DE1492042B - Process for the production of a pertussi antigen - Google Patents

Description

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The invention relates to a method for extra general pressures in the range of about 1050

here from B. pertussis antigen obtained from any up to 3550 at. Other means of Substantial content of cell material of B. pertussis Disassembly or rupture of cells and isolation

free is. the cell walls can be used as

Whooping cough is an acute, strongly excessive sound, mechanical grinding, etc., Portable, infectious disease that is caused by B. pertussis but experience shows that with the explosive disease will and in children aged compression the highest yields of clean is dangerous under 4 years of age. An immunization wall material can be obtained. Before use The above-mentioned fractionation device is previously killed against this disease by injection Bacilli or extracted antigens disadvantageously sterilized by removing all of them has been effective. The frequent occurrence of side reaction line system 24 hours of exposure to exposure to ethylene oxide, such as fever, irritation, inflammation, and then with sterile nitrogen and necrosis, as well as the rare but troubling gas purges. The printing device is 1 hour The reports of encephalitis have sterilized researches, preferably by bringing them together made necessary for a better vaccine. 15 all of their parts with / J-propiolactone (0.5%) and then

The present invention makes a B. pertussis irrigation with sterile, distilled water. For

Antigen available in a much improved form. binding the decompression chamber with a

A permanent immunity can be achieved with the antigen. Nitrogen tank serves a multiphase, bacteria

against whooping cough without the reluctant filter or other appropriate

wanted side effects of now known vaccines 20 corresponding filters.

appear. It provides an antigen that is suspended in the B. pertussis cells is free of cell substances, some of which are toxic. Compression chamber of the fractionation device and those responsible for the side reactions are introduced and in this a pressure of 1050 to that when administered commercially available 3550 at exposed, causing the cells to enter the debarer Vaccines have been observed. 25 compression chamber in which they

It has been found that a valuable initial explosive ruptures. The decompression chamber will

for immunization against whooping cough with pre-cooled nitrogen gas that is passed through the

can be obtained through a series of working steps, introduces multiphase filters into the chamber

in which cells of the B. pertussis are kept mechanically at 20 ^° C. or below. The Decom-

decomposed and the antigen from the cell wall chemically 30 30 flowing pressure chamber, the cell walls and

extracted. It has been shown that the protective antigen protoplasm is contained in one in one

Part of the cell wall (sometimes also collected as a sterile container immersed in a Zeil ice bath,

membrane or cell envelope); the proto- The outflowing, the ruptured cell material

plasma of the cell, the main part of the nitrogen-containing material is thrown and the over-

containing material, has only a small 35 standing, liquid material discarded; the remaining

until no antigenic property that can be used to immunize cell wall material with cold water

tion is suitable. However, the protoplasm contains in be washed to remove highly toxic, soluble proto-

high concentration protein that can be eliminated in laboratory plasma components. The washed ones

is toxic to animals and when present in vaccines too or unwashed cell walls are in a

can lead to undesirable side effects. 40 buffers to a concentration of about 100 to

The in the method according to the invention one 2000 bacilli / ml (B / ml) suspended, wherein in the

B. pertussis cells placed on each practice can generally have a concentration of

known, appropriate media bred 200 B / ml is preferred. The antigen will then

are extracted according to the invention, such as that described by Powell et al.

Charcoal agar medium (Public Health Reports, 66, 45 wall suspension with a salt of deoxycholic acid

P 346 [1951]), which, inter alia, of Verwey described, to a final concentration of about 0.1 to 10 ⁰ / o

liquid media (J. Bact., 58, p. 127) or the KuI deoxycholate added; to achieve a maximum

Cohen-Wheeler (Amer. J. Pub. paint extraction, however, is a final concentration

Health, 36, p. 371 [1946]). After waxing, from about 0.25 to 1% is preferred. A pH range

which the cells harvested by spinning. One can get 50 from about 8 to 10 has proven to be great for the extraction

then smash the harvested cells or those proven satisfactory.

Freeze cell paste until required and at - 30 ⁰ C. The cell wall deoxycholate mixture is continuously

store. If desired, you can move gently for just 18 to 48 hours during the

lyophilize harvested cells and then _keep the temperature between about 2 and 5 ⁰ C until

may store or the cells with thimerosal 55. The material is flung and the protruding

or by other methods by which the antigen is good against a 0.01 molar phosphate buffer

that B. pertussis is not destroyed, kill. The dialyzed to a pH in the range of 7 to 7.7

Cell concentration is intended to eliminate the deoxycholate for the purposes of the invention. The not dialyzing

preferably in the range of 100 to 1000 B / ml bare material which contains the antigen is on

(Bacilli / ml) lie. 60 pH 7.0 and adjusted to one of the prescribed

The cells are resuspended in cold, distilled water standards, such as the standards of the "National Institutes of (2 to 5 ° C), and diluted in a concentration appropriate to fractionation." The device of the type Ribi Cell Fractionator (Ivan solution of the antigen can be done with a preservative Sorvall, Inc., Norwald, Conn., V. St. A.) at 2110 atmospheres, such as thiomerral, benzethonium chloride, and a temperature of 20 ⁰ C or below 65 benzyl alcohol, y-picolinium chloride or other explosive decompression. At a known, appropriate preservation pressure of 2110 at, one obtains good results, but teln, preserved and to any desired concentration has been shown that satisfactory results can be diluted.

The antigen obtained according to the invention can be used for the preparation of an aqueous vaccine or initially adsorbed on auxiliary substances such as aluminum hydroxide or phosphate, or with aluminum potassium sulfate and then prepared for vaccine. The antigen can, since it is compatible with other antigens and / or toxoids is, with these in a conventional manner to form a polyvalent vaccine in the form of a Single dose can be combined.

The present invention offers a number of distinct advantages. The product is free from all Protoplasmic constituents, one of which, the heat-labile toxin, is known to be found in laboratory animals being extremely toxic and responsible for this or that side reaction in humans can be. The product is free from nutrient medium, cell debris and foreign chemical substances. It is combined with other antigenic substances known to be associated with the intact pertussis cell be tolerated. The process is simple and easy to carry out and with high yields reproducible on active material.

The procedure is explained in detail in the following example. The basic way of working is to use deoxycholate to isolate the protective antigen from B. pertussis cell walls, which are covered by Protoplasm as well as other foreign matter have been separated.

example

The cells of a 48 hour culture of B. pertussis grown on Cohen-Wheeler medium are harvested by adjusting the medium to pH 7.0 with concentrated hydrochloric acid and up a Sharples centrifuge. The cell paste obtained is suspended in distilled water, passed through a 200-mesh nylon screen to remove coarse particles, and the filtrate to a concentration set at 500 B / ml.

The cell concentrate (1600 ml) is introduced into a pre-sterilized fractionation device of the type Ribi Cell Fractionator and subjected to a pressure of 2110 atm. The cells are squeezed out under pressure into the decompression chamber, which is ^{kept at 20 °} C. or below with cooled nitrogen gas, and are subject to an explosive bursting therein. The cell material drains from the decompression chamber into a sterile container immersed in an ice bath, 1600 ml being obtained.

The suspension is for 3 hours at 16,000 rpm in a centrifuge of the Spinco 18000 type spun, but also other spinning devices such. B. the Sharples centrifuge, can be used are. The excess material is removed from the centrifuge by decanting and you can, if desired, fill the centrifuge one or more times and let it circulate without leaving the residue to remove. The excess material that is removed and discarded contains the highly toxic Protoplasm fraction of the cell concentrate.

The residue or sediment in the rotor is preferably removed by working on the Rotor attaches steel balls and gently moves it by placing it on a rolling mechanism. the However, the residue can also be detached from the rotor by other methods known per se respectively.

The residue from the rotor is washed with cold distilled water (2 to 5 ⁰ C, about 800 ml) from, represents the pH by the addition of 10 ⁰ / cent sodium hydroxide solution (about 1.0 ml) to 8.5 and brings the final volume ml of the cell wall suspension with cold distilled water (2 to 5 ⁰ C) to 1600th further provides to a sterile 2 ° / o solution of the Natriumdesoxycholates at pH 8.5 (adjust with In NaOH solution from the heat sterilization) forth and united 1600 ml of this solution of the wall material suspension. By adding cold, sterile, distilled water (800 ml), 4000 ml of wall material suspension with a deoxycholate concentration of 1.0% is obtained, which contains the wall material of 200 B. pertussi in 1 ml. The suspension is gently in a mechanical shaker for 18 to 24 hours at 2 to 5 ⁰ C and then moved in the rotor of a fractionating device of the type Spinco 18000 (or another centrifuge) for 1 hour at 16000 rev./min. (30100 X gravity average). The excess material is obtained by decanting; the rotor can be used repeatedly to treat other parts of the cell wall suspension without having to remove the sediment.

The obtained proportions of supernatant containing the protective antigen and sodium deoxycholate Are well combined and dialyzed against 0.01 molar phosphate buffer, pH 7.4 to 7.7, until in Dialysate no more deoxycholate can be detected when concentrated hydrochloric acid is added. Usually the end point of the deoxycholate determination is obtained with the fourth change of the buffer, whereby one allow about 135 liters of buffer to act for 48 to 72 hours with each change. The non-dialysable extract, which when diluted to a value corresponding to 32 B / ml now has a volume of 4500 ml, contains 14.7 protection units / ml (the N.I.H. No. 6 control material for comparison 8 protection units / ml) and can be used for the production of aqueous, adjuvant or multivalent vaccines can be used.

To determine the effectiveness of the cell extract and the NIH control material, differently diluted solutions of the extract and the contron material are administered to separate groups of mice and then the animals are administered intracranially with 0.03 ml of a dilution of known, virulent B. pertussis with a content of 10 ⁵ Organisms / ml infected. Results:

dilution
surviving / tested animals
Aj BIC 0.01 0.002 ED ₅₀
♦) Limit values
of reliability
° / o Protection units / ml Extract according to the example
NIH No. 6 0.05 8/32
0.02 1/32
0.0024 0.0165
0.0304 85 to 118
85 to 118 14.7
8th 29/32
0.06 4/32 0/32 25/31

*) Effective dose to protect 50% of the animals tested.

The antigen extract according to the example proves to be non-toxic according to the N.I.H. standards; these norms require that when intraperitoneal Injection of half the single human dose in mice in 3 days no weight loss, a net gain of 3 g in 7 days and in no more than 5% of the animals examined Death occurs. With intraperitoneal injection of 0.25 ml of the protective antigen extract according to the example ten mice had an average weight gain of 2.1 and am at the end of 3 days At the end of 7 days, an average increase of 6.5 g was observed without any at the end of the test period there would have been a death.

The extract according to the example also passed the animal safety test; when injected into 20 g mice each with half the individual dose for humans and three injections of two 350 g guinea pigs with a single dose for humans, there were no deaths and none Symptoms.

Conventional sterility testing has shown that the extract of molds and bacteria free is.

Electrophoretic Analysis and Ouchterlony Analysis have shown that the extract according to the example from the main portion of the protoplasmic substances and wall antigens except for the antigen were free. The final vaccine contained a third or less of the total nitrogen that has been determined with conventional vaccines.

Claims (1)

Claim: A method for producing a pertussis antigen, characterized in that the cell wall material freed from the protoplasm is suspended in water so that the suspension contains the cell wall material in an amount of 100 to 200 bacilli / ml, this suspension then by adding an alkali metal salt of deoxycholic acid to adjusts to a final concentration of 0.1 to 10% and by adding alkali to a pH value of 8 to 10, and ^{leaves at 2 to 5 0} C for up to 48 hours, separated from the cell walls and the deoxycholate from the solution of the Antigen removed.