Capture Step

Finally, there are custom two-step quantitation methods such as chromatography or ELISA that require a capture step for isolating the protein and then a quantitation step based on a standard curve of the purified target protein. The preliminary capture step may also concentrate the protein for increased sensitivity. These techniques are typically not available in a commercial kit form and may require extensive method development. They are more labor intensive and complex than the colorimetric or absorbance-based assays. In addition, recovery of the protein from and reproducibility of the capture step complicate validation. Despite these disadvantages, the custom two-step quantitation methods are essential in situations requiring protein specificity. 

After the initial isolation or capture step, the product is in a more workable... 

Case (L). It is logical to assume here that the further oxidation of an AB unit occurs through consecutive hole-capture steps, so that reaction (5) i... 

The results of this kinetic analysis have been included in Table I. It can be seen that, if both the anodic decomposition of the semiconductor and the anodic oxidation of the competing reactant would occur by irreversible hole-capture steps ((L)(H)(I) or (M)(H)(1)), as was hitherto generally accepted, the stabilization should be independent of light intensity, in contradiction with the results described above. The mechanism in which the reducing agent reacts by donating an electron to a localized surface hole ((L)(X)) leads to an expression in which s is a function of the variable (y/j) only. The three other mechanisms considered lead to the relationship of the type (18), in which s is a function of (y2/j). 

The photoanodic dissolution of n-silicon in acidic fluoride media provides an example of the complexity of multistep photoelectrochemical reactions [33, 34]. The reaction requires the transfer of four electrons, but it is clear that not all of the steps involve photogenerated holes because the photocurrent quantum efficiency is between 2 and 4. The explanation of the high quantum efficiencies is that the initial hole capture step can be followed by a series of steps in which intermediates with low electron affinity inject electrons into the conduction band. These intermediates can be assigned nominal oxidation states as shown in the following scheme. 

Fig. 8.16. Reaction scheme for photoanodic dissolution of silicon in low intensity limit illustrating the competition between hole capture steps (rate constants k to k ) and electron injection steps (rate constants k to k,). The nominal valence states of the silicon intermediates are indicated. The final product Si(IV) is the soluble hexafluorosilicate species.

Cap the tube with the Centricon retentate cup, then backspin the retentate (about 40 /zl) into the cup in a swinging-bucket rotor by bringing the speed to 1000 rpm and then braking immediately. The retentate contains the biotinylated dsDNA for use in the capture step below. 

Centricon tubes may be rinsed and reused for the same preparations later in the procedure. Fill the top of the tube (Centricon concentrator) with sterile water, cap with the retentate cup, and shake gently. Repeat a total of 3 times. The tube need not be dried, but the retentate cup must be pipetted dry before reusing it to capture the final ssDNA, so that it will not be diluted. Rinsing can conveniently be done during incubation in the capture step, below. 

TABLE 4 Capture-Step Comparison for Processing 40 Liters of C. diphtheria Culture for Production of CRM 9 Diphtheria Toxin... 

TABLE 6 Capture Step Comparison for Processing 4.5 Kg E. Co/i Cells for Production of Exotoxin A 553D... 

Most generally during elution, the liquid flow is reversed and the resin bed is therefore packed. In contrast to conventional complex initial feedstock treatments, fluidized-bed processes combine clarification, expressed product specific capture, and concentration into a single step. Residence time distribution analysis showed a small degree of axial dispersion and the generation of a few dozen theoretical plates that are enough for a good efficiency of the capture step. The efficiency of the separation is, however, dependent on the particle size of the solid phase material. 

Although most of the principles of antibody capture in packed bed mode are applicable to fluidized-bed technology, today applications are hindered by the very limited availability of sorbents specifically designed for fluidized beds. For instance, the potential applicability of a protein A affinity capture in a fluid bed seems very attractive and may become a useful operation with appropriate dense solid phases. Collected fractions rich in antibody obtained at the issue of a capture step in fluidized-bed mode can be further repurified or polished by other types of packed-bed chromatography, as described in Section V.I. 

Ion-exchange chromatography can be used as a capture step at the initial stage of antibody separation and can then be followed be one or two additional chromatography steps to remove other protein impurities. 

As with any other classical approach in downstream processing of proteins, the process is split into three steps a capture step, followed by a separation step, and finally by a polishing step to remove residual impurities originating from leached compounds of the sorbent and/or fragments and aggregates.72... 

A desirable attribute of a capture step is the high selectivity for the antibody, to eliminate bulk impurities during the early phase and, moreover, to reduce water content, resulting in a substantial concentration of the product. The requirement of such a sorbent to capture the antibody at high capacity even from feedstocks where the antibody is present at very low... 

Frequently antibodies are produced by cell culture in protein-free media. Although the expression is low, their purification becomes a simple task. When classical cation-exchangers with S groups are used for the capture step, the ionic strength of the cell culture supernatant must be decreased to at least 5 mS/cm and the pH adjusted to 5.2-5.5. Note, however, that the required... 

The goal in the capture step is to concentrate and isolate a protein product. This step is usu-... 

Although the capture step dramatically enriches a targeted protein and removes some impurities, bulk impurities such as host cell protein, DNA, endotoxin, virus, and leaching ligand remain in the eluted pool. Additional procedures are needed to eliminate these impurities. 

Depending on the required level of purity, the step used for removing impurities can be a single or multiple-step chromatography operation. Table 32.8 summarizes chromatographic methods that can be used to remove impurities following the capture step.76-78 80... 

For example, treatment of acrylate and crotonate ephedrine resins 150 and 151, with cyclohexanecarboxaldehyde 149, employing Sml2 in THF with f-butanol as a proton source, gave 152 and 153 respectively, in moderate yield and good to high enantiomeric excess (Scheme 34). The process can be considered an example of an asymmetric catch-release process, where a substrate immobilized using a chiral support captures a reactive intermediate, in this case a ketyl radical anion, from solution [23]. The chiral support controls the asymmetry of the capture step and leads to a diastereomeric, resin-bound intermediate that breaks down to release a non-racemic product. 

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