The use of different stabilizers for improving integrity of the locally prepared lyophilized Brucella melitensis Rev 1 vaccine

ARTICLE INFO Stability study of biological products especially living bacterial vaccines plays an important role for the determination of product changes in maintenance period, and ensures safety, efficacy and maintenance of biological properties of the vaccines. So, the objective of this study was to establish stability and keeping quality of the local Brucella melitensis Rev-1 vaccine using different types of stabilizers in lyophilization process. A long-term stability study was carried out for four batches of reduced-dose Brucella melitensis Rev-1 vaccine manufactured by veterinary serum and vaccine research institute using four different stabilizers. Stabilizers were: (A) sucrose and skimmed milk, (B and C) different concentrations of sucrose, sodium glutamate and gelatin, and (D) casein, sucrose and sodium glutamate. The quality control tests including colony forming unit, purity, dissociation and physicochemical tests on all batches until 12 months postproduction were performed. The obtained results indicated that in spite of collapse (shrinkage) of lyophilized cake in a number of bottles in batches prepared using stabilizer A, Brucella vaccine batches were stable and met the specification recommended by OIE 2012 for 12 months post-production in vaccine batches with stabilizers A and D. Article history: Received: 15 October 2016 Accepted: 20 November 2016 Available Online: 27 August 2017


Introduction
Brucellosis is a major bacterial zoonotic and an emerging disease of global importance caused by bacteria of the genus Brucella affecting a range of different mammals including man, cattle, sheep, goats, swine, rodents and marine mammals worldwide. Three species only of Brucella are of zoonotic importance, these species are Brucella melitensis (main host is sheep and goats), Brucella abortus (main host is cattle and buffaloes) and Brucella suis (main host is pigs) (Corbel, 2006;Mohamed et al., 2010). The bacterial pathogen is classified by the CDC as a category (B) pathogen that has potential for production of a bio-weapon. Brucella spp. is considered as the most common laboratory-acquired pathogens (Mohamed et al., 2010).
Although the efforts and works of Egyptian veterinary services to overcome brucellosis started from more than 30 years, brucellosis is still endemic in both domestic ruminants and humans and represents one of public health hazard in Egypt. National Brucella committee representing veterinary laboratories, veterinary universities and General Organization of Veterinary Services (GOVS) establishes brucellosis control programs which depend mainly on the test and slaughter policy and vaccination programs. The vaccination programs recommended by OIE depends mainly on immunization of calves (6-8 months of age) with living attenuated Brucella abortus, S19 vaccine (smooth strain) with the dose of 3-10 X10 9 CFU/dose or the living attenuated Brucella abortus RB51 vaccines (rough strain) with the dose of 1-3.4 X10 10 CFU/dose and inoculation of small ruminants (3-6 months of age) with living attenuated Brucella melitensis Rev 1 vaccine (smooth strain) with the dose of 1-3 X10 9 CFU/dose (Wareth et al., 2014).
One of the best attenuated vaccines is Rev.1 obtained by passing through Brucella mellitensis wild strain 6056 serotype 1 on the media containing streptomycine, resulting in a mutant resistant to streptomycin called strain Rev.1 (Blasco, 1997). This mutant was discovered by Elberg in the University of California in 1957 for the first time and used in the vaccine production to prevent brucellosis in sheep and goat (Alton et al., 1967). Knowledge about the stability of a vaccine especially the rate at which it loses viability (colony count) and in consequences loses of its potency at a recommended storage temperature, can be helpful in determination of vaccine shelf life. So, Stability of vaccines has a major impact on the success and effectiveness of vaccination programs worldwide and may be responsible for vaccine failures with the results of corruption of whole vaccination programs (Knezevic, 2009). According to the reports of World Health Organization "WHO", stability of Rev.1 vaccine should take at least one year, therefore attempts to establish the stability of this local vaccine can solve many problems in regard to exportation of this vaccine and to meet the specifications and the requests of General Organization of Veterinary Services (GOVS). So, the objective of this study was to establish long term stability and keeping quality of the local Brucella melitensis Rev-1 vaccine produced by VSVRI with regard of the most suitable stabilizer used in lyophilization process and also to standardize a method for production of other live attenuated Brucella vaccines.

Freeze drying stabilizer medium
In this study four stabilizers (additives preserving materials) were prepared and used in production of Brucella melitensis Rev-1 vaccine. These additives are consisting of protein or polyssacharides or both were used with the following formula: A. 7% sucrose and 7.5% skimmed milk (Modified Behroozikhah et al., 2009 (Angus, 1984).

Culture medium
Potato infusion agar, tryptose soya agar and soyabean casein digest medium (SCDM) are the reference media used for culturing and preparation of seed culture and production of vaccines (Alton et al., 1988;OIE, 2012). Media used for purity test (OIE, 2012), testing of performance, validation and verification (keeping quality) of culture media used in production of Brucella vaccines and purity test were tested according to ISO17025 and ISO11133-2014.

Preparation of master seed, working seed and vaccine batches
Master seed, working seed and vaccine batches were prepared according to (Alton et al., 1988;Kamaraj et al., 2010) using roux bottle method. Vaccine batches were adjusted to contain 6.5 X10 9 CFU/ml then they were labeled and kept at 4 °C during all period of study (12 months).

Long-term stability study
The vaccines were tested seven times (every two months) in 0, 2, 4, 6, 8, 10 and 12 months after production for long-term stability. In each period all of the quality control tests were performed as follow:

A. Colony Forming Unit
In each period of the study, viability was determined for each vial according to the OIE protocol (OIE, 2012). After 3-5 days incubation at 37 °C, CFU per each dose of vaccine was enumerated (Larry and Smith, 1998).

B. Purity test
The purity test was performed according to the OIE manual (2012).

C. Dissociation test
After counting of colonies, the cultures were tested for smoothness and roughness by using crystal violet and acriflavine test according to (white & Wilsons staining method (Alton et al., 1988;OIE, 2012). The rough colonies take up the stain (blue colonies), but the smooth colonies not absorb the stain (white colonies).

D. Physicochemical tests
Physicochemical tests were carried out according to British Pharmacopoeia (2012). In each period, physicochemical tests including appearance, negative pressure (vacuum), and solubility, and extraneous agents, vacuum and labeling test were performed for all of the samples. Color, consistency, form of lyophilized vaccine and any visible particle after reconstitution were considered for appearance and extraneous agents tests (Hasannia et al., 2015).

Results
Master seed was identified morphologically, biochemically ( Fig. 1) and serologically as a typical Brucella melitensis biovar 1 (Rev-1) and also BRUCE-LADDER (Fig. 2) assay was performed as a confirmatory test using different Brucella reference strains as control positives. Seed culture and all prepared vaccine batches were free from bacterial (aerobic and anaerobic), fungal and Mycoplasma contaminations along the entire period of the study and all colonies of seed culture and tested batches during the period of the study were 100% smooth when stained by crystal violet and on using acriflavine test (Fig. 3). Physicochemical inspection revealed that all batches met the specifications (lyophilized cream color, free of any visible particles after reconstitution in appearance, readable and stable label in label test, good soluble in saline in solubility test and prescience of negative pressure (vacuum) in each period of long-term stability study. pH of buffers, used for collection and harvesting of Brucella cultures during production and evaluation of Brucella vaccine samples, diluents and additives, used as stabilizers, were adjusted to 7.2.

Discussion
Vaccines are combination of components (biological and non-biological) that are sensitive to environmental factors and changes in non-biological ingredients of vaccines by different factors. So, biological changes may be occurred especially in live vaccine as decrease in CFU of live bacterial vaccines which consequently affects potency of vaccine. So, to determine product changes in maintenance period and ensure safety and efficacy of vaccines, stability study of biological products is needed. Stability is the ability of a vaccine to retain its chemical, physical, microbiological and biological properties within specified limits throughout its shelf life (Galazk et al., 1998;Schofield 2009).
Many factors effect stability of vaccines such as, stabilizer, heavy water, process and equipment used in production, lyophilization process, the cold chain used for maintenance and transportation of vaccines and vials or tubes of vaccines (Wang et al., 2000). In this study, Long-term stability was performed, after production of vaccine batches using roux bottles methods (Alton et al., 1988;OIE, 2012), to recommend storage conditions and to establish the shelf life and the release specifications (Knezevic, 2009).
CFU results of Rev-1 Brucella vaccines with different stabilizers in period of the 12 months longterm stability study revealed that, all of the vaccine samples of stabilizers A, C and D passed the specifications of OIE until the end of 12 months post-Production in spite of a number of vaccine samples of stabilizers A showed a collapse or shrinkage of lyophilized cake (disc) which may be due to moisture increase which may be explained by many reasons as low quality rubber used (Hasannia et al., 2015), a lyophilization program not compactable with stabilizer or may be due to low proteins or sugar content of stabilizer (Ferry, 1995;Behroozikhah et al., 2009). Anyhow, the results indicated that the increasing of moisture content do not reduce the potency of vaccine under the specifications or not greatly affect potency of vaccine and this agrees with (Hasannia et al., 2015).
Stabilizer D was the best stabilizer that maintains the viability of Brucella vaccine along the 12 months post-production and showing the least titers reduction (52.38%) but during the dry freezing process, it showed dramatically reduction in titers in comparison with other three batches. This titers reduction was from 6.5 X10 9 to 2.1 X10 9 CFU/ml i.e. 67.69%. Brucella melitensis Rev-1 Vaccine to be standard must not loss more than 2 X10 9 CFU/ml/12 months (66.66%) when colony count of tested vaccine is 3 X10 9 CFU/ml (maximum limit of OIE specification of Rev-1 vaccine) directly after dry freeze process. So, although vaccine samples prepared with stabilizer C show reduction in colony count of 35.38% after lyophilization process and met OIE specification at the end of 12 months post production, but it was not considered as a good stabilizer in maintaining the viability of Brucella along the entire period of the test as the colony count average in vaccine samples with stabilizer C was beyond the standard specification at zero day post production (4.2 X10 9 CFU/ml), colony count was 2.9. X10 9 CFU/ml at the end of the study which mean that the reduction titers in colony count was more than 2X10 9 CFU/ml. Stabilizer B was the least one in maintaining viability of Brucella vaccine with reduction titers rates of 94.28%.

Conclusion
Stabilizers D and A were the most suitable stabilizers to be used in production of Brucella Rev-1 vaccine. All vaccines especially living bacterial vaccines should be stored at temperatures recommended by national immunization programs and manufacturers.