Real‐time control of a semi‐industrial fed‐batch evaporative crystallizer using different direct optimization strategies

This article presents a model‐based control approach for optimal operation of a seeded fed‐batch evaporative crystallizer. Various direct optimization strategies, namely, single shooting, multiple shooting, and simultaneous strategies, are used to examine real‐time implementation of the control approach on a semi‐industrial crystallizer. The dynamic optimizer utilizes a nonlinear moment model for on‐line computation of the optimal operating policy. An extended Luenberger‐type observer is designed to enable closed‐loop implementation of the dynamic optimizer. In addition, the observer estimates the unmeasured process variable, namely, the solute concentration, which is essential for the intended control application. The model‐based control approach aims to maximize the batch productivity, as satisfying the product quality requirements. Optimal control of crystal growth rate is the key to fulfill this objective. This is due to the close relation of the crystal growth rate to product attributes and batch productivity. The experimental results suggest that real‐time application of the control approach leads to a substantial increase, i.e., up to 30%, in the batch productivity. The reproducibility of batch runs with respect to the product crystal size distribution is achieved by thorough seeding. The simulation and experimental results indicate that the direct optimization strategies perform similarly in terms of optimal process operation. However, the single shooting strategy is computationally more expensive. © 2010 American Institute of Chemical Engineers AIChE J, 57: 1557–1569, 2011     

Secreted production of a custom-designed, highly hydrophilic gelatin in Pichia pastoris

A custom-designed, highly hydrophilic gelatin was produced inPichia pastoris. Secreted production levels in single-copytransformants were in the range 3–6 g/l of clarified brothand purification to near homogeneity could be accomplished bydifferential ammonium sulfate precipitation. Despite the factthat gelatins are highly susceptible to proteolysis becauseof their unfolded structure, the recombinant protein was shownto be fully intact by SDS–PAGE, N-terminal sequencing,gel filtration chromatography and mass spectrometry. Owing toits highly hydrophilic nature, the migration of the syntheticgelatin in SDS–PAGE was severely delayed. Esterificationof the carboxylic amino acid side chains resulted in normalmigration. The high polarity of the synthetic gelatin also accountsfor its negligible surface activity in water at concentrationsup to 5% (w/v), as determined by tensiometry. Circular dichroismspectrometry showed that the non-hydroxylated gelatin did notform triple helices at 4°C. The spectrum was even more representativeof the random coil conformation than the spectrum of naturalnon-hydroxylated gelatins.     

Tissue-Specific Ferritin- and GFP-Based Genetic Vectors Visualize Neurons by MRI in the Intact and Post-Ischemic Rat Brain

(1) Background: Neurogenesis is considered to be a potential brain repair mechanism and is enhanced in stroke. It is difficult to reconstruct the neurogenesis process only from the histological sections taken from different animals at different stages of brain damage and restoration. Study of neurogenesis would greatly benefit from development of tissue-specific visualization probes. (2) Purpose: The study aimed to explore if overexpression of ferritin, a nontoxic iron-binding protein, under a doublecortin promoter can be used for non-invasive visualization of neurogenesis using magnetic resonance imaging (MRI). (3) Methods: Ferritin heavy chain (FerrH) was expressed in the adeno-associated viral backbone (AAV) under the doublecortin promoter (pDCX), specific for young neurons, in the viral construct AAV-pDCX-FerrH. Expression of the enhanced green fluorescent protein (eGFP) was used as an expression control (AAV-pDCX-eGFP). The viral vectors or phosphate-buffered saline (PBS) were injected intracerebrally into 18 adult male Sprague–Dawley rats. Three days before injection, rats underwent transient middle-cerebral-artery occlusion or sham operation. Animals were subjected to In vivo MRI study before surgery and on days 7, 14, 21, and 28 days after injection using a Bruker BioSpec 11.7 T scanner. Brain sections obtained on day 28 after injection were immunostained for ferritin, young (DCX) and mature (NeuN) neurons, and activated microglia/macrophages (CD68). Additionally, RT-PCR was performed to confirm ferritin expression. (4) Results: T2* images in post-ischemic brains of animals injected with AAV-pDCX-FerrH showed two distinct zones of MRI signal hypointensity in the ipsilesioned hemisphere starting from 14 days after viral injection—in the ischemic lesion and near the lateral ventricle and subventricular zone (SVZ). In sham-operated animals, only one zone of hypointensity near the lateral ventricle and SVZ was revealed. Immunochemistry showed that ferritin-expressing cells in ischemic lesions were macrophages (88.1%), while ferritin-expressing cells near the lateral ventricle in animals both after ischemia and sham operation were mostly mature (55.7% and 61.8%, respectively) and young (30.6% and 7.1%, respectively) neurons. RT-PCR confirmed upregulated expression of ferritin in the caudoputamen and corpus callosum. Surprisingly, in animals injected with AAV-pDCX-eGFP we similarly observed two zones of hypointensity on T2* images. Cellular studies also showed the presence of mature (81.5%) and young neurons (6.1%) near the lateral ventricle in both postischemic and sham-operated animals, while macrophages in ischemic lesions were ferritin-positive (98.2%). (5) Conclusion: Ferritin overexpression induced by injection of AAV-pDCX-FerrH was detected by MRI using T2*-weighted images, which was confirmed by immunochemistry showing ferritin in young and mature neurons. Expression of eGFP also caused a comparable reduced MR signal intensity in T2*-weighted images. Additional studies are needed to investigate the potential and tissue-specific features of the use of eGFP and ferritin expression in MRI studies.     

Changes in epoxy-coated aluminium due to exposure to water

An investigation was performed of the changes that occur in a typical epoxy-coated aluminium system due to exposure to water. The adhesion of the epoxy coating upon exposure to water was evaluated for different exposure temperatures and periods. The adhesion test results showed an initial loss of adhesion of the coating but after this the adhesion improved again and even significantly exceeded adhesion prior to exposure. The amount of adhesion improvement and the speed with which adhesion improvement occurred was found to be larger for higher exposure temperatures. The changes that occur in the epoxy-coated aluminium system due to exposure to water were investigated using a number of different analytical techniques. Based on this, a model was proposed for the processes that take place during exposure and which can explain the adhesion test results. First, the adhesion of the epoxy coating is lost upon exposure due to the accumulation of a significant amount of water at the interface. The water at the interface causes formation and growth of a thin oxyhydroxide layer underneath the epoxy coating. After some time, this oxyhydroxide layer re-establishes contact with the epoxy coating and forms a new, water-stable bond, hence explaining the improvement of the adhesion after its initial loss.

The temperature-dependence observed in the adhesion test results is explained by the fact that at a higher exposure temperature, more water accumulates at the interface, the oxyhydroxide layer grows faster and also attains a larger limiting thickness.     

A Conjugate of an Anti‐Epidermal Growth Factor Receptor (EGFR) VHH and a Cell‐Penetrating Peptide Drives Receptor Internalization and Blocks EGFR Activation

Overexpression of (mutated) receptor tyrosine kinases is a characteristic of many aggressive tumors, and induction of receptor uptake has long been recognized as a therapeutic modality. A conjugate of a synthetically produced cell‐penetrating peptide (CPP), corresponding to amino acids 38–59 of human lactoferrin, and the recombinant llama single‐domain antibody (VHH) 7D12, which binds the human epidermal growth factor receptor (EGFR), was generated by sortase A mediated transpeptidation. The conjugate blocks EGF‐mediated EGFR activation with higher efficacy than that of both modalities alone; a phenomenon that is caused by both effective receptor blockade and internalization. Thus, the VHH–CPP conjugate shows a combination of activities that implement a highly powerful new design principle to block receptor activation by its clearance from the cell surface.     

Modified TiAlN coatings prepared by d.c. pulsed magnetron sputtering

Coatings like TiN or TiAlN are well established as hard and wear resistant tool coatings. These coatings often are prepared by PVD techniques like arc evaporation or d.c. magnetron sputtering. Typical micro hardness values of such hard coatings are in the range of 30 GPa. Compared to d.c. magnetron sputtering processes the pulsed magnetron sputter deposition technique could be shown as a clear advancement. Furthermore pure TiAlN hard coatings as well as TiAlN coatings modified by addition of elements like Si and Cr were prepared in order to improve the coating properties using the pulsed magnetron sputter technique in a batch coater equipped with 4 targets. Coatings prepared with the pulsed sputter process showed both high hardness and high wear resistance. The application potential of pulsed sputtered TiAlN coatings is demonstrated by turning test results of coated cemented carbide cutting inserts.Beside hardness and wear, other properties like adhesion or high temperature stability were determined. Cross sectional SEM images revealed the growth structure in dependence of the applied substrate bias and of the added elements. The chemical composition of the coatings was investigated by electron microprobe analysis and the phase and crystal size were determined by X-ray diffraction. Using the pulsed magnetron sputter process the coating properties, especially the hardness and the morphology, could be significantly improved. With indentation hardness values in the range of 40 GPa the region of super hard materials could be reached.     

Molecular analysis of intact preen waxes of Calidris canutus (Aves: Scolopacidae) by gas chromatography/mass spectrometry

The intact preen wax esters of the red knot Calidris canutus were studied with gas chromatography/mass spectrometry (GC/MS) and GC/MS/MS. In this latter technique, transitions from the molecular ion to fragment ions representing the fatty acid moiety of the wax esters were measured, providing additional resolution to the analysis of wax esters. The C₂₁−C₃₂ wax esters are composed of complex mixtures of hundreds of individual isomers. The odd carbon-numbered wax esters are predominantly composed of even carbon-numbered n-alcohols (C₁₄, C₁₆, and C₁₈) esterified predominantly with odd carbon-numbered 2-methyl fatty acids (C₇, C₉, C₁₁, and C₁₃), resulting in relatively simple distributions. The even carbon-numbered wax esters show a far more complex distribution due to a number of factors: (i) Their n-alcohol moieties are not dominated by even carbon-numbered n-alcohol moieties are not dominated by even carbon-numbered n-alcohols esterified with odd carbon-numbered 2-methyl fatty acids, but odd and even carbon-numbered n-alcohols participate in approximately equal amounts; (ii) odd carbon-numbered methyl-branched alcohols participate abundantly in these wax ester clusters; and (iii) with increasing molecular weight, various isomers of the 2,6-, 2,8-, and 2,10-dimethyl branched fatty acids also participate in the even carbon-numbered wax esters. The data demonstrate that there is a clear biosynthetic control on the wax ester composition although the reasons for the complex chemistry of the waxes are not yet understood.     

Application of direct fluid flow oscillations to improve mixing in microbioreactors

This article describes an active mixing method for a microbioreactor that was designed, simulated, tested, and successfully implemented. By applying a varying pressure to a microchannel looping tangentially into a cylindrical microreactor an oscillating fluid flow was shown to occur. Such an oscillating fluid flow improved mixing, both by diffusion and convection. The oscillating fluid flow has a large impact on the ratio between the diffusion domain and the convection domain. A good match was obtained between experimental mixing results, computational fluid dynamics simulation results and the results of a simplified mixing model thus demonstrating the potential of simulation on improving the design of microreactors. © 2009 American Institute of Chemical Engineers AIChE J, 2009     

Quantitative proof of liquid penetration-involved granule formation in a high shear mixer

Previous work of the authors [K. van den Dries, H. Vromans, Qualitative proof of liquid penetration-involved nucleation in a high shear mixer, Eur. J. Pharm. Sci. 58 (2004), 551–559.] revealed that the granule formation in a high shear mixer depends on a balance between the rate of liquid penetration and binder dispersion. Three distinct nucleation mechanisms could be qualified; (I) granule formation by liquid penetration followed by granule breakage or (II) absence of granule breakage and (III) complete dispersion of the binder liquid. The aim of this study was to quantify the mechanisms of granule formation. A substandard amount (1.5% w/w) of binder liquid was added to a lactose mixture, while the mixer was operating. The powder mixture was frozen with liquid nitrogen after 15 s and analysed by sieving. The results show that, despite the minimal liquid amount, granules are formed under most conditions. It is argued granules are being formed by a liquid penetration process. These freshly formed granules are broken down at low viscosity (< 1 Pa s) and remain intact at higher viscosity (> 1 Pa s). Only at extreme conditions (viscosity > 30 Pa s) hardly any granules are formed. In this case penetration of the liquid becomes practically impossible and the binder is completely dispersed. A model based on the processes of liquid penetration, binder dispersion and granule breakage, confirms the observed nucleation behaviour. It is conclusively shown that an increase in viscosity results in a transition from nucleation mechanism I→II→III.     

Discovery of Selective Luteinizing Hormone Receptor Agonists Using the Bivalent Ligand Method

Two series of dimeric ligands for a G‐protein‐coupled receptor were prepared that differ by the interconnecting spacer system. Biological evaluation revealed that both dimeric series exhibit unique biological properties relative to their monomeric counterparts.