Process Sensing and Control

Biotechnology Progress DOI 10.1002/btpr.1990

Maximizing Recombinant Human Serum Albumin Production in a Muts Pichia pastoris Strain

Muralidhar Mallem*, Shannon Warburton*, Fang Li, Ishaan Shandil, Adam Nylen, Sehoon Kim, Youwei Jiang, Michael Meehl, Marc d’Anjou, Terrance A. Stadheim, Byung-Kwon Choi#

GlycoFi, Biologics Discovery, Merck & Co., Inc., Lebanon, New Hampshire, USA

#Address correspondence to Byung-Kwon Choi, [email protected]

*Present address: Muralidhar Mallem, Shire, Lexington, Massachusetts, USA/Shannon Warburton, Henry M Jackson foundation, San Antonio, Texas, USA

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/btpr.1990 © 2014 American Institute of Chemical Engineers Biotechnol Prog Received: Jul 25, 2014; Revised: Aug 28, 2014; Accepted: Aug 28, 2014

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2 Abstract

Human serum albumin (HSA) is a cysteine rich molecule that is most abundant in human blood plasma. To remain viable in the market due to lower marketing costs for HSA, it is important to produce a large quantity in an economical manner by recombinant technology. The objective of this study was to maximize recombinant HSA (rHSA) production using a Muts Pichia pastoris strain by fermentation process optimization. We evaluated the impact of process parameters on the production of rHSA, including induction cell density (wet cell weight, g/L) and the control of specific growth rate at induction. In this study, we demonstrated that induction cell density is a critical factor for high level production of rHSA under controlled specific growth rate. We observed higher specific productivities at higher induction cell densities (285 g/L) and at lower specific growth rates (0.0022-0.0024 h-1) during methanol induction phase, and achieved the broth titer of rHSA up to 10 g/L. The temperature shift from 24oC to 28oC was effective to control the specific growth rate at low level (≤0.0024 h-1) during methanol induction phase while maintaining high specific productivity (0.0908 mgrHSA/gwcw/h).

Keywords: human serum albumin, Pichia pastoris muts, specific growth rate, cell density

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3 Introduction

Human serum albumin (HSA) is an aglycosylated protein consisting of 585 amino acids comprising a single polypeptide chain with a molecular weight of 66.5 kDa. HSA is a cysteine rich molecule with 35 cysteinyl residues forming 17 disulfide bridges with a free cysteine at position 34.1 These disulfide bridges are responsible for folding the molecule into nine loops. These nine loops can be grouped into three domains: l (1-195), ll (196-383), lll (384-585) that bind to different physiological ligands such as metals and fatty acids. Arg 117, Lys351, and Lys475 may be binding sites for fatty acids.2 It was reported that the crystal structures of HSA derived from plasma or by recombinant Pichia pastoris expression were identical.3 HSA is a major protein component of human plasma that is produced in the liver. Approximately 35-40 g of HSA is present in a liter of blood. HSA not only maintains normal osmolality in the blood stream but also transports a variety of small molecules such as vitamins, hormones, fatty acids, amino acids, metabolites, drugs, and divalent metal ions like Zn, Cu, Co.4 HSA is also a source of reserve protein in conditions like hypoproteinemia where the blood has abnormally low levels of protein. HSA serves as a potent antioxidant by protecting the cells in the blood from reactive oxygen species.5 HSA is used for the clinical treatment of severe hypoalbuminemia and traumatic shock at a dosage of greater than 10 g per dose.6 HSA is currently produced from human plasma by fractionation technique. As a result, there is a risk of HSA contamination due to blood-derived pathogens since the blood is sourced from various people. The demand for HSA is approximately 300 tons/year while the cost of HSA per gram is lower. As a result, production of HSA by recombinant DNA technology has gained significant attention. However, its structural complexity, lower unit price and higher

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4 demand pose a major challenge for the recombinant process as HSA has to be produced in grams per liter in an industrial production scale to remain economically viable.7 It has been reported that HSA was produced in different expression systems such as methyltrophic yeast, Pichia pastoris,6,8,9 Kluyveromyces lactis,7 Saccharomyces cerevisiae,10 and Hansenula polymorpha.11 Compared to other yeast expression systems, Pichia expression system has several advantages for recombinant protein production such as high cell density, genetic stability, low endogenous protein secretion, posttranslational modification (glycosylation and disulfide bond formation), and high secretory capacity.12,13 Thus, P. pastoris has been widely used for recombinant protein production12 along with bioprocess optimization that is specific to a protein of interest.14-18 The objective of this study was to maximize the production of rHSA through fermentation process optimization to significantly higher levels (≥ 10 g/L broth titer) than the previous reports.8 A Muts P. pastoris was used as the expression host for rHSA production and process development in our study. We demonstrate that a Muts P. pastoris is an excellent recombinant production host capable of producing the highest level of rHSA that has not been achieved in any other expression systems. Here, we report for the first time the broth titer of 10 g/L rHSA (17-18 g/L supernatant titer) by optimization of induction cell density and specific growth rate in a single fed-batch process.

Materials and Methods

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5

Plasmid construction and strain generation The plasmids and strains used in this study were generated as follows. For recombinant human serum albumin production (rHSA), the open reading frame (ORF) encoding the HSA mature was codon optimized for P. pastoris and synthesized by GenScript (Piscataway, NJ, USA), where the native HSA signal sequence lacking propeptides (RGVFRR) was used. The nucleotide sequence of codon-optimized human serum albumin for P. pastoris has been deposited to GenBank under accession number KJ807834. The ORF was subcloned into pGLY5224 at EcoRI and FseI sites and was designated pGLY6660. pGLY5224 is an expression vector, where the expression of HSA is under the control of the P. pastoris alcohol oxidase 1 (PpAOX1) promoter (Figure 1A). The expression vector pGLY6660 was linearized with SpeI, and transformed into a Muts yGLY20237 or a Mut+ NRRL11430, targeting the TRP2 locus for the gene integration. YGLY20237 was generated by knocking out the AOX1 ORF in a wild-type P. pastoris NRRL11430, where the knock-out vector (pGLY3588) was transformed into YGLY1-3 (NRRL11430, ura5-) (Figure 1B). The deletion of the AOX1 ORF was confirmed by cPCR using the primers, PpAOX1/iUP (5'-CCTACCCAGTTTGCCAGGACTTCTTGAGGGC TTC-3’) and PpAOX1/iLP (5'-GGACCAGTACCATTGGCGTACCATTGGTCAAAGACTC3’). The HSA transformants were selected using the Zeocin resistance gene (Streptoalloteichus hindustanus ble) and were screened for high rHSA expressors using bioreactors (Micro24 and DasGip). High rHSA expressors were designated YGLY20284 (Mut+) and YGLY21958 (Muts), respectively and were used for fermentation process optimization.

Transformation

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6 Pichia transformation was performed as described previously.19 Briefly, 100 µL of overnight grown cells (OD600=0.2 to 2.0) were transformed with 10 µL linearized DNA (5 µg) after 5 min incubation on ice. Electroporation was in a Bio-Rad GenePulser Xcell following the preset P. pastoris protocol (2 kV, 25 µF, 200 Ω), immediately followed by the addition of 1 mL YPDS recovery media (YPD media plus 1 M sorbitol). The transformed cells were allowed to recover for 1 h at room temperature (24°C) before plating the cells on selective media.

Micro24 and DasGip screening Transformants were screened using Micro24 MicroReactor (Pall Corp., Port Washington, NY) as described previously.20 DasGip (Eppendorf, Hauppauge, NY) fermentation was performed as follows: a 48 h grown seed culture (60 mL) was inoculated aseptically into a bioreactor that was charged with 0.54 L of 0.2 µm filtered BSGY-S media (per liter of media contains 10 g of Sensient yeast extract, 20 g of soy peptone, 13.4 g of YNB w/ ammonium sulfate, 2.3 g of K2HPO4, 11.9 g of KH2PO4, 40 g of glycerol, 18.6 g of sorbitol, 8 mg biotin and 0.075 g of sigma 204 antifoam). The aeration, agitation and temperatures were set to 0.7 vvm, 500 rpm and 24oC, respectively. The pH was adjusted to and controlled at 5.85 throughout the run using 30% (v/v) ammonium hydroxide. Agitation was ramped to maintain 20% dissolved oxygen (DO) saturation. After the initial glycerol charge was consumed as indicated by a sharp increase in the DO, a 50% (w/w) glycerol solution containing 5 mg/L biotin and 12.5 mL/L PTM2 salts (per liter contains 6.5 g FeSO4.7H2O, 2 g ZnCl2, 5 mL H2SO4, 0.6 g CuSO4.5H2O, 1.8 g MnSO4.7H2O, 0.5 g CoCl2.6H2O, 0.2 g NaMoO4.2H2O, 0.2 g biotin, 80 mg NaI, 20 mg H3BO4) was triggered to feed at 7.7 g/L/h for 8 h. Completion of the glycerol fed-batch was followed by a 30-min starvation period and initiation of the induction phase. A continuous feed

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7 of a 50% (v/v) methanol solution containing 2.5 mg/L biotin and 6.25 ml/L PTM2 salts was carried out at a flat rate of 2.4 g/L/h (1.05 g/L/h methanol) until the end of fermentation.

Fermentation Each 2.8-L Fernbach baffled shake flask containing 500 mL BSGY media (per liter of media contains 10 g of Sensient yeast extract, 20 g of soy peptone, 13.4 g of YNB with ammonium sulfate, 2.3 g of K2HPO4, 11.9 g of KH2PO4, 40 g of glycerol and 8 mg Biotin) was inoculated with 2 mL frozen stock culture of YGLY21958. These shake flasks were incubated in an orbital shaker at 24oC and 180 RPM (1 inch throw) for 28-32 h before the desired inoculum volume [10% (v/v)] is transferred into the bioreactors containing BSGY-S media. The fermentation process consisted of three phases similar to traditional P. pastoris process: glycerol batch phase followed by glycerol fed-batch and methanol induction phase. The fermentation process conditions for the different experiments are described in Table 1. 6-L (F110631) and 15-L (F110611) bioreactors were started with a pre-inoculation volume of 40% of their total capacity. The dissolved oxygen concentration in the bioreactor was maintained at 20% of air saturation by cascading it to agitation and oxygen flow. The glycerol batch phase lasted for 1824 h, depending on the optical density (OD600) of the shake flask culture. Glycerol depletion was indicated by a sudden rise in the dissolved oxygen concentration (DO spike) due to the decrease in metabolic activity of the cells. Glycerol fed-batch phase was initiated immediately at a feed rate of 5.33 g/L*/h (L*: preinoculation volume) increasing exponentially at the rate of 0.08 h-1 over the desired time period. For some experiments, glycerol feed was made with basal salts media (per liter basal salts media

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8 contains 26.7 mL of 85% phosphoric acid, 0.93 g of calcium sulfate, 18.2 g of potassium sulfate, 12.3 g of magnesium chloride, 4.13 g of potassium hydroxide and 8 mg of biotin) during the glycerol fed-batch phase. The glycerol feeding was stopped after the desired feeding interval and methanol induction phase was initiated immediately with the feeding of methanol to the culture for the production of rHSA. Methanol induction phase was executed by controlling the residual methanol concentration in the reactor between 2-5 g/L* using a methanol sensor (Raven Biotech Inc., Vancouver, B.C., Canada). The induction phase was continued until a decrease in broth titer and cell viability was observed. The cell viability decrease would be evident with a decrease in oxygen uptake rate because of significant drop in metabolic activity on methanol.

Protein quantification HSA concentration was determined using Bradford method.21 Commercial HSA was used as a standard for generating standard curve data. Coomassie plus – the better Bradford assay TM

reagent (Thermo Scientific, Rockford, IL) was added to diluted and undiluted fermentation

supernatant samples according to the manufacture’s instruction. In addition, blank fermentation media sample was run to determine any background noise. Bradford analysis was performed on the samples in a 96 well plate format using a TECAN infinite M200 spectrophotometer and Magellan 6 software (TECAN, Research Triangle Park, NC) at a wavelength of 595 nm. HSA concentration was also determined by image quantification method using Image Quant 300 Capture software (GE Healthcare, Piscataway, NJ) to confirm the results from Bradford analysis.

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9 Purification P. pastoris-produced rHSA was purified using Blue Sepharose 6 Fast Flow column (GE healthcare, Piscataway, NJ). Briefly, the fermentation supernatant was harvested by centrifugation at 8500 rpm for 30 min and filtered with 0.2 µm PES membrane Nalgene filters. A 25 mL column was loaded with 200 ml of filtered supernatant and washed with 50 mM potassium phosphate buffer, pH 7.0, for 5 column volume (CV). Bound rHSA was eluted with 50 mM potassium phosphate buffer, pH 7.0, 2 M potassium chloride. The eluted rHSA was collected and used to analyze O-glycans and free thiol assay.

O-glycan determination Purified HSA was used for O-glycan occupancy assay. O-glycan content was determined as described previously.22

Free thiol assay Free thiol assay was performed as described elsewhere.23 Briefly, 40 µL (60-200 µg) of a sample was added to the solution of 150 µL of 8 M guanidine-HCl, 5µL 0.5 M EDTA, and 5 µL PBS to denature protein. Then 3 µL of 10 mM Ellman’s stock reagent (DTNB, Invitrogen) was added to the above 200 µL solution and mixed thoroughly. A control sample was run without the addition of protein. The reading was obtained immediately at 420 nm.

Results Generation of rHSA production strain

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10 For the comparison of rHSA productivity, two different phenotypes (Mut+/Muts) of wildtype P. pastoris (NRRL11430) producing rHSA were generated. In order to construct a Muts phenotype, the open reading frame encoding alcohol oxidase 1 (AOX1) was deleted so that the alcohol oxidase activity relies solely on alcohol oxidase 2 (AOX2) for methanol metabolism. It has been reported that the AOX2 activity is around 10%, compared with the AOX1 activity (90%).24 As a result, the cell growth slows down in methanol induction phase. However, the wet cell weight is not affected in glycerol phase for biomass generation. In general, the levels of protein expression are closely related with the number of a gene copy.25-28 Poorly secreted proteins are an exception where higher gene copy number is detrimental because it causes more protein aggregation and misfolding in the endoplasmic reticulum (ER). HSA is one of the well expressed proteins in yeasts, and we observed that the HSA gene copy number increased rHSA expression proportionally. To identify high rHSA producers, the multicopy integrants (Mut+ or Muts) of a HSA gene were induced using high Zeo concentration plates (300 and 500 µg/mL), and then they were screened in a Micro24 bioreactor. The resulting high producers, YGLY20284 (Mut+) and YGLY21958 (Muts), were evaluated for feasibility of prolonged fermentation process.

Optimization of temperature for minimal proteolysis rHSA degradation has been reported in some cases6,29,30, mostly due to proteolysis by native proteases from production hosts. In order to minimize the proteolysis, we tested the temperature in the range of 17-30oC at pH 6.5 because the culture temperature generally affects protein titer, and promotes proteolysis at higher temperatures (≥30C). rHSA production was reduced at the lower temperature (17oC or 20oC). It is likely that the lower temperatures are not

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11 optimal for cell growth, hence slow down protein synthesis. At higher temperature (30oC) rHSA production was improved, but more degraded products were generated (Figure 2A and B). The culture temperature was more favorable at 24oC in terms of protein production and integrity. We adjusted pH to 5.85 based on the report6, where rHSA proteolysis was minimized. In the absence of protease inhibitors, we were able to minimize the proteolysis of rHSA with higher expression levels at 24oC (Figure 2C).

Selection of Muts phenotype The major difference between Mut+ and Muts phenotype is lack of the AOX1 activity in Muts strains that comprises a majority of the AOX activity (90%). As a result of significant reduction in methanol consumption rate, the Muts strains grow much slower than the Mut+ strains on methanol. Under excess methanol and oxygen (20% dissolved oxygen) condition, we observed that the Mut+ strain consumed methanol at a faster rate than the Muts strain and resulted in cell densities closer to 50% (w/v) solids in a relatively shorter induction time. Culture would experience significant heat and mass transfer challenges at these higher cell densities as the viscosity of the broth increases with increase in cell density. Lower growth rate and higher specific productivity during induction will be the preferred way to avoid operating at the edge of boundary conditions with respect to cell density and final reactor volume. In addition, oxygen and cooling requirements for a Muts strain are significantly lower when compared to Mut+ strain due to the lack of AOX1 gene. Pla et al.24 demonstrated that protein productivity in the Muts strain was twice higher than one in the Mut+ strain. In our study, the specific productivity of the Muts strains (YGLY21958: 0.0529 mgrHSA/gwcw/h) was higher than the Mut+ strain (YGLY20284: 0.0485 mgrHSA/gwcw/h) under the same process conditions. Taken together, we

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12 selected the Muts strain for process optimization to maximize rHSA production in different scale bioreactors.

Process optimization-effect of induction cell density To investigate the impact of induction cell density on the specific productivity and broth titer, F101742 fermentation process was run without glycerol fed-batch phase. Cells were induced with methanol immediately after glycerol in the batch phase was completely consumed. The cell density at the start of induction was around 130 g/L. F101742 had the highest broth titer of 4.6 g/L at 204 h induction and afterwards the broth titer decreased to 3.8 g/L at 362 h induction even though the cell density increased from 293 to 330 g/L during this time period (Figure 3). This suggests that either the cells would have stopped producing rHSA after 204 h induction or proteolytic degradation of rHSA may be occurred at a rapid rate than production. The specific growth rate for F101742 fermentation process was 0.0024 h-1 while the specific productivity was 0.0529 mgrHSA/gwcw/h. F101909 fermentation process was run with an 8 h exponential glycerol fed-batch (50% (w/w) glycerol feed with 12.5 mL/L PTM2 salts and 5 mg/L biotin) targeting induction cell density higher than 130 g/L. The cell density at the start of induction was around 230 g/L. This was the only process change when compared to F101742. F101909 showed a broth titer of 7.7 g/L at 360 h induction while it increased marginally to 8.1 g/L at 409 h induction. The effect of induction cell density on cell growth and rHSA expression is shown in Figure 3. The specific growth rate for F101909 process was 0.0026 h-1 that is similar to F101742. However, the specific productivity (0.0616 mgrHSA/gwcw/h) and broth titer were substantially improved from 4.6 to 8.1 g/L. We observed that WCW did not increase over 314 h induction, possibly due to limitation of

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13 nutrients to support cell growth. However, the increase in broth titer indicates that the cells continue to secrete rHSA into the culture media. F102310 fermentation process was run with a 12 h exponential glycerol fed-batch (50% (w/w)) glycerol feed made with basal salts media) targeting an induction cell density higher than 230 g/L. Basal salts media was used in the glycerol feed to provide a conducive environment for the cells to reduce the growth stress that they might undergo during extended exponential glycerol-feeding period. Because of basal salts co-feeding, the higher induction cell density (285 g/L) was achieved with a broth titer of 7.4 g/L at much shorter induction time (233 h). However, the cell viability was substantially affected, resulting in a significant drop of WCW from 498 to 437 g/L at 257 h induction, thus the fermentation was terminated (Figure 3). It is possible that mass and heat transfer limitations reduce cell viability especially when cell densities are closer to or higher than 50% solids. This could be due to the significant change in fluid dynamics of the culture. This process suggests that higher specific growth rate (0.0036 h-1) should be controlled at induction phase to enhance cell viability for productive protein production. The specific productivity for F102310 fermentation process was 0.0917 mgrHSA/gwcw/h.

Process optimization-effect of specific growth rate control at induction F103031 fermentation process was run with a 12 h exponential glycerol fed-batch (50% (w/w)) glycerol feed made with basal salts media) targeting an induction cell density of 285 g/L. The culture was grown at 24oC during glycerol batch and 8 h of glycerol fed-batch phase. To better control cell growth, temperature was ramped up from 24 to 28oC during the last 4 h of glycerol fed-batch phase to carry methanol induction at 28oC. The main difference between F102310 and F103031 was the induction temperature. F103031 produced a broth titer of 7.9 g/L

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14 at 229 h induction with a cell density of 412 g/L, compared to 498 g/L for F102310 at an equivalent induction time. Induction in F103031 fermentation was extended to 405 h, where the highest broth titer of 10 g/L rHSA was achieved with 478 g/L (WCW) closer to 50% solids. Although the volumetric productivity of F103031 was comparable to F102310 at an equivalent time of 229 h induction (7.9 g/L vs 7.4 g/L), the biomass yield on methanol at 28oC (F103031) was significantly lower compared to 24oC (F102310). As a result, extended fermentation was possible which led to the production of 10 g/L rHSA broth titer. The specific growth rate of F103031 process was 39% lower (0.0022 h-1) than that of F102310 (0.0036 h-1). The resulting specific productivity was 0.0900 mgrHSA/gwcw/h that was similar to F102310 (0.0917 mgrHSA/gwcw/h). This specific productivity was 32%-42% higher when compared to F101909 and F101742 processes while maintaining low specific growth rates (0.0024-0.0026 h-1). Higher specific growth rate (0.0036 h-1) was observed with F102310 fermentation, where the process condition was the feeding of basal salts media during glycerol fed-batch phase and maintaining the temperature at 24oC during the entire fermentation. From our observation, the specific growth rates were affected by culture temperatures. That is, a higher temperature (28oC) decreased cell growth at induction, compared to the cell growth at 24oC. The temperature shift to 28oC was beneficial to keep the targeted specific growth rate with high cell density induction.

Reproducibility and scalability of high titer process After bioprocess optimization in F103031 (6 L) with 10g/L (broth titer) of rHSA production, the same process was performed in 6-L (F110631) and 15-L (F110611) bioreactors to demonstrate process reproducibility and scalability. To confirm reproducibility of F103031

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15 process, F110631 run was carried out in a 6-L bioreactor under the same process conditions. The fermentation profile for F110631 run is shown in Figure 4. Broth titer reached 10.2 g/L at 395 h induction with 415 g/L (WCW) while maintaining the similar specific growth rate (0.0023 h-1) to F103031 (0.0022 h-1). In addition, the specific productivity (0.0908 mgrHSA/gwcw/h) was almost similar to F103031 (0.0900 mgrHSA/gwcw/h). There was a 13% decrease in cell density for F110631 when compared to F103031. This may have been due to the removal of lower culture volume from the reactor during the latter part of induction. As a result, F110631 fermentation was operated at 85% capacity of total bioreactor volume that was not optimal for cell growth. For scalability of the process, F110611 run was performed in a 15-liter bioreactor. The same fermentation process (6 L) was scalable to 15 L with a broth titer of 9.8 g/L at 389h induction with 453 g/L (WCW). The specific growth rate and productivity for F110611 process was 0.0024 h-1 and 0.0887 mgrHSA/gwcw/h, respectively that were also executed similarly to F103031 (0.0022 h-1 and 0.0900 mgrHSA/gwcw/h) for high level expression of rHSA. F103031 fermentation process was successfully scaled-up from 6 L to 15 L that confirmed ~10 g/L of broth titer. The cell growth and rHSA expression by the optimized process (three-factor interaction) is shown in Figure 5, and rHSA production for F103031, F110631, and F110611 at the final induction time point is shown in Figure 6. The specific productivity was significantly improved up to 42% when compared to F101742 process. As mentioned earlier, the temperature transition from 24 to 28oC during induction was beneficial to achieve 10 g/L of broth titer by lowering biomass yield with slower growth.

Characterization of rHSA

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16 Recombinant HSA purified with Blue Sepharose column was analyzed for O-glycan occupancy and free thiol content. HSA contains 35 cysteines that make 17 disulfide bonds, and 1 free cysteine. Therefore, the presence of more free cysteines would be a result of mis-disulfide bonds on HSA. To confirm the presence of free cysteines on rHSA produced from F110631 run, the free cysteine assay was carried out. As shown in Table 2, rHSA contains free thiols at 0.11 mol/mol, compared with one (0.07 mol/mol) from plasma-derived HSA, indicating that overall free thiol content of rHSA is similar to one from plasma-derived HSA. This is in a good agreement with the report, where the crystal structure of rHSA produced in P. pastoris is identical to one derived from plasma.3 In general, yeast O-glycosylates proteins in the ER by protein O-mannosyltransferase (PMT) family and aberrant O-mannosylation may also occur.31,32 The resulting aberrant Oglycans may interrupt protein folding and generate mis-folded proteins.33 It has been demonstrated that O-glycans on rHSA produced in P. pastoris are present at low levels if it is not controlled by genetic PMT knock-outs or PMT inhibitors. Thus, to avoid any aberrant Omannosylation, PMT inhibitor (PMTi) was used for rHSA production. As a result, O-glycans were not detected on rHSA, whereas O-glycans were found in the range of 1.0-1.3 mol/mol in the absence of PMTi. Interestingly, the presence of aberrant O-glycans increased free thiol contents, and this suggests that O-glycans are likely to interfere with rHSA folding, resulting in more free thiols that are not formed in correct disulfide bonds.

Discussion rHSA has been produced at relatively high levels in various yeast strains.6-11 However, it has been a challenge to produce rHSA greater than 10 g/L (broth titer) in yeasts in a single fed-

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17 batch process due to strain robustness and maintaining high productivity required for a longer induction (>300 h). In this study, we were able to achieve 10 g/L broth titer of rHSA (17-18 g/L supernatant titer) by taking advantage of the Muts phenotype as well as by process optimization. The proteolytic degradation of rHSA has been reported in yeasts,6,29 mostly due to endogenous proteases. Genetic knock-outs of responsible protease genes would be the best strategy to keep the integrity of rHSA. However, the protease deletion is often detrimental to cell growth and robustness that affect recombinant protein expression and are not suitable for prolonged protein induction. As an alternative, we controlled the culture pH and temperature to minimize the proteolysis, where endogenous proteases are less active while maintaining high productivity. There are three different methanol utilization phenotypes (Mut+, Muts, and Mut-), depending on availability of the AOX1 or/and AOX2 activity. Their specific growth rates are different on methanol. For instance, Mut+ strains grow at 0.14 h-1 whereas Muts strains grow much slower (0.04 h-1).34,35 The specific growth rate significantly affects specific productivity so that an optimal specific growth rate should be maintained during induction. The Muts strain is much more suitable to achieve the lower specific growth rates (0.002-0.003 h-1) that maximize specific productivity.36 As a result, we were able to generate much higher specific productivity (0.0908 mgrHSA/gwcw/h) with the Muts than the Mut+ strain (0.0485 mgrHSA/gwcw/h). Along with the optimal specific growth rate, other factors such as induction cell density, pH, and temperature also affected overall protein production in a single fed-batch process. Generation of high cell density prior to methanol induction is a critical factor at a given glycerol fed-batch period for higher production of proteins. Our process optimization indicates that control of a specific

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18 growth rate during high cell density induction can maximize rHSA while minimizing the proteolytic degradation. The quality of rHSA was equivalent to plasma-derived HSA by controlling O-glycan occupancy with the use of PMTi. It would be possible to shorten the entire process time with a genetic selection of strains producing higher rHSA carrying multicopy genes from our observation of an additive effect by gene copy number. This study also demonstrates that P. pastoris is an excellent production host with a high secretory capacity even if protein secretion highly depends on the nature of an individual protein.

Conclusions We demonstrated that P. pastoris muts is an attractive strain that is suitable for a prolonged recombinant protein induction in a single fed-batch process, and by achieving the highest level of rHSA production (10g/L broth titer). In this study, the optimal control of cell density and specific growth rate during induction phase were key factors that affected specific productivity and overall recombinant protein production. The resulting optimal process conditions were robust and scalable. In addition, the use of PMTi improved the quality of rHSA comparable to that of plasma-derived HSA. The Muts phenotype would be an alternative option for recombinant protein production when the Mut+ strain is not productive.

Acknowledgements The authors would like to thank Natarajan Sethuraman for supporting this project, Heping Lin for excellent technical support, Irina Burnina for O-glycan analysis, Khanita Karaveg for free thiol analysis, Seemab Shaikh for Micro24 run, and Nathan Sharkey for DasGip run.

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19 Literature Cited 1. Kobayashi K. Summary of recombinant human serum albumin development. Biologicals 2006;34:55-59. 2. Carter DC, He XM, Munson SH, Twigg PD, Gernert KM, Broom MB, Miller TY. Threedimensional structure of human serum albumin. Science 1989;244:1195-1198. 3. Sugio S, Kashima A, Mochizuki S, Noda M, Kobayashi K. Crystal structure of human serum albumin at 2.5 A resolution. Protein. Eng. 1999;12:439-446. 4. Putnam FW. Alpha, Beta, Gamma, Omega– The structure of the plasma proteins. In:Putnam FW. The Plasma Proteins – Structure, Function, and Genetic YPS1 gene on viability and production of Control, Volume IV (2nd edition). Orlando: Academic Press, 1984:45-166. 5. Cha MK, Kim IH. Glutathione-linked thiol peroxidase activity of human serum albumin: A possible antioxidant role of serum albumin in blood plasma. Biochem. Biophys. Res. Comm. 1996;222:619–625. 6. Kobayashi K, Kuwae S, Ohya T, Ohda T, Ohyama M, Ohi H, Tomomitsu K, Ohmura T. High-level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation. J. Biosci. Bioeng. 2000;89:55-61. 7. Fleer R, Yeh P, Amellal N, Maury I, Fournier A, Bacchetta F, Baduel P, Jung G, L’Hôte H, Becquart J, Fukuhara H, Mayaux JF. Stable multicopy vectors for high-level secretion of recombinant human serum albumin by Kluyveromyces yeasts. Nat. Biotech. 1991;9: 968–975.

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20 8. Kobayashi K, Kuwae S, Ohya T, Ohda T, Ohyama M, Tomomitsu K. High level secretion of recombinant human serum albumin by fed-batch fermentation of the methylotrophic yeast, Pichia pastoris, based on optimal methanol feeding strategy. J. Biosci. Bioeng. 2000;90:280–288. 9. Maccani A, Landes N, Stadlmayr G, Maresch D, Leitner C, Maurer M, Gasser B, Ernst W, Kunert R, Mattanovich D. Pichia pastoris secretes recombinant proteins less efficiently than Chinese hamster ovary cells but allows higher space-time yields for less complex proteins. Biotechnol J. 2014;9:526-537. 10. Kang HA, Choi ES, Hong WK, Kim JY, Ko SM, Sohn JH, Rhee SK. Proteolytic stability of recombinant human serum albumin secreted in the yeast Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 2000;53:575-582. 11. Youn JK, Shang L, Kim MI, Jeong CM, Chang HN, Hahm MS, Rhee SK, Kang HA. Enhanced production of human serum albumin by fed-batch culture of Hansenula polymorpha with high-purity oxygen. J. Microbiol. Biotechnol. 2010;20:1534-1538. 12. Cereghino GPL, Cereghino JL, Christine I, Cregg JM. Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotech. 2002;13:329–332. 13. Celik E, Calik P. Production of recombinant proteins by yeast cells. Biotechnol. Adv. 2012;30:1108-1118. 14. Lopes M, Oliveira C, Domingues L, Mota M, Belo I. Enhanced heterologous protein production in Pichia pastoris under increased air pressure [published online ahead of print July 30, 2014]. Biotechnol Prog. doi: 10.1002/btpr.1964.

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21 15. Barba Cedillo V, Jesús Martínez M, Arnau C, Valero F. Production of a sterol esterase from Ophiostoma piceae in batch and fed-batch bioprocesses using different Pichia pastoris phenotypes as cell factory [published online ahead of print June 16, 2014]. Biotechnol Prog. doi: 10.1002/btpr.1939. 16. Anasontzis GE, Salazar Penã M, Spadiut O, Brumer H, Olsson L. Effects of temperature and glycerol and methanol-feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris. Biotechnol Prog. 2014;30:728-35. 17. Viader-Salvadó JM, Castillo-Galván M, Fuentes-Garibay JA, Iracheta-Cárdenas MM, Guerrero-Olazarán M. Optimization of five environmental factors to increase betapropeller phytase production in Pichia pastoris and impact on the physiological response of the host. Biotechnol Prog. 2013;29:1377-1385. 18. Viader-Salvadó JM, Fuentes-Garibay JA, Castillo-Galván M, Iracheta-Cárdenas MM, Galán-Wong LJ, Guerrero-Olazarán M. Shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris bioreactor cultures. Biotechnol Prog. 2013;29:11-16. 19. Choi BK, Bobrowicz P, Davidson RC, Hamilton SR, Kung DH, Li H, Miele RG, Nett JH, Wildt S, Gerngross TU. Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc. Natl. Acad. Sci. USA 2003;100:5022– 5027. 20. Choi BK, Warburton S, Lin H, Patel R, Boldogh I, Meehl M, d'Anjou M, Pon L, Stadheim TA, Sethuraman N. Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris. Appl. Microbiol. Biotechnol. 2012;95:671682.

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22 21. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72:248-54. 22. Stadheim TA, Li H, Kett W, Burnina IN, Gerngross TU. Use of high-performance anion exchange

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determination in yeast. Nat. Protoc. 2008;3:1026-1031. 23. Ellman GL. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 1959;82:70–77. 24. Pla IA, Damasceno LM, Vannelli T, Ritter G, Batt CA, Shuler ML. Evaluation of Mut+ and MutS Pichia pastoris phenotypes for high level extracellular scFv expression under feedback control of the methanol concentration. Biotechnol. Prog. 2006;22:881-888. 25. Lopes T, Klootwijk J, Veenstra A, van der Aar P, van Heerikhuizen H, Raué H, Planta R. High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: A new vector for high-level expression. Gene 1989;79:199–206. 26. Clare JJ, Rayment FB, Ballantine SP, Sreekrishna K, Romanos MA. High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Nat. Biotech. 1991;9:455–460. 27. Klabunde J, Diesel A, Waschk D, Gellissen G, Hollenberg C, Suckow M. Single-step cointegration of multiple expressible heterologous genes into the ribosomal DNA of the methylotrophic yeast Hansenula polymorpha. Appl. Microbiol. Biotechnol. 2002;58:797– 805. 28. Marx H, Mecklenbräuker A, Gasser B, Sauer M, Mattanovich D. Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus. FEMS Yeast Res. 2009;9:1260–1270.

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23 29. Kerry-Wiiams SM, Gilbert SC, Evans LR, Ballance DJ. Disruption of the Saccharomyces cerevisiae YAP3 gene reduces the proteolytic degradation of secreted recombinant human albumin. Yeast 1998;14:161-169. 30. Sazonova EA, Zobnina AE, Padkina MV. Effect of disruption of Pichia pastoris YPS1 gene on viability and production of recombinant proteins. Russ. J. Genet. 2013;49:602608. 31. Gentzsch

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24 FIGURE LEGENDS

Figure 1. The rHSA expression (A) and AOX1 knock-out (B) vectors.

Figure 2. Optimization of the culture temperature. YGLY20284 was fermented for 5 days in DasGip bioreactor at different temperatures, 17oC and 20oC (A), 24oC and 30oC (B) at pH6.5. (C) The strain was fermented under optimized conditions (24oC and pH5.85). The supernant samples were analyzed on reduced SDS-PAGE (4-20%) gel. MW: molecular weight marker, arrow: rHSA, asterisk: degraded rHSA.

Figure 3. rHSA broth titer in g/L (A) as a function of various induction cell densities (wet cell weight) in g/L (B).

Figure 4. Fermentation profile of F110631 (6L) run.

Figure 5. Reproducibility and scalability of rHSA production. (A) rHSA broth titer, (B) wet cell weight.

Figure 6. rHSA production under optimized process conditions. The final culture supernatant samples (Day 15) were analyzed on reduced SDS-PAGE (4-20%). The arrow indicates rHSA. Lane 1: HSA standard, lane 2: F103031, lane 3: F110631, lane 4: F110611.

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25 Table 1. Bioprocess conditions used in this study

F103031 Process parameter F101742

F101909

F102310

F110611 F110631

Induction 24

24

24

28

28

pH

5.85

5.85

5.85

5.85

5.85

Airflow rate (vvm)

0.7

0.7

0.7

0.7

0.7

0

8

12

12

12

No

No

Yes

Yes

Yes

temperature (oC)

Glycerol fed-batch duration (hr) Basal salts media feed during glycerol fed-batch

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26 Table 2. O-glycan occupancy and free thiol content on rHSA produced in P. pastoris

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Maximizing recombinant human serum albumin production in a Mut(s) Pichia pastoris strain.

Human serum albumin (HSA) is a cysteine rich molecule that is most abundant in human blood plasma. To remain viable in the market due to lower marketi...
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