Recombinant proteins product content

Separation from culture media or broth is the primary step in collecting the product found either in cells (sohd) or medium (liquid). This initial separation step is engineered based on cell size and density differences between solid and liquid (Table 4.10). In the case where the recombinant product is localized in the intracellular content such as the cytoplasm or inclusion bodies, which are highly insoluble particles found in bacteria, the cells are hrst isolated from the medium and then disrupted to collect the recombinant protein fraction. A number of cell disruption techniques have been developed to facilitate this step, and some are listed in Table 4.11. 

Methods to determine the potential biological activity of products obtained through recombinant DNA techniques are of fundamental importance. Despite the existence of numerous physicochemical techniques to characterize the protein product structure and the presence of contaminants, they provide little, if any, information about its biological potency. A bioassay is defined as a functional test, and no physicochemical test can measure the function. However, for some peptide hormones, which are less complex in structure than most cytokines, well defined physicochemical tests may be used to estimate biological activity for instance, the capillary electrophoresis analysis of a protein s isoform content if the specific activity of each one is known. 

The accumulation of recombinant proteins in an insoluble form offers even more significant benefits for downstream processing. Typically, the desired protein product represents at least 40 to 50 % of the total polypeptide content of inclusion bodies. The... 

Where the product is secreted, it should form the major proteinaceous component of the harvest broth. The best time to harvest wiU depend on the dynamics of protein expression, secretion and stability, but in general it is beneficial to seek an appropriate harvesting window in the production cycle where product expression and accumulation are maximized but degradation is Hmited. For example, in batch or fed-batch processes, peak production of the recombinant protein often occurs in the exponential phase, and begins to de-cHne at the point where the cell mass and total protein content of the culture reach... 

Compounding these ambiguities are recent observations we have made monitoring sialylation, by two different methods, of a recombinant immunoadhesion molecule, GPl-IgG [29], over the course of a CHO cell culture under high productivity conditions in which sodium butyrate is added as an inducer of protein synthesis (Figure 2). A 10 1 bioreactor culture of CHO cells secreting GPl-IgG was established and samples of the culture were withdrawn every 24 hr. The recombinant protein was isolated, the titers of the protein quantified, and the sialic acid content determined by chemical analysis. With these data the sialic acid levels of the newly synthesized protein could also be calculated for each 24 hr interval. 

Figure 2. Metabolic sialylation profile of an immunoadhesion molecule, GPl-IgG under high productivity conditions. A 10 1 bioreactor culture of CHO cells expressing GPl-IgG was sampled every 24hr. The recombinant protein was isolated and sialic acid content determined by chemical analysis ( ). Concomitantly, samples of the culture were incubated with H-ManNAc. The amount of metabolically incorporated sialic acid was determined on the recombinant protein as radioactive counts ( ). On day 3 of the culture sodium butyrate was added to be 12 mM to stimulate protein production, Up to day 8, the viability of the culture was 80% or greater. Modified from [29],...

The PEG could stabilize proteins by two different temperature-dependent mechanisms. At lower temperatures, it is preferentially excluded from the protein surface but has been shown to interact with the unfolded form of the protein at higher temperatures, given its amphipathic nature (57). Thus, at lower temperatures, it may protect proteins via the mechanism of preferential exclusion, but at higher temperatures possibly by reducing the number of productive collisions between unfolded molecules. PEG is also a cryoprotectant and has been employed in Recombinate, a lyophilized formulation of recombinant Antihemophilic Factor, which utilizes PEG 3350 at a concentration of 1.5mg/mL. The low-molecular weight liquid PEGs (PEG 300-600) can be contaminated with peroxides and cause protein oxidation. If used, the peroxide content in the raw material must be minimized and controlled throughout its shelf life. The same holds true for polysorbates (discussed below). 

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