B IO P ROCES S TECHNICAL Production of Humanlike Recombinant Proteins in Pichia pastoris From Expression Vector to Fermentation Strategy Christian Julien ver since Invitrogen of Carlsbad, effective humanlike therapeutic CA (www.invitrogen.com) protein manufacturing systems. The acquired the distribution rights goal is to achieve high fermentation Ein 1993 for the Pichia pastoris yields in processes that extend for yeast protein expression system, days instead of the weeks required P. pastoris has gained widespread for mammalian expression systems. popularity, as witnessed by an Today the P. pastoris system is licensed increasing number of publications to more than 160 companies in the (Figure 1). The expression system was biotechnology, pharmaceutical, originally developed as a single-cell vaccine, animal health, and food protein production platform by Philips industries. More than 500 Photo 1 JAMES CREGG, KECK GRADUATE INSTITUTE, Petroleum of Bartlesville, OK, and heterologous proteins have been CLAREMONT CA (WWW.KGI.EDU) later adapted for heterologous protein expressed in this host (3, 4). expression (1, 2). It is freely distributed The first of those proteins entered references 7–17 are called out in among academic research laboratories, human clinical trials in 1996, followed Table 2). The dosage/vial of 12.5 g is although its use for commercial by an increasing number of candidate extremely high compared with other production requires a license from therapeutic proteins and antigens (5). biopharmaceuticals and truly represents the current patent holder, Research All yeast-based biopharmaceutical a testimony to the high expression Corporation Technologies of Tucson, products currently on the market in levels of P. pastoris and establishment AZ (www.rctech.com). the United States and Europe are of large-scale fermentation technology: Renewed interest in yeast and manufactured in Saccharomyces up to 80,000-L reactors. fungal expression systems in general cerevisiae; however, a recombinant and the P. pastoris expression system DNA hepatitis B vaccine and AN EMERGING EXPRESSION SYSTEM in particular has been spurred by a interferon alpha derived from P. pastoris P. pastoris is a robust unicellular growing demand for scalable and cost- have been marketed in India since methylotropic yeast (Photo 1). It 1999 and 2002 respectively by Shanta combines the unique advantages of PRODUCT FOCUS: THERAPEUTIC PROTEINS Biotech (www.shantabiotech.com). prokaryotic growth characteristics and Similarly, a recombinant human insulin expression levels with the ability to PROCESS FOCUS: PROTEIN EXPRESSION was approved in India in 2003 and perform posttranslational protein (PRODUCTION), FERMENTATION, SCALE-UP marketed by the joint venture of Shanta modifications available only in WHO SHOULD READ: PROCESS Biotech and Biocon (www.biocon.com). eukaryotic systems (Table 1). Several DEVELOPMENT, MANUFACTURING, Bipha, a subsidiary of Yoshitomi heterologous proteins expressed in AUTOMATION, FACILITY PLANNERS Pharmaceutical (now Mitsubishi P. pastoris have reached expression Pharma Corporation) has been levels as high as grams per liter. KEYWORDS: P. PASTORIS, METHYLOTROPIC manufacturing recombinant Human Table 2 lists several therapeutically YEAST, METHANOL UTILIZATION PHENOTYPES, Serum Albumin (rHSA) since 2000 relevant proteins — including vaccines, AOX1 AND AOX2 PROMOTERS, using P. pastoris in its Chitose, Japan, antibody fragments, hormones, GLYCOSYLATION ENGINEERING, BATCH AND factory. The capacity of this plant is an cytokines, and matrix proteins — along FED-BATCH, DO CASCADE, METHANOL SENSOR impressive one million vials of rHSA with their achieved expression levels. a year, the eq adually increase capacity In the absence of glycerol, LEVEL: INTERMEDIATE to over 40 tons a year (6; subsequent P. pastoris uses methanol as its carbon 22 BioProcess International JANUARY 2006 Figure 1: Publications featuring the Pichia pastoris expression system from 1990 to 2005, estimated source. A strong alcohol oxidase through linear extrapolation based on actual data for the first nine months (PubMed) � promoter (alcohol oxidase 1, Aox1) is ��� induced by methanol but repressed in the presence of excess glycerol. The ��� regulation of the Aox1 gene is similar ��� to that of the gal1 gene for S. cerevisiae in that the control appears to involve ��� ������������ two mechanisms: a catabolyte �� repression/depression mechanism and an induction mechanism. � �� �� �� �� �� �� �� �� �� �� �� �� �� �� �� �� EXPRESSION VECTORS �� �� �� �� �� �� �� �� �� �� �� �� �� �� �� �� FOR HUMANLIKE PROTEIN Several genes in the methanol pathway, Table 1: Pichia pastoris expression system encompasses advantages of both prokaryotic (A–G) and eukaryotic (H) culture systems. including the highly efficient Aox1 promoter, have been cloned (18–21) to A Chemically characterized media consisting of a basal media formulation complemented with trace elements, entirely protein free, avoiding concerns with animal-derived media develop various inducible or constitutive components and offered at a fraction of the cost of mammalian cell culture media. expression vectors that produce B High growth rates from 0.04 to 0.1 μ per hour (μ = 1/x * dx/dt, where x is concentration recombinant proteins at levels equivalent and t is time). to those of alcohol oxidase (22). In C High expression levels due to its strong alcohol oxidase 1 (Aox1) promoter, which can addition, Invitrogen has developed make up as much as 35% of the soluble protein of the cell with typical expression yields multicopy expression vectors with in excess of 1 g/L. Expression levels are protein and fermentation protocol specific and various selection markers. Both have ranged from 20 mg/L to 14.8 g/L. inducible and constitutive expression D High cell densities in excess of 100 g/L dry cell weight. constructs integrate into the P. pastoris E Easy scalability due to robust nature of P. pastoris, which withstands large shear forces genome. As with S. cerevisiae, typical in large-scale industrial fermentors. P. pastoris has been scaled to 80,000-L recombination between the expression fermentors. construct and regions of homology with F Easy purification in case of secreted heterologous proteins; the purification process is P. pastoris can lead to stably transformed simplified because P. pastoris does not secrete high amounts of endogenous protein. host cells even when harboring multiple G Less expensive and easier to use than mammalian expression systems. gene copies, although the exact H Mammalian-like proteins due to posttranslational modifications, including proteolytic mechanism of multiple integration processing, folding, disulfide bonds, and O- and N-linked glycosylation, are further events is not fully understood (19, 23). enhanced by glycosylation engineering. Expression vectors with signal sequences that allow secretion of (29–32). The technological methanol at the wild-type rate. Its heterologous proteins into the culture breakthrough in incorporating a faster methanol growth rate compared medium to facilitate downstream limited humanlike glycosylation with Aox-defective strains translates processing represent a very interesting pathway in P. pastoris provides to faster production rates of prospect for integrated bioprocessing. unprecedented opportunities for heterologous protein. The methanol S This is an exciting technological link biopharmaceutical manufacturing. utilization slow (Mut ) phenotype between cell cultivation and primary Full humanization of the glycosylation contains a disrupted Aox1 gene; its downstream unit processing pathway in P. pastoris is only one step methanol metabolism depends solely operations up to and including away from completion (5). But the final on the transcriptionally weaker Aox2 purification in the manufacture of step encompasses transfer of sialic acid gene, resulting in slower growth rates. biotechnological products (24). The and promises to be challenging because The methanol utilization negative – result is a highly productive and cost- a source of endogenous sialic acid is (Mut ) phenotype has both Aox genes effective expression system that has unknown for yeasts. The importance disrupted and, as a result, cannot been successfully applied to many of terminal sialic acids is highlighted metabolize methanol. Optimal protein recombinant proteins of distinct by the often rapid clearance of expressions have been reported in all structure and function (25–27). incompletely sialylated recombinant three phenotypes, so it is difficult to It should be noted that O- and proteins by the liver. predict which phenotype will best N-linked glycosylation in native express a protein of interest. P. pastoris differ from glycosylation COMMON HOST STRAINS Therefore, multiple expression vectors occurring in mammalian cells. P. pastoris strains vary with respect to and host strains are often evaluated However, recent improvements in their ability to use methanol, leading and screened. glycosylation engineering have to three principal phenotypes. Table 3 lists several of the common produced strains that can express The methanol utilization positive P. pastoris strains. The Y-11430 wild + homogenously glycosylated and (Mut ) phenotype has functional Aox1 type is unsuitable for heterologous humanized recombinant proteins and Aox2 genes and grows on protein expression. GS115, the most 24 BioProcess International JANUARY 2006 Table 2: Examples of Pichia pastoris expression levels (g/L) obtained in fermentors for