High throughput solubility and redissolution screening for antibody purification via combined PEG and zinc chloride precipitation

As upstream product titers increase, the downstream chromatographic capture step has become a significant “downstream bottleneck.” Precipitation becomes more attractive under these conditions as the supersaturation driving force increases with the ever-increasing titer. In this study, two precipitating reagents with orthogonal mechanisms, polyethylene glycol (PEG) as a volume excluder and zinc chloride (ZnCl₂) as a cross linker, were examined as precipitants for two monoclonal antibodies (mAbs), one stable and the other aggregation-prone, in purified drug substance and harvested cell culture fluid forms. Manual batch solubility and redissolution experiments were performed as scouting experiments. A high throughput (HTP) liquid handling system was used to investigate the design space as fully as possible while reducing time, labor, and material requirements. Precipitation and redissolution were studied by systematically varying the concentrations of PEG and ZnCl₂ to identify combinations that resulted in high yield and good quality for the stable mAb; PEG concentrations in the range 7–7.5 wt/vol% together with 10 mM ZnCl₂ gave a yield of 97% and monomer contents of about 93%. While yield for the unstable mAb was high, quality was not acceptable. Performance at selected conditions was further corroborated for the stable mAb using a continuous tubular precipitation reactor at the laboratory scale. The HTP automation system was a powerful tool for locating desired (customized) conditions for antibodies of different physicochemical properties.     

Inducing activity of fractionated microsomal material from theXenopus laevis gastrula stage

Summary The postmitochondrial supernatant fromXenopus gastrulae has been fractionated on sucrose gradients. Part of the microsomal material was treated with EDTA, which dissociates most of the polysomal and monosomal material into ribosomal subunits. In addition, a series of pooled fractions from the EDTA treated gradients has been applied to discontinuous gradients in more concentrated sucrose to separate membranous material from the remaining microsomal components.Pooled fractions from all gradients have been tested for inductive activity on amphibian gastrula ectoderm. The spinocaudal (trunk and tail) inducing activity was to some extent eneriched in the membrane fractions.     

Marine invertebrate skeleton size varies with latitude,temperature and carbonate saturation: implications for global change and ocean acidification

There is great concern over the future effects of ocean acidification on marine organisms, especially for skeletal calcification, yet little is known of natural variation in skeleton size and composition across the globe, and this is a prerequisite for identifying factors currently controlling skeleton mass and thickness. Here, taxonomically controlled latitudinal variations in shell morphology and composition were investigated in bivalve and gastropod molluscs, brachiopods, and echinoids. Total inorganic content, a proxy for skeletal CaCO₃, decreased with latitude, decreasing seawater temperature, and decreasing seawater carbonate saturation state (for CaCO₃ as calcite (Ω_cal)) in all taxa. Shell mass decreased with latitude in molluscs and shell inorganic content decreased with latitude in buccinid gastropods. Shell thickness decreased with latitude in buccinid gastropods (excepting the Australian temperate buccinid) and echinoids, but not brachiopods and laternulid clams. In the latter, the polar species had the thickest shell. There was no latitudinal trend in shell thickness within brachiopods. The variation in trends in shell thickness by taxon suggests that in some circumstances ecological factors may override latitudinal trends. Latitudinal gradients may produce effects similar to those of future CO₂‐driven ocean acidification on CaCO₃ saturation state. Responses to latitudinal trends in temperature and saturation state may therefore be useful in informing predictions of organism responses to ocean acidification over long‐term adaptive timescales.     

Grand Canonical Monte Carlo Simulation for Solubility Calculation in Supercritical Extraction

Abstract

The Grand Canonical Ensemble Monte Carlo (GCEMC) technique is used to simulate highly nonideal dilute mixtures in the near vapor-liquid critical region. These systems are commonly found in supercritical fluid extraction processes. Mixtures composed of model CO₂/naphthalene/water molecules are studied. Very large and highly correlated concentration fluctuations were observed. It was found that when the total number of molecules in the system exceeded about 150, system size dependence was not significant. The GCEMC method breaks down when the system density exceeds about 1.5 times the solvent critical density due primarily to the low probability of successful addition and removal of the large naphthalene molecules. In some systems, the presence of a small amount of water caused a dramatic increase in the system density and in naphthalene solubility. By examining the radial distribution functions in these mixtures, the origin of this effect can be attributed to the preferential aggragation of the solute naphthalene molecules around the highly polar water molecules.     

Localization of acid phosphatase in lactating rat mammary glands

Summary Localization of acid phosphatase in mammary glands of lactating rats was studied by both biochemical and cytochemical methods. Cytochemically, acid phosphatase activity was detected by using lead citrate as the capture agent for the inorganic phosphate released from p-nitrophenyl phosphate. The activity was predominantly localized in the lumina of the endomembrane system and in the milk that had been secreted into the alveolar lumen. Biochemically, acid phosphatase was present in all the subcellular fractions with higher activities in the membrane-associated fractions. The localization of tartrate-resistant acid phosphatases within the endomembrane system of fully lactating rat mammary tissue suggests a possible role for these enzymes in milk secretory processes.Abbreviations ASMX 3-hydroxy-2-naphthoic acid 2,4-dimethylanilide - DMSO dimethylsulfoxide - DTT dithiothreitol - EDTA ethylenedinitrilo tetra-acetic acid - FGM fat globule membranes - MES 2-(N-morpholino) ethanesulfonic acid - PCMB p-chloromercuribenzoate - p-NPP p-nitrophenyl phosphate     

Effect of serum on organogenesis of the rat testis in vitro

Summary It was observed previously that primordia of fetal rat testes when explanted in vitro in a synthetic medium at the outset of sexual differentiation differentiate seminiferous cords during the following days, but that the addition of 15% fetal bovine serum prevents this morphogenesis. In the present study, human, horse, bovine calf, and rat sera were shown to exert the same effect. Very low concentrations of human or fetal bovine serum (0.5 or 1%) were sufficient to produce the serum effect, which was only slightly reduced when the serum was heated. The serum activity was not removed by dialysis (membrane cut-off 15 000), but it disappeared after treatment with trichloroacetic or perchloric acids or after trypsin digestion. Partial purification of the active factor(s) from human serum was achieved by successive gel filtration, affinity chromatography, and ion exchange chromatography. Analysis of the active fractions by electrofocusing and immunoelectrophoresis placed the activity within the α globulin group. Among a series of purified serum proteins tested, α₂-HS-glycoprotein was found to exhibit the serum effect, though this activity was heat labile.     

Product solubility control in cellooligosaccharide production by coupled cellobiose and cellodextrin phosphorylase

Soluble cellodextrins (linear β-1,4-d -gluco-oligosaccharides) have interesting applications as ingredients for human and animal nutrition. Their bottom-up synthesis from glucose is promising for bulk production, but to ensure a completely water-soluble product via degree of polymerization (DP) control (DP ≤ 6) is challenging. Here, we show biocatalytic production of cellodextrins with DP centered at 3 to 6 (~96 wt.% of total product) using coupled cellobiose and cellodextrin phosphorylase. The cascade reaction, wherein glucose was elongated sequentially from α-d -glucose 1-phosphate (αGlc1-P), required optimization and control at two main points. First, kinetic and thermodynamic restrictions upon αGlc1-P utilization (200 mM; 45°C, pH 7.0) were effectively overcome (53% → ≥90% conversion after 10 hrs of reaction) by in situ removal of the phosphate released via precipitation with Mg²⁺. Second, the product DP was controlled by the molar ratio of glucose/αGlc1-P (∼0.25; 50 mM glucose) used in the reaction. In optimized conversion, soluble cellodextrins in a total product concentration of 36 g/L were obtained through efficient utilization of the substrates used (glucose: 98%; αGlc1-P: ∼80%) after 1 hr of reaction. We also showed that, by keeping the glucose concentration low (i.e., 1–10 mM; 200 mM αGlc1-P), the reaction was shifted completely towards insoluble product formation (DP ∼9–10). In summary, this study provides the basis for an efficient and product DP-controlled biocatalytic synthesis of cellodextrins from expedient substrates.     

Phosphorus transformations along a soil/vegetation series of fire-prone, dolomitic, semi-arid shrublands of southern Spain Soil P and Mediterranean shrubland dynamic

Models of P transformations duringpedogenesis and with succession have developed fromstudies in temperate humid regions with neutral toacidic soils. Little is known about P biogeochemistryand P availability in semi-arid Mediterranean-typeshrublands with alkaline soils. We studied Ptransformations in a series of semi-arid, dolomiticshrublands in southeastern Spain, ranging from afrequently-burned, open gorse-scrubland on erodedTypic Xerorthents to a long-unburned, mature garrigueon Entic Haploxerolls. In contrast to the commonpattern of decreasing total P concentrations in thesoil profile with soil development due to leaching,total P increased markedly in this system. This is dueto concentration increases of relatively insolubleelements (P, Al, Fe, Ti) as karstification of parentmaterial (dolomitic marbles with up to 94% Ca-Mgcarbonate) during pedogenesis released bicarbonateand, subsequently, Ca and Mg leached from the profileat a higher rate. The total element to Ti ratiosindicated that the relative ion weathering losssequence, from easily weathered to resistant ions, wasCa>Mg > > > Fe>=Al>Ti, showing that P islost from these shrublands at an intermediate rate. Inone extreme of the series (the open gorse-scrubland),most soil P was Ca bound and organic P concentrations,organic matter content and phosphatase activity werevery low, as predicted by the model of Walker andSyers for the initial stages of soil development.However, this site showed the highest inorganic soilsolution P concentration, low soil P fixation capacityand the lowest foliar N:P ratios. Soils from theintermediate stages of the series showed the highestlabile inorganic and labile organic P concentrations.At the other extreme of the series (the maturegarrigue), a high proportion of soil P was in occludedinorganic and organic forms as predicted by the Walkerand Syers'; model. However, Ca bound P still accountedfor the largest single P fraction. Soils showed veryhigh sorption capacity (and high extractable Fe and Alconcentrations) and released very little P tosolution. Increasing values for NaOH extractable Po,organic matter and phosphatase activity indicate thatcycling of P through organic matter is increasinglyimportant with ecosystem development through theseries.