JPS6153224A - Purification of lpf-ha - Google Patents

Abstract

PURPOSE:To purify LPF-HA, by contacting a liquid containing LPF-HA and produced by Bordetella pertussis with a crosslinked polysaccharide sulfuric acid ester gel, separating the impurities, and eluting the LPF-HA from the gel. CONSTITUTION:An LPF-HA-containing liquid separated from the culture product of Bordetella pertussis is made to contact with a gel of a crosslinked polysaccharide sulfuric acid ester (e.g. epichlorohydrin-crosslinked cellulose sulfuric acid ester), and subjected to the adsorption treatment at 0-30 deg.C and a specific conductivity of 0.5-5.0ms/cm to separate the gel from impurities. The LPF-HA is eluted from the gel to obtain the LPF-HA in high purity and yield. EFFECT:The operation is extremely simple. The chromatographic adsorbent used in the above process can be prepared at a low cost, usable repeatedly without causing degradation, and is economical. Since the produced LPF-HA has high purity, it can be used in the preparation of various reagents and pharmaceuticals, and the preparation of pertussis vaccine.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides LPF-HA (Leu
cocytosis promoting factor
More specifically, a LPF-HA-containing solution obtained from a B. pertussis culture is brought into contact with a cross-linked polysaccharide sulfate ester gel to adsorb LPF-HA, and then LPF-H,A
The present invention relates to a method for purifying LPF-HA by eluting it from the gel.

Industrial applications LPF-HA is an active substance produced by B. pertussis I and II phase bacteria, and has a high virulence.
It does not produce phase III bacteria, Bordetella pertussis, or Bordetella bronchiseptica. This LPF-HA is also called pertussis toxin and is known to be a protein having various physiological activities. Its main physiological activities include leukocytosis activity,
It is known to have insulin secretion-enhancing activity, histamine-sensitizing activity, hemagglutination activity, etc., and its application as a therapeutic agent for diabetes is attracting attention based on its insulin secretion-enhancing activity.

Apart from these physiological activities, it has recently been revealed that LPF-HA plays an extremely important role in preventing Bordetella pertussis infection and disease onset, and is also used as a protective antigen against Bordetella pertussis infection. It started to attract attention [Pittm
anv M; Review of T nfectio
US D 1seas, ↓, 401-409 (
1979).

and 5ato+Y; Sem1nars in I
nfectiousDiseases IV, Ba
cterial Vaccine, 380-385 (
1982) ],.

Therefore, in order to study the physiological activity of LPF-HA and to manufacture pharmaceuticals using its physiological activity,
Furthermore, in order to industrially produce pertussis vaccines with fewer side effects, it is desired to develop a method for easily isolating and purifying LPF-HA in large quantities.

BACKGROUND ART In the conventionally known collection and purification method of LPF-HA, a culture of Bordetella pertussis is precipitated with ammonium sulfate, followed by extraction and dialysis as a starting material. , H:Biochimica
et Biophysica Acta+ 4
44. 765 (1976)] or sucrose gradient centrifugation [5ato*Y: Infect, I++
+n+un, , 6.897~704. (1972
)]. However, in such a method, high purity LPF-HA
It is difficult to obtain, and the yield is also small.

As a method for obtaining relatively large amounts of high-purity LPF-HA,
The B. pertussis culture supernatant was passed through a hydroxyapatite column to adsorb LPF-HA, and after washing and elution, concanavalin A-Sepharose (ConA-8epha) was added.
rose+ 7y Lumacia Co., Ltd. 51 methods of purifying using affinity chromatography have been proposed [
Yajima+ M: , LBiochem 83.2
95-303 (1978)]. However, when using funcanavalin A as a binder, affinity chromatography does not only have an affinity for LPF-HA, but also adsorbs sugars, glycolipids, and other glycoproteins. components of the pond, such as F-
HA(Filamentous-Hema1?glu
It also adsorbs microorganisms (tinin) and bacterial cell membrane components, creating the desired LPF.
-It is difficult to isolate HA with high purity, and it cannot be said to be an excellent affinity chromatography.

Recently, since it was discovered that human haptoglobin specifically binds to LPF-HA, LPF-HA can be extracted by affinity chromatography using this human haptoglobin as a ligand instead of huncanavalin A in the above method. A method of purification has been attempted [Iro
ns L; Biochimica et Bioph
isica Acta, 580.175-185 (1
979) Cowell, j”, Sem1na
rs in Infectious Disease
s TV, Bacterinl Vaccine+
371-379 (1982)]. When this human haptoglobin is used as a ligand, a new important problem arises in countermeasures against hepatitis viruses. That is, since human haptoglobin is collected from human blood, there is a risk of contamination with hepatitis viruses. Furthermore, concerns about contamination with other unknown infectious agents cannot be ignored, and this also applies when animal serum is used. At present, there is no absolute method to check for contamination with hepatitis viruses, etc. On the other hand, as a means to inactivate such hepatitis viruses, etc., 60°
A method of heating from 15:00 to 15:00 is known. The present inventors have discovered that when such heat treatment is performed, there is a serious defect in that the affinity of habtoglobin for LPF-HA is almost completely lost, and the desired effect is lost.

In addition, even in the purification method using hydroxyapatite del described above, since hydroxyapatite is expensive,
There are problems in extracting LPF-HA industrially and at low cost.

Purpose of the Invention The present inventors have conducted various studies in order to find an industrial method for isolating and purifying LPF-HA. As a result, the present inventors have discovered that LPF-HA-containing liquid obtained from a culture of Bordetella pertussis is treated with a cross-linked polysaccharide. By contacting with sulfate ester gel, adsorbing LPF-HA, and eluting it from the hind limb cross-linked polysaccharide sulfate ester gel, which has been separated from contaminants, highly pure L is obtained.
It was discovered that PF-HA can be obtained very easily and in a very high yield, leading to the completion of the present invention.

That is, an object of the present invention is to provide an industrially simple method for purifying LPF-) (A), which is a biologically active substance that is extremely useful medically, to extremely high purity in large quantities. .

The present invention relates to LPF-HA obtained from B. pertussis culture.
The containing liquid was brought into contact with a cross-linked polysaccharide sulfate gel to adsorb LPF-HA, and then LPF-HA was extracted from the gel.
LPF-HA characterized by eluting PF'-HA
This is a purification method.

The B. pertussis culture used as a starting material in the present invention is a culture of B. pertussis I in a conventional medium, such as a liquid medium such as a Cohen-Willer medium or a Stenner-Scholte medium, by a conventional method. Alternatively, it is a culture obtained by shaking culture or aerated agitation culture. This culture is subjected to the method of the present invention in the form of a culture supernatant from which bacterial cells have been removed by centrifugation, a centrifuged supernatant of disrupted bacterial cells, or a partially purified preparation thereof.

The crosslinked polysaccharide sulfate ester used in the present invention is a crosslinked polysaccharide such as dextran, cellulose, agarose, etc., which is obtained by crosslinking with a crosslinking agent such as epichlorohydrin, dichlorohydrin, dibromhydrin, or ethylene glycol bisepoxypropyl ether. It is obtained by converting polysaccharide into sulfuric acid ester. Cross-linked polysa tucharides are already commercially available, such as Sephadex G-10, G-25, G-50, and G-100 (manufactured by Pharmacia) as cross-linked dextran, and Sephadex CL as cross-linked 7-galose. -28, CL-48, CL-6B (manufactured by 7 Fulmacia), etc., and examples of crosslinked cellulose include Cellulofine GCL-25, GCL-90 (manufactured by Chisso), etc. By treating these gels with chlorosulfonic acid, sulfuric anhydride, or the like in the presence of an organic solvent such as pyridine, a desired crosslinked polysaccharide sulfate ester gel can be obtained.

In the present invention, LPF-HA produced by Bordetella pertussis is purified and collected using a crosslinked polysaccharide sulfate gel, for example, by the following method.

The raw material solution, LPF-HA-containing solution, is prepared by mixing the centrifuged supernatant of a B. pertussis culture with distilled water or a buffer solution to a specific conductivity of 0.
The supernatant can also be subjected to an adsorption operation after being diluted to 5-S, Oms/cm; however, this supernatant contains F-HA (Filamentous-HA), which also has an affinity for cross-linked polysaccharide sulfate ester gel. hema
glutinin), we performed chromatography using a crosslinked polysaccharide sulfate ester gel in advance under conditions that adsorbed F-HA but not LPF-HA (specific conductivity 5.0 to 25.0 ms).
/cm, a column packed with cross-linked polysaccharide sulfate ester gel equilibrated with a pH 5-9 buffer solution with a specific conductivity of 5.
θ~25.0ms/cI+1, the raw material solution adjusted to pH 5-9 is passed through), and the F which is the pass-through fraction is
Both portions containing a large amount of LPF-HA but not containing -HA may be subjected to an adsorption operation.

LPF-H to cross-linked polysaccharide sulfate gel
A series of purification operations such as adsorption of A, washing of the gel, and elution of LPF-HA can be carried out using commonly used industrial methods such as the batch method and the column method, but the column method is easier to perform. It's easy and 1) convenient. In the case of the column method, a cross-linked polysaccharide sulfate ester gel is packed in a column, and 0.02M pine kilbene (
Mcllvaine's) buffer (pH 5.2>, etc.) with a specific conductivity of 0.5-5.0 ms/cm and a pH of 5.0-9.
After equilibration is carried out by passing a suitable buffer solution having a concentration of approximately 0.0, the process moves on to adsorption of LPF-HA.

During adsorption, the LPF-HA containing solution was adjusted to pH 5.0.
~9.0, and the specific conductivity is adjusted appropriately to be 0.5 to 5.0, and the solution is passed through a crosslinked polysaccharide sulfate ester gel-filled column to adsorb LPF-HA. After this,
A buffer solution similar to that used for the above-mentioned equilibration is passed through to wash the delta and wash out contaminants.

During the elution of LPF-HA, L'(li, pH is 5.0-9
．． Elution is carried out by passing a suitable buffer solution having a specific conductivity of 0.0 or more than 5.0, and stepwise elution or salt concentration gradient elution is preferably carried out. In other words, if the diluted centrifuged supernatant of B. pertussis culture is used as the raw material solution, F-HA will be adsorbed at the same time as LPF-HA under the adsorption conditions described above. It is necessary to elute under conditions where F-HA is eluted and F-HA is not eluted. These conditions include a specific conductivity of 5-100 ms/cu+, preferably 50-60 m at pH 5-9.
A suitable buffer (for example, 0.02 M pine kilbene buffer containing 0.5 M sodium chloride) is first passed through the tube, and both portions containing LPF-HA are collected.

After this, a solution with a higher specific conductivity than the above-mentioned elution buffer (1
00-300TIIS/(!I11> Pass the buffer solution,
F-HA and other impure components are eluted, and the crosslinked polysaccharide sulfate ester gel is equilibrated and reused.

Most preferably, a salt gradient elution is performed.

LP from which F-)IA has been separated in advance as a raw material liquid
Even when using F-HA containing liquid, the specific conductivity is 0.
．． 5→300+ns/am (e.g. sodium chloride 0→4.OM salt concentration gradient 0
．． By performing elution using 02M2M Matsukiru Pen I (pH 5,2) and separating the LPF-HA-containing fraction, LPF-HAt- of extremely high purity can be obtained.

According to the purification method of the present invention, the degree of purification of LPF-HA reaches several tens of times, and the recovery rate of LPF-HA is 90% or more.
It reaches close to 00%. The specific activity of the purified LPF-HA obtained was extremely high at 9X10' LPEU/mg protein, and was analyzed by polyacrylamide disk electrophoresis (pH 4, 5).
A single band is formed in the analysis, and Bordetella pertussis endotoxin is almost completely removed.

As described above, according to the method of the present invention, the desired LPF-HA can be collected in high yield and purity from the B. pertussis culture as the starting material, and its repeated production is extremely simple. The chromatography adsorbent for purification can be prepared at low cost, shows no deterioration even after repeated use, and is extremely economical.

Therefore, the method of the present invention is an excellent method for industrially purifying high-purity LPF-HA. Furthermore, the method of the present invention is based on the conventional technique P4[! i! "It is also possible to combine it with gradient ultracentrifugation, ion exchange chromatography, etc., and in that case, results much superior to those obtained with conventional methods can be obtained.

The LPF-HA obtained by the method of the present invention is highly pure and does not aggregate with proteins, lipids, AI, etc., and endotoxins are almost completely removed. Therefore, various reagents utilizing its biological activity can be used. It is useful in the preparation of pharmaceutical products, as well as in the preparation of pertussis vaccines.

X-irradiation The present invention will now be described in more detail with reference to Preparation Examples and Examples.

Preparation Example 1 11 ml of chlorosulfonic acid is added dropwise to 200a+1 pyridine at a temperature of 0° C. or lower and mixed. After the dropwise addition is completed, the mixed liquid is heated to a temperature of 65 to 70°C. Sephadex G-, which is an epichlorohydrin cross-linked dextran,
50 (manufactured by 7 Fulmacia) 7°5 g was added, and while stirring
Hold at ~70°C for 4 hours. After the reaction is completed, it is cooled and neutralized by adding a 17um aqueous solution of hydroxide. The gel is separated by filtration and thoroughly washed with 0.01 M +J acid buffered saline to obtain a crosslinked dextran sulfate ester gel.

Preparation Example 2 To 21 Qml of the pyridine-chlorosulfonic acid mixture prepared in the same manner as in Preparation Example 1, 7.58 g of dried cellulofine GCL-25 (manufactured by Chisso Corporation), which is a cross-linked cellulose gel, was added to 65-70 g. React for 4 hours at °C. After the reaction is completed, it is cooled and neutralized by adding an aqueous sodium hydroxide solution. The gel was separated by filtration and 0. Wash thoroughly with OIMIJ acid buffered saline to obtain 7.2 g of crosslinked cellulose sulfate gel.

Preparation Example 3 To 21 Qml of the pyridine-chlorosulfonic acid mixture prepared in the same manner as in Preparation Example 1, 30 ml of pyridine inclusions of Sepha 0-S CL-6B (manufactured by Pharmacia), which is a cross-linked agarose del, was added, and React at 70°C for 4 hours. After the reaction is completed, it is cooled and neutralized by adding an aqueous sodium hydroxide solution.

The gel was separated by filtration and 0. Wash thoroughly with OIMIJ acid-buffered saline solution and remove 23 ml of cross-linked agarose sulfate gel.
get.

Example 1 5ml of crosslinked cellulose sulfate gel prepared in the same manner as in Preparation Example 1 was added to a column (40m+oφX 200
+nm), and 200 ml of distilled water was passed through it. A solution obtained by diluting 100 ml of the centrifuged supernatant of Pertussis I Ainae Higashihama fi device culture solution seven times with distilled water (specific conductivity 3.0 ms/am) is passed through this column.

Approximately 200 ml of 0.02M pine kirbene buffer 1 (pH 5)
, 2) through the column, washed the column, and then heated to 0°02
Using 50 IIll of pine kilbene buffer solution (specific conductivity 2.Oms/am, pH 5,2) supplemented with M sodium chloride, sodium chloride O→4. Elution was performed using a salt concentration gradient of OM, and after fractionation into approximately 1+ ol portions, LPF-
Pool the 6 ml aliquots containing HA.

Table 1 shows the analysis results and experimental results of the raw material solution and purified LPF-HA fraction.

Table 1 1) In vitro test of LPF-HA: Hapto-
ELISA method [LPF-1 according to Sumigome et al., see Proceedings of the 28th Toxin Symposium 141-144 (1981)
Unit of HA 2) Displayed as protein content using Kirbourg method protein nitrogen measurement value x 6,25 3) After dilution to a content of 6.25 μg protein/ml, it was determined to be non-defective in accordance with the Biological Products Standards (Yakuhatsu No. 287, 1981). It was revitalized and implemented.

Claims (8)

[Claims] (1) When purifying and obtaining LPF-HA produced by Bordetella pertussis, the LPF-HA-containing solution is brought into contact with a cross-linked polysaccharide sulfate ester gel to adsorb LPF-HA and separate it from impurities. A method for purifying LPF-HA, which comprises eluting LPF-HA from the crosslinked polysaccharide sulfate gel. (2) The method according to item (1) above, wherein the crosslinked polysaccharide sulfate is one selected from crosslinked cellulose sulfate, crosslinked agarose sulfate, and crosslinked dextran sulfate. (3) The method according to item (2) above, wherein the crosslinked cellulose sulfate is epichlorohydrin crosslinked cellulose sulfate. (4) The method according to item (2) above, wherein the crosslinked agarose sulfate is epichlorohydrin crosslinked agarose sulfate. (5) Item (2) above, wherein the crosslinked dextran sulfate is epichlorohydrin crosslinked dextran sulfate.
The method described in section. (6) The adsorption treatment is carried out at a temperature of 0 to 30°C and a specific conductivity of 0.5.
The method according to item (1) or item (2), which is carried out under conditions of ~5.0 ms/cm. (7) The method according to any one of items (1) to (6) above, wherein elution from the gel adsorbing LPF-HA is performed using a buffer solution with a specific conductivity of 5.0 to 100.0 ms/cm. . (8) Prior to the elution process, the adsorbent gel has a specific conductivity of 0.
．． The above (7) washing with a buffer solution of 5 to 5.0 ms/cm
) Method described in section.