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Membrane adsorbers provide an attractive alternative to traditional bead-based chromatography columns used to remove trace impurities in downstream applications. A linearly scalable novel membrane adsorber family designed for the efficient removal of trace impurities from biotherapeutics, are capable of reproducibly achieving greater than 4 log removal of mammalian viruses, 3 log removal of endotoxin and DNA, and greater than 1 log removal of host cell protein. Single use, disposable membrane adsorbers eliminate the need for costly and time consuming column packing and cleaning validation associated with bead-based chromatography systems, and minimize the required number and volume of buffers. A membrane adsorber step reduces process time, floor space, buffer usage, labor cost, and improves manufacturing flexibility. This "process compression" effect is commonly associated with reducing the number of processing steps. The rigid microporous structure of the membrane layers allows for high process flux operation and uniform bed consistency at all processing scales.

A procedure for the immobilization of rhizomycelia of two strains of anaerobic fungi, namely Piromyces sp. KSX1 and Orpinomyces sp. 478P1 using Ca-alginate beads has been developed. Zoospores were found to be suitable propagules for the immobilization of the monocentric strain KSX1 giving rise to colonization in 100% of beads. For polycentric strain 478P1, which produced very few zoospores, partially homogenized rhizomycelia proved a suitable inoculum for the beads. The distribution of rhizomycelial growth inside the beads and on the surface depended on which fungal strain was immobilized. Strain 478P1 grew as a mycelial network inside the beads whereas strain KSX1 grew in microcolonies throughout the bead and on the surface, and was unable to evenly colonize the entire Ca-alginate matrix.

Isothermal annealing and continuous cooling experiments have been performed on synthetic orthopyroxene crystals with compositions En81Fs19 and En49Fs51. Their intracrystalline Fe2+, Mg-partitioning was determined by X-ray structure analysis. En81Fs19 was equilibrated at 550°C, 675°C and 850°C, En49Fs51 at 550°C, 650°C, 675°C, 800°C, and 850°C. The Fe2+, Mg distribution coefficients vary with temperature according to \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{In(K\_{D})}\ =\ 0.633(31)\ {-}\ 2625(31)/\mathrm{T[K]\ (En\_{81}Fs_{19})}\] \end{document} \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{In(K\_{D})}\ =\ 0.594(52)\ {-}\ 2581(51)/\mathrm{T[K]\ (En\_{49}Fs_{51})},\] \end{document} suggesting that in this compositional range In(KD) values do not significantly depend on the Fs content. Rate constants were determined in ordering runs at 550°C: \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{k}\_{\mathrm{dis}}\ =\ 0.00165(37)\ \mathrm{[h^{{-}1}]\ (En\_{81}Fs_{19})}\] \end{document} \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{k}\_{\mathrm{dis}}\ =\ 0.0080(18)\ \mathrm{[h^{{-}1}]\ (En\_{49}Fs_{51})}.\] \end{document} The results closely agree with predictions from Arrhenius equations given by Ganguly & Tazzoli (1994) and Kroll et al. (1997). Non-linear continuous cooling experiments were performed on both orthopyroxenes to test the ability of the Mueller rate equation (Mueller, 1967, 1969) to correctly predict the evolution of ordering. The crystals were cooled from 850°C to 250°C at an average rate of 13°C/day. The frozen site occupancies could be fully reproduced by the calculated ordering paths when the Mueller equation was run with the temperature dependencies of KD given above and Arrhenius parameters for kdis, taken from Kroll et al. (1997) and slightly adjusted within their error limits. Site refinements were also performed under the assumption that an error of ±1% total Fe had occurred in the microanalysis. This resulted in In (KD) lines which almost coincide for En49Fs51, but are clearly different for En81Fs19. Consequently, the error of calculated cooling rates is negligible for En49Fs51, but becomes significant for En81Fs19.

The understanding of variation in sieving properties of membranes is of great importance for the successful development of ultrafiltration applications. A liquid porosimetry technique is presented to quantify the sieving variation among several polyethersulfone ultrafiltration membranes. Observed sieving coefficients were measured with proper precautions taken to control and minimize fouling. These data were translated to intrinsic sieving coefficients using a stagnant film model. The intrinsic membrane sieving coefficient correlated well with the liquid porosimetry data. This liquid porosimetry technique can distinguish between membranes of different molecular weight cut-off and is sensitive enough to capture slight changes in the sieving coefficient of variants of the same cut-off membrane. This technique has several attractive features: it is non-destructive, independent of the module configuration and relatively simple to perform. Two potential applications of this technique are also examined: (1) quantification of the effect of membrane variation on high performance tangential flow filtration (HPTFF) for protein separations and (2) development of a membrane integrity test to ensure batch-to-batch consistency. This technique has the potential for use in membrane quality control, membrane selection, and validation of industrial ultrafiltration processes.

Experiments conducted on a natural kaersutitic amphibole, combined with statistical analysis of high-quality chemical analyses reported in the literature, document that both Fer+ and Ti in this group of Ti-rich hornblendes are accommodated by the oxy substitutions Fe3*O2 Felj(OH)-, and Ti4+O2-Rli(OH) ,. The valence of the cation Rli can be variable, provided that the sum of all substitutions maintains charge balance within the amphibole crystal structure. If the OH lost as a result of the two oxy substitutions is accounted for, the mean amphibole 03 site occupancies (i.e., OH + F+ Cl+ O'-) of both the experimentally treated and naturally occurring sample populations sum to the theoretical value of 2.00, within the statistical uncertainty. The unusually low H contents reported for kaersutite from Martian meteorites are concluded to result from their unusually high Ti and total Fe contents relative to terrestrial kaersutite.

Experiments have been carried out from 500 to 1200 °C, 1 atm to 10 kbar, and fH2from that of the IQF buffer to air, to quantify the variation of Fe oxy-component content in a titanian pargasite megacryst amphibole from Vulcan's Throne, Arizona. The results document the operation of the following substitution mechanism in the amphibole crystal structure: FeH + OH - = Fe3+ + 02- + I/zH2whereby the Fe3+/Fetot of the amphibole is controlled by T, P, and fH2' For the amphibole composition that was investigated, there is a linear variation of log fH2 as a function of log (Fe3+ /FeH) at fixed T and P of the form log fH2 = a + b 10g(Fe3+ /FeH ). Values of a and bare

Anaerobic chytridiomycete fungi are found in the gastrointestinal tracts of sheep, cattle and goats, as well as in many other domesticated ruminant and nonruminant herbivores and a wide variety of wild herbivorous mammals. They are principally found associated with the fibrous plant particles of digesta and as free swimming zoospores in the fluid phase. The presence of large fungal populations in animals consuming mature pasture or diets largely composed of hay or straw together with the production of highly active fibre degrading enzymes lead to' the belief that anaerobic fungi may have a significant role to play in the assimilation of fibrous feeds by ruminants. While many early studies focused on anaerobic fungi because of their unusual biology and metabolism, the large part of subsequent research has emphasized the biotechnological potential of their cellulases, xylanases and phenolic esterases. In recent years, the extent of the contribution of anaerobic fungi to the nutrition of ruminants has also been established through studies of fungal populations in the rumen and the dietary factors which influence them, as presented in this review. Further, we discuss the evidence supporting an important contribution of anaerobic fungal populations in the rumen to feed intake and digestion of poor quality feed by domesticated ruminants. In conclusion, the review explores some different methods for manipulating fungi in the rumen for increased feed intake and digestion.

Product . . . . . . . . . 363 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364Virus filtration is one component in the overall strategy to minimize transmission of infectious pathogens for biological products. Recombinant products such as monoclonal antibodies are expressed by mammalian, bacteria or yeast cells in fermentors, and within ascites fluid, or fluids from transgenic mammals. Mammalian cell lines may contain endogenous viruses that are generated in the bioreactor. Endogenous retroviruses are expressed because the retroviral genome is integrated into the cell line and cannot be screened-out during the creation of the Master Cell Bank. This causes retrovirus like particles, or RVLP’s, to be produced within the bioreactor. Products can also become contaminated by adventitious viruses which enter the process streams.

Two anaerobic fungi, one a monocentric strain ( Piromyces sp. KSX1) and the other a polycentric strain ( Orpinomyces sp. 478P1), were immobilised in calcium alginate beads and cultured in sequential batches where spent medium (containing 0.25% cellobiose) was repeatedly drained and replaced. beta-Glucosidase production with KSX1 was maintained for 45 days over six repeated batch cultures yielding a maximum level of 107 mIU/ml. For 478P1, beta-glucosidase production was maintained for 30 days over four repeated batches yielding a maximum level of 34 mIU/ml. Although repeat-batch cultures of KSX1 produced more beta-glucosidase than strain 478P1, the maximum specific beta-glucosidase produced from these immobilised cultures was similar. The immobilised polycentric strain proved to be operationally superior to strain KSX1, as strain 478P1 did not produce any growth in the culture liquor.

The Mobius® CellReady bioreactor product platform incorporates novel disposable technologies that provide optimal performance for suspension mammalian cell culture. Here we show the utility of EMD Millipore's 3L and 50L CellReady single use bioreactors for the cultivation of adherent mammalian cells on microcarriers. Cytodex3® and Solohill® collagen microcarriers were first tested in a mixing study to assess feasibility. We evaluated the normalized mixing speed required in the 3L and 50L to achieve a suspension of the microcarriers and enable growth of the cells.

Abstract. The verification of membrane integrity for filtration processes specifically designed for the removal of adventitious virus from biotherapeutics is of the utmost importance to both biomanu-facturers and regulatory agencies. Although conventional bubble-point and air-diffusion tests are widely accepted for integrity testing of bacteria-retentive membranes, these tests have severe limitations in their ability to assess the integrity of virus-retentive membranes. A novel membrane integrity test based upon liquid—liquid porosimetric principles (CorrTest™) has been specifically designed to correlate and predict the virus retention capabilities of Viresolve™ virus removing membranes. To optimize test sensitivity for both Viresolve/70™ and Viresolve/180™ membrane types, two distinct porosimetric correlations at different transmembrane pressures have been developed. Based upon an 80% prediction interval, an integrity test performed at either test pressure can reliably predict the ability of Viresolve™ membranes to remove the bacteriophage φ X174 to within 0·4 log removal value (LRV) units. To maintain test sensitivity and provide greater flexibility for conducting the liquid—liquid intrusion integrity test, appropriate pressure- and temperature-correction equations have been established. The two immiscible fluids employed in the developed technology are easily flushed from the membrane structure and are generally regarded as acceptable, non-toxic reagents for pharmaceutical applications. Consequently, the CorrTest™ integrity test can reliably and non-destructively measure both pre- and post-use membrane integrity to verify virus removal performance with the Viresolve™ module.

The excess Gibbs free energy due to non-convergent ordering is described by a Landau expansion in which configurational and non-configurational entropy contributions are separated: $$G^L = - hQ_t + \tfrac{1}{2}a^* (T - T_c^* )Q_t^2 + \tfrac{1}{n}e_n Q_t^n - TS_{conf \cdot }^{ord} $$ Neglecting higher order terms in Q t t, this expansion is formally equivalent to the reciprocal solution model for the distribution of Fe2+ and Mg over the non-equivalent M1 and M2 sites of orthopyroxenes: $$\begin{gathered} G^{ord} = - \tfrac{1}{2}[\Delta G_{exch}^0 - (L_{M1}^G - L_{M2}^G )X] + Q_t \hfill \\ {\text{ + }}\tfrac{1}{4}[\Delta G_{rec}^0 - (L_{M2}^G - L_{M1}^G )] + Q_t^2 {\text{ - }}TS_{{\text{conf}}^ \cdot }^{{\text{ord}}} \hfill \\ \end{gathered} $$ The Q t term describes a temperature and composition-dependent thermodynamic field that prevents the crystal from attaining full disorder at a finite temperature. The X term models the dependence of the field on composition. It causes the isotherms in a Roozeboom diagram X Fe M2 vs. X Fe M1 to be asymmetric. The Q t 2 term incorporates nearest-neighbour interactions. Higher order interactions are accounted for by the Q t n term, which is not routinely foreseen in the reciprocal solution model. The critical temperature T c * is interpreted as a ratio of enthalpy and entropy contributions to the free energy, ΔG rec 0 , of a reciprocal reaction $$T_c^* = \frac{{\Delta H_{rec}^0 - (L_{M1}^H + L_{M2}^H )}}{{\Delta S_{rec}^0 - (L_{M1}^S + L_{M2}^S )}}.$$ The comparison of Landau and classical approaches is extended to convergent ordering models which are shown to be incorporated in expressions for non-convergent ordering.

Fructose, glucose and xylose were the only monosaccharides to be fermented by the polycentric fungi, Orpinomyces joyonii (three cattle isolates) and O. intercalaris (two cattle isolates) and Anaeromyces spp. (four cattle isolates and two water buffalo isolates). Both Orpinomyces spp. utilised a similar range of oligosaccharides and polysaccharides by fermenting cellobiose, gentiobiose, lactose, maltose, sucrose, cellulose, glycogen, starch and xylan. In contrast, there was considerable variation in carbohydrate fermentation amongst Anaeromyces spp., with only cellobiose, gentiobiose and cellulose being fermented by all strains. Formate, acetate and ethanol were the major fermentation end-products formed from glucose by all polycentric fungi. In addition, Anaeromyces spp. produced considerable amounts of lactate, although only small amounts were formed by Orpinomyces spp. This difference was explained by the low specific activity for lactate dehydrogenase in Orpinomyces spp. Several Anaeromyces spp. also produced malate as a significant end-product of glucose fermentation. Fermentation of specifically-labelled Z14C]glucose molecules by polycentric fungi showed that hexose was catabolised by both polycentric and monocentric fungi via the glycolysis pathway with end-products being derived from the following carbon atoms: lactate and malate (C1-C3; C4-C6), acetate and ethanol (C1-C2; C5-C6), CO2 and formate (C3; C4). The results were compared to those obtained for monocentric and polycentric fungi isolated from temperate climate ruminants.

A mathematical model was developed for the simulation of non-ideal displacement chromatography. The model incorporates finite mass transport to the solid adsorbent by using a linear driving force approximation with a coupled external film and internal pore mass transfer coefficient. Equilibrium adsorption at the fluid—solid interface is described using competitive langmuirian adsorption isotherms. A finite difference numerical technique was employed to approximate the system of coupled, non-linear partial differential equations. The model was used to simulate the effluent concentration profiles under various displacement chromatographic conditions. The effects of axial dispersion and finite mass transport were examined by varying the Peclet and Stanton numbers, respectively. Slow mass transfer rates were shown to have a dispersive effect on the shock waves generated in displacement chromatography, resulting in greater zone overlap. Constant pattern formation was observed under non-ideal conditions. The throughput obtained in displacement chromatography was examined as a function of feed load, flow velocity, and displacer concentration. For non-ideal systems, the throughput was shown to exhibit a maximum at unique values of these operating parameters. The effects of particle diameter and solute diffusivity on the throughput were also examined. Model predictions indicate that the use of large particles could be detrimental to the performance of displacement systems when high velocities are employed. For macromolecular separations by displacement chromatography, small particles are required regardless of the linear velocity. The model presented here is a useful tool for the optimization and scale-up of displacement chromatographic processes.

Displacement chromatography was employed for the preparative-scale separation of peptides and proteins using large particle diameter chromatographic systems. Peptide displacements were successfully scaled-up with respect to particle and column diameter with no adverse effects on product recovery. Protein displacements on 30- and 90-μm agarose-based adsorbent systems resulted in well separated displacement zones of pure material. The present work extends the scope of biopolymer displacement chromatography to large particle diameter systems and is expected to further increase the distinct economic advantages associated with preparative-scale displacement chromatography.

Mulliken bond overlap populations calculated for beryllium and boron oxyanions isolated from a number of minerals and synthetic compounds correlate with observed bond lengths with shorter bonds tending to involve larger overlap populations. As the populations were calculated assuming constant bond lengths and observed valence angles, the correlations are asserted to reflect the angular geometry impressed on the oxyanions by neighboring cations. Extended Huckel theory shows that the dependence of overlap population upon valence angles is greater for boron than for beryllium oxyanions. This is consistent with the observation that bond length variations in boron oxyanions are greater than those in corresponding beryllium oxyanions. As expected, observed bond lengths correlate with valence angles with the shorter bonds tending to be involved in wider angles.

Multiple linear regression analysis has been applied to the geometric and chemical variables in sodic plagioclases in order to determine their relative effects on individual T-O bond lengths in the Al1+xSi3−xO8 tetrahedral framework. Using data from crystal structure analyses of low and high albite, An16 and An28, and assuming that low albite is completely ordered, $$\begin{gathered} {\text{T}} - {\text{O = 1}}{\text{.568}} + {\text{[(0}}{\text{.122) x (Al content of the T site)]}} \hfill \\ {\text{ }} - {\text{[(0}}{\text{.037) x (}}\Delta {\text{{\rm A}l}}_{{\text{br}}} )] + [0.063){\text{ x }}(\Sigma {\text{[}}q{\text{/(Na,Ca}} - {\text{O)}}^{\text{2}} ])] \hfill \\ {\text{ }} + {\text{[(0}}{\text{.029) x (}} - {\text{1/cosT}} - {\text{O}} - {\text{T)]}} \hfill \\ \end{gathered}$$ (1) where the Al content of a particular tetrahedral (T) site can be estimated from empirically-derived determinative curves, where Δ Albr is a linkage factor to account for the Al content of adjacent tetrahedral sites, where the formal charge on the (Na1−xCax) atom is q=1+x, and where T-O-T is the inter-tetrahedral angle involving the T-O bond. For sodic plagioclases it is essential to know only the anorthite content and the 2Θ131-2Θ1¯31 spacing (CuKα radiation) in order to determine the independent variables in this equation and thus to evaluate the individual T-O distances.

Nine strains of anaerobic fungi, assigned to the genera Neocallimastix and Piromonas , have been isolated from samples of ruminal digesta obtained from sheep and cattle in temperate Australia. Two strains of Sphaeromonas were also isolated from sheep. The patterns of utilization of mono-, oligo- and polysaccharides were determined for these fungi, four Neocallimactix spp. from Britain and New Zealand, and two Piromonas spp. from Britain. All 17 strains utilized cellobiose, fructose, gentiobiose, glucose and lactose. The seven Neocallimastix spp., whether from sheep or cattle, also fermented cellulose, glycogen, inulin, maltose, raffinose, starch, sucrose, xylan and xylose. Both Sphaeromonas isolates also fermented xylan and xylose. The eight Piromonas strains displayed a diversity in carbohydrate utilization, and could not be formed into a cohesive group. The metabolic endproducts of one strain each of Neocallimastix, Sphaeromonas and Piromonas were determined. They all produced acetate, formate, d (−)-lactate, ethanol and CO 2 during glucose fermentation.

Displacement chromatography was used for the preparative-scale separation of peptides, antibiotics, and proteins. The feed components were both purified and concentrated during the separation processes. The components of a peptide mixture were separated on a reverse-phase analytical column using 2-(2-butoxyethoxy) ethanol as the displacer. The use of organic modifiers in the carrier along with an elevated column temperature of 45 degrees C enabled the efficient separation of relatively hydrophobic peptides by displacement chromatography. In addition, the throughput of the process was significantly increased by carrying out the separation at an elevated flow-rate with no adverse effect on product purity. The antibiotic cephalosporin C was isolated from impurities in a fermentation broth using 2-(2-butoxyethoxy)ethanol as the displacer along with a step change in column temperature. The proteins cytochrome c and lysozyme were purified on a weak cation-exchanger column using cationic polymers as the displacers. While polymers of 60 and 20 kilodaltons were both found to be good displacers for these proteins, only the lower molecular weight polymer was readily removed from the column by standard regeneration techniques.