Chromatographic Steps

No Technique used X-fold increase Reference Source 

Purification step Nature of chromatography Specific activity (Umg- ) % Activity recovery Purification fold 

Hydrophobic-interaction chromatography (HIC) and affinity chromatography (AFC) [29-31] have been used in lipase purification. The ligands are very specific and each ligand is only applicable for the separation of the lipase from a certain source. Polypropylene glycol is reported to be a suitable ligand for the fractionation of Chromohacterium viscosum lipase [29]. 

Lipases are interphase-active enzymes with hydrophobic domains. The hydro-phobic surface (loop) on lipase is thought to enable lipophilic interfacial binding with substrate molecules that actually induces the conformational changes in lipases. The open conformation will provide substrate with access to the active site, and vice versa. In certain types of lipases, the movements of a short a-hehcal hydrophobic loop in the lipase structure cause a conformational change that exposes the active sites to the substrate. This movement also increases the nonpolarity of the surface surrounding the catalytic site [30, 32, 34, 35]. Obviously, the hydrophobic surface plays an important role in the activity of lipase as an enzyme. 

Thermophilic bacteria, and their possible practical use, have attracted much interest, as is reflected in the research available for the purificahon of thermally stable lipases from such bacteria [9, 13, 15, 19, 45, 46]. The microbial sources give extremely low purification levels after the precipitahon steps. Sugihara et al. [15] attribute this to a viscous material excreting into the culture fluid that made the salting out of the enzyme incomplete. However, ion-exchange chromatography followed by gel filtration chromatography results in about a 200-fold purification of hpase. 

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Because temperature shifts may also influence the packing quality, the temperature should not be changed during the chromatographic step and the packing of the column should be done at the operation temperature. To prevent the denaturation of sensitive proteins, the chromatography is carried out in a cold chamber (or cabinet). For this purpose the column packing has to be performed at the same ambient temperature (store the gel before use at the same temperature ). 

The purification of value-added pharmaceuticals in the past required multiple chromatographic steps for batch purification processes. The design and optimization of these processes were often cumbersome and the operations were fundamentally complex. Individual batch processes requires optimization between chromatographic efficiency and enantioselectivity, which results in major economic ramifications. An additional problem was the extremely short time for development of the purification process. Commercial constraints demand that the time interval between non-optimized laboratory bench purification and the first process-scale production for clinical trials are kept to a minimum. Therefore, rapid process design and optimization methods based on computer aided simulation of an SMB process will assist at this stage. 

A. tubingensis strain NW756 was cultivated for 55 h at 30 C in minimal medium according to Pontecorvo et al [1] supplemented with yeast extract (2g/l) and 10 g/1 endo-PG II digested polygalacturonic acid (PGA). Complete hydrolysis of PGA by endo-PG II resulted in a mixture of mono, di and trigalacturonate [2]. Four subsequent colunm chromatographic steps were used for purification of the enzyme for which the data are summarized in Table 1. 

Finally, the integration of biochemical or biosensor methods with conventional chromatographic analyses should not be overlooked. For example, the use of im-munoaffinity columns prior to chemiluminescence or the use of biosensor detection systems following the chromatographic step may provide useful solutions to speciflc analytical needs. 

Retrospective validation uses historical information gathered in actual process runs to evaluate the process. For example, batch records can provide extensive data on column performance and analytical data of fractions and final product can provide valuable information on the efficiency of the chromatographic steps in removing contaminants. Chapman67 cautions that while retrospective validation is a valid and valuable approach, it is not meant to be retroactive — validation must be done before product is released to market. 

SFE-GC is an attractive approach to coupling the extraction, concentration and chromatographic steps for the analysis of samples containing analytes that can be analysed using capillary GC. Often it is difficult to identify all the components which are extracted from samples by FID alone. This is a particular problem when the sample history and/or the identity of the compounds of interest are not known. When SFE-GC is combined to powerful spectroscopic detectors, unique data can be obtained, allowing their use as routine tools in the analytical laboratory. For positive identification of components of interest, multihyphenated techniques such as SFE-GC-AED, SFE-GC-MS, SFE-GC-FUR-MS are employed [46]. 

Applications If an extract needs further cleanup, it is possible to couple it with multidimensional chromatographic techniques such as LC-LC or LC-GC. The first chromatographic step can then be used for the on-line cleanup and concentration of the extract, and the second one for the final separation. Large-volume, on-column injection (LVI-COC) is particularly useful for coupled LC-GC in which 100-350 xL fractions of eluent from the NPLC cleanup separation step are transferred on-line to the GC column. For example, on-line removal of high-MW interfering material, such as polymers from a polymer/additive dissolution, can be achieved easily by using SEC before the fraction containing additives is transferred to the GC. 

Alternatives to off- and on-line chromatography are desirable in order to avoid the time-consuming chromatographic step, and are imperative for polymer-bound additive functionalities. 

Chong, S., Montello, G.E., Zhang, A., Cantor, E.J., Liao, W., Xu, M.-Q., and Benner, J. (1998) Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucl. Acids Res. 26, 5109-5115. 

Liquid chromatography is the core preparative technique in protein purification, and all supplementary procedures like extraction, centrifugation and filtration, ultimately serve to condition the protein solution for chromatography. A series of chromatographic steps, usually termed capture, intermediate purification and polishing, mak-... 

The greatest area of applications of this type of ECL has been in the analysis of pharmaceutical compounds with amine functionality. The reader is directed toward the previously mentioned review articles and Table 1 for further details [12, 14-16], Many methods have also been successfully applied to real samples in the form of body fluids or pharmaceutical preparations, although sample pretreatment such as deproteinization, centrifugation, and neutralization followed by a chromatographic step to remove interfering species is often required. Limits of detection are typically in the range 10-9—10 12 M. Figure 4 shows examples of some classes of pharmaceutical compounds that have been determined by Ru(bpy)32+ ECL. 

The physicochemical and other properties of any newly identified drug must be extensively characterized prior to its entry into clinical trials. As the vast bulk of biopharmaceuticals are proteins, a summary overview of the approach taken to initial characterization of these biomolecules is presented. A prerequisite to such characterization is initial purification of the protein. Purification to homogeneity usually requires a combination of three or more high-resolution chromatographic steps (Chapter 6). The purification protocol is designed carefully, as it usually forms the basis of subsequent pilot- and process-scale purification systems. The purified product is then subjected to a battery of tests that aim to characterize it fully. Moreover, once these characteristics have been defined, they form the basis of many of the QC identity tests routinely performed on the product during its subsequent commercial manufacture. As these identity tests are discussed in detail in Chapter 7, only an abbreviated overview is presented here, in the form of Figure 4.5. 

Most of the chromatographic steps undertaken during downstream processing are specifically included to separate the protein of interest from additional contaminant proteins. This task is not an insubstantial one, particularly if the recombinant protein is expressed intracellularly. 

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