MUHAMMAD AHSAN RAFI

0039-BH-BIO-T-19

ASSIGNMENT TOPIC: AVA-ANTHRAX VACCINE ADSORBED

ASSIGNMENT SUBMITTED TO: DR. M USMAN AHMAD

COURSE TITLE: IMMUNOLOGY

COURSE CODE: BIOTECH-3202

DATED: 09-MAY-2022

INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY

 TABLE OF CONTENT

 AVA- (ANTHRAX VACCINE ADSORBED) PREPARATION

 INTRODUCTION
 AVA DEVELOPMENT
 AVA MENUFECTURING PROCESS
 AVA- (ANTHRAX VACCINE ADSORBED) PREPARATION

Introduction:

Anthrax vaccine adsorbed is the first and only FDA approved vaccine for human
anthrax that’s been used in United States. The cell filtrate of an avirulent named Bacillus
anthracis is being used in the preparation of this particular vaccine. This avirulent contains a
protective antigen along with some other cell products which are being adsorbed in aluminum
hydroxide and works as an adjuvant. The standard vaccination procedure for pre-exposed
patient contains doses at 0, 1, 6, 12 and 18 months along with some booster doses which are
injected annually. In case of post exposure, the doses are applied at 0, 2 and 4 weeks and
additionally some antimicrobial therapy.

AVA- DEVELOPMENT:

First research for the development of anthrax vaccine was conducted at Fort Detrick (Camp
Detrick at that time). This study was based upon the cultures of Bacillus anthracis which was
grown inside synthetic medium in the absence of protein or any other macromolecules
(Turnbull, 2000). In 1954, scientists were able to formulate a chemically defined media in which

they might be able to grow bacteria and proved that the protective antigens present inside the
culture filtrates can actually be stabilized, concentrated and purified by combining them with
alum and can be used for vaccine development. This vaccine would require some further
refinement to ensure the large-scale production. The refinement procedures used included
adsorption into aluminum hydroxide gel, microaerophilic incubation, use of different growth
medium and use of different strains of B. anthracis.

AVA Manufacturing process:

In the manufacturing process of licensed anthrax vaccine, an

inoculum containing non proteolytic, non-encapsulated strain of avirulent B. anthracis called
(V770-NP1-R) is used. Initially, it is placed inside a sterile growth medium named 1095, this
growth medium contains minerals, amino acid and glucose along with some other specific
ingredients which are known to be optimal for growth of B. anthracis. Microaerophilic
conditions are used for bacterial growth through fermentation process. This inoculated medium
is then passes through further agitation and held at the strict temperature of 37  ± 0.5 0C for 23-
24 hours. This agitation process is done within two fermenters with the volume of 10 and 100
liters. During this 24-hour duration, the glucose level and temperature is strictly monitored
(Puziss & Wright, 1963).

After is incubation process is complete, the mixture is run through filtration process with the
help of low protein binding hydrophobic filters. After the filtration is complete, sterile form of
aluminum hydroxide gel is added and then for next 17-18 hours, the mixture is stirred. After the
mixture is settled, the supernatant is is removed (this is done after 73 hours period of settling
process). Mixture is further subjected to centrifugation process for the removal of any left
supernatant. The antigen adsorbing aluminum hydroxide is then suspended in formaldehyde
containing saline solution with the concentration of 0.01 %. This saline solution works as a
stabilizer and in next step preservative (benzathine chloride) is used for preservation of
mixture. Finally, the tests for potency, purity and stability is carried out and vaccine is filled and
packed inside 10 dose vials, stoppered and sealed. After that, vaccine is labeled and packaged
for use.

The total time consumed in preparation of AVA vaccine from initial step to distribution is 22
weeks. The table showing timelines for AVA production is given below:
 Figure 1(Timeline for AVA production)

The license ship of a certain vaccine is defined by its dating period but FDA is able to grant the
extension for expiration date if it passes all the sterility test and potency test. Previously AVA
was licensed for the period of 36 months while stored in 2–8-degree centigrade temperature.

The evolution of vaccine quality over the past 30 years highly depends upon the
characterization of components of vaccine. When anthrax vaccine was first developed in 1950
and 1960s, only a few immunological and protein characterization techniques were there, so
the evaluation of protein quality depended highly upon its potency to effect animals. For
example, the production of effective immunity of a vaccine in pigs, rabbits’ monkeys and guinea
provided the data for formulation of its success rate. Wright and Puziss did research on
protective antigen activity in 1954 by the immunization experiment on guinea pig. They used
complement fixation titration technique in their study for estimation of protective antigens.

Anthrax vaccine was officially licensed in 1970 and the detail of vaccine constituents was
formulated which was mainly adjuvant, preservatives and stabilizers. Protective antigen of any
kind was not originally specified in vaccine licensed details neither maximum and minimum
number of other components of interest or concern were discussed. These components might
be lethal factor (LF) or edema factor (EF). But still the vaccine was able to pass required purity,
safety and potency tests.

In today’s research, when the details of every component of this vaccine has successfully been
formulated, BioPort has taken charge to characterize AVA validation studies.
One big hurdle in separation and formulation of vaccine components is Alhydroxygel and
because of its difficulty in conducting desorption researchers at BioPort aliquots of
fermentation filtrate before they could perform adsorption. Bradford method was used for
protein determination, ELISA, western blotting and SDS PAGE separation. 10 micrograms of
Bradford protein per one milliliter was separated in a typical fermentation filtrate. No
noticeable EF was found by using western blotting but ELISA was able to determine LF with the
range of 10-30 nanograms per milliliter.

Due to these results, FDA and BioPort has been able to change standard manufacturing
parameters for AVA to provide a greater product consistency. The changes shown in the figure
2 has been applied to improve the production quality and rate by varying various factors
including the temperature to time of fermentation (Myers, 2002).
 Figure 2 (Changes applied to improve production quality)

REGULATORY ACTIONS IN AVA PRODUCTION:

MDPH, a state-owned and -run facility, received the license to manufacture AVA in 1970 and
produced vaccine until the facility was transferred to MBPI in 1995. In September 1998 the
facility was sold to the private company BioPort, but the existing MBPI management team was
retained

FDA conducted an inspection of the MDPH anthrax vaccine manufacturing facility in January
1993, which was followed in July 1993 by FDA approval of amendments to the license
application for new equipment and facilities (Donlon, 1993). Meanwhile, FDA inspections of
MDPH product lines other than AVA in 1993 and 1995 resulted in findings of significant
deviations from GMP and an official warning letter.

During a follow-up inspection of MBPI conducted in November 1996 that did not include the
facilities for manufacture of AVA, FDA documented many deviations from the Food, Drug, and
Cosmetic Act, FDA regulations, and current GMPs for the manufacture of blood-derived
products and bacterial vaccines other than AVA (Zoon 1997). FDA issued a Notice of Intent to
Revoke (NOIR) letter to MBPI in March 1997, stating that if MBPI's corrective actions proved to
be inadequate, FDA might revoke its licenses. The letter did not mandate closure of the facility
or involve seizure of finished product. MBPI's response to the NOIR letter was a Strategic Plan
for Compliance, provided to FDA in April 1997. Periodic updates to the strategic plan that
reported on MBPI's progress were also provided to FDA (Michigan Biologic Products Institute,
1997–1998). In January 1998, after completion of renovation planning, MBPI shut down the
anthrax vaccine production facility for planned renovations.

FINDINGS AND RECOMMENDATIONS:

1. FDA's process of plant inspection and FDA's validation of the vaccine manufacturing

process have changed and have become more stringent with time.

2. With high-priority efforts by the manufacturer and FDA, the manufacturing process for
AVA has been validated so that vaccine manufactured post renovation has been
approved for release and distribution.

3. AVA will now be produced by a newly validated manufacturing process under strict
controls, according to current FDA requirements. As a result, the post renovation
product has greater assurance of consistency than that produced at the time of original
licensure. (Masiello, 2001)
REFRENCES:

1. Turnbull, P. (2000). Current status of immunization against anthrax: old vaccines may be

here to stay for a while. Current Opinion In Infectious Diseases, 13(2), 113-120.

2. Puziss, M., & Wright, G. (1963). STUDIES ON IMMUNITY IN ANTHRAX X. Journal Of

Bacteriology, 85(1), 230-236

3. Turnbull PCB. 2000. Current status of immunization against anthrax: old vaccines may be here to

stay for a while. Current Opinion in Infectious Diseases.

4. Myers R. 2001. Technical review. E-mail to Joellenbeck L, Institute of Medicine, Washington,

D.C., December 15.

5. Donlon JA. 1993. FDA approval of Michigan Department of Public Health amendment to

establishment license application. Letter to Myers RC, Michigan Department of Public Health,
Lansing, Mich.

6. Zoon KC. 1997. Inspections of Michigan Biologic Products Institute, November 18 and 27, 1996.
Letter to Myers R, Michigan Biologic Products Institute, Lansing, Mich.

7. Masiello SA. 2001. FDA approval of a BioPort Corporation biologics license application

supplement. Letter to Giri L, BioPort Corporation, Lansing, Mich.