Protein products, purification

Protein processing systems for concentration and/or purification offer a very broad range of application which may be tailored to a given protein product purification scheme. The particular apparatus and operating parameters employed must be selected and refined to the unique requirements of any given protein as well as those of the broth in which it occurs. 

PVDF-based microporous filters are in use at wineries, dairies, and electrocoating plants, as well as in water purification, biochemistry, and medical devices. Recently developed nanoselective filtration using PVDF membranes is 10 times more effective than conventional ultrafiltration (UF) for removing vimses from protein products of human or animal cell fermentations (218). PVDF protein-sequencing membranes are suitable for electroblotting procedures in protein research, or for analyzing the phosphoamino content in proteins under acidic and basic conditions or in solvents (219). 

Purification. Enzyme purity, expressed in terms of the percent active enzyme protein of total protein, is primarily achieved by the strain selection and fermentation method. In some cases, however, removal of nonactive protein by purification is necessary. The key purification method is selective precipitation of the product or impurities by addition of salt, eg, sodium sulfate, or solvent, eg, ethanol or acetone by heat denaturation or by isoelectric precipitation, ie, pH adjustments. Methods have been introduced to produce crystalline enzyme preparations (24). 

From an economic point of view, the number of sequential operations necessary to achieve the desired purity of a protein product contributes significantly to the overall cost of the downstream process. This is due to the capital investment and amount of consumables needed for each step as well as the individual time required for each operation. Additionally, the overall yield of the purification is reduced with each additional process step as a result of inherent handling losses of product and/or product activity. It has been estimated that the overall cost of the downstream process is closely correlated with the number of purification steps involved and that cost may account for up to 80% of the final process investment.26... 

Downstream purification and isolation of proteins and biomolecules is often the most expensive and challenging aspect of their production [91]. Many of the downstream separation processes used by industry today, e.g., ultraliltration, chromatography, and centrifugation, are slow, inherently batch, nonspecific, expensive, overconsume energy, and generate wastes, particularly for downstream product purification, an important cate-... 

Bio-Research Products Inc., was founded in 1975, and specialized in the isolation, purification and characterization of enzymes and proteins. The company is well known for its production of wheat germ phosphoenolpyruvate carboxylase (PEPC). Currently, it is involved in finished goods and raw material production, through a biomedical contract. Bio-Research Products runs custom services on enzymes, proteins production, diagnostic assays, and other goods for industry, governments, or academia. Bio-Research Products, Inc. also markets a number of enzymes and associated products, and carries out custom synthesis projects. 

Extensive research has been carried out into the molecular aspects of foreign protein production in whole plants to enhance the yield, quality and stability of the product and to facilitate protein separation and purification from the biomass [3, 6, 9], In contrast, comparatively little research has been undertaken to investigate the... 

Biopharm production can be divided into upstream and downstream processing (Figure 5.5). Upstream processing refers to the initial fermentation process that results in the initial generation of product, i.e. the product biosynthesis phase. Downstream processing refers to the actual purification of the protein product and generation of finished product format (i.e. filling into its final product containers,... 

Nucleic acids may also be removed by treatment with nucleases, which catalyse the enzymatic degradation of these biomolecules. Indeed, nuclease treatment is quickly becoming the most popular method of nucleic acid removal during protein purification. This treatment is efficient, inexpensive and, unlike many of the chemical precipitants used, nuclease preparations themselves are innocuous and do not compromise the final protein product. 

In addition to the direct absorbance methods, colorimetric methods are suited for relatively pure proteins as purification progresses. They are accurate if calibrated from a standard curve of the test protein reference sample and fast if automated. However, they are not as simple to perform as direct absorbance methods. Hence they are not as suitable for production as direct absorbance methods. The relative simplicity of colorimetric methods makes them more suited to automated formulation and stability studies and total-protein assays of complex mixtures. Microtiter plate versions of colorimetric assays allow for automation and consumption of relatively small sample sizes while requiring little specialized equipment or training. 

The MS techniques described previously for characterization of the final recombinant protein product can be applied at all stages during process development. MS might be used upstream to define clone selection, processing format, and purification steps, and downstream to characterize the final product, ascertain lotto-lot reproducibility, determine stability, and define the formulation of biopharmaceutical molecules. Presented here are some examples found either in the literature or from our own experience in which MS has been found to be a useful or necessary tool. Potential limitations of MS methods are discussed, and when appropriate, other analytical methods are mentioned that can be alternatives to MS and are also efficient tools for biopharmaceutical development. 

The ELISA can be used for identification and quantitation of the protein product (biopharmaceutical) of interest throughout the development, production, and manufacturing process. For example, in the initial development phase, ELISAs can aid in the selection of the best cell line. In the early manufacturing steps, it can be used to identify the appropriate product-containing pools or fractions in process to be subjected to further purification. Because of the selectivity of ELISA, it is a suitable tool to select out the protein of interest from complex protein mixtures, such as cell culture fermentation media or product pools in early steps of protein recovery as well as downstream processing. Even complex mixtures do not require much sample preparation. It is important to determine... 

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