Posttranslational Modifications Of Recombinant Proteins In Animal Cell Lines

Recombinant DNA technology opened the field for the production of an unlimited variety of clinically relevant therapeutics in various host cell lines. The majority of proteins applied undergo more or less extensive posttranslational modifications in their natural counterparts, including mainly glycosylation, but also phosporylation, carboxyla-tion, and signal-peptide processing. These modifications affect the biological activity of the heterologous protein by being involved in solubility, pharmacokinetics, antigeneic-ity, circulating half-life, secretion, protein folding, oligomer assembly, and susceptibility to proteolytic attack (79,80).

Choosing a host cell line able to fulfill these criteria is, therefore, important. Regulatory authorities such as the FDA demand a comprehensive analysis of carbohydrate structures and consistency in the production process. Because prokaryotes are not capable of performing posttrans-lational modifications observed in eukaryotic cells, they cannot be utilized for the production of recombinant proteins, where bioactivity relies heavily on correct glyco-sylation, for example. These unique advantages of animal cell technology inspired research directed toward overcoming process or regulatory difficulties.

Research has increasingly revealed already substantial differences in the glycosylation of recombinant proteins using several host cell lines or even transgenic animals. Variations can also stem from different environmental factors, such as media or bioreactor configuration, or the internal cell status at the time of production (80).

A few examples illustrate the importance of choosing a host system able to produce heterologous proteins with appropriate characteristics for clinical applications. Although CHO and BHK lack certain functional glycosyl-transferases found in human cells (80), they still represent the most favorable host cell lines for large-scale manufacture of protein-based pharmaceuticals such as erythropoi-etin or tissue-type plasminogen activator (t-PA). Studies on product consistency revealed an independence of gly-cosylation pattern by applying varied process parameters (8) and showed that glycosylation was identical to natural sources (81). Constant improvement in the methods available for the analysis of carbohydrate structures allow a detailed pattern identification (79) and have been used to detect species-specific oligosaccharide variations of recombinant human IFN-y produced in CHO and SF cells and transgenic mice (82).

The glycosylation status of recombinant proteins synthesized in baculovirus-infected insect cells can vary quite considerably, as shown by Ogonah et al. (55). Estigmena cells are capable of producing complex oligosaccharides, whereas the still more frequently used SF 9 cell line is restricted to the performance of only simple glycosylation reactions.

These findings demonstrate the importance of investigating and identifying an animal cell system best suited for the production of therapeutics. Even shortcomings, such as the lack of glycosyltransferases in CHO cells, can be overcome by transfection of appropriate enzymes (80), indicating the potential for future processes.

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