# Patent

## Title
Alkaline feed

## Abstract

A method for cultivating a bacterial cell comprising the addition of an amino acid in an alkaline solution used for pH regulation. Also an aspect is a method for producing a polypeptide comprising the steps of a) providing a bacterial cell comprising a nucleic acid encoding the polypeptide, b) cultivating the provided cell, c) adjusting the pH value during the cultivating with a basic solution comprising an amino acid, d) recovering the polypeptide from the cell or the cultivation medium and thereby producing the polypeptide.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This Application is a continuation of U.S. application Ser. No. 13/217,540 filed on Aug. 25, 2011, now granted as U.S. Pat. No. 8,951,756, which claims priority under 35 U.S.C. §119 to European Application No. EP 10008997.8 filed Aug. 30, 2010, the disclosures of which are incorporated herein by reference.


FIELD OF THE INVENTION
Herein is reported a method for high cell density cultivation of a prokaryotic cell, such as an Escherichia coli strain, in a chemically defined medium for the production of a polypeptide wherein an amino acid is fed by a concentrated alkaline solution simultaneously regulating the pH of the cultivation medium and acting as a nitrogen source.
BACKGROUND OF THE INVENTION
In recent years the production of proteins has steadily increased and it is likely that proteins will become the biggest group of therapeutics available for the treatment of various diseases in the near future. The impact of proteins emerges from their specificity, such as the specific target recognition and binding function.
Cell cultures are used in fermentative processes to produce substances and in particular proteins. A distinction is made between processes in which the cell cultures are genetically unmodified and form their own metabolic products and processes in which the organisms are genetically modified in such a manner that they either produce a larger amount of their own substances such as proteins or produce foreign substances. The organisms producing the substances are supplied with a nutrient medium which guarantees the survival of the organisms and enables the production of the desired target compound. Numerous culture media are known for these purposes which enable an optimal cultivation of the specific host.
High-cell-density cultivation of Escherichia coli is reported by Riesenberg (Riesenberg, D., et al., Curr. Opin. Biotechnol. 2 (1991) 380-384) and Horn (Horn, U., et al., Appl. Microbiol. Biotechnol. 46 (1996) 524-532). Riesenberg, D. and Guthke, R. (Appl. Microbiol. Biotechnol. 51 (1999) 422-430) reported the high-cell-density cultivation of microorganisms. Growing E. coli to high cell density is reviewed by Shiloach, J. and Fass, R. (Biotechnol. Advances 23 (2005) 345-357).
In WO 91/10721 a process for high cell density fermentation of Escherichia coli in an agitated boiler fermenter is reported. A method of plasmid DNA production and purification is reported in WO 97/29190. The controlling of growth of aerobic submerged microorganism cultures by controlling dissolved oxygen concentration and oxygen transfer rate is reported in DD 295867. In EP 0 866 876 a process for the preparation of recombinant proteins in E. coli by high cell density fermentation is reported.
In WO 03/048374 a process for the production of aromatic amino acid metabolite or derivative thereof is reported. A process for the preparation of recombinant proteins in E. coli by high cell density fermentation is reported in WO 97/21829.
SUMMARY OF THE INVENTION
It has been found that a prokaryotic cell, especially an amino acid auxotrophic E. coli K12 strain, can be cultivated on chemically defined medium at high cell densities if an amino acid is added to the cultivation medium in an alkaline solution.
One aspect as reported herein is a method for cultivating a bacterial cell, especially an E. coli cell, at high cell densities, wherein the cell expresses a recombinant polypeptide, wherein the cultivating comprises the adding of an alkaline solution of an amino acid selected from aspartate, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan and tyrosine during the cultivating, wherein the amino acid has a concentration in the alkaline solution that is higher than its solubility in water at 20° C. and at neutral pH, and wherein the dry cell weight of the cultivated bacterial cell is at one point in the cultivating at least 20 g/l.
One aspect as reported herein is a method for producing a polypeptide comprising:

    
    
        a) providing a bacterial cell, especially an E. coli cell, comprising a nucleic acid encoding a polypeptide,
        b) cultivating the provided cell,
        c) adjusting the pH value during the cultivating with an alkaline solution comprising an amino acid selected from aspartate, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan and tyrosine,
        d) recovering the polypeptide from the cell or the cultivation medium and thereby producing the polypeptide.
    
    


Another aspect as reported herein is the use of an alkaline solution comprising an amino acid for adjusting the pH value during the cultivation of a bacterial cell.
Also an aspect as reported herein is the use of an alkaline solution of an amino acid as feed in the cultivation of a bacterial cell, wherein the amino acid is selected from aspartate, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan and tyrosine and the cultivation is up to a dry cell weight of 20 g/l or more.
The following are specific embodiments of all aspects as outlined before.
In one embodiment the amino acid is a poorly water soluble amino acid. In one embodiment the bacterial cell is an amino acid auxotrophic cell and the auxotrophy is for an amino acid selected from aspartate, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan and tyrosine. In another embodiment the bacterial cell is an Escherichia coli cell or mutant thereof. In a further embodiment the amino acid has a solubility in water at 20° C. of 50 g/l or less. In a further embodiment the amino acid has a solubility in water at 20° C. of 40 g/l or less. In also an embodiment the amino acid is selected from aspartate, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan and tyrosine. In another embodiment the amino acid is selected from aspartate, aspartic acid, glutamine, glutamic acid, histidine, leucine, tryptophan and tyrosine. In one embodiment the amino acid is leucine. In still another embodiment the amino acid has a concentration in the alkaline solution that is higher than its solubility in water at 20° C. In one embodiment the solubility is two times higher, in another embodiment three times higher than the solubility in water at 20° C. In another embodiment the solubility is higher than the solubility in water at 20° C. and at a pH value of pH 6 to 8. In one embodiment the amino acid has a concentration of 25 g/l or more in the alkaline solution, or in a further embodiment of 30 g/l or more, or in still another embodiment of 35 g/l or more. In one embodiment the amino acid has a concentration of 45 g/l or more in the alkaline solution. In one embodiment the amino acid has a concentration of about 50 g/l in the alkaline solution. In a further embodiment the alkaline solution has a pH value of 9 or more, in a further embodiment of 10 or more, and still in another embodiment of 10.5 or more. In one embodiment the alkaline solution is an ammonia solution of more than 5% (w/v) or of 10% (w/v) or more or of 15% (w/v) or more. In one embodiment the alkaline solution is an ammonia solution of about 12.5% (w/v) in water. In also an embodiment the polypeptide is human apolipoprotein A1 or a derivative thereof. In a further embodiment the apolipoprotein A1 has an amino acid sequence selected from of SEQ ID NO: 01 to SEQ ID NO: 35.
In another embodiment, the invention provides a method for cultivating an Escherichia coli cell expressing a polypeptide, characterized in that the cultivating comprises the adding of an alkaline solution of an amino acid selected from aspartate, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan and tyrosine during the cultivating,
wherein the amino acid has a concentration in the alkaline solution that is higher than its solubility in water at 20° C. and at neutral pH, and the amino acid has a concentration of 30 g/l or more, and
wherein the alkaline solution is an ammonia solution of 10% (w/v) or more, and
wherein the dry cell weight of the cultivated bacterial cell is at one point in the cultivating at least 20 g/l.
In another embodiment, the invention provides a method for producing a polypeptide comprising:

    
    
        a) cultivating an Escherichia coli cell comprising a nucleic acid encoding the polypeptide,
        c) adjusting the pH value during the cultivating with an alkaline solution comprising an amino acid selected from aspartate, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan and tyrosine, and
        d) recovering the polypeptide from the cell or the cultivation medium and thereby producing the polypeptide,
        wherein the amino acid has a concentration in the alkaline solution of 30 g/l or more, and
        wherein the alkaline solution is an ammonia solution of 10% (w/v) or more.
    
    


In certain embodiments, the method is characterized in that the bacterial cell is an amino acid auxotrophic cell and the auxotrophy is for an amino acid selected from aspartate, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan and tyrosine.
In certain embodiments, the method is characterized in that the alkaline solution has a pH value of 9 or more.
In certain embodiments, the method is characterized in that the polypeptide is human apolipoprotein A1 or a derivative thereof.
In certain embodiments, the method is characterized in that the apolipoprotein A1 has an amino acid sequence selected from SEQ ID NO: 01 to 35.
In certain embodiments, the method is characterized in that the apolipoprotein A1 has an amino acid sequence selected from SEQ ID NO: 01, 02, 34, and 35.
In certain embodiments, the method is characterized in that the amino acid has a concentration of about 50 g/l.
In certain embodiments, the method is characterized in that the alkaline solution is an ammonia solution of about 12.5% (w/v) of ammonia in water.
In certain embodiments, the method is characterized in that the amino acid is leucine.
In certain embodiments, the method is characterized in that the alkaline solution comprises leucine and proline.
In certain embodiments, the method is characterized in that the alkaline solution is an ammonia solution of about 12.5% (w/v and comprises the amino acids leucine and proline each at a concentration of about 50 g/l.
In certain embodiments, the invention provides for use of an alkaline solution of an amino acid as feed in the cultivation of a bacterial cell, wherein the amino acid is selected from aspartate, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan and tyrosine and the cultivation is up to a dry cell weight of 20 g/l or more and the amino acid has a concentration of 30 g/l or more in the alkaline solution and the alkaline solution is an ammonia solution of 10% (w/v) or more.
In certain embodiments, the invention provides for use according to embodiment 13, characterized in that the bacterial cell is an amino acid auxotrophic cell and the auxotrophy is for an amino acid selected from aspartate, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan and tyrosine.
In certain embodiments, the method is characterized in that the Escherichia coli cell is an amino acid auxotrophic Escherichia coli cell.



DESCRIPTION OF THE FIGURES
FIG. 1 Parameter plot Example 3—separate amino acid feeding.
FIG. 2 Parameter plot Example 3—separate amino acid feeding.
FIG. 3 Parameter plot Example 4—combined amino acid feeding.
FIG. 4 Parameter plot Example 4—combined amino acid feeding.



DETAILED DESCRIPTION OF THE INVENTION
Herein is reported a method for cultivating a prokaryotic cell, e.g. an amino acid auxotrophic bacterial cell, wherein at least one amino acid, e.g. that for which the cell is auxotrophic, is added in an alkaline solution.
It has been found that a prokaryotic cell, e.g. an amino acid auxotrophic E. coli K12 strain, can be cultivated at high cell densities if a feed comprising at least one amino acid, such as that for which the cell has an auxotrophy, is added to the cultivation medium in an alkaline solution. This is especially advantageous when the amino acid is poorly soluble in water and the solubility can be increased by dissolving the amino acid in an alkaline solution. At the same time the alkaline solution can be used to adjust the pH value of the cultivation medium. By combining the amino acid solution and the pH adjustment solution in a highly concentrated single feed solution the added volume can be reduced, thus, allowing for a high cell density cultivation of the prokaryotic cell. Additionally it has been found that a concentration of at least 45 g/l of the amino acid in the alkaline feed solution results in an increased production of a recombinant polypeptide.
In one embodiment the method for cultivating a prokaryotic cell comprises the following steps

    
    
        a) providing a prokaryotic cell,
        b) cultivating the prokaryotic cell,
        c) adjusting the pH value during the cultivating of the prokaryotic cell with an alkaline solution comprising an amino acid selected from aspartate, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan and tyrosine.
    
    


An “amino acid auxotrophic prokaryotic cell” is a prokaryotic cell that cannot synthesize an essential amino acid e.g. due to a mutation or deletion within a gene locus comprising the structural gene encoding the proteins of the corresponding biosynthetic pathway. Without the addition of the respective amino acid to the cultivation medium the cell cannot proliferate. The auxotrophy can be for any amino acid. The prokaryotic cell can also be auxotrophic for more than one amino acid. Thus, in one embodiment the amino acid auxotrophic prokaryotic cell is auxotrophic for at least one amino acid. In another embodiment the amino acid auxotrophic prokaryotic cell is auxotrophic for at least one, at least two, at least three, at least four, at least five amino acids. In a further embodiment the amino acid auxotrophic prokaryotic cell is auxotrophic for up to 5, or up to 10, or up to 15 amino acids. In another embodiment the amino acid auxotrophic prokaryotic cell is auxotrophic for one to five amino acids, or one to three amino acids, or for one to two amino acids, or for one amino acid, or for two amino acids, or for three amino acids, or for four amino acids. The amino acid auxotrophic prokaryotic cell is in one embodiment a bacterial cell.
In one embodiment the bacterial cell is an Escherichia cell, or a Bacillus cell, or a Lactobacillus cell, or a Corynebacterium cell, or a Yeast cell (Saccharomyces, Candida, or Pichia). In a further embodiment the cell is an Escherichia coli cell, or a Bacillus subtilis cell, or a Lactobacillus acidophilus cell, or a Corynebacterium glutamicum cell, or a Pichia pastoris yeast cell.
The term “adjusting a value” denotes that the respective value is maintained at a predetermined level throughout a cultivating, i.e. the value is checked continuously or at predetermined constant time intervals and change by the addition of a correction fluid if the value is outside a preset acceptance range. For example, the term “adjusting the pH value” denotes that the pH value of a cultivation medium is determined periodically at fixed time points, i.e. with fixed time intervals, and if the determined pH value is outside an acceptance range, such as e.g. 0.1 pH units or 0.15 pH units or 0.2 pH units, the pH value is re-adjusted to the predetermined pH value by the addition of a correction fluid, such as an acid or an alkaline solution.
Methods for cultivating a prokaryotic cell and also for cultivating an amino acid auxotrophic prokaryotic cell are known to a person of skill in the art (see e.g. Riesenberg, D., et al., Curr. Opin. Biotechnol. 2 (1991) 380-384). The cultivating can be with any method. In one embodiment the cultivating is a batch cultivating, a fed-batch cultivating, a perfusion cultivating, a semi-continuous cultivating, or a cultivating with full or partial cell retention.
The only requirement for the cultivating is that an alkaline solution has to be added. This addition can be a sole feed solution or as a combined feed and pH adjustment solution.
The cultivation medium used for the start of the cultivating of the cell can be any medium known to a person skilled in the art, whereby the concentration of the amino acid to be fed is in the medium less than 5 g/l, or less than 7.5 g/l, or less than 10 g/l. It has to be pointed out that the concentration of the respective compounds has to be chosen in a way that no negative interference with the growth of the cell is to be expected. In one embodiment is the medium a defined glucose-mineral salt medium.
In one embodiment the cultivating is a high cell density cultivating. The term “high cell density cultivating” denotes a cultivating method wherein the dry cell weight of the cultivated prokaryotic cell is at one point in the cultivating at least 10 g/l. In one embodiment the dry cell weight is at one point in the cultivating at least 20 g/l, or at least 50 g/l, or at least 100 g/l, or more than 100 g/l. In order to reach such a high cell density state the volume of feed and/or adjustment solutions added during the cultivating has to be as small as possible. Methods for the determination of dry cell weight are reported e.g. in Riesenberg, D., et al., Appl. Microbiol. Biotechnol. 34 (1990) 77-82.
The nutrients in the provided medium will be metabolized during the cultivation and have to be replenished in order to avoid a limitation. If an amino acid has a poor solubility only a lowly concentrated feed solution can be prepared and added. To provide the required amount of the amino acid a large volume of the feed solution has to be added. This results in an increase in the total cultivation volume, a dilution of the culture broth and, thus, is disadvantageous for a high cell density processes.
The solubility of the 20 naturally occurring amino acids is listed in the following Table.











TABLE









solubility in water

solubility












amino acid
[g/l]
at [° C.]
[good/acceptable/poorly]
















alanine
166.5
25
good



arginine
150
20
good



aspartate
22
20
poorly



aspartic acid
4
20
poorly



cysteine
280
20
good



glutamine
26
18
poorly



glutamic acid
11
25
poorly



glycine
225
20
good



histidine
38
20
poorly



isoleucine
40
20
poorly



leucine
24
20
poorly



lysine
300
20
good



methionine
48
20
poorly



phenylalanine
27
20
poorly



proline
1500
20
good



serine
364
20
good



threonine
90
20
acceptable



tryptophane
10
20
poorly



tyrosine
0.4
20
poorly



valine
88
20
acceptable










The solubility of the amino acids aspartate, aspartic acid, glutamine, glutamic acid, histidine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine is below 50 g/l and, thus, these amino acids are termed to have a poor solubility in water.
For example, the amino acid leucine has a solubility in water at 20° C. of 24 g/l and, thus, is poorly soluble. In an alkaline solution comprising 12.5% (w/v) ammonia the solubility is increased to 76 g/l and, thus, is more than three times the solubility in water. At the same time the required feed volume is reduced by more than 60%. If at the same time the alkaline solution is also used to adjust the pH value of the cultivation the added volume can be reduced even more. For example, the amino acid tyrosine has a solubility in water at 20° C. of 0.4 g/l and, thus, is poorly soluble. In an alkaline solution comprising 12.5% (w/v) ammonia the solubility is increased to 39 g/l and, thus, is about one hundred times the solubility in water.
In one embodiment the amino acid is aspartate, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan and/or tyrosine. In also an embodiment the amino acid is aspartate, aspartic acid, glutamine, glutamic acid, histidine, leucine, tryptophan and/or tyrosine. In one embodiment the amino acid is leucine. In one embodiment the amino acid is an amino acid that it poorly soluble in water of a pH value of about 7 at 20° C. In also an embodiment the amino acid is leucine and proline, or the amino acid is leucine and proline and tryptophan. In a further embodiment the amino acid has a solubility in the alkaline solution that is higher than the solubility in water at 20° C. In a further embodiment the solubility in the alkaline solution is of from two times to ten times the solubility in water at 20° C. In one embodiment the amino acid has a solubility in water of 40 g/l or less. In another embodiment the amino acid has a solubility in water of 30 g/l or less. In also an embodiment the amino acid has a solubility in the alkaline solution of 25 g/l or more. In a further embodiment the amino acid has a concentration in the alkaline solution of 30 g/l or more. In another embodiment the amino acid has a concentration in the alkaline solution of 35 g/l or more. In also an embodiment the amino acid has a solubility in the alkaline solution of 50 g/l or more.
In the following Table the cultivation results in a cultivation vessel with 10 l working volume of the same Leucine and Proline auxotrophic E. coli cell obtained with different feeding modes are shown.











TABLE







total






biomass

final




(dry
recombinant
cultivation




matter)
protein yield
volume


experiment
feed
[g/l]
[g/l]
[l]



















1
separate feeds:
49.4
10.0
11.8



20 g/l L-Leucine



100 g/l L-Proline


2
separate feeds:
50.6
9.0
12.2



20 g/l L-Leucine



100 g/l L-Proline


3
combined feed:
75.7
16.5
10.2



12.5% (w/v) NH3



solution containing



50 g/l L-Leucine



and 50 g/l L-Proline


4
combined feed:
56.5
13.5
9.3



12.5% (w/v) NH3



solution containing



33 g/l L-Leucine



and 33 g/l L-Proline









It can be seen that in Experiments 1 and 2, in which the two amino acids are added as separate feeds to the cultivation medium, the total biomass obtained at the end of the cultivation and the yield of recombinant protein is lower compared to Experiments 3 and 4, in which the amino acids are added as a combined alkaline feed that at the same time is used to adjust the pH value of the cultivation medium. Also the final cultivation volume in Experiments 3 and 4 does not exceed the working volume of the cultivation vessel as in Experiments 1 and 2.
In one embodiment the alkaline solution is a 12.5% (w/v) ammonia solution in water and comprises at least one amino acid at a concentration of about 50 g/l or more. In one embodiment the alkaline solution comprises Leucine and Proline at a concentration of about 50 g/l.
Prokaryotic cells that can be used in the method as reported herein can comprise one or more amino acid auxotrophies. For example, E. coli cells deficient in the Leucine biosynthetic pathway can be selected from the LeuB6 deficient cells 13-6, χ148, χ156, χ2224, χ462, χ463, χ474, χ478, χ515, χ65, χ697, χ760, 2000k MSE248, 342-167, 342MG, 679-680, A586, A592, A593, AA100, AA7852, AA787, AB1102, AB1111, AB1115, AB1122, AB1129, AB113, AB1132, AB1133, AB114, AB1157, AB1157-D, AB1314, AB1330, AB1331, AB1881, AB1884, AB1885, AB188, CP78, CP79, CR34 Thy-, CR34 Thy-SR, CR34/308, CR34/313, CR34/399, CR34/43, CR34/454, CR34/500, CR34/7a, CS130, CS312, CS419, CS425, CS426, CS460, CS471, CS472, CS50, CS81, CS85, CSR06, CSR603, CSR603/pDR1996, CT28-3b, DA10, DA11, DB1161, DB1257, DE1878, DE1882, DE2345, DF225, DF41, JRG94, JS10 C600r-m-, T6R, P678SSR pro-, PA20SR, PA200 SR, PA201 SR, PA214SRT6R, PA265 SR, PA309, PDE70, PA340, PA340/T6, PA360, PA414, PAM161, PAM162, PAM163, PAM164, PAM660, PAT84, PB349, PB69, PC1, PC2, PC3, PC5, PC6, PC8, PJ1, PJ2, PJ3, PJ4, PJ5, PJ C600 (=CRSR), W208 SR AzR, W2660, LAM-, W945his, WA2127, WA2379, WA2548, WA2552, WA2574, WA2899, WA921, WA946, WA960, Y10, Y46, Y53, Y70, YYC100.
In one embodiment the prokaryotic cell is an E. coli K12 cell or an E. coli B cell.
In one embodiment the alkaline solution is a strongly alkaline solution. In another embodiment the alkaline solution has a pH value of pH 9 or more, or pH 10 or more, or pH 10.5 or more. In a further embodiment the solubility of the amino acid in the alkaline solution is at least twice the solubility of the amino acid in water.
In one embodiment the method for producing a polypeptide as reported herein comprising the following steps

    
    
        a) providing an amino acid auxotrophic bacterial cell comprising a nucleic acid encoding the polypeptide,
        b) cultivating the provided cell,
        c) adjusting the pH value during the cultivating with an alkaline solution comprising an amino acid for which the bacterial cell is auxotrophic,
        d) recovering the polypeptide from the cell or the cultivation medium and thereby producing the polypeptide.
    
    


The following examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.
Material and Methods
Optical density of the cultivations was measured with a DR2800 photometer (Hach-Lange, Dusseldorf, Germany) at 578 nm.
Protein concentration was determined densitometrically by comparing the volumes of a standard protein band with the band volumes of the produced protein within the fermentation samples on a SDS-Page gel.
Example 1
Determination of Solubility of Leucine in an Ammonia Solution
The calculated amount of Leucine was weighted into a 500 ml flask. After addition of 250 ml deionized water the solution is sterilized by autoclavation. Thereafter 250 ml of a 25% (w/v) ammonia solution is added and determined whether the Leucine was dissolved or not. After dissolution of the Leucine the final volume of the solution was determined. If it deviates notably from 500 ml the solution was prepared again with a reduced amount of water (see Table 3).









TABLE 3









Leucine concentration [g/l]

















17
18
19
20
21
25
30
40
50





water[ml]
250
250
250
250
250
250
250
250
250


ammonia
250
250
250
250
250
250
250
250
250


solution [ml]


soluble
yes
yes
yes
yes
yes
yes
yes
yes
yes


final volume
500
500
500
500
500
500
500
500
500


[ml]












Leucine concentration [g/l]

















60
70
71
72
73
74
75
76
77





water[ml]
250
250
250
250
250
250
250
250
250


ammonia
250
250
250
250
250
250
250
250
250


solution [ml]


soluble
yes
yes
yes
yes
yes
yes
yes
yes
no


final volume
500
500
500
500
500
500
500
500
500


[ml]









Example 2
Making and Description of the E. coli Expression Plasmids
The tetranectin-apolipoprotein A-I fusion polypeptide was prepared by recombinant means. The amino acid sequence of the expressed fusion polypeptide in N- to C-terminal direction is as follows:

    
    
        the amino acid methionine (M),
        a fragment of an interferon sequence that has the amino acid sequence of CDLPQTHSL (SEQ ID NO: 36),
        a GS linker,
        a hexa-histidine tag that has the amino acid sequence of HHHHHH (SEQ ID NO: 37),
        a GS linker,
        an IgA protease cleavage site that has the amino acid sequence of VVAPPAP (SEQ ID NO: 38), and
        a tetranectin-apolipoprotein A-I that has the amino acid sequence of SEQ ID NO: 02.
    
    


The tetranectin-apolipoprotein A-I fusion polypeptides as described above are precursor polypeptides from which the tetranectin-apolipoprotein A-I fusion polypeptides was released by enzymatic cleavage in vitro using IgA protease.
The precursor polypeptide encoding fusion gene was assembled with known recombinant methods and techniques by connection of appropriate nucleic acid segments. Nucleic acid sequences made by chemical synthesis were verified by DNA sequencing. The expression plasmid for the production of tetranectin-apolipoprotein A-I was prepared as follows.
Making of the E. coli Expression Plasmid
Plasmid 4980 (4980-pBRori-URA3-LACI-SAC) is an expression plasmid for the expression of core-streptavidin in E. coli. It was generated by ligation of the 3142 bp long EcoRI/CelII-vector fragment derived from plasmid 1966 (1966-pBRori-URA3-LACI-T-repeat; reported in EP-B 1 422 237) with a 435 bp long core-streptavidin encoding EcoRI/CelII-fragment.
The core-streptavidin E. coli expression plasmid comprises the following elements:

    
    
        the origin of replication from the vector pBR322 for replication in E. coli (corresponding to by position 2517-3160 according to Sutcliffe, J. G., et al., Quant. Biol. 43 (1979) 77-90),
        the URA3 gene of Saccharomyces cerevisiae coding for orotidine 5′-phosphate decarboxylase (Rose, M., et al., Gene 29 (1984) 113-124) which allows plasmid selection by complementation of E. coli pyrF mutant strains (uracil auxotrophy),
        the core-streptavidin expression cassette comprising
        
            the T5 hybrid promoter (T5-PN25/03/04 hybrid promoter according to Bujard, H., et al., Methods. Enzymol. 155 (1987) 416-433 and Stueber, D., et al., Immunol. Methods IV (1990) 121-152) including a synthetic ribosomal binding site according to Stueber, D., et al., (see before),
            the core-streptavidin gene,
            two bacteriophage-derived transcription terminators, the λ-T0 terminator (Schwarz, E., et al., Nature 272 (1978) 410-414) and the fd-terminator (Beck, E., and Zink, B., Gene 1-3 (1981) 35-58),
        
        
        the lacI repressor gene from E. coli (Farabaugh, P. J., Nature 274 (1978) 765-769).
    
    


The final expression plasmid for the expression of the tetranectin-apolipoprotein A-I precursor polypeptide was prepared by excising the core-streptavidin structural gene from vector 4980 using the singular flanking EcoRI and CelII restriction endonuclease cleavage site and inserting the EcoRII/CelII restriction site flanked nucleic acid encoding the precursor polypeptide into the 3142 bp long EcoRI/CelII-4980 vector fragment.
Example 3
Feeding of Leucine and Proline as Separate Solutions
In this reference example the cultivation of an auxotrophic E. coli strain with a high cell density cultivation method as reported by Riesenberg, et al., (1991, supra) in combination with separate feeding of the amino acids L-Leucine and L-Proline was performed.
The E. coli K12 strain CSPZ-2 (leuB, proC, trpE, th-1, ΔpyrF) was used. The strain was transformed with an expression plasmid for the production of a therapeutic protein and maintained as primary seed bank in ampoules containing 1 ml of the strain grown on defined pre-culture medium to an optical density (determined at 578 nm) of approximately 1 and 1 ml of glycerol 85% (v/v) and stored at −80° C.
The defined pre-culture medium was a M9 medium according to Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) supplemented with

    
    
        1.0 g/l L-Leucine,
        1.0 g/l L-Proline, and
        1.0 mg/l Thiamine-HCl.
    
    


For fermentation a batch medium according to Riesenberg, et al., (1991, supra) was used:

    
    
        27.6 g/l Glucose,
        13.3 g/l KH2PO4,
        4.0 g/l (NH4)2HPO4,
        1.7 g/l Citrate,
        1.2 g/l MgSO4*7H2O,
        60 mg/l Iron(III)citrate,
        2.5 mg/l CoCl2*6H2O,
        15 mg/l MnCl2*4H2O,
        1.5 mg/l CuCl2*2H2O,
        3 mg/l H3BO3,
        2.5 mg/l Na2MoO4*2H2O,
        8 mg/l Zn(CH3COO)2*2H2O,
        8.4 mg/l Titriplex III, and
        1.3 ml/l Synperonic 10% anti foam agent.
    
    


The batch medium was supplemented with

    
    
        5.4 mg/l Thiamin-HCl, and
        1.2 g/l l-Leucine and l-Proline respectively.
    
    


The feed 1 solution contained

    
    
        700 g/l Glucose, and
        19.7 g/l MgSO4*7H2O.
    
    


The feed 2 solution contained

    
    
        20 g/l l-Leucine.
    
    


The feed 3 solution contained

    
    
        100 g/l l-Proline.
    
    


Feed 2 and 3 were prepared by weighting the amino acid, dissolving the amino acids in water, and autoclaving the solution. Afterwards the pH value of the solution has been determined to be about 6.15 for feed 2 and about 6.43 for feed 3.
The alkaline solution employed for pH regulation was an aqueous 12.5% (w/v) NH3 solution.
All components were dissolved in deionized water.
For pre-culture 300 ml M9-medium in a 1000 ml Erlenmeyer-flask with 3 baffles was inoculated with 2 ml of primary seed bank ampoule. The cultivation was performed on a rotary shaker for 13 hours at 37° C. until an optical density (determined at 578 nm) of 1-3 was reached.
The main fermentation was carried out in 10 l Biostat C DCU3 fermenter (Sartorius, Melsungen, Germany). Starting with 6.4 l sterile batch medium plus 300 ml pre-cultivation the batch fermentation was carried out at 37° C., pH 6.9±0.2, 500 mbar and an aeration rate of 10 l/min. After the initially supplemented glucose was depleted the temperature was shifted to 28° C. and the fermentation entered the fed-batch mode with dissolved oxygen (pO2) kept at 50% (DO-stat, see e.g. Shay, L. K., et al., (1987, infra)) and by adding feed 1 in combination with constantly increasing stirrer speed (550 rpm to 1000 rpm within 10 hours and from 1000 rpm to 1400 rpm within 16 hours) and aeration rate (from 10 l/min to 16 l/min in 10 hours and from 16 l/min to 20 l/min in 5 hours). The supply with additional amino acids was started when the pH reached the lower regulation limit, i.e. pH 6.70, with the addition of feed 2 (starting with 33.8 ml/h for 14 hours, and then increased to 97.6 ml/h) and feed 3 (starting with 6.8 ml/h for 14 hours, and then increased to 19.5 ml/h). The flow rates were calculated from a separate fermentation run (see example 4), to respectively apply exactly the same amount of amino acids to the cultivations independently from the feeding strategy. The expression of recombinant therapeutic protein was induced by the addition of 1 mM IPTG at an optical density of 70.
The parameter plots of this fermentation are shown in FIGS. 1 and 2.
After inoculation followed by a short lag-phase, the cells were growing at maximum specific growth rate of μ max=0.30 l/h. After 8 hours of cultivation the lower pH regulation limit was reached and the pH was controlled at 6.70 with the addition of a 12.5% NH4OH solution. Simultaneously the amino acid feeding was started by adding feed 2 and feed 3. After 16 hours the glucose was used up, indicated by the steep increase in pO2-value. At this time the cultivation temperature was shifted from 37° C. to 28° C. After additional 15 minutes the pO2-feed control was started and pO2 was controlled at 50% by the addition of feed 1 while continuously increasing agitation and aeration rate to their respective maxima of 1400 rpm and 20 l/min. The growth rate was continuously decreasing from 0.15 to approximately 0.05 l/h. Concomitantly the stirrer speed was reduced stepwise after 36 hours of cultivation. When no further increase in optical density could be determined the fermentation was terminated and bacteria cells where cooled to 4° C. over night before harvest.
At fermentation end the total biomass yield was 49.4 g/l (dry matter). During the fermentation almost no acetate was excreted but towards the end concentrations increased steeply to 7 g/l. The recombinant protein formation yielded 9.96 g/l. The volume of the culture broth exceeded the normal working volume of the fermenter vessel and increased to 11.8 l.
The fermentation was repeated and resulted in an end optical density of 130, end biomass yield of 50.6 g/l, and recombinant protein yield of 9.0 g/l, culture broth volume at the end of fermentation was 12.2 l.
Example 4
Feeding of Leucine and Proline Incorporated in Alkali Solution for pH Regulation
In this fermentation amino acid feeding was incorporated in the alkaline pH control solution. The basis of this fermentation is the same high cell density cultivation method according to Riesenberg, et al., (1991, supra) as used in Example 3. The amino acids L-Leucine and L-Proline were incorporated in the aqueous 12.5% NH3 solution and fed with alkali addition during pH control.
The E. coli K12 strain CSPZ-2 (leuB, proC, trpE, th-1, ΔpyrF) was used. The strain was transformed with an expression plasmid for the production of a therapeutic protein and maintained as primary seed bank in ampoules containing 1 ml of the strain grown on defined pre-culture medium to an optical density (determined at 578 nm) of approximately 1 and 1 ml of glycerol 85% (v/v) and stored at −80° C.
The defined pre-culture medium was a M9 medium according to Sambrook, J., et al., (Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) supplemented with

    
    
        1.0 g/l L-Leucine,
        1.0 g/l L-proline, and
        1.0 mg/l Thiamine-HCl.
    
    


For fermentation a batch medium according to Riesenberg, et al., (1991, supra) was used:

    
    
        27.6 g/l Glucose,
        13.3 g/l KH2PO4,
        4.0 g/l (NH4)2HPO4,
        1.7 g/l Citrate,
        1.2 g/l MgSO4*7H2O,
        60 mg/l Iron(III)citrate,
        2.5 mg/l CoCl2*6H2O,
        15 mg/l MnCl2*4H2O,
        1.5 mg/l CuCl2*2H2O,
        3 mg/l H3BO3,
        2.5 mg/l Na2MoO4*2H2O,
        8 mg/l Zn(CH3COO)2*2H2O,
        8.4 mg/l Titriplex III, and
        1.3 ml/l Synperonic 10% anti foam agent.
    
    


The batch medium was supplemented with

    
    
        5.4 mg/l Thiamin-HCl, and
        1.2 g/l l-Leucine and l-Proline respectively.
    
    


The feed 1 solution contained

    
    
        700 g/l Glucose, and
        19.7 g/l MgSO4*7H2O.
    
    


The alkaline solution employed for pH regulation was an aqueous 12.5% (w/v) NH3 solution supplemented with 50 g/l L-Leucine and 50 g/l L-Proline respectively.
All components were dissolved in deionized water.
The main fermentation was carried out in 10 l Biostat C DCU3 fermenter (Sartorius, Melsungen, Germany). Starting with 6.4 l sterile batch medium plus 300 ml pre-cultivation the batch fermentation was carried out at 37° C., pH 6.9±0.2, 500 mbar and an aeration rate of 10 l/min. After the initially supplemented glucose was depleted the temperature was shifted to 28° C. and the fermentation entered the fed-batch mode with dissolved oxygen (pO2) kept at 50% (DO-stat, see e.g. Shay, L. K., et al., (Shay, L. K., et al., J. Indus. Microbiol. 2 (1987) 79-85) and by adding feed 1 in combination with constantly increasing stirrer speed (550 rpm to 1000 rpm within 10 hours and from 1000 rpm to 1400 rpm within 16 hours) and aeration rate (from 10 l/min to 16 l/min in 10 hours and from 16 l/min to 20 l/min in 5 hours). The supply with additional amino acids resulted from the addition of alkali, when the pH reached the lower regulation limit of pH 6.70. From the time course of alkali addition the flow rates for feed 2 and 3 within example 2 were calculated, to respectively apply exactly the same amount of amino acids to the cultivations independently from the feeding strategy. The expression of recombinant therapeutic protein was induced by the addition of 1 mM IPTG at an optical density of 70.
The typically parameter plots of this fermentation are shown in FIGS. 3 and 4.
After inoculation followed by a short lag-phase the cells were growing at maximum specific growth rate of μ max=0.30 l/h. After 8 hours of cultivation the lower pH regulation limit was reached and the pH was controlled at pH 6.70 with the addition of aqueous 12.5% NH3 solution supplemented with 50 g/l L-Leucine and 50 g/l L-Proline respectively. This simultaneously starts the amino acid feeding. After 16 hours the provided glucose was used up. At this time the cultivation temperature was shifted from 37° C. to 28° C. After additional 15 minutes the pO2-feed control was started and pO2 was controlled at 50% by the addition of feed 1 while continuously increasing agitation and aeration rate to their respective maxima of 1400 rpm and 20 l/min. The growth rate was continuously decreasing from 0.15 to approximately 0.05 l/h. When no further increase in optical density was recognized, the fermentation was terminated and bacteria cells where cooled to 4° C. over night before harvest.
The optical density at fermentation end was 169 and the total biomass yield was 75.7 g/l (dry matter). During the fermentation almost no acetate was excreted and towards the end concentrations increased to 1 g/l. The recombinant protein formation yielded 16.5 g/l. The volume of the culture broth was 10.2 l.
The fermentation was repeated with different amounts of amino acids dissolved within the alkali solution (33 g/l of L-Leucine and L-Proline). The amount of fed amino acids was lower and resulted in an optical density of 145, end biomass yield of 56.5 g/l, and recombinant protein yield of 13.5 g/l, culture broth volume at the end of fermentation was 9.3 l.

## Claims

1. A method for producing a polypeptide comprising:
a) cultivating an Escherichia coli cell comprising a nucleic acid encoding the polypeptide, the peptide comprising an apolipoprotein A1 having an amino acid sequence selected from SEQ ID NO: 01, 02, 34, and 35,
b) adjusting the pH value during the cultivating with an alkaline solution comprising an amino acid selected from aspartate, aspartic acid, glutamine, glutamic acid, histidine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine,
c) recovering the polypeptide from the cell and thereby producing the polypeptide,
wherein the amino acid has a concentration in the alkaline solution of 30 g/l or more,
wherein the alkaline solution is an ammonia solution of 10% (w/v) or more, and
wherein the dry cell weight of the cultivated bacterial cell is at one point in the cultivating at least 20 g/l.

2. The method of claim 1, wherein the bacterial cell is an amino acid auxotrophic cell and the auxotrophy is for an amino acid selected from aspartate, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan and tyrosine.

3. The method of claim 1, wherein the alkaline solution has a pH value of 9 or more.

4. The method of claim 1, wherein the amino acid has a concentration of about 50 g/l.

5. The method of claim 1, wherein the alkaline solution is an ammonia solution of about 12.5% (w/v) of ammonia in water.

6. The method of claim 1, wherein the amino acid is leucine.

7. The method of claim 1, wherein the alkaline solution comprises leucine and proline.

8. The method of claim 1, wherein the alkaline solution is an ammonia solution of about 12.5% (w/v) and comprises the amino acids leucine and proline each at a concentration of about 50 g/l.