Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells

In this study we describe optimization of polyethylenimine (PEI)-mediated transient production of recombinant protein by CHO cells by facile manipulation of a chemically defined culture environment to limit accumulation of nonproductive cell biomass, increase the duration of recombinant protein production from transfected plasmid DNA, and increase cell-specific production. The optimal conditions for transient transfection of suspension-adapted CHO cells using branched, 25 kDa PEI as a gene delivery vehicle were experimentally determined by production of secreted alkaline phosphatase reporter in static cultures and recombinant IgG(4) monoclonal antibody (Mab) production in agitated shake flask cultures to be a DNA concentration of 1.25 mu g 10(6) cells(-1) mL(-1) at a PEI nitrogen: DNA phosphate ratio of 20:1. These conditions represented the optimal compromise between PEI cytotoxicity and product yield with most efficient recombinant DNA utilization. Separately, both addition of recombinant insulin-like growth factor (LR3-IGF) and a reduction in culture temperature to 32 degrees C were found to increase product titer 2- and 3-fold, respectively. However, mild hypothermia and LR3-IGF acted synergistically to increase product titer 11-fold. Although increased product titer in the presence of LR3-IGF alone was solely a consequence of increased culture duration, a reduction in culture temperature post-transfection increased both the integral of viable cell concentration (IVC) and cell-specific Mab production rate. For cultures maintained at 32 degrees C in the presence of LR3-IGF, IVC and qMab were increased 4- and 2.5-fold, respectively. To further increase product yield from transfected DNA, the duration of transgene expression in cell populations maintained at 32 C in the presence of LR3-IGF was doubled by periodic resuspension of transfected cells in fresh media, leading to a 3-fold increase in accumulated Mab titer from similar to 13 to similar to 39 mg L-1. Under these conditions, Mab glycosylation at Asn297 remained essentially constant and similar to that of the same Mab produced by stably transfected GS-CHO cells. From these data we suggest that the efficiency of transient production processes (protein output per rDNA input) can be significantly improved using a combination of mild hypothermia and growth factor(s) to yield an extended activated hypothermic synthesis.

Measurement and stability of FTY720 in human whole blood by high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry

We report here a validated method for the quantification of a new immunosuppressant drug FTY720, using HPLC-tandem mass spectrometry. Whole blood samples (500 mu l) were subjected to liquid-liquid extraction, in the presence of an internal standard (Y-32919). Mass spectrometric detection was by selected reaction monitoring with an atmospheric pressure chemical ionization source in positive ionization mode (FTY720: m/z 308.3 -> 255.3). The assay was linear from 0.2 to 25 mu g/l (r(2) > 0.997, n = 5). The inter- and intra-day analytical recovery and imprecision for quality control samples (0.5, 7 and 15 mu g/l) were 95.8-103.2 and < 5.5%, respectively. At the lower limit of quantification (0.2 mu g/l) the interand intra-day analytical recovery was 99.0-102.8% with imprecision of < 7.6% (n = 5). The assay had a mean relative recovery of 100.5 +/- 5.8% (n = 15). Extracted samples were stable for 16 h. IFTY720 quality control samples were stable at room temperature for 16 h at 4 degrees C for at least 8 days and when taken through at least three freeze-thaw cycles. In conclusion, the method described displays analytical performance characteristics that are suitable for pharmacokinetic studies in humans. (c) 2006 Elsevier B.V. All rights reserved.

Chemical deterrence of a marine cyanobacterium against sympatric and non-sympatric consumers

This study investigates the influence of mesograzer prior exposure to toxic metabolites on palatability of the marine cyanobacterium, Lyngbya majuscula. We examined the palatability of L. majuscula crude extract obtained from a bloom in Moreton Bay, South East Queensland, Australia, containing lyngbyatoxin-a (LTA) and debromoaplysiatoxin (DAT), to two groups: (1) mesograzers of L. majuscula from Guam where LTA and DAT production is rare; and (2) macro- and mesograzers found feeding on L. majuscula blooms in Moreton Bay where LTA and DAT are often prevalent secondary metabolites. Pair-wise feeding assays using artificial diets consisting of Ulva clathrata suspended in agar (control) or coated with Moreton Bay L. majuscula crude extracts (treatment) were used to determine palatability to a variety of consumers. In Guam, the amphipods, Parhyale hawaiensis and Cymadusa imbroglio; the majid crab Menaethius monoceros; and the urchin Echinometra mathaei were significantly deterred by the Moreton Bay crude extract. The sea hares, Stylocheilus striatus, from Guam were stimulated to feed by treatment food whereas S. striatus collected from Moreton Bay showed no discrimination between food types. In Moreton Bay, the cephalaspidean Diniatys dentifer and wild caught rabbitfish Siganus fuscescens were significantly deterred by the crude extract. However, captive-bred S. fuscescens with no known experience with L. majuscula did not clearly discriminate between food choices. Lyngbya majuscula crude extract deters feeding by most mesograzers regardless of prior contact or association with blooms.

Different routes to pure food allergens: extraction, recombinant techniques and total chemical synthesis for obtaining the plant-food pan-allergen LTP

La richiesta di allergeni puri è in continuo aumento per scopi diagnostici, come standard per metodi di rilevamento e di quantificazione, per l'immunoterapia e per lo studio a livello molecolare dei meccanismi delle reazioni allergiche, al fine di facilitare lo sviluppo di possibili cure. In questa tesi di dottorato sono descritte diverse strategie per l’ottenimento di forme pure di non-specific Lipid Transfer Proteins (nsLTPs), le quali sono state riconosciute essere rilevanti allergeni alimentari in molti frutti e verdure comunemente consumati e sono state definite come modello di veri allergeni alimentari. Una LTP potenzialmente allergenica, non nota in precedenza, è stata isolata dalle mandorle, mentre una LTP dall’allergenicità nota contenuta nelle noci è stata prodotta mediante tecniche di DNA ricombinante. Oltre a questi approcci classici, metodi per la sintesi chimica totale di proteine sono stati applicati per la prima volta alla produzione di un allergene, utilizzando Pru p 3, la LTP prototipica e principale allergene della pesca nell'area mediterranea, come modello. La sintesi chimica totale di proteinepermette di controllarne completamente la sequenza e di studiare la loro funzione a livello atomico. La sua applicazione alla produzione di allergeni costituisce perciò un importante passo avanti nel campo della ricerca sulle allergie alimentari. La proteina Pru p 3 è stata prodotta nella sua intera lunghezza e sono necessari solo due passaggi finali di deprotezione per ottenere il target nella sua forma nativa. Le condizioni sperimentali per tali deprotezioni sono state messe a punto durante la produzione dei peptidi sPru p 3 (1-37) e sPru p 3 (38-91), componenti insieme l'intera proteina. Tecniche avanzate di spettrometria di massa sono state usate per caratterizzare tutti i composti ottenuti, mentre la loro allergenicità è stata studiata attraverso test immunologici o approcci in silico.

Degradable poly(ethylene glycol)-based hydrogels:synthesis, physico-chemical properties and in vitro characterization

Designing degradable hydrogels is complicated by the structural and temporal complexities of the gel and evolving tissue. A major challenge is to create scaffolds with sufficient mechanical properties to restore initial function while simultaneously controlling temporal changes in the gel structure to facilitate tissue formation. Poly(ethylene glycol) was used in this work, to form biodegradable poly(ethylene glycol)-based hydrogels with hydrolyzable poly-l-lactide segments in the backbone. Non-degradable poly(ethylene glycol) was also introduced in the formulation to obtain control of the degradation profile that encompasses cell growth and new tissue formation. The dependence on polymer composition was observed by higher degradation profiles and decreased mechanical properties as the content of degradable segments was increased in the formulation. Based on in vitro tests, no toxicity of extracts or biomaterial in direct contact with human adipose tissue stem cells was observed, and the ultraviolet light treatment did not affect the proliferation capacity of the cells.

Control of photomagnetic behavior in heterobimetallic Prussian Blue analogs

A new challenge in the field of molecular magnetism is the design of optically and thermally switchable solid state magnetic materials for which various kinds of application may be feasible. Our research activities involve preparative methods, the study of the physical properties and associated mechanisms, as well as the exploration of further possibilities. Particular focus is on heterobimetallic Prussian Blue analogs, such as on RbMn[Fe(CN)6], in which the interplay between the two different adjacent metal ions is crucial for the observation of photo-induced phenomena. Our studies revealed that modification of the preparative conditions lead to differences in structural features that allowed tuning of the magnetic and electron transfer properties of RbxMn[Fe(CN)6]y.zH2O.

Spatially orthogonal chemical functionalization of a hierarchical pore network for catalytic cascade reactions

The chemical functionality within porous architectures dictates their performance as heterogeneous catalysts; however, synthetic routes to control the spatial distribution of individual functions within porous solids are limited. Here we report the fabrication of spatially orthogonal bifunctional porous catalysts, through the stepwise template removal and chemical functionalization of an interconnected silica framework. Selective removal of polystyrene nanosphere templates from a lyotropic liquid crystal-templated silica sol–gel matrix, followed by extraction of the liquid crystal template, affords a hierarchical macroporous–mesoporous architecture. Decoupling of the individual template extractions allows independent functionalization of macropore and mesopore networks on the basis of chemical and/or size specificity. Spatial compartmentalization of, and directed molecular transport between, chemical functionalities affords control over the reaction sequence in catalytic cascades; herein illustrated by the Pd/Pt-catalysed oxidation of cinnamyl alcohol to cinnamic acid. We anticipate that our methodology will prompt further design of multifunctional materials comprising spatially compartmentalized functions.

Optimization of chemical plant simulation using double collocation

A method has been constructed for the solution of a wide range of chemical plant simulation models including differential equations and optimization. Double orthogonal collocation on finite elements is applied to convert the model into an NLP problem that is solved either by the VF 13AD package based on successive quadratic programming, or by the GRG2 package, based on the generalized reduced gradient method. This approach is termed simultaneous optimization and solution strategy. The objective functional can contain integral terms. The state and control variables can have time delays. Equalities and inequalities containing state and control variables can be included into the model as well as algebraic equations and inequalities. The maximum number of independent variables is 2. Problems containing 3 independent variables can be transformed into problems having 2 independent variables using finite differencing. The maximum number of NLP variables and constraints is 1500. The method is also suitable for solving ordinary and partial differential equations. The state functions are approximated by a linear combination of Lagrange interpolation polynomials. The control function can either be approximated by a linear combination of Lagrange interpolation polynomials or by a piecewise constant function over finite elements. The number of internal collocation points can vary by finite elements. The residual error is evaluated at arbitrarily chosen equidistant grid-points, thus enabling the user to check the accuracy of the solution between collocation points, where the solution is exact. The solution functions can be tabulated. There is an option to use control vector parameterization to solve optimization problems containing initial value ordinary differential equations. When there are many differential equations or the upper integration limit should be selected optimally then this approach should be used. The portability of the package has been addressed converting the package from V AX FORTRAN 77 into IBM PC FORTRAN 77 and into SUN SPARC 2000 FORTRAN 77. Computer runs have shown that the method can reproduce optimization problems published in the literature. The GRG2 and the VF I 3AD packages, integrated into the optimization package, proved to be robust and reliable. The package contains an executive module, a module performing control vector parameterization and 2 nonlinear problem solver modules, GRG2 and VF I 3AD. There is a stand-alone module that converts the differential-algebraic optimization problem into a nonlinear programming problem.

Egg quality and fecundity in rainbow trout: the determining factors and mechanisms of control

This thesis considers the factors involved in the determination of egg quality and fecundity in farmed stocks of rainbow trout ( Salmo gairdneri R) • Measurements of egg quality, ie. percentage survivals of eggs and fry, from the production batches of eggs of seven fish farms, showed mean survivals of 70% to eying but levels of only 35% to 4.5g fry (approx. 130 days post-fertilisation). Under optimum conditions survivals may reach 85% suggesting that husbandry methods exert significant influences on egg quality. Chemical analyses of the protein, fat, vitellogenin, ash, amino acids, free fatty acid and mineral levels of eggs of varying quality and from parents of different strains showed compositional differences even between individuals of the same stock. However, none of these differences were correlated with egg quality. Egg size showed similar variations but, again under hatchery conditions there was no correlation with differences in egg quality. The only factor which has been shown to exert a significant influence on egg quality is the time of stripping after ovulation. At 1 0°C eggs should be removed from gravid females within ten days of ovulation to achieve optimum egg and fry survival. Studies of egg production from approximately 10,000 broodstock revealed that total fecundity and egg size increased and relative fecundity decreased with increasing fish size. In general, most fish appeared to produce a constant volume of eggs. This is consistent with a hypothesis that egg size can only be increased by parallel reductions in fecundity. Feeding broodstock at half-ration (0.35% body weight day- 1 ) did not affect egg quality but reduced total fecundity and egg size and increased relative fecundity when compared with eggs produced by fish on full-ration. Comparisons of regressions of total fecundity against fish weight for three strains using ANOCO revealed that one strain was significantly more fecund than two other strains considered. Trout of the same strain maintained on different farms behaved similarly suggesting there was some reproducibility of strain characteristics.

The environmental control of reproduction in the female dace, Leuciscus leuciscus

An investigation was made into the nature and control of the annual reproductive cycle of the dace, Leuciscus leuciscus. It includes 1) a study of the natural reproductive cycle, 2) the use of Carp Pituitary Extract (CPE) to induce final maturation and ovulation in captive fish, 3) the effect of artificial light treatments on ovarian development and 4) the measurement of serum melatonin levels under different photoperiod regimes. Ovarian development was monitored by endocrinological data, notably serum cycles of 17-oestradiol (E2), testosterone (T), and calcium (as an index of vitellogenin), oocyte diameter, the gonadosomatic index and histological studies of the ovary. Under natural conditions, ovarian development can broadly be divided into 4 stages: 1) oogenesis which occurs immediately after spawning; 2) a primary growth phase (previtellogenic growth) prevalent between spawning and June; 3) a secondary growth phase (yolk vesicle plus vitellogenic growth) occurring between June and December and 4) final maturation and ovulation which occurs in mid-March. During the annual ovarian cycle, the sex steroids E2 and T showed two clear elevations. The first occurred initially in April followed by a rise in serum calcium levels. This subsequently initiated the appearance of yolk granules in the oocytes in June. The second rise occurred in September and levels were maintained until December, after which there was a decline in serum E2 levels. It is proposed that in the dace, high serum E2 levels between September and December were required to maintain vitellogenin production and therefore its uptake into the developing oocytes which occurred during this time, albeit at a slower rate than in the summer months. After December, prior to final maturation, whereas serum E2 and calcium levels declined, serum T levels remained elevated. In captivity, final maturation beyond the germinal vesicle migration stage failed to occur suggesting that the stimuli required for these events were absent. However ovulation could be induced by a single injection of CPE, which induced ovulation between 6 and 14 hours after treatment. Endocrine events associated with the artificial induction of spawning included a rise in serum levels of E2, T and the maturation inducing steroid 1720-dihydroxy progesterone. Photoperiodic manipulation demonstrated that whereas short or increasing daylengths were stimulatory to ovarian development, long days delayed development. Changes from long to short and constant short daylengths early in the reproductive cycle advanced maturation (up to 5 months), suggesting that the stimulus for ovarian development and maturation was a short day. However, experiments conducted later in the reproductive cycle demonstrated that only a simulated ambient photoperiod could induce final maturation. It is proposed therefore that under natural conditions the environmental stimulus for ovarian development and final maturation are short and increasing daylengths respectively. Further support that photoperiod is the dominant timing cue in this species was provided by the pattern of serum melatonin levels. Under different photoperiod treatments, serum melatonin, which is believed to be the chemical transducer of photoperiodic information (similar to other photoperiodic species) was elevated for the duration of the dark phase, indicating that the dace at least has the ability to `measure' changes in daylength.