The high cell density fermentation of recombinant Pichia pastoris for human serum albumin (HSA) production is a high oxygen demand process. The oxygen demand is usually met by increased agitation rate and use of oxygen-enriched air. Microbubble fermentation however can supply adequate oxygen to the microorganisms at relatively low agitation rates because of improved mass transfer of the microbubbles used for the sparging. Conventionally sparged fermentations were conducted for the production of HSA using P. pastoris at agitation rates of 350, 500, and 750 rpm, and were compared to MBD sparged fermentation at 150, 350, and 500 rpm agitation rates. The MBD improved the volumetric oxygen transfer coefficient (kLa) and subsequently increased the cell mass and protein production compared to conventional fermentation. Cell production in MBD fermentation at 350 rpm was 4.6 times higher than that in conventional fermentation at 350 rpm, but similar to that in the conventional 750 rpm. Maximum cell mass productivity in the conventional 350 rpm was only 0.37 g / (L·h), while the maximum value in MBD 350 rpm was 2.0 g / (L·h), which was similar to 2.2 g / (L·h) in the conventional 750 rpm. Biomass yield on glycerol Ys (g cell/ g glycerol) was 0.334 g / g in the conventional 350 rpm, 0.431 g / g in MBD 350 rpm and 0.438 g / g in the conventional 750 rpm. Protein production in MBD 350 rpm was 7.3 times higher than that in the conventional 350 rpm, but similar to the conventional 750 rpm. Maximum protein productivity in the conventional 350 rpm was 0.37 mg / (L·h), 2.8 mg / (L·h) in MBD 350 rpm, and 3.3 mg / (L·h) in the conventional 750 rpm. Protein yield on methanol Yp (mg protein / g methanol) was 1.57 mg /g in the conventional 350 rpm, 5.02 in MBD 350 rpm, and 5.21 in the conventional 750 rpm. The volumetric oxygen transfer coefficient kLa was 1011.9 h-1 in MBD 350 rpm, which was 6.1 times higher than that in the conventional 350 rpm (164.9 h-1) but was similar to the conventional 750 rpm (1098 h-1). Therefore, MBD fermentation results at low agitation of 350 rpm were similar to those in the conventional fermentation at high agitation of 750 rpm. There was considerable improvement in oxygen transfer to the microorganism using MBD sparging relative to the conventional sparging. Conventional fermentations were conducted both in a Biostat Q fermenter (small) at 500 rpm, 750 rpm, and 1000 rpm, and in a Bioflo III fermenter (large) at 350 rpm, 500 rpm, and 750 rpm. At the same agitation rate of 500 rpm, cell production in the large reactor was 3.8 times higher than that in the small one, and no detectable protein was produced in the small reactor at 500 rpm. At the same agitation rate of 750 rpm, both cell production and protein production in the large reactor were 4.6 times higher than the small reactor. Thus, the Bioflo III fermenter showed higher oxygen transfer efficiency than the Biostat Q fermenter, because of the more efficient aeration design of the Bioflo III fermenter. / Master of Science

In this study, it was aimed to develop a bioprocess using the Pichia pastoris expression system as an alternative to the mammalian system used in industry, for production of the therapeutically important glycoprotein, erythropoietin, and to form stoichiometric and kinetic models. Firstly, the human EPO gene, fused with a polyhistidine-tag and factor-Xa protease target site, in which cleavage produces the native termini of EPO, was integrated to AOX1 locus of P. pastoris. The Mut+ strain having the highest rHuEPO production capacity was selected. The glycosylation profile of rHuEPO was characterized by MALDI-ToF MS and Western blotting. The native polypeptide form of human EPO was obtained for the first time in P. pastoris expression system, after affinity-purification, deglycosylation and factor-Xa protease digestion. Thereafter, effects of medium components and pH on rHuEPO production and cell growth were investigated in laboratory-scale bioreactors. Sorbitol was shown to increase production efficiency when added as a co-substrate. Moreover, a cheap alternative nutrient, the byproduct of biodiesel industry, crude-glycerol, was suggested for the first time for P. pastoris fermentations. Furthermore, methanol feeding strategy was investigated in fed-batch pilot-scale bioreactors, producing 70 g L-1 biomass and 130 mg L-1 rHuEPO at t=24h. Moreover, metabolic flux analysis by using the stoichiometric model formed, which consisted of m=102 metabolites and n=141 reactions, proved useful in further understanding the P. pastoris metabolism. Finally, the first structured kinetic model formed for r-protein production with P. pastoris successfully predicted cell growth, substrate consumption and r-product production rates, where rHuEPO production kinetics was associated with AOX production and proteolytic degradation.

Recombinant Pichia pastoris is one of the important methylotropic yeast due to its robustness and ability to produce hormones like human chorionic gonadotropin (hCG), luteinizing hormone (LH) extracellularly. High growth on glycerol and strong protein expression on methanol by insertion of alcohol oxidase (AOX) promoter demand the fermentation to be a multistage operation. Methylotropic pathway demands more oxygen as methanol has to be converted to formaldehyde with half mole of oxygen. Moreover as fermentation progresses cell density in the reactor also increases. In case of Pichia pastoris fermentation cell density usually reaches very high (above 100 gm/lit) at the end of fermentation. Both these contribute in the increased oxygen demand in the fermentation and oxygen transfer turns out to be a limiting step. The present study focuses on the oxygen transfer process and its improvement in the fermentation. Oxygen transfer in bioreactor is a multistep process and involves different kinetic as well as mass transfer steps. In case of fermentation especially at high cell densities, oxygen transfer from bubbles to the broth becomes limiting step. The interface transport is governed by many physical as well as kinetic parameters. It is essential to screen these parameters from the whole set to identify the key parameters. Sensitivity analysis is carried out by using Metabolic Control Analysis (MCA) to quantify the effects of different parameters. It is found that bubble size and oxygen partial pressure are two such key parameters which can be manipulated. Use of pure oxygen to increase partial pressure and thereby solubility of oxygen in broth is a common approach. This work focuses on bubble size manipulation to increase the oxygen transfer rates.The idea behind this work is on to generate micron sized bubbles and utilize them effectively in the fermentation. There are many techniques reported to generate microbubble dispersions. In this work ’Spinning Disc microbubble Generator’ is fabricated to generate microbubbles. A flat disc surrounded by baffles with 5 mm gap in between, when subjected to 5000 rpm generates microbubbles. Some modifications are done to the set up to achieve desired properties of the bubbles. The bubbles generated fall in the range of 30-300 micron with mean size of about 60 micron. Use of Tween-20 surfactant stabilize the bubbles and hence offer a good resistance to coalescence and breakage. The liquid fraction in the bubbles can be as high as 40%. Contineous addition of this dispersion unnecessarily can dilute the fermentation broth. To overcome this volume constrain, a recirculation system is designed. Microbubble dispersion is added contineously to the reactor and equivalent fermentation broth is pumped back to the microbubble generator to achieve steady state to the liquid volume in both the vessels. Mass transfer studies with microbubbles show the potential of microbubble dispersion (MBD) to enhance mass transfer significantly. Decrease in volumetric mass transfer coefficient (KLa) due to surfactant is overcompensated by the increase in the interfacial area and net effect is, potential enhancement in KLa. The enhance- ment factor, that is, ratio of mass transfer coefficient with MBD to mass transfer coefficient with conventional sparging, is obtained to be about 4 to 5. Prior to utilization of bubbles in the recirculation system, cells are checked for the shear sensitiveness. Negligible lysis losses and almost no effect on growth patterns in shake flask culture confirm that the cells used are mechanically stable at operating conditions. Better growth patterns in shake flask are observed when microbubbles are pumped for predetermined duration in the broth. It shows possible use of MBD as oxygen carriers. Glycerol batch phase with MBD and conventional sparging is studied at different initial cell densities. Conventional sparging fails to grow the cells and Dissolved Oxygen (DO) levels close to zero suggest high oxygen demands which can not be sustained by conventional sparging. The same batch is run using MBD. Reasonably good growth patterns are observed. DO levels are well above 70% for most of the time during operation. High oxygen demand which can not be sustained by conventional sparging alone can be sustained by MBD. In this way in high den- sity cultures utilization of MBD can be a good alternative to fulfill required oxygen demand in fermentation.

In recent years, several industrial yeasts, owing to their robust growth and certain other characteristics, have been developed as recombinant host systems for commercial production of heterologous proteins. One such yeast Pichia pastoris has proven to be an excellent host for production of secreted and intracellular proteins (Cereghino and Cregg. 2000). The increasing popularity of this particular expression system can be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the molecular genetic manipulation of Pichia pastoris and their similarity to those of Saccharomyces cerevisiae, one of the most well-characterized experimental systems in modern biology;(2) the ability of Pichia pastoris to produce foreign proteins at high levels, either intracellularly or extracellularly; and (3) the capability of performing many eukaryotic post-translational modifications, such as glycosylation, disulfide bond formation and proteolytic processing. The expression level for a given recombinant protein produced by Pichia pastoris seems to be determined largely by its inherent properties such as amino acid sequence, the tertiary structure and the site for expression (Sreekrishna et al.,1997). The attempts on increasing the protein expression levels by far are focused on genetic manipulations to enhance the gene expression and protein stability. Although this is crucial, there is ample scope to improve the productivity of Pichia pastoris fermentations by undertaking a systematic program of optimizing the entire fermentation process. This work aims at undertaking such a program by focusing on strategy to identify and to characterize trends in the behavior of the system. It can be expected that by addressing the process as a whole, rather than narrowly focusing on the protein expression alone, the methodology proposed here can simplify process scale-up and can be applied to several products made by the same host. Pichia pastoris is methylotrophic yeast. In the Pichia pastoris fermentation, the limiting carbon source is glycerol, method or mixture of both. It can grow on methanol as a sole carbon and energy source. It possesses a highly inducible methanol utilization pathway. The first step in the metabolism of methanol is the oxidation of methanol to formaldehyde using molecular oxygen by alcohol oxidase (AOX). AOX, the first enzyme of the pathway, accounts for up to 35% of the total protein in cells grown on limited amounts of methanol. The enzymes undetectable in cells grown on glucose, ethanol or glycerol. There are two genes in Pichia pastoris that code for AOX: AOXI. The AOXI gene product accounts for the majority of alcohol oxidase activity in the cell. This highly inducible and stringently regulated AOXI promoter has been used to construct expression vectors for the production of heterologous proteins in Pichia pastoris. Although some foreign proteins have expressed well in shake-flask cultures, expression levels are typically low compared to fomenter cultures. There are several key aspects of Pichia pastoris fermentations: 1. Fed-batch operation – Controlled addition of glycerol, methanol or mixture thereof. In general, strains are grown initially in a defined medium containing glycerol as its carbon source (growth phase). During this phase, biomass accumulates but heterogonous gene expression is fully repressed. Upon depletion of glycerol, a transition phase is initiated in which additional glycerol is fed to the culture at a growth-limiting rate. Finally, method a mixture of glycerol and methanol is fed to the culture to induce expression (induction phase). The duration of individual substrate feeds, the amount and mode of feeding are critical to optimal fermentation performance. 2.Online measurement and control-One of the most important key parameters in Pichia pastor is expression system is the methanol concentration. Monitoring and controlling this variable are important because high levels of this inductor substrate can be toxic to the cells and low levels of methanol may not be enough to initiate the AOX transcription (Cereghino and Cregg, 2000) This research work aims at investigation the above mentioned aspects by conduction an in depth engineering analysis of the Pichia Pastoris fermentations.