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The most important properties of a protein are determined by its primary structure, its amino acid sequence. However, protein features can be also modified by a large number of posttranslational modifications. These modifications can occur during or after the synthesis process, and glycosylation appears as the most common posttranslational modification. It is estimated that 50% of human proteins have some kind of glycosylation, which has a key role in maintaining the structure, stability, and function of the protein. Besides, glycostructures can also influence the pharmacokinetics and immunogenicity of the protein. Although the glycosylation process is a conserved mechanism that occurs in yeast, plants, and animals, several studies have demonstrated significant differences in the glycosylation pattern in recombinant proteins expressed in mammalian, yeast, and insect cells. Thus, currently, important efforts are being done to improve the systems for the expression of recombinant glycosylated proteins. Among the different mammalian cell lines used for the production of recombinant proteins, a significant difference in the glycosylation pattern that can alter the production and/or activity of the protein exists. In this context, human cell lines have emerged as a new alternative for the production of human therapeutic proteins, since they are able to produce recombinant proteins with posttranslational modifications similar to its natural counterpart and reduce potential immunogenic reactions against nonhuman epitopes. This chapter describes the steps necessary to produce a recombinant glycoprotein in a human cell line in small scale and also in bioreactors.

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The industrial production of recombinant proteins preferentially requires the generation of stable cell lines expressing proteins in a quick, relatively facile, and a reproducible manner. Different methods are used to insert exogenous DNA into the host cell, and choosing the appropriate producing cell is of paramount importance for the efficient production and quality of the recombinant protein. This review addresses the advances in recombinant protein production in mammalian cell lines, according to key patents from the last 30 years.

ObjectiveNowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO) and baby hamster kidney cells (BHK). Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K.MethodsThis study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones.ResultsThe retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes) was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293.ConclusionThe results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins.

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