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 various Temperature Control: heterologous proteins of biopharmaceutical importance The heat Expressed Protein Titer (g/L) Reference production rates for glycerol and Vaccines methanol substrates, respectively, are Tetanus toxin fragment C 2.6 and 4.5 kcal/g substrate consumed/ 12 7 Heavy chain botulinum neurotoxin serotype A 1.72* 8 hour. To remove excess heat, especially Antibody fragments during the induction phase in high cell A33scFv 4 9 density cultures, autoclavable laboratory Anti-HB Fab 0.05 10 fermentors must have temperature con- Anti-HbsAg Fab 0.46 11 trol systems using jacketed culture vessels Hormones and closed-loop recirculation systems, Human parathyroid hormone 0.3 12 with provisions to connect an external Cytokines chiller when needed. For sterilizable- Human tumor necrosis factor 10 13 in-place fermentors, a dual heat- Bovine interferon gamma 1.0 14 exchanger design with a connection to Ovine interferon tau 0.4 15 a chilled water or glycol loop is Matrix proteins preferred. Mouse gelatin Oxygen supply 14.8 16 is achieved by proper Human collagen I–III 0.2–0.6 17 blending of air and oxygen using either * Intracellular protein, expression level per gram of cell paste gas flow ratio control (GFRC) or conventional oxygen supplementation. Table 3: Common Pichia pastoris strains used for heterologous protein expression as they differ GFRC allows the total gas flow rate to in genotype, phenotype, promoter, and induction be fixed and the ratio between air and Strain Genotype Phenotype Promoter Induction oxygen to vary depending on the Y-11430 Wild-type NRRL* Aox1 and Aox2 Methanol oxygen demand of a culture. The GS115 his4 Mut+ His– Aox1 and Aox2 Methanol oxygen consumption rates for glycerol KM71 Aox1::Sarg4 his4 arg4 MutS His– Aox1 disrupted Methanol and methanol substrates are – – MC100-3 Aox1::Sarg4 Mut His Aox1 and Aox2 Glycerol and respectively 13 and 35 mM O2/g of Aox2::Phis4 his4 arg4 disrupted methanol substrate consumed/hour. SMD1168 pep4 his4 Mut+ His– Aox1 and Aox2 Methanol The common method of adding protease A deficient methanol using a septum and needle is SMD1165 prb1 his4 Mut+ His– Aox1 and Aox2 Methanol not recommended because methanol protease B will then pool and release in large deficient droplets, leading to erratic dissolved * Northern Regional Research Laboratories, Peoria, IL oxygen (DO) profiles. That can easily be avoided by using a dip tube that is commonly used expression host, has small percentages of lysed cells, fully immersed in the culture. For the functional Aox1 and Aox2 genes and released proteases appear to be a same reason, a variable-speed pump is grows on methanol at the wild-type significant factor in protein recommended over fixed-speed pumps rate. The popularity of GS115 is based degradation. The use of host strains for dosing methanol because it does on its ability to grow to high cell deficient in such proteases helps not need pulsing to achieve the densities in large-scale fermentors. alleviate such enzymatic degradation. required volumetric quantities. All unique His– phenotypes were For example, the SMD1168 and Ammonium hydroxide is used to developed for selection purposes and are SMD1165 strains are deficient, control pH and also serves as the auxotrophic for histidine. They therefore respectively, in protease A, which is nitrogen source. Ammonium require medium supplementation for encoded by pep4, and in protease B, hydroxide and methanol are volatile. growth unless His+ expression cassettes which is encoded by prb1. They diffuse readily through silicone are used. In the KM71 host, a derivative and other types of tubing, causing of GS115, the chromosomal Aox1 gene FERMENTOR DESIGN AND gaps of air. That can result in irregular is largely deleted and replaced with the OPERATIONAL PARAMETERS methanol dosing leading to erratic S. cerevisiae arg4 gene resulting in The use of P. pastoris for high cell DO profiles. We recommend the use dependence on the much weaker Aox2 density culture and protein expression of polytetrafluoroethylene (PTFE) or gene. The MC100-3 strain is deficient requires careful evaluation of various equivalent tubing throughout the for both Aox1 and Aox2 and cannot fermentor design aspects and operating process except for a small piece of grow on methanol. parameters as described in Table 4 (28, chemically compatible flexible tubing Some secreted heterologous 33). Photo 2 shows a typical system to fit in the peristaltic pump head. proteins are unstable in culture media suitable for process development and where they are rapidly degraded by small-scale production. Two critical INOCULUM PREPARATION proteases. In high-density fermenta- design aspects are temperature control AND GROWTH MEDIUM tion cultures, which typically contain and oxygen supply. The initial inoculum, 5–10% of the 26 BioProcess International JANUARY 2006 Table 4: Fermentor design aspects and operational parameters for Pichia pastoris initial culture volume, is typically (vvm = volume of gas/volume of culture/min) prepared in baffled shake flasks Inoculation 5–10% of Total Fermentor Volume containing minimal glycerol (MGY) Agitation Up to 1000 RPM for autoclavable lab-scale systems; requires Rushton or buffered minimal glycerol (BMGY) impellers and four removable baffles to support high oxygen transfer rate. medium. MGY medium contains Temperature 30 °C optimal; requires jacketed vessel for efficient heat removal using high primarily a yeast nitrogen based cell density processes using Mut+ strains and eventually additional chiller. (YNB) medium, typically 1% glycerol Single wall vessel and cooling finger recommended only for low cell density of Mut+ and for MutS and Mut– strains only. Growth above 32 °C is and small amounts of biotin. BMGY detrimental to protein expression. medium contains peptone and yeast Gassing rate 1–1.25 vvm total gas flow; requires multiple large-orifice ring sparger. extract, which may help stabilize Higher gas flow rates are not recommended due to increased methanol secreted proteins. evaporation. A culture is initiated with Gas Mix Air and oxygen; through either gas flow ratio control using one mass flow P. pastoris cells derived from a frozen controller per gas or oxygen supplementation control with one mass flow vial retrieved from the cell bank and is meter for total gas flow incubated in an orbital shaker at Dissolved 35% air saturation; requires sequential cascade control programming to oxygen agitation, gas mix, and substrate. 30 °C, 250–300 rpm, for 16–24 hours until OD of 2–6 has been reached pH 5.0 ± 0.1 in glycerol batch and fed-batch phase, 2−5 in methanol induction 600 phase depending on protein stability (27). This culture provides inoculum Substrate 1 Glycerol 50%, feed rate 15 mL/hr/L culture volume; requires fixed or for the seed train, whereby each variable speed peristaltic pump for volumetric feed, and optional weighing fermentor in turn inoculates the next scale for gravimetric feed, or pressure addition system. larger reactor, providing a total of Substrate 2 Methanol 100%, feed rate 1–12 mL/hr/L culture volume for Mut+ and 5–10% of the fermentation volume in 1–6 mL/hr/L culture volume for MutS strains; requires variable speed each step and representing scale-up peristaltic pump with PTFE tubing or equivalent for volumetric feed, optional weighing scale for gravimetric feed, or pressure addition system. factors in the range of 10–20 up to the Fully immersed dip tube in culture medium is required. production 80-m3 fermentor. Methanol 0.4–4% for Mut+ and MutS strains, 0.2-0.8% for Mut– strains and 0.5% for The preferred fermentation growth Mut–. On-line methanol sensor or off-line analytical analyzer is medium consists of a basal salts (BS) recommended. medium containing 4% glycerol Antifoam 5% Struktol JA 673 in 100% methanol, KF0673 Kabo Jackson or equivalent; complemented with a trace element requires fixed-speed peristaltic pump. solution (27). The trace element solution Base 30% NH4OH; requires fixed-speed peristaltic pump and PTFE tubing or is filter sterilized before use, whereas the equivalent. BS medium is heat sterilized in the fermentor. The ability of P. pastoris to host phenotype. Sampling is typically strategy can be used with one mass grow in simple, inexpensive, chemically performed twice daily for analysis of flow controller for total gas flow. For characterized media without animal- cell growth at OD600 and recombinant most installations, the agitation range derived growth factors or other protein concentration or activity set in the DO cascade controller is troublesome media components (e.g., during phase III. typically between 30% of minimum serum) alleviates some of today’s Phase I is the glycerol batch culture and maximum rpm. At the end of the regulatory concerns and makes it to create biomass. A maximum initial batch phase, when the initial amount of considerably more cost effective than concentration of 4% glycerol is recom- glycerol in the fermentor is completely complex chemically defined (CD) mended because of cellular toxicity exhausted, oxygen demand will sharply mammalian cell culture media. associated with higher levels. The decline. Setting the DO controller to initial culture volume is 65–75% or “off” will result in a sharp rise of the FERMENTATION PROTOCOL 45–60% of the total reactor volume for DO process value (DO spike). But AND CONTROL STRATEGIES induction phases of less or more than with the DO controller active, the The standard culture protocol is to 40 hours, respectively. DO is typically DO spike will be masked by a rapid grow P. pastoris in excess glycerol to controlled through a sequential control decline of the amount of oxygen in repress protein expression, followed by cascade programmed first to agitation the gas mixture, which is an essential methanol induction to initiate and subsequently to either GFRC or attribute of modern PID (proportional- heterologous protein expression as the oxygen supplementation, depending integral-derivative) controllers. glycerol becomes exhausted. Figure 2 on the preferred air and oxygen gas Nevertheless, the oxygen decline can shows a schematic lay-out for a typical blending strategy. GFRC gas blending be readily monitored in the GFRC fermentor set-up. Protein expression is often preferred because it not only configuration. A cellular yield of 90– with all three phenotypes is performed maintains a constant total gas flow, 150 g/L wet cells is expected at the using a culture strategy in three but also monitors and controls both air end of phase I. phases (Figure 3). Note that the first and oxygen individually through mass Phase II is the glycerol fed-batch two phases are the same for all flow controllers. culture, which limits glycerol, thereby phenotype expression hosts, with Alternatively, a more conventional derepressing the methanol metabolic phase III depending on the specific oxygen supplementation gas mixing pathways and allowing cells to 28 BioProcess International JANUARY 2006 Photo 2: Biostat B Plus laboratory fermentorin a twin configuration with gas Once DO has raised by 10%, the preprogrammed dosing profile, or flow ratio control for air and oxygen suitable for Pichia pastoris process development and glycerol feed pump can be turned on manually by adjusting set points) in small-scale production. SARTORIUS BBI SYSTEMS, again. If a DO cascade control for increments of 1 mL/hr/L of culture BETHLEHEM, PA (SARTORIUS-BBI-SYSTEMS.COM) glycerol is unavailable, it is also possible volume every one to two hours until to add glycerol at a fixed rate of achieving a feed rate of 11 to typically 15 mL/hr/L of culture 12 mL/hr/L. To maximize protein volume. As long as glycerol is added at expression levels, the methanol feed a fixed rate, the rate of oxygen use will rate should be brought to maximum as also be fixed, allowing DO control quickly as possible, but optimum through either GFRC or oxygen induction time profiles can be derived supplementation as described earlier, only through analysis of time-based with the exception of deleting agitation samples (35). For many recombinant from the control cascade. At the end of proteins, a direct correlation between phase II, a cellular yield of 180–220 the amounts of methanol consumed g/L wet cells should be achieved. and protein produced has been Phase III is the methanol fed-batch observed (36). That observation can be culture or induction phase, which extrapolated to the consumption of Table 5: Selected physical properties of differs according to each expression other carbon sources as well as to methanol host phenotype. In all cases, glycerol cases of mixed feed strategies. Boiling point 64.7 ºC must be exhausted before methanol In recent years sensor technology Flash point, closed cup 12.2 ºC feed starts. This can be accomplished has become available to directly Flash point, open cup 15.6 ºC by turning off the glycerol pump and measure methanol in situ, thereby Explosion limits, vol% in air 5.5–44 DO cascade control allowing the DO preventing starvation or intoxication of to rise from 30% to 100% (DO spike). cultures (9, 37, 38). In this way a specific Relative density to water 0.7915 Note that in the case of a Mut+ host methanol concentration of 0.4–4.0% Solubility in water Miscible strain it may take one to three hours can be maintained in culture using a Vapor pressure at 20 ºC 12.3 kPa for DO to stabilize back at 30%. If the feedback control loop with a methanol Relative vapor density to air 1.1 DO cascade remains active, the substrate pump. Mut+ strains are very amount of oxygen in the blend mixture sensitive to higher methanol will decrease over time. To ensure that concentrations, and if the DO drops transition smoothly from glycerol to methanol does not accumulate during below 15% due to an excess of methanol growth (34). Because it is the induction phase, frequent DO methanol, the methanol feed pump critical that DO and oxygen flow rate spikes can be performed manually if must be turned off to allow the culture are properly balanced in phase II, the needed. The DO cascade control to metabolize it. An intuitive operator DO cascade control should be changed strategies used in phase II with air and response would be to increase the only at the end of phase I either after a oxygen gas blending through either oxygen flow rate, but a sudden increase DO spike or in its absence if the GFRC or oxygen supplementation can in oxygen in the presence of an excess oxygen flow rate has sufficiency still be used, but it is quite common to amount of methanol (1–2%) can result declined. At this point the DO cascade maintain fixed set points for air and in accumulation of toxic levels of can be changed to substrate (glycerol) oxygen gas flow rate and blend ratio to formaldehyde (the first product of supplementation at a growth-limiting ensure that oxygen is in abundant methanol metabolism in the rate, while new fixed set points for supply and methanol will be added at a peroxisomes), leading to cell death (21). both air and oxygen flow rates and gas growth limiting rate. It is also possible The induction protocol for the MutS ratios ensure that oxygen is in to program a cascade to substrate strains is the same as for the Mut+ abundant supply. The total gas flow (methanol). In any case, methanol strains except for a lower methanol feed remains at 1–1.25 vvm. The agitation levels in the fermentor are critical and rate. That rate is initiated at 1 mL/hr/L speed is typically set between 80 to must be carefully controlled. At the of culture volume and subsequently 100% of maximum rpm. end of the induction phase, a final increased in 10% increments every 30 DO should never be allowed to cellular yield of 350–450 g/L of wet minutes to a maximum of 3 mL/hr/L drop below 20% of air saturation; cells can be obtained. of culture volume. The methanol therefore, appropriate alarm thresholds The most common induction concentration must remain within 0.2– should be programmed in the protocol is to use a progressive 0.8% for MutS strains (39, 40). DO controller. Or to ensure growth- methanol dosing profile. For Mut+ spikes cannot be used effectively to limiting feed of glycerol, feed pumps strains this feed rate is typically evaluate cultures because oxygen can be shut off manually and the time initiated at 3.5 mL/hr/L of culture consumption is too slow with methanol to allow DO to rise by 10% measured. volume. Once a culture adapts to its as the sole carbon source. An alternative If the lag time for the DO spike is current level of methanol, which may induction strategy for MutS strains is to short, generally less than one minute, take two to five hours, the feed rate is use a mixed methanol/glycerol glycerol is assumed to be limiting. increased (using either a (delivered in g/hr) feed, e.g., in ratios Figure 2: Schematic layout of a P. pastoris fermentation system. The Figure 3: Model growth curve of Pichia pastoris Mut+ host strain in optical fermentor is equipped with a powerful motor (1), exhaust condenser (2), foam density (OD) units/mL at 600 nm (left) and dissolved oxygen (DO) set point probe (3), multiple Rushton impellers (4), a multiorifice ring sparger (5), and in % air saturation (right) over the time course of the fermentation in hours; sensors for temperature, pH, DO, and methanol (6). Air and oxygen are added graph features three distinct culture phases: glycerol batch (16–24 hours), through individual solenoid valves (7) and mass flow controllers (8). The liquid glycerol fed-batch (four hours), and methanol fed batch (18–96 hours)

substrates methanol (MeOH) and glycerol as well as base (NH4OH) and along with a typical methanol feed profile in mL/hr per L culture volume antifoam (AF) are added through peristaltic pumps (9). Glycerol and methanol for a Mut+ host strain. The DO value is basically kept constant at 35% except substrate addition bottles and/or the entire culture vessel are put on weighing for multiple DO spikes to evaluate the state of the culture. scales (10). Fixed lines (—): both actual soft or hard piping; dotted lines (----): � electrical wiring from actuators and sensors to the controller.

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ranging from 4/1 to 1/4 (34). Some its important physical properties. guidelines from industry empirically determined protocols The typical methanol organizations, and in-house achieve the same effect by gradually concentrations in fermentation broth occupational health and safety changing the proportion of methanol in are too low to raise any particular programs (43–45). a feedstream. Because mixed feed safety concerns, leaving the actual strategies reduce the overall cooling and transport, handling, and storage of A COST-EFFECTIVE HOST oxygen requirements for fermentation, large amounts as the predominant Significant improvements in this feature may become of particular hazard. Because methanol is widely glycosylation engineering have further interest in evaluating fermentor design. used as an industrial and laboratory propelled P. pastoris as a cost-effective Mut– strains require an alternative solvent, many safety recommendations expression host for the manufacture of carbon source — glycerol or sorbitol, and best practices have originated in various vaccines and for example (41). Despite the fact that the chemical industry. Methanol biopharmaceuticals, including methanol cannot be metabolized by should be stored in clean containers biogenerics. Understanding and Mut– strains, its presence will still made from carbon steel, stainless steel, applying the molecular biology and induce transcription of the Aox1 high-density polyethylene (HDPE), or protein chemistry foundations to promoter and expression of genes vulcanized natural rubber. Storage select expression vectors and hosts is regulated by it (42). However, excess containers should be constructed with prerequisite to successful glycerol can repress the Aox1 gene, and an internal floating roof and an inert implementation of this fermentation the induction strategy for Mut– strains gas pad to minimize vapor emissions. strategy, which in itself cannot be follows the mixed feeding strategy for Compressed air should never be used developed without a careful evaluation MutS strains. Glycerol is fed at 1 g/hr/ for filling, discharging, or handling. of the design and operational L of culture volume under growth- Inert gas (e.g., nitrogen) can be used, parameters of fermentor systems. limiting conditions, and methanol is but pumping is generally preferred. Critical design considerations are maintained at 0.5% during the Standard preventive measures to temperature control, oxygen induction phase (40). An on-line alleviate explosion risk are the use of requirements, and methanol feeding methanol sensor is required here closed fermentation systems, algorithms, along with facility because DO spikes cannot effectively ventilation and explosion-proof considerations associated with the use measure methanol limitation. electrical equipment and lighting, and of large quantities of methanol. nonsparking hand tools. The explosion FACILITY CONSIDERATIONS risk can also be addressed by using ACKNOWLEDGMENTS I would like to thank Dr. Scott M. Brown at The most important facility well-mixed methanol–water feeds with Biologics Process Development, Inc. and William consideration that arises when using increased the flash points. The Whitford at HyClone Research and Product P. pastoris involves the safe use of principal disadvantage of that would be Development for their suggestions and methanol. A clear, volatile, colorless, increased feed volumes during comments in the preparation of this manuscript. toxic, flammable liquid with a faint odor fermentation. REFERENCES like alcohol, methanol can pose a serious For further information please fire hazard. It mixes well with air, easily consult international, federal, state, 1 Wegner GH. Emerging Applications of the Methylotrophic Yeasts. FEMS Microbiol. forming explosive mixtures. Table 5 lists and local regulations, best practice 30 BioProcess International JANUARY 2006 Rev. 7(3–4): 1990: 279–283. 17 Myllyharju J, et al. Expression of Development of Recombinant Pichia pastoris 2 Hagenson MJ. Production of Recombinant Human Type I-III Collagens in Expressing the Heterologous Gene: Bovine Recombinant Proteins in the Methylotrophic the Yeast Pichia pastoris. Biochem. Soc. Trans. Lysozyme. Ann. NY Acad Sci. 589, 1990: 350–363. Yeast Pichia pastoris. Bioprocess Technol. 12, 28(4) 2000: 353–357 35 Sheldon JB, et al. 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