Plasma fractions, preparation

Human blood plasma contains over 700 different proteins (qv) (1). Some of these are used in the treatment of illness and injury and form a set of pharmaceutical products that have become essential to modem medicine (Table 1). Preparation of these products is commonly referred to as blood plasma fractionation, an activity often regarded as a branch of medical technology, but which is actually a process industry engaged in the manufacture of speciaUst biopharmaceutical products derived from a natural biological feedstock (see Pharmaceuticals). 

Plasma fractionation is unusual in pharmaceutical manufacturing because it involves the processing of proteins and the preparation of multiple products from a single feedstock. A wide range of unit operations are utilized to accompHsh these tasks. They are Hsted in Table 3 some are common to a number of products and all must be closely integrated. The overall manufacturing operation can be represented as a set of individual product streams, each based on the processing of an intermediate product derived from a mainstream fractionation process (Fig. 1). 

A. General description Prolastin is prepared from pooled human plasma of normal donors it has a molecular weight of 52kDa. To reduce the potential risk of transmission of infectious agents, Prolastin has been heat-treated in solution at 60 + 0.5°C for not less than 10 hours. However, no procedure has been found to be totally effective in removing viral infectivity from plasma fractionation products. 

Plasma separation for preparing plasma fractionates should be performed at low temperatures to prevent bacterial contamination and protein denaturation. Sakai et al. [1989] found that organic polymer membranes, when used for low-temperature plasma... 

In the past, hemophiliacs were treated with transfusions of a concentrated plasma fraction containing factor VIII. This therapy carried the risk of infection. Indeed, many hemophiliacs contracted hepatitis and AIDS. A safer preparation of factor VIII was urgently needed. With the use of biochemical purification and recombinant DNA techniques, the gene for factor VIII was isolated and expressed in cells grown in culture. Recombinant factor VIII purified from these cells has largely replaced plasma concentrates in treating hemophilia. 

Simple systems (with a single defined additive) were produced with each of the following materials. Calf thymus DNA, polymerized, was obtained from Sigma. Protein sources were prepared in-house and subsequently dialyzed into low salt solutions. Human serum albumin and immunoglobulin G (IgG) were plasma-derived. Human immunoglobulin M (IgM) was produced by tissue culture fermentation and purified. A defined complex system consisted of both albumin and IgM together. An undefined complex system was set up with an intermediate material of Cohn plasma fractionation containing alpha-1 antitrypsin (alpha-1), albumin, and other contaminants. 

There are a number of in vitro and in vivo biotransformation techniques available to generate metabolites. The in vitro techniques include the use of subcellular fractions prepared from cells that mediate drug metabolism, intact cell-based systems, intact organs, and isolated expressed enzymes. In vivo methods involve the use of biological fluids (plasma, bile, urine, etc.) obtained from laboratory animals or humans dosed with the parent molecule. Microbial methods and biomimetic systems based on metalloporphyrin chemistry can also be used as bioreactors to produce metabolites. 

In this chapter, the preparation of synaptosomal plasma membranes using centrifugation techniques will be described in detail. The method here is based on that described previously by Kristjansson et al. (14) with only slight modifications. Section 3.1. outlines protocols for dissection and homogenization of the brain, indicating the parameters most important to obtain synaptosomal plasma membrane preparations of reproducibly high quality. Section 3.2. describes the subcellular fractionation procedure itself, and Section 3.3. outlines a protocol for assessment of yield of the synaptosomal plasma membranes employing a protein determination assay described by Bradford (15). 

The addition of water to a crude P2 fraction changes the osmotic pressure of the medium. The synaptosomes will easily lyse as a result of this osmotic shock, whereas the mitochondria and vesicles are largely unaffected. The lysis efficiency of the synaptosomes determines the yield of the synaptosomal plasma membrane preparation. 

All the following bilayer experiments were conducted in presence of 1% 1,2-dipalmitoyl (diC16) Ptdlns(4,5)P2, which resulted in higher stability of the planar lipid bilayers in comparison to the short chain diC8 PtdIns(4,5)P2. No menthol-activated channels were observed in presence of PtdIns(4,5)P2 on plasma membrane fractions from HEK-293 cells not expressing TRPM8, total 11 experiments were conducted from three different plasma membrane preparations (data not shown). 

Antibody preparation starts from the plasma pool prepared by removing the cellular components of blood. Cold ethanol is added in increments to precipitate fractions of the blood proteins, and the precipitate containing IgG antibodies is collected. This is further redissolved and purified by ultrafiltration, which also exchanges the buffer to the stabilizer formulation. Sometimes ion-exchange chromatography is used for further purification. Although the plasma is screened for viral contamination prior to pooling, all three purification techniques remove some virus. 

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