Title of Invention	AN IMPROVED FERMENTATION PROCESS FOR PRODUCING HIGH LEVELS OF RECOMBINANT HUMAN PROINSULIN IN METHYLOTROPHIC YEAST
Abstract	wherein Rl represents a carboxy-protecting group viz., substituted methyl group, which can be deprotected easily, such as t-bUtyl group, diphenylmethyl, 4- methoxybenzyl, 2-methoxybenzyl, 2-chlorobenzyl or benzyl group; R2 represents hydrogen, (C1-C4)alkyl, substituted or unsubstituted phenyl or substituted or unsubstituted phenoxy group. 11 .The chloromethylcephem derivatives of the formula (I) prepared according to the process of the present invention are useful for the preparation of cephalosporin antibiotics of the -

Title of Invention

AN IMPROVED FERMENTATION PROCESS FOR PRODUCING HIGH LEVELS OF RECOMBINANT HUMAN PROINSULIN IN METHYLOTROPHIC YEAST

Abstract

wherein Rl represents a carboxy-protecting group viz., substituted methyl group, which can be deprotected easily, such as t-bUtyl group, diphenylmethyl, 4- methoxybenzyl, 2-methoxybenzyl, 2-chlorobenzyl or benzyl group; R2 represents hydrogen, (C1-C4)alkyl, substituted or unsubstituted phenyl or substituted or unsubstituted phenoxy group. 11 .The chloromethylcephem derivatives of the formula (I) prepared according to the process of the present invention are useful for the preparation of cephalosporin antibiotics of the -

Full Text	AN IMPROVED FERMENTATION PROCESS FOR PRODUCING HIGH LEVELS OF RECOMBINANT HUMAN PROINSULIN IN METHYLOTROPHIC YEAST. Field of invention: The invention is generally related to a novel fermentation process wherein a methylotrophic yeast transformant containing a target gene encoding an insulin precursor (proinsulin) is used. It is more particularly related to an improved fermentation process for producing high levels of human proinsulin in methylotrophic yeasts such as Pichia pastoris (P. pastoris) as host cells under the effect of variation of process parameter such as pH and nitrogenous feed. Background of the invention: Recombinant DNA (r-DNA) technology has enabled the expression of foreign (heterologous) protein in microbial and other host cells. A vector containing genetic material directing the host cell to produce a protein encoded by a portion of the heterologous DNA sequence is introduced into the host, and the transformant host cells can be fermented and subjected to conditions which facilitate the expression of the heterologous DNA, leading to the formation of large quantities of the desired protein. The advantages in producing protein by using r-DNA technology in lieu of isolation of protein from natural sources include the ready availability of raw materials, high expression levels, which is especially useful for proteins of low natural abundance and the ease with which a normally intracellular protein can be secreted into the fermentation medium facilitating the purification process. There are three major methods used for the production of human insulin in microorganisms. Two involve E. coli with either the expression of a large fusion protein in the cytoplasm (see reference 1) or use a signal peptide to enable secretion into the periplasmic space (see reference no. 2). A third method utilizes S. cerevisiae to secrete an insulin precursor into the medium (see reference no. 3). The prior art discloses a limited number of insulin precursors, which are expressed in either E. coli or Saccharomyces cerevisiae (S. cerevisiae), vide patent numbers US 5,962,267, WO 95/16708, EP 0055945, EP0163529, EP0347845 and EP 0741188. E. coli has been the host system of choice for the expression of many proteins because its genome has been fully mapped and the organism is easy to handle; grows rapidly; requires an inexpensive, easy-to-prepare medium for growth. However, E. coli, which is a prokaryote, does not have intracellular organelles such as endoplasmin reticulum and the Golgi apparatus, mitochondria, which are present in eukaryotes. Many eukaryotic proteins can be produced in E. coli but a disadvantage of this is that they are produced in a non-native conformation, as post-translational modifications (glycosylation) do not take place when E. coli is used as host system. Moreover, it is difficult to process it to its native form and as a result the yield is less. Another disadvantage of using E. coli as host is the formation of high molecular weight aggregates, commonly known as inclusion or refractile bodies. These result from the inability of the expressed proteins to fold correctly in an unnatural cellular environment. The recombinant protein cannot be secreted into the culture media upon formation of inclusion bodies. There are other associated problems related to E, coli when it is used as expression system like endotoxin presence, plasmid instability, difficulty in reaching very high cell density, use of antibiotic, costly isopropyl-beta-D-thiogalacto pyranoside (IPTG), deleterious effect of acetate accumulation (greater than 5 g/L) on cell growth and recombinant protein expression. Therefore researchers turned to eukaryotic host systems and mammalian expression of protein production. Yeasts, which are eukaryotes, are well suited to the expression of heterologous protein such as insulin precursor. Approximately half of the world's need for insulin is met by production processes that employ S. cerevisiae or the baker's yeast as it is popularly known. There are many advantages of using S. cerevisiae as host for expression of heterologous proteins over E. coli. These include ease of genetic manipulation, ability to carry out post-translational modifications, ease of growth, lack of endotoxin production, and well established industrial processes. However, there are some major disadvantages including low levels of expression of foreign genes and an inefficient secretion apparatus. Furthermore, the Crabtree-effect exhibited by S. cerevisiae necessitates fed-batch cultivation, which has to be operated at low dilution rates to prevent reduction in biomass yield and build-up of toxic levels of metabolites. There are other drawbacks using S. cerevisiae, which include difficulty in reaching high cell density, and instability of clone. Also, while using S. cerevisiae normally mannose-rich glycosylated protein is produced. The use of S. cerevisiae has been well documented in the literature. While attempting to discover host systems that yield high expression levels without encountering the disadvantages offered by process using E. coli or the eukaryotic yeasts described earlier, researchers have turned to methylotrophic yeasts for production of recombinant heterologous proteins (see reference 4). Among these are species like P. pastoris, Hansenulla polymorpha, P. methanolica and the budding yeast Kluyveromyces lactis (see references 5-10). In particular, the yeast P. pastoris has become successful in the production of high level of a broad range of heterologous proteins (see references 6, 7, 11, 12). The advantages of P. pastoris as expression system are: clone stability (the gene is integrated into host's genome), very high cell density, easy scale up, strong and tightly regulated promoter, limited or absent hyperglycosylation, absence of endotoxin and choice of intracellular or extra cellular expression. Y. Wang et al. (see reference 13) used P. pastoris for over-expression of human insulin precursor. In this high cell density fermentation using a simple culture medium composed mainly of salts and methanol, the expression level reached was 1.5 g/L. However, processes using P. pastoris as host cells need to be tightly monitored during the induction stage when methanol is used as an inducer. This is because any excess methanol converts the culture to a 'pickling' stage. P. pastoris also requires high concentration of dissolved oxygen (DO) for high cell density growth, as compared to other hosts. Human insulin is produced using recombinant microorganisms that produce intact proinsulin (insulin precursor) instead of the methods wherein A and B chains of human insulin are produced separately. The proinsulin route is the current method of choice for insulin production (see reference 14) and entails one sequence of fermentation rather than two sets of sequences, that is one for the A-chain and one for the B-chain. Thus P. pastoris is able to express human proinsulin as a single chain polypeptide and export into the media. P. pastoris offers many advantages over E. coli which is a more widely used host for protein expression. First, unlike E. coli, it does not have the endotoxin problem associated with bacteria or the viral contamination problem of proteins produced in animal cell culture. Furthermore, P. pastoris can utilize methanol as a carbon source in the absence of glucose. The P. pastoris expression system uses the methanol-induced alcohol oxidase (AOX1) promoter, which controls the gene that codes for the expression of human pro-insulin. This promoter has been characterized and incorporated into a series of P. pastoris expression vectors. Since the proteins produced in P. pastoris are typically folded correctly and secreted into the medium, the fermentation of genetically engineered P. pastoris provides an excellent alternative to E. coli expression systems. Another advantage of P. pastoris seems to be a Kex2 endoprotease enzyme analogue that is significantly more efficient that the S. cerevisiae Kex2 endoprotease in processing the pro-leader/insulin precursor fusion protein (see reference 10). An important aspect of any cellular production system is its easy and efficient adaptation to large-scale fermentation conditions used for the production of recombinant proteins. Clearly, the potential of P. pastoris for easy up scaling and high cell density is an attractive feature in the production of recombinant proteins. European patent EP1211314 and Canadian patent CA 2357072 by Beta Lab SA (AR) describe the extra cellular expression of human insulin precursor in P. pastoris. These patents describe the use of methylotrophic recombinant yeast for producing human insulin precursor, the strain comprising in its genome, a copy of a DNA construction and a second DNA construction, wherein the constructions are capably directing the expression of secretion of human insulin precursor of the formula B (1-30) - Y1 - Y2- A (1-21). Despite its documented advantages over comparable host systems, P. pastoris has not become a commercially viable host system for expression of heterologous proteins, particularly the human insulin proteins. Main reason behind this is the relative newness of P. pastoris as a host system. The Japanese Patent JP 02222685 describes the provision of 5'- regulatory region of a high - productivity fermentation yeast, Pichia Pastoris, transcripted in high efficiency responding to various carbon sources such as glucose, glycerol and methanol. This gene is a DNA fragment containing the total or a part of glyceraldehyde - 3 -phosphate dehydrogenase gene of Pichia Pastoris. The gene is ligated to a DNA coding for a peptide such as streptokinase, tissue plasminogen activation factor, hepatitis surface antigen and bovine lysozyme and capable of producing the peptide in high efficiency by transforming procaryotic cell or eucaryotic cell or eukaryotic cell. Thus this Japanese Patent stresses the use of their invention of a DNA -fragment. The patent does not teach about modifying the process parameters like pH and addition of neutrients during methanol induction phase, in a fermentation process for producing high levels of recombinant human proinsulin in methylotrophic yeast like Pichia Pastoris, whereas the invention of the present patent application relate to the modification of these process parameters, for producing high levels of recombinant human proinsulin. The Japanese Patent JP 11290673 describe the use of glycerol as carbon source and use of pH value upto 8. However, the invention of this Japanese Patent realates to the emulsification approach of the water - in - oil type insolubility micell especially by rhamno lipid about the emulsification approach of the oil by the surfactant. According to the solution suggested by this invention, a water - in - oil water droplet - type micelle in a crude oil generated in an oil well or a waste oil generated by a spillage accident or the like, is intended for emulsification. Thus the field of this invention is totally unrelated to the field of the present invention. This Japanese Patent therefore does not teach or suggest the use of these factors in a fermentation process for producing high levels for recombinant human proinsulin in methylotrophic yeast. There is a perceived need for cheaper processes using P. pastoris that will make the use of P. pastoris commercially viable. There is also a need for improved fermentation process for producing high levels of recombinant human proinsulin in methylotrophic yeast. Objects and advantages of the invention Accordingly, an object of the present invention is to develop a cost-effective fermentation process with improved expression level of proinsulin in a extra cellular manner using methylotrophic yeast, P. pastoris, by optimizing parameter such as pH during induction phase. A further object of the present invention is to improve the expression of the heterologous protein, in particular human insulin, by a methylotrophic yeast such as P. pastoris using nitrogenous supplements during induction phase. Yet another object of the present invention is to develop a fermentation process that gives intact proinsulin rather than separate A and B chains. Still another object of the present invention is to develop a fermentation process for producing proinsulin, which can be easily scaled-up. Summary of the invention: The present invention provides a process for producing high levels of recombinant human proinsulin in a methylotrophic yeast, P. pastoris, which is gaining increasing recognition in the industry as a viable host to produce recombinant protein, and has been used as yeast host to produce human insulin precursor with a recombinant DNA technology. A normal fermentation process, referred to as control process for the purpose of this invention is where no nitrogenous component is added during induction phase but induction is at pH 5.0. An improved process is described wherein some of the key parameters of a control process, namely, pH and substrate, have been varied and a novel combination of these parameters has been provided. The resulting yields of the product are significantly higher than those obtained by conventional fermentation methods. Brief description of the figures: Figure 1 shows comparison of specific productivities of improved and control batches. Figure 2 shows the fermentation time vs. dry cell wt, pH, glycerol and methanol feed rate, cumulative feed of glycerol and methanol, air saturation (%), back pressure and agitation speed of an improved process. Figure 3 compares the volumetric productivities of improved and control batches. Figure 4 shows comparison of specific activities of improved and control batches. Figure 5 shows the comparison of dry cell weight of improved and control batches during induction phases Figure 6 depicts estimation of pro-insulin by HPLC (High Pressure Liquid Chromatography by BP method, British Pharmacopoiea), SDS-PAGE (Sodium Dodecyl Sulphate - Polyacrylamide Gel Electrophoresis) and HPLC chromatograms for standard and fermentation samples. Detailed description of the invention: Before describing the preferred embodiments and providing illustrations, the general principles and practices followed in the invention are described. Accordingly the present invention describes an improved fermentation process for producing high levels of recombinant human pro-insulin in methylotrophic yeast. The invention describes an improved fermentation process comprising inoculation of seed, followed by growth phase comprising batch and fed-batch stages where high cell density of the yeast is achieved. Glycerol is used as a single carbon source during the batch and fed-batch growth stages. This is followed by a fed-batch induction/production phase where methanol is used as a single carbon source. The fermentation process is conducted in an in-situ sterilizable fermenter equipped with controller/instrumentation to control pH, temperature, dissolved oxygen, agitation and methanol feed. In the present invention, a methylotrophic recombinant yeast P. pastoris Mut (methanol utilizing fast) clone is used for the production of pro-insulin into the fermentation media. Human pro-insulin gene used for expression is constructed synthetically by annealing and ligation of oligonucleotides. A novel feature of this invention is that the high cell density was achieved using fed-batch strategy with precise profiling of feed along with the backpressure used in the fermenter. Seed development: Seed required to start the biomass generation is obtained by growing the yeast cells on suitable media at suitable temperature under continuous stirring. It is important to ensure that the seed should not reach the stationary stage of its growth phase. Similarly, it needs to be ensured that seed is at an active growing stage in order to reduce the lag phase period during the biomass generation stage of fermentation. The present invention uses frozen glycerol stock of P. Pastoris yeast cells that are grown on minimal glycerol (MGY) medium, at 30°C temperature to mid-logarithmic growth stage to an optical density (OD6oo) of 10 to 16 under continuous stirring. Growth phase: The growth phase is carried out in two steps. First of these is the glycerol batch (GB) phase, where the substrate comprises basal salt media. The main objective of this phase is to grow the culture to achieve cell density up to a suitable fraction of the required total cell density. A substrate is added in such amount so as not to cause substrate inhibition towards growth. The carbon source added at the start of this phase is consumed by growing number of cells in this phase. pH level of the culture solution is maintained at 5.0 throughout the GB phase. The completion of this phase of growth phase is indicated by a sudden rise in the DO level or the DO spike. Further increase in cell density leading to the desired levels is achieved under fed-batch conditions in a phase called glycerol fed-batch phase (GFB). A programmed glycerol feed is initiated under carbon limiting condition. The programmed feeding strategy is conducted through a computer-controlled fermenter in auto or profile mode. In order to keep the DO level at suitable dissolved oxygen concentration of P. pastoris, feeding profile of glycerol is adjusted in combination with backpressure, agitation speed, and aeration rate. The pH of this fermentation growth phase was also controlled via a pH control loop using suitable alkali solutions, such as ammonia, and maintained at 5.0 A novel aspect of the present invention is the adjustment of pH to a higher level towards the end of the GFB phase. This adjustment is carried out at the end of the GFB itself and is linked to the starvation phase. The residual glycerol level in the fermentation broth is maintained at very low predetermined level indicating carbon-limiting conditions. As the required cell density of the fed-batch growth phase is reached, the glycerol feed is stopped and pH adjusted automatically to the pH required for the induction/production phase that follows. The cell density referred to in the foregoing discussion is calculated based on the specific productivity, defined as amount of protein expressed per gram of dry cell mass, and is to be calculated instead of volumetric productivity, which is defined as the amount of expressed protein per litre of culture supernatant. Improvement in specific productivity during improved process can be seen from Figure 1. The high cell density is achieved using fed-batch strategy with precise profiling of 50% glycerol feed. Required levels of DO are maintained by supply of air and maintaining a suitable backpressure in the fermenter. Adjustment in backpressure for adjusting DO levels is only resorted to once the maximum airflow rate and agitation rate is reached. Induction: Once glycerol addition stops, the carbon source gets depleted gradually. This results in a sudden spike in DO, indicating the exhaustion of residual glycerol carbon source. Simultaneously, pH is adjusted to 6.3 using liquid ammonia in automatic mode. After reaching the pH at 6.3 in this stage of fermentation, the actively grown cells are subjected to methanol induction thereby diverting the process towards the expression of protein by fully inducing the AOX1 promoter on methanol. Fed-batch addition of methanol is designed so that the entire induction phase (expression of human pro-insulin) has residual methanol between 0.02% (v/v) to 0.4% (v/v), more preferably less than or equal to 0.1% (v/v). Required levels of DO are maintained by supply of air or pure oxygen or combination of these two and maintaining a suitable backpressure in the fermenter. When the culture at the end of the growth phase is subjected to the induction phase, it takes some time for the cells to adapt to the conditions of the induction phase. During this stage of adaptation, it is very important to introduce methanol slowly. High levels of methanol are toxic to the existence of the cells, and may lead to the death of cells. The residual methanol is maintained at a very low predetermined level indicating a virtual equilibrium between demand for and supply of methanol. This addition of methanol can be done in auto mode (stepwise manually) or by on-line methanol sensor (feedback control, make: Raven Biotech Inc., Canada) based on a particular set-point value (mV) equivalent to a particular residual methanol concentration that is estimated off-line using gas chromatography (GC). It is better to maintain the residual methanol by using on-line methanol sensor but in auto mode, it is incremented manually based on required residual methanol concentration. It is important to use DO spike to analyze the metabolic state of culture and toxic time point over the course of methanol induction to optimize protein expression. An important aspect of the present invention is the impact of pH on the yield of the target protein. Conventional fermentation techniques use pH levels of up to 5.0. In the present invention, a pH value between 5.6 to 7.0 was used. This shows significant enhancement over the yield of target protein as obtained using pH of 5.0 and below, for example, all other conditions remaining same. The variation of the process parameters such as pH, methanol feed, and glycerol feed, and backpressure adjustment, as well as process output such as dry cell weight, and volume of the culture are seen in Figure 2. Another important aspect of the present invention is addition of nitrogenous compounds along with methanol. It must be noted that the general process of fermentation will work satisfactorily without any addition of nitrogenous compounds. However, according to the present invention, when certain predetermined nitrogenous compounds are administered in predetermined proportions and in certain manner, yield of the target protein is enhanced substantially. Along with methanol, nitrogenous source is added optionally. Addition of nitrogenous source prevents proteolytic degradation of recombinant protein secreted into the media. The degradation of protein is caused by extra cellular proteases. Nitrogenous source will act as substrate for the extra cellular protease thereby preventing degradation of the expressed protein. This ultimately results in the enhancement of the expressed protein levels. Nitrogenous source can be in different combinations and strengths of yeast extract, peptone, skimmed milk powder, and casamino acid. Nitrogenous compounds such as peptone and yeast extract are added in a certain proportion with respect to each other. Their total quantity is determined based on the volume of culture at the start of the induction phase. What is important is that this quantity is administered over the entire induction phase. The nitrogenous compounds are mixed with suitable amount of water and sterilized. The sterilized solution containing nitrogenous compound and water is administered continuously throughout the induction phase. The amount of water used for dilution is of secondary importance and is known to any person skilled in the art. Although it is important to ensure that the solution must be applied continuously throughout the induction phase, the rate of administration of the nitrogenous compound solution can vary. It can be administered at a constant or a variable rate or in a stepwise or exponential variation, or in a repeated or continuous manner. The specific details of administration of nitrogenous compounds are considered in the examples. The rate of expression of protein is somewhat slow at the start of the induction/production phase. It increases and reaches plateau towards the end of the induction/production phase. Volumetric productivity, also defined as amount of proinsulin expression per litre has been plotted against time in Figure 3. Another advantage of the present invention is the efficiency of the process defined in terms of the specific activity, which is defined as the proportion of the expressed proinsulin in the total protein of the culture. The higher the proportion, the better is the efficiency (as seen in Figure 4). Culture is harvested and cell-free supernatant is collected for further downstream processing. Various embodiments of the present invention are now described. Each embodiment uses the general principles and practices of the invention described hereinbefore. In a preferred embodiment of the present invention, the pro-insulin gene was designed such that the protein coded by it could have an amino acid sequence identical to human insulin B chain (amino acid 1-29, B threonine is added by enzymatic conversion of human insulin precursor); a spacer with sequence Ala, Ala, Lys followed by the human insulin A chain (amino acids 1-21). The synthetic pro-insulin gene obtained as described above is prepared for expression in P. pastoris. For this, S. cerevisiae alpha-mating secretory signal coding sequence is amplified by polymerase chain reaction (PCR) and ligated in front of human pro-insulin gene. Seed is obtained by growing P. pastoris on MGY media up to 10 to 16 OD600-The seed is in mid-logarithmic growth stage. In glycerol batch phase, fermentation media consisting of complete synthetic media along with trace elements and D-biotin is inoculated at a seed concentration of 5% (v/v). During the glycerol fed-batch phase, glycerol of 50% concentration is set at a rate from 5 ml/hour/L to 45 ml/hour/L of initial fermenter batch volume, more preferably between 10 ml/hour/L and 35 ml/hour/L, and most preferably between 15 ml/hour/L and 30 ml/hour/L. During the induction phase of the preferred embodiment, the methanol feed rate profile is from 2.5 ml/hour/L to 35 ml/hour/L more preferably between 4 ml/hour/L and 28 ml/hour/L, and most preferably between 5 ml/hour/L and 25 ml/hour/L of the initial fermenter batch volume, based on residual methanol, determined by GC (off-line) and on-line methanol sensor. The entire methanol fed-batch process lasts for approximately 108 to 120 hours. This preferred embodiment uses a nitrogenous source as a substrate in the form of the yeast extract and peptone (YEP). Yeast extract and peptone are used in a predetermined proportion to each other and administered continuously over the entire induction stage. In this embodiment the proportion of yeast extract: peptone is important, the preferred proportion being 1:2. In another embodiment, the conditions are same as the preferred embodiment except the composition of the nitrogenous source. In this embodiment skimmed milk powder is used along with the YEP. In order that the individual components of the mixture do not precipitate, skimmed milk powder and YEP are sterilized separately and then mixed together. The strategy of administering this mixture during the induction phase is same as that adopted for administering the nitrogenous source in the first preferred embodiment. The preferred proportions of yeast extract, peptone, and skimmed milk powder are 1:2:2. Specifics of this embodiment are considered in Example 3. In yet another embodiment of the present invention the nitrogenous source used in the induction phase of the invention is casamino acid. The strategy of administering casamino acid during the induction phase is same as that adopted for administering the nitrogenous source in the first preferred embodiment. A solution of unit strength (IX) is prepared by dissolving in required quantities of water, casamino acid powder, the amount of which was calculated as a certain proportion of the fermentation culture volume as measured at the end of GFB. Solution of unit strength is prepared by using 0.015 g of casamino acid powder per 100 ml of the fermentation culture volume, as measured at the end of GFB. Example 4 considers the specifics of this embodiment. All of the above embodiments describe various forms of the improved process as defined earlier in the summary of invention section of this specification. Figure 2 illustrates one of the improved processes. In a still further embodiment of the present invention the pH was increased to 6.3 without the addition of nitrogenous source in the induction phase of the invention. All other steps of the preferred embodiment remain same. This case is covered more specifically in Example 1. It is observed that the expressed protein yield using the present invention (improved process) is higher by 10% to 15% (w/v) at pH 6.3 over the control process at pH 5.0 or below, when no nitrogenous source is added at the induction stage (see Figure 3). It is further observed that the expressed protein yield increases by 35% to 45% (w/v) over control process when nitrogenous source is added at the induction stage at pH 6.3. Therefore overall improvement in pro-insulin expression level of improved fermentation process is significant as compared to control process where induction is at pH 5.0 and without addition of any nitrogenous supplement such as yeast extract, peptone, skimmed milk powder, or casamino acid, or a combination of any two or more of these supplements. A better understanding of the present invention and the enhancement of expression level of pro insulin by submerged fermentation will be had from the following examples, given by way of illustrations. Example 1: yield improvement with pH variation Seed required to start the biomass generation was in the form of frozen glycerol stock of P. Pastoris yeast cells. The seed was grown on MGY medium at 30°C temperature to mid-logarithmic growth stage to an OD600 value of 10 to 16 under continuous stirring for 22 hours at 250 rpm and at 30°C. The grown seed was transferred at a rate of 5% (v/v) to the fermenter containing fermentation media comprising a synthetic media along with trace elements and D-biotin. The biomass generated at the end of the GB phase was 33-38 g dry cell weight/L of culture broth and cellular yield coefficient was of 0.65 to 0.75 g of dry cell weight per gram of glycerol. During the fed-batch growth phase, the profile of glycerol feed was set at a rate from 5 to 45 ml/hour/L of initial fermenter batch volume. The pH of the fermentation growth phase was controlled at 5.0 automatically via a pH control loop using 30% ammonia. A spike in DO in a range of 70-80% of air saturation was observed, indicating the exhaustion of residual glycerol carbon source. Sampling was performed in growth phase at 6-hour intervals and analyzed for cell growth, OD6oo5 NTU (Nephleometric Unit), wet and dry cell weight, pH and contamination. During the induction fed-batch phase the methanol feed rate profile was from 2.5 ml/hour/L to 35 ml/hour/L of the initial fermenter batch volume. The residual methanol was maintained at 0.02 (v/v) to 0.4% (v/v). The variation of dry cell weight with time is shown in Figure 5. The yield was measured in mg/L of culture supernatant in mg/L of culture supernatant using HPLC (BP method) as is indicated in Figure 6. Just before induction phase starts (at the end of GFB phase) pH was adjusted to 6.3 using 30% Ammonia. The improvement in yield was 10% to 15% as compared to the control process. Example 2: yield improvement with pH variation and addition of combination of nitrogenous sources: This example illustrates the use of nitrogenous compounds for enhancement of yield. In one case, yeast extract and peptone of a proportion 1:2 respectively were used. A solution of unit strength (IX) was prepared by mixing (in required quantities of water), yeast extract and peptone, amounts of which were calculated as a certain proportion of the culture broth volume as measured at the end of GFB. For this example, 0.045 g of yeast extract and 0.09 g of peptone were used per 100ml of the culture broth volume, as measured at the end of GFB. Batches were carried out with solutions of strength IX to 8X, after sterilizing (in a required amount of water), administered during the entire induction phase. All other conditions of this example are same as Example 1. The improvement in yield as compared to the control process was 20% to 25%. Example 3: yield improvement with pH variation and addition another combination of nitrogenous sources: This example also illustrates the use of nitrogenous compounds for enhancement of yield. In this case, yeast extract, peptone and skimmed milk powder of a proportion 1:2:2 respectively were used. A solution of unit strength (IX) was prepared by mixing (in required quantities of water), yeast extract, peptone and skimmed milk powder, amounts of which were calculated as a certain proportion of the fermentation culture volume measured at the end of GFB. For this example, 0.045g of yeast extract, 0,09 g of peptone, and 0.09 g of skimmed milk powder were used for every 100 ml of the fermentation culture volume, as measured at the end of GFB. Batches were carried out with solutions of strength IX to 8X, after sterilizing (in a required amount of water), administered during the entire induction phase. All other conditions of this example are same as Example 1. The improvement in yield as compared to the control process was 35% to 45%. Example 4: yield improvement with pH variation and addition of casamino acid as a nitrogenous source: This example also illustrates the use of nitrogenous compounds for enhancement of proinsulin yield. In this case, casamino acid was added. A solution of unit strength (IX) was prepared by dissolving in required quantities of water, casamino acid powder, the amount of which was calculated as a certain proportion of the fermentation culture volume as measured at the end of GFB. For this example, 0.015g of casamino acid powder was used per lOOmL of the fermentation culture volume, as measured at the end of GFB. Batches were carried out using solutions of strengths IX to 16X, after sterilizing (in a required amount of water), was administered during the entire induction phase. The improvement in yield as compared to the control process was 15% to 20%. All other conditions of this example are same as Example 1. While the above description contains many specificities, these should not be construed as limitations in the scope of the invention, but rather as an exemplification of the preferred embodiments thereof. Accordingly, the scope of the invention should be determined not by the embodiments illustrated, but by the appended claims and their legal requirements. References US 5,962,267 HANIL SYNTHETIC FIBER CO., LTD. WO95/16708 NOVO NORDISK A/S EP0055945 GENENTECH, INC EP0163529 NOVO NORDISK A/S EP0347845 NOVO NORDISK A/S EP0741188 ELI LILLY AND COMPANY EP1211314 LABORATORIOS BETA S.A. CA 2357072 LABORATORIOS BETA S.A. Other References 1. Frank, et al. 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An improved fermentation process for producing high levels of recombinant human pro-insulin in a genetically engineered methylotrophic yeast, such as Pichia Pastoris comprising the steps of: a developing a seed which starts biomass generation, by growing the Pichia Pastoris on minimum glycerol medium at 30 °C optical density 600 value in the range of 10 to 16, said seed being grown to mid-logarithmic growth stage under continuous stirring; b. forming an aqueous fermentation medium comprising substrate of salts and glycerol, wherein said substrate is of such amount so as not to cause substrate inhibition towards grovv1:h and wherein said glycerol acts as a single carbon source; c. inoculating said seed which is grown, into said aqueous fermentation medium of step (b) in a fermenter at a rate of 5% v/v, thereby forming a cell culture; d. growing said cell culture in glycerol batch cell growth phase achieving cell density up to suitable fraction of the required total cell density, wherein pH level of solution of said cell culture is maintained at 5 and the completion of said glycerol batch cell growth phase is indicated by a sudden rise in the dissolved oxygen level; e. further growing said cell culture of step (d) in glycerol fed-batch cell growth phase up to said required total cell density by initiating a programmed glycerol feed and maintaining residual glycerol at very low level indicating carbon limiting condition and wherein pH level of said solution of said cell culture is maintained at 5.0; and wherein the glycerol concentration is 50% v/v in said fed-batch cell growth phase, and wherein the rate of feeding said glycerol is varied between 5.0 ml/hour/L and 45 ml/hour/L, more preferably between 10 ml/hour/L and 35 ml/hour/L, and most preferably between 15 ml/hour/L and 30 ml/hour/L of the initial fermenter batch volume under backpressure in said fermenter; f. stopping glycerol feed after reaching said required total cell density and adjusting pH level of said solution of said cell culture in the range of 5.6 to 7.0, most preferably at 6.3, which is required for fed-batch methanol induction phase; g. subjecting the actively grown cells of said cell culture to said fed-batch methanol induction phase for protein expression, wherein methanol and sterilized aqueous solution of a nitrogenous compound such as casamino acid or of a combination of nitrogenous compounds such as yeast extract, peptone and skimmed milk powder is added to said solution of said cell culture and pH level of resultant solution of said cell culture is maintained in the range of 5.6 to 7.0, most preferably at 6.3; h. harvesting said cell culture; and i. collecting cell-free supernatant containing proinsulin for further dovrastream processing. 2. An improved fermentation process, as claimed in claim 1, wherein in said step (g) of claim 1, the rate of feeding said methanol in said fed-batch methanol induction phase is varied between 2.5 ml/hour/L and 35 ml/hour/L, more preferably between 4 ml/hour/L and 28 ml/hour/L, and most preferably between 5 ml/hour/L and 25 ml/hour/L of the initial volume of the batch; and wherein the residual methanol is maintained in the range of 0.02% v/v to 0.4% v/v, more preferably 0.1% v/v. 3. An improved fermentation process, as claimed in any of claims 1 and 2, wherein said sterilized aqueous solution in said step (g) of claim 1 is administered continuously over said induction phase till completion thereof; and wherein said combination of nitrogenous compounds is selected from a group of combinations such as, a combination of yeast extract and peptone or a combination of yeast extract, peptone, and skimmed milk powder. 4. An improved fermentation process as claimed in claim 3, wherein, in said combination of yeast extract and peptone, the proportion of said yeast extract and peptone is preferably 1:2 w/w and the sterilized solution being in the range of strength IX to 8X, preferably in the range of 4X to 5X, and wherein IX is obtained by adding, for every 100 ml volume of said cell culture volume measured at the end step (f) of claim 1,0.045 g of said yeast extract and 0.09 g of said peptone to a predetermined amount of water. 5. An improved fermentation process as claimed in claim 3, wherein, in said combination of yeast extract, peptone and skimmed milk powder, the proportion of said yeast extract, said peptone and said skimmed milk powder is preferably 1:2:2 w/w and the sterilized solution being in the range of strength IX to 8X, preferably in the range of 4X to 5X, wherein IX is obtained by adding, for every 100 ml volume of said cell culture volume measured at the end step (f) of claim 1, 0.045 g of said yeast extract, 0.09 g of said peptone, and 0.09 g of said skimmed milk powder to a predetermined amount of water. 6. An improved fermentation process, as claimed in claim 3, wherein, while using said casamino acid alone as said nitrogenous compound, concentration of said casamino acid added is in the range of 0.015% to 0.25% w/v, more preferably 0.15% w/v of said cell culture volume measured at the end of said step (f) of claim 1. 7. An improved fermentation process, as claimed in any of claims 1 to 6, wherein said administering of said sterilized aqueous solution is carried out at any one of constant, variable, repeated, continuous, stepwise increment, or exponential flow rate or at any combination thereof. 8. An improved fermentation process for producing high levels of recombinant human proinsulin in a genetically engineered methylotrophic yeast, substantially as hereinbefore described with reference to the examples illustrated and accompanying drawings.

Full Text

AN IMPROVED FERMENTATION PROCESS FOR PRODUCING HIGH LEVELS OF RECOMBINANT HUMAN PROINSULIN IN METHYLOTROPHIC YEAST.
Field of invention:
The invention is generally related to a novel fermentation process wherein a methylotrophic yeast transformant containing a target gene encoding an insulin precursor (proinsulin) is used. It is more particularly related to an improved fermentation process for producing high levels of human proinsulin in methylotrophic yeasts such as Pichia pastoris (P. pastoris) as host cells under the effect of variation of process parameter such as pH and nitrogenous feed.
Background of the invention:
Recombinant DNA (r-DNA) technology has enabled the expression of foreign (heterologous) protein in microbial and other host cells. A vector containing genetic material directing the host cell to produce a protein encoded by a portion of the heterologous DNA sequence is introduced into the host, and the transformant host cells can be fermented and subjected to conditions which facilitate the expression of the heterologous DNA, leading to the formation of large quantities of the desired protein.
The advantages in producing protein by using r-DNA technology in lieu of isolation of protein from natural sources include the ready availability of raw materials, high expression levels, which is especially useful for proteins of low natural abundance and the ease with which a normally intracellular protein can be secreted into the fermentation medium facilitating the purification process.

There are three major methods used for the production of human insulin in microorganisms. Two involve E. coli with either the expression of a large fusion protein in the cytoplasm (see reference 1) or use a signal peptide to enable secretion into the periplasmic space (see reference no. 2). A third method utilizes S. cerevisiae to secrete an insulin precursor into the medium (see reference no. 3). The prior art discloses a limited number of insulin precursors, which are expressed in either E. coli or Saccharomyces cerevisiae (S. cerevisiae), vide patent numbers US 5,962,267, WO 95/16708, EP 0055945, EP0163529, EP0347845 and EP 0741188.
E. coli has been the host system of choice for the expression of many proteins because its genome has been fully mapped and the organism is easy to handle; grows rapidly; requires an inexpensive, easy-to-prepare medium for growth.
However, E. coli, which is a prokaryote, does not have intracellular organelles such as endoplasmin reticulum and the Golgi apparatus, mitochondria, which are present in eukaryotes. Many eukaryotic proteins can be produced in E. coli but a disadvantage of this is that they are produced in a non-native conformation, as post-translational modifications (glycosylation) do not take place when E. coli is used as host system. Moreover, it is difficult to process it to its native form and as a result the yield is less.
Another disadvantage of using E. coli as host is the formation of high molecular weight aggregates, commonly known as inclusion or refractile bodies. These result from the inability of the expressed proteins to fold correctly in an unnatural cellular environment. The recombinant protein cannot be secreted into the culture media upon formation of inclusion bodies.
There are other associated problems related to E, coli when it is used as expression system like endotoxin presence, plasmid instability, difficulty in

reaching very high cell density, use of antibiotic, costly isopropyl-beta-D-thiogalacto pyranoside (IPTG), deleterious effect of acetate accumulation (greater than 5 g/L) on cell growth and recombinant protein expression.
Therefore researchers turned to eukaryotic host systems and mammalian expression of protein production. Yeasts, which are eukaryotes, are well suited to the expression of heterologous protein such as insulin precursor. Approximately half of the world's need for insulin is met by production processes that employ S. cerevisiae or the baker's yeast as it is popularly known.
There are many advantages of using S. cerevisiae as host for expression of heterologous proteins over E. coli. These include ease of genetic manipulation, ability to carry out post-translational modifications, ease of growth, lack of endotoxin production, and well established industrial processes. However, there are some major disadvantages including low levels of expression of foreign genes and an inefficient secretion apparatus. Furthermore, the Crabtree-effect exhibited by S. cerevisiae necessitates fed-batch cultivation, which has to be operated at low dilution rates to prevent reduction in biomass yield and build-up of toxic levels of metabolites. There are other drawbacks using S. cerevisiae, which include difficulty in reaching high cell density, and instability of clone. Also, while using S. cerevisiae normally mannose-rich glycosylated protein is produced. The use of S. cerevisiae has been well documented in the literature.
While attempting to discover host systems that yield high expression levels without encountering the disadvantages offered by process using E. coli or the eukaryotic yeasts described earlier, researchers have turned to methylotrophic yeasts for production of recombinant heterologous proteins (see reference 4). Among these are species like P. pastoris, Hansenulla polymorpha, P. methanolica and the budding yeast Kluyveromyces lactis (see references 5-10). In particular,

the yeast P. pastoris has become successful in the production of high level of a broad range of heterologous proteins (see references 6, 7, 11, 12).
The advantages of P. pastoris as expression system are: clone stability (the gene is integrated into host's genome), very high cell density, easy scale up, strong and tightly regulated promoter, limited or absent hyperglycosylation, absence of endotoxin and choice of intracellular or extra cellular expression.
Y. Wang et al. (see reference 13) used P. pastoris for over-expression of human insulin precursor. In this high cell density fermentation using a simple culture medium composed mainly of salts and methanol, the expression level reached was
1.5 g/L.
However, processes using P. pastoris as host cells need to be tightly monitored during the induction stage when methanol is used as an inducer. This is because any excess methanol converts the culture to a 'pickling' stage. P. pastoris also requires high concentration of dissolved oxygen (DO) for high cell density growth, as compared to other hosts.
Human insulin is produced using recombinant microorganisms that produce intact proinsulin (insulin precursor) instead of the methods wherein A and B chains of human insulin are produced separately. The proinsulin route is the current method of choice for insulin production (see reference 14) and entails one sequence of fermentation rather than two sets of sequences, that is one for the A-chain and one for the B-chain. Thus P. pastoris is able to express human proinsulin as a single chain polypeptide and export into the media.
P. pastoris offers many advantages over E. coli which is a more widely used host for protein expression. First, unlike E. coli, it does not have the endotoxin problem associated with bacteria or the viral contamination problem of proteins

produced in animal cell culture. Furthermore, P. pastoris can utilize methanol as a carbon source in the absence of glucose. The P. pastoris expression system uses the methanol-induced alcohol oxidase (AOX1) promoter, which controls the gene that codes for the expression of human pro-insulin. This promoter has been characterized and incorporated into a series of P. pastoris expression vectors. Since the proteins produced in P. pastoris are typically folded correctly and secreted into the medium, the fermentation of genetically engineered P. pastoris provides an excellent alternative to E. coli expression systems.
Another advantage of P. pastoris seems to be a Kex2 endoprotease enzyme analogue that is significantly more efficient that the S. cerevisiae Kex2 endoprotease in processing the pro-leader/insulin precursor fusion protein (see reference 10). An important aspect of any cellular production system is its easy and efficient adaptation to large-scale fermentation conditions used for the production of recombinant proteins. Clearly, the potential of P. pastoris for easy up scaling and high cell density is an attractive feature in the production of recombinant proteins.
European patent EP1211314 and Canadian patent CA 2357072 by Beta Lab SA (AR) describe the extra cellular expression of human insulin precursor in P. pastoris. These patents describe the use of methylotrophic recombinant yeast for producing human insulin precursor, the strain comprising in its genome, a copy of a DNA construction and a second DNA construction, wherein the constructions are capably directing the expression of secretion of human insulin precursor of the formula B (1-30) - Y1 - Y2- A (1-21).
Despite its documented advantages over comparable host systems, P. pastoris has not become a commercially viable host system for expression of heterologous proteins, particularly the human insulin proteins. Main reason behind this is the relative newness of P. pastoris as a host system.

The Japanese Patent JP 02222685 describes the provision of 5'- regulatory region of a high - productivity fermentation yeast, Pichia Pastoris, transcripted in high efficiency responding to various carbon sources such as glucose, glycerol and methanol. This gene is a DNA fragment containing the total or a part of glyceraldehyde - 3 -phosphate dehydrogenase gene of Pichia Pastoris. The gene is ligated to a DNA coding for a peptide such as streptokinase, tissue plasminogen activation factor, hepatitis surface antigen and bovine lysozyme and capable of producing the peptide in high efficiency by transforming procaryotic cell or eucaryotic cell or eukaryotic cell. Thus this Japanese Patent stresses the use of their invention of a DNA -fragment. The patent does not teach about modifying the process parameters like pH and addition of neutrients during methanol induction phase, in a fermentation process for producing high levels of recombinant human proinsulin in methylotrophic yeast like Pichia Pastoris, whereas the invention of the present patent application relate to the modification of these process parameters, for producing high levels of recombinant human proinsulin.
The Japanese Patent JP 11290673 describe the use of glycerol as carbon source and use of pH value upto 8. However, the invention of this Japanese Patent realates to the emulsification approach of the water - in - oil type insolubility micell especially by rhamno lipid about the emulsification approach of the oil by the surfactant. According to the solution suggested by this invention, a water - in - oil water droplet - type micelle in a crude oil generated in an oil well or a waste oil generated by a spillage accident or the like, is intended for emulsification. Thus the field of this invention is totally unrelated to the field of the present invention. This Japanese Patent therefore does not teach or suggest the use of these factors in a fermentation process for producing high levels for recombinant human proinsulin in methylotrophic yeast.
There is a perceived need for cheaper processes using P. pastoris that will make the use of P. pastoris commercially viable. There is also a need for improved fermentation process for producing high levels of recombinant human proinsulin in methylotrophic yeast.

Objects and advantages of the invention
Accordingly, an object of the present invention is to develop a cost-effective fermentation process with improved expression level of proinsulin in a extra cellular manner using methylotrophic yeast, P. pastoris, by optimizing parameter such as pH during induction phase.
A further object of the present invention is to improve the expression of the heterologous protein, in particular human insulin, by a methylotrophic yeast such as P. pastoris using nitrogenous supplements during induction phase.
Yet another object of the present invention is to develop a fermentation process that gives intact proinsulin rather than separate A and B chains.
Still another object of the present invention is to develop a fermentation process for producing proinsulin, which can be easily scaled-up.
Summary of the invention:
The present invention provides a process for producing high levels of recombinant human proinsulin in a methylotrophic yeast, P. pastoris, which is gaining increasing recognition in the industry as a viable host to produce recombinant protein, and has been used as yeast host to produce human insulin precursor with a recombinant DNA technology. A normal fermentation process, referred to as control process for the purpose of this invention is where no nitrogenous component is added during induction phase but induction is at pH 5.0. An improved process is described wherein some of the key parameters of a control process, namely, pH and substrate, have been varied and a novel combination of these parameters has been provided. The resulting yields of the product are significantly higher than those obtained by conventional fermentation methods.
Brief description of the figures:

Figure 1 shows comparison of specific productivities of improved and control batches.
Figure 2 shows the fermentation time vs. dry cell wt, pH, glycerol and methanol feed rate, cumulative feed of glycerol and methanol, air saturation (%), back pressure and agitation speed of an improved process.
Figure 3 compares the volumetric productivities of improved and control batches.
Figure 4 shows comparison of specific activities of improved and control batches.
Figure 5 shows the comparison of dry cell weight of improved and control batches during induction phases
Figure 6 depicts estimation of pro-insulin by HPLC (High Pressure Liquid Chromatography by BP method, British Pharmacopoiea), SDS-PAGE (Sodium Dodecyl Sulphate - Polyacrylamide Gel Electrophoresis) and HPLC chromatograms for standard and fermentation samples.
Detailed description of the invention:
Before describing the preferred embodiments and providing illustrations, the general principles and practices followed in the invention are described.
Accordingly the present invention describes an improved fermentation process for producing high levels of recombinant human pro-insulin in methylotrophic yeast. The invention describes an improved fermentation process comprising inoculation of seed, followed by growth phase comprising batch and fed-batch stages where high cell density of the yeast is achieved. Glycerol is used as a single carbon source during the batch and fed-batch growth stages. This is followed by a fed-batch induction/production phase where methanol is used as a single carbon source. The fermentation process is conducted in an in-situ sterilizable fermenter equipped with controller/instrumentation to control pH, temperature, dissolved oxygen, agitation and methanol feed.

In the present invention, a methylotrophic recombinant yeast P. pastoris Mut (methanol utilizing fast) clone is used for the production of pro-insulin into the fermentation media. Human pro-insulin gene used for expression is constructed synthetically by annealing and ligation of oligonucleotides.
A novel feature of this invention is that the high cell density was achieved using fed-batch strategy with precise profiling of feed along with the backpressure used in the fermenter.
Seed development:
Seed required to start the biomass generation is obtained by growing the yeast cells on suitable media at suitable temperature under continuous stirring. It is important to ensure that the seed should not reach the stationary stage of its growth phase. Similarly, it needs to be ensured that seed is at an active growing stage in order to reduce the lag phase period during the biomass generation stage of fermentation. The present invention uses frozen glycerol stock of P. Pastoris yeast cells that are grown on minimal glycerol (MGY) medium, at 30°C temperature to mid-logarithmic growth stage to an optical density (OD6oo) of 10 to 16 under continuous stirring.
Growth phase:
The growth phase is carried out in two steps. First of these is the glycerol batch (GB) phase, where the substrate comprises basal salt media. The main objective of this phase is to grow the culture to achieve cell density up to a suitable fraction of the required total cell density. A substrate is added in such amount so as not to cause substrate inhibition towards growth. The carbon source added at the start of this phase is consumed by growing number of cells in this phase. pH level of the culture solution is maintained at 5.0 throughout the GB phase. The completion of this phase of growth phase is indicated by a sudden rise in the DO level or the DO spike.
Further increase in cell density leading to the desired levels is achieved under fed-batch conditions in a phase called glycerol fed-batch phase (GFB). A

programmed glycerol feed is initiated under carbon limiting condition. The programmed feeding strategy is conducted through a computer-controlled fermenter in auto or profile mode. In order to keep the DO level at suitable dissolved oxygen concentration of P. pastoris, feeding profile of glycerol is adjusted in combination with backpressure, agitation speed, and aeration rate.
The pH of this fermentation growth phase was also controlled via a pH control loop using suitable alkali solutions, such as ammonia, and maintained at 5.0
A novel aspect of the present invention is the adjustment of pH to a higher level towards the end of the GFB phase. This adjustment is carried out at the end of the GFB itself and is linked to the starvation phase. The residual glycerol level in the fermentation broth is maintained at very low predetermined level indicating carbon-limiting conditions. As the required cell density of the fed-batch growth phase is reached, the glycerol feed is stopped and pH adjusted automatically to the pH required for the induction/production phase that follows.
The cell density referred to in the foregoing discussion is calculated based on the specific productivity, defined as amount of protein expressed per gram of dry cell mass, and is to be calculated instead of volumetric productivity, which is defined as the amount of expressed protein per litre of culture supernatant. Improvement in specific productivity during improved process can be seen from Figure 1.
The high cell density is achieved using fed-batch strategy with precise profiling of 50% glycerol feed. Required levels of DO are maintained by supply of air and maintaining a suitable backpressure in the fermenter. Adjustment in backpressure for adjusting DO levels is only resorted to once the maximum airflow rate and agitation rate is reached.

Induction:
Once glycerol addition stops, the carbon source gets depleted gradually. This results in a sudden spike in DO, indicating the exhaustion of residual glycerol carbon source. Simultaneously, pH is adjusted to 6.3 using liquid ammonia in automatic mode. After reaching the pH at 6.3 in this stage of fermentation, the actively grown cells are subjected to methanol induction thereby diverting the process towards the expression of protein by fully inducing the AOX1 promoter on methanol.
Fed-batch addition of methanol is designed so that the entire induction phase (expression of human pro-insulin) has residual methanol between 0.02% (v/v) to 0.4% (v/v), more preferably less than or equal to 0.1% (v/v). Required levels of DO are maintained by supply of air or pure oxygen or combination of these two and maintaining a suitable backpressure in the fermenter. When the culture at the end of the growth phase is subjected to the induction phase, it takes some time for the cells to adapt to the conditions of the induction phase. During this stage of adaptation, it is very important to introduce methanol slowly. High levels of methanol are toxic to the existence of the cells, and may lead to the death of cells. The residual methanol is maintained at a very low predetermined level indicating a virtual equilibrium between demand for and supply of methanol.
This addition of methanol can be done in auto mode (stepwise manually) or by on-line methanol sensor (feedback control, make: Raven Biotech Inc., Canada) based on a particular set-point value (mV) equivalent to a particular residual methanol concentration that is estimated off-line using gas chromatography (GC). It is better to maintain the residual methanol by using on-line methanol sensor but in auto mode, it is incremented manually based on required residual methanol concentration.

It is important to use DO spike to analyze the metabolic state of culture and toxic time point over the course of methanol induction to optimize protein expression.
An important aspect of the present invention is the impact of pH on the yield of the target protein. Conventional fermentation techniques use pH levels of up to 5.0. In the present invention, a pH value between 5.6 to 7.0 was used. This shows significant enhancement over the yield of target protein as obtained using pH of 5.0 and below, for example, all other conditions remaining same.
The variation of the process parameters such as pH, methanol feed, and glycerol feed, and backpressure adjustment, as well as process output such as dry cell weight, and volume of the culture are seen in Figure 2.
Another important aspect of the present invention is addition of nitrogenous compounds along with methanol. It must be noted that the general process of fermentation will work satisfactorily without any addition of nitrogenous compounds. However, according to the present invention, when certain predetermined nitrogenous compounds are administered in predetermined proportions and in certain manner, yield of the target protein is enhanced substantially. Along with methanol, nitrogenous source is added optionally.
Addition of nitrogenous source prevents proteolytic degradation of recombinant protein secreted into the media. The degradation of protein is caused by extra cellular proteases. Nitrogenous source will act as substrate for the extra cellular protease thereby preventing degradation of the expressed protein. This ultimately results in the enhancement of the expressed protein levels. Nitrogenous source can be in different combinations and strengths of yeast extract, peptone, skimmed milk powder, and casamino acid.

Nitrogenous compounds such as peptone and yeast extract are added in a certain proportion with respect to each other. Their total quantity is determined based on the volume of culture at the start of the induction phase. What is important is that this quantity is administered over the entire induction phase. The nitrogenous compounds are mixed with suitable amount of water and sterilized. The sterilized solution containing nitrogenous compound and water is administered continuously throughout the induction phase. The amount of water used for dilution is of secondary importance and is known to any person skilled in the art. Although it is important to ensure that the solution must be applied continuously throughout the induction phase, the rate of administration of the nitrogenous compound solution can vary. It can be administered at a constant or a variable rate or in a stepwise or exponential variation, or in a repeated or continuous manner. The specific details of administration of nitrogenous compounds are considered in the examples.
The rate of expression of protein is somewhat slow at the start of the induction/production phase. It increases and reaches plateau towards the end of the induction/production phase. Volumetric productivity, also defined as amount of proinsulin expression per litre has been plotted against time in Figure 3.
Another advantage of the present invention is the efficiency of the process defined in terms of the specific activity, which is defined as the proportion of the expressed proinsulin in the total protein of the culture. The higher the proportion, the better is the efficiency (as seen in Figure 4).
Culture is harvested and cell-free supernatant is collected for further downstream processing.
Various embodiments of the present invention are now described. Each embodiment uses the general principles and practices of the invention described hereinbefore.

In a preferred embodiment of the present invention, the pro-insulin gene was designed such that the protein coded by it could have an amino acid sequence identical to human insulin B chain (amino acid 1-29, B threonine is added by enzymatic conversion of human insulin precursor); a spacer with sequence Ala, Ala, Lys followed by the human insulin A chain (amino acids 1-21). The synthetic pro-insulin gene obtained as described above is prepared for expression in P. pastoris. For this, S. cerevisiae alpha-mating secretory signal coding sequence is amplified by polymerase chain reaction (PCR) and ligated in front of human pro-insulin gene.
Seed is obtained by growing P. pastoris on MGY media up to 10 to 16 OD600-The seed is in mid-logarithmic growth stage. In glycerol batch phase, fermentation media consisting of complete synthetic media along with trace elements and D-biotin is inoculated at a seed concentration of 5% (v/v).
During the glycerol fed-batch phase, glycerol of 50% concentration is set at a rate from 5 ml/hour/L to 45 ml/hour/L of initial fermenter batch volume, more preferably between 10 ml/hour/L and 35 ml/hour/L, and most preferably between 15 ml/hour/L and 30 ml/hour/L.
During the induction phase of the preferred embodiment, the methanol feed rate profile is from 2.5 ml/hour/L to 35 ml/hour/L more preferably between 4 ml/hour/L and 28 ml/hour/L, and most preferably between 5 ml/hour/L and 25 ml/hour/L of the initial fermenter batch volume, based on residual methanol, determined by GC (off-line) and on-line methanol sensor. The entire methanol fed-batch process lasts for approximately 108 to 120 hours.
This preferred embodiment uses a nitrogenous source as a substrate in the form of the yeast extract and peptone (YEP). Yeast extract and peptone are used in a

predetermined proportion to each other and administered continuously over the entire induction stage. In this embodiment the proportion of yeast extract: peptone is important, the preferred proportion being 1:2.
In another embodiment, the conditions are same as the preferred embodiment except the composition of the nitrogenous source. In this embodiment skimmed milk powder is used along with the YEP. In order that the individual components of the mixture do not precipitate, skimmed milk powder and YEP are sterilized separately and then mixed together. The strategy of administering this mixture during the induction phase is same as that adopted for administering the nitrogenous source in the first preferred embodiment. The preferred proportions of yeast extract, peptone, and skimmed milk powder are 1:2:2. Specifics of this embodiment are considered in Example 3.
In yet another embodiment of the present invention the nitrogenous source used in the induction phase of the invention is casamino acid. The strategy of administering casamino acid during the induction phase is same as that adopted for administering the nitrogenous source in the first preferred embodiment. A solution of unit strength (IX) is prepared by dissolving in required quantities of water, casamino acid powder, the amount of which was calculated as a certain proportion of the fermentation culture volume as measured at the end of GFB. Solution of unit strength is prepared by using 0.015 g of casamino acid powder per 100 ml of the fermentation culture volume, as measured at the end of GFB. Example 4 considers the specifics of this embodiment.
All of the above embodiments describe various forms of the improved process as defined earlier in the summary of invention section of this specification. Figure 2 illustrates one of the improved processes.

In a still further embodiment of the present invention the pH was increased to 6.3 without the addition of nitrogenous source in the induction phase of the invention. All other steps of the preferred embodiment remain same. This case is covered more specifically in Example 1.
It is observed that the expressed protein yield using the present invention (improved process) is higher by 10% to 15% (w/v) at pH 6.3 over the control process at pH 5.0 or below, when no nitrogenous source is added at the induction stage (see Figure 3). It is further observed that the expressed protein yield increases by 35% to 45% (w/v) over control process when nitrogenous source is added at the induction stage at pH 6.3. Therefore overall improvement in pro-insulin expression level of improved fermentation process is significant as compared to control process where induction is at pH 5.0 and without addition of any nitrogenous supplement such as yeast extract, peptone, skimmed milk powder, or casamino acid, or a combination of any two or more of these supplements.
A better understanding of the present invention and the enhancement of expression level of pro insulin by submerged fermentation will be had from the following examples, given by way of illustrations.
Example 1: yield improvement with pH variation
Seed required to start the biomass generation was in the form of frozen glycerol stock of P. Pastoris yeast cells. The seed was grown on MGY medium at 30°C temperature to mid-logarithmic growth stage to an OD600 value of 10 to 16 under continuous stirring for 22 hours at 250 rpm and at 30°C.
The grown seed was transferred at a rate of 5% (v/v) to the fermenter containing fermentation media comprising a synthetic media along with trace elements and

D-biotin. The biomass generated at the end of the GB phase was 33-38 g dry cell weight/L of culture broth and cellular yield coefficient was of 0.65 to 0.75 g of dry cell weight per gram of glycerol.
During the fed-batch growth phase, the profile of glycerol feed was set at a rate from 5 to 45 ml/hour/L of initial fermenter batch volume. The pH of the fermentation growth phase was controlled at 5.0 automatically via a pH control loop using 30% ammonia. A spike in DO in a range of 70-80% of air saturation was observed, indicating the exhaustion of residual glycerol carbon source. Sampling was performed in growth phase at 6-hour intervals and analyzed for cell growth, OD6oo5 NTU (Nephleometric Unit), wet and dry cell weight, pH and contamination. During the induction fed-batch phase the methanol feed rate profile was from 2.5 ml/hour/L to 35 ml/hour/L of the initial fermenter batch volume. The residual methanol was maintained at 0.02 (v/v) to 0.4% (v/v). The variation of dry cell weight with time is shown in Figure 5.
The yield was measured in mg/L of culture supernatant in mg/L of culture supernatant using HPLC (BP method) as is indicated in Figure 6. Just before induction phase starts (at the end of GFB phase) pH was adjusted to 6.3 using 30% Ammonia. The improvement in yield was 10% to 15% as compared to the control process.
Example 2: yield improvement with pH variation and addition of combination of nitrogenous sources:
This example illustrates the use of nitrogenous compounds for enhancement of yield. In one case, yeast extract and peptone of a proportion 1:2 respectively were used. A solution of unit strength (IX) was prepared by mixing (in required quantities of water), yeast extract and peptone, amounts of which were calculated as a certain proportion of the culture broth volume as measured at the end of GFB. For this example, 0.045 g of yeast extract and 0.09 g of peptone were used per

100ml of the culture broth volume, as measured at the end of GFB. Batches were carried out with solutions of strength IX to 8X, after sterilizing (in a required amount of water), administered during the entire induction phase. All other conditions of this example are same as Example 1. The improvement in yield as compared to the control process was 20% to 25%.
Example 3: yield improvement with pH variation and addition another combination of nitrogenous sources:
This example also illustrates the use of nitrogenous compounds for enhancement of yield. In this case, yeast extract, peptone and skimmed milk powder of a proportion 1:2:2 respectively were used. A solution of unit strength (IX) was prepared by mixing (in required quantities of water), yeast extract, peptone and skimmed milk powder, amounts of which were calculated as a certain proportion of the fermentation culture volume measured at the end of GFB. For this example, 0.045g of yeast extract, 0,09 g of peptone, and 0.09 g of skimmed milk powder were used for every 100 ml of the fermentation culture volume, as measured at the end of GFB. Batches were carried out with solutions of strength IX to 8X, after sterilizing (in a required amount of water), administered during the entire induction phase. All other conditions of this example are same as Example 1. The improvement in yield as compared to the control process was 35% to 45%.
Example 4: yield improvement with pH variation and addition of casamino acid as a nitrogenous source:
This example also illustrates the use of nitrogenous compounds for enhancement of proinsulin yield. In this case, casamino acid was added. A solution of unit strength (IX) was prepared by dissolving in required quantities of water, casamino acid powder, the amount of which was calculated as a certain proportion

of the fermentation culture volume as measured at the end of GFB. For this example, 0.015g of casamino acid powder was used per lOOmL of the fermentation culture volume, as measured at the end of GFB. Batches were carried out using solutions of strengths IX to 16X, after sterilizing (in a required amount of water), was administered during the entire induction phase. The improvement in yield as compared to the control process was 15% to 20%. All other conditions of this example are same as Example 1.
While the above description contains many specificities, these should not be construed as limitations in the scope of the invention, but rather as an exemplification of the preferred embodiments thereof. Accordingly, the scope of the invention should be determined not by the embodiments illustrated, but by the appended claims and their legal requirements.
References
US 5,962,267 HANIL SYNTHETIC FIBER CO., LTD. WO95/16708 NOVO NORDISK A/S EP0055945 GENENTECH, INC EP0163529 NOVO NORDISK A/S EP0347845 NOVO NORDISK A/S EP0741188 ELI LILLY AND COMPANY EP1211314 LABORATORIOS BETA S.A. CA 2357072 LABORATORIOS BETA S.A.
Other References
1. Frank, et al. (1981) in peptides: proceedings of the 7 American peptide chemistry symposium, Rich & Gross, eds., Pierce chemical co., Rockford, IL pp 729-739.
2. Chan et. al. (1981) Proceedings of National Academy of Sciences, 78; pp 5401-5404.
3. Thim et al. (1986) Proceedings of National Academy of Sciences, 83: pp6766-6770.
4. Thomas Kjeldsen, Annette Frost Pettersson and Marten Hach (1999) Biotechnol. Appl. Biochem. 29, pp79-89.
5. Weydemann, U., Keup, P., Piontek, M., Strasser, A. W., Schweden, J., Gellissen, G and Janowicz, Z. A (1995) Appl. Microbiol. Biotecnol. 44, pp377-385.

6. Romanos, M. (1995) Curr. Opin. Biotechnol. 6, pp527-533
7. Faber, K. N., Harder, W., Ab, G. and Veenhuis, M. (1995), Yeast 11, ppl331-1344.
8. Hollenberg, C. P. and Gellissen, G. (1997) Curr. Opin. Biotechnol. 8, pp554-560.
9. Raymond, C. K., Bukowski, T. Holderman, S. D., Ching, A. F. T., Vanja, E. and Stamm, M. R. (1998) Yeast 14, ppl 1-23.
10. Hollenberg, C. P. and Gellissen, G. (1997) Gene 190, pp87-97.
11. Cregg, J. M., Vedvick, T. S. and Raschke, W. C. (1993) Bio/technology ll,pp905-910.
12. Scorer, C. A., Clase, J. J., McCombie, W. R., Romanos, M. A. and Sreekrishna, K. (1994) Bio/Technology 12, ppl81-184.
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14. Kroeff, E. P., Owens, R. A., Campbell, E. L., Johnson, R.D. and Marks, H. I. (1989) J. Chromatgr., 461, 45-61.

We claim:
1. An improved fermentation process for producing high levels of recombinant human pro-insulin in a genetically engineered methylotrophic yeast, such as Pichia Pastoris comprising the steps of: a developing a seed which starts biomass generation, by growing the Pichia Pastoris on minimum glycerol medium at 30 °C optical density 600 value in the range of 10 to 16, said seed being grown to mid-logarithmic growth stage under continuous stirring;
b. forming an aqueous fermentation medium comprising substrate of salts
and glycerol, wherein said substrate is of such amount so as not to
cause substrate inhibition towards grovv1:h and wherein said glycerol
acts as a single carbon source;
c. inoculating said seed which is grown, into said aqueous fermentation
medium of step (b) in a fermenter at a rate of 5% v/v, thereby forming
a cell culture;
d. growing said cell culture in glycerol batch cell growth phase achieving
cell density up to suitable fraction of the required total cell density,
wherein pH level of solution of said cell culture is maintained at 5 and
the completion of said glycerol batch cell growth phase is indicated by
a sudden rise in the dissolved oxygen level;
e. further growing said cell culture of step (d) in glycerol fed-batch cell
growth phase up to said required total cell density by initiating a
programmed glycerol feed and maintaining residual glycerol at very
low level indicating carbon limiting condition and wherein pH level of
said solution of said cell culture is maintained at 5.0; and wherein the
glycerol concentration is 50% v/v in said fed-batch cell growth phase, and
wherein the rate of feeding said glycerol is varied between 5.0 ml/hour/L
and 45 ml/hour/L, more preferably between 10 ml/hour/L and 35
ml/hour/L, and most preferably between 15 ml/hour/L and 30 ml/hour/L

of the initial fermenter batch volume under backpressure in said fermenter;
f. stopping glycerol feed after reaching said required total cell density
and adjusting pH level of said solution of said cell culture in the range
of 5.6 to 7.0, most preferably at 6.3, which is required for fed-batch
methanol induction phase;
g. subjecting the actively grown cells of said cell culture to said fed-batch
methanol induction phase for protein expression, wherein methanol
and sterilized aqueous solution of a nitrogenous compound such as
casamino acid or of a combination of nitrogenous compounds such as
yeast extract, peptone and skimmed milk powder is added to said
solution of said cell culture and pH level of resultant solution of said
cell culture is maintained in the range of 5.6 to 7.0, most preferably at
6.3;
h. harvesting said cell culture; and
i. collecting cell-free supernatant containing proinsulin for further dovrastream processing.
2. An improved fermentation process, as claimed in claim 1, wherein in said step (g) of claim 1, the rate of feeding said methanol in said fed-batch methanol induction phase is varied between 2.5 ml/hour/L and 35 ml/hour/L, more preferably between 4 ml/hour/L and 28 ml/hour/L, and most preferably between 5 ml/hour/L and 25 ml/hour/L of the initial volume of the batch; and wherein the residual methanol is maintained in the range of 0.02% v/v to 0.4% v/v, more preferably 0.1% v/v.
3. An improved fermentation process, as claimed in any of claims 1 and 2, wherein said sterilized aqueous solution in said step (g) of claim 1 is administered continuously over said induction phase till

completion thereof; and wherein said combination of nitrogenous compounds is selected from a group of combinations such as, a combination of yeast extract and peptone or a combination of yeast extract, peptone, and skimmed milk powder.
4. An improved fermentation process as claimed in claim 3, wherein, in said combination of yeast extract and peptone, the proportion of said yeast extract and peptone is preferably 1:2 w/w and the sterilized solution being in the range of strength IX to 8X, preferably in the range of 4X to 5X, and wherein IX is obtained by adding, for every 100 ml volume of said cell culture volume measured at the end step (f) of claim 1,0.045 g of said yeast extract and 0.09 g of said peptone to a predetermined amount of water.
5. An improved fermentation process as claimed in claim 3, wherein, in said combination of yeast extract, peptone and skimmed milk powder, the proportion of said yeast extract, said peptone and said skimmed milk powder is preferably 1:2:2 w/w and the sterilized solution being in the range of strength IX to 8X, preferably in the range of 4X to 5X, wherein IX is obtained by adding, for every 100 ml volume of said cell culture volume measured at the end step (f) of claim 1, 0.045 g of said yeast extract, 0.09 g of said peptone, and 0.09 g of said skimmed milk powder to a predetermined amount of water.
6. An improved fermentation process, as claimed in claim 3, wherein, while using said casamino acid alone as said nitrogenous compound, concentration of said casamino acid added is in the range of 0.015% to 0.25% w/v, more preferably 0.15% w/v of said cell culture volume measured at the end of said step (f) of claim 1.

7. An improved fermentation process, as claimed in any of claims 1 to 6,
wherein said administering of said sterilized aqueous solution is carried
out at any one of constant, variable, repeated, continuous, stepwise
increment, or exponential flow rate or at any combination thereof.
8. An improved fermentation process for producing high levels of
recombinant human proinsulin in a genetically engineered methylotrophic
yeast, substantially as hereinbefore described with reference to the
examples illustrated and accompanying drawings.

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Patent Number

211888

Indian Patent Application Number

1129/CHE/2004

PG Journal Number

02/2008

Publication Date

11-Jan-2008

Grant Date

13-Nov-2007

Date of Filing

01-Nov-2004

Name of Patentee

M/S. SHANTHA BIOTECHNICS LIMITED

Applicant Address

SERENE CHAMBERS ,3RD FLOOR, BANJARA HILLS ,ROAD NO.7, HYDERABAD 500 034,

Inventors:

#	Inventor's Name	Inventor's Address
1	SWAPAN K.JANA	SERENE CHAMBERS ,3RD FLOOR, BANJARA HILLS ,ROAD NO.7, HYDERABAD 500 034,

PCT International Classification Number

C 12 N 9/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA