WO2000024430A2

WO2000024430A2 - Caracterization and preservation of vaccine formulations

Info

Publication number: WO2000024430A2
Application number: PCT/CU1999/000005
Authority: WO
Inventors: Tomás Marcelino MOREIRA HERNANDEZ; Herminía de la Caridad DELGADO RODRIGUEZ
Original assignee: Centro Nacional De Investigaciones Cientificas
Priority date: 1998-10-22
Filing date: 1999-10-20
Publication date: 2000-05-04
Also published as: AU6459699A; WO2000024430A3; CU22847A1

Abstract

System for the thermophysical caracterization of formulations and method for the preservation of Vibrio cholerae independently of the biotype, serotype or serogroup, intended to the preparation of oral vaccines. It comprises the use of formulations with adequate proportions of at least three components which are representative of the following groups: proteins, peptides, polymers, skimmed milk, glycin and sorbitol which guarantee an incipient fusion temperature in excess -20°C. Viability losses are lower than 0.7 in logarithmic order.

Description

CHARACTERIZATION AND CONSERVATION OF VACCINE FORMULATIONS.

Field or branch of the technique with which the invention relates The present innovation relates to biotechnology and in particular to a method to conserve Vibrio Cholerae strains and prepare vaccines with them.

Known level of technique

In recent years, cholera has been a major cause of death in many developing countries. Taking into account that Vibrio cholerae is a non-invasive microorganism, which generates an immune response at the level of intestinal mucosa, and that the disease itself confers a lasting response, a vaccine preparation against cholera should be oral, preferably with a live bacteria, so that it effectively stimulates the immune system at that level. There are several options to prepare oral vaccines. The preparation of the vaccine in lyophilized form, besides presenting the advantage of improving the conservation of the strain, facilitates the preparation of the doses, guarantees a prolonged storage, limits the risks of contamination and facilitates the commercialization and distribution when dispensing with a chain from cold to very low temperatures, generally not available in underdeveloped countries where the disease proliferates.

Vibrio cholerae has long been considered a microorganism very sensitive to the lyophilization process, as shown by the works of S. A. Sturdza, T. Vasilescu, G. Boltasu and E. Barlacu published in Arch. Roum. Path Exp. Microbiol. 38, (2), 227-249, 1979 and is summarized in the book "La Lyophilisation, Principes et Applications", Collection de l'ANRT, by D. Simatos, G. Blond, P. Dauvois and F. Sauvageot.

In recent years, the effect of additives has been deepened and viability has increased. Thus, in works aimed at preserving the Inaba CVD Hg-R Classical vaccine strain of the Center for Vaccine Development, University of Maryland, USA, the Swiss Institute of Serums and Vaccines, of Bern (ISSVB), developed a formulation Of which, in a paper published in Vaccine, 8, 577-580, 1990, SJ Cryz Jr, M. M. Levine, J. B. Kaper, E. Fürer and B. Althaus point out that it is mainly composed of sugars and amino acids. Later,

H JA SUBSTITUTE RULE 26 in a paper published in Dev. Biol. Stand., Basel Karger, 1996, Vol 87, pp 277-281 under the authorship of SJ Cryz, O. Pasteris, SJ Varaliyay and E. Fürer, although neither the proportions nor all the components of the formulation, mention is made of the presence of sucrose, lactose and sorbitol and it is shown that the lyophilized strain used in the vaccine, when stored between 22 and 25 ° C, maintains cell levels up to 7 days feasible necessary for vaccination and 1 day when storage is done at 37 ° C.

In a work on lyophilization conservation of the 569B Classical Inaba wild strain, published in Cryo-Letters, 16, 91-101 (1995) by H. Delgado, T. Moreira, L. Luis, H. García, T.K. Martino and A. Moreno, the effect of different additives on post-freeze-drying viability and after storage at different temperatures is compared. It is demonstrated that using a formulation based on skim milk, peptone and inositol, it is possible that after 3 days of storage at 45 ° C, the loss of viability is equal to 1 logarithmic order, although inositol crystallization is mentioned in the frozen phase

However, the crystallization of some of the components of the lyophilized formulations is a phenomenon that according to Pikal, MJ should be avoided. This author has shown that such crystallization significantly affects the stability of lyophilized proteins (Biopharm, 3, 26-30, 1990). In addition to looking for those formulations that guarantee viability, it is of the utmost importance to guarantee the economy of any freeze-drying process. The chemical composition of a formulation determines its freezing temperature, and to some extent the water desorption capacity, thus establishing the ease of the system to be lyophilized, and its residual moisture content. The thermophysical characterization during the freezing process, allows to select those formulations with better characteristics to perform lyophilization at the highest possible temperatures and accelerate the sublimation and desorption kinetics, reducing the duration of the process without risks of causing the structural sinking. The temperature that characterizes any formulation is the beginning of the softening of the hyperconcentrated substance contained in the interstices formed between the ice crystals (T _g ') and is determined by equipment based on the principle of differential scanning calorimetry or thermal analysis differential, which are of high price, for their high precision. However, the determination of the temperature at which the melting of the ice crystals becomes evident or incipient melting temperature (TFI), although somewhat higher than T _g ', is very convenient for comparisons between the behavior of different formulations and can be determined by systems of differential thermal analysis much simpler. This requires a system capable of reading and recording the temperature difference between the frozen solution and an inert reference, when the two undergo the same heating regime. Such a system was partially described in a paper published in Cryo-Letters, 16, 91-101 (1995) by H. Delgado, T. Moreira, L. Luis, H. García, TK Martino and A. Moreno, obtaining maximum cooling and heating speeds of 6 and 1.5 ° C min ^"1 respectively that allow obtaining acceptable thermograms. In this sense, HM Hatley (Develop. Biol. Standard. Vol 74 pp 105-122, Karger, Basel, 1991) He pointed out the inaccuracies that can be committed by not having heating rates of the order of 10 ° C min ^"1 in terms of the resolution of thermograms.

Essence of the invention

The present invention aims at developing a method to conserve strains of Vibrio Cholerae by lyophilization in order to prepare vaccines with them. It includes the design, construction and use of a measuring cell which, when placed in a commercial freezer unit (NICOOL LM-10 mini freezer, CFPO), achieves heating and cooling speeds of up to 10 ° C min ^"1 , guarantees the good resolution of the thermograms and thus allows to accurately determine the incipient melting temperatures (TFI) corresponding to different formulations to select, those with values between -10 ° C and -20 ° C, capable of allowing adequate lyophilization and a rapid post-lyophilization solution without affecting its viability the fact of being dissolved in a solution of sodium bicarbonate at 1.33% or in the traditional way in 0.9% saline solution. It is the object of the invention that, by means of the appropriate component selection, lyophilized formulations ensure that the viability losses of Vibrio cholerae strains are reduced to less than 1 log order as a consequence of storage, regardless of serogroup, serotype or

two biotype or having been lyophilized separately or joined as part of the same preparation.

The formulations must be composed of at least one of the following components: purified proteins (Pr), skim milk (Ld) and polymers (Pm), with the addition of peptides (Pd) or glycine (Gl) and sorbitol (So). The total concentration should not exceed 10%.

Specifically, among the proposed components are: (Pr): gelatin or casein,

(Pm): dextrana or Ficoll, (Pd): hydrolyzed casein or bacteriological peptone.

Figure 1 shows a general scheme of the system and in particular of the measuring cell, where you can see:

1: Sample holder (aluminum cylinder φ 10 mm H 40 mm with threaded bottom cap, polypropylene) where the sample is placed (10 to 20μL). Include the reference

5 (brass ring φ 10 mm, H 5 mm)

2: Copper-constantán thermocouple system (φ 0.2 mm) to measure the temperature and ΔT (which is read properly, is the difference in electrical potential ΔV, but using the table corresponding to the thermocouple, it is possible to determine the temperature). The thermocouple system is connected to a potentiometric recorder capable of recording 10 μV cm ^'1 in the registration channel of ΔT and 250 μV cm ^"1 for the temperature recording. 3: Aluminum cylinder of de 20 mm and 100 mm of length within which the sample holder is placed, which is filled with 10 mL of methanol (4) to ensure good heat transfer.

With this system, variable cooling and heating rates can be established, up to 10 ° C min ^'1 , by operating the extractor (6). It consumes a maximum of 200 mL of liquid nitrogen per experimental run and provides an accuracy of ± 0.2 ° C in TFI determinations. For heating, the upper body of the equipment is placed on a heat source.

Figure 2 illustrates the record of 5 different formulations, where the difference between the TFI values of each can be seen:

A) skim milk + casein hydrolyzate + sorbitol

B) skim milk + peptone + glycine + sorbitol

C) gelatin + peptone + sorbitol

To 26 D) dextran + peptone + sorbitol

E) skim milk + dextran + casein hydrolyzate + sorbitol

In the examples that follow, the culture of the microorganism was carried out in Luria broth at 37 ° C in thermostated orbital zaranda, with a stirring between 150 and 250 rpm. Subsequently, the biomass was separated until it reached the logarithmic phase, using centrifugation between 5000 and 8000 rpm at 4 ° C for 10-20 minutes and mixed with the formulations that showed high TFI values and good characteristics for the protection of the microorganism, so that the cell concentration was between 10 ⁸ and 10 ⁹ mL ^"1 cells, at a rate of 2mL per bottle of type 10R. The lyophilization cycle included deep freezing of the material, the realization of primary drying keeping each product between -20 ° C and -32 ° C for 5 to 12 hours and for secondary drying the product temperature was maintained between 18 ° C and 28 ° C for no more than 12 hours The cycle duration never exceeded 24 hours. defined the loss of viability as the logarithmic difference of the CFU / mL before and after lyophilization or before and after storage of lyophilized material when the product is dissolved used in a 1.33% solution of sodium bicarbonate. Table 1 illustrates the viability behavior of different strains during storage by using one of the preferred embodiments of the following invention, where the general composition of the formulation was Ld 4%, Pd 2%, So 2%, TFI was located between -10 ° C and -15 ° C, guaranteed the completion of a lyophilization in less than 22 hours, presented a good appearance, easy dissolution in a solution of sodium bicarbonate at 1.33% and immediate recovery of the cells, with a loss minimum viability.

ORIA RULE 26 TABLE 1: Concentration of viable cells (logUFC / mL) of different strains, before (A) and after freeze-drying (DL) as well as after 6 and 12 months of storage at 8 ° C.

Strain Source Serogroup Serotype log log log

CFU / mL CFU / mL CFU / mL

AL DL, 6 M DL.12 M

CVD103-HgR Vaccine USA 01 Classic Inaba 9.12 8.76 _

413 CNIC Vaccine El Tor Inaba 8.96 8.72 8.00

417 ^« 8.87 8.72 8.00

417-10 - 9.58 9.16 8.67

C7258-1 The Tor Ogawa 9.56 9.12 8.71

638 _" " ^» 8.28 8.00 7.32

68 " ^» 8.96 8.51 8.15

251a "0139 9.07 8.51 8.10

The following examples are intended to illustrate this invention, either in general terms or in detail. However, it should be understood that the invention is not limited thereto, both in scope and in purposes.

Example 1: In an embodiment of the present invention, using a formulation of the Pr 2%, Pd 2%, So 2% type, whose TFI was found to be -17 ° C, were subjected to the same lyophiolization process, the CVD strains 103-HgR, (Inaba Classic), 417 (The Inaba Tor), 638 (The Ogawa Tor) and 251a (0139). Freezing was carried out at -70 ° C. During primary drying, the temperature of the product was kept at -25 ° C for 10 hours and in secondary drying the temperature of the product was 22 ° C for 12 hours. The solution in a 1.33% solution of sodium bicarbonate was instantaneous. The loss of viability calculated immediately after dissolution, with respect to the concentration of living cells before lyophlization was found to be 0.60, 0.53, 0.45 and 0.68 logarithmic order respectively. When the dissolution was effected

RULE 26 in a 0.9% solution of sodium chloride there were no significant differences in the counts in relation to the sodium bicarbonate solution.

Example 2 A mixture was lyophilized with similar proportions of strain 251a (0139) and 638 (Tor Ogawa). The latter has integrated the cell A gene of Clostridium thermocellum, which codes for the enzyme endoglucanase A and can easily be identified as a red colony surrounded by a transparent halo when covered with a thin layer of agar containing carboxymethyl cellulose and subsequently stained with Congo Red, which allowed his count in the mix. For the lyophilization, the formulation of the Pr 2%, Pm 2%, Pd 2%, So 2% type was used, whose TFI was found to be -11 ° C. Freezing was carried out at -70 ° C. During the primary drying the temperature of the product was kept at -20 ° C for 10 hours and in the secondary drying the temperature of the product was 24 ° C for 8 hours. The solution in a 1.33% solution of sodium bicarbonate was instantaneous and the loss of viable in logarithmic terms was 0.45 for 251a while for 638 it was 0.42. When the solution was carried out in 0.9% sodium chloride solution there were no significant differences in the counts in relation to the solution in sodium bicarbonate.

Example 3 A mixture was lyophilized with similar proportions of strain 413 (Tor Inaba) and 638 (Tor Ogawa). The same property of strain 638 indicated in the previous example was used to perform the counts in the mixture. For the lyophilization the formulation was used: Ld 2%, Pm 2%, Pd 2%, Gl 2% and So 2%, whose TFI was found to be -12 ° C. Freezing was carried out at -70 ° C. During the primary drying the temperature of the product was kept at -20 ° C for 8 hours and in the secondary drying the temperature of the product was 20 ° C for 12 hours. The dissolution in a 1.33% solution of sodium bicarbonate was instantaneous and the loss of viable expressed in logarithmic terms was 0.65 for 413 while for 638 it was 0.62. When the solution was carried out in 0.9% sodium chloride solution there were no significant differences in the counts in relation to the solution in sodium bicarbonate.

IA RULE 26

Claims

CHARACTERIZATION AND CONSERVATION OF VACCINE FORMULATIONS.

1. System for thermophysically evaluating formulations, characterized by the construction of an appropriate cell to be adapted to a NICOOL LM-10 mini freezer, CFPO, which converts it into a differential thermal analysis system (ATD) that allows cooling and heating speeds to be reached up to 10 ° C min ^"1 and with them, high resolution thermograms.

2. System according to claim 1, characterized in that it contains a sample holder (aluminum cylinder of φ 10 mm H 40 mm with threaded lower cover, made of polypropylene) where the sample is placed (10 to 20μL). Includes reference (brass ring φ 10 mm, H 5 mm).

3. System according to claim 1 characterized by containing copper-constantán thermocouples (φ 0.2 mm) for measuring temperature and ΔT.

4. System according to claim 1 characterized in that it contains an aluminum cylinder of φ 20 mm and 100 mm in length within which the sample holder is located. It is filled with 10 mL of methanol to ensure a good heat transfer.

5. Conservation method by lyophilization of Vibrio cholerae, intended for the development of oral live vaccines, characterized by the use of formulations with high TFI (-10 ° C to -20 ° C), containing at least one of the following components : purified proteins (gelatin or casein), skim milk and polymers (dextran or ficoll), in a concentration equal to 2% or multiple, with the addition of peptides (casein hydrolyzate or bacteriological peptone) or glycine and sorbitol, all at 2%. The total concentration should not exceed 10%.

6. Conservation method according to claim 5, characterized by enabling lyophilization processes whose duration is less than 24 hours.

7. Conservation method according to claim 5, characterized in that the lyophilized material has an instant solution in a solution of sodium bicarbonate at 1.33%.

SUBSTITUTE SHEET RULE 26

8. Conservation method according to claim 5, characterized in that the viability of the strains is not affected by the dissolution of the lyophilized material in the manner expressed in claim 7.

9. Conservation method according to claim 5, characterized by protecting strains of Mibrio cholerae from the effect of lyophilization and storage regardless of the biotype, serogroup and serotype used.

10. Conservation method according to claim 5, characterized in that the loss of post-lyophilization viability of Vibrio cholerae strains is less than 0.7 logarithmic order.

11. Conservation method according to claim 5, characterized by allowing lyophilization in the form of a mixture of strains of different biotype, serogroup or serotype and ensuring that the loss of viability of each of them is less than 0.7 logarithmic order.

RIA RULE 26