Title of Invention	"A PROCESS FOR THE PRODUCTION OF PROTEINS AND HORMONES IN LARGE VOLUMES"
Abstract	A process for the production of proteins and hormones in large volumes, said process comprising the steps of: i) culturing recombinant Escherichia coli cells in a medium containing glucose and yeast extract in the proportion 1:0.75, upto 11 hours at 37°C, at near neutral pH, under aerobic conditions until the cell concentration reaches an optimum density of about 86, ii) adding 1-3 mM isopropyl ß-D-thiogalactopyranoside to the medium to induce expression of the recombinant E. coli cells, iii) adding nutrients to the medium based on the specific growth rate of the cells, in a manner such as hereindescribed, iv) harvesting the .. cells 5 hours after induction of isopropyl ß-D-thiogalactopyranoside, and v) isolating the protein produced in the medium by a method known per se.

Title of Invention

"A PROCESS FOR THE PRODUCTION OF PROTEINS AND HORMONES IN LARGE VOLUMES"

Abstract

A process for the production of proteins and hormones in large volumes, said process comprising the steps of: i) culturing recombinant Escherichia coli cells in a medium containing glucose and yeast extract in the proportion 1:0.75, upto 11 hours at 37°C, at near neutral pH, under aerobic conditions until the cell concentration reaches an optimum density of about 86, ii) adding 1-3 mM isopropyl ß-D-thiogalactopyranoside to the medium to induce expression of the recombinant E. coli cells, iii) adding nutrients to the medium based on the specific growth rate of the cells, in a manner such as hereindescribed, iv) harvesting the .. cells 5 hours after induction of isopropyl ß-D-thiogalactopyranoside, and v) isolating the protein produced in the medium by a method known per se.

Full Text	FIELD OF THE INVENTION The present invention relates to a process for achieving high volumetric productivity of a recombinant protein in High Cell Density Fed-Batch culture. Preferably, the invention provides a process for the production in large quantities, recombinant oGH (Ovine Growth Hormone) expressed in Escherichia coll. BACKGROUND In, the field of fermentation research, more specifically in the study of recombinant proteins, Escherichia coli is a favourite host for protein synthesis, mainly because it is a well-characterised system. A primary goal of fermentation research is to develop cost effective and better methods to maximise the volumetric productivity of the proteins expressed in E.coli i.e., units of products formed per volume and unit time). As proteins are intracellularly accumulated in recombinant E coli, productivity of proteins is proportional to the final cell-density and specific productivity (i.e., the amount of proteins formed per unit cell mass per unit time). Fed-batch processes primarily focus on increasing the cell mass as compared to simple batch process. High Cell Density Fed-Batch fermentation processes are generally used to enhance the production of recombinant proteins expressed in E coli. Due to the intracellular nature of the expression system in Escherichia coli, the volumetric yield of the recombinant protein depends on both the final cell concentration and the specific cellular protein yield (mg. Protein/ unit cell concentration). Increasing the cell concentration during fed-batch fermentation helps in achieving higher volumetric productivity of the recombinant protein expressed in Escherichia coli. Volumetric productivity of many recombinant proteins have been enhanced by using high cell density fermentation process. However, in most these cases, though maximising cell concentration helps in increasing of the volumetric productivity of recombinant proteins, it is usually at the cost of lower specific cellular protein yield. Lower specific cellular protein yield during high cell density fermentation using E.coli in comparison to that obtained in simple batch fermentation have been reported using for many recombinant proteins like trypsin, porcine growth hormone recombinant ß-galactoside fusion protein and recombinant insecticidal protein. Ideally, to maximise the volumetric productivity of the expressed protein, the specific cellular protein yield obtained in batch culture should be maintained during high cell density growth though a process in respect thereof has not been reported so far. Prior art relating to the invention: Recombinant protein expression using E. coli as host is frequently associated with formation of intracellular aggregates as inclusion bodies. Thus, the volumetric yield of a recombinant protein depends on both the final cell concentration as well as the specific cellular protein yield (Yee and Blanch, 1992). In high cell density fermentation, maximizing the cell concentrations help in increasing the volumetric yield of recombinant proteins, usually at the cost of lower specific cellular protein yield (Kleman and Strohl, 1994). Lower specific cellular protein yields during high cell density fermentation in comparison to that obtained in simple batch fermentation have been reported for recombinant trypsin (Yee and Blanch, 1993), recombinant porcine growth hormone (Chen et al., 1992); recombinant ß-gal fusion protein (Strandberg and Enfors, 1991), recombinant insecticidal protein expressed hi E coli (Shimizu et al., 1992) and recombinant human growth hormone (Shin et al., 1998). This is primarily because most of the investigations related to high cell density fermentation have been focused on increasing unit cell concentrations with very little attention to the specific cellular yield of recombinant proteins. As the ultimate goal of the recombinant fermentation research is to maximize volumetric productivity, i.e. to obtain highest amount of product in given volume in least amount of time, it is essential that the specific cellular protein yield should be maintained during high cell density fermentation. A major constraint on both cell growth and recombinant protein expression during growth of E. coli on glucose as a carbon source has been the secretion of acetic acid (Majewski and Domach, 1990; Han et al., 1992; Lee, 1996). To reduce the secretion of acetic acid during fed-batch fermentation, cells are generally grown at lower specific growth rates to achieve high cell concentrations (Yoon et al., 1994; Korz et al, 1995). Operation of fed-batch fermentation at lower specific growth rate of E, coli extends the duration of batch time and hence affects the volumetric productivity of recombinant proteins. Induction of foreign protein synthesis exerts metabolic burden on the cell physiology, which is associated with reduction in specific growth rates of growing cells (Ryan et ah, 1989; Bentley et al., 1991); Dong et al., 1995; Lim and jung, 1998). If the nutrient feeding during protein expression phase is not adjusted according to the reduction in specific growth rate of the culture, it will lead to accumulation of toxic byproducts particularly acetic acid and build-up of high concentration of nutrients during fed-batch operation. This will have deleterious effect on cell physiology resulting in lowering of the specific cellular protein yield. Further, specific cellular protein yield also depend on concentration of nutrients which favour not only growth but also provide essential nitrogenous source i.e. amino acids for protein synthesis or precursors for macromolecular synthesis. This is particularly true during induction of foreign gene expression at a very high cell concentration, where the nutrient demand increases suddenly for the synthesis of plasmid derived protein resulting in the drainage of amino acid pools of the cell (Harcum and Bentley 1993; Ramirez and Bently 1993). It is expected that feeding complex nitrogenous nutrients along with glucose Will provide precursors to meet the demand for high level synthesis of the expressed protein so that the specific cellular yield can be maintained during high cell density fermentation. The applicants have investigated the kinetics of recombinant Ovine growth hormone (r-oGH) expression as inclusion bodies during the batch and fed-batch fermentation of E. coh in their studies. Ovine growth hormone was expressed as fusion protein containing histidine tag at the N terminal end of the peptide using pQE expression vector in Ml 5 E. coli cells (Appa Rao et al., 1997). The metabolic burden during expression of r-oGH in terms of reduction in the specific growith rate of E. coli was investigated during batch fermentation and correspondingly the cultures were suitably induced to maximize protein expression. The feeding strategy of nutrients during post induction period of fed-batch fermentation process was designed in tune with the reduction in specific growth rate of the culture to maximize the volumetric productivity of r-oGH. In short, the ultimate goal of recombinant fermentation research is to maximize the volumetric productivity of the expressed protein. In high cell density fed-batch fermentation process, where the cell mass is increased considerably in comparison to that obtained during simple batch process is generally used for maximizing the production of recombinant protein expressed in E.coli. OBJECTS OF THE INVENTION Accordingly, the main object of the invention is to develop a novel process for achieving high volumetric productivity of a recombinant protein. One object of the invention relates to a process for maintaining specific cellular protein yield during high cell density fed-batch fermentation for production of recombinant protein in E coli. Another object of the invention is to provide a process for achieving high volumetric productivity of the recombinant protein expressed in E.coli. Yet another object of the invention is to provide a process for production of large quantities of recombinant ovine growth hormone expressed in E.coli, Still, another object of the invention relates to the process of achieving the highest volumetric productivity of any recombinant protein expressed with help of IPTG (isopropyl p- D-thiogalactopyranoside) inducible promoter using single stage fermentation process. One more object of the invention relates to a process for high cell density growth of E.coli in glucose containing medium with reduced secretion of acetic acid. DESCRIPTION OF THE INVENTION In accordance with the above and other objectives, the applicants have developed a novel process for maintaining the specific cellular yield of a recombinant protein with simultaneous increase in the host cell concentration, comprising the steps of: In short, the ultimate goal of recombinant fermentation research is to maximize the volumetric productivity of the expressed protein. In high cell density fed-batch fermentation process, where the cell mass is increased considerably in comparison to that obtained during simple batch process is generally used for maximizing the production of recombinant protein expressed in E.coli. OBJECTS OF THE INVENTION Accordingly, the main object of the invention is to develop a novel process for achieving high volumetric productivity of a recombinant protein. One object of the invention relates to a process for maintaining specific cellular protein yield during high cell density fed-batch fermentation for production of recombinant protein in E.coli. Another object of the invention is to provide a process for achieving high volumetric productivity of the recombinant protein expressed in E.coli. Yet another object of the invention is to provide a process for production of large quantities of recombinant ovine growth hormone expressed m E.coli. Still, another object of the invention relates to the process of achieving the highest volumetric productivity of any recombinant protein expressed with help of IPTG (isopropyl p- D-thiogalactopyranoside) inducible promoter using single stage fermentation process. One more object of the invention relates to a process for high cell density growth of E.coli in glucose containing medium with reduced secretion of acetic acid. STATEMENT OF INVENTION: The invention provides a process for the production of recombinant proteins in large volumes, said process comprising the steps of: i) cultunng recombinant Escherichia colt cells in a medium containing glucose and yeast extract in the proportion 1:0.75, for about 11 hours at 37°C, at near neutral pH, under aerobic conditions until the cell concentration reaches an optimum density of about 86, ii) adding 1-3 mM isopropyl ß-D-thiogalactopyranoside to the medium to induce expression of the recombinant E. coli cells, iii) adding nutrients to the medium based on the specific growth rate of the cells, in a manner such as hereindescribed, iv) harvesting the cells 5 hours after induction of isopropyl p-D- thiogalactopyranoside, and v) isolating the protein produced in the medium by a method known per se. DESCRIPTION OF THE INVENTION In accordance with the above and other objectives, the applicants have developed a novel process for maintaining the specific cellular yield of a recombinant protein with simultaneous increase in the host cell concentration, comprising the steps of: i) culturing the recombinant E.coli cells in a medium containing glucose and yeast in the proportion of about 1:0.75, for about 11 hours at 37°C, in near neutral pH, under aerobic conditions to achieve cell concentration of about 86 OD. ii) adding 2mM IPTG to the culture to induce expression of the recombinant gene; iii) controlling the nutrient feeding rate according to the fall in the specific growth rate of cells in the culture; iv) harvesting the cells 5 hours after induction of IPTG, and v) estimation of the protein expressed, by conventional methods. In an embodiment, the recombinant protein is selected from ovine growth hormone, human growth hormone, and zona pellucida protein (bonnet monkeys). In another embodiment, the preferred recombinant protein is ovine growth hormone (oGH). In another embodiment high cell growth of transformed E. coli cells is possible while maintaining recombinant plasmids. In yet another embodiment, the recombinant protein is expressed as inclusion bodies in E.coli. In still another embodiment, isopropyl-ß-thiogalactopyranoside (IPTG) inducible expression system is used to achieve high volumetric productivity of the recombinant protein. In an embodiment, volumetric productivity up to 0.2g/L/hr. of recombinant ovine growth hormone is obtained using a single stage fermentation process. In an embodiment, the volumetric yield of the recombinant protein is 3-5 gm/litre In another embodiment, Escherichia coli cells are cultured to high density preferably by using the oxidative pathways of glucose metabolism. In another embodiment, the E.coh cells are grown by supplying the nutrients for energy generation and precursors for building blocks of the cells. In yet another embodiment, the accumulation of acetic acid is low during high growth rate of the E.coli cells. In a further embodiment, the acetic acid accumulation during aerobic growth of Escherichia coli in glucose containing medium is maintained below a concentration inhibitory for cell growth. In still another embodiment, the specific cellular yield of the recombinant protein during high cell density fermentation is maintained by addition of complex nutrients like yeast extract. In an embodiment, the acetic acid secretion by E.coli is lowered due to the presence of optimal quantity of yeast extract during the fermentation. In another embodiment, the presence of yeast extract in the medium leads to high level expression of the recombinant protein in the medium. In yet another embodiment, IPTG induction causes high level expression of recombinant protein in Escherichia coli with reduction in specific growth rate of the culture. In still another embodiment, the nutrient feeding strategy during the post induction period is controlled so as to maintain the specific cellular yield of the recombinant protein expressed in Escherichia coli. In a further embodiment, induction of IPTG causes reduction in Escherichia coli cell growth with concomitant increase in percent expression of the recombinant protein. In an embodiment, the gene expression through IPTG induction leads to an irreversible arrest of cell growth. In another embodiment, the specific cellular yield of the recombinant protein expressed intracellularly in Escherichia coli during high cell density fermentation is maintained. In yet another embodiment, high volumetric productivity of recombinant protein expression in E.coli is achieved. In another embodiment, the specific cellular yield of a recombinant protein is expressed intracellularly in E.coli during high cell density fermentation. In particular, the applicants have developed a high cell density fed-batch fermentation process for high specific cellular protein yield. The expression of the gene cloned in E.coli was induced by addition of IPTG to the medium. Parameters optimal for achieving high specific cellular product yield such as optimal induction strategy, inducer concentration and supplementation of complex nitrogenous sources during the high level synthesis of the foreign protein in the fed-batch fermentation process were designed after careful investigation of the protein expression dynamics. The information on kinetics of protein expression and its effect on specific growth rate of the culture was used to design the feeding strategy of the nutrients for high cell density fed batch culture. The nutrient feed policy were optimised particularly during the high cell density fed-batch fermentation process to maintain the specific cellular product yield that is achieved during simple batch fermentation. The invention is described in detail with the aid of the following examples and the accompanying drawings. Various modifications that may be apparent to one in the art are intended to fall within the scope of the invention. BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS Figure 1. Relates to the growth kinetics of E. coli expressing r-oGH during induction with 1 mM IPTG at different stages of growth. Induction at early log phase at cell OD of 2, induction at mid log phase at cell OD of 4, induction at late log phase at cell OD of 10, induced culture. Figure 2. Relates to batch fermentation kinetics of E. coli expressing r- oGH. Cells were induced with 1 mM IPTG at cell OD of 4 residual glucose concentration. Acetic acid concentrations, cell OD at 600 nm. and r-oGH concentrations. Figure 2 B. Relates to batch fermentation kinetics of uninduced E. coli cells. Residual glucose concentration, acetic acid concentrations and cell OD at 600 nm. Figure 3. Relates to the kinetics of Inclusion body formation and its effect on specific growth rates of the culture. Reduction in the specific growth rate of E. coli cells and increase in specific r-oGH yield as a function of post induction time is presented, specific growth rate and specific r-oGH yield during induction at early log phase. Specific growth rate and specific r-oGH yield during induction at mid log phase. Figure 4. Relates to the High cell density fed-batch fermentation of E, coli for the expression of r-oGH. Cells were grown in fermented in fed-batch mode till OD of 86 and then induced with 2 mM IPTG residual glucose concentration, cell OD at 600 nm, r-oGH concentrations and acetic acid concentrations. Figure 5. Relates to the effect of r-oGH inclusion body formation on specific growth rate of E. coli cells during high cell density fermentation. Specific growth rate and specific r-oGH yield during the post induction period. Example: Use of the process of the invention for expression of oGH The high cell density fermentation process was applied for the expression of Ovine growth hormone (oGH). Ovine growth hormone was expressed using pQE expression vector in E.coli Ml5 cells as a fusion protein containing histidine tag at the N-terminal end. Ecoli cells were grown in fermenter using fed-batch operation and high cell concentration was achieved using specific growth rate controller feeding strategy of the nutrients. Expression of the cloned gene was induced by addition of IPTG to the medium. Parameters which influence the specific cellular product yield during the expression of protein in Escherichia coli such as optimal induction strategy, effect of gene expression on the physiological status of the host cells and the requirement of complex nitrogenous sources during the high level synthesis of the foreign protein in fed-batch fermentation process were investigated in detail. The metabolic burden during the expression of protein in Escherichia coli was manifested in terms of reduction of the specific growth rate of the culture during batch fermentation and correspondingly the culture were suitably induced to maximize protein expression. The information on kinetics of the protein expression and its effect on specific growth rate of the culture was used to design the feeding strategy of the nutrients for high cell density fed batch culture. The nutrient feed policy was optimised particularly during the expression phase to maintain specific cellular product yield during high cell density fed-batch fermentation. In batch fermentation, 400 mg of the recombinant oGH was obtained at a cell OD (Optimal Density) of 15, the specific yield was around 26mg/OD of IL culture. During high cell density fermentation, the specific yield was maintained while increasing the cell concentration. A maximum of 3.2 grams/L of the recombinant oGH was produced at a cell OD of 120 (~ 50 gm/L dry cell mass) in 16 hours. During the entire period of fermentation process, the secretion of acetic acid was very low and was below the inhibitory concentration for E.coh cells ( The invention is described in detail hereinbelow: Material and methods Chemicals Pituitary derived Ovine growth hormone and its poiyclonai antibody were obtained fiom NIH (MDDK. Bethesda). USA. Urea, deoxycholate. SDS. isopropyl-P-D thiogalactopyranoside (IPTG) were from Amresco, USA. All other reagents were of analytical grade. Bacterial strains and plasmid A c-DNA fragment coding for ovine growth hormone (oGH) was cloned in-frame downstream of the histidine tag under T5 promoter-lac operator control using pQE-30 expression vector (Qiagen, USA) in E. colt cells (Appa Rao et al . 1997) Transformed E. coli M15 cells containing the recombinant expression plasmid (pQE 30-oGH) were grown in Luna Bertani (LB) or complex medium in the presence of kanamycin (25 µg ml "' ) and ampicillm (50 µg ml"1 ) The grown cultures, when induced with 1 mM IPTG expresses r-oGH as inclusion bodies. Medium composition and inoculum preparation Stock cultures (-70° C) were activated at 37° C in LB broth in an orbital shaker at a speed of 250 rpm. For fermenter experiments, 200 ml of the grown cultures were harvested, centrifuged, dissolved in 25 ml of fresh medium and used as inoculum for the two liter fermenter. The composition of the complex medium was as described in Mori et al 1979, except that the initial glucose and yeast extract concentration were 10 g L-1. The medium composition used for fermentation is given in table 1 The fermentation medium was supplemented with 50 µg ml " of ampicillm and 25 µg ml"' of kanamycm Fermentation conditions A 3 5 liter Chemap fermenter (FZ3000, ChemapAG. Switzerland) with 2 L working volume was used for batch and fed-batch experiments The agitation rate was from 400-900 rpm and the cultivation temperature was 37°C. The pH was maintained at 7.0. by automatic pH controller with addition of alkah f5M NaOH). Dissolved oxygen and pH were monitored by steam stenhzable electrodes (Ingold, Wilminton, MA) Air and/or pure ox\gen was supplied at a rate of 1-2 v.v m. to maintain dissolved oxygen concentration above 40 % air saturation.-The initial glucose and yeast extract concentration was 10 g L"! for batch as well as for fed-batch operation. To understand the kinetics of the inclusion body formation after induction with IPTG, series of batch fermentations were earned out in fermenter. The cultures were induced at early log phase (OD 600 at 2), mid log phase (OD 600 at 4) and late log phase (OD 600 at 10) with 1 mM IPTG and fermentations were carried out for further 5 hours. Samples were taken from the fermenter at regular interval to check cell mass, glucose, acetic acid and r-oGH concentrations. For fed-batch fermentation, E. coli cells were grown for three hours in batch mode in fermenter and then nutrient feeding was initiated to obtain high cell density. The initial working volume of the fermenter during fed-batch operation was 1.5 L and 600 ml of concentrated feed solution containing glucose, yeast extract and magnesium sulfate was supplied at a predetermined rate to maintain a particular specific growth rate. The culture OD was monitored at regular Interval and concentrated nutrient feed solution (40 % glucose, 30 % of yeast extract and 2.5 gram magnesium sulfate per 100 gram of glucose) was fed by peristaltic pump. The yeast extract to glucose ratio in the feed was 0.75. During fed-batch operation, concentrated salt solution were also added to promote better cell growth (Mori et ai , 1979) In order to achieve high volumetnc productivity of r-oGH during fed-batch operation, the cultures were allowed to grow at a moderately high growth rates (0 6 h"1) for two hours, at 0 25 h'! for 2 hours and then the growth rate was decreased to0.15h~' by controlling the feed rate of glucose Cell yield of 1.3 OD L' h"1 glucose was used to fix the pump flow rate during fed-batch operation. The cultures were induced with 2 rnM IPTG at the cell concentration of 86 OD (specific growth rate at the time of induction was 0 15 If1 ). Samples during fed-batch fermentation were taken at regular interval to check cell OD, glucose, acetic acid and r-oGH concentrations. The expression of r-oGH was also checked in SDS-PAGE ( figure not shown). Isolation, purification and estimation ofr-oGH from inclusion bodies Induced cells sampled from the fermenter at different time points were centnfuged at 4000 g for 30 mm and the cell pellet was dissolved in 50 mM Tns-HCl buffer (pH 8.0) containing 5 mM EDTA and I mM PMSF. The cell suspension was sonicated for lysis and were further centnfuged at 8000 g for 30 mm to isolate the inclusion bodies from the cell debris. The inclusion bodies thus obtained were washed with 50 mM Tns-HCl buffer (pH 8 0) containing 5 mM EDTA and 2 % deoxycholate solution. Finally the inclusion bodies were washed with distilled water to remove contaminating salt and detergent, centrifuged at 8000 g for 30 min and pellet containing proteins was used for estimation of r-oGH. At this stage, inclusion bodies were more than 95 % pure as determined by SDS-PAGE, containing mostly monomenc r-oGH with 5-10 % of high molecular aggregates (Khan et al., 1998). The inclusion bodies were completely soluble in 50 mM Tris-HCI buffer (pH 10) containing 1% SDS and the solubilized r-oGH was detectable by radioimmunoassay (RIA). The details of the RIA estimation of r-oGH has been described elsewhere (Appa Rao et al., 1997). The clear samples were diluted appropriately and also assayed using BCA protein assay. Analysis of fermentation parameters The cell density was determined by measuring the culture optical density (OD) at 600 nm with a Kontron (Kontron AG, Switzerland) UV-visible spectrometer. Higher QD samples were diluted suitably to have an absorbance in the range of 0.2 - 0.6. Dry cell weight was determined by centnfuging the sample broth at 4000 g for 20 min and drying the washed cell to constant weight at 105°C. One absorbance unit was equivalent to 0.35 g L"1 of dry cell weight for the uninduced culture and 0.4 g L"1 dry cell weight for induced culture. The specific growth rate ((i = 1/x dx/dt, where x is the cell concentration) was calculated from the slope of the straight line obtained by plotting In x vs time t during the exponential growth phase Residual glucose in the fermentation broth was measured by glucose kit (Sigma, USA), and the acetic acid concentration was monitored by acetic acid kit (Boreigner Mannheim, Germany). Plasmid stability Plasmid stability was measured by direct plating onto selective (50 µg ml"1 ampicillin and 25 µg ml"1 kanamycm) and non-selective LB agar plates and was calculated by taking the ratio of the average number of colonies from three selective plates to the average from three non-selective plates, Results and discussion Batch fermentation Transformed E. coli cells upon induction with IPTG expressed r-oGH as inclusion bodies and the expression plateued within 2-4 hours of induction in shaker flask (Appa Rao et al., 1997). Series of batch fermentation were carried out to evaluate the effect of IPTG induction on cell growth and r-oGH expression. It was observed that induction of E. coli ceils with ImM IPTG was associated with reduction in cell growth (Figure 1). Reduction in cell growth was more profound during IPTG induction at the early log phase of the cultures where the final cell concentrations achieved was almost half of the unmduced cultures. In case of induction at mid log phase, reduction in cell growth was low and the final biomass was same as achieved for uninduced culture. In both the cases, four hours after IPTG induction, cell growth almost stopped. During induction at late log phase, reduction in cell growth was not observed as the cells were approaching stationary growth phase. Uninduced E. coli cells grew exponentially and maximum cell OD at 600 nm of 15 was achieved in 5 hours of batch fermentation. The specific cellular r-oGH yield as well as the volumetric productivity of r-oGH also varied depending on time of induction (Table 2). In the case of IPTG induction at an early and mid log phase, specific r-oGH yield was same (- 66 mg g'1 of cells), whereas the volumetric productivity was higher only in case of induction at mid log phase. This was because, high cell concentrations were obtained while inducing at mid log phase indicating the importance of both high specific cellular protein yield and high cell concentrations for maximizing volumetric productivity of r-oGH. In case of the induction at late log phase, the specific yield of r-oGH was low (50 mg g"1 of ell;;} and correspondingly the volumetric productivity was low in spite of high cell concentrations At the end of the log phase, cells were growing at a lower growth rate and due to the build-up of by-products, were less efficient to express the recombinant protein The low specific cellular protein yield obtained while inducing at late log phase can be attributed to the lower biosynthettc capacity of the growing cells. Induction at late tog phase also increased the time of batch fermentation in comparison to others, thereby reduced the volumetric productivity of r-oGH In all the cases, expression of r-oGH was associated with accumulation of acetic acid Highest protein concentration and high volumetric productivity was achieved, when cells were induced at exponential growth phase. In batch fermentation process, a maximum of 400 mg L"1 of the r-oGH was produced when cells were induced at mid log phase. The specific cellular yield of r-oGH was around 66 mg g"1 of dry cell weight (26.6 mg L"1 OD"5 of cells). The cell yield was 0.60 g g'1 glucose indicating the contribution of yeast extract in the medium for better cell growth. These results are in indication that for maximizing volumetric productivity of r-oGH, it is essential that both the specific cellular protein yield as well as cell mass should be high. As expression of r-oGH was associated with reduction in the specific growth rate of host cells, it is essential to induce actively growing culture at a higher cell concentration so that high volumetric productivity of the expressed protein can be achieved. Kinetics of inclusion body production during batch fermentation. To understand in detail the kinetics of r-oGH expression as inclusion bodies and its effect on specific growth rate of E. colif batch fermentation data of the culture induced with 1 mM IPTG at mid log phase (OD of 4) were analyzed and compared with that of uninduced culture (Figure 2), Induction of E. coli cells with IPTG produced 400 mg L-1 of r-oGH in four hours after which the cell growth and protein expression platued (Figure 2 A). Cell concentration of 15 OD and acetic acid concentration of 2 g L"1 were achieved m 4-5 hours of post induction period Uninduced culture grew exponentially and reached a cell OD of 15 in 5 hours of fermentation (Figure 2B). Expression of r-oGH was not observed in uninduced culture, indicating tight regulatory nature of the promoter system. Glucose utilization was faster and accumulation of acetic acid was lower in uninduced culture in comparison to induced culture- Thus, it was concluded that expression of r-oGH results in reduction of cell growth during batch fermentation Such reduction in cell growth due to r-oGH expression was also observed during IPTG induction at early log phase of the culture (Figure 1). The kinetics of inclusion body formation and its effect on specific growth rate of the culture was evaluated during post-induction period. To delineate the effect of gene expression on specific growth rate from that due to decrease in substrate concentrations, cultures induced with IPTG at early log phase (OD of 2) and mid log phase (OD of 4) were analyzed. It was observed that in both the cases, r-oGH expression was associated with reduction in specific growth rate of E. coli cells (Fig. 3). Trends in reduction in specific growth rate of the cultures upon induction with IPTG were similar for induction at early as well as mid log phase culture. In both the cases, immediately after the addition of IPTG, the cells grew with the same specific growth rate till the inclusion bodies were detected m the eel!. In one hour post induction time, the reduction in specific growth rate was very little (5% of the original value). Maximum reduction in the specific growth rate of the cultures was observed during 2-3 hours of post induction period where the specific growth rate decreased from 0.55 hr"1 to 0.1 hr"1. In terms of doubling time of the organism, it was - observed that cells upon induction with IPTG grew with almost same specific growth rate for one cycle after which the effect of IPTG on specific growth rate was observed. The specific growth rate of cells reduced to 40 % of its original value in two hours and to 80 % in three hours of post induction, complete cessation of growth occurred at four hours of IPTG induction. Irrespective of the specific growth rate at which the culture were growing, specific growth rate decreased almost in similar manner with time upon induction with IPTG : 5 % reduction in first hour, 40 % reduction in 2nd hour, 80 % reduction in 3rd hour and complete cessation of growth after 4 hours of induction. Specific cellular yield of r-oGH also increased with time after mduction with IPTG and platued in four hours (Figure 3) Maximum increase in specific cellular yield of r-oGH was achieved during 1-3 hours of post induction period where the yield increased from !5 to 62 mg g ' of cells. The increase In the specific cellular yield of r-oGH was simultaneously associated with the reduction in the specific growth rate of the culture, indicating the metabolic burden exhibited on the growing cells due to gene expression Such effect of increasing protein expression on reduction of the specific growth of the growing culture has been quantitatively shown recently in £. coli (Dong et al., 1995. Lim and Jung, 1998), This reduction of specific growth rate during expression of the recombinant protein is of prime importance particularly during the high cell density fermentation with controlled feeding of glucose. During such process, if glucose feeding is not lowered in accordance with the specific growth rate of the culture, there will be excess glucose build-up resulting in the secretion of acetic acid which will affect the metabolic activity of the growing cells (Neubauer et al t 1995) Kinetics ofr-oGH expression in high cell density fed-batch fermentation High cell density fed-batch fermentations were carried out to increase the volumetric productivity of r-oGH. It has been reported that organic nitrogen sources like yeast extract (Jung et al , 1988). casamino acids (Shimizu et al., 1987) and soybean hydrolysate (Shimizu et al., 1992) enhance the specific cellular yield of the expressed protein particularly during high cell density fermentation where the demand for nitrogenous nutrients become very high following induction. Incorporation of yeast extract in the feeding medium along with glucose at a ratfo of 0.75 '1 during the fed-batch operation helped in achieving high cell growth with very little secretion of acetic acid (yeast extract optimization data not shown). The kinetics of i-oGH expression in the optimized high cell density fermentation is presented in Figure 4 Cell concentration of 86 OD (34 g L"1 dry cell weight) were achieved in 11 hours of fed-batch fermentation, after which the cultures were induced with 2 mM IPTG (optimum IPTG for induction was 0 02 mM L"! OD"1 of culture) More than 95 % of the cultures had plasmids before IPTG mduction. indicating high stability of the recombinant plasmids during extended fed-batch fermentation The cultures were grown for another 6 hours and the batch was terminated at a cell OD of 124. The nutrient feeding rate during the expression period was decreased according to the fall of specific growth rate of the culture due to r-oGH expression. With the use of optimal glucose and yeast extract feeding, a maximum of 3.2 g L "' of r-oGH were produced in 16 hours of fed-batch fermentation The final cell OD was 124 (49 g dry cell weight L"1 ) and the specific cellular r-oGH yield was 65 mg g "' dry cell weight (25.8 mg L"1 OD "' of cells). Expression of r-oGH as insoluble aggregates in the form of inclusion bodies helped in achieving high specific yield during the fed-batch fermentation process. The specific • growth rate of the culture during the induction period declined as experienced in batch fermentation process (Figure 5). The specific growth rate remained little affected during 1st hour of post induction period after which it decreased with concomitant increase in the specific cellular r-oGH yield. Kinetics of inclusion body production also followed almost the same pattern as it was observed during batch fermentation. However, during high cell density fermentation, maximum expression of r-oGH was observed in 5 hours after IPTG induction instead of 4 hours observed during batch fermentation. This specific cellular r-oGH yield was 65.5 g g"1 cells and was very close to that obtained in batch fermentation. Presence of optimal amount of yeast extract during the entire period of the fed-batch fermentation lowered glucose uptake without compromising the cell growth, produced less acetic acid and helped in maintaining the specific cellular yield of r-oGH. Apart from this, presence of yeast extract in the medium also helps in lowering the inhibitory effect of acetic acid (Koh et al„ 1992) and also works as a better physiological buffer in comparison to the minimal medium (George el al, 1992). In sixteen hours of fed-batch fermentation, a maximum of 3.2 g L'1 of r-oGH were produced at a cell concentration of 124 OD, which is the highest amount of recombinant ovine growth hormone reported to date using E coli based expression system. The volumetric productivity r-oGH was 0.2 g LT1 H"', which is higher than the recently reported high cell density human growth hormone expression (Shin, et al. 1998) This indicated that with the use of yeast extract in the feed medium alongwith glucose during high cell density fermentation, not only the specific cellular protein vield is maintained but also the duration of the process is reduced which results in high volumetric productivity of the expressed protein. The volumetric productivity of r-oGH is the highest value reported so far for any therapeutic protein using IPTG based induction system with single stage fed-batch fermentation system. Higher volumetric productivity of recombinant protein with IPTG based induction system have been reported only m the cases where high cell density fed-batch fermentations were carried out along with cross flow filtration system (Shimiju et al., 1992 ) or in two stage operation (Shin et al., 1997). In conclusion, we have described a strategy by which the volumetric productivity of a r-oGH expressed in E. coli was maximized by maintaining the specific cellular protein yield during high cell density fermentation It was observed that during gene expression particularly from a strong promoter, the specific growth rate of the culture became an intrinsic property of the cells which reduced in a programmed manner upon induction. Unlike the fed-batch strategy used for IGF-1 expression (Wangsa-Wirawan et al., 1997) using the shock-recovery model proposed by Lee and Ramirez 1992, expression of r~oGH was associated with reduction in specific growth rate of the culture analogous to the shock model proposed by Bentley et al , 1991. Hence it was essential to design the feeding strategy of nutrients according to the reduction in specific growth rate of the culture during post induction period for maximizing expression of recombinant ovine growth hormone. A maximum of 3 2 g L" of r-oGH were produced in fed-batch operation in sixteen hours time which is the highest value ever reported for the hormone using E. coli based expression system. Such nutrient feeding strategy can be used for enhancing the volumetric productivity of recombinant proteins expressed as inclusion bodies m E. coli. Table 1. Composition of the medium used for fermentation (Table Removed) Table 2. Effect of IPTG induction at various stages of growth on cell yield and r-oGH expression. a = batch harvest time is indicated in parenthesis WE CLAIM 1. A process for the production of proteins and hormones in large volumes, said process comprising the steps of: i) culturing recombinant Escherichia coli cells in a medium containing glucose and yeast extract in the proportion 1:0.75, upto 11 hours at 37°C, at near neutral pH, under aerobic conditions until the cell concentration reaches an optimum density of about 86, ii) adding 1-3 mM isopropyl ß-D-thiogalactopyranoside to the medium to induce expression of the recombinant E. coli cells, iii) adding nutrients to the medium based on the specific growth rate of the cells, in a manner such as hereindescribed, iv) harvesting the cells 5 hours after induction of isopropyl ß-D-thiogalactopyranoside, and v) isolating the protein produced in the medium by a method known per se. 2. A process as claimed in claim 1 wherein the proteins and hormones are selected from ovine growth hormone, human growth hormone, or zona pellucida protein. 3. A process as claimed in claim 1 wherein the amount of isopropyl ß-D-thiogalactopyranoside added to the medium is 2mM. 4. A process as claimed in claim 1 wherein the amount of nutrients added to the medium in the fed batch operation is about 1.5mL to 600ml. 5. A process as claimed in claim 1 wherein the cells are cultured for about 2 hours at the rate of 0.6 per hour in the fed batch operation. 6. A process as claimed in claim 1 wherein the cells are cultured for 1 to 4 hours at the rate of in the batch fermentation operation. 7. A process as claimed in claims 1 and 2 wherein the volumetric productivity of recombinant ovine growth hormone ' in batch fermentation method is upto 0.2g/L/hr. 8. A process as claimed in claims 1 and 2 wherein the volumetric yield of the recombinant protein is 3-5 gm/litre 9. A process as claimed in claim 1 wherein the acetic acid is secreted by E.coli in the medium. 10. A process as claimed in claim 1 and 9 wherein the acetic acid secretion by E.coli is lowered during the fermentation. 11. A process for the production of proteins and hormones in large, volumes, substantially as hereindescribed and illustrated.

Full Text

FIELD OF THE INVENTION
The present invention relates to a process for achieving high volumetric productivity of a recombinant protein in High Cell Density Fed-Batch culture. Preferably, the invention provides a process for the production in large quantities, recombinant oGH (Ovine Growth Hormone) expressed in Escherichia coll.
BACKGROUND
In, the field of fermentation research, more specifically in the study of recombinant proteins, Escherichia coli is a favourite host for protein synthesis, mainly because it is a well-characterised system.
A primary goal of fermentation research is to develop cost effective and better methods to maximise the volumetric productivity of the proteins expressed in E.coli i.e., units of products formed per volume and unit time). As proteins are intracellularly accumulated in recombinant E coli, productivity of proteins is proportional to the final cell-density and specific productivity (i.e., the amount of proteins formed per unit cell mass per unit time). Fed-batch processes primarily focus on increasing the cell mass as compared to simple batch process. High Cell Density Fed-Batch fermentation processes are generally used to enhance the production of recombinant proteins expressed in E coli.
Due to the intracellular nature of the expression system in Escherichia coli, the volumetric yield of the recombinant protein depends on both the final cell concentration and the specific cellular protein yield (mg. Protein/ unit cell concentration). Increasing the cell concentration during fed-batch fermentation helps in achieving higher volumetric productivity of the recombinant protein expressed in Escherichia coli. Volumetric productivity of many recombinant proteins have been enhanced by using high cell density fermentation process.
However, in most these cases, though maximising cell concentration helps in increasing of the volumetric productivity of recombinant proteins, it is usually at the cost of lower specific cellular protein yield. Lower specific cellular protein yield during high cell density fermentation using E.coli in comparison to that obtained in simple batch
fermentation have been reported using for many recombinant proteins like trypsin, porcine growth hormone recombinant ß-galactoside fusion protein and recombinant insecticidal protein. Ideally, to maximise the volumetric productivity of the expressed protein, the specific cellular protein yield obtained in batch culture should be maintained during high cell density growth though a process in respect thereof has not been reported so far.
Prior art relating to the invention:
Recombinant protein expression using E. coli as host is frequently associated with
formation of intracellular aggregates as inclusion bodies. Thus, the volumetric yield of a recombinant protein depends on both the final cell concentration as well as the specific cellular protein yield (Yee and Blanch, 1992). In high cell density fermentation, maximizing the cell concentrations help in increasing the volumetric yield of recombinant proteins, usually at the cost of lower specific cellular protein yield (Kleman and Strohl, 1994). Lower specific cellular protein yields during high cell density fermentation in comparison to that obtained in simple batch fermentation have been reported for recombinant trypsin (Yee and Blanch, 1993), recombinant porcine growth hormone (Chen et al., 1992); recombinant ß-gal fusion protein (Strandberg and Enfors, 1991), recombinant insecticidal protein expressed hi E coli (Shimizu et al., 1992) and recombinant human growth hormone (Shin et al., 1998). This is primarily because most of the investigations related to high cell density fermentation have been focused on increasing unit cell concentrations with very little attention to the specific cellular yield of recombinant proteins. As the ultimate goal of the recombinant fermentation research is to maximize volumetric productivity, i.e. to obtain highest amount of product in given volume in least amount of time, it is essential that the specific cellular protein yield should be maintained during high cell density fermentation.
A major constraint on both cell growth and recombinant protein expression during growth of E. coli on glucose as a carbon source has been the secretion of acetic acid (Majewski and Domach, 1990; Han et al., 1992; Lee, 1996). To reduce the secretion of acetic acid during fed-batch fermentation, cells are generally grown at lower specific growth rates to achieve high cell concentrations (Yoon et al., 1994; Korz et al, 1995).
Operation of fed-batch fermentation at lower specific growth rate of E, coli extends the duration of batch time and hence affects the volumetric productivity of recombinant proteins. Induction of foreign protein synthesis exerts metabolic burden on the cell physiology, which is associated with reduction in specific growth rates of growing cells (Ryan et ah, 1989; Bentley et al., 1991); Dong et al., 1995; Lim and jung, 1998). If the nutrient feeding during protein expression phase is not adjusted according to the reduction in specific growth rate of the culture, it will lead to accumulation of toxic byproducts particularly acetic acid and build-up of high concentration of nutrients during fed-batch operation. This will have deleterious effect on cell physiology resulting in lowering of the specific cellular protein yield. Further, specific cellular protein yield also depend on concentration of nutrients which favour not only growth but also provide essential nitrogenous source i.e. amino acids for protein synthesis or precursors for macromolecular synthesis. This is particularly true during induction of foreign gene expression at a very high cell concentration, where the nutrient demand increases suddenly for the synthesis of plasmid derived protein resulting in the drainage of amino acid pools of the cell (Harcum and Bentley 1993; Ramirez and Bently 1993). It is expected that feeding complex nitrogenous nutrients along with glucose Will provide precursors to meet the demand for high level synthesis of the expressed protein so that the specific cellular yield can be maintained during high cell density fermentation.
The applicants have investigated the kinetics of recombinant Ovine growth hormone (r-oGH) expression as inclusion bodies during the batch and fed-batch fermentation of E. coh in their studies. Ovine growth hormone was expressed as fusion protein containing histidine tag at the N terminal end of the peptide using pQE expression vector in Ml 5 E. coli cells (Appa Rao et al., 1997). The metabolic burden during expression of r-oGH in terms of reduction in the specific growith rate of E. coli was investigated during batch fermentation and correspondingly the cultures were suitably induced to maximize protein expression. The feeding strategy of nutrients during post induction period of fed-batch fermentation process was designed in tune with the reduction in specific growth rate of the culture to maximize the volumetric productivity of r-oGH.
In short, the ultimate goal of recombinant fermentation research is to maximize the volumetric productivity of the expressed protein. In high cell density fed-batch fermentation process, where the cell mass is increased considerably in comparison to that obtained during simple batch process is generally used for maximizing the production of recombinant protein expressed in E.coli.
OBJECTS OF THE INVENTION
Accordingly, the main object of the invention is to develop a novel process for achieving high volumetric productivity of a recombinant protein.
One object of the invention relates to a process for maintaining specific cellular protein yield during high cell density fed-batch fermentation for production of recombinant protein in E coli.
Another object of the invention is to provide a process for achieving high volumetric productivity of the recombinant protein expressed in E.coli.
Yet another object of the invention is to provide a process for production of large quantities of recombinant ovine growth hormone expressed in E.coli,
Still, another object of the invention relates to the process of achieving the highest volumetric productivity of any recombinant protein expressed with help of IPTG (isopropyl p- D-thiogalactopyranoside) inducible promoter using single stage fermentation process.
One more object of the invention relates to a process for high cell density growth of E.coli in glucose containing medium with reduced secretion of acetic acid.
DESCRIPTION OF THE INVENTION
In accordance with the above and other objectives, the applicants have developed a novel process for maintaining the specific cellular yield of a recombinant protein with simultaneous increase in the host cell concentration, comprising the steps of:
In short, the ultimate goal of recombinant fermentation research is to maximize the volumetric productivity of the expressed protein. In high cell density fed-batch fermentation process, where the cell mass is increased considerably in comparison to that obtained during simple batch process is generally used for maximizing the production of recombinant protein expressed in E.coli.
OBJECTS OF THE INVENTION
Accordingly, the main object of the invention is to develop a novel process for achieving high volumetric productivity of a recombinant protein.
One object of the invention relates to a process for maintaining specific cellular protein yield during high cell density fed-batch fermentation for production of recombinant protein in E.coli.
Another object of the invention is to provide a process for achieving high volumetric productivity of the recombinant protein expressed in E.coli.
Yet another object of the invention is to provide a process for production of large quantities of recombinant ovine growth hormone expressed m E.coli.
Still, another object of the invention relates to the process of achieving the highest volumetric productivity of any recombinant protein expressed with help of IPTG (isopropyl p- D-thiogalactopyranoside) inducible promoter using single stage fermentation process.
One more object of the invention relates to a process for high cell density growth of E.coli in glucose containing medium with reduced secretion of acetic acid.
STATEMENT OF INVENTION:
The invention provides a process for the production of recombinant proteins in large
volumes, said process comprising the steps of:
i) cultunng recombinant Escherichia colt cells in a medium containing glucose and yeast extract in the proportion 1:0.75, for about 11 hours at 37°C, at near neutral pH, under aerobic conditions until the cell concentration reaches an optimum density of about 86,
ii) adding 1-3 mM isopropyl ß-D-thiogalactopyranoside to the medium to induce
expression of the recombinant E. coli cells, iii) adding nutrients to the medium based on the specific growth rate of the cells, in a
manner such as hereindescribed, iv) harvesting the cells 5 hours after induction of isopropyl p-D-
thiogalactopyranoside, and v) isolating the protein produced in the medium by a method known per se. DESCRIPTION OF THE INVENTION
In accordance with the above and other objectives, the applicants have developed a novel process for maintaining the specific cellular yield of a recombinant protein with simultaneous increase in the host cell concentration, comprising the steps of: i) culturing the recombinant E.coli cells in a medium containing glucose and yeast in the proportion of about 1:0.75, for about 11 hours at 37°C, in near neutral pH, under aerobic conditions to achieve cell concentration of about 86 OD. ii) adding 2mM IPTG to the culture to induce expression of the recombinant gene; iii) controlling the nutrient feeding rate according to the fall in the specific growth rate
of cells in the culture; iv) harvesting the cells 5 hours after induction of IPTG, and v) estimation of the protein expressed, by conventional methods. In an embodiment, the recombinant protein is selected from ovine growth hormone, human growth hormone, and zona pellucida protein (bonnet monkeys). In another embodiment, the preferred recombinant protein is ovine growth hormone (oGH). In another embodiment high cell growth of transformed E. coli cells is possible while maintaining recombinant plasmids. In yet another embodiment, the recombinant protein is expressed as inclusion bodies in E.coli.
In still another embodiment, isopropyl-ß-thiogalactopyranoside (IPTG) inducible expression system is used to achieve high volumetric productivity of the recombinant protein. In an embodiment, volumetric productivity up to 0.2g/L/hr. of recombinant ovine growth hormone is obtained using a single stage fermentation process. In an embodiment, the volumetric yield of the recombinant protein is 3-5 gm/litre
In another embodiment, Escherichia coli cells are cultured to high density preferably by using the oxidative pathways of glucose metabolism.
In another embodiment, the E.coh cells are grown by supplying the nutrients for energy generation and precursors for building blocks of the cells.
In yet another embodiment, the accumulation of acetic acid is low during high growth rate of the E.coli cells.
In a further embodiment, the acetic acid accumulation during aerobic growth of Escherichia coli in glucose containing medium is maintained below a concentration inhibitory for cell growth.
In still another embodiment, the specific cellular yield of the recombinant protein during high cell density fermentation is maintained by addition of complex nutrients like yeast extract.
In an embodiment, the acetic acid secretion by E.coli is lowered due to the presence of optimal quantity of yeast extract during the fermentation.
In another embodiment, the presence of yeast extract in the medium leads to high level expression of the recombinant protein in the medium.
In yet another embodiment, IPTG induction causes high level expression of recombinant protein in Escherichia coli with reduction in specific growth rate of the culture.
In still another embodiment, the nutrient feeding strategy during the post induction period is controlled so as to maintain the specific cellular yield of the recombinant protein expressed in Escherichia coli.
In a further embodiment, induction of IPTG causes reduction in Escherichia coli cell growth with concomitant increase in percent expression of the recombinant protein.
In an embodiment, the gene expression through IPTG induction leads to an irreversible arrest of cell growth.
In another embodiment, the specific cellular yield of the recombinant protein expressed intracellularly in Escherichia coli during high cell density fermentation is maintained.
In yet another embodiment, high volumetric productivity of recombinant protein expression in E.coli is achieved.
In another embodiment, the specific cellular yield of a recombinant protein is expressed intracellularly in E.coli during high cell density fermentation.
In particular, the applicants have developed a high cell density fed-batch fermentation process for high specific cellular protein yield. The expression of the gene cloned in E.coli was induced by addition of IPTG to the medium. Parameters optimal for achieving high specific cellular product yield such as optimal induction strategy, inducer concentration and supplementation of complex nitrogenous sources during the high level synthesis of the foreign protein in the fed-batch fermentation process were designed after careful investigation of the protein expression dynamics. The information on kinetics of protein expression and its effect on specific growth rate of the culture was used to design the feeding strategy of the nutrients for high cell density fed batch culture. The nutrient feed policy were optimised particularly during the high cell density fed-batch fermentation process to maintain the specific cellular product yield that is achieved during simple batch fermentation.
The invention is described in detail with the aid of the following examples and the accompanying drawings. Various modifications that may be apparent to one in the art are intended to fall within the scope of the invention.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
Figure 1. Relates to the growth kinetics of E. coli expressing r-oGH during induction with 1 mM IPTG at different stages of growth. Induction at early log

phase at cell OD of 2, induction at mid log phase at cell OD of 4, induction at late log phase at cell OD of 10, induced culture.
Figure 2. Relates to batch fermentation kinetics of E. coli expressing r- oGH. Cells were induced with 1 mM IPTG at cell OD of 4 residual glucose concentration. Acetic acid concentrations, cell OD at 600 nm. and r-oGH concentrations.
Figure 2 B. Relates to batch fermentation kinetics of uninduced E. coli cells.
Residual glucose concentration, acetic acid concentrations and cell OD at 600 nm.
Figure 3. Relates to the kinetics of Inclusion body formation and its effect on specific growth rates of the culture. Reduction in the specific growth rate of E. coli cells and increase in specific r-oGH yield as a function of post induction time is presented, specific growth rate and specific r-oGH yield during induction at early log phase. Specific growth rate and specific r-oGH yield during induction at mid log phase.
Figure 4. Relates to the High cell density fed-batch fermentation of E, coli for the expression of r-oGH. Cells were grown in fermented in fed-batch mode till OD of 86 and then induced with 2 mM IPTG residual glucose concentration, cell OD at 600 nm, r-oGH concentrations and acetic acid concentrations.
Figure 5. Relates to the effect of r-oGH inclusion body formation on specific growth rate of E. coli cells during high cell density fermentation. Specific growth rate and specific r-oGH yield during the post induction period.
Example: Use of the process of the invention for expression of oGH
The high cell density fermentation process was applied for the expression of Ovine growth hormone (oGH). Ovine growth hormone was expressed using pQE expression vector in E.coli Ml5 cells as a fusion protein containing histidine tag at the N-terminal end. Ecoli cells were grown in fermenter using fed-batch operation and high cell concentration was achieved using specific growth rate controller feeding strategy of the nutrients. Expression of the cloned gene was induced by addition of IPTG to the medium. Parameters which influence the specific cellular product yield during the expression of
protein in Escherichia coli such as optimal induction strategy, effect of gene expression on the physiological status of the host cells and the requirement of complex nitrogenous sources during the high level synthesis of the foreign protein in fed-batch fermentation process were investigated in detail. The metabolic burden during the expression of protein in Escherichia coli was manifested in terms of reduction of the specific growth rate of the culture during batch fermentation and correspondingly the culture were suitably induced to maximize protein expression. The information on kinetics of the protein expression and its effect on specific growth rate of the culture was used to design the feeding strategy of the nutrients for high cell density fed batch culture. The nutrient feed policy was optimised particularly during the expression phase to maintain specific cellular product yield during high cell density fed-batch fermentation. In batch fermentation, 400 mg of the recombinant oGH was obtained at a cell OD (Optimal Density) of 15, the specific yield was around 26mg/OD of IL culture. During high cell density fermentation, the specific yield was maintained while increasing the cell concentration. A maximum of 3.2 grams/L of the recombinant oGH was produced at a cell OD of 120 (~ 50 gm/L dry cell mass) in 16 hours. During the entire period of fermentation process, the secretion of acetic acid was very low and was below the inhibitory concentration for E.coh cells ( The invention is described in detail hereinbelow:
Material and methods
Chemicals
Pituitary derived Ovine growth hormone and its poiyclonai antibody were obtained fiom NIH (MDDK. Bethesda). USA. Urea, deoxycholate. SDS. isopropyl-P-D thiogalactopyranoside (IPTG) were from Amresco, USA. All other reagents were of analytical grade.
Bacterial strains and plasmid A c-DNA fragment coding for ovine growth hormone (oGH) was cloned in-frame downstream of the histidine tag under T5 promoter-lac operator control using pQE-30 expression vector (Qiagen, USA) in E. colt cells (Appa Rao et al . 1997) Transformed E. coli M15 cells containing the recombinant expression plasmid (pQE 30-oGH) were grown in Luna Bertani (LB) or complex medium in the presence of kanamycin (25 µg ml "' ) and ampicillm (50 µg ml"1 ) The grown cultures, when induced with 1 mM IPTG expresses r-oGH as inclusion bodies.
Medium composition and inoculum preparation Stock cultures (-70° C) were activated at 37° C in LB broth in an orbital shaker at a speed of 250 rpm. For fermenter experiments, 200 ml of the grown cultures were harvested, centrifuged, dissolved in 25 ml of fresh medium and used as inoculum for the two liter fermenter. The composition of the complex medium was as described in Mori et al 1979, except that the initial glucose and yeast extract concentration were 10 g L-1. The medium composition used for fermentation is given in table 1 The fermentation medium was supplemented with 50 µg ml " of ampicillm and 25 µg ml"' of kanamycm
Fermentation conditions A 3 5 liter Chemap fermenter (FZ3000, ChemapAG. Switzerland) with 2 L working volume was used for batch and fed-batch experiments The agitation rate was from 400-900 rpm and the cultivation temperature was 37°C. The pH was maintained at 7.0. by automatic pH controller with addition of alkah f5M NaOH). Dissolved oxygen and pH were monitored by steam stenhzable electrodes (Ingold, Wilminton, MA) Air
and/or pure ox\gen was supplied at a rate of 1-2 v.v m. to maintain dissolved oxygen concentration above 40 % air saturation.-The initial glucose and yeast extract concentration was 10 g L"! for batch as well as for fed-batch operation. To understand the kinetics of the inclusion body formation after induction with IPTG, series of batch fermentations were earned out in fermenter. The cultures were induced at early log phase (OD 600 at 2), mid log phase (OD 600 at 4) and late log phase (OD 600 at 10) with 1 mM IPTG and fermentations were carried out for further 5 hours. Samples were taken from the fermenter at regular interval to check cell mass, glucose, acetic acid and r-oGH concentrations.
For fed-batch fermentation, E. coli cells were grown for three hours in batch mode in fermenter and then nutrient feeding was initiated to obtain high cell density. The initial working volume of the fermenter during fed-batch operation was 1.5 L and 600 ml of concentrated feed solution containing glucose, yeast extract and magnesium sulfate was supplied at a predetermined rate to maintain a particular specific growth rate. The culture OD was monitored at regular Interval and concentrated nutrient feed solution (40 % glucose, 30 % of yeast extract and 2.5 gram magnesium sulfate per 100 gram of glucose) was fed by peristaltic pump. The yeast extract to glucose ratio in the feed was 0.75. During fed-batch operation, concentrated salt solution were also added to promote better cell growth (Mori et ai , 1979) In order to achieve high volumetnc productivity of r-oGH during fed-batch operation, the cultures were allowed to grow at a moderately high growth rates (0 6 h"1) for two hours, at 0 25 h'! for 2 hours and then the growth rate was decreased to0.15h~' by controlling the feed rate of glucose Cell yield of 1.3 OD L' h"1 glucose was used to fix the pump flow rate during fed-batch operation. The cultures were induced with 2 rnM IPTG at the cell concentration of 86 OD (specific growth rate at the time of induction was 0 15 If1 ). Samples during fed-batch fermentation were taken at regular interval to check cell OD, glucose, acetic acid and r-oGH concentrations. The expression of r-oGH was also checked in SDS-PAGE ( figure not shown).
Isolation, purification and estimation ofr-oGH from inclusion bodies
Induced cells sampled from the fermenter at different time points were centnfuged at 4000 g for 30 mm and the cell pellet was dissolved in 50 mM Tns-HCl buffer (pH 8.0) containing 5 mM EDTA and I mM PMSF. The cell suspension was sonicated for lysis and were further centnfuged at 8000 g for 30 mm to isolate the inclusion bodies from the cell debris. The inclusion bodies thus obtained were washed with 50 mM Tns-HCl buffer (pH 8 0) containing 5 mM EDTA and 2 % deoxycholate solution. Finally the inclusion bodies were washed with distilled water to remove contaminating salt and detergent, centrifuged at 8000 g for 30 min and pellet containing proteins was used for estimation of r-oGH. At this stage, inclusion bodies were more than 95 % pure as determined by SDS-PAGE, containing mostly monomenc r-oGH with 5-10 % of high molecular aggregates (Khan et al., 1998). The inclusion bodies were completely soluble in 50 mM Tris-HCI buffer (pH 10) containing 1% SDS and the solubilized r-oGH was detectable by radioimmunoassay (RIA). The details of the RIA estimation of r-oGH has been described elsewhere (Appa Rao et al., 1997). The clear samples were diluted appropriately and also assayed using BCA protein assay.
Analysis of fermentation parameters The cell density was determined by measuring the culture optical density (OD) at 600 nm with a Kontron (Kontron AG, Switzerland) UV-visible spectrometer. Higher QD samples were diluted suitably to have an absorbance in the range of 0.2 - 0.6. Dry cell weight was determined by centnfuging the sample broth at 4000 g for 20 min and drying the washed cell to constant weight at 105°C. One absorbance unit was equivalent to 0.35 g L"1 of dry cell weight for the uninduced culture and 0.4 g L"1 dry cell weight for induced culture. The specific growth rate ((i = 1/x dx/dt, where x is the cell concentration) was calculated from the slope of the straight line obtained by plotting In x vs time t during the exponential growth phase Residual glucose in the fermentation broth was measured by glucose kit (Sigma, USA), and the acetic acid concentration was monitored by acetic acid kit (Boreigner Mannheim, Germany).
Plasmid stability
Plasmid stability was measured by direct plating onto selective (50 µg ml"1 ampicillin and 25 µg ml"1 kanamycm) and non-selective LB agar plates and was calculated by taking the ratio of the average number of colonies from three selective plates to the average from three non-selective plates,
Results and discussion
Batch fermentation
Transformed E. coli cells upon induction with IPTG expressed r-oGH as inclusion bodies and the expression plateued within 2-4 hours of induction in shaker flask (Appa Rao et al., 1997). Series of batch fermentation were carried out to evaluate the effect of IPTG induction on cell growth and r-oGH expression. It was observed that induction of E. coli ceils with ImM IPTG was associated with reduction in cell growth (Figure 1). Reduction in cell growth was more profound during IPTG induction at the early log phase of the cultures where the final cell concentrations achieved was almost half of the unmduced cultures. In case of induction at mid log phase, reduction in cell growth was low and the final biomass was same as achieved for uninduced culture. In both the cases, four hours after IPTG induction, cell growth almost stopped. During induction at late log phase, reduction in cell growth was not observed as the cells were approaching stationary growth phase. Uninduced E. coli cells grew exponentially and maximum cell OD at 600 nm of 15 was achieved in 5 hours of batch fermentation.
The specific cellular r-oGH yield as well as the volumetric productivity of r-oGH also varied depending on time of induction (Table 2). In the case of IPTG induction at an early and mid log phase, specific r-oGH yield was same (- 66 mg g'1 of cells), whereas the volumetric productivity was higher only in case of induction at mid log phase. This was because, high cell concentrations were obtained while inducing at mid log phase indicating the importance of both high specific cellular protein yield and high cell concentrations for maximizing volumetric productivity of r-oGH. In case of the induction at late log phase, the specific yield of r-oGH was low (50 mg g"1 of
ell;;} and correspondingly the volumetric productivity was low in spite of high cell concentrations At the end of the log phase, cells were growing at a lower growth rate and due to the build-up of by-products, were less efficient to express the recombinant protein The low specific cellular protein yield obtained while inducing at late log phase can be attributed to the lower biosynthettc capacity of the growing cells. Induction at late tog phase also increased the time of batch fermentation in comparison to others, thereby reduced the volumetric productivity of r-oGH In all the cases, expression of r-oGH was associated with accumulation of acetic acid Highest protein concentration and high volumetric productivity was achieved, when cells were induced at exponential growth phase. In batch fermentation process, a maximum of 400 mg L"1 of the r-oGH was produced when cells were induced at mid log phase. The specific cellular yield of r-oGH was around 66 mg g"1 of dry cell weight (26.6 mg L"1 OD"5 of cells). The cell yield was 0.60 g g'1 glucose indicating the contribution of yeast extract in the medium for better cell growth. These results are in indication that for maximizing volumetric productivity of r-oGH, it is essential that both the specific cellular protein yield as well as cell mass should be high. As expression of r-oGH was associated with reduction in the specific growth rate of host cells, it is essential to induce actively growing culture at a higher cell concentration so that high volumetric productivity of the expressed protein can be achieved.
Kinetics of inclusion body production during batch fermentation. To understand in detail the kinetics of r-oGH expression as inclusion bodies and its effect on specific growth rate of E. colif batch fermentation data of the culture induced with 1 mM IPTG at mid log phase (OD of 4) were analyzed and compared with that of uninduced culture (Figure 2), Induction of E. coli cells with IPTG produced 400 mg L-1 of r-oGH in four hours after which the cell growth and protein expression platued (Figure 2 A). Cell concentration of 15 OD and acetic acid concentration of 2 g L"1 were achieved m 4-5 hours of post induction period Uninduced culture grew exponentially and reached a cell OD of 15 in 5 hours of fermentation (Figure 2B). Expression of r-oGH was not observed in uninduced
culture, indicating tight regulatory nature of the promoter system. Glucose utilization was faster and accumulation of acetic acid was lower in uninduced culture in comparison to induced culture- Thus, it was concluded that expression of r-oGH results in reduction of cell growth during batch fermentation Such reduction in cell growth due to r-oGH expression was also observed during IPTG induction at early log phase of the culture (Figure 1).
The kinetics of inclusion body formation and its effect on specific growth rate of the
culture was evaluated during post-induction period. To delineate the effect of gene
expression on specific growth rate from that due to decrease in substrate
concentrations, cultures induced with IPTG at early log phase (OD of 2) and mid log
phase (OD of 4) were analyzed. It was observed that in both the cases, r-oGH
expression was associated with reduction in specific growth rate of E. coli cells (Fig.
3). Trends in reduction in specific growth rate of the cultures upon induction with
IPTG were similar for induction at early as well as mid log phase culture. In both the
cases, immediately after the addition of IPTG, the cells grew with the same specific
growth rate till the inclusion bodies were detected m the eel!. In one hour post
induction time, the reduction in specific growth rate was very little (5% of the
original value). Maximum reduction in the specific growth rate of the cultures was
observed during 2-3 hours of post induction period where the specific growth rate
decreased from 0.55 hr"1 to 0.1 hr"1. In terms of doubling time of the organism, it was -
observed that cells upon induction with IPTG grew with almost same specific growth
rate for one cycle after which the effect of IPTG on specific growth rate was
observed. The specific growth rate of cells reduced to 40 % of its original value in
two hours and to 80 % in three hours of post induction, complete cessation of
growth occurred at four hours of IPTG induction. Irrespective of the specific growth
rate at which the culture were growing, specific growth rate decreased almost in
similar manner with time upon induction with IPTG : 5 % reduction in first hour, 40
% reduction in 2nd hour, 80 % reduction in 3rd hour and complete cessation of
growth after 4 hours of induction. Specific cellular yield of r-oGH also increased
with time after mduction with IPTG and platued in four hours (Figure 3) Maximum increase in specific cellular yield of r-oGH was achieved during 1-3 hours of post induction period where the yield increased from !5 to 62 mg g ' of cells. The increase In the specific cellular yield of r-oGH was simultaneously associated with the reduction in the specific growth rate of the culture, indicating the metabolic burden exhibited on the growing cells due to gene expression Such effect of increasing protein expression on reduction of the specific growth of the growing culture has been quantitatively shown recently in £. coli (Dong et al., 1995. Lim and Jung, 1998), This reduction of specific growth rate during expression of the recombinant protein is of prime importance particularly during the high cell density fermentation with controlled feeding of glucose. During such process, if glucose feeding is not lowered in accordance with the specific growth rate of the culture, there will be excess glucose build-up resulting in the secretion of acetic acid which will affect the metabolic activity of the growing cells (Neubauer et al t 1995)
Kinetics ofr-oGH expression in high cell density fed-batch fermentation High cell density fed-batch fermentations were carried out to increase the volumetric productivity of r-oGH. It has been reported that organic nitrogen sources like yeast extract (Jung et al , 1988). casamino acids (Shimizu et al., 1987) and soybean hydrolysate (Shimizu et al., 1992) enhance the specific cellular yield of the expressed protein particularly during high cell density fermentation where the demand for nitrogenous nutrients become very high following induction. Incorporation of yeast extract in the feeding medium along with glucose at a ratfo of 0.75 '1 during the fed-batch operation helped in achieving high cell growth with very little secretion of acetic acid (yeast extract optimization data not shown).
The kinetics of i-oGH expression in the optimized high cell density fermentation is presented in Figure 4 Cell concentration of 86 OD (34 g L"1 dry cell weight) were achieved in 11 hours of fed-batch fermentation, after which the cultures were induced with 2 mM IPTG (optimum IPTG for induction was 0 02 mM L"! OD"1 of culture) More than 95 % of the cultures had plasmids before IPTG mduction.
indicating high stability of the recombinant plasmids during extended fed-batch fermentation The cultures were grown for another 6 hours and the batch was terminated at a cell OD of 124. The nutrient feeding rate during the expression period was decreased according to the fall of specific growth rate of the culture due to r-oGH expression. With the use of optimal glucose and yeast extract feeding, a maximum of 3.2 g L "' of r-oGH were produced in 16 hours of fed-batch fermentation The final cell OD was 124 (49 g dry cell weight L"1 ) and the specific cellular r-oGH yield was 65 mg g "' dry cell weight (25.8 mg L"1 OD "' of cells). Expression of r-oGH as insoluble aggregates in the form of inclusion bodies helped in achieving high specific yield during the fed-batch fermentation process. The specific • growth rate of the culture during the induction period declined as experienced in batch fermentation process (Figure 5). The specific growth rate remained little affected during 1st hour of post induction period after which it decreased with concomitant increase in the specific cellular r-oGH yield. Kinetics of inclusion body production also followed almost the same pattern as it was observed during batch fermentation. However, during high cell density fermentation, maximum expression of r-oGH was observed in 5 hours after IPTG induction instead of 4 hours observed during batch fermentation. This specific cellular r-oGH yield was 65.5 g g"1 cells and was very close to that obtained in batch fermentation.
Presence of optimal amount of yeast extract during the entire period of the fed-batch fermentation lowered glucose uptake without compromising the cell growth, produced less acetic acid and helped in maintaining the specific cellular yield of r-oGH. Apart from this, presence of yeast extract in the medium also helps in lowering the inhibitory effect of acetic acid (Koh et al„ 1992) and also works as a better physiological buffer in comparison to the minimal medium (George el al, 1992). In sixteen hours of fed-batch fermentation, a maximum of 3.2 g L'1 of r-oGH were produced at a cell concentration of 124 OD, which is the highest amount of recombinant ovine growth hormone reported to date using E coli based expression system. The volumetric productivity r-oGH was 0.2 g LT1 H"', which is higher than the
recently reported high cell density human growth hormone expression (Shin, et al. 1998) This indicated that with the use of yeast extract in the feed medium alongwith glucose during high cell density fermentation, not only the specific cellular protein vield is maintained but also the duration of the process is reduced which results in high volumetric productivity of the expressed protein. The volumetric productivity of r-oGH is the highest value reported so far for any therapeutic protein using IPTG based induction system with single stage fed-batch fermentation system. Higher volumetric productivity of recombinant protein with IPTG based induction system have been reported only m the cases where high cell density fed-batch fermentations were carried out along with cross flow filtration system (Shimiju et al., 1992 ) or in two stage operation (Shin et al., 1997).
In conclusion, we have described a strategy by which the volumetric productivity of a r-oGH expressed in E. coli was maximized by maintaining the specific cellular protein yield during high cell density fermentation It was observed that during gene expression particularly from a strong promoter, the specific growth rate of the culture became an intrinsic property of the cells which reduced in a programmed manner upon induction. Unlike the fed-batch strategy used for IGF-1 expression (Wangsa-Wirawan et al., 1997) using the shock-recovery model proposed by Lee and Ramirez 1992, expression of r~oGH was associated with reduction in specific growth rate of the culture analogous to the shock model proposed by Bentley et al , 1991. Hence it was essential to design the feeding strategy of nutrients according to the reduction in specific growth rate of the culture during post induction period for maximizing expression of recombinant ovine growth hormone. A maximum of 3 2 g L" of r-oGH were produced in fed-batch operation in sixteen hours time which is the highest value ever reported for the hormone using E. coli based expression system. Such nutrient feeding strategy can be used for enhancing the volumetric productivity of recombinant proteins expressed as inclusion bodies m E. coli.
Table 1. Composition of the medium used for fermentation
(Table Removed)
Table 2. Effect of IPTG induction at various stages
of growth on cell yield and r-oGH expression.

a = batch harvest time is indicated in parenthesis

WE CLAIM
1. A process for the production of proteins and hormones
in large volumes, said process comprising the steps of:
i) culturing recombinant Escherichia coli cells in a medium containing glucose and yeast extract in the proportion 1:0.75, upto 11 hours at 37°C, at near neutral pH, under aerobic conditions until the cell concentration reaches an optimum density of about 86, ii) adding 1-3 mM isopropyl ß-D-thiogalactopyranoside to the medium to induce expression of the recombinant E. coli cells, iii) adding nutrients to the medium based on the specific growth rate of the cells, in a manner such as hereindescribed, iv) harvesting the cells 5 hours after induction of
isopropyl ß-D-thiogalactopyranoside, and v) isolating the protein produced in the medium by a method known per se.
2. A process as claimed in claim 1 wherein the proteins and hormones are selected from ovine growth hormone, human growth hormone, or zona pellucida protein.
3. A process as claimed in claim 1 wherein the amount of isopropyl ß-D-thiogalactopyranoside added to the medium is 2mM.
4. A process as claimed in claim 1 wherein the amount of nutrients added to the medium in the fed batch operation is about 1.5mL to 600ml.
5. A process as claimed in claim 1 wherein the cells are cultured for about 2 hours at the rate of 0.6 per hour in the fed batch operation.
6. A process as claimed in claim 1 wherein the cells are cultured for 1 to 4 hours at the rate of in the batch fermentation operation.
7. A process as claimed in claims 1 and 2 wherein the volumetric productivity of recombinant ovine growth hormone ' in batch fermentation method is upto 0.2g/L/hr.
8. A process as claimed in claims 1 and 2 wherein the volumetric yield of the recombinant protein is 3-5 gm/litre
9. A process as claimed in claim 1 wherein the acetic acid is secreted by E.coli in the medium.
10. A process as claimed in claim 1 and 9 wherein the acetic acid secretion by E.coli is lowered during the fermentation.
11. A process for the production of proteins and hormones in large, volumes, substantially as hereindescribed and illustrated.

Documents:

594-del-1998-abstract.pdf

594-del-1998-claims.pdf

594-del-1998-correspondence-others.pdf

594-del-1998-correspondence-po.pdf

594-del-1998-description (provisional).pdf

594-del-1998-description (complete).pdf

594-del-1998-drawings.pdf

594-del-1998-form-1.pdf

594-del-1998-form-2.pdf

594-del-1998-form-26.pdf

594-del-1998-form-4.pdf

594-del-1998-form-5.pdf

594-del-1998-form-6.pdf

594-del-1998-form-9.pdf

594-del-1998-petition-others.pdf

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Patent Number

189312

Indian Patent Application Number

594/DEL/1998

PG Journal Number

6/2003

Publication Date

08-Feb-2003

Grant Date

03-Feb-2004

Date of Filing

06-Mar-1998

Name of Patentee

NATIONAL INSTITUTE OF IMMUNOLOGY

Applicant Address

ARUNA ASAF ALI MARG, NEW DELHI-110067, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	SATISH MAHADEO RAO TOTEY	NATIONAL INSTITUTE OF IMMUNOLOGY, ARUNA ASAF ALI MARG, NEW DELHI-110067, INDIA.
2	KUMMARAPURUGU BALACHANDRA APPA RAO	NATIONAL INSTITUTE OF IMMUNOLOGY, ARUNA ASAF ALI MARG, NEW DELHI-110067, INDIA.
3	AMULYA KUMAR PANDA	NATIONAL INSTITUTE OF IMMUNOLOGY, ARUNA ASAF ALI MARG, NEW DELHI-110067, INDIA.

PCT International Classification Number

C07G 15/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA