WO1997017427A1 - Novel process for the propagation of b. pertussis - Google Patents

Abstract

The invention relates to a process for culturing bacteria from the genus Bordetella from which antigens useful in an acellular vaccine may be isolated. The invention results in improved yields of FHA and PT in the culture media and involves culturing the bacteria with specific grades of Polyvinyl alcohol.

Description

NOVEL PROCESS FOR THE PROPAGATION OF B. PERTUSSIS

This invention relates to a process for culturing bacteria belonging to the genus Bordetella for isolation of antigenic proteins useful in the production of whooping cough vaccine. In particular, the invention relates to a process for improving the yield of the major protein components FHA and PT which are secreted into the culture medium during bacterial growth of Bordetella pertussis .

Whooping cough, or pertussis, is a highly-infectious disease which primarily affects children. In addition to causing respiratory complications, whooping cough may result in nerve damage and a high incidence of mortality, particularly in children from low socioeconomic groups and in newborn infants who do not possess maternal anti-pertussis antibodies. The etio logic agent of pertussis is the Gram- negative coccobacillus, Bordetella pertussis . The bacteria are believed to invade the respiratory tract and induce a toxic state which remains even after their disappearance, several days later.

For many years the disease has been controlled through immunisation with "whole cell" vaccine prepared by growing the Bordetella pertussis organism in fermenters and then inactivating the resulting cells by heat treatment and/or addition of chemical agents. Although the World Health Organisation presently recommends the immunisation of infants to prevent the incidence and spread of pertussis, concern has arisen over the reported adverse events resulting from various vaccine formulations. The consequent reduced usage of conventional B. pertussis vaccine has resulted in an increase in the incidence of pertussis infections. In order to avoid the reported adverse events from whole cell vaccine, considerable research effort has been put into the development of an efficaceous acellular vaccine comprising a small number of highly-purified antigenic proteinaceous components.

A number of antigenic proteins have been identified as having utility as vaccine components, including for example lymphocytosis promoting factor (LPF), also known as histamine sensitising factor, islet activating protein or, more commonly, pertussis toxin (PT); filamentous hemagglutinin (FHA); fimbrial agglutinogens; and the outer membrane protein of the bacterium, having a molecular weight of approximately 69,000 Daltons, known as pertactin .

The Bordetella pertussis bacterium is fastidious in its growth requirements. It is unable to utilise carbohydrates, pyruvate, lactate and intermediates of the gylcolysis pathway, relying instead on amino acids, particularly glutamate for its carbon and energy source in culture media. A suitable glutamate-containing culture medium is described by Stainer and Scholte (J. J. Gen. Microbiol. , 63, 211-220, 1971).

It has been shown that the production of antigenic materials for use in a vaccine against whooping cough involving growth of its etiological agent, Bordetella pertussis, in a suitable liquid culture media can be enhanced by the addition of growth promoting agents to the culture medium.

EP-A-0 077 646 describes a method for enhancing the amount of antigenic materials secreted into the cell-free broth, comprising supplementing the culture medium, suitably Stainer-Scholte medium, with cyclodextrin, in panicular β-2,6-0-dimethyl cyclodextrin.

US 4,551,429 describes the use of an alternative growth promoting agent in B. pertussis propagation under submerged aerobic conditions with shaking of the culture medium. According to US 4,551,429, addition of polyvinyl alcohol (PVA) having a molecular weight (MW) from about 1,500 to 16,000, suitably a 10,000 MW material, at the level of 0.5 to 2.0g/l of medium, also substantially enhances levels of PT and FHA.

Propagation using submerged aerobic conditions with shaking, as described in EP- A-0 077 646 and US 4,551,429, is suitable for small-scale production, eg. in shake flasks with volumes up to about 5 litres. These conditions are not however representative of large-scale, commercial production where large volume fermenters requiring mechanical agitation are required.

A problem arises when the B. pertussis organism is propagated in a large scale fermenter suitable for commercial production of whooping cough vaccine. The problem lies in the tendency for the B. pertussis organism to accumulate at the surface of the liquid culture medium as a foam layer. This results in removal of the bacteria from the growth medium and premature termination of the culture process. A high proportion of the FHA protein secreted by the bacterium also accumulates in this foam layer.

The problem caused by foam production is not solved by addition of growth promoting agents to the culture medium and has moreover been found to be exacerbated when PVA is used in place of cyclodextrin, especially when PVA is present at the upper end of the concentration range disclosed in US 4,551 ,429. Foam production thus counteracts the beneficial effect of growth promoters, in particular the use of PVA as a growth promoter.

An economic solution to the foaming problem with B. pertussis, using methods commonly used in culture fermentation, is not readily available. Chemical antifoaming agents have not been found to provide a practical solution under normal cultivation conditions. A number of agents have been tested; they were either not tolerated by the B. pertussis bacterium or were found to be effective only for short time periods after which the foam soon returned.

Foambreakers which supress foaming mechanically are commercially available. Because of the susceptibility of the FHA protein to physical degradation if high shear conditions are generated by the foambreaker, particular attention must be given to the design of mechanical foambreakers for large scale B. pertussis culture fermentation.

EP-A-0 121 249 discloses an improved method for production of the protective antigens of B. pertussis on an industrial scale when using a cyclodextrin as a growth promotor in the culture medium. The method requires fermentation to be carried out under defoaming conditions with control of the culture temperature and the amount of dissolved oxygen. The defoaming conditions may be chemical or mechanical, preferably mechanical. EP-A-0 121 249 describes a fermenter producing suitable mechanical defoaming conditions, manufactured by Marubishi Rika KK (Japan). A defoaming vane for a mechanical defoaming apparatus is described in Japanese Patent Publication No. J55-11011 (Marubishi Rika KK).

Large scale propagation of B. pertussis, when carried out according to the method described in EP-A-0 121 249 and using PVA as a growth promotor in the culture medium in place of cyclodextrins, gave variable and disappointing results in terms of reduction in the level of foam production and isolation of acceptable yields of antigenic proteins, especially FHA.

It has now been found that the problem of excessive foam production and the associated decrease in the level of FHA secretion from B. pertussis in large scale fermenters if PVA is used in the culture medium can be overcome by selecting both the MW and the degree of hydrolysis of the PVA added to the culture medium. Accordingly, the present invention provides a process for commercial scale culture of B. pertussis, comprising propagating the bacterium in an aqueous nutrient medium under submerged aerobic conditions with mechanical agitation and supplementing the nutrient medium with PVA having a molecular weight (MW) in the range 10,000 to 50,000 and a degree of hydrolysis in the range 80 to 90 mol % . Preferably the degree of hydrolysis is 85 to 90 mol % .

According to the invention, commercial scale culture of B. pertussis is suitably carried out in fermenters with a capacity of at least 10 litres, for example in the range 20 to 1000 litres. Foam production is suitably controlled using a mechanical foambreaker which disperses foam generated during fermentation but does not generate high shear conditions in the fermenter, for example a foambreaker of the type described in Japanese Patent Publication No. J55-11011.

Suitable strains of Bordetella pertussis for use in the invention are described in the literature and are readily available in commercial collections such as the American Type Culture Collection, Rockville, Maryland, USA. Any of the available strains may be used in the process of the invention provided it produces at least one, and preferably both of the antigenic proteins PT and FHA in liquid culture medium.

Examples of strains which may be employed include B. pertussis phase I, B. pertussis phase II, B. pertussis phase I CS, B. pertussis Tohama, B. pertussis strain 185-30, B. pertussis strain 18323, B. pertussis strain 134, B. pertussis strain 509, B. pertussis strain Wellcome 28, and Office of Biologies B. pertussis strain 165. A preferred strain for use in the present invention is B. pertussis phase I, Tohama, which is available from the Institute of Fermentation, Osaka, Japan under accession number IFO- 14073.

The selected B. pertussis strain may be grown in a variety of ways known to persons skilled in the art. Cultivation methods are known which employ different cultivation steps, and liquid or solid media, depending on the quantity and origin or conservation methods of the seed culture. However, any known method will suffice for use in the present invention which provides an inoculum of a conventionally- acceptable size for large-scale production.

A suitable medium for growth of a B. pertussis inoculum may be selected by any person skilled in the art. Suitable media include, without limitation, Gengou medium (EP-A-0 077 646); the media described in N. Andorn et al. (Appl. Microbiol. Biotechnol., 28, 356-360, 1988) and references cited therein; Stainer- Scholte medium (J. Gen. Microbiol. , 63, 211-220, 1971); modified Stainer-Scholte medium described in A. Imaizumi et al (Infect. Immun., 41, 3, 1138-1143, 1983 and J. Microbiol. Methods, 2, 339-347, 1984); Verway medium (US 4,784,589); synthetic medium B2 (P. Van Hemert; Prog. Indust. Microbiol.; (Bull, M. J. ed.), Vol 13, p.151, Elsevier Sci., Amsterdam (1977)) or described modifications thereof.

For growth of B. pertussis culture according to the present invention, an inoculum is added to a suitable liquid medium and fermentation is conducted as hereinbefore described. Persons skilled in the art will appreciate that results may vary depending upon fermenter design, the selected fermentation nutrient medium supplemented with PVA, method and fermentation parameters. The preferred nutrient medium for use in the present invention is Stainer-Scholte medium.

A PVA concentration in the range 0.5 to 2.5 g/1, suitably 1 or 2 g/1 has been found to be most effective in enhancing propagation of PT and FHA.

PVA having the requisite MW and degree of hydrolysis is commercially available from a number of manufacmrers. For example, Aldrich supplies PVA having a MW range 31,000 to 50,000 and a degree of hydrolysis in the range 87 to 89 mol %; Sigma supplies PVA having an average MW of 30,000 to 70,000 and a degree of hydrolysis in the range 87 to 89 mol %; Fluka supplies PVA having a MW of 15,000 and a degree of hydrolysis in the range 86 to 89 mol % and Janssen offers PVA having a specified MW of 22,000 and a specified degree of hydrolysis of 88 mol %.

The PT and FHA may be isolated from the culmre supernatant and purified by known methods. The purified antigen can then be combined optionally with an adjuvant, typically aluminium hydroxide and formulated as a vaccine. The vaccine may advantageously contain pertactin (69kDa omp), which can for example be prepared from the B. pertussis cell grown in accordance with the method of the invention.

The following Examples illustrate the invention, without limitation. Example 1

Preparation of Master/Working Seeds ) Master Seed

The contents of an ampoule containing Bordetella pertussis Tohama strain (Teijin Ltd., Japan) were reconstituted with deionised water (0.5 mL). The resulting suspension was streaked onto five Petri dishes (0.1 mL/dish) containing B.G. agar (Bordet Gengou, Institut Pasteur, Paris) supplemented with 2% defibrinated sheepblood. After incubation at 35 °C for 24 to 48 hours, the surface growth from each Petri dish was resuspended with 3-5 mL of modified Stainer-Scholte medium (J. Clin. Microbiol., 17(5), 781-786, 1983) supplemented with 10% (v/v) glycerol, and the suspensions were pooled. Aliquots (0.3 mL) of the pooled suspension were placed in plastic cryotubes and immediately frozen and stored at -70°C.

h) Working Seed A plastic cryotube containing Master Seed was thawed to room temperamre and the contents were streaked into each of two glass tubes containing modified S.S. medium supplemented with agar. After incubation at 35 °C for 24 hours, the surface growth from each tube was streaked into two larger glass tubes, each containing modified S.S. medium (35 mL) supplemented with agar. After further incubation at 35 °C for 24 hours, the surface growth from each tube was resuspended with modified S.S. medium (3 mL) and added to an Erlenmeyer flask (IL) containing modified S.S. medium (150 mL). Each flask was placed on an orbital shaker and incubated at 35 °C for 24 hours. The contents of the four flasks were pooled and the suspension was centrifuged at 8000 rpm at 4°C for 15 minutes. The supernatant was discarded and the pellet was resuspended in modified S.S. medium (150 mL) supplemented with 10% (w/v) glycerol. Aliquots (0.4 mL) of the suspension were placed in plastic cryotubes and immediately frozen and stored at -70°C.

F amp.e 2 Preparation of Inoculum Liquid Preculture

Working Seed as prepared in Example 1 (0.08 ml) was incubated at 35°C for 24 hours on solid medium (10 ml) in the form of modified Stainer-Scholte medium supplemented with agar. Surface growth was streaked onto the same medium (35 ml) and incubated at 35°C for 24 hours. Surface growth was suspended with modified Stainer-Scholte medium (3 ml) and added to a Roux bottle (1 litre) containing modified Stainer-Scholte medium (100 ml) supplemented with agar. The Roux bottle was incubated at 35 °C for 24 hours. The surface growth from each Roux bottle was resuspended in modified Stainer- Scholte medium (6 mL) and added to an Erlenmeyer flask (5L) containing modified S.S. medium. PVA having a MW and degree of hydrolysis according to the process of the invention was present in the medium in place of β-2,6-0 dimethyl cyclodextrin, at a final concentration of 2 g/litre. The flasks were incubated at 35 °C for 24 hours. The contents of the flasks were pooled to provide the inoculum for 20 L fermenters.

Example 3 Growth of Bordetella pertussis in 20 Litre Fermenter

Inoculum as prepared in Example 2 was added to a fermenter (Biolafitte, Poissy, France) containing 13 litres of filter-sterilised modified Stainer-Scholte medium containing an appropriate PVA, at a final concentration of 2 g/litre. The pH was adjusted to and maintained at 7.2 by periodic addition of acetic acid (50 %) and the temperamre was adjusted to and maintained at 35°C. Dissolved oxygen was maintained at 20-30 % of saturation by adjustment of the agitation speed and aeration rate. A mechanical foambreaker was used to disperse foam generated during the fermentation. Samples of culmre (10-20 ml) were removed periodically from the fermenter throughout the duration of the cultivation (30-40 hours) to determine the mrbidity (optical density at 650 nm) and to follow the growth kinetics.

The concentrations of both PT and FHA in pre-filtered samples of culture were determined in duplicate by the ELISA technique. The test was performed in the presence of two reference samples of PT and FHA of known concentration in order to verify the accuracy of the test. The precision of the test was, on average +/- 20 % of the correct value.

The identity and quality of PT and FHA in pre-filtered samples of culture were determined by Western blot. The blot of PT showed the 5 PT sub-unit bands as major components at apparent MW's 29 kDa, 25 kDa, 24 kDa, 14 kDa and 9 kDa. The blot of FHA showed the major band at an apparent MW of 220 kDa together with several degradation bands in the region 100 to 200 kDa.

Example 3

Concentrations of PT and FHA using PVA (2 g/litre^ in a 20 litre Fermenter

The results obtained using a range of commercially available PVA materials having different MW's and degrees of hydrolysis are given in the following Table. Results from PVA materials falling outside the scope of invention are included for comparison. The concentrations of PT and FHA are those measured in the final sample from the fermenter in mid-stationary phase, ie. at the end of the cultivation period.

Production of PT and FHA with PVA (2 g/L) in 20 L fermenter

MW Degree of Origin Ferm Final Final Average Average range hydrolysi id PT FHA cone final PT final FHA (OOO) s (mol%) BPC cone (mg/L) cone cone

(mg L) (mg/L) (mg L)

9-10 80 Aldrich 435 22 187 22 187

15 86-89 Fluka 269 26 70

425 19 124 22.5 97

13-23 87-88 Aldrich 437 15 179 15 179

13-23 99 Aldrich 436 15 1 1 15 1 1

22 88 Janssen 418 18 106

422 24 182 21 144

22 97.5-99.5 Fluka 415 13 3 416 14 7 13.5 5

22 >98 BDH 438 19 4 19 4

31-50 87-89 Aldrich 419 22 119 421 21 116 21.5 1 17.5

31-50 99 Aldrich 434 22 9 22 9

49 86-89 Fluka 406 8 30 8 30

30-70 87-89 Sigma 264 21 95 384 13 90 17 92.5

50 99 Aldrich 427 10 3 10 3

72 97.5-99.5 Fluka 423 1 1 5 1 1 5

95 85 Janssen 424 14 25 14 25

100 86-89 Fluka 407 8 20 8 20

124-186 99 Aldrich 408 18 3 18 3

Claims

1. A process for commercial scale production of B. pertussis, comprising propagating the bacterium in an aqueous nutrient medium under submerged aerobic conditions with mechanical action and supplementing the media with polyvinyl alcohol (PVA) having a molecular weight in the range of 10,000 to 50,000 and a degree of hydrolysis in the range 80 to 90 mol% .

2. A process as claimed in claim 2, wherein the degree of hydrolysis is in the range 85 to 90%.

3. A process for the production of high yields of B. pertussis Pertussis Toxin (PT) and Filamentous hemagglutinin (FHA), comprising cultivating B. pertussis according to claim 1 and isolating the PT and FHA.

4. A process for the production of a vaccine comprising Pertussis Toxin and Filamentous hemagglutinin capable of affording protection against B. pertussis infections, comprising propagating B. pertussis in an aqueous nutrient medium under submerged conditions with mechanical action and supplementing the media with polyvinyl alcohol having a molecular weight in the range of 10,000 to 50,000 and a degree of hydrolysis in the range 80 to 90 mol%, isolating the Pertussis Toxin and Filamentous hemagglutinin, and formulating as a vaccine, optionally with one or more of pertactin, or fimbrial agglutinogens.