PREVENTING NORVALINE AND NORLEUCINE MISINCORPORATION IN RECOMBINANT PROTEINS
FIELD OF THE INVENTION
[01] The invention relates to methods of preventing norvaline raisincorporation in recombinant proteins.
BACKGROUND
[02] Previous efforts to prevent norleucine misincorporation into recombinant proteins involved supplementing medium with leucine and/or methionine (e.g., U.S. Patent 5,599,690; Apostol et ah, J. Biol. Chem. 272, 28980-88, 1997) or supplementing the medium with combinations of leucine, methionine, and cysteine (e.g., U.S. Patent 5,622,845). There is a need in the art for methods of preventing misincorporation of norvaline and norleucine in recombinant proteins.
SUMMARY OF THE INVENTION
[03] The invention provides a method for preventing misincorporation of norvaline in place of leucine in recombinant proteins. The method can be utilized in the production of any recombinant protein, and may be used with both large and small scale production. The method involves feeding the culture with the amino acids leucine, isoleucine, and methionine and is particularly suited for the prevention of norvaline and norleucine misincorporation in recombinant proteins without compromising cell densities and titers.
[04] Accordingly, in one embodiment, the invention is directed to a method for preventing norvaline and norleucine misincorporation into a recombinant protein comprising providing a culture of host cells, which inducibly express the recombinant protein, an amount of amino acids leucine, isoleucine, and methionine sufficient to maintain a constant amino acid concentration of each of the amino acids throughout induction of recombinant protein expression.
[05] In certain embodiments of the method, the leucine, isoleucine, and methionine are present in a glucose feed. [06] In additional embodiments of the method, the glucose feed comprises 5 g/L leucine, 6.3 g/L isoleucine, and 4 g/L methionine.
[071 I*1 still further embodiments, the method further comprises supplying leucine and isoleucine to the culture at glucose exhaustion.
[08] In other embodiments, the recombinant protein is TFPI or ala-TFPI.
[09] In yet an additional embodiment, the culture is in a fermentor.
[10] In certain embodiments of the subject invention, the host cells are E. cailL
[11] These and other embodiments of the subject invention will readily occur to those of skill in the art in view of the disclosure herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[12] FIG. 1. Map of plasmid pMON37621.
DETAILED DESCRIPTION OF THE INVENTION
[13] The invention provides a method for preventing misincorporation of norvaline in place of leucine in recombinant proteins. The method involves feeding the culture with leucine, isoleucine, and methionine. Addition of methionine prevents incorporation of norleucine. Leucine inhibits and represses the enzymes involved in the synthesis of both norvaline and norleucine. However, leucine also affects other cellular functions and results in lower cell densities and titers at the end of fermentation. Addition of isoleucine results in greatly increased amount of recombinant protein production. Thus, the present method prevents norvaline and norleucine incorporation without compromising on cell densities and titers achieved in the absence of leucine.
[14] Methods of the invention can be used in the production of any recombinant protein and can be used with both large and small scale production. It is particularly useful for use with high density fermentation, for example, of E. coli host cells. One embodiment of the invention is illustrated in the examples below for the production of recombinant ala-TFPI (TFPI as shown in SEQ ED NO:1 comprising an N-terminal alanine). These examples are provided for purposes of illustration only and are not intended to limit the scope of the invention.
[15] All patents, patent applications, and references cited in this disclosure are expressly incorporated herein by reference.
EXAMPLE l
Fermentation Conditions
[16] The following procedures were used in Example 2, below.
[17] Preparation of the Master and Working Cell Bank, Master and Working Cell Banks were prepared from production strains as follows for use in the experiments described below. See also US 2005/0037475. To prepare a Master Cell Bank, a frozen vial of parent MON210/pMON37621 cells (ATCC Accession No. PTA-5564) is thawed and grown in a shake flask for approximately 9 generations in the defined production seed medium with spectinomycin. Vials of cells with 10% glycerol are frozen and maintained at —70° C. Working Cell Banks are prepared by thawing a master stock vial and growing the cells as described for the Master Cell Bank. MON210/pMON37621 cells comprise pMON37621, shown in FIG. 1, which comprises a coding sequence for ala-TFPI,
[18] Fermentation Conditions. Growth of seed cultures for inoculation of the 10,000 L fermentor comprises two stages: (1) Seed 1 shake flask and (2) Seed 2 fermentor. The composition of the media used during the fermentation process are listed in Table 1, which also lists the components of medium used for large scale production of ala- TFPI. KOH and H2SO4 are used to adjust the medium pH of the Seed 1 and Seed 2 media. NH4OH and H2SO4 are used to adjust pH of fermentor medium. NH4OH also is used to control pH during the fermentation.
[19] USP purified water is used throughout the fermentation process. The selective antibiotic spectinomycin is used during the preparation of the Working Cell Bank and is not used during the inoculum preparation or fermentation process. Table 1. Fermentation Media Composition and Concentration
1 The initial volume in the fermentor is 6400 L.
2Actual glucose concentration at start is 34-42 g/L due to moisture content
(<9%) of Cerelose.
33.5 L of Ucon antifoam added at start. Additional antifoam (up to 12.5 L) may be added during the fermentation.
[20] Seed 1 — Shake Flask. The fermentation process begins by thawing a frozen vial of a Working Cell Bank. The contents of this vial (1 mL) is used to inoculate 0.5 L of seed 1 medium in a seed 1 shake flask. The flask is incubated at 37±2° C. and mixed at 200 RPM. The culture is grown for approximately 9 generations until a cell density of 0.9- 1.7 OD is reached. The Seed 1 culture is then transferred to the Seed 2 fermentor.
[21] Seed 2-30 L Fermentor. The 30 L of Seed 2 medium is inoculated with the 0.5 L contents of the seed 1 culture. The seed 2 medium is essentially the same as the Seed 1 medium, except for the addition of 0.1 mL/L Ucon antifoam. The Seed 2 fermentation preferably is carried out at a temperature of 37±2° C, with an air sparge of >2 LPM to maintain the dissolved oxygen above 10% of air saturation. The initial pH of the medium preferably is 7.2±0.2. When the cells have grown approximately 6 generations to a density of 0.9-1.7 OD, the Seed 2 culture is transferred to the 10,000 L fermentor.
[22] 10,000 L Production Fermentor. The entire contents of the Seed 2 fermentor are transferred to the 10,000 L fermentor containing approximately 6,400 L of production medium. The production medium composition is shown in Table 3. The production fermentor is controlled for the following parameters. The temperature of the growth phase preferably is 37±2° C. The temperature set point is changed from 37° C. to 30° C. approximately 0.5 hours before the induction of TFPI or TFPI analog expression, and the temperature of the expression phase preferably is 30±2° C. pH is controlled by the addition of concentrated NH40H and preferably is maintained at 6.9±0.2. A temporary pH spike to approximately pH 7.4±0.2 occurs when the initial supply of glucose is depleted and just prior to the start of the nutrient feed. Finally, dissolved oxygen (preferably 0.1-0.5 atm) is controlled by adjusting agitation rate, sparge rate, and the proportion of oxygen in the sparge gas.
[23] Cell growth in the 10,000 L production fermentation begins as a simple batch culture, using the glucose of the starting medium. Glucose is depleted when the cell density reaches approximately 40 OD, as indicated by a pH increase to 7.4±0.2. At that time, a glucose/nutrient feed is started. The nutrient feed contains 550 g/L glucose, 18 g/L sodium polyphosphate, 6.65 g/L magnesium sulfate, and 4 g/L methionine. The nutrient feed rate is increased exponentially.
[24] When the cell density reaches approximately 100 OD, the culture is induced to produce TFPI or TFPI analog by the addition of IPTG (e.g., 187+/-3 g of IPTG per fermentation, nominal volume=9500 L) and the glucose/nutrient feed rate is reduced to limit the glucose level during the expression phase. The temperature set point is changed so that the culture reaches 30±2° C. within one hour after induction. The culture is harvested approximately 12 hours after induction. The TFPI or TFPI analog concentration at harvest is approximately 5 g/L TFPI as determined by SDS-PAGE analysis.
[25] Cell Harvest, Dissolution ofRefractile Bodies, and Refolding of the Expressed TFPI Cell Harvest. For cell harvest, the fermentation broth is adjusted to pH 5.5-6 and the delivery of oxygen and glucose are discontinued. Agitation is reduced, and the broth temperature is decreased to 5-10° C. The chelator DTPA is added to a final concentration of 1 mM. The DTPA addition is made using a stock solution that has been pH adjusted to 5.5-6.0 with citric acid. The harvest culture is fed to a BTUX-510 centrifuge operated at a flow rate to minimize loss of solids in the supernatant. Solids containing the harvested cells are continually pumped to a tank until the fermentor is empty. Purified water is added to the collected cells to a volume of ~10,000 liters, DTPA is added to a final concentration of 1 mM and processed through the BTUX- 510 centrifuge as previously described. This wash step is repeated a total of two times.
EXAMPLE 2
[26] Fermentation Conditions. Growth of seed cultures for inoculation of the 10 L fermentor comprises two stages: (1) Expansion of working cell bank and freezing of development cell bank and (2) Seed 1 shake flask. The composition of the media used during the fermentation process are the same as listed in Table 1. KOH and H2SO4 are used to adjust the medium pH of the Seed 1 media. NH4OH and H2SO4 are used to adjust pH of fermentor medium. NH4OH also is used to control pH during the fermentation. Seed 1 — Shake Flask. The fermentation process begins by thawing a frozen vial of a Working Cell Bank. The contents of this vial (1 mL) is used to inoculate 0.5 L of seed 1 medium in a seed 1 shake flask. The flask is incubated at 37±2° C. and mixed at 200 RPM. The culture is grown for approximately 9 generations until a cell density of 0.9-1.7 OD is reached. A development seed stock is then frozen and used for inoculation of the seed culture of all 10 L fermentations. The contents of the development stock are used to inoculate 200 mL of culture in a 1 L shake flask. The flask is incubated at 37±2° C. and mixed at 200 RPM. The culture is grown for approximately 9 generations until a cell density of 0.9-1.7 OD is reached.
[27] 10 L Production Fermentor. A volume of the seed flask is transferred to the 10 L fermentor containing 6.9 L of medium to obtain an intial cell density of 0.006 OD550. The production medium composition is the same as shown in Table 3 for the 10,000 L fermentor. The 10 L production fermentor is controlled and operated the same as the 10,000 L fermentor as described in sections 13, 14 and 15 above.
Effects of supplementation fermentation
[28] Four types of experiments were carried out to determine the effect of supplementing the fermentation runs with isoleucine, leucine, and methionine, added either in the feed only or in the feed and also as a bolus at the beginning of stage 2.
[29] Experiment type 1. Addition of the Ile/Leu/Met as a bolus in second stage of fermentation and in the feed, or addition of Ile/Leu as a bolus and in the feed. The data is presented in Table 2. Table 2.
[30] Experiment type 2. Addition of Ile/Leu/Met or Leu/Ile in the feed only in stage 2. The data is shown in Table 3.
Table 3.
[31] Experiment type 3. In these experiments the fermentor was run with an initial seed in the presence of Leu/Ile/Met/Val/Thr. The use of Leu, He, and Met from the beginning prevents any norvaline or norleucine from being produced at all, so there is none to be incorporated even at a very low level in the recombinant protein product. The addition of the valine and the threonine were used to help cell growth.
[32] Experiment type 4. These experiments demonstrate the benefit of adding the lie into the feed in terms of the final amount of TFPI produced.