Biology论文模板 – Biopharmaceuticals Production Using Pichia Pastoris


Biopharmaceutical productions which are mainly protein based are slowly gaining market within the biotechnology and medical industries. Some of the approved biopharmaceutical products include anticoagulants, hormones vaccines, blood factors and monoclonal antibodies with a majority being glycoproteins. The glycosylation, biosynthesis and covalent bonding of the oligosaccharides to polypeptides are tissue and species specific with the control of the production of carbohydrates and glycosylation being very crucial for the generation of the therapeutic glycoproteins. The product of these glycoproteins can either occur in the yeast or in mammalian cells. However, the mammalian system of production is faced with numerous challenges making it to have vast number of disadvantages. Some of these disadvantages are low protein titres, long production time, viral containment and product heterogeneity which thereby hinder the therapeutic glycoprotein production in large scale. These challenges, therefore, create a gap for the search and development of an alternative system with improved quality performance.

Methylotrophic yeast Pichia pastoris, on the other hand, presents vast number of attractive features that suit it for the production of heterologous protein. For instance, the ability to secrete large quantity of recombinant protein, ability to undergo post-translational alterations and the protein antigens glycosylated by it shows enhanced immunogenicity thus can offer a platform for the recombinant vaccines priduction hence qualifying it as an alternative system of glycoprotein production.

The purpose of the dissertation is to develop bioprocesses that can be employed for the efficient synthesis of P-selectin glycoprotein ligand/mouse immunoglobulin G2b (PSGL-1/mIgG2b) and  Alpha1-acid glycoprotein/mouse immunoglobulin G2b (AGP-1/mIgG2b).This is to provide adequate materials necessary to characterize and to illustrate the various functions of PSGL-1/mIgG2b and AGP-1/mIgG2b within the boundary of MR, DC-SIGN, and Mannose binding lectin (MBL) mannose specific receptors bindings.

This research was also aimed at determining the various crucial bioprocess parameters that are relevant for the large scale generation of the recombinant glycoproteins. From the study, it was realised that methanol feeds, pH and some media components played a critical role in the production, productivity and homogeneity of most recombinant proteins. Additionally, the biocore analysis showed that both PSG-1/mlgG2b and AGP-1/mlgG2b had high binding affinity to all the receptors.


Recombinant protein therapy is a slowly broadening concept in the medical and biotechnology institutions. Nearly, all of the accredited biopharmaceuticals are mainly protein based such as blood hormones, monoclonal antibodies, vaccines, blood factors and anticoagulants (Lua et Al, 2012). According to Fu et Al, (2012), The majority of these protein based biopharmaceuticals are glycoproteins which for correct folding, stability in the circulation or biological activities require specific carbohydrate structures linked together with certain amino acids.

The bonding and biosynthesis of these special carbohydrates attached to amino acid i.e. oligosaccharides and polypeptide core are both species and tissue specific (Walsh, 2013).

The eukaryotic cells may bond the glycoproteins either to the aspargine (N-linked) side or through the threonine/serine (O-linked) sides (Salgado et Al, 2014). Glycosylation forms the key component in the development of therapeutic glycoproteins and presently, mammalian cell culture system is mainly used for production of the cultured cells since the possess the capability to perform and behave like human glycosylation (Mulder et Al, 2015).

Narum, (2008) in his findings showed thatPichia pastoris was widely accepted as an alternative for the production of recombinant protein due to the following factors

The technique required for its molecular modifications is similar to that of Saccharomyces cerevisiaet. The promoter generated from the alcohol oxidase I (AOX1) gene of pastoris is uniquely suited for monitored expression of foreign genes, the strong preference of the yeast for the respiratory growth which is a vital physiological trait that primarily promotes its culturing at a high cell densities relative to fermentive yeast. Consequently, the decision of Philips Petroleum Company in Bartlesvile, Okla of 1993 that released Pichia pastoris expression system to the research farms, is also attributed to the explosion of knowledge about the yeast and its acceptance (Berlec & Strukel, 2013).

Problem Statement

The recombinant proteins, in general, are produced from the mammalian cell system and the Escherichia coil (Gormordo et Al, 2010).Bacterial system on the other hand Bill, (2014) has decribed to be showing a rapid and robust growth in the bioreactors through the application of simple media.Culture processes of the mammalian cells, however, are relatively slow, highly susceptible to viral infection and require complex media (Maccani et Al, 2014). Maccani et Al, 2014) further explained that the mammalian systems of production are frequently hindered by factors such as low protein titres and product heterogeneity which hamper and most at times complicate the entire production process especially the large scale therapeutic production of glycoproteins. Also, Gullissen et Al, (2005) in their work enumerated that the bioprocess parameters were also capable of having influence on the glycosylation profile of the recombinant glycoproteins which on the other hand may affect the biological properties of the therapeutic glycoproteins. Therefore, Gullissen et Al, (2005) suggested that there was a need to develop a bioprocess that could be employed for mass production of the glycoproteins and also offer a substantial ground for functional studies for the glycoproteins. Sommer et Al, (2014) clarified that the development of the bioprocess entailed frequent analysis of the productivity, analysis of the glycan structure, bioactivity of the therapeutic recombinant glycoprotein under various culture conditions and proteolytic degradation. However, the development of an efficient culture conditions for the industrial purposes is time consuming and just but an expensive task. Quantification of the recombinant protein on the other side is a potential challenge in a high throughput protein production (Huang et Al, 2013). The quantification is usually ascertained by Bradford chemical analysis or through immunological methods such as Western blot and ELISA. These methods are slow and most at times require sampling together with sampling preparation before the actual assay (Dingermann, 2008). Therefore, to realize a high throughput protein production, there is the need to come up with faster and efficient assay procedures.

It is these disadvantages and many more that has foster the continual search and need for another alternative expression system with better features and of high performance characteristics.Pichia pastoris offer a highly and successful system of production of a wide range of varieties of the recombinant proteins. P. pastoris being a yeast is single-celled thus is easy to manipulate and culture. Consequently, pastoris also belongs to the eukaryotes family and thus is having the capability of exhibiting numerous post-translational alterations that are performed by the higher eukaryotic cells. These modifications include folding, secretion of large quantity of the therapeutic protein, proteolytic processing and disulfide bond formation (Wang et Al, 2001).


The primary ojectives for the study included;

  1. To produce biopharmaceuticals using yeast Pichia pastoris as a platform of production.
  2. To construct or develop an efficient bioprocess that can be used for the production of P-selectin glycoprotein ligand/mouse immunoglobulin G2b and Alpha1-acid glycoprotein/mouse immunoglobulin G2b in order to provide adequate amount of materials that are necessary for their characterization with respect to their binding properties to mannose receptor, DC-SIGN and mannose binding lectin
  3. To determine the most vital procedures in the bioprocess for large production of biopharmaceuticals.
  4. To come up with the necessary techniques that can be applied for the quantification of the biopharmaceuticals.
  5. To relate certain specific glycan structures to their biological activities.


The therapeutic recombinant glycoproteins produced by the yeast have exhibited indication of improved immunogenicity when compared to their counterparts which have not undergone glycosylation process. Also, the great mannose substance of yeast that is generated from the N- and O-linked glycans has been proposed to have target on the recombinant protein (antigen) so as to immunoregulates the specific mannose receptors. These regulators upon binding facilitate improved immune responses. Therefore, these findings suit the methylotrophic yeast Pichia pastoris to be used as a platform for the production of the recombinant vaccines. Consequently, using yeast gives room for the combination of the robust growth on a simple media i.e. in large scale bioreactors through a readily achievable genetic alterations and the introduction of the desired post translational modifications (Soetaert & Waegeman, 2011). In short, the Pichia pastoris system is faster, easier and more economical to use than other expressions generated from higher eukaryotes and does provides higher expression level (Lualdi et Al, 2015).

Literature Review

Biopharmaceuticals [Fig.1] is an essential development in the pharmacy and biotechnology industries that is indispensable in the modern medicine, diagnostics, food, nutrition polymers, detergents among others with an estimated annual growth of up to 15% (Roque et Al, 2004). Werner et Al, (2007) in their work described biopharmaceuticals as recombinant therapeutic protein and nuclei acid based products which may also entail biologically engineered products on a larger scale. Agarwal et Al, (2013) enumerated the examples of the therapeutic proteins as vaccines, antibodies, interferon and hormones such as insulin, and human growth hormones. Typically, recombinant therapeutic proteins are produced in the Escherichia coli and mammalian cells with the bacterial cell system indicating a robust and faster growth in biorectors through the use of simple media (Lehmann et Al, 2009). Castilho,(2015) explained that since 1970s, the scope of both the medicine, diagnostic, food among others have experienced dramatical changes by the onset of recombinant DNA technology.

Fig,1: Biopharmaceutical production, (Soetaert & Waegeman, 2011).

According to Nordon et Al, (2013), the recombinant proteins are generated from systems such as bacteria, filamentous fungi, yeast, insect cells, mammalian cross transgenic animals and transgenic plants which were also inline with (Zhang et Al, 2015) arguments. A total of around 39% of all the recombinant proteins are mainly produced by the E.coli, 35% being from CHO cells, 15% from yeast, 10% from the mammalian cells and around 1% of the proteins produced by other bacteria as well as other systems (Maity et Al, 2016). Usually, proteins which are greater than 100kD are expressed in eukaryotic systems. However, those of smaller sizes of less than 30kD are typically expressed in prokaryotic systems. Bacterial cells, on the other hand, offer great simplicity, shorter generation time, and higher product yields at a lower cost of productivity (Herwing & Spadiut, n.d). Typically, yeast is a unicellular microorganism and therefore are easily manipulated and cultivated (Fazenda et Al, 2013). Fazenda et Al, (2013) in their work identified that the presence of the bacteria in the yeast also provided the eukaryotic medium that is usually crucial for the production of broad and complex proteins. According to Adnan & Ahmed, (2007), The use of the methylotrophic yeast Pichia pastoris began in the early 1970’s when the yeast was applied for the production of a single cell protein thereby providing a cheap and a high cell density fermentation process. Adnan & Ahmed, (2007) findings were in agreement with those of Castilho (2015). They all together stated that this production process of protein was enhanced by Philips Petroleum Company and that by around 1980’s, researchers mainly from Salk Institute Biotechnology isolated the AOX1 gene coding for alcohol oxidase together with its promoters. They also developed strain vectors and the procedure for molecular genetic alterations of the Pichia pastoris, whose expression system is highly attractive for the recombinant glycoprotein generation.

Methylotrophic yeast Pichia pastoris exhibits all the favourable yeast characteristic (Mhatre et Al, 2009).It has successfully been employed to generate higher titer quantities for some heterologous proteins with the approval of the FDA. Ahmad et Al, (2014) stated that the expression system of the yeast was of great importance. Ahmad et Al, (2014) showed that the expression was essential because it could be used as an alternative whenever the E.coil protein machinery failed to deliver a correctly folded functional protein. Again the rate of productivity of the Pichia pastoris is much faster compared to others (Herwig & Spadist, 2014).

Pichia pastoris as a yeast species is capable of metabolizing methanol (Harms et Al, 2008). The metabolism of methanol begins by the oxidation of the methanol compound to form formaldehyde thereby generating hydrogen peroxide in the process. This process is catalysed by enzyme alcohol oxidase, AOX. The hydrogen peroxide produced is toxic and therefore the metabolism of methanol occurs in a specialized organelle known as peroxisome [Fig.2] which can sequester the hydrogen peroxide formed out of the cell (Harms et Al, 2008). According to Krainer et Al, (2013), the peroxisomes contains three crucial enzymes that are necessary for the complete metabolism of methanol. These enzyme are alcohol oxidase, catalase and dihydroxyacetone synthase. From the first stage of metabolism, the formaldehyde enters the cytosol from where the final stage occurs (Sallach et Al, 2009). Sallach et Al, (2009) in their study showed that this final stage results in energy and biomass constituents. AOX have low affinity for oxygen and therefore the methylotrophic yeast compensate for the deficiency by producing large quantity of this enzyme.

Fig.2: schematic illustration of peroxismal reaction in methanol, (Berlec & Strukel, 2013).

Pichia pastoris posses two genes that are capable of coding with the enzyme AOX. According to Mhatre et Al, (2009), these genes are AOX1 and AOX2. However, AOX1 is mostly responsible for the the numerous majority of the oxidase enzyme activities in the cell (Mhatre et Al, 2009).

Sallach et Al, (2009) in their work showed that the expression of AOX1 is mainly regulated and induced by methanol to high level of transcription. Sallach et Al, (2009) stressed that it was the tight regulations of the AOX1 promoter gene that facilitate the production the proteins. The possibility of the pastoris to make use of methanol is depicted by the three phenotypic types of expression of the host strains (Berlec & Strukel, 2013).

These phenotypic expression includes Must+ referring to methanol utilization plus, Muts which means methanol utilization slow and Mut- meaning methanol utilization minus

The host strain that contain alcohol oxidase gene is capable of growing on the methanol at the fullest rates of growth and hence are referred to as Must+ phenotype. Muts strain on the other hand have AOX1 gene removed thereby making the cells to rely on the relatively weaker AOX2 gene thus slower growth rate on methanol.

According to Berlec & Strukel, (2013), Mut- strain have all the genes deleted hence are unable to germinate and grow on methanol. This conditions, therefore, places the Mut- as the most preferred whenever methanol is not the suitable source of carbohydrate. In order to control the phenotypic trait of pastoris, Mulder et Al, (2015) indicates that its many cassettes of expression should include the various elements that enhance removal of alcohol oxidase 1 or all genes during the recombinant gene insertion process. Even though the Muts and Mut- have progressively displayed numerous advantages, the Mut+ strain with strong alcohol oxidase 1 is still the most common promoter (Mulder et Al, 2015). The control regime of the alcohol oxidase one promoter may, therefore, indicate depression and induction principles which have sole purpose in the formation or generation of pichia in the biopharmaceuticals production process

Some different techniques and method according to Adnan & Ahmed, (2007) have previously been employed for the recombinant protein production using Pichia expression system. However, the standardized technique that is mainly made use of is the methanol limited fed batch (Adnan & Ahmed, 2007). This technique typically is as a result of the repression, depression, or induction process of the AOX1 gene. At the start, the cells are given time to germinate on carbon sources such as glycerol in order to produce cell mass with absence of heterologous protein production (Soetaert & Waegeman, 2011). This process thereafter is followed by the limited glycerol feed phase which limitss the alcohol oxidase 1 gene. The transcription process of the alcohol oxidase and the recombinant genes as a result begins and thereby ensures a smoother transition to the induction phase (Mulder et Al, 2015). At introduction phase, pure methanol is made use of as the source of carbon and to fully initiate the AOX1 promoter. The induction phase, therefore, can be utilized for the generation of vast number of cell masses.

Harms et Al, (2008) showed that the initial stage of induction is of great importance as the cells haven’t fully cope up with the breakdown of the methanol, thus a little supply of methanol should be kept constant for a given period. Agarwal et Al, (2013) argued that since oxygen is of high demand during the cell density fermentation, methanol feed should be limited to help curb the oxygen limitations. Soetaert & Waegeman, 2011) However, cautioned that the oxygen limited fed batch procedures has to be properly developed and put in place. Krainer et Al, (2013) showed that the recombinant protein production process, on the other hand, is influenced by factors such as specific growth and the specific methanol uptake rates. Krainer et Al, (2013) therefore, suggested that in order to attain a given specific growth rates and methanol uptake rates, methanol feed, oxygenation and agitation parameters are to be employed.


In order to produce large quantities of the recombinant protein, a high cell density bioreactor processes were made use of. These processes, however, were used in parallel with those of the shakeflask cultivation process. The shake flask was, employed since one of the proteins showed little fragmentation whenever high cell density bioreactor technique was applied. The PSGL-1/ mlgG2b was in most case expressed as a 250-300 kDa protein which is usually consistent with its types of dimer (Mhatre et Al, 2009). Consequently, AGP-1/mlgG2b was typically expressed as 68 kDa protein in the bioreactor culture at a pH 6.0 since it is a monomer of the AGP-1/mlgG2b protein (Mhatre et Al, 2009). Majority of the AGP-1/mlgG2b in the shakeflask expression were however expressed as 160 kDa protein which was also consistent with its dimeric.

The fragmentation process, on the other hand, was believed to occur due to of proteolytic degradation or due to various glycosylation processes (Adnan & Ahmed, 2007). In order to monitor and accurately control the preteolysis pricess in the bioreactor, a pH reduction of upto3.5 was induced into the system. Western blot analysis was conducted on the PSGL-1/mIgG2b component generated from the bioreactor cultures which was of a lower pH value. The result, therefore, showed that at a lower pH, the protein constituent exhibited a suddenly lower fragmentation process. Additionally, AGP-1/mlgG2b developed from the bioreactor cultivation process at a lower pH of 3.5 exhibited little degradation than when was placed at a higher pH of 6.0 however, these findings were in contrast to those of shakeflask cultivation at a pH 6.0 which showed little fragmentation.

Concanavaline A, which is a lectin binding mannose, was employed for the investigation of the presence of mannose in the O-glycans (Adnan & Ahmed, 2007). The Concanavaline bonded with PSGL-1/mIgG2b that were the release of its N-glycans,  during the Western blotting analyses. Despite removing N-glycans by the PNGase F treatment, Concanavaline A remained bound to PSGL-1/mIgG2b. The binding clearly depicts the fact that some determinants having the mannose were still present.

The ability of the therapeutict protein to binding to the receptor mannose receptor (MR),DC-SIGN and mannose binding lectin (MBL) at a high affinity was also determined by the Biacore analysis. A fragmentation constant ranging between 4.23-84 nM was an indication of high affinity binding.


The general productivity in the bioreactor was seen to be superior to those cultivated in the shakeflask producing a total of 21-200 mg/L recombinant proteins in comparison to 3.5-15 mg/L produced from the shakeflask cultivation. However, the bioreactor suffered from recombinant protein degradation at pH 6. Consequently, the therapeutic recombinant protein production did not show any significant change whenever the pH was varied from 6.0-3.5. Results from the biocore analysis revealed that PSGL-1/ and AGP-1/mIgG2b, which are two recombinant fusion proteins secreted by P. pastoris and have distinct glycosylation, features exhibited high affinity level of binding to the recombinant mannose specific receptors MR, DC-SIGN, and MBL. These recombinant proteins are thus expected to efficiently and effectively targets these receptors in vivo and also promotes immune response. The multivalent bonding among the glycoproteins and receptors, therefore, is a major factor for high affinity realization between the interacting recombinant protein and receptors. Free mannose, on the other hand, did not stick to or bind to any of the receptors. This finding was in contrast to those of oligomannose-9 that weakly bound.

The result from the experiment also indicated that the recombinant proteins of the Pichia pastoris are much better and more superior to the mammalian cell lines. However, they are less superior when compared to other expressed protein in the fermenter

The simplicity of expression system of the Pichia pastoris together with high production capacity in combination with PSGL-1/mIgG2b  and AGP-1/mIgG2b depicts an efficient system of production of the adjuvant vaccines. Industrial manufacturing of adjuvant vaccines should, therefore, consider incorporating the Pichia pastoris concept into their production practices.

The versatile host strain of the yeast p. Pastoris together with appropriate vector coded and expressed many different types of protein. Also, significant amount of vaccines, enzymes, human proteins and other biopharmaceuticals were synthesized by the Pichia pastoris as a platform. Furthermore, the AOX1 promoter provided enough room for the desirable control and separation of growth as well as production.

Advantages of Pichia pastoris

  1. It can be easily cultured to a high cell density in a bioreactor.
  2. It possesses good secretion capability for the heterologous proteins.
  3. It has less extensive glycosylation features compared to S.cerevisiae.
  4. It has simpler molecular genetic manipulation, and the protocols are available.
  5. It has higher tendency for the respiratory growth.
  6. It can be cultured in a simple salt media with secretion of a little endogenous proteins which after that simplifies the product the product purification and recovery.
  7. The heterologous gene expression can easily be controlled by the use of a tightly regulated and efficient promoter


Glycoproteins and proteins are found either in the cell, within the surrounding of the cell or as a transmembrane proteins in which its carbohydrate section take part in many function (Castilho, 2015). These functions includes cell-cell signalling, cell-cell adhesion, intracellular trafficking and targeting of some specific receptors (Fazenda et Al, 2015). The oligosaccharides of the glycoproteins, on the other hand, play a significant role of directing the protein folding, the thermostability of the proteins as well as its half life among others (Sallach et Al, 2009). According to Herwig & Spadist, (2014), the diversity in the function of the glycoproteins is what makes them suitable and the most popular elements of the immune systems.

Glycosylation is the process by which carbohydrate structures found on the backbones of polypeptides are synthesized in the presence of enzymes.(Maity et Al, 2016). It is the existence of enzymes that speed up the rate of synthesis. The glycosylation process, therefore, results in the modification of the polypeptide structures and occurs in the endoplasmic reticulum or golgi bodies resulting formation glycoprotein (Legmann et Al, 2009). Maity et Al, (2016) indicated in their work that usually, the N-glycans are linked to the amide nitrogen of an aspargine(Asn) remnants by an N-glycosyl attachment with the consensus sequence of Asn-X-Ser/Thr in which X is an amino acid other than proline. However, Nordon et Al, (2013) on the other hand showed that the O-glycans were linked to the hydroxyl ion of the Ser or Threonine containing its anomeric carbon through the glycosidic linkage. Nevertheless, no consensus sequence have so far been reported (Maity et Al, 2016). In order to produce certain oligosaccharide structure on a growing polypeptide, an orderly fashion of monosaccharides addition should be employed. The addition demands for some donors of sugar and the necessary machinery for locating these requirements in their right position within the golgi and endoplasmic (Zhang et Al, 2015).

The Biosynthesis of the N-glycan involves a series of events that occurs between yeast and eukaryotes thereby giving rise to the covalent attachment of the Glc3Man9GlcNAc2  oligosaccharide to any suitable aspargine residue (Krainer et Al, 2013).  Krainer et Al, (2013) suggested the covalent bonding process to be taking place on the protein that is being translated. The product of the covalent bonding i.e. The Glc3Man9GlcNAc2-Asn is shaped by glucosidase and mannosidase that eliminate glucose and certain specific D-1,2 linked mannose thereby resulting in formation of Man8GlcNAc-Asn structure. This structure formed is made available for Golgi that further processes it (Krainer et Al, 2013).

The oligosaccharide [Fig.3] used for bonding in the above case is mainly common in both yeast and eukaryotes of higher order. It is, therefore, this fact that makes the mammalian cells and yeast to take different glycoslation paths. In mammalian cell, the production process continues in the golgi apparatus to produce either Man5GlcNAc2-Asn or Man3GlcNAc2-Asn structures that participates in the complex types of N-glycans and hybrid generation methods (Krainer et Al, 2013).

Fig.3: showing Oligosaccharides, (Zhang et Al, 2015

S.cerevisiae together with Pichia pastoris contains D-1,6-mannosyltransferase. The D-1,6 is responsible for the addition of a very crucial mannose that trimmes Mannose8N-acetyl glucose mine 2-Asn structure. It is, therefore, this feature that makes the N- glycan to act as a substrate that is subject for the additional D-1,2, D-1,3, D-1,6-mannosyltransferases and as a phosphor mannosyltransferases (Krainer et Al, 2013). These, therefore, gives rise to a highly mannosylated structures that are easily applicable to the generated yeasts glycoproteins. Mannosyltransferases D-1,3, however, has not been reported in Pichia pastoris. The formation of the highly mannosylated structure is followed by the diversification and elongation of its core through the cis-, medial, and trans-golgi sections. Legmann et Al, (2009) research showed that the N- glycan distal parts are mostly formed in the later golgi compartments. It is, therefore, apparent that the enzymes responsible for the termination of the synthesis of oligosaccharide is only found in the trans-golgi compartment.


Large number of human proteins, vaccines, enzymes and other biopharmaceuticals can sufficiently be synthesized by Pichia pastoris as a platform. The adaptive features of the Pichia yeast to a high mannose form of glcoslylation to a more complex human like glycoslation is indeed a great achievement that will see uniform glycoforms in the microbial productions.Additionally, the versatile host strains of the yeast Pichia pastoris when used together with appropriate vectors is capable of coding different proteins of interest, and expression of genes since they are readily available. Both in simple mineral salt media and under controlled bioreactor environment, the Pichia pastoris is capable of growing to a high cell density easily. Again its efficient and tightly controlled promoter, AOX1 gives room for the desirable control and separation of growth together with production. Consequently, the secretion of the endogenous recombinant protein by the yeast Pichia pastoris is highly efficient, and the separation of the recombinant glycoprotein is quite simple even in salty medium. These factors together with many more, therefore, makes Pichia pastoris as the ideal and most preferred platform for the efficient formation, synthesis, and purification of recombinant proteins.


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