Plant-specific N-glycosylation can represent an important limitation for the use of recombinant glycoproteins of mammalian origin produced by transgenic plants. Comparison of plant and mammalian N-glycan biosynthesis indicates that ␤1,4-galactosyltransferase is the most important enzyme that is missing for conversion of typical plant N-glycans into mammalian-like N-glycans. Here, the stable expression of human ␤1,4-galactosyltransferase in tobacco plants is described. Proteins isolated from transgenic tobacco plants expressing the mammalian enzyme bear N-glycans, of which about 15% exhibit terminal ␤1,4-galactose residues in addition to the specific plant N-glycan epitopes. The results indicate that the human enzyme is fully functional and localizes correctly in the Golgi apparatus. Despite the fact that through the modified glycosylation machinery numerous proteins have acquired unusual N-glycans with terminal ␤1,4-galactose residues, no obvious changes in the physiology of the transgenic plants are observed, and the feature is inheritable. The crossing of a tobacco plant expressing human ␤1,4-galactosyltransferase with a plant expressing the heavy and light chains of a mouse antibody results in the expression of a plantibody that exhibits partially galactosylated N-glycans (30%), which is approximately as abundant as when the same antibody is produced by hybridoma cells. These results are a major step in the in planta engineering of the N-glycosylation of recombinant antibodies.

Plants are regarded as a promising system for the production of heterologous proteins. However, little is known about the influence of plant physiology and plant development on the yield and quality of the heterologous proteins produced in plants. To investigate this, tobacco (Nicotiana tabacum cv Samsun NN) was transformed with a single construct that contained behind constitutive promotors the light- and heavy-chain genes of a mouse antibody. The in planta stability of the antibody was analyzed in transgenic plants that were grown under high and low irradiation at 15 degrees C and 25 degrees C. High-light conditions favored the production of biomass, of total soluble protein, and of antibody. The plants grown at 25 degrees C developed faster and contained less antibody per amount of leaf tissue than the plants grown at 15 degrees C. Both endogenous protein and antibody content showed a strong decline during leaf development. The heavy chains of the antibody underwent in planta degradation via relatively stable fragments. In vitro incubations of purified plantibody with leaf extracts of wild-type tobacco indicated the involvement of acidic proteases. It is interesting that the same antibody produced by mouse hybridoma cells exhibited higher stability in this in vitro assay. This may be explained by the assumption that the plant type of N-glycosylation contributes less to the stability of the antibody than the mouse-type of N-glycosylation. The results of this study indicate that proteolytic degradation during plant development can be an important factor affecting yield and homogeneity of heterologous protein produced by transgenic plants.

Plants are regarded as a promising system for the production of heterologous proteins. However, little is known about the influence of plant development and growth conditions on N-linked glycosylation. To investigate this, transgenic tobacco (Nicotiana tabacum cv Samsun NN) plants expressing a mouse immunoglobulin G antibody (MGR48) were grown in climate rooms under four different climate conditions, i.e. at 15°C and 25°C and at either low or high light conditions. N-glycans on plantibodies and soluble endogenous proteins were analyzed with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). Antibodies isolated from young leaves have a relatively high amount of highmannose glycans compared with antibodies from older leaves, which contain more terminal N-acetylglucosamine. Senescence was shown to affect the glycosylation profile of endogenous proteins. The relative amount of N-glycans without terminal N-acetylglucosamine increased with leaf age. Major differences were observed between glycan structures on endogenous proteins versus those on antibodies, probably to be attributed to their subcellular localization. The relatively high percentage of antibody N-glycan lacking both xylose and fucose is interesting.

The stability of an antibiotic is a very important characteristic, especially in the field of antibiotic residue analysis. During method development or validation, the stability of the antibiotic has to be demonstrated no matter if the method is used for screening, confirmation, qualitative or quantitative analysis. A procedure for testing the stability of antibiotics in solutions and food samples using LC-MS/MS is described. The procedure is based on the assumption that the antibiotics are stable when stored at -70 °C. Representative solutions or spiked samples containing the antibiotic were stored at the temperature to be tested (-18 or 4 °C) and at -70 °C. After a selected storing time samples were moved from the chosen storage temperature to -70 °C. At the end of the study, all samples--per class of antibiotic--were analysed in one batch. By applying statistical models, it was finally concluded in which circumstances the antibiotic is stable. The stability of 60 antibiotics belonging to the classes of tetracyclines, sulphonamides, quinolones, penicillins, macrolides and aminoglycosides were tested. The stability of solutions containing tetracyclines and penicillins is only guaranteed for 3 months while stored at -18 °C. Solutions of all other antibiotics tested are stable for at least 6 or 12 months when stored at 4 °C. In muscle tissue stored at -18 °C no severe degradation of the tested antibiotics was observed, with the exception of the penicillins. The stability data reported here are useful as a reference for laboratories carrying out validation studies of analytical methods for antibiotic (residue) detection. The data should save the time needed for long-term stability testing of solutions and samples.