An overview of mechanistic modeling of liquid chromatography

Abstract

Liquid chromatography is considered to be the bottleneck for purification of therapeutic proteins. Development and optimization of chromatography process is a cumbersome activity due to the increasing complexities in the types and content of impurities present in the high product titer cell culture harvest obtained from the upstream processing. Further, regulatory expectations are continuously rising with the recent initiatives of quality by design and process analytical technology expecting the manufacturer to have a deeper understanding of the process and the product. Mechanistic modeling is one approach to gain this deeper understanding of a process step. It involves modeling of the underlying physicochemical processes. A well calibrated model with acceptable predictability can be very effective in both process optimization and process characterization activities. In this paper we provide an overview of mechanistic modeling of liquid chromatography. We discuss the various components that such a model entails and also presents the status quo of this area.     

Uptake,Transport and Storage of Cardenolides in Foxglove. Cardenolide Sinks and Occurrence of Cardenolides in the Sieve Tubes of Digitalis lanata1

Cardiac glycoside transport was investigated on the organ and whole plant level. Uptake experiments were carried out with shoot and root cultures of Digitalis lanata. In both systems primary cardenolides, i.e., those with a terminal glucose in their oligosaccharide side chain, were taken up against their concentration gradient, whereas the glucose-free secondary cardenolides were not. Active uptake of primary cardenolides was further evidenced by KCN inhibition of uptake. Using plantlets grown in vitro the long-distance transport of primary cardenolides from the leaves to the roots was demonstrated. Cardenolides were also detected in etiolated leaves, induced on plants with green leaves, which are supposed to be unable to synthezise cardenolides de novo, providing further evidence for long-distance transport. Several primary cardenolides were detected in the honeydew excreted by aphids fed on Digitalis lanata leaves, indicating that the phloem is a transporting tissue for cardenolides. On the other hand, the xylem sap obtained by applying the pressure-chamber technique was cardenolide-free. It was concluded that in Digitalis primary cardenolides serve as both the transport and the storage form of cardenolides. After their synthesis they are either stored in the vacuoles of the source tissue or loaded into the sieve tubes, from which they are unloaded at other sites where they are trapped in the vacuoles of the respective sink tissue.     

Anterograde Axonal Transport of Peptidylglycine α-Amidating Monooxygenase in Rat Sciatic Nerves

Axonal transport of peptidylglycine alpha-amidating monooxygenase (PAM) activity was studied in rat sciatic nerves from 12 to 120 h after double ligations. The anterograde axonal transport increased and reached a plateau between 48 and 72 h and then decreased. The flow rate was 100 mm/day, and the molecular mass of the active entity was 70 kDa, which was determined by gel filtration. In contrast, there was no evidence for significant retrograde axonal transport. Anterograde axonal transport of immunoreactive cholecystokinin, a carboxy-terminal-amidated putative neuropeptide, was also found. These results suggest that PAM is transported by a rapid axonal flow and may play a role as a processing enzyme during transport or in the terminals of rat sciatic nerves.     

Structural and Functional Impact of SRP54 Mutations Causing Severe Congenital Neutropenia

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Translocation - A key factor limiting the efficiency of nitrogen fixation in legume nodules

A hypothesis is presented that the availability of water for export of nitrogenous products from legume nodules is a major factor limiting the efficiency of symbiotic nitrogen fixation. Water for export of solutes in the xylem probably depends largely on the import of water and reduced carbon in the phloeum, and one function of respiration may be to dispose of reduced carbon in order to increase the supply of water. A second hypothesis presented is that control of gas diffusion in soybean nodules is largely restricted to the cortex nearby the vascular bundles, thus making possible the linkage of solute balances in xylem and phloem with resistance to diffusion. These concepts are used in a re-examination of literature on manipulations of nodules and nodulated plants such as lowering of light levels, water stress, defoliation, stem girdling, and alteration of oxygen supply. The concept of translocation as a major factor limiting efficiency of symbiotic fixation is consistent with the failure of superior rhizobial isolates to improve N input significantly, and this limitation could also prevent exploitation of superior bacterial symbionts in the future     

Conflicting effects of BMI and waist circumference on iron status

The association between obesity and iron status has a long history and is still receiving attention. However comparative analysis of the association between general obesity (BMI) and visceral obesity (waist circumference) with iron status has not been extensively researched. The aim of the present study is thus to determine if body mass index and waist circumference have the same correlation with iron status. One thousand one hundred and thirty people (225 men and 905 women) aged 30 years and above participated in this study. Anthropometric parameters, haemoglobin, iron and total iron binding capacity concentrations were measured using standard methods. Percentage transferrin saturation was calculated and ferritin concentrations were measured using an enzyme linked immunosorbent assay. Obese or overweight women had significantly lower iron and transferrin saturation concentration when compared to non-obese women. In contrast, women with high waist circumference had comparable plasma iron and transferrin saturation to women with normal waist circumference. Partial correlation analysis and linear regression analysis showed that BMI is negatively and significantly associated with plasma iron, transferrin saturation, Hb and ferritin concentration, whilst waist circumference is positively but insignificantly associated with plasma iron, transferrin saturation, Hb and ferritin concentration. Binary regression analysis showed that obese or overweight people are more likely to have iron deficiency, whilst those with raised waist circumference are more likely to have iron overload. Multivariate analysis showed that body mass index is negatively and significantly associated with low iron status, while waist circumference is positively and insignificantly associated with iron status. This is supported by a comparison of plasma iron, transferrin saturation and ferritin concentrations in participants with high body mass index and normal waist circumference and participants with normal body mass index and high waist circumference to those participants having normal body mass index and normal waist circumference. The present study suggests that in women body mass index is associated with low plasma iron, transferrin saturation and ferritin concentrations, while waist circumference is associated with high plasma iron, transferrin saturation and ferritin concentrations.     

Phospholipid dependence of the anion transport system of the human erythrocyte membrane. Studies on reconstituted band 3/lipid vesicles

Band 3 protein extracted from human erythrocyte membranes by Triton X-100 was recombined with the major classes of phospholipid occurring in the erythrocyte membrane. The resulting vesicle systems were characterized with respect to recoveries, phospholipid composition, protein content and vesicle size as well as capacity and activation energy of sulfate transport. Transport was classified into band-3-specific fluxes and unspecific permeability by inhibitors. Transport numbers (sulfate ions per band 3 per minute) served as a measure of functional recovery after reconstitution. The transport properties of band 3 proved to be insensitive to replacement of phosphatidylcholine by phosphatidylethanolamine, while sphingomyelin and phosphatidylserine gradually inactivated band-3-specific anion transport when present at mole fractions exceeding 30 mol%. The activation energy of transport remained unaltered in spite of the decrease in transport numbers. The results, which are discussed in terms of requirements of band 3 protein function with respect to the fluidity and surface charge of its lipid environment, provide a new piece of evidence that the transport function of band 3 protein depends on the properties of its lipid environment just as the catalytic properties of some other membrane enzymes. The well-established species differences in anion transport (Gruber, W. and Deuticke, B. (1973) J. Membrane Biol. 13, 19–36) may to some extent reflect this lipid dependence.     

Changes in DTPA-iron and management of iron chlorosis in rice nurseries

Summary On a Typic Ustochrept soil incorporation of 10 tons/ha of a green manure plus submergence for 10 days followed by raising upland nursery checked iron chlorosis. In contrast, presubmergence with and without FYM and iron sulfate or pyrite were a failure. Nor weekly sprays with 3.0% iron sulfate were found very effective. The success of green manure plus submergence was associated with the mobilization of soil iron as a result of intense reduction and its subsequent retention in available form at a sufficient high level during the growth of upland nursery.     

Mössbauer spectroscopy of iron-containing dermal granules from Molpadia intermedia

Dermal granules containing hydrous ferric oxide cores from Molpadia intermedia were studied by Mössbauer spectroscopy from 1.5 t0 300 K and in magnetic fields up to 80 kOersted at 4.2 K. A magnetic phase transition to an antiferromagnetically ordered state is observed at 10 K. The results are compared with the magnetic behavior of micellar cores of ferritin from eukaryotes and iron-storage materials from prokaryotes.