Abstract:
This invention discloses a process for preparing an acellular Bordetella pertussis vaccine by passing culture supernatants or cellular extracts of B. pertussis through a column containing an ion exchange resin. The eluate from the column is detoxified and can then be employed by conventional means as a vaccine. The vaccine so produced has reduced toxicity due to the removal of endotoxin.

Description:
BACKGROUND OF THE INVENTION 
     One of the more serious diseases of infancy is whooping cough, which is an acute, highly communicable, infectious disease characterized by a paroxysmal or spasmodic cough which usually ends with a prolonged, high-pitched crowing inspiration (the whoop). In infants, choking spells may be more common than whoops. The disease is caused by the organism Bordetella pertussis. Immunization against this disorder has been accomplished in the past by injection of either killed-cell vaccine or extracted antigen. Until the present time, however, the available preparations, which contain a large amount of bacterial cells, have had certain shortcomings. In particular, such preparations have a tendency to produce side reactions such as fever, irritation, redness and the like. 
     Currently, B. pertussis vaccine is composed of the whole bacteria of B. pertussis, or of the whole bacteria and an adjuvant such as aluminum phosphate, aluminum hydroxide or a mixture thereof. A description of said vaccine production may be found in U.S. Pat. Nos. 3,577,319 and 4,429,046 and in the concurrently filed patent application of Wenlii Lin and William A. Griffith entitled: &#34;Enhanced Large Scale Cultivation of Bordetella Pertussis Cells for Vaccine Production&#34;, the teachings of which are incorporated herein by reference. 
     Japanese Pat. No. 55-141416, entitled: &#34;A Triple Vaccine Prepared by Adding Diphtheria Toxoid and Tetanus Toxoid to a Pertussis HA Fraction,&#34; employs the use of sucrose gradient centrifugation to remove endotoxin from Phase I B. pertussis hemagglutinin (HA) fraction after precipitating the culture filtrate with ammonium sulfate, then detoxifying the histamine-sensitizing factor (HSF) and leukocytosis promoting factor (LPF) in the HA fraction thus obtained. The shortcoming of this technology is its low capacity and high time consumption. Thus, it would require twenty hours to process up to 200 ml of a preparation using the largest Beckman zonal centrifuge. 
     U.S. Pat. No. 3,897,309 describes the removal of trace amounts of pyrogens from aqueous solutions by passing a strongly ionic solution thereof through a column of a basic anion exchange resin. The invention describes a specific process for the removal of pyrogens from L-asparaginase. When impure L-asparaginase is dissolved in a strongly ionic buffer solution and passed through a column of a basic anion exchange resin the pyrogens are selectively and irreversibly adsorbed onto the anion exchange resin while the L-asparaginase solution, free of pyrogens passes through the column. Examples of the resins cited in said patent are: Sephadex® DEAE-A-25, Sephadex® DEAE-A-50, Dowex® 1X2, Amberlite® IRA-938 and Whatman DE-52. 
     The chemical properties of endtoxins and proteins of different sources vary greatly, thus making it impossible for those familiar with the art to anticipate from the immediately preceding patent that a particular ion-exchange resin can separate endotoxin from the active components of B. pertussis vaccines. In fact, when the procedure of Example 4 in the above last cited patent was studied, using DE-52 resin, it was concluded to be inapplicable to B. pertussis vaccine preparation. 
     It is an object of the present invention to provide a B. pertussis vaccine of substantially improved form, having a markedly reduced tendency to produce side effects, while providing long-term immunity against whooping cough. 
     SUMMARY OF THE INVENTION 
     In the present invention, culture supernatants or cellular extracts of B. pertussis are passed through a column of an anion exchange resin of sufficient capacity to adsorb endotoxin and permit active, desired substances to flow through or otherwise be separated from the endtoxin. Alternatively, the supernatant or cell extract is mixed with an anion exchange resin and then the mixture is loaded into a column for elution. In addition, the present invention permits the separation of β-lactoglobulin, which may be present in the culture supernatants or cellular extracts, from the active substances. The anion exchange resin of this invention is equilibrated with 0.005M sodium phosphate buffer, pH 7.2, containing 0.05-0.1M sodium chloride. By way of example and not of limitation such anion exchange resins include Whatman DE-52 and Whatman DE-53 (Whatman Chemical Separation, Inc., 9 Bridewell Place, Clifton, N.J. 07014 U.S.A.) and Sephadex® DEAE-A-50 (Pharmacia AB, Upsala, Sweden). The active substances, including fimbrial hemagglutinin (FHA) and leucocytosis promoting toxin (LPT), also known as leucocytosis promoting factor (LPF) or pertussis toxin (PT), flow freely through the column. The effluents are then detoxified using, for example, lysine and formalin , formaldehyde or glutaraldehyde and treated with aluminum phosphate as adjuvant. These vaccine preparations are effective when tested by the specific mouse potency test based on the U.S. Standard Pertussis Vaccine. 
     DETAILED DESCRIPTION OF THE INVENTION 
     As demonstrated by the Examples hereinbelow, endotoxin is removed by the resins employed but with varying effect on the amount of active substance and, therefore, potency resulting. In a preferred embodiment of the present invention, Whatman DE-53 is employed. Whatman DE-53 yields the unexpected result of excellent separation of endotoxin with a much higher level of active substances recovered than with Whatman DE-52. This result is unanticipated since the DE-53 resin would be expected to bind much more of the active substances than DE-52, due to the nature of the resins. 
    
    
     The invention will be further illustrated and the methods of using the invention will be described in conjunction with the following examples which are to be taken as illustrative only and not by way of limitation. 
     EXAMPLE 1 
     The Separation of Bordetella pertussis Antigens from Endotoxin in a Culture Supernatant Using an Anion Exchange Resin 
     A 10 ml amount of a B. pertussis culture supernatant, concentrated by ultrafiltration, in 0.005M sodium phosphate buffer, pH 7.2, containing 0.05M sodium chloride (Buffer A) was mixed with 3 g of Whatman DE-53 anion exchange resin equilibrated in Buffer A. After 2.5 hours binding at 22° C. the supernatant was aspirated and the resin was poured into a 1.5 cm×10 cm column. The resin was washed step-wise with 15 ml volumes of 0.005M sodium phosphate buffer, pH 7.2, containing the following increasing concentrations of sodium chloride: 0.05M, 0.10M, 0.15M, 0.20M, 0.25M. Each 15 ml eluate was assayed for (a) total LPT units which equal (white blood cells count of the sample÷white blood cell count of the control)-1; (b) total hemagglutination (HA) units of goose red blood cells, where one unit equals the reciprocal of the last dilution causing hemagglutination; and (c) total endotoxin units determined by the Limulus ameobocyte lysate assay according to the printed procedure available from Associates of Cape Cod, Incorporated, 10 Rose Morin Drive, Falmouth, Mass., mailing address: P.O. Box 224, Woods Hole, Mass. 02543, release date Jan. 7, 1981. Under the above conditions pertussis antigens were separated from endotoxin as summarized below in Table I. 
     
                       TABLE I______________________________________Separation of Bordetella pertussis Antigens FromEndotoxin in a Culture Supernatant Using Whatman DE-53 Resin         Units      NaCl     Total    Total  TotalFraction   Molarity (a) LPT  (b) HA (c) Endotoxin______________________________________Starting Material      0.05     15,930   810,920                               7,500,000Unbound    0.05     *8,311   *810,920                               *750SupernatantEluate 1   0.05     *3,116   *38,400                               *1.125Eluate 2   0.10     *812     *3,600 *1,125Eluate 3   0.15     0        600    1,125,000Eluate 4   0.20     0        600    7,500,000Eluate 5   0.25     0        450    1,125,000______________________________________ *Recovery of (a) LPT in unbound supernatant plus eluates 1 and 2 was 77% (12,289 units/15,930 units × 100); recovery of (b) HA in the above was 105% (854,570 units/810,920 units × 100); while (c) Endotoxin i these samples was reduced nearly 4,000 fold. Differences of less than one (1) log in the endotoxin assay results are not considered significant. 
    
     EXAMPLE 2 
     Endotoxin Binding on Whatman DE-53 Resin 
     The Whatman DE-53 resin used in Example 1 was regenerated in the 1.5 cm×10 cm column using 4 volumes of high salt wash containing 4.0M sodium chloride, 0.005M phosphate buffer, pH 7.2. Eighty ml of concentrated B. pertussis culture supernatant as used in Example 1 was loaded onto the resin bed. Eight fractions of 10 ml each were collected and assayed for endotoxin by the Limulus ameobocyte lysate assay to determine the capacity of the DE-53 resin to bind endotoxin. The results of this test, listed below in Table II, show that one gram of resin bound essentially 40×10 6  endotoxin units. 
     
                       TABLE II______________________________________Endotoxin Binding Capacity ofWhatman DE-53 ResinFraction          Total Endotoxin Units______________________________________Starting Material 120,000,000Eluate 1          150Eluate 2          150Eluate 3          600Eluate 4          600Eluate 5          1,500Eluate 6          1,500Eluate 7          15,000Eluate 8          150,000______________________________________ 
    
     EXAMPLE 3 
     The Separation of Endotoxin and Pertussis Antigens in a Cellular Extract Using an Anion Exchange Resin 
     A cellular extract made from high salt extraction of B. pertussis cells were clarified and dialyzed by conventional means against a buffer consisting of 0.005M sodium phosphate, pH 7.2 containing 0.09M sodium chloride and 0.01% thimerosal (Buffer B). The sample was applied to a 5 ml column of DE-53 equilibrated in Buffer B. After washing the column in the same buffer, the resin was regenerated using 4.0M sodium chloride in 0.005M sodium phosphate buffer, pH 7.2. Table III shows the separation of endotoxin from pertussis antigens. The majority of the antigens were found to be present in Fraction 1. 
     
                       TABLE III______________________________________Separation of Endotoxin and Pertussis Antigensin a Cellular Extract Using DE-53 Resin      Molarity  Total HA  Total EndotoxinFraction   NaCl      Units*    Units*______________________________________Starting Material      0.09      51,200    7.5 × 10.sup.6Fraction 1 0.09      30,720     9.0Fraction 2 0.09      1,920      4.5Fraction 3 0.09      960       45.0Fraction 4 0.09      480       45.0Fraction 5 4.00      640         3 × 10.sup.5______________________________________ *Determinations for hemagglutination (HA) units and endotoxin units were carried out as described in Example 1. 
    
     EXAMPLE 4 
     Large-scale Separation of B. pertussis Antigen From Endotoxin 
     A large scale batch of concentrated B. pertussis culture supernatant (8.1 L) in Buffer B was processed using 4.5 liters of packed Whatman DE-53 resin in an Amicon GA 180 column (Amicon Corporation, 21 Hartwell Avenue, Lexington, Mass. 02173) with a cross-sectional area of 254.5 cm 2 . The resin was equilibrated in Buffer B and packed at a flow rate of 8.7 L/hour. The supernatant was loaded onto the 4.5 L column of DE-53 resin at a flow rate of 5.88 L/hour. The first 1.8 L of effluent was discarded and the remainder collected as Fraction 1. The column was washed with 1.5 L of Buffer B and the effluent added to Fraction 1. Three more washes of 1.5 L each of Buffer B were collected as Fractions 2, 3 and 4. Fractions 1-4 were assayed for B. pertussis antigens and endotoxin was described in Example 1. The results of these tests are shown below in Table IV. 
     
                       TABLE IV______________________________________Large-scale Separation of B. pertussis AntigensFrom Endotoxin Using Whatman DE-53 Resin      Volume              TotalFraction   Liters    Total HA  Endotoxin Units______________________________________Starting Material      8.1       2.6 × 10.sup.6                          4.9 × 10.sup.8Fraction 1 8.1       *1.3 × 10.sup.6                          5.0 × 10.sup.6Fraction 2 1.5       9.0 × 10.sup.4                          0.9 × 10.sup.3Fraction 3 1.5       3.0 × 10.sup.4                          0.9 × 10.sup.4Fraction 4 1 5       1.5 × 10.sup.4                          0.9 × 10.sup.3______________________________________ *The majority of the HA antigen was eluted in Fraction 1. 
    
     EXAMPLE 5 
     The Separation of B. pertussis Antigens and Endotoxin Using Whatman DE-52 Anion Exchange Resin 
     Anion exchange resin Whatman DE-52 was substituted for Whatman DE-53 anion exchange resin and the procedure of Example 1 was followed. Although the separation of B. pertussis antigens and endotoxin was similar to that obtained in Example 1, the recovery of antigens were considerably lower as shown below in Table V. 
     
                       TABLE V______________________________________Separation of B. pertussis Anitgens and EndotoxinUsing Whatman DE-52 Resin        NaCl      Total   TotalFraction     (molarity)                  HA*     Endotoxin Units*______________________________________Starting Material        0.05      810,920 7,500,000Unbound Supernatant        0.05      102,400 75Eluate 1     0.05      7,200   112.5Eluate 2     0.10      3,600   1,125,000Eluate 3     0.15      1,800   1,125,000Eluate 4     0.20      600     1,125,000Eluate 5     0.25      150     112,500______________________________________ *Determinations for hemagglutination (HA) units and endotoxin units were carried out as described in Example 1. 
    
     EXAMPLE 6 
     The Separation of B. pertussis Antigens and Endotoxin Using Sephadex® DEAE-A-50 Anion Exchange Resin 
     A 10 ml bed volume of Sephadex® DEAE-A-50 anion exchange resin (Pharmacia), equilibrated in Buffer A was mixed with 25 ml of the same concentrated B. pertussis culture supernatant used in Example 1. After adsorption and elution from a 2.5 cm diameter column following the procedure of Example 1, the pertussis antigens and endotoxin were separated as shown in Table VI. 
     
                       TABLE VI______________________________________Separation of Pertussis Antigens and EndotoxinUsing Sephadex ® DEAE-A-50      NaCl       Total   TotalFraction   (molarity) HA*     Endotoxin Units*______________________________________Starting Material      0.05       96,000  37,500,000Eluate 1   0.05       9,600   300Eluate 2   0.10       1,600   3,000Eluate 3   0.15       800     30,000Eluate 4   0.20       800     300,000Eluate 5   0.25       400     3,000,000______________________________________ *Determinations for hemagglutination (HA) units and endotoxin units were carried out as described in Example 1. 
    
     EXAMPLE 7 
     Preparation of a Component Vaccine 
     The acellular B. pertussis material obtained from Fraction 1 in Example 4 was concentrated using 50% saturated ammonium sulfate precipitation. The resuspended pellet was detoxified using formalin and lysine according to procedures well known to those skilled in the art. After dialysis to remove excess formalin, the material was stored at 4°-8° C. The soluble vaccine was adsorbed to aluminum phosphate before combining with diphtheria and tetanus adsorbed toxoids. Pertussis potency of the adsorbed vaccine thus obtained was 11.85 units/total immunizing dose (TID) based upon the standard U.S. dosage of 1.5 ml. 
     EXAMPLE 8 
     The Separation of Endotoxin and β-lactoglobulin from B. pertussis Antigens 
     A 10 ml aliquot of concentrated B. pertussis culture supernatant in Buffer B containing 0.1% β-lactoglobulin was passed through a 5 ml bed volume of DE-53 equilibrated in Buffer B. The column was washed with Buffer B and then regenerated with 4M NaCl, 0.005M phosphate buffer, pH 7.2. Under these conditions the column had the capacity to separate both endotoxin and β-lactoglobulin from pertussis antigens. Endotoxin and β-lactoglobulin which bound to the DE-53 resin were removed by the high salt regeneration step. See Table VII and FIG. 1 below. Table VII provides comparative separation data which resulted from the hereinabove described procedure. 
     
                       TABLE VII______________________________________Separation of Endotoxin and β-lactoglobulinFrom Pertussis Antigens      NaCl       Total   TotalFraction   Molarity   HA*     Endotoxin Units*______________________________________Starting Material      0.09       80      7.5 × 10.sup.5Fraction 1 0.09       80      7.9 × 10.sup.1Fraction 2 0.09       0       7.5 × 10.sup.1Fraction 3 0.09       0       &lt;1.0 × 10.sup.1Fraction 4 4.00       0       &gt;7.5 × 10.sup.5______________________________________ *Determinations for HA units and endotoxin units were carried out as described in Example 1. 
    
     FIG. 1 is a photograph of a 0.2% sodium dodecyl sulfate-10% polyacrylamide gel run according to Laemmli, U.K., Nature, 227:680 (1970) depicting the effect of column chromatography on an acellular B. pertussis sample containing 0.1% β-lactoglobulin while using Whatman DE-53 as the anion exchange resin as described in Example 8. In particular, Lane A is 0.1% β-lactoglobulin; Lanes B and K are molecular weight makers; Lane D is acellular pertussis containing 0.1% β-lactoglobulin; Lane E is Fraction 1 from the DE-53 column; Lane F is Fraction 2 from the DE-53 column; Lane G is Fraction 3 from the DE-53 column; Lane H is Fraction 4 from the DE-53 column; and Lane I is acellular pertussis without β-lactoglobulin. 
     The procedures described in the preceding Examples were carried out under the customary aseptic conditions and the final material in each case was uncontaminated by undesired organisms. The Examples do, in fact, demonstrate that endotoxin is removed from concentrated B. pertussis culture supernatants or cellular extracts when column chromatography with an anion exchange resin equilibrated with a neutral phosphate buffer of a low salt concentration is employed. The procedures also demonstrates that certain anion exchange resins are more preferred than others. In conclusion, vaccines produced by incorporating the process procedures described by the present invention constitute improved products due to the removal of some of the impurities which tend to produce side effects upon injection into mammals.