Chapter 1: Introduction and review of literature
1. Interferons
1.3. P. pastoris as expression system for the production of huIFNα2b
Inorder to overcome the disadvantages of other expression platforms (bacteria, mammalian cell lines, insect cell lines, and plants), yeasts are preferred as the most suitable and attractive hosts for the production of various recombinant proteins/biopharmaceuticals among the different eukaryotic microorganisms. They offer numerous advantages over other expression systems and include superior fermentation characteristics, such as high cell density cultivation, lower nutritional demands, extracellular secretion of produced recombinant proteins, and high protein yields, which make the production process more economical [60]. Besides, the recombinant products produced through yeast platforms are free of endotoxins, oncogenic, and viral DNA.
Yeast species used for recombinant huIFNα2b production includes S. cerevisiae, P. pastoris, and Y. lipolytica. Though studies have shown the successful expression of huIFNα2b in S. cerevisiae is reported [61], it is associated with various metabolic bottlenecks, such as inefficient secretion of the produced protein, protein misfolding in the endoplasmic reticulum, low protein yields (because of its crab tree–positive nature), hyperglycosylated and the presence of terminal allergic α-1,3 mannose in attached N-glycan. These disadvantages have led to the development of alternate nonconventional yeasts (P. pastoris and Y. lipolytica) for the expression of huIFNα2b. On the other hand, Y. lipolytica offers many advantages over the conventional yeast (S. cerevisiae) but the protein titers are far lower as compared to P. pastoris and require further investigation enhance huIFNα2b yield.
It offers numerous advantages, which include
Strongly and tightly regulated inducible AOX1 (alcohol oxidase) promoter which is well suited for controlled expression of foreign genes.
Ease molecular genetic manipulation similar to technique adapted for S. cerevisiae.
Availability of well developed P. pastoris strains and vectors as expression kits to academic research laboratories.
The strong preference of P. pastoris for respiratory growth, a key physiological trait that greatly facilitates its culturing at high-cell densities relative to fermentative yeasts.
Ease of purification of the extracellular expressed recombinant protein, since P. pastoris do not secrete much of its native proteins outside cells.
It has many advantages over conventional yeast (S. cerevisiae), including less extensive hypermannosylation, absence of immunogenic α-1,3 mannose, crabtree negative, and high protein yields. The success of P. pastoris in the production of recombinant proteins is directly linked to the very strong and tightly regulated promoter of the gene encoding the first enzyme of the methanol utilization pathway, AOX1. P. pastoris contains two alcohol oxidase promoters, AOX 1 and AOX 2, of which AOX 1 is more tightly regulated and expressed when compared to AOX 2. Based on the AOX promoter‟s activity for methanol utilization, Mut+ (methanol utilization positive), Mut- (methanol utilization negative), and Muts (methanol utilization slow) are the three available Pichia phenotypes. Expression level of AOX 1 is about 30 % of total soluble protein [63]. Apart from AOX 1 and AOX 2 promoters, alternative promoters are also present for the expression of recombinant proteins, which include GAP, FLD1, PEX8, and YPT1;
however, AOX 1 remains the best option. There are different secretion signals, such as
PHO1 and α-pre pro sequence (S. cerevisiae), available for P. pastoris to facilitate the release of recombinant proteins either in the intracellular or the extracellular environment. Of all the secretion signals available for Pichia vectors, α-pre pro sequence has been reported as the best for the extracellular secretion of recombinant protein.
The first report on the expression of recombinant huIFNα2b showed an expression level of 400mg/L with a biological activity of 1x107 IU/mg, [53] which is lower than natural human interferon (2x108 IU/mg) [64]. In another study, authors showed an expression level of 298 mg/L with activity levels of 1.9x109 IU/mg using P. pastoris Mut+ and pPICZα vector. Also, a decrease in temperature from 30ºC to 20ºC during the methanol feed phase increased the yield of protein dramatically [65]. The use of three different secretion signals viz., native secretion signal, α-pre pro sequence, and mutated α-pre pro sequence addressed the for efficient expression of recombinant huIFNα2b [55]. Plasmid with a huIFNα2b gene along with a mutated α-pre pro sequence showed a single band of 19.2 kDa by MALDI-TOF. Conversely, plasmids with a huIFNα2b gene along with a α- pre pro sequence showed two distinct bands at 19.8 and 20.2 kDa, of which the latter was a co-contaminant [55]. Shardul Salunkhe et al., (2010) depicted the strategies to maximize the expression of recombinant huIFNα2b in P. pastoris [57]. Dimethyl sulphoxide as added as a media component in order to reduce the expression of the isoform contaminant of 20 kDa and 93 % recovery of the protein was achieved using single-step anion exchange chromatography. Atef Ayed et al., (2008) demonstrated that huIFNα2b is highly sensitive to the protease activity during high cell density culture of P. pastoris [56] and employed a strategy of adding casamino acids at 0.1 % (w/w) and 10 mM EDTA, which significantly improved the huIFNα2b expression and prevented proteolysis. In order to improve the expression level, three different methanol feeding strategies were adapted and achieved a yield of 600 mg/L using a smooth increase in
methanol feeding. All the above reported literature for huIFNα2b production using different P. pastoris strains have focused on different strategies viz., molecular startegies, medium formulation and bioprocess strategies for proper and enhanced expression. None of these literature have studied the importance of glycosylation for enhancing the serum/plasma half-life of the huIFNα2b. The present thesis work addresses this gap and focuses on the production of human-like glycosylated huIFNα2b using glycoengineered P. pastoris. The thesis also focuses on the development of an effective process control strategy using PAT tools for enhanced production of huIFNα2b while maintaining the product quality.