Chapter 2 Review of Literature
2.7. Bioprocessing of human interferon gamma (hIFN-γ)
2.7. Bioprocessing of human interferon gamma (hIFN-γ)
productivity of biomass as well as protein. In order to obtain the maximum specific productivity high cell density cultivation (HCDC) is often used. Fed-batch cultivation using high feeding strategies are critical for achieving HCDC, because of effects on maximum attainable cell concentration and formation of byproducts(Babaeipour et al., 2013).
2.7.2. Expression of recombinant hIFN-γ in other protein production systems
Many studies revealed that the glycosylation affects the half-life, protease resistance and solubility of the hIFN-γ(Bocci, 1992). To overcome these barrier other expression systems are used apart from E.coli (Leister et al., 2013). Various production studies were carried out using multicellular eukaryotic expression system. Multicellular eukaryotes provide the advantage of product homogeneity and hence the required biological activity.
hIFN-γ produced in monkey COS 1 cell line were reported to be 40% homologous to native hIFN-γ (Gray PW G. D., 1983). Human proteins were reported to be most similar to their native structures when cloned in CHO cell line (Utsumi J, 1999). However the production level of CHO cell lines were very less compared to E.coli and moreover the CHO cell lines requires serum containing medium which increases the production cost.
Zamani A., 2006, found very low production of hIFN-γ (1337 ng/ml), using CHO cell lines used as expression host. Rodrigues et al., 2013 reported the high cell density growth of CHO cell lines in serum free medium, which significantly reduces the process cost. Recently Chung et al., 2013, used codon optimized gene based on two selected design parameters, codon context (CC), and individual codon usage (ICU). They showed that CC optimized gene resulted in 13 fold increase in hIFN-γ production. While ICU gene resulted in 10 fold increase in hIFN-γ production compared to control.
Glycosylation plays a major roles in stability of hIFN-γ, the differences in the
glycoprotein sialic acid content that may occur in CHO cells leading to extensive charge differences (Curling et al., 1990) along with altered clearance rates in vivo(Ashwell and Harford, 1982), due to unrecognizable specific receptors of the altered glycoproteins in the liver or also due to proteolytic activities (Sandeberg H, 2006). Also eukaryotic systems such as Xenopus laevis oocytes was reported to be used in heterologous expression of hIFN-γ(Taya et al., 1982).
Virus vectors such as Adenovirus(Xu et al., 1997) and Baculovirus (Chen et al., 2005) were reported to produce hIFN-γ. Biological activity of the recombinant adenovirus hIFN-γ was tested by co-transfection into human embryo kidney cell line (Xu R, 1997).
Baculovirus infected Trichoplusia ni and Spodoptera exigua insect larval cells were found to be biologically active against dengue serotype(CHEN et al., 2011). Bacterial and other prokaryotic systems were unable to undergo the post-translational modifications associated with it. These altered structures may thus lead to various complications including altered immunogenicity(Honda et al., 1987), clearance rates (Ashwell G, 1982) and biological activity(Curling et al., 1990). However Baculovirus infected cell lines exhibit low secretion of recombinant protein
Yeasts are suitable host organisms for the production of recombinant proteins since they combine the ease of genetic manipulation, rapid growth at high yield on inexpensive media, and ability to perform complex posttranslational modifications. Yeast expression system such as S. cerevisae (Fieschko et al., 1987) and P. pastoris (Fang, 2013) were also used for the heterologous production of hIFN-γ. These systems, being unicellular provides the advantage of easy and fastidious production and maintenance along with the provision of the required post-translational modifications. These systems are being studied increasingly due to the above mentioned advantages. Some of the bottlenecks are hyper-glycosylation(Grinna and Tschopp, 1989), overflow metabolism leading to altered
or inhibited production (Puxbaum et al., 2015; Zhang et al., 2007). Unsatisfactory yield from mostly S. cerevisae has been reported due to poor secretion in the culture medium, improper folding of the protein along with hyper-glycosylation(Ogrydziak, 1993).
However, recent advances have been made including codon optimized strains concerning the above drawbacks, for the proper and maximized production of the protein, thus making yeast as one of the most promising expression systems (Bretthauer, 2003;
Iliopoulos et al., 2003; Sreekrishna et al., 1997).
Plant expression systems have also been used for the expression of hIFN-γ with an attempt of producing high levels of recombinant proteins. Advantages of using plant systems also include safety against human-animal diseases such as HCV, HIV etc(Memari et al., 2010). Plants such as Lycopersicon esculentum (Ebrahimi et al., 2012) and transgenic rice and barley (Hordeum vulgare) have been seen to successfully express the protein. However, disadvantages such as higher cost of purification and low level expression of some plant expression systems have been observed. Plants such as barley, on the other hand, are used for hIFN-γ on a commercial basis (Isokine), (Biomol).
Various host platform and the production yield of hIFN-γ are shown in Table 2.2.
Table 2.2. Expression system used for the production of hIFN-γ production (Razaghi et al., 2016)
Expression system Yield
[mg L−1] Activityb Molecular
size [kDa] Reference
(Mus spp.) Mouse mammary gland
23 × 10−6 1 × 107 IU mg−1 20–25 (Bagis et al., 2011) 350–570 1 × 107–
5 × 107 IU mL−1 a (Lagutin et al., 1999)
(Rattus spp.) Rat cells a 4 × 105 IU mL−1 22–25 (Nakajima et al., 1992)
(Cricetulus sp.) Chinese hamster ovary cells
a 2.0 × 104–
1.0 × 105 IU mL−1 22–23 (Haynes and Weissman, 1983)
a 5.5 × 104 IU mL−1 21–25 (Scahill et al., 1983) a 1–2 × 108 IU mg−1 20–26 (Mory et al., 1986)
15 a a (McClain, 2010)
Spodoptera spp. (BIIC) 2 Activea 18–23 (Chen et al., 2011) Solanum lycopersicum
(Tomato) a Activea a (Ebrahimi et al.,
2012) Oryzea sativa
17 × 10−3 Activea 24–27 (Chen et al., 2004) (Rice)
Bacillus sp. (Bacteria) 2–20 Activea 17 (Rojas Contreras et al., 2010)
Leishmania sp.
(Protozoa) 9.5 Activea 17 (Davoudi et al.,
2011) Saccharomyces
cerevisiae (Baker‘s yeast)
a 2.5 × 104 IU mL−1 Detecteda (Derynck et al., 1983)
Pichia pastoris (Methylotrophic yeast)
1–
16 × 10−3 Activea a
(Razaghi et al., 2015; Razaghi et
al., 2016)
2.5 Activea 17 (Prabhu et al.,
2016) 300 1-1.4 × 107 IU mg−1 15 (Wang et al.,
2014) Monkey cells a 6.2 × 10−2 IU mL−1 a (Gray et al., 1982) Homo sapiens
6 1.93 × 107 IU mg−1 a (Leister et al., 2014) (Human tissue culture)
E. coli 1700 9 × 107 IU L−1 17 (Huang et al., 2013)
a No data.
b The antiviral assay for quantifying biological activity of human IFNs is based on the induction of a cellular reaction in the transformed human cell line (WISH); the effectiveness of interferon is assessed by comparing its protective effect against a viral cytopathic effect (usually vesicular stomatitis virus) against a calibrated reference in international unit (IU) (Petrov et al., 2009).