JP2869417B2 - Human serum albumin obtained by genetic manipulation - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a human serum albumin obtained by gene manipulation.

[0002]

BACKGROUND OF THE INVENTION Albumin, particularly human serum albumin (hereinafter also referred to as "HSA") is a major protein component of plasma. This protein is made in the liver and is primarily responsible for maintaining normal osmolality in the bloodstream. It also functions as a carrier for various serum molecules. HSA is administered in various clinical settings. For example, in patients with shock or burns, H is usually used to restore blood volume, thereby improving some of the symptoms associated with trauma.
Requires frequent administration of SA. Patients with hypoproteinemia or fetal erythroblastosis may also require treatment with HSA. Thus, the basic therapeutic significance of administering HSA lies in treating conditions where there is fluid loss from the blood vessels, such as in surgery, shock, burns, and hypoproteinemia causing edema. .

[0003] Currently, HSA is produced primarily as a product from a fraction of the collected blood. Disadvantages of this process are that it is uneconomical and that the supply of blood is difficult. Blood may also contain undesired substances such as hepatitis virus. Therefore, HSA
It would be beneficial to develop alternative feedstocks.

By the way, the advent of recombinant DNA technology has enabled the production of a wide variety of useful polypeptides by microorganisms, and many mammalian polypeptides have already been produced by various microorganisms. About HSA,
The technology of mass production by the technology of genetic manipulation and the highly purified technology is being established.

However, in genetic engineering, when culturing a host microorganism or further purifying HSA, certain coloring components in the raw material or substances secreted by the microorganism are contaminated. It seems that HSA itself is colored by binding to HSA. In addition, these contaminants are conventional plasma-derived HSA
Can not be sufficiently removed by the purification method of
No HSA from genetic engineering without quality has been obtained
Is the current situation.

[0006]

OBJECTS OF THE INVENTION It is therefore an object of the present invention relates to HSA obtained by gene manipulation, produce
An object of the present invention is to provide HSA which is substantially free of proteins and polysaccharides other than host-derived HSA and whose coloring is sufficiently suppressed.

[0007]

Means for Solving the Problems The present inventors have made intensive studies in view of the above circumstances, and as a result, when obtaining HSA obtained by genetic manipulation, particularly preferably at the end of the purification step of the HSA, coloring material by treating the HSA with a chelating resin is adsorbed by the chelate resin, it is possible to reduce the coloration of HSA, the production host
It found to be able to provide a high purity HS A the protein and polysaccharide other than HSA derived is sufficiently removed, thereby completing the present invention.

[0008] That is, the present invention provides a method obtained by genetic manipulation.
From HSA and producing host; HS A having the features of hereinafter that
Depending on the content ratio with proteins and polysaccharides other than HSA
The purity of SA is 99.9999999% or more.

[0009] HS A having the above characteristics, the HSA obtained by gene manipulation, in the purification step,
Particularly preferably, it can be obtained by treating at the end with a chelate resin, preferably a chelate resin having, as a ligand, an exchange group having a chelating ability selected from a polyol group, a polyamine group or a thiourea group.

[0010] The present invention relates to a method for obtaining H by genetic engineering.
The HSA is related to SA, and the HSA is obtained by culturing cells (eg, Escherichia coli, yeast, Bacillus subtilis, koji, etc.), animal cells, etc., which express HSA through genetic manipulation, and expressing the cells extracellularly (secretion expression) Produced by

(1) HSA obtained by genetic manipulation The HSA-producing host prepared by genetic manipulation used in the present invention is not particularly limited as long as it is prepared through genetic manipulation. In addition to the above, even those that will be developed in the future can be used as appropriate. Specific examples include bacteria (eg, Escherichia coli, yeast, Bacillus subtilis, etc.) and animal cells that have been made HSA-producing through genetic manipulation. In particular, in the present invention, as a host, yeast, especially Saccharomyces (eg, Saccharomyces cerevisiae)
Or the genus Pichia (eg, Pichia pastoris) is preferably used. In addition, auxotrophic strains and antibiotic-sensitive strains can be used. Furthermore, Saccharomyces cerevisiae AH22 strain (a, his 4, leu 2, can 1), Pichia pastoris GT
S115 strain (his 4) and the like are preferably used.

A method for preparing these HSA-producing hosts, a method for producing HSA by culturing them, and a method for preparing HS from cultures
All the methods for separating and collecting A are carried out by employing a known method or an equivalent method. For example, HSA
Examples of the method for preparing a production host (or HSA-producing strain) include, for example, a method using a normal human serum albumin gene (JP-A-58-56684, 58-90515, and 58-150517) and a novel human serum. A method using an albumin gene (JP-A-62-29985 and JP-A-1-98486), a method using a synthetic signal sequence (JP-A-1-240191), and a method using a serum albumin signal sequence (JP-A-2-294). 167095
Japanese Patent Application Laid-Open No. 3-72889), a method of fusing hosts with each other (Japanese Patent Application Laid-Open No. 3-53877), a method of causing mutation in a methanol-containing medium, a method using a mutant AOX ₂ promoter (Japanese Patent Application No. 3-63598, the 3-
No. 63599), HSA expression by Bacillus subtilis
No. 2-25133), expression of HSA by yeast (JP-A-60-41487, JP-A-63-39576 and JP-A-63-57676).
74493) and the expression of HSA by Pichia yeast (JP-A-2-104290).

Among them, a method for causing a mutation in a methanol-containing medium is specifically performed as follows. That is, first, an appropriate host, preferably Pichia yeast, specifically, the GTS115 strain (NRRL accession number Y-15851)
Obtain a transformant HSA by a conventional method to AOX ₁ gene region under AOX ₁ promoter governing of introducing a plasmid having a transcription unit to express (JP 2-104
290). This transformant has a low ability to grow in a methanol medium. Therefore, this transformant is cultured in a medium containing methanol to cause mutation, and only a viable strain is recovered. At this time, the methanol concentration is, for example, about 0.0001 to 5%. The medium may be an artificial medium or a natural medium. Culture conditions are 1
5 to 40 ° C. and about 1 to 1000 hours are exemplified.

As a method for culturing an HSA-producing host (that is, a method for producing HSA), in addition to the methods described in each of the above publications, fed batch culture is used to supply a high concentration of glucose in an appropriate small amount. A method of obtaining a high concentration of cells and a product by avoiding high-concentration substrate inhibition of the producing cells (Japanese Patent Application No. 1-219561), a method of enhancing the production of HSA by adding a fatty acid to the medium ( Japanese Patent Application 3-81
719) and the like. Further, as a method for separating and collecting HSA, in addition to the methods described in the above publications, inactivation of protease by heat treatment (Japanese Unexamined Patent Publication (Kokai) No. 3-1031)
No. 88), HSA using at least one selected from the group consisting of an anion exchanger, a hydrophobic carrier and activated carbon
And a coloring component by separating the coloring component from the coloring component (JP-A-4-54198).

The medium used for culturing the transformed host is as follows:
Usually, a culture medium known in this field to which a fatty acid having 10 to 26 carbon atoms or a salt thereof has been added is used, and the culture is carried out in accordance with a general ordinary method. The medium may be a synthetic medium or a natural medium, and a liquid medium is preferred. For example,
As a synthetic medium, generally, various sugars as a carbon source, urea, ammonium salt, nitrate, and the like as a nitrogen source, various vitamins and nucleotides as a micronutrient, and Mg, Ca, Fe, Na, K, Mn, Co as inorganic salts. , Cu
And the like. YNB liquid medium [base of 0.7% yeast nitrogen (manufactured by Difco), 2% glucose] and the like. As a natural medium, YPD
A liquid medium [1% yeast extract (manufactured by Difco), 2% bactopeptone (manufactured by Difco), 2% glucose] is exemplified. The pH of the medium may be neutral or weakly basic or weakly acidic. In the case of a methanol-assimilating host, a medium containing methanol can be used. In this case, the methanol concentration is about 0.01 to 5%.

The culture temperature is 15 to 43 ° C. (for yeast, 20 to 43 ° C.)
30 ° C, and 20-37 ° C for bacteria). The culture time is about 1 to 1000 hours, and the culture is allowed to stand or shake,
It is carried out by batch culture, semi-batch culture or continuous culture under stirring and aeration. Preferably, pre-culture is performed prior to the culture. As the medium at this time, for example, a YNB liquid medium or a YPD liquid medium is used. The culture conditions for the preculture are as follows. That is, the culturing time is preferably 10 to 100 hours, and the temperature is preferably about 30 ° C. for yeast and about 37 ° C. for bacteria.

After completion of the culture, HSA is collected from the culture filtrate, the cells, and the cells by known separation means.

(2) Purification of HSA As the purification step of HSA of the present invention, known methods such as various fractionation methods, adsorption chromatography, affinity chromatography, gel filtration, density gradient centrifugation, and dialysis are employed. You. As the purification step, for example, a step including the following to is preferably mentioned. The culture supernatant of the human serum albumin producing host is treated using an ultrafiltration membrane having a molecular weight cut-off of 100,000 to 500,000 and 1,000 to 50,000. The heat treatment is performed at 50 to 70 ° C. for 30 minutes to 5 hours. Acid treatment at pH 3-5. The treatment is performed using an ultrafiltration membrane having a molecular weight cutoff of 100,000 to 500,000. After contacting with a cation exchanger under conditions of pH 3 to 5 and salt concentration of 0.01 to 0.2 M, elution is performed under conditions of pH 8 to 10 and salt concentration of 0.2 to 0.5 M. The non-adsorbed fraction is collected by contacting with a carrier for hydrophobic chromatography under conditions of pH 6 to 8 and a salt concentration of 0.01 to 0.5M. The non-adsorbed fraction is recovered by contacting with an anion exchanger under conditions. Also, instead of the above steps, pH 6-8, salt concentration 1 ~
After contacting with a carrier for hydrophobic chromatography under 3M conditions, elution is performed under the conditions of pH 6 to 8 and a salt concentration of 0.01 to 0.5M.
8. After contacting with an anion exchanger under the condition of salt concentration 0.001 to 0.05M, pH 6 to 8, salt concentration 0.05
Eluting under a condition of １1 M, and before, during or after the above step, salting out under a condition of pH 3 to 5 and a salt concentration of 0.5 to 3 M; May be further included.

(3) Decolorization of HSA The decolorization step of HSA of the present invention comprises the following steps:
It is particularly preferably carried out by contacting HSA with a chelating resin which is incorporated at the end and has a specific ligand moiety. The carrier portion of the chelate resin is preferably a hydrophobic carrier, and examples thereof include a copolymer of styrene and divinylbenzene, and a copolymer of acrylic acid and methacrylic acid. On the other hand, the ligand part is a polyamine group having a plurality of polyol groups such as N-methylglucamine group, imino group, amino group, ethyleneimino group and the like in the molecule (including polyalkylene polyamine groups such as polyethylene polyamine). And thiourea groups. Commercially available chelate resins having the above-mentioned carrier portion and ligand portion include DIAION CRB02 (ligand portion; N-methylglucamine group, manufactured by Mitsubishi Kasei) whose carrier portion is a copolymer of styrene and divinylbenzene, and DIAION.
CR20 (ligand _{moiety; -NH (CH 2 CH 2 NH} ) n H, manufactured by Mitsubishi Kasei), LEWATIT TP (ligand portion; -NHCSN
H _2, manufactured by Bayer), used Amberlite CG4000 suitably.

The conditions for the treatment with the chelate resin are preferably as follows. pH conditions: acidic or neutral (3-9, preferably 4-
7) Time: at least 1 hour or more, preferably 6 hours or more Ionic strength: 50 mmho or less, preferably 1 to 10 m
mho Mixing ratio: 0.1 to 100 resin for 250 mg of HSA
g, preferably 1 to 10 g (wet weight)

The degree of coloration of HSA that can be obtained through the above steps (including and salting-out treatment, and further including chelating resin treatment) is _{500 nm} / A in the case of a 25% HSA solution.
_{280 nm} is about _0.001 to 0.005 . HSA coloring degree is 1/2 to 1/10 by chelating resin treatment
To be reduced. In particular, the absorption wavelength near 500 nm, that is, the degree of red coloring is reduced to 1/3 to 1/10.

[0022] (4) HS A formulation present invention, known methods (ultrafiltration, addition of stabilizers, sterile filtration, dispensing, lyophilization and the like) can be formulated by. The HSA preparation thus prepared can be used clinically as an injection as in the case of the plasma-derived HSA preparation. Furthermore, stabilizers or carriers pharmaceutical, Ru also available Der as carrier.

[0023] (5) HSA of properties present invention HS A from gene manipulation of the present invention has a molecular weight of about 67,000, an isoelectric point 4.6 to
It is a single substance of 5.0. The HSA is composed of a monomer and is substantially free of a dimer, a polymer, or a decomposition product.
Specifically, the total content of the dimer, polymer, and decomposition product is about 0.01% or less. The HSA of the present invention does not contain contaminating components derived from the production host (the proteins other than H SA and multi <br/> ToNaru min) substantially of HSA calculated from the content ratio of the該夾miscellaneous ingredients Purity is 99.9999999% or more,
It is preferably at least 99.999999%. Specifically, in the case of a 25% HSA solution, protein components other than HSA are 1 ng / ml or less, preferably 0.1 ng / ml.
The following are exemplified. Also, the polysaccharide component is 10 ng /
ml or less, preferably 1 ng / ml or less.
In addition, the purity of HSA referred to in the present invention means the purity of HSA calculated from the content ratio with contaminant components (proteins other than HSA and polysaccharide components) derived from the production host. The degree of coloring was 0.050 for A ₃₅₀ / A _{280 in} the case of a 25% HSA solution.
1 to 0.05, A ₄₅₀ / A ₂₈₀ is 0.001 to 0.
02, A ₅₀₀ / A ₂₈₀ is approximately 0.001 to 0.005. In addition, the amount of the fatty acid bound to HSA is 1 molecule or less, preferably 0.1 molecule or less per HSA molecule.

[0024]

Industrial Applicability The present invention is characterized in that it is substantially free of contaminant components derived from a production host, and is characterized by being derived from genetic engineering.
Is S A, According to the present invention, even though a material Sanseiyado main secreted is not contaminated because, they are mixed coloring and other contaminants caused by binding is sufficiently suppressed to HSA it is possible to provide a high purity HS a.

[0025]

The present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Reference Example 1 Cultivation of HSA-producing host (1) Strain used: Pichia pastoris GCP101 strain Pichia pastoris GTS11 according to the method described in JP-A-2-104290.
By replacing the AOX1 gene region of No. 5 (his4) with a NotI fragment of plasmid pPGP1 having a transcription unit for expressing HSA under the control of the AOX1 promoter, PC4130 was obtained. This strain is AOX1
Due to the absence of the gene, the ability to grow on a medium using methanol as a carbon source is low (Mut- strain).

PC4130 was inoculated into 3 ml of YPD medium (1% yeast extract, 2% bactopeptone, 2% glucose), and 24 hours later, the initial OD _{540 was} 0.1.
Was inoculated into 50 ml of YPD medium. 3 days 30
YPD so that the initial OD ₅₄₀ = 0.1 after culturing at
The cells were inoculated into 50 ml of the medium. The same passage was repeated every three days. The cells were diluted with sterile water to 10 ⁷ cells / plate for each passage, and were diluted with 2% MeOH-YNBw / oa.
a. Plate (0.7% yeast nitrogen base with out amino acid, 2% methanol, 1.5%
% Agar powder, and cultured at 30 ° C. for 5 days to determine the presence or absence of colonies. As a result, 2
% MeOH-YNBw / oa. a. The plate gave rise to 20 colonies. Mut- strains can hardly grow on this plate, whereas Mut + strains can. That is, the formation of colonies in this plate indicates that the assimilation of methanol was increased and a strain converted to Mut + was obtained. One of the resulting colonies was appropriately diluted with sterile water to give 2% MeOH-YNBw / o.
a. a. It was spread on a plate and isolated into a single colony.
One of them was named GCP101.

(2) Culture of the strain (pre-culture) 1 ml of the frozen glycerol stock strain was added to 20 ml.
1,000 ml with baffle containing 0 ml YPD medium (Table 1)
The cells were inoculated into a ml Erlenmeyer flask and cultured with shaking at 30 ° C. for 24 hours.

[0029]

[Table 1]

(Pre-cultivation) A 10 L jar fermenter containing 5 L of YPD medium was inoculated with the pre-pre-culture liquid and cultured with aeration and stirring for 24 hours. The culture temperature was 30 ° C and the aeration rate was 5 L /
Minutes. In the pre-culture, the pH was not controlled.

(Main culture) Medium for batch culture (Table 2) 25
0 L of the preculture was inoculated and cultured with aeration and agitation using a 1,200 L fermenter. The tank pressure is 0.5kg / c
Batch culture was started while controlling the stirring speed so that the dissolved oxygen concentration was maintained at about 50% to 30% of the saturated dissolved oxygen concentration with the maximum aeration rate of 800 N-L / min and m ² .
At the time when glycerol in the medium was consumed in the batch culture, addition of the feed medium (Table 3) was started. A computer was used to add this feed medium, and high-density culture was performed while controlling so that methanol did not accumulate in the medium. The pH was controlled at a constant value of 5.85 by adding 28% aqueous ammonia. The defoaming is performed by adding an antifoaming agent (Adecanol, manufactured by Asahi Denka Kogyo) to 0.3% at the start of batch culture.
This was carried out by adding 0 ml / L and then adding a small amount as needed.

[0032]

[Table 2]

[0033]

[Table 3]

Reference Example 2 AOX2 promoter isolated from GCP101 strain of Reference Example 1 [mutant type. Native AOX2 promoter (TEAST,
5, 167-177 (1988) or Mol.Cell, Biol., 9, 1316-1.
323 (1989)), in which the 255th base upstream of the initiation codon was mutated from T to C], to construct a plasmid pMM042 for HSA expression, and to obtain Pichia pastoris (Pichia pastoris).
pastoris) The transformant UHG was introduced into the GTS115 strain.
42-3 strains were obtained (Japanese Patent Application No. 3-63599). This UHG42-3 strain was cultured according to Reference Example 1 to produce HSA.

Reference Example 3 Purification of HSA [i] Separation of culture supernatant to membrane fractionation (II) The culture supernatant was separated by pressing about 800 L of the culture solution obtained in Reference Example 1. Culture supernatant was fractionated with molecular weight cutoff of 30.
Treated with 10,000 ultrafiltration membranes. Subsequently, the liquid volume was concentrated to about 80 L using an ultrafiltration membrane having a molecular weight cut-off of 30,000 [membrane fractionation (I)]. After heat treatment at 60 ° C. for 3 hours, the concentrated solution was rapidly cooled to about 15 ° C., adjusted to pH 4.5, and again treated with an ultrafiltration membrane having a molecular weight cut off of 300,000 [membrane fraction]. (II)]. Next, the buffer in the albumin solution was adjusted to 50 mM using an ultrafiltration membrane having a molecular weight cut off of 30,000.
50 mM acetate buffer containing sodium chloride, pH 4.5
Was replaced.

[Ii] Cation exchanger treatment 50 mM acetate buffer containing 50 mM sodium chloride, p
After adsorbing albumin to the S-Sepharose packed column equilibrated with H4.5 and washing well with the same buffer,
0.1 M phosphate buffer containing 0.3 M sodium chloride,
Albumin was eluted at pH9.

[Iii] Hydrophobic chromatographic treatment The albumin solution eluted from the S-Sepharose packed column was applied to a column packed with phenylcellulofine equilibrated with 50 mM phosphate buffer containing 0.15 M sodium chloride, pH 6.8. Was added. Under these conditions, the fraction that passed through the column was collected without adsorbing phenylcellulofine. The volume of albumin that passed through the column was concentrated to about 50 L using an ultrafiltration membrane having a molecular weight cut off of 30,000, and the buffer in the albumin solution was replaced with a 50 mM phosphate buffer, pH 6.8.

[Iv] Anion exchanger treatment After the hydrophobic chromatography treatment, the concentrated and buffer-exchanged albumin solution was added to a column packed with DEAE-Sepharose equilibrated with 50 mM phosphate buffer, pH 6.8. . Under these conditions, albumin passed through the column without adsorption to DEAE-Sepharose.

[V] Salting-out treatment of HSA A solution having a final concentration of 1 M by adding sodium chloride to 5% HSA was adjusted to pH 3.5 with acetic acid,
Was precipitated. This precipitate was separated from the supernatant by centrifugation to remove impurities.

[Vi] Decolorization treatment DIAION CRB was added to 1 ml of purified HSA having a concentration of 25%.
02 (a carrier portion is a styrene-divinylbenzene copolymer, a ligand portion is a chelate resin comprising an N-methylglucamine group, manufactured by Mitsubishi Kasei Co., Ltd.), and the mixture is added at pH 6.8 and an ionic strength of 5 mmho for 24 hours at room temperature. Stirred. The resin was washed with distilled water, the non-adsorbed fraction was collected as a HS A.

[0041] The HSA completing the purification steps up to HPLC analysis hydrophobic chromatography treatment of property (1) purification step of purifying HS A in Example 1 the invention was analyzed by HPLC gel filtration. Gel filtration analysis was performed under the following conditions. Column: TSK gel G3000SW (manufactured by Tosoh Corporation) Developing solution: 0.1 M KH ₂ PO ₄ /0.3 M NaC
l Buffer detection: Absorbance at 280 nm Purified HSA was a single peak of HSA monomer.

(2) Analysis of Yeast-Derived Components The culture supernatant of albumin non-producing yeast was roughly purified in the same manner as in the present method, and a rabbit was immunized, and the yeast present in purified HSA was obtained using the obtained antiserum. Attempt to detect derived components
Was . The measurement was performed by an enzyme immunoassay (EIA method). The results of detection of yeast-derived components for each sample are shown. The sample was measured using an albumin concentration adjusted to 250 mg / ml.

[0043]

[Table 4]

(3) Molecular Weight The molecular weight was measured by the aforementioned HPLC gel filtration method. The molecular weight of the purified HS A of the present invention is about 67000, was similar to plasma-derived HSA.

(4) Isoelectric point The isoelectric point was measured using a thin-layer polyacrylamide gel.
n et al. [J. Chromatog. , 146,1
(1978)]. Purified HS A of the present invention
Had an isoelectric point of about 4.9, comparable to that of plasma-derived HSA.

(5) Degree of coloring Degree of coloring is 280 nm, 350 nm, 450 nm, 500
The absorbance at nm was measured, and A ₃₅₀ / A ₂₈₀ , A
₄₅₀ / _A280 and _A500 / _A280 were calculated. The coloring degree of the purified HS A of the present invention is about 0.02 for A ₃₅₀ / A ₂₈₀ , about 0.01 for A ₄₅₀ / A ₂₈₀ , and about A _500.
/ A ₂₈₀ was about 0.002, comparable to plasma-derived HSA.

(6) Bound Fatty Acid Content NFEA-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) was used for measurement. As a result, it was 1.6 mol (per mol of HSA) before the chelate resin treatment, but 0.03 mol after the treatment.
It was greatly reduced to 7 moles (per mole of HSA).

Example 2 The culture solution obtained in Reference Example 2 was treated in the same manner as in Reference Example 3. The properties of the purified HSA were comparable to those of the molecular weight, isoelectric point and coloring degree, polysaccharide content, gel filtration pattern and yeast-derived component disclosed in Example 1.

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:84) (C12P 21/02 C12R 1: 865) (72) Inventor Munehiro Noda 2-25, Shodai Otani, Hirakata City, Osaka Prefecture No. 1 Inside the Green Cross Research Institute Co., Ltd. (72) Takao Omura Inventor 2-25-1, Invitation Otani, Hirakata-shi, Osaka Prefecture Inside the Midori Cross Research Institute Co., Ltd. No. 25-1, Examiner at Midori Cross Research Institute, Inc. Takeshi Ukai (56) References Special Table Hei 6-501033 (JP, A)

Claims (3)

(57) [Claims] 1. A human serum albumin having the following characteristics obtained by genetic manipulation; human serum albumin based on the content ratio of human serum albumin to a protein and a polysaccharide having antigenicity other than human serum albumin derived from a production host. The purity is not less than 99.999% and not more than 99.99999999% , and contamination of human serum albumin with proteins and polysaccharides other than human serum albumin derived from the production host is determined by enzyme immunoassay (EI).
Below the detection limit of Method A). 2. The purity of human serum albumin is 99.99.
The human serum albumin obtained by the genetic manipulation according to claim 1, which is at least 999%. 3. The purity of human serum albumin is 99.99.
The human serum albumin obtained by the genetic manipulation according to claim 1, which is 9999% or more.