IL24946A - Process for preparing b. pertussis antigens - Google Patents

Description

o'nons ?w o"»3TA3 man? v nn — at—a«►-s—=«· «·-a«■s—ss—ss·»-a«·a«■e=—=3·»s—a—a«· Process for preparing B. pertussis antigens MERCK & CO., INC J C:-23864 I I This invention is concerned with a novel method for extracting B. pertussis antigen and the use of this ■ antigen in the preparation of monovalent or polyvalent ... vaccines that are devoid of any substantial quantity of ..

B. pertussis cellular material. i ' Whooping cough is defined as an acute, highly communicable, Infectious disease caused by B. pertussis^ which in children under years of age is dangerous* and probably ranks first as the cause of infant mortality.

Immunization against this disorde^has^been accomplished in the past by injection of killed cell vaccine or extracted antigen. However, the. frequent incidence of side reactions such as fever, irritability, inflammation · and necrosis and the rare but disheartening report of encephalitis has constituted a potent stimulus for research leading to a better vaccine. 1 An object of the present invention is to provide a B, pertussis antigen of substantially improved form which is capable of providing a lasting immunity to whooping .cough and which .does not produce the undesirable side effects of currently known vaccines.

Another object is to provide a vaccine which,is free of cellular substances, some of which are toxic and may be responsible for the side reactions observed upon administration of commercially available vaccines.

In accordance with the above and other objects we have discovered that a useful vaccine for immunization against whooping cough can be prepared by a combination n me hanical disru tion of B. ertussie cells and chemical extraction of the antigen from the cell wall. It has been established that the protective antigen is an integral part of the cell wall (sometimes referred to as cell membrane or cell envelope), the protoplasm of the cell containing the major bulk of nitrogenous material, possesses little to no protective activity. The protoplasm does, however, contain a high concentration of toxic protein which can cause death in laboratory animals and undesirable side effects when present in vaccines that are administered clinically.

A feature of this invention, therefore, resides in disrupting the intact B. pertussis cell and isolating the cell walls by centrifugetion or by other mechanical means. The isolated cell walls then are washed substantially free of cellular material, and extracted with a solution of sodium or potassium chloride which solubilizes the protective antigen and allows it to be separated mechanically from the cell wall. .,- The B. pertussis cells used in the process of this invention can be grown in any one of the known, suitable media, such as the charcoal agar medium,, the liquid media or -in Cohen-Wheeler culture medium. After the growth period, the cells are harvested fey centrlfugation and can be used as harvested in the process of this invention or the cell paste can be frozen and stored at -30eC. until needed.

If desired, the harvested cells can be lyophilized and then stored until needed, or the cells may be killed with thimerosal or by other methods that are known not to destroy the protective antigen of B. pertussis.

The cells are resuspended in cold ( 2-5*C) distilled water and subjected to explosive decompression in a Ribi Cell Practionator at 30 , 000 p.s.i. and a temperature of 20eC. or less. While good results are obtained by employing 30 , 000 p.s.i. pressure it has been found that satisfactory results are obtained when pressures ranging between about 15, 000-50 , 000 p.s.i. were used.

Other means of disrupting cells and isolating the cell walls also can be used such as by sonic oscillation, mechanical mill, etc., but experience indicates that the Ribi Pressure Cell produces the highest yields of clean wall material. Before use, the Ribi Cell Practionator is sterilized advantageously by exposing all of its tubing to ethylene oxide over a 2ij.-hour period and then flushing with sterile nitrogen gas. The pressure unit is sterilized for one hour preferably by bringing all of its parts into contact with β-propiolactone {0,$%) and then flushing with sterile distilled water. A multiphase bacterial retaining filter or other suitable filter .connects the decompression chamber with a nitrogen tank.

The B. pertussis cell suspension is fed into the compression chamber of the Practionator where they are subjected to a pressure of 15, 000-50, 000 p.s.i. which forces the cells into the decompression chamber where they explosively rupture. The decompression chamber is maintained at 20° C. or less by precooled nitrogen gas which is passed through the multiphase filter and into the chamber. The effluent from the decompression chamber containing the cell walls and protoplasm is collected in a sterile container immersed in an ice bath.

The effluent containing the disrupted cell material is centrifuged, the supernatant fluid discarded : and the cell wall material remaining can bedas ed with cold distilled water to eliminate highly toxic, "soluble ; ;' protoplasmic constituents. Either washed or'unwashed cell walls are suspended in a buffer to give .a concen-. tra,tion between about 100 B/ml. to 2,000 B/ml., although for practical purposes a~concentra ■t»■·i■on * of 500 B/ml. gen- ■ «r* ■ « erally is used. The protective antigen then is extracted " by adding to the cell wall suspension sufficient sodium or potassium chloride to produce a final concentration of 0.5 to 1.5 Molar, the pH adjusted to from 8.5 to 10 by the addition of a basic salt, such as-trlsodium ortho- -phosphate, sodium or potassium carbonate, and sodium hexametaphosphate or tris(hydroxymethyl)aminomethane along with sodium or potassium hydroxide, and the volume adjusted v/ith distilled water so as to produce a cell wall equivalence of 200 B/ml.

The cell wall-salt mixture is gently agitated , continuously for a period of from 18-48 hours while , maintaining the temperature at between about 2-5* C.

The material is centrifuged and the supernate ma be dialyzed against buffer (0.01M phosphate buffer) !at a pH The process is described in detail in the following example. It is to be understood that modifications can be made in the various techniques employed in carrying out the process of this invention; the important_contri-bution being the extraction of the protective" antigen1 from B. pertussis cell walls that have' een isolated from protoplasm as well as from other extraneous substances.

** J~nr~ ......

EXAMPLE 1 The cells from a 48-hour culture of B. pertussis, grown on Cohen-Wheeler medium, -ore harvested by adjusting the medium to pH 7.0 with concentrated hydrochloric acid, and centrifuglng in a Sharpies centrifuge. The cell paste obtained is resuspended-in. distilled water, passed through a 200-mesh nylon screen to remove gross particles and the filtrate is adjusted to a concentration . o ^ 500 B/ml. The cell concentration ; preferably should be within the range of 100-1000 B/ml. for the purposes of this invention.

-The cell concentrate (l600 ml,.) is fed into a pre-sterilized Ribi Cell Fractlonator and subjected to a pressure of 30,000 p. s.i. The cells under pressure are extruded into the decompression chamber, maintained at 20° C. or less by cooled nitrogen gas, where the , cells rupture explosively. The cellular material drains from the decompression chamber into a sterile vessel immersed in an ice bath, yielding 1600 ml. of effluent.

The effluent is centrifuged in a Spinco 18,000 Other centrifuge equipment can be used, such as the Sharpies Centrifuge, for example. The supernate is decanted from the rotor head and the head may be refilled and spun one or more times, if desired, withou removing the residue. The supernate that is--removed , and discarded contains the highly toxic protoplasmic fraction of the cell concentrate.

The residue or sediment remaining n½ie rotor head is removed preferably by adding steel beads to the rotor head and gently agitating the head .by placing it on a rolling mechanism. Other known .methods also can be used to dislodge the residue from the rotor head.

I ,The residue is washed out of the rotor head , with cold distilled water (2-5° C. ) (approx. 1600 ml.). Qne liter of sterile sodium chloride Containing 213.8 grams) is added, the pHjLs adjusted to 10.0 by the-.addition._of.J;rispdij^pJiosphate (66.6 ml. of 0.6M) and 2.0 ml. of 10$ hydrochloric acid and the volume adjusted to 4000 ml. with distilled water giving a mixture . , containing a pertussis concentration equivalent to 200 B/ml. and sodium chloride and trlsodium orthophoephate concentrations of 1.0 M and 0.01 M respectively.

The concentrations of cell wall can as well be adjusted to a pertussis concentration equivalent to 100-2,000 B/ml. and sodium chloride between 0.5-1.5M. The suspension is gently agitated in a mechanical shaker at a temperature of 2-5* C. for 18-24 hours, and jbherv centrifuged in a Spinco 18,000 batch rotor head (or other centrifuge) at 16,000 r.p.m. ( 30,100 x gravity; ; f a single human dose into mice, there be no loss of weight in 3 days, a net gain of 3 grams in 7 days, and no ·· more than 5$ mortality in; the animals used in the study; Upon Ϊ.Ρ. injection of 0.25 ml. of the protective antigen extract from Example 1 into 10 mice, an average weight gain of 2.5 g. was observed at the end of 3 days, and an average gain of 6.5 g. was observed at the end- of >i: 7 days, with no deaths at the end of the test period.

The extract of Example 1 also passed the animal safety test in that no deaths andT noTsymp'toms occurred, when 1/2 of a single human dose was Injected into each of two 20 g. mice and 3 times a single human dose was injected into each of two 350 g. guinea pigs.

Conventional sterility tests ^established that the extract was free of mold and bacteria.

Electrophoretic and Ouchterlony analysis established —~the extract of Example—1-^ as- devoid of the major portion of protoplasmic and wall antigenic substances other ...... than protective antigen. The final vaccine contains. one third or less of the total nitrogen found in conventional yaccines.

Vaccine Preparation ... Aqueous vaccine. ■ he protective antigen extract concentrate from Example 1 is adjusted with buffered saline (pH 7.2) containing thimerosal 1:10,000 to a final concentration of 32 B/ml. or less. This vaccine remains stable [when ' stored at 2-5° C.

Adjuvant vaccine: To 4000 ml. of protective antigen concentrate from Example 1 is added ml. sterile 10$ potassium alum and the pH adjusted to 7.0 with cold (2-5° C.) $ sodium- hydroxide solution. The material. is*~stored at 2-5* C. for 2 hours and then centrifuged at 2000 r.p.m. for about 15 minutes in an International centrifuge maintained at 2-5° C. The supernate<4a^dl carded.

The sediment is resuspended in buffered saline, pH 7.2 to give a final volume of 3,000 ml."at a concen- · · " tration of 267 B/ml equivalent. e vaccine concentration may, be adjusted to 32 B/ml. equivalent by addition of : ' sterile, saline, pH 7.2, or 0.3M glyqine buffer. Thimero-sal is added as a preservative in an, amount sufficient to produce a concentration of 1:10,000 when diluted.

This vaccine remains stable when stpred at 2-5° C, Multivalent vaccines: Toxoids, such as diphtheria and tetanus toxoids, can be added to either of"the"above aqueous or adjuvant vaccines to give a multivalent vaccine wherein the toxoids are present in concentrations normally recommended.

The invention contemplates the extraction ^ of protective antigen from B. pertussis cell walls by employing modifications of the procedures Illustrated In Example 1 that have been described above. In particular, protective antigen can be extracted in concentrations useful for preparing vaccines by admixing under alkaline conditions, preferably at pH sus ension of tvom about 100 to 2000 B/ml. of B.|portua0ia cell wall material and a sufficient quantity of sodium or potassium chloride to give a final salt concentration of 0.5-1.5M. The protective antigen thus obtained cart" "be separated from wall debris by centrlfugation*. The concentrate of protective antigen thus obtained can be adjusted to any desired concentration and made up into a monovalent or polyvalent vaccine by any of the methods conventionally employed for preparing vaccines particularly those containing B. pertussis antigen. Additional , examples to illustrate working"*wl hi the above preferred ranges are not provided as it will be evident to those skilled in the biological, sciences how to make the various modifications specifically taught "herein.

I Therefore, while the invention has been illustrated

Claims (13)

HAVING NOW particularly described and aeolertalned the nature of our said invention and in vfha manner the; same is to be performed, we declare that what we claim is

1. The process for extracting protective antigen from isolated B. pertussis cell wall material by admixing under alkaline conditions "and at a temperature ' between about 2-5° C. a suspension of from 100-2000 B/ml. of said cell wall material and sufficient solution of 'a salt selected, from sodium chloride and potassium chloride to give a final concentration of between 0.5-1.5M. ·> ·-·

2. The process as claimed in Claim 1. herein sodium chloride is employed and"£xtr#ction is carried out at a pH between about 8.5-10. '

3. The process as claimed' ¾' Claim 1 wherein following extraction the material is,,centrifuged, the supernate collected and dlalyzed against phosphate buffer, pH 7.0-7.2, to remove substantially all salt,

4. The process as claimed 1η. CJLaim'l wherein the concentration of B. pertussis cell wall material is about 500 B/ml.

5. The process for extracting protective antigen from isolated B. pertussis cell wall material wherein a suspension of cell wall material having a concentration of between about 100-2,000 B/ml. is admixed with a solution 5 of 0.5-1.5 of a salt selected from sodium chloride and potassium chloride at a pH between about 8.5-10 and gently agitating the mixture for at least 'about- 18 hours at a temperature between about 2-5° C. then centrifuging at about 16,000 r.p.m. for about 1 hour 'and collecting 10 the protective antigen containing supernate. ·

6. The process as claimed.,in Claim 1 wherein B. pertussis cell wall material is obtained by mechanically rupturing B. pertussis cells and separating the dell wail material from protoplasmic material. ' | - ■ I- '

7. The process as claimed in Claim 1 wherein B. pertussis cell wall material is separated from all other cellular material by extruding. B. pertussis under 15,000-50,000 p.s.i. pressure into a cooled decompression 5 chamber, and then mechanically harvesting the cell wall material. . / .

8. The process as claimed in Claim 1 wherein B. pertussis cell wall material is separated from all other cellular material by extruding virulent B. pertussis under 15,000-50,000 p.s.i. pressure into a decompression chamber maintained at a maximum of 20° C, centrifuging the disrupted cell mass obtained at about 16,000 r.p.m. (30,100 x gravity average) and selectively harvesting cell wall material.

9. The process for preparing a vaccine effective upon parenteral administration in eliciting protective antibodies to B. pertussis comprising coprecipitating the protective antigen prepared by the process claimed in Claim 3 with an adjuvant selected from the class consisting of aluminum hydroxide, aluminum phosphate and alum, resuspend-ing the adjuvant adsorbate in physiological saline solution at a concentration of at least 8 protective units of B. pertussis antigen per ml. and adding thimerosal to a final concentration of at least about 1:10,000.

10. A process as claimed in Claim 9. herein there is added to the vaccine at least one member selected from a bacterial toxoid and a compatible antigen.

11. A vaccine effective upon parenteral administration in eliciting protective antibodies to B. pertussis containin at least 8 protective units per ml. of B. pertussis antigen prepared by the process claimed in Claim 1.

12. A vaccine effective upon parenteral administration in eliciting protective antibodies to B. pertussis containing at least 8 protective units per ml. of B. pertussis antigen prepared by the process claimed. in Claim 3·

13. A vaccine effective upon parenteral administration in eliciting protective antibodies to B. pertussis containing at least 8 protective units per ml. of B. pertussis antigen prepared by the process claimed in Claim 5. ,-! . Dated this 9th day of January, 1966