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  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/30—Organic components
  • C12N2500/32—Amino acids

Definitions

• the invention relates to methods of preventing norvaline raisincorporation in recombinant proteins.
• 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.
• 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.
• the leucine, isoleucine, and methionine are present in a glucose feed.
• the glucose feed comprises 5 g/L leucine, 6.3 g/L isoleucine, and 4 g/L methionine.
• the method further comprises supplying leucine and isoleucine to the culture at glucose exhaustion.
• the recombinant protein is TFPI or ala-TFPI.
• the culture is in a fermentor.
• the host cells are E. cailL
• FIG. 1 Map of plasmid pMON37621.
• 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.
• 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.
• the present method prevents norvaline and norleucine incorporation without compromising on cell densities and titers achieved in the absence of leucine.
• 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.
• the initial volume in the fermentor is 6400 L.
• 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.
• 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.
• 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.
• dissolved oxygen preferably 0.1-0.5 atm
• 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.
• 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.
• 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.
• 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 OD 550 .
• 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.

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• 2007
  • 2007-03-07 WO PCT/US2007/005963 patent/WO2007103521A2/en not_active Ceased
  • 2007-03-07 EP EP07752647A patent/EP1996697A2/de not_active Withdrawn

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