ReviewHuman cells: New platform for recombinant therapeutic protein production
Highlights
► Development of novel expression systems to face the therapeutics demands is necessary. ► Human cell lines are a powerful alternative for the production of therapeutic proteins. ► Murine cells do not possess the “machinery” required for human-type glycosylation. ► Non-human glycosylation pattern may be highly immunogenic. ► Proteins with murine glycosylation pattern may be rapidly cleared from circulation.
Introduction
Therapeutic proteins were initially extracted from human tissue or blood; for example, blood clotting factors and human serum albumin from plasma, insulin from pancreas, and glucocerebrosidase from placenta. However, the protein extraction from tissue (animal or human) has several disadvantages such as not having the biological material available in the quantity required for an industrial production and it can be contaminated by pathogens (viruses and/or prions contamination). Even when a protein could be obtained from human tissue in sufficient quantities, it can be much safer to use genetically engineered-derived products. In addition to safety issues, a major benefit of recombinant proteins is that they serve as a platform for the development of more advanced products that are engineered for an improved therapeutic profile, such as enhanced safety, lower immunogenicity, increased half-life and improved bioavailability [1].
Recombinant therapeutic proteins have been traditionally produced in mammalian, insect cells, bacteria and yeast. Mammalian cells are the preferred expression systems to produce complex glycosylated proteins. Among the 27 lasted FDA approved biopharmaceuticals (2008–2011), 18 are proteins derived from cultivated cells, microorganisms or transgenic animals (Table 1). The other nine are vaccines and therapeutics manufactured from natural product sources. Considering the 18 recombinant products, 12 are produced using mammalian expression systems, three are produced in Escherichia coli and the others are produced in baculovirus, yeast and transgenic goat [2].
The preference for the mammalian expression system is due to their ability to synthesize proteins that are similar to those naturally occurring in human with respect to molecular structures and biochemical properties [2]. Mammalian cell lines have the basic machinery to express and secrete recombinant protein, and huge number of cell lines from various tissues and species with suitable growth properties is available. Two hamster cells lines, Chinese hamster cell ovary cells (CHO)1 and BHK (Baby hamster kidney cells) and two mouse cell lines, NS0 (myeloma) and SP2-0 (hybridoma), supply most of the biopharmaceutical industry. Research and clinical studies have provided substantial safety information about the use of these cells [3].
Despite the safety and efficacy of recombinant protein expression in murine cells, the glycosylation pattern is of profound importance when considering their potential as expression systems for biosynthesis of human recombinant glycoproteins. Murine cells simply do not possess the “machinery” required for human-type glycosylation: specific glycosidases, glycosyltransferases and specific sugar donors are absent. These differences in glycosylation pattern may be highly immunogenic in humans, and/or may be rapidly cleared the recombinant protein from circulation [4].
In spite the great interest of human cell lines as a new production platform, currently, little information about the cultivation of human cells for the production of biopharmaceuticals are available. These cells have shown efficient production in laboratory scale, however the application for commercial manufacturing is still limited. The human cell lines that are been considered for recombinant proteins production are HEK293, HKB11, PER.C6 and CAP cells [5]. These cell lines will be further described in this review as well as the commercial recombinant products related.
It is worth highlighting that for many years, there was an issue regarding the use of human cell lines for recombinant therapeutic protein production: the regulatory hurdles. The lack of species barrier allowing easier transfer of adventitious agents was considered as a major limitation. Rose et al. [3] argue that infection with human pathogenic agents is likely to result in a full-blown pathogenic effect in human cells that is easy to detect, whereas the agent may be dormant in rodent cells. For all new cell lines, whether of animal or human origin, the risk of transmission of prion-based diseases is addressed with strict documentation requirements and the lack of contact with any potentially infected bovine material.
Section snippets
Prokaryotic expression system
Prokaryotic recombinant protein expression systems have several advantages. It is an easy system of culture, and very rapid cell growth, the expression can be induced easily using IPTG and the purification is quite simple in prokaryotic expression systems.
On the other hand, some recombinant proteins can became insoluble in inclusion bodies and are very difficult to recover as functional proteins. Furthermore, most of post-translational modifications are not added by bacteria system and
Human cell lines
Biopharmaceutical companies and researchers are evaluating the best expression system to produce human recombinant proteins and today most companies are considering the clinical, quality, and productivity implications of their expression system selections [12]. Due to its glycolyation machinery, human cell lines have emerged as a new and powerful alternative for the production of human therapeutic proteins. However, only few cell lines have been used to produce recombinant proteins in a
Advantages of human cells for recombinant therapeutic protein production
Protein glycosylation is fundamental to the efficacy of therapeutic glycoproteins. The knowledge about the precise carbohydrate structures present in the recombinant proteins has revealed the importance of posttranslational modifications for functionality of therapeutic proteins. These glycosylated biotherapeutics (antibodies, growth factors, cytokines, hormones and clotting factors) often need to be produced in mammalian expression systems (as cited above), because the location, number, and
Comparison with other expression systems
The technology for production of recombinant therapeutic proteins has not dramatically changed since the first product approval, 25 years ago. The major host for the production of recombinant proteins is still the CHO cell line and the preferred culture system for this cell line is large-scale stirred tank bioreactors [48]. Therefore, what will be the contribution of the human cells production platform and the differences between this and the traditional expression systems?
The main difference of
Conclusion
The therapeutic protein production using mammalian cell lines have become the dominant production platform, producing more than half of the biopharmaceutical products on the market. Among the mammalian cell lines available, human cells lines have emerged as the most promising alternative to the substitute the main production platform based on murine CHO cells. Despite the great promise, it is worth point out that there is no universal production platform. An optimized expression system has to
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