JPS5982095A - Preparation of human gamma-interferon - Google Patents

Abstract

PURPOSE:To produce gamma-interferon from human leukocyte or human leukocyte- relating cell, economically, in high yield and purity, by using a serumfree medium and using plural interferon-inducing substances in combination. CONSTITUTION:Human gamma-interferon is prepared from human leukocyte or human T-cell, by using a medium containing restricted amount of human serum albumin as the only protein component, and using Staphylococcus enterotoxin B in combination with OK432 (picibanil) to culture and induce human gamma-interferon having high titer, which is separated and purified by affinity column chromatography or chromotofocussing method.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing γ-type interferon, a non-specific virus inhibitor, from human leukocytes or human leukocyte-derived cells at low cost, in good yield, and with high purity. More specifically, the present invention is directed to the production of human r-type interferon from human leukocytes or human T cells by using a non-depleted medium as a medium and using multiple interferon-inducing substances in combination, thereby producing high units of γ-type interferon. The purpose of the present invention is to provide a method for easily producing r-type interferon on an industrial scale with high cylindrical yield and high purity by inducing culture and separating and purifying the target product from the obtained crude r-type interferon liquid.

Currently, six types of interferon are known: α-type, β-type, and γ-type, each of which differs in molecular weight, isoelectric point, and other physicochemical properties.

All have the common property of non-specifically suppressing the intracellular proliferation of viruses every second. γ-type interferon is a substance different from α-type and (2) β-type interferon, which are induced by nucleic acids or viruses, and are distinguished from each other by specific neutralizing antibodies, stability at acidic pH, heat resistance, SDS resistance, etc. Ru. γ-type interferon, also called immune interferon, is a substance with non-specific virus-suppressing activity that is mainly produced by thymus-derived lymphocytes. bacteria or toxins, calcium ionophores.

A general term for protein-like substances induced by anti-lymphocyte antibodies, etc. γ-type interferon has stronger cell-suppressive and immunomodulatory activities than α-type and β-type interferon, and in cancer disease models using animal models, it suppresses cancer growth more strongly than α-type and β-type interferon. A synergistic anticancer effect can be seen when used in combination with R-type and R-type. Although γ-type interferon is a highly active substance in extremely small amounts, it has not yet been used as a medical treatment for various diseases.

The biggest reason for this is that γ-type interferon is more species-specific than α- and β-types (3) and is less stable, making it difficult to obtain extremely small amounts. In other words, interferons intended for use in the treatment of various viral diseases and cancer in humans are not effective unless they are induced in human cells. #l IJil that can induce human γ-type interferon production! The end is a human white catch ball and a human T lymphocyte.

Human T lymphoblasts and human tile 1i = W & JU cells are known, but among these, γ cells can be efficiently grown on an industrial scale.
At present, the only known cell that can produce interferon-type interferon is the white one derived from human peripheral blood.

rBρ interferon is considered a glycoprotein.

It is currently impossible to produce glycoproteins identical to natural ones using recombinant DNA methods using microorganisms.

The method for producing crude yr-type interferon liquid from human Shiratsukuda bulbs is as follows: ) (, A. Johnson (IRC3Me
dicalScience 7 559 (1979)
), Marcdeley et al. (Eur.

J, Lmnaunol, 10871 883 (19
) and Y, YIP et al. (Proc, N, A
, S, 78Ld 1601-161J5 (1981)
) etc. have been reported. These methods involve purifying mononuclear cells, including lymphocytes and macrophages, using human peripheral blood buffy coats (4) or by specific gravity centrifugation using Ficoll, and adding 5 to 10 cells of human plasma or serum. 1 in RPML1640 medium etc.
06-107 cells/m7! each research group's own gamma interferon inducer (e.g., hemagglutinin, staphylococcal enterotoxin A or B).

After adding anti-lymphocyte antibodies, etc.) and culturing at 37 C for 1 to 4 days, the culture solution was centrifuged to obtain a crude γ-interferon solution. This crude solution contains foreign proteins added as inducers, toxins, and many serum proteins, so it must be sufficiently purified before it can be administered to the human body.

The conventional method for extracting γ-type interferon from a citrate-type interferon solution is to adsorb FR-type interferon on control topoglass or silica gel, elute with a phosphate buffer containing ethylene glycolone, and perform affinity extraction with concanavalin A and Sepharose.
This is a method of obtaining a partially purified sample of about 10 to 10 units/~ protein culm by chromatography and gel permeation fractionation.
Currently, there is no established assay method or international standard product for interferon, and although it varies from researcher to researcher, there are many cases where the assay method for α-type interferon, for which there is an international standard, is applied mutatis mutandis for convenience. The amount of γ-type interferon in the crude r-type interferon solution obtained as a culture solution of the IJ cell line is very small, and it can be said that the majority of the protein is derived from the serum added to the medium.
It is extremely difficult to collect only type interferon from the culture filtrate.

Compared to human α-type interferon, γ-type interferon is inferior in stability in acidic regions, thermal stability, and freeze-thaw resistance, making it even more difficult to purify γ-type interferon, and it takes a great deal of effort to purify it to the extent that it can be administered to the human body. It takes 7. To overcome these difficulties, γ at the end of lymphocyte culture
To obtain a crude gamma interferon solution that does not contain any impurities other than r-type interferon (6) using an induction culture method that brings the titer of type interferon to 100%, and to purify it easily and with high yield. things are desired.

In order to solve these problems, the present invention does not use the 5-15% human or animal serum normally added to lymphocyte culture, but instead uses a limited amount of human serum albumin as shown in Table 1. By using a serum-free medium containing Miwo protein as a component, we succeeded in inducing γ-type interferon at a level equal to or greater than that of a medium supplemented with serum serum. Furthermore, γ
Among the various intubators of type interferon (Staphylococcus enterotoxin B and 0K462
When used in combination with the drug, a high titer of γ-type interferon, which could not be obtained by using each drug, was induced, and a remarkable synergistic effect was observed due to the combined use.

Furthermore, a higher titer of γ-interferon activity can be obtained by adding an appropriate amount of transferrin (diderophilin), which is one of the blood protein components, to the III-free supernatant medium (see Table 3). still.

Transferrin alone does not induce γ-type interferon. In other words, in this case, transfer(7) phosphorus acts auxiliary to the induction of γ-type interferon.
Think about it.

Although there are individual differences in the ability to induce γ-type interferon from the provided lymphocytes, the synergistic effect described above is particularly noticeable in low-titer producers, and it should be used effectively with human leukocytes.

The inventors succeeded in obtaining a high-potency crude interferon solution Z with few impurities by combining the use of a bloodless medium and the initial interferon inducer, and completed the present invention. According to the method of the present invention, the purification process is significantly simplified, and the purity can be easily and quickly reduced to 100/mg or more (converted to international units of α-type interferon).
It is possible to obtain R-type interferon with a yield of 80% or more.

Furthermore, the present inventor performed affinity chromatography of two S species, that is, using a carrier bound to Cibacron Blue and a carrier bound to concanavalin A'(!1').
The crude γ-interferon solution obtained by induction using the above serum-free medium has a strong effect and high yield.

We discovered that it is possible to fractionate and purify Renkyu.

(8) As shown in the flow sheet of purified fractionation in FIG. 1, interferon is divided into four fractions, which are referred to as 'iA, B, C and D fractions, respectively. The total recovery rate for all activities is 8.
It is 0% or more. Among these, the recovery rate of total activity in the Cibacron Blue column was more than 8 degrees, and the recovery rate in the Concanavalin A Sepharose column was approximately 100 degrees.

Details of the purification fractionation method #1 that can be used in the present invention will be described below (see FIGS. 3 and 4).

Crude γ-type interferon was added to Matrix Blue A agarose (Amicon) or Blue Sepharose 0L.
-6B (Pharmacia), the interferon activity is fractionated into a non-adsorbed fraction and an adsorbed fraction, and both fractions exhibit γ-type content.1 Total recovery rate of the combined activity of both. is 80% or more. On average, the yield of the non-adsorbed fraction (MBi) relative to the activity of the crude extract is about 16%, and the yield of the adsorbed fraction (MBi) is about 16%.
B II ) is approximately 64 chi. Adsorption fraction (MBII)
Bathed out, and further added Concanavalin A and Sepharose 4B (
Pharmacia Co., Ltd.'s Affinity Column (9) When performing chromatography, interferon activity is divided into a non-adsorbed fraction and an adsorbed fraction. Both parts? The recovery ratio of combined activity to M B 11 activity is approximately 100 range. The activity of the concanavalin A/Sepharose 4B non-adsorbed fraction (fraction C) is about 2.3 times the activity of the crude extract. On the other hand, the r-type interferon activity of the filtrate fraction obtained by ignition elution from the adsorption part was approximately 61.7% of the crude extract activity.
%. After elution, column 7 was further eluted with different eluents, but interferon activity was not eluted, and it was concluded that β-type interferon was not contained. Also.

Human serum albumin added to the medium has been completely removed at this stage.

The γ-type interferon contained in the non-adsorbed fraction (MB) of the Blue Sepharose or Matrix Blue A column is also fractionated into a non-adsorbed part and an adsorbed part by the Concanavalin A-Sepharose column, and the non-adsorbed fraction (fraction A)
The activity is about 1 6% of the crude extract activity. The adsorbed fraction B is eluted, and the proportion (10) of fraction B activity in the crude extract is about 16, and is neutralized by the γ-interferon-specific antibody.

Specific eluents that can be used in the chromatography for fractionation and purification of the present invention are described in the following examples, but can be changed as appropriate within the scope of achieving the objectives of the present invention.

γ-type interferon ultrogen) LlACA54 (
Gel permeation chromatography of the MBI fraction and the MB [fraction] by LKB Co., Ltd. revealed that each molecular weight was estimated to be approximately 43,000 daltons. The specific activity of γ-type interferon in main fraction D (MBI-ConAll) is 28 [llnm1 unit absorbance is 0 in protein aqueous solution.
．． Assuming 7 mq/ml, it was more than 106V9 protein. The specific activity of the extract before purification is approximately 3X10 U/
~It was protein.

When the purification process of the present invention was applied to cases cultured in a serum-supplemented medium, no increase in specific activity was easily observed, and on the contrary, there were many cases in which the specific activity decreased. From this.

While it can be said that the purification process described here is specifically effective (11) for the production of crude gamma interferon solutions obtained by using the serum-free medium of the present invention, it lacks affinity for serum albumin. Using Remazol Blue Sepharose 4) and the above crude interferon # solution, the specific activity level was 10” U/m in one step.
y Highly purified γ-type interferon on protein 9 can be purified in one step (see Figure 5). This operation can be performed by either a column method or a batch method.

Also suitable for industrial scale operations.

Another purification method is fractionation using a chromatofocusing column, which is a fractionation method based on a 1-$ point difference, which allows at least Aa γ-interferon to be fractionated in one step from a crude interferon solution (Figure 6 reference).

The two fractionation methods described below may be operated alone or in combination with the method described in Mi+. Methods of the Invention 79 are explained in more detail by the Examples below.

Example 1: γ-type interferon was produced using a blood-free swab in combination with a fat transfer agent. (2) Mix an equal amount of Hank's balanced salt solution with a fresh human individual's buffy coat. Then, it was layered with Ficoll-Punque solution (Pharmacia) in an amount equal to that of the buffy coat. 19
Centrifuge at 2,000 rpm for 20 minutes at
I collected ain. Lymphocytes washed twice with PBS(-) and once with medium were suspended in medium at 5×10/d, and Staphylococcal enterotoxin B and 0K432 (bisibanyl) were each added to a final concentration of 50 n'! '/r
rtl, 0.025KE/m? It was added as a Turferon inducer. The composition of the serum-free medium is shown in Table 1. Cultured in the presence of 67C 5% CO□ for 42 hours, and culture supernatant '21.5 Q Or, p, rn.

Obtained by centrifugation for 5 minutes. The interferon titer in the supernatant is expressed as the number of sample dilutions to inhibit Sindbis virus cytotoxicity of human FL cells' PI: 50%. The titer of the crude interferon solution obtained as above was up to 1.6 × i 0 ' U/tnl.
Y Koshi F'j.

The sensitivity of this assay ranges from 7 to 24 1 international units of alpha interferon. Since there is no international standard or common assay system for r-type interferon (13), α-type interferon was used as the standard in accordance with conventional practice. F
The combination of L cells and Sindbis virus is )], A, Jo
Staphylococcus enterotoxin B and OK4 on serum-free medium showed approximately 10 times more sensitivity than the combination of i cells and 77 Dobis virus.
The concentration dependence of interferon titer on interferon titer 2 is shown in Table 2 and FIG.

As seen in Table 2 and FIG. 2, the two inducers used act synergistically on the induction of interferon. The synergistic effect was particularly evident for samples with low interferon-inducing ability. In samples with high induction ability, even a single inducer showed sufficiently high interferon titers. Even in comparison of the same individual, cases in which 0K432 and Staphylococcus enterotokin were used in combination showed a higher sieve titer of interferon than cases in which they were used alone. Induced interferon activity When treated with YpH 2.0 for 17 hours, the residual oiliness of interferon almost disappeared, and γ
It was confirmed that the induced interferon was of the γ type, as it was almost completely neutralized with a type interferon-specific neutralizing antibody (14).

RPMI-1640 (manufactured by Hinaga Pharmaceutical Co., Ltd.)
96ml essential amino acid mixture (x50)
2ml non-essential amino acid mixture ('X100) 1
mi human serum albumin: 650m9H
EPES 10nM 7% Sodium Bicarbonate 2ml.

Table 2. Staphylococcal enterotoxin B (sE
B) and r-type interferon by 0K432 (1
5) Furthermore, interferon activity was increased by adding transferrin to the above serum-free medium. Using the above serum-free medium supplemented with an appropriate amount of transferrin, #End 1! [Staphylococcal enterotoxin B (SIEB)'&'50nVrrt1.0K432
v0.025KE/ml"Q, r-type interferon was induced according to the procedure described above (7". The results are shown in Table 3. As is clear from Table 3, transfer7"; s: 1
5 pFj per ml, 10. When editing ttP,
The activity was about 4 times higher than when no i was added.

In addition, transferrin 'Y10μ'/-/ml addition 1~
When human lymphocytes were cultured in IfIlff-free medium under the same conditions as described above without using the above two inducers, no interferon activity was observed.

Furthermore, the addition of transferrin did not affect the separation and purification of human Φγ-type interferon, which will be described below. In other words, transferrin exhibited the same 'I', r behavior as serum albumin in the following purification process.
l) (u7y)0
7,281 0.5 7.2811.0
7.2815.0 29
,12710.0 29,127*
Staphylococcal enterotoxin B50 nft
/ml + 0K432 (Hishiha=-v)0.0
γ-type interferon was induced at 25 kg/a.

Example 2: Matrix Blue A column and G o
n Purification by Sepharose Column (Part I) The culture supernatant obtained in Example 1 is purified as a crude γ-interferon solution. A matrix blue-A column (5tMT, height 6cm
The pass-through fraction (MBI) added to ) also contained γ-type interferon, so it was stored in 4C and further purified. 'r II! 11 minutes (MB II
) contains 1.5M1iaC6 P B S (-)
It was obtained by detaching it at. P B with 50% ethylene glycol and 1,5 M Na C1yf5
No interferon activity was recovered in the fraction eluted with S (-), and the total recovery rate of MBI and MBII by Matrix Blue A-column chromatography was over 80.
Since the activity was not recovered after dialysis, it seems that most of the interferon contained in both components is γ-type (see Figure 6). Similar results were obtained with Blue Sepharose CL-6B manufactured by Pharmacia.

After dialyzing M'BII in PBS (-1), a Gon A e Sepharose column (1,5X
5t-rn) (see Figure 4). The pass-through fraction (Fraction C: (18) MB [-ConA I) contained γ-type interferon activity at a ratio of 2.6% to the recovered crude extract activity. Both adsorbed portions also contain interferon activity,
P containing 0.1M α-methylmannoside
eluted by B S (-) (Fraction D: MB1-
ConAll). The recovery rate of the fraction relative to the activity of the crude extract was 61.7%, and it was confirmed that it was of the γ type by pH 2.0 treatment and a neutralization test with an anti-γ type interferon antibody.

Example 3: Matrix Blue A and ConA filtered through sepharose garum 1Fi3 interferon crude interferon solution 1tvMixed with Matrix Blue 1ooy flattened with PBS(-) at 4C.

After standing for 4 hours, fill the column and wash thoroughly with PBS (-). PBS containing adsorbed fraction Y 1.0 M 5aGI Y
(It was eluted with -1 solution. The non-adsorbed fraction (MBI: 2t) was left as is, and the adsorbed fraction (MBII: 1.2/=) was diluted 9 times with water, and then normalized with PBS (-). Concanavalin A/Sepharose 4B (non-adsorbed fraction is 200ml 1゜(
19) The adsorbed fraction was mixed with 250ml and 4t? After being left standing for 4 hours, the column was filled and thoroughly washed with PBS (-).

Elution of the adsorbed fraction was carried out with PBS (-147) containing 0.1 M α-7 termannoside.

Thus, in both the M B I -Gon A I1 fraction and M B I1-Con A1 fraction, 106 U/m9
6,660 ml of liquid containing γ-type interferon activity on proteins.

The combined interferon activity recovery for both 750rd and 750th lapii samples was just over 75%.

Example 4: Chromatofocusing method Chromatography using Polybuffer TM96 (manufactured by γ-Pharmacia) and RehabSofa exchanger PBS TM94' was applied to the fractionation and purification of γ-type interferon.

'IOme to crude interferon-6 p[-]B,
The solution was added to a PBETM94 column (1.5 cm x 12 m) and extracted with 250 ml of polybuffer (20) at pH 6.0.

432 in Figure 110. ! : Shows the separation pattern by chromatofocusing of a crude interferon solution induced by simultaneous addition of Staphylococcus enterotoxin B (referred to as SEB). As shown in the figure, interferon activity is divided into three parts in the gradient from pH 9 to p) 16, and their 'J point pI is 8.5, 8.0, respectively.
7.8 (each referred to as CP-1°c p -11 and CP-1). Furthermore, after fractionation Pr, 1.5 M NaC
Interferon activity was also found in both fractions eluted at 6:2 (fraction S), which had an isoelectric point of 6:2 (CPI) and an isoelectric point of 5.2 (CPV) in a lower pH gradient.
), the interferon activity peak lf J was obtained.

All fractions of Kore et al. were neutralized with anti-human γ-interferon antibody. The molecular weight of each fraction was calculated using Ultroagel AcA.
Estimated by gel permeation chromatography (o, 5x 50ctn) using No. 34 (LKB), CP-1, CP-11, CP-I and CP■ correspond to a molecular weight of approximately 43,000 daltons (2I). CP-V shows a single peak, whereas CP-V shows a single peak of 30.00
It was drained in the range of 0 to 40,000 Daltons.

Example 5: Purification of r-type interferon using Remazol Blue column (see Figure 5) 5ml of the crude interferon solution shown in Example 1, P
Remazol Blue column (
IMX 9 cm). a 50% of the interferon activity and most of the protein were obtained in the flow-through fraction (R-1), or as shown in Example 6, both fractions were obtained in Example 2.
It can be used for further purification as follows. The adsorption fraction (R-1) is PBS containing 1.5 M NaGt←)#
This one-step operation yielded r-type interferon with a yield of 50% and a specific activity of 105 U/~protein or higher.

Unlike other Blue Sepharose-adsorbed fractions, these two fractions had the characteristic that they did not contain serum albumin protein added to the medium.

Example 6: Remazol Blue power (22
) Remazol Blue column non-adsorbed fraction (Rl) obtained in Example 5 9.5 ml, Matrix Blue A column shown in Example 2 (and 6) (1 crn x 9 c
m) passed. As shown in Figure 7, the non-adsorbed fraction (RI
-MBI') and adsorbed fraction (fraction RL -MB l', which is bathed in PBS (-1) containing 1,5M NaC1'fc)
Interferon activity was obtained at a ratio of yl:1.

The activity recovery rate is R1-MB l', R1-MB [1'
The total percentage was over 80%. That is, when combined with the Remazol Blue adsorption fraction shown in Example 5 (having an activity of 105 U/Ing protein or more) and the R1-MBl' fraction in this example, the recovery rate from the crude interferon solution was 70 factors or more. The specific activity of γ-type interferon was obtained. This is a different combination of the methods shown in Example 5 and this example.
can be separated separately, indicating that it is extremely effective in purifying γ-type interferon.

These purification methods (Examples 5 and 6) are particularly useful when γ-type interferon is produced using a bloodless septic medium (23); was difficult.

[Brief explanation of the drawing]

FIG. 1 is a flow sheet showing an example of the purification of γ-type interferon that is effective in the method of the present invention. FIG. 2 is a graph showing the synergistic induction of γ-type interferon by Staphylococcus enterotoxin B and OK4-2 in the serum-free medium of the present invention. FIG. 6 is a diagram showing the separation pattern of the crude γ-interferon solution obtained in Example 1 by Matrix Blue A column chromatography. FIG. 4 is a diagram showing separation pattern 7 of the Matrix Blue A column adsorption fraction (MBll) in FIG. 6 by Con A Sepharose nonoramic chromatography. FIG. 5 is a diagram showing the separation pattern of the crude γ-interferon solution obtained in Example 1 by Remazol Blue column chromatography. FIG. 6 is a diagram showing the separation pattern of the sum γ-type in(24) terferon solution induced by the method of the present invention by the chromatofocusing method. FIG. 7 is a diagram showing the separation pattern of the Remazol Blue column non-adsorbed fraction (R-1) obtained in Example 5 by IJ Tux Sprue A column chromatography. Patent applicant Suntory Co., Ltd. 3-'-". Agent Patent attorney Kyozo Yuasa, -1'-fu! (4 others) (25) Procedural amendment Commissioner of the Japan Patent Office Kazuo Wakasugi 1, Display of the case Patent Application No. 193146 of 1982 2, Name of the invention Process for producing human gamma interferon 6, Person making the amendment Relationship to the case Patent applicant address name (190) Suntory Ltd. 4, Agent 5, Column 6 of [Claims] and [Detailed Description of the Invention] of the specification subject to amendment, contents of the amendment as shown in the attached sheet (Attachment) (1) The scope of claims is corrected as follows. "1) Using a serum-free medium, human leukocytes, mononuclear cells, lymphoblasts, leukemia cells, splenic cells, etc. are cultured with the addition of Staphylococcus enterotoxin B and 0K432 (Picibanil) as a γ-type interferon derivative. A method for producing human γ-type interferon, which is characterized in that human γ-type interferon is highly produced, separated and purified by 2) remazol blue agarose column, matrix blue A column, or concanavalin A column chromatography. The manufacturing method according to claim 1, characterized in that human γ-type interferon is separated and purified using one or more of them. The manufacturing method described in Scope 1.'' (1) (2) The statement in the specification is corrected as follows. Page Line Before correction After correction 2
3 Human leukocyte system Human leukocyte system 4 10
White blood cells Mononuclear cells 5 6 For example,
For example, 6 7 White blood cells Mononuclear cells 8 4 Inducibility Inducibility 9 17
Approximately 16% Approximately 20%9 18 Approximately 64%
Approximately 60%10 6 Approximately 2.3% Approximately 2% 10 8 Approximately 61.7% Approximately, 60%11 2
0 Purification Purification 12 12 Type A 4 types l 4 4 Joh, n5o
n et al. Johnson et al. 15 8 10n
M 10mM19 16 5a (J7
Na(J20 11 Focusing Focusing 20 14 Rebuffer Polybuffer 20 14 PBS PBE20
15 Focusing Focusing 21 1 Elution Elution 2116-17
Ultroa Ultokuchi 2117-18
Gel Bar Mini-Gel Permeability Shin 22 2 Elution Elution 22 8-9a
Interferon 26 15 Combination talent 2 depending on the combination
3 16 5ubpopuj ationsubp
opulation

Claims (1)

[Claims] 1) Using a serum-free medium, human leukocytes, lymphoblasts, leukemia cells or spleen cells, etc., Staphylococcus enterotoxin B Oyohi 0K462 (bisibanil) as a gamma-type interferon derivative 1. A method for producing human r-type interferon, which comprises adding and culturing human r-type interferon to produce a high level of human r-type interferon, and separating and purifying the human r-type interferon. 2) Remazol Blue Agarose-Column. Human gamma interferon is separated using one or more of Matrix Blue A column or Concanavalin A column chromatography. 2. The manufacturing method according to claim 1, which comprises refining. 6) The manufacturing method according to claim 1, characterized in that (1) human γ-type interferon is separated and purified by a chromatography method.