l. INTRODUCTION

In 1969, the first report of methylotrophic yeast being able to use methanol as single carbon and energy source was published (Ogata et al., 1969). Due to its ability for growing at very high cellular densities (up to 130 gDW/L, where DW refers to dry weight), Pichia pastoris was used for the development of the single cell protein industry, at the end of the last century (Sreekrishna & Kropp, 1996; Aw & Polizzi, 2013). The Phillips Petroleurn Company tried to produce biornass for feedstock, but it resulted in a non-economically viable process. This led the company to start researching about the usage of P. pastoris for recombinan! protein production with good results and the subsequent release of the expression system for academic research laboratories in 1993 (Sreekrishna & Kropp, 1996; Cereghino & Cregg, 2000). Currently, P. pastoris has been a big interest in academic and industrial purposes, mainly due to its promoter derived from Alcohol Oxidase gene (AOXl) and the similarity of genetic modification techniques with those of the well-known and well-studied yeast Saccharomyces cerevisiae (Cregg et al., 1985; Cregg et al., 2000). Other interesting features that P. pastoris has, are the ease for the excretion of products, its promoter strength, the simple media in which it can be cultured, the integration of multi-copies of foreign DNA, yielding in more stable transforrnations and the easiness of recovering proteins due to its well-studied secretion systems (Li et al., 2007; Demain & Vaishnav, 2009). Unlike the bacterial systems, eukaryotes, such as P. pastoris can make post-translational modifications, which include protein processing, folding, and N-glycane addition. This is importan!because most of the biological activity of recombinan !proteins (as those for medical purposes) depends on modifications carried out after the translation process (Baghban et al., 2016). One importan!advantage of using P. pastoris over the traditional yeast, such as S. cerevisiae, is the avoidance of hyperglycosylation patterns, especially for the healthcare industry (Demain & Vaishnav, 2009). Inthis paper, importan!aspects of P. pastoris related to the recombinan !protein production, by using it as a biological platforrn, are reviewed, from cell features to the culture techniques and metabolic aspects to produce yeast strains suitable for biotechnological processes. The relevance of P. pastoris is also highlighted for the developrnent of the biotechnological industry, in Colombia, based on the studies conducted in the country with this expression system in the production of recombinan!proteins.

2. METABOLIC ASPECTS

2.1. Methanol metabolism

Pichia pastoris results in a good expression platform under certain non-conventional conditions, such as methanol, by increasing medium concentration in which the majority of microorganisms will not be able to grow, by overcoming these milestones and, by becoming this organism a really good standpoint for industrial applications (Duan et al., 2018). These features can also be found in other yeast genera, such as Hansenula, Pichia, Candida, and Torulopsis (Lee & Komagata, 1980).Thus, it would be useful to look at the genetic relationship that this microorganism has with other genera, such as Candida or Saccharomyces. Not only is determining the phylogenetic position useful for further phylogenetic description purposes, but also it can be useful for determining further genetic elements search, such as ribosomal binding sites, promoter sites, initiation, and stop codons, among others. This has been proven as shown by De Schutter et al. (2009) where a similarity analysis found that the codon usage for this yeast was like the one for S. cerevisiae, which turas out to be attractive for further genetic modifications.

The utilization of a certain number of carbon and nitrogen sources for growth by yeasts is characteristically associated with the development of unique subcellular compartments in the cell (Van Dijkenet al., 1975).Pre­ viously, sorne authors have discovered that the adaptation process that this kind of microorganisms suffer, by growing them on methanol, is associated with the proliferation of large microbodies in the cell

(Avers & Federman, 1968).Thus, a key physiological feature that methylotrophic microorganisms have, is a set of particular inner structures, which are called peroxisomes. It can be defined as intracellular compartrnents that have certain special enzymes, such as catalases or oxidases that help in the detoxification processes within the cell (Titorenko & Rachubinski, 2004). This complex arrangement allows to carry out reactions with a specialized target without affecting the other cellular processes (Gabaldón & Carreté, 2015). lt has been noted that during the exponential phase of growing methylotrophic yeasts fed with glucose, peroxi­ somes present within the cell, are generally very difficult to detect, as well as their physiological function (Veenhuis et al., 1983). Thus, it is known that there is a considerable increase in the number and size of sorne cellular compartrnents, such as mitochondrial structures, as well as lipid droplets, under a glucose fed regi­ me in P. pastoris. This result is contrary to the methanol feeding case in which mitochondrial structures are hardly visible regarding peroxisomal structures that suffer a dramatic expansion (RuBmayer et al., 2015). It has been proven that peroxisomal structures can adopt different shapes, ranging from rectangular shapes to complex crystalline structures that may fulfill around 80% of the total intracellular volume depending on the environmental conditions of the media and the kind of substrate with which the cell is fed (Veenhuis et al., 1983). Thus, it is crucial to understand how different substrates enter into the cell, as well as their further transformation. Let us analyze the most important substrate for the methylotrophic yeast: methanol. Singh & Narang (2019) have assumed that methanol enters into the cell by diffusion rather than by active transport. This assumption, often made implicitly, is probably motivated by the observation that in Saccharomyces cerevisiae, in which ethanol enters into the cell by diffusion, which suggests that methanol, a similar and even smaller compound, also enters into P. pastoris cells by diffusion (Loureiro & Ferreira, 1983).

According to different authors (Cereghino & Cregg, 2000; Gao et al., 2016; Krainer et al., 2012; Lin et al., 2000) the use of methanol as a sole carbon source by this yeast bas sorne drawbacks during its develop­ ment process. Methanol is potentially bazardous due to its toxicity and flammability capacity, which could be an issue under fermentation process conditions. Also, it is known that these cells take oxygen at a high rate so that the expression of foreign genes can be negatively affected by oxygen limitation. Finally, a con­

siderable amount of heat is produced due to the large amounts of oxygen that is consumed through cellular growth, which causes extra difficulties in the stability of the cellular system.

About the enzymology of methanol oxidation in yeasts, it is fundamentally different from those found in bacteria (Veenhuis et al., 1983).It appears most likely that P. pastoris and other methylotrophic yeasts, have evolved a specialized set of enzymes for sugar phosphate rearrangements that shows high transcript levels. These enzymes are located in peroxisome and are specifically induced by growth in methanol (RuBmayer et al., 2015). The initial methanol assimilation step is carried out within the peroxisome where methanol is transformed into formaldehyde as a result of the alcohol oxidase (EC 1.1.3.13) enzyme action, by using oxygen as electron acceptor and, by generating hydrogen peroxide as a product (Yurimoto et al., 2011; Van der Klei et al., 2006).

As it is indicated in Figure 1, the formed formaldehyde is a central intermediate situated at the branch point between the assimilation and dissimilation pathways (Yurimoto et al., 2005).Formaldehyde must be rapidly assimilated into biomass or dissimilated to C02 due to its toxicity. A portion of formaldehyde is fixed to xylulose 5-phosphate (Xu5P) by dihydroxyacetone synthase (DAS) (EC 2.2.1.3), by forming dihydroxyacetone (DHA) and glyceraldehyde 3 phosphate (GAP), which are used for the synthesis of cell constituents and the generation of Xu5P through Xylulose Monophosphate (XuMP) pathway (Yurimoto et al., 2011; Zhang et al., 2017).Another portion of formaldehyde is further oxidized to C02 by the cytosolic dissimilation pathway. At this point, it would be useful to recognize the crucial role that peroxisome has in methanol assimilation, by giving methylotrophic yeasts their main feature. However, peroxisomes not only allow alcohol degradation, but they can also assimilate nitrogen sources, such as tertiary amines like

methylamine into formaldehyde through other peroxisomal enzymes called amine oxidases in order to be assimilated by the cell as it was explained earlier (Shiraishi et al., 2015; Siverio, 2002; Zwart et al., 1983).

Figure 1: Pichia pastoris assimilation alcohol and amine pathway. Source: Own elaboration

2.2. Promoters

There are two main promoters to produce recombinant proteins: AOXl, which is induced by methanol and GAP, which is the promoter for the glyceraldehyde-3-phosphate dehydrogenase.

AOXI

The AOXl system regulates the production of Alcohol Oxidase along with AOX2, but it is known that AOXI is responsible for most of the enzyme activity in the cell. The promoters are so active when wild-type yeast grown in methanol as sole carbon source and energy, the enzyme can reach approximately 30 % of the total soluble cell proteins and 5 % of the mRNA. That is because, surprisingly, the enzyme has a low affinity for its substrates, methanol, and oxygen. Although AOX2 is less common and has a lower expression leve!than AOXI. There are studies in which truncated versions of this system are used for the production of recombi­ nant proteins (Mochizuki et al., 2001;Kuwae et al., 2005; Potvin et al., 2012; Vogl & Glieder, 2013).

AX02 is activated under higher methanol concentration than AOXl (Mayson et al., 2003). The regulation of AOXI is at transcriptional leve!and the promoter is repressed by glucose, glycerol, and ethanol. lt is neces­ sary to eliminate repressor carbon sources of the broth before starting the induction process with methanol, so cultures must be carried out in two stages, one of growth and the other one of induction.

The preference of AOX for small alcohols is explained by the presence of conserved bulky aromatic residues nearby the active site in which FAD cofactor is present near the active site (Vonck et al., 2016).As mentioned above, methanol metabolism has undesirable consequences. To overcome the issues, mutants have been developed: the mutant S. denoted by MutS (AOXI-) and the mutant denoted by Mut- (-AOX1-AOX2) which cannot use methanol a subtrate. In general terms, the advantage of these modifications is less methanol consuption (Singh & Narang 2019).

Figure 2: Methanol assimilation mechanism in P. pastoris. Source: adapted from Singh et al. (2019)

The previous figure clearly shows that, under the same methanol concentration condition, each mutant has a different behavior: the Mut-si not able to assimilate methanol due to alcohol oxidases absence so that methanol diffuses freely wothout decreasing its concentration. On the other hand, the MutS has cretain methanol consumption rate thanks to the presence of some alcohol oxidase enzymes, but it is not so much. Certainly, its assimilation is slow.That is why this mutant has the suffix S. Finally, the mutant Mut+ presents the higher methanol consumption because of a considerable presence of alcohol oxidase enzymes, which allow better methanol assismilation (Lin-Cereghino et al.,2006;Singh & Narang, 2019).

GAP

GAP promoter is active in a constitutively way because it regulates the production of an important enzyme involved in the glyconeogenesis. It was first isolated in 1993 (Waterham et al., 1997). This is an advantage with respect to AOX1 because it is possible to produce foreign proteins as the cell grows. Moreover, some products are affected by what is produced in the methanol metabolism due to the increase of the oxidative stress in the broth and, of course, the danger associated with working with a flammable and toxic substance as methanol, which requires a special care (Waterham et al., 1997; Shen et al., 2016; Yang & Zhang, 2018). GAP promoter is optimal for continuous production processes because it allows to avoid the shift in the growth and induction phases. As Potvin et al. (2012) mentioned, there are some studies in which the effeciency of the AOX1 promoter is greater than GAP, but other studies claim just the opposite.

Yang & Zhang (2018) reported that the expression of an alkaline phytase, by using the GAP system, was eight-fold higher than that under the AOX1-based system. More studies are necessary to define under which culture conditions and products, the use of each one, is more suitable.

Alternative promoters

Despite GAP and AOX promoters are more common, there is an interest in developing alternative promoters, especially to replace the AOX J production processes because large volumes of methanol are required when bioprocess are scaled-up, besides the disadvantages mentioned above. Menéndez et al. (2003) reported the isolation and sequencing of the P. pastoris isocitrate lyase gene (ICLJ ). They tested this promoter system for the expression of dextranase from Penicillium minioluteum under the control of p/CLJ in P. pastoris. The ICLI system is based on the gene used to the Isocitrate lyase (EC 4.1.3.1) production, fundamental for ethanol metabolism in order to produce Acetil-Coa, which was used before in order to generate gluco­ se metabolic intermediates (Dunn et al., 2009). In 1998 Shen et al. (1998) isolated and characterized the gene coding for the glutathione-dependent formaldehyde dehydrogenase (FDLI ), (EC 1.2.1.1) of P. pas­ toris, which was an important enzyme involved in the metabolism for methanol, as an alternative for the AOX J expression system. They showed that Fl.DI can produce heterologous proteins at the same level as AOX J system, but with the difference that Fl.DI can be induced both of them for methanol as carbon source and methylamine as nitrogen source. FDLJ expression systems needs lower oxygen supply, too (Resina et al., 2005). A comparison of yields between GAP and AOX promoters is presented below.

Table 1: Expression of codon-optimized genes in P. pastoris, adapted from Yang et al., 2018.

*N.A.: Not available.

3. CULTIVATION TECHNIQUES

Due to P. pastoris has gained attention for the biotechnological production of different chemical compounds, companies such as lnvitrogen have developed expression kits with defined culture conditions. Other condi­ tions available in the literature are quite similar for !hose presented by Invitrogen but new trends go towards the development of specific culture conditions for every product and genetic construct (Looser et al., 2014).

A table with culture conditions of different products is sbown below (Table 2).

Table 2: Culture conditions comparison in various production processes with P. pastoris.

*N.A.: Not available.

Inthe culture technique, FB: Fed batch, and CC: Continuous culture.

P. pastoris grows well in different carbon sources as glucose, glycerol, fructose, sorbitol, ethyl amine and mannitol, among others. The doubling time depends on the carbon source, and it is typically around 90 minutes in glucose, and 6 hours in methanol. The normal growth temperature is 30ºC and 250 rpm when growing in liquid media. Por plate cultures, it is necessary to consider the evaporation of substrate if it is methanol (Sreekrisbna & Kropp, 1996).

3.1. Fed-Batch culture

Fed-Batch culture is maybe the most common production system for high-density cultures of microorga­ nisms, such as P. pastoris. The process starts with culture in a carbon source efficient for the accumulation of biomass, typically glucose or glycerol. The depletion of carl>on source is measured, and the flux of indu­ cer is started to activate the promoter chosen (Yang & Zhang, 2018). The recommended process is made up of 3 phases: 1) Glycerol or glucose batch phase, 2) or glucose fed-batch phase, 3) lnduction phase, typically with methanol. The objective of the two initial phases is to increase biomass concentration in order to reach high-cell densities. lt is important to know that reaching high yields in dense cultures is complex because the feeding of methanol processes have to be controlled in order to avoid toxicity levels for cells and to maintain an adequate flow for its consumption as an inducer and carbon source (Liu et al., 2019).

Mayson et al. (2003) used glucose as substrate for both batch and first fed-batch phases in a Mut+ system of

P. pastoris for production of transferrin in a 3L bioreactor. When induction started, the culture density was 200 g/L of wet cell weight. Glucose flow did not stop during methanol induction. 450 mg of transferrin per liter after 72 h of elapsed fermentation time was obtained as a maximum. Rodrigues et al. (2019) used the AOXJ Muts system for production of L-asparaginase from S. cerevisiae in a 3L bioreactor under substrate

limit conditions. The batch step was performed with glycerol followed by a starvation period of 1-2 hours, as well as the subsequent fed-batch step with methanol at different feeding regimes. The overall enzyme volumetric productivity obtained was 31 U/L h.

Fed-batch cultures can be performed with the GAP or other alternative promoter systems, too, for recom­ binant protein production. As GAP promoter is expressed during cell growth, it is desirable to maintain an exponential growth of biomass in order to keep the product generation in the same way. It is necessary to obtain the optimum specific growth rate (µ) in order to keep it constant with the adequate feeding flow rate (Yang & Zhang, 2018). Garcia-Ortega et al. (2013) used a GAP system in P. pastoris in order to produce a human antigen-binding fragment (Fab) in a 5L bioreactor operated in fed-batch with an initial media volume of 2L and 4L approximately at the end of the feeding. The process consisted of two phases, a batch process followed by a feeding step, both used alternatively glucose or glycerol in different fermentations and the fee­ ding regime was programmed in order to keep a specific value of µ. The overall product substrate yield was

0.081 and 0.073 for glucose and glycerol, respectively. Resina et al. (2009) used the FLDJ promoter system in P. pastoris to produce a Rhiwpus oryzae lipase-encoding gene in a 5L bioreactor operated in fed-batch. The culture was carried out in three steps: a batch growth phase, by using glycerol as carbon source and ammonium sulfate as nitrogen source, a sorbitol-batch phase when glycerol was depleted, and the addition of methylarnine as nitrogen source and a fed-batch induction phase with exponential feed of sorbitol and methylarnine, as well. Different mutant strains were evaluated, by yielding productivity levels from 602 to 3274 AU/L h (lipolytic activity units per liter per hour).

Anane et al. (2016) used an oxygen tension strategy in order to program the feeding rate in 10.5 L bio­ reactor with 8L of working volume to compare the AOX J and GAP promoter systems in the production of b-fructofuranosidase (FFase) enzyme. In GAP fermentation system, glycerol was used for both batch and fed-batch steps while in AOX J fermentation system glycerol was used for batch phase and methanol for fed-batch phase. They found that the AOX J system was better for the FFase production compared with GAP, but it required an important externa! cofactor supply not needed under the GAP promoter system.

3.2. Continuous culture

Continuous process has the advantage of further controlling fermentation parameters, especially the speci­fic growth rate if the process is carried out under chemostat conditions (Harmand et al., 2017). lt can be considered that reaching low-growth rates and high-substrate consumption can improve product yields, but decreasing feed rate can increase the possibility of cell wash if the value of µ decreases nearly to zero. Con­tinuous culture then, is less common in P. pastoris due to the limited range of dilutions rates available to get a true continuous feed to the bioreactor (Rebnegger et al., 2016). The retentostat system, described for the first time by Herbert et al. (1956) is an interesting alternative in which biomass is retained inside the bioreactor while liquid media flows through. This kind of culture, theoretically speaking, allows to main-

tain low-growth rates, but it avoids the cell damage caused by substrate depletion in long-tenn cultures.

Rebnegger et al. (2016) also demonstrated a viability of 97 % in retentostat cultures of P. pastoris when very low-growth rates (µ < O,OOlh- 1) were reached. They also demonstrated a decrease in the value of cell maintenance and a transcriptional re-programming.

Rahimi et al. (2019) carried out a comparison between fed-batch and continuous culture strategies in MUT+

P. pastoris in order to produce recombinant hepatitis B surface antigen in a 10 L bioreactor. Initially, two­ stage batch cultures were carried out, the first one with glycerol and the second one with methanol. This was done to malee a transition before feeding. The third step was a fed-batch process with methanol, and finally, the system was switched to a continuous process, by maintaining the same volume (5L). They observed a specific and volumetric productivity of 0,00468 mg HBsAg/g cell/h and 1,699 mg HBsAg/Uh, respectively, for chemostat fennentation and 0,00375 mg HBsAg/g cell/h and 1,13 mg HBsAg/IJh, respectively, for fed batch, by considering harvesting and next, run initiating time. HBsAg refers to Hepatitis B surface Antigen.

4. PROTEIN SECRETION

A key aspect that makes P. pastoris a very interesting biological platfonn for the production of recombinant proteins, is the possibility of introducing a group of specialized signals called secretion signals, which allow the product to be recovered from the broth media much more easily so that higher titers can be achieved due to the lower secretion levels of endogenous proteins (Chahal et al., 2017). Secretion signaling can be made either by native signal peptides as that of fungal xylanase or the commercial a-mating factor (MF), which is very used in P. pastoris systems (Yang & Zhang, 2018).

The first step of the classical secretory pathway is the translocation of the polypeptide in the endoplas­ mic reticulum (ER) to malee post-translational modifications (Zahrl et al , 2018). This is an important step because the overload of proteins translocating to the ER can cause an intracellular accumulation, and of­ ten, also, degradation, which results in yield decrease (Garcia-Ortega et al., 2016) even when cells as P. pastoris as other eukaryotes have the unfolded protein response (UPR) in the ER. lt is a system, which detects disturbances in the ER normal functionality and augment the secretion capability with the upre­gulation of more than 300 genes involving protein folding, glycosylation, translocation, trafficking, and degradation (Taylor, 2016; Bankefa et al., 2018). Despite that the composition of amino acids and length of signal peptides among the secreted proteins in same species are highly diverse, and it would be hard to predict whether a secretion sequence will be able to translocate a protein to secretion pathway (Karaoglan et al., 2014). There are few sequences, which have been characterized and applied for secretion of proteins in addition to the Saccharomyces cerevisiae a-mating factor prepropeptide (MATa) (See Table 3). The S. cerevisiae SUC2 gene signal sequence, the bovine J3-casein, and the native acid phosphatase (PHOI), (EC 3.1.3.2) signal peptide was occasionally used only (Kang et al., 2016). This last secretion signal sequence shows significant hornology to repressible PHO from other yeast species, such as Saccharomyces cerivisae.

Payne et al. (1995) dernonstrated that PHOl is translocated into the ER, glycosylated, and transported to the cell surface in about 5 minutes. Regarding AOXl promoter, the PHOl promoter has the advantage that it can be induced about 100-fold under conditions that do not significantly alter cell growth or morphology, so that the regulated PHOl promoter will be a useful alternative tool for heterologous gene expression in

P. pastoris. (Payne et al., 1995). Sorne proteins have been produced, by using this secretion systern. Por example, Ben et al. (2016) showed that in the production ofrabies virus, glycoprotein (RABV-G) in P. pas­ toris, by using PHOl and the arnating factor despite that the expression leve!of secreted RABV-G was similar, the PHOl signal presented higher protein degradation rates in comparison to a-mating factor (Ben et al., 2016). A similar result was obtained by (Tanaka et al., 2004), who demonstrated that amazing factor signal peptide resulted in two-fold higher xylanase yield than PHOl signal sequence into growth media. Therefore, it is evident the necessity to explore the a-mating factor further because of its capacity to export different proteins as it has been previously shown.

The MKf a signal leader contains a preregion with 19 amino acids and a pro-region with 67. This is the most widely used secretion signal used in P. pastoris, being, even in sorne cases, better than the leader sequence of the native signal peptide. However, variability in the N-terminal amino acids are reported in heterologous protein secreted with this signaling systern and, in sorne cases, the protein is retained in the ER or Golgi apparatus, by impeding transport, and triggering degradation (Macauley-Patrick et al., 2005; Chahal et al , 2017). Modifications have been proposed in order to enhance the secretory efficiency of the MATa, by using different approaches as codon optimization, directed evolution, in addition to spacer sequences, and site-directed mutagenesis (Duan et al., 2019).

Table 3: Signa! peptides used for secretion of enzymes in P.pastoris, adapted from Kang et al., 2016

5. RECOMBINANT PROTEINS IN COLOMBIA

According to Zapata et al. (2012) the Colombia's health system faces some issues that the government should solve first in the near future. Within the main topics, it was included the price of some drugs, which are included in the Mandatory Health Plan (POS for its acronym in Spanish "Plan Obligatorio de Salud"). Among the highest prices registered in it, there were drugs developed by biotechnological processes, which represents a 12 % of the global drug sales, according to a market study published in Colombiacompra.gov.co (2019). The same market study shows that, the National Cancerology Institute in Colombia registered a ca­ pital expenditure in the 2016-2017 period up to 5,7-dollar millions for the purchase of the 10most expensive drugs. It is estimated that, in 2013, approximately, 36 % of the total pharmaceutical market, in Colombia, corresponded to drugs of biological origin, which included sorne for cancer treatment, such as breast cancer and other expensive diseases. As for biosimilars, it is very important to highlight that the National lnstitute of Cancerology of Colombia is currently conducting ali the conventional tests for radiopharmaceuticals, by using nuclear medicine and Positron Emission Tomography (PET), (Vaca-González et al., 2019).

For the reasons mentioned above, not only has the government of Colombia, but also ali governments around the world, begun the search for affordable treatments, which allows a more integral health coverage (Blanco­ García et al., 2018). A very attactive solution is the use of biosimilar drugs, discussed broadly by

(Uhlig & Goll, 2017; Kaida-Yip et al., 2018; Nabhan et al., 2018). However, still some concems remain in the sense that these alternatives should be overcome. The topics may vary from technical issues like adverse effects (Odinet et al., 2018; Colloca et aL, 2019) to legal process related to the specific legislation, which varies from country to country (Tesar et al., 2019; Joung, 2015), as well as patents and intellectual property (Moorkens et al., 2016).

Broadly speaking, a biosimilar drug should have a high degree of similarity with regard to the reference me­dicinal product in terms of quality features, biological activity, safety, and efficacy, based on a comprehen­sive comparability exercise, which needs to be established (European Medicines Agency, 2014). These requirements should be regulated by specialized institutions for drug control and distribution, such as the Food and Drugs Administration (FDA) for the United States, the European Medicines Agency for Europe, in Colombia, one remarkable institution with regard to drug control is the National Food and Drug Survei­llance Institute (INVIMA, which has been recognized as a national drug regulatory authority of Reference for Drugs and Biological components (Ojeda et al., 2016). This latter entity plays an important role because it is in charge of allowing or denying the circulation of ali bioproducts developed or distributed throughout the Colombian territory. According to the resolution 1782 as of 2014, Article 6, published by INVIMA, there are 3 routes in which a drug can be approved, in Colombia, for new or pioneer biological drugs. The so-called ful!dossier route was established, which requires the submission of pre-clinical studies (in-vivo and/or in-vitro) and clinical trials. In addition to the fact that they must comply with the common require­ments for their relevant authorizations (Bernal-Carnargo et al., 2018).

In the biotechnological drugs case, there are some expensive ones, such as transcription factors and mo­noclonal antibodies, which have high usage rates (Manrique-López & Jiménez-Barbosa, 2012). Ithas been seen that these molecules are extremely complex, and their biological activity is because of a spatial complex arrangement, which allows to carry out a reaction with a specialized target (Putz et al., 2016). These mole­cules can be synthesized by biological platforms as is shown in Meza-Gutierrez et al. (2019). This will allow that recombinant proteins, which are produced throughout microorganisms can be used for biosimilar drug production (Baeshen et al., 2014) as is the case of the recombinant insulin (Heinemann & Hompesch, 2014), which is likely to become widely available in the coming years, and are expected to have a major impact on diabetes care (American Diabetes Association, 2019) . However, the economic assessment of the impact that these drugs could have on the market remains unclear due to the fact that there are considerable limitations in terms of the range of included costs and reliance on assumptions canied out in the economic analysis instead of robust data for feeding the models (Simoens et al., 2017).

Likewise, it has been demonstrated that P. pastoris can be used into the recombinant insulin production, by gaining a save in time and economic terms, by yielding an overall output from insulin precursor to human insulin of 51% (Polez et al., 2016). Rodríguez-López et al. (2019) and Rodríguez-López et al. (2019) con­ ducted two quite similar studies in which N-acetylgalactosarnine-6-sulfate sulfatase (GALNS), an enzyme used to treat the Morquio A syndrome, was produced in a 3,7L bioreactor. Also, Espejo-Mojica et al. (2016) showed that the enzyme can be internalized and reach the lysosome in mammalian cells while Rodríguez­ López et al. (2019) achieved an important improvement in the enzyme activity after removing a native signa! peptide and also observed that human skin fibroblasts took it up in a dose-dependent manner through a process potentially mediated by an endocytic pathway without any additional protein or host modification. Finally, Iduronate-2-sulfate sulfatases, a group of enzymes linked to the Hunter syndrome, have been ex­ pressed in a lOOmL of media culture and its activity was reported as 4,213 nmol/mg h of total protein-1 at 72 h (Landázuri et al., 2009). The same k:ind of enzyme was cultured in 3L bioreactor by Poutou-Piñales et al. (2005), by yielding activities between 25,4 y 29,36 nmol/mg h and by Córdoba-Ruiz et al. (2009) who obtained an activity between 7,3 and 29,5 nmol/mg h. Pimentel et al. (2018) studied this enzyme, too, in a 3,7L bioreactor, by yielding 12,45 nmol/mg h, showing a dose-dependent cell uptake.

Regarding enzymes with environmental applications, laceases, originally, produced from different orga­nisms, have also been studied and produced, by using P. pastoris at different volume scales. Sorne authors fo­ cus on the improvement of culture conditions (Ardila-Leal et al., 2019; Gouzy-Olmos et al., 2018; Morales­ Álvarez et al., 2017); while others research different applications, such as the detoxification of pulping black liquor, and still sorne others, in water treatments (Rivera-Hoyos et al , 2018). Other lysosomal products as

¡3-hexosarninidases are important for Tay Sachs or Sandhoff disease treatment, and they have been produ­ced and characterized in P. pastoris, by yielding important results that demonstrated the potential of this expression system (Espejo-Mojica et al., 2020). Table 4 surnmarizes sorne of the Colombian research papers related to P. pastoris applied to recombinant proteins, most of them mainly conducted by "Pontificia Universidad Javeriana".

Table 4: Colombian research papers developed with P. pastoris as expression system

Therefore, the national panorama shows that there are few research groups actively working with P. pastoris and many of them are oriented towards enzyme production of whose altered function generates diseases. In Colombia, there are still many approaches and developments that can be made in order to establish solid stu­dies on this important system of expression, which, as has been mentioned, presents great advantages, which could direct research to the industrial sector that has been little addressed in the country. It is important to emphasize that a great part of the research is concentrated on "Pontificia Universidad Javeriana" because it has a long-standing tradition, and severa! publications related to P. pastoris as a system of expression. At this moment, the country is putting efforts to produce biosimilar drugs inside the country to grapple with the high cost of imported health treatments, There are few papers available oriented towards the production of biosimilars in Colombia, but some of them are starting to medium and large-scale production, in which marnmalian cell cultures are being used (Díaz et al,, 2020).

6. CONCLUSIONS

This paper reviewed the features, which make P. pastoris a suitable model for the development of recombi­ nant proteins. From the advantages, which were presented by this kind of system, it was possible to see how the well-studied promoter system can be used with fed-batch (maybe the best option) or continuous culture in order to produce different heterologous proteins, by taking advantage of the secretion feature, which can contribute to easily recover proteins after culture. AOX promoters are the most used, as well as the most studied since they represent a very good option for the development of expression systems.

References

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