Blood plasma and serum are very useful samples for the detection, identification and quantitation of proteins associated with both health and disease. However, analysis of plasma and serum is a challenge because traces of interesting polypeptides and proteins can be dominated by the very high concentration of albumin present. Albumin may be depleted by adsorption to immunoaffinity columns or to columns containing dyes such as Cibacron Blue, or by ultrafiltration, but these methods are far from ideal. We describe a new peptide-based affinity medium which is effective for removing albumin and is very specific. The albumin-binding capacity is at least 14 mg per mL of gel. The material may be reused hundreds of times after a simple regeneration step involving NaOH, with full retention of specificity and capacity. The material was tested with human and monkey plasma and serum and rat serum, and has been used to deplete litre volumes of human plasma. The development of other peptide-based affinity media to deplete abundant proteins is briefly discussed.
In biphasic cultivations, the culture conditions are initially kept at an optimum for rapid cell growth and biomass accumulation. In the second phase, the culture is shifted to conditions ensuring maximum specific protein production and the protein quality required. The influence of specific culture parameters is cell line dependent and their impact on product quality needs to be investigated. In this study, a biphasic cultivation strategy for a Chinese hamster ovary (CHO) cell line expressing an erythropoietin fusion protein (Epo-Fc) was developed. Cultures were run in batch mode and after an initial growth phase, cultivation temperature and pH were shifted. Applying a DoE (Design of Experiments) approach, a fractional factorial design was used to systematically evaluate the influence of cultivation temperature and pH as well as their synergistic effect on cell growth as well as on recombinant protein production and aggregation. All three responses were influenced by the cultivation temperature. Additionally, an interaction between pH and temperature was found to be related to protein aggregation. Compared with the initial standard conditions of 37°C and pH 7.05, a parameter shift to low temperature and acidic pH resulted in a decrease in the aggregate fraction from 75% to less than 1%. Furthermore, the synergistic effect of temperature and pH substantially lowered the cell-specific rates of glucose and glutamine consumption as well as lactate and ammonium production. The optimized culture conditions also led to an increase of the cell-specific rates of recombinant Epo-Fc production, thus resulting in a more economic bioprocess.
The 2-aminoethyl glycoside of 0 -a-L-f u cop yr anos y 1-(1 + 2 ) -0 -p -Dgalactopyranosyl-( 1+3)-[ O-a-L-fucopyranosyl-( 1+4)]-2-acetamido-2-deoxy-p-Dglucopyranose (Lewis B tetrasaccharide) was synthesized on a large scale and acryloylated with acryloyl chloride. The obtained oligosaccharide 2-acrylamidoethyl glycoside was then copolymerized with acrylamide to form a water-soluble, high molecular weight polymer, suitable for use in adhesion inhibition studies with HeZicobacter pylori. Also synthesized were the corresponding derivatives of 0 -a-L-f ucop yr anosyl-( 1 -+ 2)-0-p-D-gal a c topyranosyl-(l+3)-2-acetamido-2-deoxy-p-D-glucopyranose and O-a-L-fucopyranosyl-( 1+2)-p-D-galactopyranose.
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