Freeze-dried live Bordetella vaccine

Abstract

This invention provides a method for producing a freeze-dried vaccine containing attenuated Bordetella bacteria. [Solution] A method for producing a freeze-dried vaccine includes the steps of: collecting Bordetella bacteria from a culture; mixing the collected Bordetella bacteria with a freeze-drying buffer; freeze-drying the mixture of Bordetella bacteria and the freeze-drying buffer, wherein the holding time between the collection step and the freeze-drying step is less than 48 hours, and the freeze-drying step includes a pre-crystallization holding step, in which the mixture of Bordetella bacteria and the freeze-drying buffer is held for 0.5 to 10 hours at a temperature 0.1 to 10°C higher than the crystallization temperature of the mixture before further cooling; and collecting the freeze-dried Bordetella bacteria.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 066,020, filed on 14 August 2020.

[0002] Description of research funded by the federal government. Not applicable

[0003] The present invention relates as a whole to the fields of microbiology, vaccines, and lyophilization, and more particularly to a method for lyophilizing Bordetella bacteria and a lyophilized preparation produced according to such a method. [Background technology]

[0004] BPZE1 is a live attenuated Bordetella pertussis strain previously developed for use in a pertussis vaccine. (See U.S. Patent No. 9,180,178). This vaccine strain was constructed by genetically removing cutaneous necrotic toxin, reducing tracheal cytotoxin to background levels, and inactivating pertussis toxin. In non-human primate models, a single intranasal dose of BPZE1 was found to provide robust protection against both pertussis disease and infection after exposure (challenge) to a recently clinically isolated, highly pathogenic Bordetella pertussis strain. BPZE1 is currently in clinical development, and two Phase I trials have already been successfully completed, demonstrating that the vaccine is safe in adult volunteers, capable of transient colonization in the human nasal cavity, and capable of inducing an antibody response to Bordetella pertussis antigens. The liquid formulation of BPZE1 used in these previous studies needs to be stored at -70°C to maintain bacterial survival. Since most point-of-care facilities do not have ultra-low temperature freezers, this requirement is hindering the future commercialization of BPZE1-based vaccines. [Overview of the Initiative]

[0005] This specification describes a lyophilized Bordetella preparation and a method for producing such preparation, which is stable for at least two years when stored at a temperature of -20°C to 22.5°C and exhibits sufficient bacterial viability and efficacy for use as a live vaccine. Prior to the research described herein, it was unclear whether such lyophilized preparations could even be produced, as successfully lyophilizing biological molecules, particularly live bacteria, is a challenging endeavor for several reasons. Firstly, components used in bacterial culture may destabilize bacterial molecules even after lyophilization. Secondly, bacterial viability may be impaired during the lyophilization process due to interactions at the air / liquid interface and the solution / ice interface. Thirdly, bacterial aggregation / clumping often occurs, leading to reduced function or viability. Fourthly, crystal (ice) formation may kill bacteria. And fifthly, dehydration may destabilize the structure and activity of proteins.

[0006] Furthermore, large-scale lyophilization of Bordetella-based vaccines (e.g., BPZE1-based vaccines) presents additional challenges. For example, Bordetella species produce numerous virulence factors that enable binding to epithelial cells, but these factors also cause bacteria to adhere to each other when grown at high cell density in a bioreactor, thereby exacerbating the decline in function / viability due to aggregation and biofilm formation. Aggregation or biofilm formation can result in heterogeneous products, leading to significant loss of product on the filter during the tangential flow filtration (TFF) step. Aggregation can be avoided by increasing agitation in the bioreactor, but the resulting increase in shear stress can lead to a decline in viability. Also, the longer the time from the collection step to the start of lyophilization, the greater the loss of viability. In large-scale production where collection, concentration, formulation, and subsequent filling into lyophilized vials may take longer than 20 hours, a significant decline in viability generally occurs. Moreover, Gram-negative bacteria such as Bordetella are particularly susceptible to viability decline during the freezing step of the lyophilization process. In particular, BPZE1 has a thinner cell wall than its parent wild-type strain, possesses a mutated pertussis toxin gene (ptx), lacks a skin necrosis gene (dnt), and has a heterologous ampG gene that replaces the native Bordetella ampG gene, which can affect the bacterium's ability to withstand freeze-drying. See U.S. Patents 9,180,178.

[0007] Therefore, described herein is a method for producing a lyophilized vaccine containing attenuated live Bordetella bacteria as the active agent. The method is based on an OD of 0.4 to 1.6 600Steps of collecting Bordetella bacteria from the culture; mixing the collected Bordetella bacteria with a lyophilization buffer containing 5 - 65% by weight of cryoprotective sugar and having a temperature of 2 - 35°C, wherein the ratio of the collected Bordetella bacteria to the lyophilization buffer is 5:1 to 1:5 by volume; freeze-freezing the mixture of Bordetella bacteria and the lyophilization buffer, wherein the holding time between the collection step and the lyophilization step is less than 48 hours (e.g., less than 36 hours); and collecting the lyophilized Bordetella bacteria can be included. The Bordetella bacteria can be a pertussis strain such as the BPZE strain (e.g., BPZE1). In some variants of the method, the Bordetella bacteria are derived from a culture with an OD 600 of 0.4 - 1.0, or less than 1.0. The cryoprotective sugar can be sucrose, and the lyophilization buffer can contain a nutrient substrate such as glutamate.

[0008] The lyophilization step can include a pre-crystallization holding step, wherein the mixture of Bordetella bacteria and the lyophilization buffer is held at a temperature 0.1 - 10°C higher than the crystallization temperature of the mixture for 0.5 - 10 hours before being further cooled. The method of the present invention can also be characterized by a step of concentrating the collected Bordetella bacteria to an OD 600 of 1.0 - 30.0.

[0009] Also described herein is a lyophilized vaccine product containing live attenuated Bordetella bacteria produced according to the methods described above and elsewhere in this specification. The lyophilized vaccine product can have a shelf life of at least 2 years when stored at 22.5°C, and at least 20% of the cells in the product are maintained in a viable state even after the lyophilization step. Further, the collected lyophilized bacteria in the vaccine product can be characterized by the ability to prevent or suppress the infection of the respiratory tract of a subject (e.g., a mammalian subject such as a human or a mouse) by a pathogenic strain of Bordetella pertussis.

[0010] Unless otherwise defined, all technical terms used herein have the same meaning as those understood by those skilled in the art in which the invention pertains. Similar or equivalent methods and materials to those described herein may be used in carrying out or testing the invention, but preferred methods and materials are described below. All publications, patents, and patent applications referenced herein are incorporated in their entirety by reference. In the event of any conflict, this specification shall prevail, including definitions. Furthermore, the specific embodiments discussed below are illustrative and not intended to be limiting. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 is a series of gel photographs showing PCR analysis of loci of lyophilized Bordetella bacteria (BPZE1 strain of Bordetella pertussis) preparations compared with liquid BPZE1 preparations. Two lots of liquid BPZE1 preparations (lanes 1 and 2) and two lots of lyophilized BPZE1 preparations (lanes 3 and 4) show Escherichia coli (Panel A), Bordetella pertussis ampG (Panel B), Bordetella pertussis dnt flanking region (Panel C), and BPSM wild-type control (Lane 5). [Figure 2] Figure 2 is a graph showing the results of quantitative PCR (q-PCR) amplification of DNA encoding the pertussis toxin (PTX) S1 subunit. The results for the S1 subunit gene of liquid BPZE1 preparation (BPZE1 liquid), lyophilized BPZE1 preparation (BPZE1 lyo), BPSM, and lyophilized BPZE1 (spiked BPSM) are shown. [Figure 3] Figure 3 is a graph showing the microbiological stability (measured in units of CFU) of the liquid BPZE1 formulation at various time points over a two-year storage period at -70°C. The results for the liquid BPZE1 formulation at 107 CFU / dose (middle line, low dose), 108 CFU / dose (top line, middle dose), and 109 CFU / dose (bottom line, high dose) are shown. [Figure 4]Figure 4 is a graph showing the microbiological stability (measured in units of CFU) of the lyophilized BPZE1 preparation over time. A lyophilized BPZE1 preparation with 109 CFU / dose was stored for two years at -20°C ± 10°C (top line), 5°C ± 3°C (middle line), and 22.5°C ± 2.5°C (bottom line), and the CFU was quantified at the indicated points in time. The dotted and solid lines represent the upper and lower limits of the specifications shown in Table 1 below. [Figure 5] Figure 5 is a series of graphs showing the in vivo colonization dynamics of lyophilized BPZE1 formulation compared to liquid formulation in sacrificial BALB / c mice that were administered intranasally with 105 CFU of liquid BPZE1 formulation (black bar) or redissolved lyophilized BPZE1 formulation (gray bar) at 3 hours (day 0), 1 day, or 3 days after administration. Graph A shows a comparison of the CFU count of liquid BPZE1 formulation with the CFU count of redissolved lyophilized BPZE1 formulation administered immediately after lyophilization. Graph B shows a comparison of the CFU count of liquid BPZE1 formulation with the CFU count of lyophilized BPZE1 formulation redissolved after 6 months of storage at -20°C±10°C (light gray bar), 5°C±3°C (medium gray bar), or 22.5°C±2.5°C (dark gray bar). Graph C compares the CFU count of liquid BPZE1 formulations with the CFU count of lyophilized BPZE1 formulations redissolved after 24 months of storage at -20°C±10°C (light gray bars), 5°C±3°C (medium-dark gray bars), or 22.5°C±2.5°C (dark gray bars). Results are expressed as mean + / - SEM. *, p<0.05; **, p<0.01; ***, p<0.005; ns, not significant. [Figure 6]Figure 6 is a series of graphs showing the efficacy of lyophilized BPZE1 compared to liquid formulations in BALB / c mice that were intranasally administered 105 CFU of liquid BPZE1 (black bar), redissolved lyophilized BPZE1 (gray bar), or PBS (white bar) as a mock control, followed by intranasal exposure to 106 CFU of Bordetella pertussis pathogenic strain (BPSM) four weeks later. CFU present in the lungs was quantified at 3 hours (D0) and 7 days (D7) after exposure. Graph A shows a comparison of the efficacy of liquid BPZE1 and redissolved lyophilized BPZE1 administered immediately after lyophilization. Graph B shows a comparison of the efficacy of the liquid BPZE1 formulation with that of the lyophilized BPZE1 formulation redissolved after 6 months of storage at -20°C±10°C (light gray bars), 5°C±3°C (medium-dark gray bars), or 22.5°C±2.5°C (dark gray bars). Graph C shows a comparison of the efficacy of the liquid BPZE1 formulation with that of the lyophilized BPZE1 formulation redissolved after 24 months of storage at -20°C±10°C (light gray bars), 5°C±3°C (medium-dark gray bars), or 22.5°C±2.5°C (dark gray bars). Results are expressed as mean + / - SEM. *, p<0.005. [Figure 7] Figure 7 is a graph showing a comparison of the CFU counts of three different GMP runs after freeze-drying using different methods, as described in the Examples section below. [Modes for carrying out the invention]

[0012] This specification describes lyophilized formulations containing attenuated live Bordetella bacteria as an active agent, which are stable for at least two years when stored at a temperature of -20°C to 22.5°C and exhibit sufficient bacterial viability and efficacy for use as a live vaccine. Methods for producing these lyophilized formulations are also described. The embodiments described below provide representative examples of these formulations and methods. Nevertheless, other aspects of the present invention can be carried out and / or implemented based on the description below, derived from the description of these embodiments.

[0013] A general method for producing a lyophilized preparation containing attenuated live Bordetella bacteria suitable for use as a vaccine. A lyophilized preparation containing attenuated Bordetella bacteria is produced by collecting Bordetella bacteria from a culture during the appropriate growth phase, concentrating the optionally collected Bordetella bacteria from the culture, mixing the concentrated Bordetella bacteria with a lyophilization buffer containing freeze-protecting sugars, and then lyophilizing the mixture of Bordetella bacteria and lyophilization buffer.

[0014] Bacteria of the genus Bordetella The Bordetella bacteria used in the compositions and methods described herein may be any suitable species or strain of the genus Bordetella. Examples of Bordetella species include Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchoseptica. Preferred Bordetella bacteria are those that exhibit vaccine activity against infectious diseases (e.g., pertussis) or have other beneficial prophylactic or therapeutic effects (e.g., reduction of inflammation or treatment of allergies). Numerous attenuated Bordetella pertussis strains have been developed that are effective in preventing or mitigating pathological conditions associated with pertussis or other infectious diseases, or that have other beneficial prophylactic or therapeutic effects, and are preferred for use in the methods and compositions described herein.These include BPZE1 (described in U.S. Patent No. 9,180,178 and deposited on March 9, 2006, at the Collection Nationale de Cultures de Microorganismes in Paris, France, under CNCM I-3585) and its variants, such as BPZE1 (described in U.S. Patent No. 9,528,086) modified to express a hybrid protein containing the N-terminal fragment of filamentous hemagglutinin (FHA) and a heterologous epitope or antigenic protein or protein fragment, and BPAL10 (described in U.S. Patent No. 10 / 369,207 and deposited on October 23, 2015, at the National Measurement Institute, 1 / 153 Bertie Street, Port Melbourne, Victoria, Australia). Adenylyl cyclase-deficient BPZE strains such as 3207 (deposited with accession number V15 / 032164) and BPZE1AS (described in International Publication No. 2020049133 and deposited with the Collection Nationale de Cultures de Microorganismes on September 4, 2018, with accession number CNCM 1-5348), partactin-deficient BPZE strains such as BPZE1-P (described in U.S. Patent No. 11,065,276 and deposited with the Collection Nationale de Cultures de Microorganisms on December 12, 2016, with accession number CNCM-I-5150), and BPZE1f3 (described in U.S. Patent Application No. 16 / 848,793 and deposited with the Collection Nationale de Cultures de Microorganisms on October 11, 2017, with accession number CNCM This includes various BPZE strains, such as Fim2- and Fim3-producing BPZE strains (deposited under I-5247).

[0015] Pretreatment of Bordetella bacteria for freeze-drying The method for producing a lyophilized vaccine product containing attenuated Bordetella bacteria begins with culturing, followed by collecting Bordetella bacteria from a bioreactor. Suitable culture media and conditions are described in the Examples section below. The collection of cultured bacteria is performed using standard methods. Unexpectedly from early studies, Bordetella bacteria such as BPZE1 have been shown to be particularly prone to aggregation / clumping during culture. Therefore, to avoid the decrease in viability due to this aggregation / clumping, the culture is OD (Oxidation Discharge). 600 However, it is preferable to collect when the concentration reaches 0.4-1.6; 0.5-1.5, 0.6-1.4, 0.7-1.3, 0.8-1.2, 0.9-1.1, 1.0, or less than 1.0 (e.g., 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9). After collecting Bordetella bacteria, they may be optionally concentrated (e.g., to meet final CFU / dosage requirements) and / or subjected to diafiltration for desalting or buffer exchange. For example, the collected Bordetella bacteria should be subjected to an OD of 1.0-30.0 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30+ / -0, 0.1, 0.2, 0.3, 0.4, or 0.5) before the mixing step. 600It can be concentrated. After collection and concentration / diafiltration (if carried out), the bacteria are mixed with a suitable lyophilization buffer. When mixing with the bacteria, the lyophilization buffer is generally at a temperature of 2 to 35 °C (e.g., 4 to 30 °C, 8 to 25 °C, 10 to 20 °C, or 4 ± 1, 2, or 3 °C). A suitable cryoprotectant is included in (or added in the mixing step to) the lyophilization buffer at a weight ratio of 5 to 65% (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 ± 0, 1, 2, 3, 4, or 5%). Based on the comparison of various cryoprotective substances, cryoprotective sugars (especially sucrose) are preferred. The ratio of the Bordetella bacteria in the mixture to the lyophilization buffer is 5:1 to 1:5 (e.g., 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 4:1 to 1:4, 3:1 to 1:3, or 2:1 to 1:2) by volume. The time from collection to the start of lyophilization should be less than 48 hours (e.g., less than 44 hours, 40 hours, 36 hours, 32 hours, 28 hours, 24 hours, 20 hours, or 16 hours) to avoid a significant decrease in viability.

[0016] Lyophilization Next, the prepared mixture of bacteria and lyophilization buffer is dispensed into a lyophilization container (e.g., a glass vial), 5 × 10 6 ~1 × 10 10 (e.g., 1 × 10 6 , 5 × 10 6 , 1 × 10 7 , 5 × 10 7 , 1 × 10 8 , 5 × 10 8 , 1 × 10 9 , 2 × 10 9 , or 3 × 10 9Bacteria in a CFU of + / - 10, 20, 30, 40, or 50%) are added. The filled container is then placed in a freeze-dryer to initiate the freeze-drying process. Primary drying can be carried out at a suitable pressure (e.g., 50–250 microbars or 100+ / - 0, 10, 20, 30, 40, 50, 60, 70, 80, or 90 microbars) in the range of -40–0°C (e.g., 34°C). Typically, this primary drying step is continued until the readings of the Pirani analyzer and capacitance manometer converge, indicating that sublimation is complete. After primary drying, the temperature can be increased over several hours (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 hours) to a secondary drying temperature of, for example, +20 to +40°C (e.g., +30 ± 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C) from the primary drying temperature, and then the temperature can be held at the secondary drying temperature until the pressure rise increases to less than 10 microbars (indicating that the product has dried) after the valve is closed to the condenser chamber. The container is then stoppered, cooled (e.g., to +4°C), unloaded, and then capped (e.g., with an aluminum cap).

[0017] For large-scale production, the freeze-drying step preferably includes a pre-crystallization holding step to reduce vial-to-vial viability. Ice crystal formation means an increase in the molar concentration of dissolved components in the freeze-drying buffer, including salts. High salt concentrations are more likely to damage the outer membrane of Bordetella pertussis or other bacteria, yeasts, fungi, or viruses, and therefore the time spent in the phase with high salt concentrations should be kept as short as possible. Glass vials are very poor conductors of heat during heating / cooling and typically only contact the freeze-drying rack at three points. During freezing, this poor conductivity leads to uneven cooling of the vials, where some vials begin crystallization, while others remain liquid for longer periods. As described later in the Examples section, very slow cooling of the freeze-drying buffer resulted in rapid ice crystal formation in the vials at a specific temperature above the glass transition temperature (Tg'). Holding at this specific temperature (crystallization point) resulted in most vials exhibiting rapid crystallization within minutes of each other. On the other hand, if the cooling step is not included in the retention period, ice crystal formation may differ by more than an hour among various vials, resulting in significant differences in survival rates between vials.

[0018] The introduction of a pre-crystallization holding step before freezing to Tg' is as follows: The crystallization temperature of a given freeze-drying buffer is determined by slowly cooling the buffer and recording the temperature at which crystallization begins. The pre-crystallization holding step can be defined as a holding step at a temperature 0.1 to several (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8)°C higher than the crystallization point, depending on the size of the freeze-dryer, for 30 minutes to several (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8) hours, depending on the size of the freeze-dryer. [Examples]

[0019] Example 1 - Materials and Methods

[0020] Bacterial strains and growth conditions

[0021] Pathogenic Bordetella pertussis BPSM (Menozzi et al., Infect Immun 1994, 62:769-778) was cultured at 37°C on Bordeaux-Jang (BG) agar containing 100 μg / ml streptomycin, supplemented with 1% glycerol and 10% defatted sheep's blood, as described (Mielcarek et al., PLoS Pathog 2006;2:e65). After growth, the plates were scraped and the bacteria were collected by resuspending them in phosphate-buffered saline (PBS) at the desired density. A BPZE1 vaccine strain (Mielcarek et al., PLoS Pathog 2006;2:e65) Working Cell Bank (WCB) was cultured in fully synthetic Thijs medium (Thalen et al., Biologicals 2006, 34:213-220) with agitation. 20% (vol / vol) of 86% glycerol was added, and 1.5 mL aliquots were filled into cryovials. The whole-body cubic cells (WCBs) were then stored at -70°C until further use, as described (Thorstensson et al., PLoS ONE 2014;9,e83449; and Jahnmatz et al., Lancet Infect Dis 2020,20:1290-1301).

[0022] Fermentation of BPZE1

[0023] 1.5 ml of WCB was inoculated into an Erlenmeyer flask containing 28.5 ml of Thijs medium (Thalen et al., Biologicals 2006, 34:213-220). A second pre-culture, consisting of a 2 L Erlenmeyer flask containing 0.5 L of Thijs medium, was prepared in OD. 600 The cultures were inoculated to a value of 0.1, and then used as inoculum in 5 × 2 L flasks each containing 0.5 L of Thijs medium. The five cultures were pooled and added to a 50 L bioreactor (Sartorius, 50 L SUB) containing 20 L of Thijs medium, with the bioreactor being inoculated to a value of 0.1.600 The process was initiated using [specific method / tool]. Fermentation was carried out at 35°C, with dissolved oxygen controlled to 20% using compressed air supplied from a sparger, and pH controlled to 7.5 using 0.2M lactic acid. All product contact materials, including culture and medium flasks, containers, tubing, filters, connectors, and the bioreactor, were single-use. Target OD 600 After reaching 1.1-1.4, the 8L culture sample is filtered through hollow fiber tangential flow filtration (TFF; 750kDA mPES membrane 1400cm²) at a maximum intermembrane pressure of 0.3 bar. 2 Using Spectrum, the specified OD 600 It was concentrated and / or diafiltration.

[0024] Freeze-drying of BPZE1

[0025] Initial culture and lyophilization development led to the development of lyophilization buffers and small-scale lyophilization cycles. In all large-scale cultures, lyophilization buffer cooled to +4°C was added to the bacterial suspension in a 1:1 ratio using 100 g / L sucrose as the primary cryoprotectant. The resulting formulation was then filled into 13 mm DIN 2R vials with bromobutyl lyophilization stoppers and lyophilized using a conservative cycle consisting of primary drying at -34°C and 100 microbars until the readings on the Pirani meter and capacitance manometer converged, indicating that sublimation was complete. After primary drying, the temperature was raised from -34°C to +30°C over 12 hours, followed by holding the temperature at 30°C until the pressure rise increased to less than 10 microbars after closing the valve to the condenser chamber, indicating that the product was dry. After stoppering, the vials were cooled to +4°C until unloaded, and then capped with aluminum caps.

[0026] Plate count

[0027] Colony-forming units (CFUs) were counted by plating 1, 2, and 5 10-fold dilutions of the formulation sample onto Bordeaux-Jang agar plates supplemented with 15% sheep blood. All dilutions were plated in triplicates, resulting in an average of 9 plates counted to obtain one result. The specification of the formulation after lyophilization was 0.2–4.0 × 10⁶. 9 Set to CFU / ml.

[0028] Microbiological safety testing of active pharmaceutical ingredients (APIs) and drug formulations. The absence of Staphylococcus aureus, Pseudomonas erginosa, and bile acid-resistant organisms was confirmed in the United States Pharmacopeia, Test 62 (USP). <62> The purity of both the active pharmaceutical ingredient and the finished product was tested according to the United States Pharmacopeia (USP42-NF37, 2019). <61> The tests were conducted according to the specifications. All runs met both USP safety tests.

[0029] Implantation and efficacy assay in mice

[0030] BALB / c mice were purchased from Charles Rivers and reared in an animal facility under conditions free from specific pathogens. In the colonization assay, various BPZE1 suspensions were administered at a rate of 10 per 20 μl. 5 Diluted in CFU, it was administered intranasally to 6-week-old BALB / c mice. Three hours, 24 hours, or three days after infection, mice were sacrificed, and nasal homogenates were prepared as described (Solans et al., Mucosal Immunol 2018, 11:1753-1762). These were then plated onto BG blood agar plates in 10-fold serial dilutions and incubated at 37°C for 3-5 days, with colonization quantified by CFU counting. To determine the efficacy of various BPZE1 preparations, 10 doses were administered to 6-week-old wild BALB / c mice as described (Debrie et al., Vaccine 2018, 36:1345-1352). 5CFU BPZE1 was administered intranasally, or PBS was administered intranasally. After 4 weeks, mice were given 10 6 Intranasal exposure was administered to pathogenic BPSM from CFU. Lung colonization was assessed 3 hours and 7 days after exposure.

[0031] Genetic stability assay

[0032] The genetic stability of various BPZE1 preparations was evaluated by polymerase chain reaction (PCR) targeting the dnt and ampG genes, as described (Feunou et al., Vaccine 2008, 26:5722-5727). The pertussis toxin (PT) S1 subunit gene ptxA was analyzed by quantitative PCR (Mielcarek et al., PLoS Pathog 2006;2:e65) to ensure there was no reversal of the two codon changes introduced to inactivate PT. Approximately 10 10 The BPZE1 preparation of CFU was collected by centrifugation, suspended in buffer B1 (Qiagen, #19060) containing RNase A and proteinase K, and incubated at 37°C for 30 minutes. The bacteria were then lysed in lysis buffer at 50°C for 30 minutes and loaded onto a Qiagen genomic-tip100 / G column.

[0033] After washing and eluting according to the manufacturer's recommendations, the DNA was precipitated with isopropanol (CarloErba), centrifuged at 5,000 × g for 15 minutes, washed with ice-cold 70% ethanol, air-dried for 10 minutes, and resuspended in 100 μl of redistilled water. DNA concentration was measured using a NanoDrop2000c spectrophotometer. 7BPZE1, BPSM, or BPZE1 DNA spiked with BPSM, equivalent to a genome copy, was mixed in 19 μl of LightCycler480 SYBR GreenI master mix containing 0.5 μM primer pairs in a 96-well LightCycler480 plate. The plate was sealed with a dedicated plastic film and transferred to a LightCycler 480. The cells were incubated at 95°C for 15 minutes, followed by denaturation at 95°C for 15 seconds, annealing at 68°C for 8 seconds, and extension at 72°C for 18 seconds, repeated 1 to 40 times. The data was then analyzed using LightCycler480 software release 1.5.0. 10 6 To adjust the sensitivity of an assay that can detect one potential reversion from a genome copy, 10 copies of BPSM DNA were used. 7 I mixed in copies of BPZE1 DNA. All primers were purchased from Eurogentec (Liège, Belgium).

[0034] Example 2 - Results

[0035] Development of BPZE1 formulations

[0036] Bordetella pertussis has the ability to form biofilms by producing numerous virulence factors that enable not only binding to each other but also to binding to epithelial cells. In bioreactors, biofilm formation leads to bacterial aggregation, and therefore to inherently inhomogenous vaccine formulations. Aggregation in bioreactors can be avoided by increasing agitation, but excessive shear stress during fermentation or ultrafiltration can lead to cell damage and low viability after lyophilization. In a 20L bioreactor with 8L of medium and a 6-blade Rushton impeller running at 400 RPM, the viability after lyophilization did not exceed 45%, while in a 50L bioreactor with 20L of medium and a 3-blade Marine impeller running at 150 RPM, the viability after lyophilization was up to 65% under similar conditions (data not shown).

[0037] In an 8L bioreactor, the suspension OD was 0.5. 600 Although almost no aggregation was observed, OD >1.0 600 In comparison, the survival rate after freeze-drying was poor. Therefore, all subsequent cultures were OD 1.1-1.6. 600 These were collected. 600 This is the maximum OD, well before all the culture medium substrate is consumed. 600 This represents approximately 50-80% of the total, and as a result, the bacteria were in a physiological state that resulted in a high survival rate after freeze-drying. It was found that adding cold freeze-drying buffer is preferable to stop cellular metabolism on the freeze-drying shelf during the period from collection to freezing.

[0038] To minimize the influence of bioreactor and TFF geometry on post-freeze drying viability, all 50L bioreactors were operated under the same conservative conditions during fermentation and ultrafiltration, with a compromise to minimize shear stress while avoiding aggregation.

[0039] Development of freeze-drying buffers

[0040] The development of a manufacturing method for the formulation consisted of developing a lyophilized formulation including a lyophilized buffer and a matching lyophilized cycle, and verifying that the developed method does not inhibit the biological activity of the BPZE1 formulation. It was particularly important that the formulation maintain its ability to reduce the bacterial load in the lungs by at least two orders of magnitude in mouse defense assays. The attributes of the target formulation are shown in Table 1.

[0041] TIFF2026086426000001.tif170170

[0042] The lyophilization buffer formulations were based on commonly used cryoprotective agents containing 5–10% sucrose or trehalose, sometimes combined with other cryoprotective agents such as hydroxyethyl starch (HES) or monosodium glutamate (MSG). All formulations shown in Table 2 were generated using a single bacterial suspension. All formulations exhibited the target residual water content (RMC) of less than 2.5% and the target glass transition temperature (Tg) above 35°C. Sucrose appeared to be a superior cryoprotective agent to trehalose when used alone. Adding HES or MSG to trehalose or sucrose did not increase survival rates. Repeated experiments using sucrose and trehalose showed similar results, although absolute survival percentages varied between experiments. Therefore, 10% sucrose was selected for further development.

[0043] TIFF2026086426000002.tif96170 1 HES, hydroxyethyl starch; RMC, residual water content; Tg, glass transition temperature 2 The survival rate is expressed as a percentage of CFU, comparing the contents of the vial before and after lyophilization.

[0044] Table 3 outlines various runs performed using the same type of bioreactor. It describes manufacturing methods including direct dilution of the culture in lyophilized buffer, concentration and diafiltration of the culture, followed by dilution and concentration in lyophilized buffer, and further dilution in lyophilized buffer. Various diafiltration buffers, including Thijs medium without NaCl or Tris and those without Thijs supplements, were tried and achieved varying degrees of success for washing the concentrated bacterial suspension. The primary reason for diafiltration of the BPZE1 drug substance was to reduce the salt content from the medium, specifically 1.66 g / L NaCl and 0.765 g / L Tris. The presence of these salts slowed the lyophilized cycle compared to the salt-free state. However, some degree of agglutination was observed in all concentrated and diafiltrationd drug substances (Table 3).

[0045] TIFF2026086426000003.tif195170 1 Run 1 involved filling <50 vials and initiating lyophilization with a holding time of <6 hours. 2 Run 2 involved filling <700 vials and initiating lyophilization with a holding time of <16 hours. 3 Runs 3-7 were collected in 2000-7000 vials per formulation, lyophilized, and lyophilization was initiated after a holding period of 24-36 hours. 4 ) Direct dilution: 1:1 dilution of the culture with lyophilized buffer. 5 Concentration and diafiltration: Concentration of the culture, followed by diafiltration and 1:1 dilution with lyophilized buffer. 6 Concentration: Concentration of the culture followed by a 1:1 dilution with lyophilized buffer.

[0046] Thijs medium is chemically defined and consists of components generally considered safe. Therefore, from a quality standpoint, it is not necessary to remove these components from the BPZE1 formulation. Cultures directly diluted with lyophilized buffer (Table 3, Runs 1a and 6b), or cultures concentrated and then diluted with lyophilized buffer (Table 3, Run 7), showed no signs of agglutination immediately after collection or immediately before packing. To achieve the CFU target of the formulation, the cultures were prepared with an OD of 5.0. 600 The bacterial suspension was concentrated to this extent, and then diluted 1:1 with cold lyophilized buffer (Table 3, Run 7).

[0047] The retention time from collection to the start of lyophilization significantly affected bacterial viability both before and after lyophilization. In the first run, a high post-lyophilization viability of 64% was observed using a 1:1 direct dilution of the culture with lyophilization buffer (Table 3, Run 1a), while diafiltration cultures showed viability of 46% and 47% (Table 3, Runs 1b and 2). These formulations were lyophilized within 16 hours of collection and formulation, whereas all subsequent runs were lyophilized 26–32 hours after collection. In Runs 6b and 7, bacterial viability in the formulations was tested immediately after formulation and after storage at +4°C for 48 hours. Both formulations showed a reduction of approximately half in CFU, which explains the relatively low viability of 18% and 23% in Runs 6b and 7, respectively (Table 3). Therefore, the pre-lyophilization storage period had a significant impact on post-lyophilization viability, as otherwise, the viability between Run 1a and Run 7 would likely have been more similar.

[0048] Genetic comparison of liquid BPZE1 formulations and lyophilized BPZE1 formulations

[0049] The lyophilized formulation was compared with the liquid formulation stored at -70°C to confirm the conservation of mutations introduced into the Bordetella pertussis genome to produce BPZE1, particularly the deletion of the dnt gene, the substitution of the Bordetella pertussis ampG gene with the E. coli ampG gene, and the presence of two mutant codons in the PT S1 subunit gene. The first two gene modifications were verified by PCR as described in Feunou et al., Vaccine 2008, 26:5722-5727. The presence of the E. coli ampG gene was detected by amplification of a 402 bp fragment corresponding to the internal fragment of the E. coli ampG gene. The two lyophilized BPZE1 formulations and the two liquid BPZE1 formulation controls produced the expected 402 bp fragment, which was not observed in the BPSM control sample (Figure 1A). Conversely, a 659 bp fragment corresponding to the Bordetella pertussis ampG gene was amplified in the BPSM control sample but not in any of the BPZE1 formulations (Figure 1B). This indicates that both the liquid and lyophilized BPZE1 formulations lacked Bordetella pertussis ampG, while containing Escherichia coli ampG. The deletion of the dnt gene was indicated by amplification of a 1,511 bp fragment obtained from PCR using primers adjacent to the deleted dnt gene. The expected 1,511 bp fragment was obtained in the two lyophilized BPZE1 formulations and the two liquid BPZE1 control samples, but not in the BPSM control sample (Figure 1C).

[0050] To verify the presence of two mutant codons in the PT S1 gene, 10 6 We developed a quantitative PCR method that can detect one copy of the wild-type gene among the mutant genes. For this purpose, 10 7 10 copies of BPZE1 DNA and 10 copies of BPSM DNA were spiked. 7 Copies of BPZE1 DNA were subjected to qPCR using BPSM or BPZE1-specific oligonucleotides. 10 7A copy of BPSM DNA was used as a control. The positivity threshold was set at 35 cycles of qPCR. Lyophilized and liquid BPZE1 formulations showed indistinguishable amplification patterns. Specifically, no amplification was observed with BPSM-specific primers, but when BPZE1-specific primers were used, the amplified product was detected with a Cp value of 12.21–13.32. In contrast, BPSM DNA was amplified with BPSM-specific primers but not with BPZE1-specific primers, while spiked BPZE1 DNA was amplified with both primer pairs (Figure 2). These results suggest that BPZE1 preserves codon modifications and is 1 / 10 6 This indicates that there was no reversal at a higher frequency.

[0051] Microbiological stability

[0052] The stability of the liquid BPZE1 formulation stored at -70°C was tested for 10 7 (Low dose), 10 8 (Medium dose), and 10 9 Three different formulations with varying CFU / dose (high dose) were stored at -70°C for two years and tracked. As shown in Figure 3, the liquid BPZE1 formulations stored at -70°C remained stable for at least two years at each tested dose.

[0053] 10 9 The microbiological stability of lyophilized BPZE1 formulations prepared at CFU / dose was tested at -20°C±10°C, 5°C±3°C, and 22.5°C±2.5°C. As shown in Figure 4, at all tested temperatures, 10 9The lyophilized BPZE1 formulations, measured by CFU / dose, met the CFU specifications and remained suitable even after storage at 22.5°C ± 2.5°C for at least two years. No decrease in CFU was observed in formulations stored at -20°C ± 10°C or 5°C ± 3°C. However, formulations stored at 22.5°C ± 2.5°C showed a slight decrease in viability during the first few months of storage, but remained stable for at least two years thereafter. Even in this case, the CFU count remained within specifications. Stability data from Run 7, obtained by concentrating the culture and diluting it with lyophilized buffer, were similar to those from Run 6, despite higher CFU counts due to the concentration step before adding the lyophilized buffer.

[0054] biological stability

[0055] The biological stability of lyophilized BPZE1 formulations was evaluated using two different mouse assays: an in vivo immobilization assay and a efficacy assay. In each of these assays, the performance of BPZE1 formulations stored at various temperatures was compared to the performance of the original liquid BPZE1 formulation stored at -70°C.

[0056] To quantify the dynamics of in vivo colonization, mice were subjected to approximately 10 doses of either a redissolved lyophilized BPZE1 preparation stored at various temperatures or a liquid BPZE1 preparation control. 5CFU was inoculated intranasally. Mice were sacrificed 3 hours, 1 day, and 3 days after administration, and the CFU present in nasal homogenates was counted. First, the effects of lyophilization and the composition of the lyophilization buffer were investigated by comparing the liquid formulation with the lyophilized formulation immediately after lyophilization. As shown in Figure 5A, there was no statistically significant difference between the liquid and lyophilized formulations, so both formulations immobilized equally in the mouse nasal cavity. Next, the lyophilized formulations were stored for 2 years at -20°C±10°C, 5°C±3°C, or 22.5°C±2.5°C, and the pharmacokinetics of immobilization after 6 months (Figure 5B) and 24 months (Figure 5C) were evaluated and compared with the pharmacokinetics of the liquid formulation. After 6 months of storage, the substance stored at -20°C±10°C showed slightly better adhesion on day 0 and immobilized earlier than the substance stored at other temperatures, and this difference was no longer detectable after 3 days (Figure 5B). However, after 24 months of storage, the lyophilized formulations stored at 5°C±3°C and 22.5°C±2.5°C showed slightly less adhesion on day 0 and slightly slower fixation on days 1 and 3 after administration compared to the formulation stored at -20°C±10°C (Figure 5C).

[0057] To evaluate the efficacy of BPZE1 formulations after storage at various temperatures, 10 5Mice were intranasally immunized with a lyophilized BPZE1 formulation or a BPZE1 liquid formulation control containing rehydrated CFUs, followed by intranasal exposure to pathogenic BPSM. Lung bacterial loads were assessed in sacrificial mice 3 hours or 7 days after BPSM exposure. First, the liquid formulation was compared to the lyophilized formulation tested immediately after lyophilization. Both formulations provided equivalent protection to mice, with lung CFUs decreasing by two orders of magnitude between day 0 (3 hours) and day 7 after exposure, while the cell load in the lungs of unvaccinated mice increased between day 0 and day 7 (Figure 6A). Storage of the lyophilized formulations for 6 months at all tested temperatures did not affect vaccine efficacy, and 7 days after exposure, unvaccinated mice carried approximately 10 times more BPSM bacteria in their lungs than 3 hours after infection, while all vaccinated mice had approximately 100-fold reductions in lung CFUs compared to unvaccinated controls (Figure 6B). No statistically significant difference was observed between mice immunized with liquid BPZE1 and those immunized with lyophilized BPZE1, and no effect of storage temperature was detected. Therefore, lyophilized BPZE1 preparations stored at 5°C±3°C or 22.5°C±2.5°C showed slightly lower adhesion on day 0 and slower colonization in the mouse nasal cavity on day 1 compared to preparations stored at -20°C±10°C, but after 6 months of storage, there was no effect on the ability of the lyophilized preparation to provide protection against BPSM exposure.

[0058] After 24 months of storage, lyophilized formulations stored at 5°C±3°C or 22.5°C±2.5°C showed a slight but significant decrease in efficacy compared to lyophilized formulations stored at -20°C±10°C (Figure 6C). However, compared to unvaccinated mice, mice that received formulations stored at 5°C±3°C or 22.5°C±2.5°C still showed an almost 1000-fold reduction in lung bacterial load.

[0059] In summary, these data indicate that after storing the lyophilized BPZE1 formulation between -20°C ± 10°C and 22.5°C ± 2.5°C for at least two years, the lyophilized BPZE1 maintained within specifications its ability to colonize the nasal cavity and protect mice from exposure to pathogenic Bordetella pertussis.

[0060] Consideration

[0061] Previous studies have shown that a single intranasal administration of BPZE1 provides protection against Bordetella pertussis exposure in mice (Mielcarek et al., PLoS Pathog 2006;2:e65; and Solans et al., Mucosal Immunol 2018,11:1753-1762) and non-human primates (Locht, et al., J Infect Dis 2017,216:117-124), and that it is safe even in severely immunodeficient animals such as IFN-γ receptor knockout mice and MyD88-deficient mice. In two Phase 1 clinical trials, BPZE1 has been shown to be safe and immunogenic in humans.

[0062] All previous preclinical and clinical trials have been conducted using a liquid formulation of BPZE1, and that formulation is 10 7 CFU / ml, 10 8 CFU / ml and 10 9 The drug had to be stored at a temperature stable for at least two years at CFU / ml, ≤-70°C (Figure 3). However, storage at -70°C is unsuitable for further clinical and commercial development. This specification states that lyophilized BPZ1 formulations can be obtained that are stable for at least two years at -20°C±10°C, 5°C±3°C, or 22.5°C±2.5°C.

[0063] Before commencing BPZE1 process development, several target product characteristics were established, as shown in Table 1. The target lyophilized survival rate was 20%, as it was the same as the survival rate of the liquid BPZE1 formulation used in Phase 1 trials [11, 12]. The target for the lyophilized BPZE1 formulation was a CFU number of 0.2–4 × 10⁶ over a storage period of at least 2 years at +4°C. 9 It was determined that the level should be maintained at CFU / ml.

[0064] The survival rate of living organisms after freeze-drying depends on the freeze-drying cycle, freeze-drying buffer, and the physiological state of the organism before freeze-drying. These parameters are likely to be interdependent. However, the freeze-drying survival rate of BPZE1 also depends on the culture and harvesting conditions, and it has become clear that shear stress and harvest optical density, in particular, have a significant impact on the freeze-drying survival rate.

[0065] The critical importance of the retention time of the liquid bacterial suspension from collection to the start of freeze-drying also became clear during actual production runs. In the first run, where freeze-drying was started within 16 hours of collection, a viability rate of 46–64% was achieved, while with a retention time of 26–32 hours, the viability rate consequently dropped to approximately 20%. Since the collection, concentration, and formulation of bacterial suspensions, and especially the filling of >200,000 vials per batch, can easily take 24–48 hours, evaluating viability after a 24–48 hour retention time is particularly important in large-scale production.

[0066] The RMC of lyophilized formulations has consistently been less than 2.5%, which is generally compatible with long-term stability below 5°C. However, the relationship between temperature and viability after lyophilization is determined by Tg, which is the temperature at which water remaining in the lyophilized product becomes mobile again, accelerating the decline in viability. As confirmed by the 2-year stability of lyophilized formulations at +22.5±2.5°C, exposing the formulations to controlled temperatures, albeit ambient temperatures, for relatively short periods (a few hours to a few days) does not significantly affect the formulations. Therefore, for logistical and supply chain reasons, the target Tg was set to ≥35°C.

[0067] The method for producing the freeze-dried BPZE1 product involves the attenuation of key molecular characteristics of the attenuated BPZE1 vaccine, namely, the substitution of the Bordetella pertussis ampG gene with the E. coli ampG gene, the deletion of the dnt gene as evaluated by specific PCR, and 10 6The qPCR method, which can detect one predicted reversion from a genome-equivalent sequence, did not affect the modification of the PT S1 subunit gene that results in the inactivation of PT by genetic manipulation.

[0068] While RMC and Tg are generally expected indicators of stability, there is no substitute for real-time stability. Therefore, lyophilized BPZE1 formulations were subjected to real-time stability tests at -20°C±10°C, 5°C±3°C, and 22.5°C±2.5°C. Lyophilized BPZE1 formulations prepared by direct dilution, concentration, and diafiltration showed a CFU count of 0.2 to 4 × 10⁶ during storage when stored at -20°C±10°C, 5°C±3°C, and 22.5°C±2.5°C. 9 Since the CFU / ml level did not fall below the specified value, the formulation remained stable even after being stored for at least 24 months.

[0069] The adhesion and colonization kinetics of liquid and lyophilized formulations were evaluated in mice using a liquid formulation containing 5% sucrose in PBS and stored at -70°C. Phase 1b clinical trials showed that the liquid formulation resulted in colonization in >80% of subjects, despite PBS being hypertonic compared to the salinity of the airways. A reduction in salinity from PBS + 5% sucrose to hypotonic Thijs medium + 10% sucrose in the liquid formulation did not affect adhesion or colonization in the mouse nasal cavity. Furthermore, the kinetics of in vivo colonization and protective efficacy were evaluated for storage up to 24 months at three different temperatures. Two years of storage at +5°C ± 3°C or +22.5°C ± 2.5°C appeared to slightly but significantly reduce the rates of adhesion and colonization, but this had minimal impact on vaccine efficacy. Lyophilized BPZE1 formulations stored for 24 months at either of the test temperatures continued to provide protection, showing a more than 100-fold reduction in cell mass compared to unvaccinated controls 7 days after exposure.

[0070] Glass vials typically have very poor heat transfer during heating / cooling because contact between the glass bottom and the freeze dryer shelf is limited to three points. During freezing, this poor conductivity leads to uneven cooling of the vials; that is, some vials will begin crystallizing, while others will remain liquid for longer periods. In large freeze dryers in particular, these problems of uneven heating / cooling heat transfer can result in relatively large time differences between the first and last vials.

[0071] It was hypothesized that the difference in time from the start of ice crystal formation to reaching Tg', the temperature at which water stops moving, affects the survival rate of bacteria after freeze-drying is complete. Ice crystal formation means an increase in the molar concentration of dissolved components in the freeze-drying buffer, including salt. High salt concentrations are more likely to damage the outer membrane of Bordetella pertussis or other bacteria, yeasts, fungi, or viruses, and therefore the time spent in the phase with high salt concentrations should be kept as short as possible. Small-scale studies have shown that, with the freeze-drying buffer used, very slow cooling causes rapid ice crystal formation in the vial at the crystallization point of -5.8°C, with most vials showing rapid crystal formation within minutes, while ice crystal formation between the first and last vials can typically take more than an hour.

[0072] In the case of this formulation, Tg' only reaches -34°C, but since crystal formation in the vials begins almost simultaneously at -5.8°C, the starting point before crystallization is the same for all vials, meaning high uniformity between vials. As an example, Figure 7 compares three runs (GMP runs 1, 2, and 3) of freeze-drying performed in a production-scale freeze-dryer. Vials for GMP runs 1 and 2 were cooled from ambient temperature to -50°C using a gradient of 1°C / min. Vials for GMP run 3 were cooled from ambient temperature to -5°C at a rate of 1°C / min, held for 1.5 hours, and then frozen to -50°C at a rate of 1°C / min. Comparing the CFU counts for GMP runs 1, 2, and 3 in Figure 7, there is a difference of 3 to approximately 6 times between the highest and lowest CFU counts in GMP runs 2 and 1, respectively. In GMP run 3, the difference between the highest and lowest vials is less than 2 times. The same information is shown in Table 5, normalized to the highest CFU count for each batch as 100%. TIFF2026086426000004.tif120170

[0073] The introduction of a pre-crystallization holding step before freezing to Tg' is as follows: The crystallization temperature of a given freeze-drying buffer is determined by slowly cooling the buffer and recording the temperature at which crystallization begins. The pre-crystallization holding step can be defined as a holding step of 30 minutes to several hours, depending on the size of the freeze-dryer, at a temperature 0.1°C to several degrees higher than the crystallization temperature, depending on the temperature variation of the coolant passing through the freeze-dryer shelves.

[0074] Other Embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to describe, and not to limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method for producing a freeze-dried vaccine containing attenuated metaphytic Bordetella bacteria, 0.4-1.6 OD ６００ Steps to collect Bordetella bacteria from the culture; The collected Bordetella bacteria are mixed with a freeze-drying buffer containing 5-65% by weight of freeze-protecting sugar and having a temperature of 2-35°C, wherein the ratio of the collected Bordetella bacteria to the freeze-drying buffer is 5:1 to 1:5 by volume; The mixture of Bordetella bacteria and the freeze-drying buffer is frozen, wherein the holding time between the collection step and the freeze-drying step is less than 48 hours; and Step of collecting the freeze-dried Bordetella bacteria Methods that include...

2. The method according to claim 1, wherein the Bordetella bacterium is a strain of Bordetella pertussis.

3. The method according to claim 2, wherein the Bordetella pertussis strain is the BPZE strain.

4. The method according to claim 3, wherein the BPZE strain is BPZE1.

5. The aforementioned Bordetella bacteria have an OD of 0.4 to 1.0 ６００ The method according to claim 1, wherein the culture is derived from the culture of the

6. The aforementioned Bordetella bacteria have an OD of less than 1.0 ６００ The method according to claim 1, wherein the culture is derived from the culture of the

7. The method according to claim 1, wherein the freeze-protected sugar is sucrose.

8. The method according to claim 1, wherein the freeze-dried buffer contains a nutrient substrate.

9. The method according to claim 8, wherein the nutritional substrate is a glutamate salt.

10. The method according to claim 1, wherein the holding time between the collection step and the freeze-drying step is less than 36 hours.

11. The method according to claim 1, wherein the freeze-drying step includes a pre-crystallization holding step, in which the mixture of Bordetella bacteria and the freeze-drying buffer is held for 0.5 to 10 hours at a temperature 0.1 to 10°C higher than the crystallization temperature of the mixture before further cooling.

12. The Bordetella bacteria collected above were subjected to an OD of 1.0 to 30.0 ６００ The method according to claim 1, further comprising the step of concentrating.

13. The following steps: 0.4-1.6 OD ６００ Steps to collect Bordetella bacteria from the culture; Bordetella bacteria from the collected cultures were divided into OD 1.0 to 30.0 ６００ A step to concentrate it; The concentrated Bordetella bacteria are mixed with a freeze-drying buffer containing 5-65% by weight of freeze-protecting sugar at a temperature of 2-35°C, wherein the ratio of the concentrated Bordetella bacteria to the freeze-drying buffer is 5:1 to 1:5 by volume; The step of freeze-drying the mixture of Bordetella bacteria and the freeze-drying buffer, wherein the holding time between the collection step and the freeze-drying step is less than 48 hours; and Step of collecting the freeze-dried Bordetella bacteria A lyophilized vaccine product containing attenuated live Bordetella bacteria, manufactured according to a method including the following.

14. The freeze-dried vaccine product according to claim 12, wherein when stored at 22.5°C, it has an expiration date of at least two years, and at least 20% of the bacteria in the product survive after the freeze-drying step.

15. The freeze-dried vaccine product according to claim 12, wherein, after the freeze-drying step, the collected freeze-dried bacteria can prevent or suppress infection of the target respiratory tract by pathogenic strains of Bordetella pertussis.

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