No abstract

The production of recombinant glycoprotein therapeutics requires characterization of glycosylation with respect to the lot-to-lot consistency. Here we introduce the ¿hypothetical N-glycan charge Z' as a parameter that allows to characterize the protein glycosylation in a simple, however, efficient manner. The hypothetical N-glycan charge of a given glycoprotein is deduced from the N-glycan mapping profile obtained via HPAE-PAD. In HPAEC, N-glycans are clearly separated according to their charge, i.e., their number of sialic acid residues, providing distinct regions for neutral structures as well as for the mono- di-, tri, and tetrasialylated N-glycans (Hermentin et al., 1992a). Z is defined as the sum of the products of the respective areas (A) in the asialo, monosialo, disialo, trisialo, tetrasialo, and pentasialo region, each multiplied by the corresponding charge: [formula: see text] Thus, a glycoprotein with mostly C4-4* structures will provide Z approximately equal to 400 (e.g., rhu EPO (CHO), Z = 361), a glycoprotein carrying largely C3-3* structures will amount to Z approximately equal to 300 (e.g., bovine fetuin, Z = 290), a glycoprotein with mostly C2-2* structures will have Z approximately equal to 200 (e.g., human serum transferrin, Z = 207, or human plasma AT III, Z = 180), and a glycoprotein carrying only high-mannose type or trunkated structures will provide Z approximately equal to 0 (e.g., bovine pancreas ribonuclease B, Z = 15, and hen ovomucoid, Z = 15, respectively). The determination of Z was validated in multiple repetitive experiments and proved to be highly accurate and reliable. Z may therefore be regarded as a new and characteristic parameter for protein N-glycosylation.

The oxygen supply of cell cultures with the aid of free gas bubbles is an efficient process strategy in pharmaceutical production. If the cell-damaging impact of gas bubbles is reduced, direct aeration becomes a practical solution with scale-up potential and comparatively high oxygen transfer rates. In this paper a microsparging aeration system made of porous ceramic was compared with bubble-free membrane aeration. The sparging system was used for the long-term cultivation of mammalian cells in 2- to 100-L scale bioreactors and produced bubble sizes of 100-500 microm in diameter. Using a scale of 2.5 and 30 L, a cell density of 2.6 x 10(6) cells/mL was attained. When a 100-L scale was used, a density of 1.1 x 10(6) cells/mL was achieved, whereas a comparable membrane-aerated system showed a cell density of 2.2 x 10(6) cells/mL. At relatively low agitation rates of less than 70 rpm in the sparged bioreactors, a homogeneous and constant oxygen concentration was kept in the medium. As a result of the different foam-forming tendency caused by the lower gas flow of the ceramic sparger compared to that of the standard aeration systems, we were able to develop an appropriate process control strategy. Furthermore, oxygen transfer measurements for the common stainless steel sparger and the ceramic sparger showed a 3-fold higher oxygen transfer coefficient for the ceramic sparger.

The increasing demand for products from mammalian cells has prompted the authors to develop a new type of bioreactor. Its significant features include the supply of oxygen, homogeneous distribution of microcarrier suspensions and process control. Media with high protein contents, required for mammalian cell cultures, tend to generate foam. This causes the flotation of solid particles. The reactor was equipped with a system of porous hydrophobic Accurel hollow fibre membranes in order to prevent the formation of bubbles. The membrane is coiled in the form of a basket, or fixed on several carriers. If the liquid pressure is higher than that of the gas phase inside the membrane, a bubble-free oxygen supply to the culture broth can be achieved. The problem of axial mixing of microcarrier suspensions was solved by the use of a spiral agitator, attached underneath the aeration system at the bottom of the reactor. The combined aeration and mixing system, which is driven by an eccentric motor, undergoes a tumbling motion. Sufficiently homogeneous suspensions are produced in this system at low membrane velocities, i.e. in presence of low shear forces.

A vortex chamber for continuous adsorption of the antibiotic Myxovirescin A on XAD resins was developed. In this paper the design and use of the vortex chamber in an external bypass of a continuous process is described. Compared with the normal continuous process, the specific production rate of the antibiotic is four to five times higher when the antibiotic is continuously adsorbed. A semicontinuous process could be performed by using two chambers for adsorption and regeneration alternatively.