytosine, thymine, and uracil, which all are nucleobases in DNA and RNA nucleotides.

Energy saving processes of biofuel production from fermentation broth

The energy used for distillation in bioethanol production reaches the 40 % of the total energy demand. The pervaporation is an important alternative process to distillation that can be applied as a hybrid process or even as a single process to produce high quality biofuel. It will be shown how the energy demand, MJ/kgEthanol energy, can be saved applying pervaporation process with different separation factors and operating modes. It is stated that relatively high separation factor is needed to lower the energy demand below a simple distillation column.

Glycosimilarity assessment of biotherapeutics 1: Quantitative comparison of the N-glycosylation of the innovator and a biosimilar version of etanercept

HighlightsGlycosimilarity is introduced to quantitatively address N‐glycosylation differences.Practical examples of glycosimilarity assessment are given (innovator and biosimilar).Quantitative differences between the N‐glycan profiles are discussed. Abstract The carbohydrate moieties on the polypeptide chains in most glycoprotein based biotherapeutics and their biosimilars play essential roles in such major mechanisms of actions as antibody‐dependent cell‐mediated cytotoxicity, complement‐dependent cytotoxicity, anti‐inflammatory functions and serum clearance. In addition, alteration in glycosylation may influence the safety and efficacy of the product. Glycosylation, therefore, is considered as one of the important critical quality attributes of glycoprotein biotherapeutics, and consequently for their biosimilar counterparts. Thus, the carbohydrate moieties of such biopharmaceuticals (both innovator and biosimilar products) should be closely scrutinized during all stages of the manufacturing process. In this paper we introduce a rapid, capillary gel electrophoresis based process to quantitatively assess the glycosylation aspect of biosimilarity (referred to as glycosimilarity) between the innovator and a biosimilar version of etanercept (Enbrel® and Benepali®, respectively), based on their N‐linked carbohydrate profiles. Differences in sialylated, core fucosylated, galactosylated and high mannose glycans were all quantified. Since the mechanism of action of etanercept is TNF&agr; binding, only mannosylation was deemed as critical quality attribute for glycosimilarity assessment due to its influence on serum half‐life. Abbreviations: APTS: 8‐aminopyrene‐1,3,6‐trisulfonic acid; CGE‐LIF: capillary gel electrophoresis – laser induced fluorescence; CQA: critical quality attributes; ADCC: antibody‐dependent cell‐mediated cytotoxicity; CDC: complement‐dependent cytotoxicity; Fc: fragment crystallizable; TNF&agr;: tumor necrosis factor alpha; MOA: mechanism of action.

Modelling and Prediction of Renewable Energy Generation by Pressure Retarded Osmosis

Abstract A more general mass transport model of pressure retarded osmosis has been developed and will be presented in this lecture. Essential of this model is that it does not have any simplifications and/or neglects for description of the mass transport process. It takes into account the effect of the external boundary layers on both sides of the membrane, the dense and the sponge layers of an asymmetric membrane used, applying the diffusive-convective mass transport equation for every sub-layer except of the skin/dense layer of the membrane. A widely applied, “diffusive” transport equation was used for the dense layer, for the salt transport through it. Accordingly this model enables the user to calculate the membrane performance under all possible operating conditions. Thus, it can be used to optimize the operating conditions in order to get efficient energy generation unit. The energy density obtained by means of the presented and the literature model have been compared in the paper and showed the process performance under different conditions.

Tilted pillar array fabrication by the combination of proton beam writing and soft lithography for microfluidic cell capture Part 2: Image sequence analysis based evaluation and biological application

As a continuation of our previously published work, this paper presents a detailed evaluation of a microfabricated cell capture device utilizing a doubly tilted micropillar array. The device was fabricated using a novel hybrid technology based on the combination of proton beam writing and conventional lithography techniques. Tilted pillars offer unique flow characteristics and support enhanced fluidic interaction for improved immunoaffinity based cell capture. The performance of the microdevice was evaluated by an image sequence analysis based in‐house developed single‐cell tracking system. Individual cell tracking allowed in‐depth analysis of the cell–chip surface interaction mechanism from hydrodynamic point of view. Simulation results were validated by using the hybrid device and the optimized surface functionalization procedure. Finally, the cell capture capability of this new generation microdevice was demonstrated by efficiently arresting cells from a HT29 cell‐line suspension.