Protein purification homogenization

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. 

Upon completion of the homogenization step, cellular debris and any remaining intact cells can be removed by centrifugation or by microfiltration. As mentioned previously, these techniques are also used to remove whole cells from the medium during the initial stages of extracellular protein purification. 

Claims to products defined by a particular state of purity may cause difficulties at the European Patent Office where it has been held that a known product does not necessarily acquire novelty merely by virtue of the fact that it is prepared in a purer form However, if the claim is directed to product defined in terms of a technical feature e g. specific activity not present in the less pure known product, the potential difficulty may be overcome. The situation may be different in the USA where it has been held that purification of a protein to homogeneity was enough to distinguish a claim over a prior disclosure of only unpurified mixtures of native protein. 

To some individuals, especially those who recall their experiences in organic chemistry laboratory, the ultimate step in purification of a molecule is crystallization. The desire to obtain crystalline protein has long been strong and many proteins have been crystallized. However, there is a common misconception that the ability to form crystals of a protein ensures that the protein is homogeneous. For many reasons (entrapment of contaminants within crystals, aggregation of protein molecules, etc.), the ability to crystallize a protein should not be used as a criterion of purity. The interest in pro-... 

L Hall and P Campbell, in Essays in Biochemistry, Vol 22, R. Marshall and K Tip-ton, Editors (1988), Academic Press (London), pp 1-26. A review on a-lactalbumin E Harris and S. Angal, Editors, Protein Purification Applications A Practical Approach, Vol 2 (1990), IRL Press (Oxford) An excellent book for undergraduates B. Johnson, The Scientist, November 9, 1998 A review of cell homogenizers A Lehmnger, D. Nelson, and M Cox, Principles of Biochemistry, 3rd ed (1999), Worth Publishers (New York), pp 130-133 Working with proteins. 

Acid phosphatase of S. aureus PS 55 is eluted from the cell surface by 1.0 M KC1 at pH 8.5. Gel filtration of this material gave a 44-fold purification. The protein seems homogeneous by gel filtration, starch block electrophoresis, and analytical ultracentrifugation with the weight of approximately 58,000 (12a). 

The detailed study of enzyme mechanisms requires the use of purified if not homogeneous enzymes. This experiment presents three procedures commonly used in protein purification ammonium sulfate precipitation, heat denaturation, and ion-exchange chromatography. Although the purification procedure outlined in this experiment is useful in the isolation of glutamate-oxaloacetate transaminase (GOT), the same techniques can be modified to aid in the purification of many other proteins of interest. 

HPLC can be useful for the purification of proteins to homogeneity. It can also be useful as an analytical tool to monitor the purification of proteins using other more preparative procedures. 

It is the intention of this article to survey the literature on hemicellulases from 1950 to 1973, and to deal, in detail, only with those enzyme preparations that have, by the usual criteria of purity employed in protein purification procedures, been shown to be homogeneous. The article will, therefore, exclude most of the work published on hemicellulases in which crude or partially purified enzyme prepara-... 

When purified enzymes are used, basically the same requirements have to be met. The purification may cause additional costs, but contrary to a biochemical characterization it is not necessary to purify the protein to homogeneity. On the contrary, the remaining protein content in a partly purified enzyme may increase its stability. The only requirement is to have a functional pure enzyme, meaning that activities... 

Differential Centrifugation The most common initial step in protein purification is the separation of soluble proteins from Insoluble cellular material by differential centrifugation. A starting mixture, commonly a cell homogenate, is poured into a tube and spun at a rotor speed and for a period of time that forces cell organelles such as nuclei to collect as a pellet at the bottom the soluble proteins remain in the supernatant (Figure 3-3la). The supernatant fraction then is poured off and can be subjected to other purification methods to separate the many different proteins that it contains. 

Removal of cell debris, DNA and proteases, constitute the major problems faced during the large-scale isolation of intracellular proteins. This has emphasized the need for rapid prodedures for protein purification. The fundamental studies on separations in aqueous two-phase systems suggest that it is possible to spontaneously partition the protein of interest into one of the phases by selecting the suitable conditions. The method is very efficient and convenient when set up. However, it may be very laborious to find the proper conditions for a favourable partitioning. Furthermore, such a system may be influenced by variations in parameters such as composition of the cell homogenate. Several examples on large scale isolation of proteins have been reported. Some of these are listed in Table II. 

Recently, another variation on this theme was presented. By introducing PEG-modified affinity sorbents (Sepharose beads carrying the immobilized ligand) a new process configuration for protein purification was achieved as shown in Figure 2 (32). The beads were exposed to the cell homogenate before the phase components were added. After phase separation the particles were recovered from the top phase, as a layer on top of the interface. The beads were collected and transferred to a column, where they were washed free of the phase polymers, and then the elution was carried out according to conventional procedures. 

Most reported applications have been for protein purification (28-30). One example (31) was for direct recovery of Annexin V, an intracellular recombinant protein, from E. coli homogenate, using the Streamline DEAE adsorbent (Pharmacia). The procedure was developed in a laboratory-scale column (50 mm diameter by 1000 mm length) and then successfully scaled to a pilot-plant column (200 mm diameter by 950 mm length). The yield was 95% for both laboratory and pilot-plant runs and achieved a threefold reduction in volume compared to the homogenate. 

V3 does not elute until a concentration of 300 mM of KCl is reached. By this purification process, it was possible to increase the specific activity of V 3 to 20 units per milligram of protein. A homogenous protein is therefore present. VI, V2 were purified to the point of homogeneity, in the same manner. 

Protein purification and antibody preparation. FNR from A. variabilis was purified to homogeneity using a procedure based on the use of... 

PURIFICATION OF HEAT SHOCK ACTIVATOR PROTEIN TO HOMOGENEITY... 

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