Ultrafiltration step

Until this point, the sample preparation techniques under discussion have relied upon differences in polarity to separate the analyte and the sample matrix in contrast, ultraflltration and on-line dialysis rely upon differences in molecular size between the analyte and matrix components to effect a separation. In ultrafiltration, a centrifugal force is applied across a membrane filter which has a molecular weight cut-off intended to isolate the analyte from larger matrix components. Furusawa incorporated an ultrafiltration step into his separation of sulfadimethoxine from chicken tissue extracts. Some cleanup of the sample extract may be necessary prior to ultrafiltration, or the ultrafiltration membranes can become clogged and ineffective. Also, one must ensure that the choice of membrane filter for ultrafiltration is appropriate in terms of both the molecular weight cut-off and compatibility with the extraction solvent used. 

Diafiltration is a process whereby an ultrafiltration system is utilized to reduce or eliminate low molecular mass molecules from a solution and is sometimes employed as part of biopharmaceuti-cal downstream processing. In practice, this normally entails the removal of, for example, salts, ethanol and other solvents, buffer components, amino acids, peptides, added protein stabilizers or other molecules from a protein solution. Diafiltration is generally preceded by an ultrafiltration step to reduce process volumes initially. The actual diafiltration process is identical to that of ultrafiltration, except for the fact that the level of reservoir is maintained at a constant volume. This is achieved by the continual addition of solvent lacking the low molecular mass molecules that are to be removed. By recycling the concentrated material and adding sufficient fresh solvent to the system such that five times the original volume has emerged from the system as permeate, over 99... 

The general method for ASMS is shown in Fig. 4.1. In ASMS, the target concentration is generally set at 5-10 xM, so that at equilibrium, ligands with affinities of no weaker than Ku 10 xM will be significantly bound and, therefore, retained in the ultrafiltration steps. The minimal concentration of each small molecule is dictated by the eventual need to detect ligands by mass spectrometry after several cycles of ultrafiltration and subsequent extraction. In order to ensure detection just above baseline for the vast majority of compounds, which vary in inherent ionization properties and efficiency of mass spectrometric visibility, the starting compound concentration is set at 1.5 pM per compound. The mixture... 

Figure 3.17. A likely purification procedure for tPA produced in recombinant E. coli cells. The heterologous product accumulates intracellularly in the form of inclusion bodies. In this particular procedure, an ultrafiltration step is introduced on several occasions to concentrate the product stream, particularly prior to application to chromatographic columns. Lysine affinity chromatography (Lys-chromatography) is employed as tPA is known to bind immobilized lysine molecules. Adapted with permission from Datar et al. (1993)...

Two of the more recent such approvals are that of Ovitrelle and Luveris. Ovitrelle is the trade name given by Serono to its recombinant hCG-based product. The producer is an engineered CHO cell line that has been co-transfected with the genes coding for both the hCG a- and P-subunits. Downstream processing entails a combination of several chromatographic and ultrafiltration steps and the final product is presented in freeze-dried form. Each vial of product contains 285 fig of active substance (hCG) and the product has been assigned a 2 year shelf-life. It is reconstituted with water for injections (WFI) immediately before use. 

Optimal fermentation parameters have been well established and air-lift, stirred tank, and hollow fibre systems have all been used. At commercial scale, fermentation volumes in excess of 1000 litres can be used, which can yield 100 g or more of final product. While hybridoma growth is straightforward, production levels of antibody can be quite low compared with ascites-based production systems. Typically, fermentation yields antibody concentrations of 0.1-0.5 mg/ml. Removal of cells from the antibody-containing media is achieved by centrifugation or filtration. An ultrafiltration step is then normally undertaken in order to concentrate the filtrate by up to 20-fold. 

ESR examination of nonchemically isolated fulvic acids showed that Mn2+ was the primary paramagnetic species observable (60, 61). Most likely, the soluble-colloidal fraction we identified in the speciation studies consisted primarily of such complexes. Because the ESR spectral characteristics of Mn in fulvic acid complexes are quite similar to Mn(H20)62+, Alberts et al. (62) suggested that the metal-fulvate interaction was weak. Stronger interaction would be expected to lead to changes in peak shape. This view leaves unexplained the ability of the complexes to survive the isolation procedure s long ultrafiltration steps, because weak interactions are usually associated with reversible complexation. 

During the semibatch experiments, vacuum filtration was applied at 4 d and 8 d after the start of saccharification, to remove the sugar product as filtrate. In selected semibatch experiments, ultrafiltration was applied to the vacuum filtrate to recover soluble enzymes. In other semibatch experiments, the vacuum filter cake was washed extensively with deionized water to remove any enzymes not bound to the solids. After filtration, pretreated corn stover slurry and 7 mL of solution (the ultrafiltration filtrate, or citrate buffer when ultrafiltration was not used) were added to the residual solids and bound enzymes, to replace the volume removed as filtrate during the ultrafiltration step. The additional substrate promotes further saccharification by reusing the cellulase enzymes. To promote further saccharification in a final set of semibatch experiments, additional cellulase at a specific activity of 5 FPU/g of fresh cellulose was added along with the fresh corn stover after vacuum filtration. 

The simultaneous determination of 3TC and AZT in human serum and seminal plasma was reported [41-42]. The sample pretreatment consisted of an automated ultrafiltration step. For isocratic LC, a 150x2-mm-ID Cig colunrn was used and 15% acetonitrile in water at 0.3 ml/min. The eluate of the first 2 min after injection were directed to waste. ESI-MS in SRM mode was apphed in the positive-ion mode for 3TC and negative-ion mode for AZT. Polarity switching was done in between the two peaks. The LOQ was 2.5 ng/ml. 

Numerous descriptions of methods of analysis of the acidic functional groups of humic substances have been published (e.g., Stevenson and Butler, 1969 Schnitzer and Khan, 1972 Stevenson, 1982). The published methods include direct titrations, discontinuous titrations, indirect titrations, indirect titrations coupled with either a distillation or ultrafiltration step, thermometric titration methods, nonaqueous titrations, irreversible reactions of acidic hydrogens with various organic and inorganic reagents, and so on. The two most commonly described methods are the barium hydroxide... 

The ultrafiltration step was developed with smaller-scale disposable devices (Centra-... 

Product development in water-based paints must consider how paint residues are to be recycled in the user s painting facilities. In certain paint systems, such as auto body paint booths, overspray is caught in circulating aqueous liquors and reconcentrated by ultrafiltration (see Fig. 123). Concentrate solutions are added to the appropriate paint formulations. In a special stove-drying paint system, the material recycling rate is ca. 95%. The special membranes needed for the ultrafiltration step were developed by Hoechst subsidiaries Herberts and Hoechst Celanese Corporation, together with plant construction specialists. 

Thus, the gain in entropy and the partial relief of the osmotic pressure by the released ions present a strong driving force for adsorption despite the fact that protein and carrier are like-charged. In the ultrafiltration step shown in Figure 32, the unbound protein is flushed away, in addition to the... 

High solids content sludge for example, waste material from the centrifuge or ultrafiltration steps. This material may require separate handling depending on its viscosity. Often, it is sufficiently liquid to be pumped and combined with the lower viscosity material for make-safe treatment. If not, separate treatment may be necessary, and for small quantities autoclaving or incineration may be adequate. 

The use of ultrafiltration steps as compared with conventional cheese making is shown in Fig. 10.26. Alternatively, conventionally produced whey can be concentrated by ultrafiltration and then added to the curd or milk can be soured with starter culture and/or rennet... 

First, an ultrafiltration membrane with a cut off of 5 kDa was used to remove salts and small molecular weight inq)urities. In a second ultrafiltration step using a 30 kDa mend rane die eluate was collected. In this step high molecular weight contaminant and endotoxins were removed. As in the procedure described above, the aqueous solution was treated alternately with cation and anion exchange resins in order to remove charged by-products. Finally, 1 was isolated by freeze drying and obtained as a white fluffy powder. 

The repeated use of the biocatalyst is often necessary to reduce the catalyst costs. Soluble enzymes can be easily recovered from the reaction mixture using an ultrafiltration step [18,96,97], thereby combining homogeneous catalysis wifli selective enzyme recovery and reuse. Ultrafiltration can be performed from a microliter to a multi cubic meter scale with commercially available ultrafiltration membranes and equipment. For first experi-... 

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