1.

Company Profile
Panacea Biotec is Indias highly progressive, Innovative health management company based
on Research & Development, Manufacturing and marketing of Pharmaceuticals,
Biopharmaceuticals, Vaccines and natural / indigenous products.

The company is witnessing a period of expansion across every aspect of our business from
innovative products to customers in market, from manufacturing to regulatory approvals and
thereby laying the foundation for translation of our vision in becoming greatest, largest and
most admired biotechnology company in the World by 2020.
Ardent Research and Development efforts have always been a great strength of the company.
The main research areas are New Chemical Entities (NCE), New Biological Entities (NBE)
Novel Drug Delivery System (NDDS) based pharmaceutical formulations, Novel peptides &
human monoclonal antibodies and Vaccine development. The company has developed four
distinguished, ultra-modern, state-of-art R&D centers in different locations, having internal
capabilities for constant research, with over 300 highly professional and skilled scientists
engaged in various aspects of research.
Focused research efforts have led to grant of worldwide product patents valid in over 68
countries for Panacea Biotec. As on 31st March 2011, the company had filed over 1400
patent applications in various parts of the world including India. Of these, 382 have been
granted patent and others are under various stages of examination or publication by the patent
authorities. Some of these countries are USA, U.K., France, Germany, Italy, Sweden,
Denmark, Spain, Finland, Switzerland, The Netherlands, New Zealand, Mexico, Brazil,
Nigeria, Zimbabwe, Australia, South Africa, Japan, Russia, Canada, Ukraine, Korea and
China,
The company has ultra-modern, state-of-art production facilities at Baddi (Himachal
Pradesh), Larlu (Punjab) & Delhi for manufacturing tablets, capsules (including soft gelatin),
ointments (transgel formulation) liquids, herbal formulations and vaccines. The facilities are
WHO cGMP compliant.
The product portfolio includes highly innovative prescription products in niche therapeutic
areas like pain management, diabetes & cardiovascular management, Oncology, renal disease
management, osteoporosis management, anti-tubercular, gastro-intestinal care products and
vaccines. Our current business stems from our leadership segments in India i.e.
Nephrologicals, Anti Diabetes & Pain with some exclusive products based on patented Drug
Delivery System.

Panacea Biotec is the largest Vaccine manufacturing Company in India and is well
acknowledged by the UN Health Agencies in partnering the Polio eradication initiative with
supplies of millions of doses of WHO Pre-qualified Polio vaccine. As a sequel to the
completion of full range of Oral polio vaccines (tOPV, mOPV1, mOPV3 & bOPV) the
company has introduced the next generation Inactivated Polio vaccine (eIPV) vide a
collaboration with the Netherland Vaccine Institute. This vaccine has found extensive usage
in India for more than 3 years now and is registered in Bangladesh. IPV is also in advanced
stage of registration in 10 countries word wide with the target of being in more than 30
countries in a couple of years and has been put up for WHO prequalification which is
expected soon.
It is the first company in to have developed fully liquid Pentavalent vaccine (DTwP+Hep
B+Hib) Easy Five. Easy Five is a WHO Prequalified vaccine being used in more than 25
countries worldwide. The vaccines portfolio also consists of Enivac-HB (Hepatitis B
vaccine), Enivac-HB Safsy, Ecovac-4 (DTwP+Hep B), Easyfour (DTwP+Hib), which are also
WHO prequalified. Vaccines in the offing are- Anthrax, Dengue, Japanese encephalitis and
several others. A strong innovative vaccine pipeline is on the anvil which would pave the way
for a consistent introduction of Next Gen Vaccines in future.
Panacea Biotec is the third largest biotechnology company (as per ABLE Survey, 2011), as
well as among the top 50 pharmaceutical companies (as per ORG IMS March 2010) of India.
To tap newer opportunities, the company has organized its formulation marketing into Six
SBUs - PRO Care, Diacar Alpha, Diacar Delta, GROW Care, Onco Trust, and Critical Care,
which enables it to respond to changes in the industry and marketplace. The vaccines are
marketed through Chiron Panacea Vaccines, a 50:50 joint venture with Novartis Vaccines,
U.K
The company has tie up with National Institute of Immunology, India for Japanese
Encephalitis candidate vaccine, Biotech Consortium India Ltd. for the development,
manufacture and marketing of Anthrax vaccine, worldwide and with National Institute of
Health, USA, for use of a peptide based product for generation of hair follicles and hair
growth.
Panacea Biotec has also collaborated with Netherlands Vaccine Institute for Inactivated Polio
Vaccine; NRDC- India for Foot & Mouth Disease vaccine for veterinary use and Bio FarmaIndonesia for Measles vaccine.

2. Introduction
2.1 Vaccines
A vaccine is a biological preparation that improves immunity to a particular disease. A
vaccine typically contains an agent that resembles a disease-causing microorganism, and is
often made from weakened or killed forms of the microbe. The agent stimulates the body's
immune system to recognize the agent as foreign, destroy it, and "remember" it, so that the
immune system can more easily recognize and destroy any of these microorganisms that it
later encounters.
The term ''vaccine'' derives from Edward Jenner's 1796 use of the term ''cow pox'' (Latin
''variol vaccin'', adapted from the Latin ''vaccn-us'', from ''vacca'' cow), which, when
administered to humans, provided them protection against smallpox.
The immune system recognizes vaccine agents as foreign, destroys them, and 'remembers'
them. When the virulent version of an agent comes along the body recognizes the protein coat
on the virus, and thus is prepared to respond, by (1) neutralizing the target agent before it can
enter cells, and (2) by recognizing and destroying infected cells before that agent can
multiply to vast numbers. Vaccines have contributed to the eradication of smallpox, one of
the most contagious and deadly diseases known to man. Other diseases such as rubella,
polio, measles, mumps, chickenpox, and typhoid are nowhere near as common as they were a
hundred years ago.

2.2 Immunization
Immunization can be achieved in two stages- active or passive. In each case immunity can be
acquired either by natural processes (usually by transfer from mother to fetus or by previous
infection by the organism) or by artificial means such as injection of antibodies or vaccines.
The types of immunization include:

Passive Immunization
AIM- Transient protection or alleviation of an existing condition

In passive immunization, preformed antibodies are transferred to a recipient; in the past,

passive immunization provided a major defense against various infectious diseases.
Currently, passive immunization is used in several conditions like:
i.
ii.
iii.
iv.

Deficiency in synthesis of antibody.

When a susceptible person is exposed to a disease.
When sufficient immunization is not provided by active immunization.
When a disease is already present

Active Immunization
AIM- To elicit protective immunity and immunogenic memory

It can be achieved by natural infection with a microorganism or it can be acquired artificially

by administration of a vaccine. After active immunization, a subsequent exposure to a
pathogenic agent elicit`ts a heightened immune response that successfully eliminates the
pathogen and prevents the disease.

2.3 Types of Vaccines

Attenuated Viral or Bacterial vaccines - Microorganisms can be attenuated so
that they lose their ability to cause significant disease but retain their capacity for transient
growth within an inoculated host. Attenuation often can be achieved by growing a pathogenic
bacterium or virus for prolonged periods under abnormal conditions.
The ability of many attenuated vaccines to replicate within host cells makes them particularly
suitable for inducing a cell-mediated response.
Inactivated Viral or Bacterial vaccines - Another approach in vaccine
production is to inactivate the pathogen by heat or by chemical means so that it is no longer
capable of replication in the host. Chemical inactivation with formaldehyde or various
alkylating agents has been successful.

2.3.1 Purified Macromolecules as Vaccines

Polysaccharide vaccine: The virulence of some pathogenic bacteria depends
primarily on the anti-phagocytic properties of their hydrophilic polysaccharide capsule.
Coating of the capsule with antibodies and/or complement greatly increases the ability of
macrophages and neutrophils to phagocytose such pathogens. These findings provide the
rationale for vaccine consisting of purified capsular polysaccharide.

Toxoid Vaccine: Some bacterial pathogens, including those that cause diphtheria and
tetanus, produce exotoxins. These exotoxins produce many of the disease symptoms that
result from infection. Diphtheria and tetanus vaccines, for example, can be made by purifying
the bacterial exotoxin and then inactivating the toxin with formaldehyde to form a toxoid.
Vaccination with toxoid induces antitoxoid antibodies, which are also capable of binding to
the toxin and neutralizing its effect. So formed toxoid are called formol toxoid.
Recombinant Antigen Vaccines: The first recombinant antigen vaccine approved
for human use is the Hepatitis B vaccine. This vaccine was developed by cloning the gene for
hepatitis B virus (HBsAg) in yeast cells (Pichia pastoris). This recombinant Hepatitis B
vaccine has been shown to induce the production of protective antibodies.

2.3.2 Recombinant-Vector Vaccines:

It is possible to introduce genes that encode major antigens of especially virulent pathogens
into attenuated viruses or bacteria. The attenuated organism serves as a vector, replicating
within the host and expressing the gene products of the pathogen.
DNA Vaccines: Plasmid DNA encoding antigenic proteins is injected directly into the
muscle of recipient. Muscle cells take up the DNA and encoded protein is expressed, leading
to both humoral antibody response and a cell-mediated response.
Synthetic Peptide Vaccines: Peptides are not as immunogenic as proteins and it is
difficult to elicit both humoral and cellular immunity to them. The use of conjugates and
adjuvants can assist in raising protective immunity to peptides, but barriers to the widespread
use of peptide vaccines remain and pose an interesting problem for immunologists.

2.4 Some Processes associated with

preparation of Vaccines
Attenuation
To "attenuate" is to weaken a live micro-organism by ageing it or altering its growth
conditions. This is accomplished by serial passage, that is, passing the live micro-organism
through animal tissue several times to reduce its potency. For example, measles virus is
passed through chick embryos, poliovirus through monkey kidneys, and the rubella virus
through human diploid cells - the dissected organs of an aborted foetus.
Vaccines made in this way are often the most successful vaccines, probably because they
multiply in the body thereby causing a large immune response. However, these live,
attenuated vaccines also carry the greatest risk because they can mutate back to the virulent
form at any time.

Detoxification
Some vaccines are made from toxins. In these cases, the toxin is often treated with aluminium
or adsorbed onto aluminium salts to decrease the toxin's harmful effects. After the treatment,
the toxin is called a "toxoid". Examples of toxoids are the diphtheria and the tetanus vaccines.
Vaccines made from toxoids often induce low-level immune responses and are therefore
sometimes administered with an "adjuvant", an agent that increases the immune response.

HEPATITIS-B VACCINE
Hepatitis - B vaccine (rDNA) is a non-infectious recombinant DNA Hepatitis B vaccine. It
contains purified surface antigen of the virus obtained by culturing genetically-engineered
Pichia pastoris yeast cells having the surface antigen gene of the Hepatitis B virus. The
Hepatitis-B surface antigen (HBsAg) expressed in the cells of Pichia pastoris is purified
through several chemical steps and formulated as a suspension of the antigen adsorbed on
aluminium hydroxide and thiomersal is added as preservative.

2.5 Good Manufacturing Practices

Good manufacturing practice (GMP) (also referred to as cGMP or Current Good
Manufacturing Practices)is that part of quality assurance which ensures that products are
consistently produced and controlled to the quality standards appropriate to their intended use
and as required by the marketing authorization. GMP is aimed primarily at diminishing the
risks inherent in any pharmaceutical/biotechnological production, which may broadly be
categorized in two groups: cross contamination/mix-ups and false labelling. Above all,
manufacturers must not place patients at risk due to inadequate safety, quality or efficacy; for
this reason, risk assessment has come to play an important role in WHO quality assurance
guidelines.
A good manufacturing practice (GMP) is a production and testing practice that helps to
ensure a quality product. Many countries have legislated that pharmaceutical and medical
device companies must follow GMP procedures, and have created their own GMP guidelines
that correspond with their legislation. Basic concepts of all of these guidelines remain more
or less similar to the ultimate goals of safeguarding the health of the patient as well as
producing good quality medicine, medical devices or active pharmaceutical products. In the
U.S. a drug may be deemed adulterated if it passes all of the specifications tests but is found
to be manufactured in a condition which violates current good manufacturing guidelines.
Therefore, complying with GMP is a mandatory aspect in pharmaceutical manufacturing.
Although there are a number of them, all guidelines follow a few basic principles:

Manufacturing processes are clearly defined and controlled. All critical processes are
validated to ensure consistency and compliance with specifications.

Manufacturing processes are controlled, and any changes to the process are evaluated.
Changes that have an impact on the quality of the drug are validated as necessary.

Instructions and procedures are written in clear and unambiguous language.

Operators are trained to carry out and document procedures.

6

Records are made, manually or by instruments, during manufacture that demonstrate

that all the steps required by the defined procedures and instructions were in fact taken
and that the quantity and quality of the drug was as expected. Deviations are investigated
and documented.

Records of manufacture (including distribution) that enable the complete history of a

batch to be traced are retained in a comprehensible and accessible form.

The distribution of the drugs minimizes any risk to their quality.

A system is available for recalling any batch of drug from sale or supply.

Complaints about marketed drugs are examined, the causes of quality defects are
investigated, and appropriate measures are taken with respect to the defective drugs and
to prevent recurrence.

GMP guidelines are not prescriptive instructions on how to manufacture products. They are a
series of general principles that must be observed during manufacturing. When a company is
setting up its quality program and manufacturing process, there may be many ways it can
fulfill GMP requirements. It is the company's responsibility to determine the most effective
and efficient quality process.

3. Clean Rooms
A clean room is a controlled environment where products are manufactured. It is a room in
which the concentration of airborne particles is controlled to specified limits. Eliminating
sub-micron airborne contamination is really a process of control. These contaminants are
generated by people, process, facilities and equipment. They must be continually removed
from the air. The level to which these particles need to be removed depends upon the
standards required. The only way to control contamination is to control the total environment.
Air flow rates and direction, pressurization, temperature, humidity and specialized filtration
all need to be tightly controlled. And the sources of these particles need to controlled or
eliminated whenever possible. There is more to a clean room than air filters. Clean rooms are
planned and manufactured using strict protocol and methods. They are frequently found in
electronics, pharmaceutical, biopharmaceutical, medical device industries and other critical
manufacturing environments.

3.1 Clean Room Operating Parameters

Room Cleanliness: Expressed as a classification per Fed. Std. 209E (e.g., Class 10, Class
100, Class 1,000, Class 10,000, Class 100,000), room cleanliness levels are the basis of any
clean room design. It is important to know what classification you require, since an overdesigned room can be costly to build and costly to operate.
Room Temperature/Humidity: If the process is not affected by temperature/humidity
conditions which fall outside of human comfort ranges, do not specify tight tolerances. A
typical specification is 70 F2 and 30-70% RH. Tighter tolerance will result in unnecessarily
escalated costs.
Lighting Level: Level of 100 foot candles at the work surface is more than adequate for
close assembly work. Higher lighting levels are costly in terms of energy consumption and
initial cost. Lower levels can be maintained by control switching.
Process Flow: This deals with the process being carried out within the room. The process
will dictate the materials of construction as well as the layout of the facility. If the process is
general in nature, a more flexible layout is recommended. The process should be defined as
completely as possible before any clean room is laid out. This sounds elementary, but a
poorly defined process will result in an unsatisfactory clean room, or one in which
modifications are being made before the paint is dry. A knowledgeable clean room consultant
will have experience in process flow and will be able to offer valuable assistance.

SOURCES

ISSUES

Facility

Dust, bacteria, non-viable particulates

Bacteria,

Utilities

non-viable

particulates,

hydrocarbons, aerosols
Bacteria, non-viable particles, hard to clean

Equipment

areas
Non-sterile

connections,

growth

supporting

process parameters, aerosols, metallic silvers,

Process

fibers, glass and rubber particles

People

Viable and non-viable particles, skin cells,

bacterial

4 .Key Elements of Contamination Control

HEPA (High Efficiency Particulate Air Filter) - These filters are extremely important for
maintaining contamination control. They filter particles as small as 0.3 microns with a
99.97% minimum particle-collective efficiency.
Clean Room Architecture - Clean rooms are designed to achieve and maintain a airflow in
which essentially the entire body of air within a confined area moves with uniform velocity
along parallel flow lines. This air flow is called laminar flow. The more restriction of air flow
the more turbulence. Turbulence can cause particle movement.
Filtration - In addition to the HEPA filters commonly used in cleanrooms, there are a number
of other filtration mechanisms used to remove particles from gases and liquids. These filters
are essential for providing effective contamination control.
Clean Room Garments- Before entering the clean rooms a person should remove his street
footwear and street clothes and wear cleanroom garments. The garments should be non-static
and should shed minimum number of particles. Clean room garments also include Gloves,
face masks and head covers

Limits For Particle Count:

Grades

A
B

At Rest

In Operation

Maximum no. of Particles

Permitted per m3

Maximum no. of Particles

Permitted per m3

0.5-5m

>5m

0.5-5m

>5m

3500
3500

0
0

3500
350000

0
2000

C
D

2000
20000

35000
350000

3500000
ND

20000
ND

4.1Enviromental Monitoring Program

An appropriate microbial monitoring program should be established and used in
production and laboratory facilities for both sterile and non-sterile products. The
complexity of testing performed and the frequency of testing may vary
depending upon the type of product, i.e. sterile versus non-sterile, the type of
sterilization process, e.g., terminal sterilization versus aseptic filling and even
within processes, e.g., overkill sterilization cycles versus combined biological
indicator bioburden based cycles and absolute bioburden based cycles. Aseptic
processing is the most stringent application of these principles.

Limits for Microbial Contamination:

SETTLE
GRADE
S

AIR

PLATES

SAMPLES

DIA

cfu/m3

90cm/4hr
s

CONTACT
PLATES,
D55mm
cfu/plate

GLOVE
PRINTS (5
FINGERS)

<3

<3

<3

<3

10

100

50

25

200

100

50

10

5. The Vaccine Production Plant

The vaccine production plant has three departments:
1. Production Department
2. Quality Control Department
3. Quality Assurance Department

The production of vaccine proceeds in two stages: Firstly upstream processing (up till
harvesting
of the
desired protein
UPSTREAM
PROCESSING
AREA produced by fermentation). Secondly downstream
processing
whichfermentation,
involves purification
of that
protein.
After Acid
purification,
the vaccine
Inoculation,
Harvesting,
Cell
disruption,
precipitation
of theisdesired prote
formulated in the formulation area. In addition to these three units, there is a support area
that supplies all the solutions, chemicals and sterilized equipments to the above three units.
To summarize, the production unit can be divided into four divisions:

DOWNSTREAM PROCESSING AREA

Protein is purified here up to 98% with the help of chromatographic techniques.

Production Area

FORMULATION AREA
The active raw material is mixed up with thiomersal solution in an Aseptic Formulation System

SUPPORT AREA
Provides media, buffers, glasswares and solutions to the above areas. Sterilization by autoclaving and
11

5.1 Critical Systems

HVAC System
HVAC system is short for Heating, Ventilation and Air Conditioning system. This system
generates high quality air and controls concentration of air borne particles. It is constructed
and used in the manner which minimizes the introduction, generation and retention of the
particles in the rooms and in which the relevant parameters e.g., temperature, humidity and
pressure are controlled as necessary

Water System
The water used for production process is provided by the critical system
area. Water for Injection (WFI), Purified water (PW), clean steam
generation and distribution come under the preview of water system.

Purified
Water

WFI

PURE
STEAM

Conductivi
ty

1.3S/cm 1.3S/cm

1.3S/cm

pH

57

57

57

12

TOC

<500
ppb

<500 ppb

<500 ppb

Microbiolo
gy

100
cfu/ml

10cfu/100 10cfu/100
ml
ml

5.2 Quality Control (QC)

Quality control (QC) department has the responsibility to conduct the various tests that deal
with the checking and maintenance of the quality of the products being synthesized. In
vaccine production utmost importance has to be given to the product purity as well as to its
ability to mount the immune response. To ensure this the quality of all the materials coming
in contact with the product or the processes directly involved with the production must be
ascertained.
The QC Department examines various factors like Potency, Viability, Contaminations,
Water Purity, Sterility, Raw Materials etc.; all these factors determine the success of the
product in the market.

5.3 Quality Assurance (QA)

The Quality Control (QA) is an independent department which is not under the influence of
either the production, QC or any other department of the company. The release of any batch
of vaccine is the responsibility of the Quality Assurance (QA) department only. Thus, QA has
to make sure that the product is safe for human consumption and it complies with all the
guidelines of the regulatory authorities. It keeps a check on all the tests involved whether in
process, or on the raw material or the bulk. The QC has the job of conducting the tests. Also,
it is the responsibility of QA to check the records of all the tests which are done to produce a
vaccine.

13

6. Hepatitis B
The term 'hepatitis' simply means inflammation of the liver. Hepatitis may be caused by a
virus or a toxin such as alcohol. Other viruses that can cause injury to liver cells include
the hepatitis A and hepatitis C viruses. These viruses are not related to each other or to
hepatitis B virus and differ in their structure, the ways they are spread among individuals, the
severity of symptoms they can cause, the way they are treated, and the outcome of the
infection.
Hepatitis B is an infectious hepatitis caused by the hepatitis B virus (HBV). This infection
has two possible phases; 1) acute and 2) chronic.
1. Acute hepatitis B refers to newly acquired infections. Affected individuals notice
symptoms approximately 1 to 4 months after exposure to the virus. In most people
with acute hepatitis, symptoms resolve over weeks to months and they are cured of
the infection. However, a small number of people develop a very severe, lifethreatening form of acute hepatitis called fulminant hepatitis.
2. Chronic hepatitis B is an infection with HBV that lasts longer than 6 months. Once
the infection becomes chronic, it may never go away completely.
Approximately 90% to 95% of infected adults are able to fight off the virus so their infection
is cured. Only about 5% to 10% of adults infected with HBV go on to develop chronic
infection. Children are at much higher risk for chronic infection. Up to 90% of infected
young children will fail to clear the virus from their bodies and go on to develop chronic
infection.
About two-thirds of people with chronic HBV infection are chronic carriers. These people do
not develop symptoms, even though they harbor the virus and can transmit it to other people.
The remaining one third develop "active" hepatitis, a disease of the liver that can be very
serious.

Transmission and Causes

The hepatitis B virus is known as a blood-borne virus because it is transmitted from one
person to another via blood or fluids contaminated with blood. Another important route of
transmission is from an infected mother to a new-born child, which occurs during or shortly
after birth.

Direct contact with blood may occur through the use of dirty needles during illicit
drug use, inadvertent needle sticks experienced by healthcare workers, or contact with
blood through other means. Semen, which contain small amounts of blood, and saliva that
is contaminated with blood also carry the virus.

14

The virus may be transmitted when these fluids come in contact with broken skin or a
mucous membrane (in the mouth, genital organs, or rectum) of an uninfected person.

Symptoms
Half of all people infected with the hepatitis B virus have no symptoms and may never realize
that they have been infected. Adults are more likely to develop symptoms than children. For
those who do get sick, symptoms usually develop within 1 to 4 months after exposure to the
virus. The initial symptoms are often similar to the flu.
Common symptoms of hepatitis B include:
Appetite loss; Feeling tired (fatigue); Nausea and vomiting, Itching all over the body; Pain
over the location of the liver (on the right side of the abdomen, under the lower rib cage);
Jaundice (a condition in which the skin and the whites of the eyes turn yellow in color); Dark
urine; Pale-colored stools (grayish or clay colored)

6.1 Hepatitis B Virus

The hepatitis B virus is a DNA virus, meaning that its genetic material is made up of
deoxyribonucleic acids. It belongs to a family of viruses known as Hepadnaviridae. The virus
is primarily found in the liver but is also present in the blood and certain body fluids.
Hepatitis B virus consists of a core particle (central portion) and a surrounding envelope (outer
coat). The core is made up of DNA and the core antigen (HBcAg). The envelope contains the
surface antigen (HBsAg). These antigens are present in the blood and are markers that are
used in the diagnosis and evaluation of patients with suspected viral hepatitis.
Hepatitis B virus, is composed of several layers. Outermost, there is a membrane envelope,
picked up when the virus buds from an infected cell. The membrane is studded with three
similar forms of an envelope protein. These envelope proteins search for cells to infect,
attaching to a yet-unknown receptor on the liver cell surface. Inside the membrane, there is a
protein capsid. As with many viral capsids, it is formed of a single type of protein, which
associates to form a hollow shell with icosahedral symmetry

15

Outer
Envelope
Reverse
Transcriptase
DNA
Capsid
.

The most common form of hepatitis B virus contains a capsid composed of 240 capsid
proteins, but about 10% have a smaller capsid comprised of 180 proteins.

Genome
It is unusual because the DNA is not fully double-stranded. One end of the full length strand is
linked to the viral DNA polymerase. The genome is 30203320 nucleotides long (for the fulllength strand) and 17002800 nucleotides long (for the short length-strand). The negativesense, (non-coding), is complementary to the viral mRNA. The viral DNA is found in
the nucleus soon after infection of the cell. The partially double-stranded DNA is rendered
fully double-stranded by completion of the (+) sense strand and removal of
a protein molecule from the (-) sense strand and a short sequence of RNA from the (+) sense
strand. Non-coding bases are removed from the ends of the (-) sense strand and the ends are
rejoined. There are four known genes encoded by the genome, called C, X, P, and S. The core
protein is coded for by gene C (HBcAg), and its start codon is preceded by an upstream inframe AUG start codon from which the pre-core protein is produced. HBeAg is produced

by proteolytic processing of the pre-core protein. The

DNA polymerase is encoded by gene P. Gene S is the gene that codes for the
surface antigen (HBsAg). The HBsAg gene is one long open reading frame but contains three
in frame "start" (ATG) codons that divide the gene into three sections, pre-S1, pre-S2, and S.
Because of the multiple start codons, polypeptides of three different sizes called large, middle,
and small (pre-S1 + pre-S2 + S, pre-S2 + S, or S) are produced. The function of the protein
coded for by gene X is not fully understood but it is associated with the development of liver
16

cancer. It stimulates genes that promote cell growth and inactivates growth regulating
molecules.

Replication
The life cycle of hepatitis B virus is complex. Hepatitis B is one of a few
known pararetroviruses: non-retroviruses that still do use reverse transcription in their
replication process. The virus gains entry into the cell by binding to an unknown receptor on
the surface and being endocytosed in. Because the virus multiplies via RNA made by a host
enzyme, the viral genomic DNA has to be transferred to the cell nucleus by host proteins
called chaperones. The partially double stranded viral DNA is then made fully double stranded
and transformed into covalently closed circular DNA (cccDNA) that serves as a template for
transcription of four viral mRNAs. The largest mRNA, (which is longer than the viral
genome), is used to make the new copies of the genome and to make the capsid core protein
and the viral DNA polymerase. These four viral transcripts undergo additional processing and
go on to form progeny virions that are released from the cell or returned to the nucleus and recycled to produce even more copies. The long mRNA is then transported back to the
cytoplasm where the virion P protein synthesizes DNA via its reverse transcriptase activity.

Pathogenesis
Hepatitis B virus primarily interferes with the functions of the liver by replicating in liver
cells, known as hepatocytes. The receptor is not yet known, though there is evidence that the
receptor in the closely related duck hepatitis B virus is carboxypeptidase D. The virions bind
to the host cell via the preS domain of the viral surface antigen and are subsequently
internalized by endocytosis. PreS and IgA receptors are accused of this interaction. HBVpreS-specific receptors are expressed primarily on hepatocytes; however, viral DNA and
proteins have also been detected in extrahepatic sites, suggesting that cellular receptors for
HBV may also exist on extrahepatic cells
During HBV infection, the host immune response causes both hepatocellular damage and
viral clearance. Although the innate immune response does not play a significant role in these
processes, the adaptive immune response, in particular virus-specific cytotoxic T
lymphocytes(CTLs), contributes to most of the liver injury associated with HBV infection.
CTLs eliminate HBV infection by killing infected cells and producing antiviral cytokines,
which are then used to purge HBV from viable hepatocytes, Although liver damage is
initiated and mediated by the CTLs, antigen-nonspecific inflammatory cells can worsen CTLinduced immunopathology, and platelets activated at the site of infection may facilitate the
accumulation of CTLs in the liver.

17

6.3 Hepatitis-B Vaccine

The hepatitis B virus was discovered in 1965 by Dr. Baruch Blumberg who won the Nobel
Prize for his discovery. Originally, the virus was called the "Australia Antigen" because it was
named for an Australian aborigine's blood sample that reacted with an antibody in the serum
of an American haemophilia patient.

First Commercial Hepatitis B Vaccine

In 1981, the FDA approved a more sophisticated plasma-derived hepatitis B vaccine for
human use. This inactivated type of vaccine involved the collection of blood from hepatitis
B virus-infected (HBs Ag-positive) donors. The pooled blood was subjected to multiple steps
to inactive the viral particles that included formaldehyde and heat treatment (or
pasteurization). Merck Pharmaceuticals manufactured this plasma vaccine as "Heptavax"
which was the first commercial hepatitis B virus vaccine. The use of this vaccine was
discontinued in 1990 and it is no longer available in the U.S.

Drawbacks of the Plasma Derived Vaccine.

Unfortunately, although plasma derived vaccines have repeatedly been shown to be safe and
effective, they several drawbacks:
(i)

The manufacturing process must involve tedious, stringent and time consuming
procedures to inactivate infectious hepatitis B virus and often living pathogens
that might be present in plasma and are responsible for blood-transmitted diseases
such as acquired immune -deciency syndrome;

(ii)

The cost of production is relatively high, which is against the implementation of

mass immunization programs by health services of poor and even medium-level
countries;

(iii)

The availability of suitable amounts of human plasma is limited; and

(iv)

A lengthy (about 6 months) innocuity test in chimpanzees is required.

18

7. Current Recombinant Hepatitis B Vaccines

In 1986, research resulted in a second generation of genetically engineered (or DNA
recombinant) hepatitis B vaccines. These new approved vaccines are synthetically prepared
and do not contain blood products.
Virus recovered from the plasma of a hepatitis B carrier has been used to prepare viral DNA;
that DNA has been cloned in Escherichia coli and the gene coding for HBsAg has been
isolated.
This gene has been inserted into yeast and mammalian cells by means of appropriate
expression vectors. Purified antigens obtained from transfected cultures containing it have
been shown to induce antibodies in mice and guinea-pigs and have been formulated into
vaccines. Electron microscopy has revealed that the purified HBsAg used for these vaccines
exists as particles 1530 nm in diameter, with the morphological characteristics of free
surface antigen in plasma and of the purified antigen now used in plasma-derived hepatitis B
vaccines. Some of the vaccine formulations containing these materials have already been
shown to be immunogenic in mice, chimpanzees and other monkeys, and human beings, with
antigenic potencies similar to those of vaccine made from plasma-derived antigen. The yeast
used for this purpose is Pichia pastoris.

7.1 Pichia pastoris

Yeast offers certain over prokaryotic hosts, and as eukaryotes, the intracellular environment is
generally more suitable for correct folding of eukaryotic proteins. Yeast also has the ability to
glycosylate proteins, which may be crucial for biological activity. Saccharomyces cerevisiae
was the first eukaryotic expression system to be used, and remains the most common due to
the vast amount of information available on its genetics and physiology. However, expression
of heterologous proteins in Saccharomyces is not always optimal for large-scale production
due to problems such as loss of the plasmid during scale-up, hyperglycosylation, and low
protein yield. The methylotrophic yeast, Pichia pastoris, has been developed for expression
as an alternative to S. cerevisiae. Advantages of the Pichia expression system include: growth
to very high cell densities in a simple defined medium, strongly inducible promoters, and
commercially available methods, host strains, and expression vectors for genetic
manipulations.

7.1.1 Genome and Recombinant Strains of Pischia

pastoris

19

The genome of P. pastoris contains two copies of the alcohol oxidase gene, AOX1 and AOX2,
which allow for growth on methanol as the sole carbon source. The AOX1 promoter regulates
85% of the alcohol oxidase activity in the cell, and is the promoter used to drive heterologous
protein expression in Pichia. The AOX1 promoter-Gene X expression cassette is inserted
into the Pichia genome along with a histidinol dehydrogenase gene (HIS4) or a drug resistant
gene such as zeosin, for selection of transformed cells in his- host strains, i.e. GS115 (his4).
Insertion of the expression cassette into the HIS4 or AOX1 locus, by single crossover
integration, generates a Mut+ strain (methanol utilization plus), a phenotype whose growth
characteristics are indistinguishable from wild type P. Pastoris. Alternatively, when the
expression cassette is inserted within the AOX1 locus by double crossover gene
transplacement, the Muts strain (methanol utilization slow) is generated. Another way of
obtaining a Muts phenotype is by disruption of the AOX1 gene via gene insertion i.e. KM71.
The P. pastoris KM71 strain grows very slowly in media containing methanol as the sole
carbon source because of the defective AOX1 gene [5]. A third host strain used for
heterologous protein expression is the Mut (methanol utilization minus) strain in which both
the AOX1 and AOX2 genes are disrupted i.e. MC100-3. The alcohol oxidase defective strain,
MC100-3, cannot utilize methanol as its sole carbon source. The inability to grow on
methanol requires the use of alternate carbon source, such as glycerol, for growth and
recombinant protein production. However non-limiting glycerol concentrations in shake flask
culture can cause repression of the AOX1 promoter and may result in production of ethanol,
also a strong repressor of the AOX1 promoter.
Protease deficient strains of P. pastoris (SMD series) have been developed because some
secreted foreign pro teins are unstable in the P. pastoris culture medium. Although native
proteases of P. pastoris are not secreted into the fermentation medium, cell lysis can occur,
especially at high cell densities, releasing proteases. This problem may be overcome by using
protease deficient host strains. For secretion of foreign proteins, vectors contain a DNA
sequence immediately following the AOX1 promoter that encodes a secretion signal.
Examples of secretion signals in Pichia are the S. cerevisiae -factor prepro signal sequence,
and the P. pastoris acid phosphotase gene (PHO1)

8. Support Area
Support area deals with initial preparation of production like
preparing media, autoclaving etc
8.1 Mechanism of killing by dry heat
Dry heat kills the organisms by destructive oxidation of
essential cell constituents
Killing of most resistant spores by dry heat requires a
temperature of about

20

160 C for 60 mins

Dry heat is employed for glasswares, syringes, metal
instruments and paper wrapped goods, which are not spoil by
high temperature
It is also use for anhydrous ,oils and powder that are
impermeable to moisture

8.2 Mechanism of killing by moist heat

Moist heat kills the organism by coagulating and denaturing
their enzymes and structural proteins.
Sterilization by moist heat of the most resistant spores
generally requires 121 C for 15- 30 mins.
Moist heat is used for the sterilization of culture media and all
other materials steam can penetrate
Moist heat is more effective than dry heat .
Sterilization can be done at lower temperature in the given
time at a shorter duration at the same temperature.
Upon heating wet proteins release free-sh groups and form
smaller peptide chain which in turn form new complexes
different from original protein molecules.

21

22

8. Upstream Processing
Upstream processing (USP) involves inoculation and then fermentation of the organism
(Pichia pastoris) to produce the desired protein (HBs Ag). Since this process deals with the
live organism, it is also called live area. The area also performs the operations of harvesting,
centrifugation, preparation of the disruption cream, acid precipitation and centrifugation.

STEP 1: GPT and Sterility of Plate and Flask

5 Petri Plates containing solid culture media and 7 Erlenmeyers flasks with 500ml liquid
culture media each are received from the support area. Vial containing the cell seed lot for
inoculation is received from the Working Cell Bank (WCB). One of the Erlenmeyer flasks is
checked for Sterility by incubating the flask in a BOD incubator for 72 hours at 30C. The
flasks are checked for any growth in the culture media. For the media to be sterile, no growth
should be observed in the flasks.
The culture media is again tested through a Growth Promotion Test (GPT). In this test one
petri plate and a flask are inoculated with yeast cells from the vial. The petri plate is
incubated in the BOD incubator for 5 days and flask is incubated in a shaker for 24 hours at
30C. These are then checked for growth and physiology under a stereoscope.

STEP 2: Pre- Inoculum Preparation

Streaking:
The plates containing media are streaked with WCB (Working Cell Bank which is preserved
in quality control at -70oC). Then the plates are incubated at 30oC till individual colonies of
Pichia pastoris are observed.

500 ml flask inoculation:

Inoculation is the placement of something to where it will
grow or reproduce. When growth is observed on plate. 2-5
colonies are inoculated in 500 ml flask containing 50 ml
media and incubated at 30oC for appropriate time at 250 rpm.

2L flask inoculation:
50 ml of previously grown inoculum (of 500 ml flasks) is
added to 2L flask and incubated at 30oC for appropriate time
at 250 rpm.
The process of pre- inoculation is carried out under a Bio-Safety Cabinet. A biological safety
cabinet is a ventilated cabinet, which uses a variety of combinations of ULPA (ultra-low
penetration air) filtration, laminar air flow and containment to provide personnel, product or
23

environmental protection or protection of all components against particulates or aerosols

from bio hazardous agents. An ULPA filter can remove from the air at least 99.999% of dust,
pollen, mold, bacteria and any airborne particles with a size of 120 nanometres (0.12 micron)
or larger. The pre-inoculum is then also prepared in Erlenmeyer Flasks: the flasks containing
500ml media are inoculated with 50 ml culture media and the flasks containing 2.0L culture
media are inoculated with 450ml culture media.

STEP 3: Fermentor Preparation

Before carrying out fermentation in a fermentor the system needs to be prepared as follows:

Sterilization In Place (SIP) or In situ Sterilization:

Steam is an effective sterilant for two reasons. First, saturated steam is an extremely effective
carrier of thermal energy. Second, steam is an effective sterilant. This is because any
resistant, protective outer layer of the microorganisms can be softened by the steam, allowing
coagulation of the sensitive inner portions of the microorganism.
Requirements: Steam sterilization requires four conditions:
Adequate contact, sufficiently high temperature, correct time and sufficient moisture

a) Empty vessel Sterilization: In it the fermentor is sterilized by directly

passing the pure steam into the vessel (and not jacket) and all the addition lines.
During the sterilization all the filters being used are also sterilized. The
sterilization temperature is maintained at 121C for 30 minutes after which the
system is allowed to cool to normal temperature. To help maintain the temperature
and also to maintain the efficiency of steam, steam traps are used. It is basically a
device used to discharge condensate and non-condensable gases while not
permitting the escape of live steam.
b) Full Vessel Sterilization with Culture Media : After empty vessel
sterilization the mechanical seal is sterilized using steam and the condensate is
used to lubricate it. Then pH probe is calibrated using buffers of pH 4.01 and 7.0
and the pH probe and DO probe are inserted into the fermentor. The support area
provides the media for the fermentor. The media is charged into the fermentor
along with antifoam.
Then media is steam sterilized by heating it to a temperature of 121C for 30 minutes. Since
vitamins and trace elements degrade at high temperature so these are added afterwards
through filters and sterilized at a lower temperature.

24

STEP4: Inoculum Preparation

Prior to this step the yeast culture is checked for its purity by Gram Staining. As the cell
wall of Pichia pastoris is peptidoglycan rich, so it retains crystal violet stain. After staining,
the culture is checked for any bacterial contamination under a stereoscope. The culture is then
is transferred aseptically to the inoculum fermentor(22L) and the fermentation parameters
are set. The working volume of the fermentation is 15L. During fermentation the growth is
accessed by taking sample at various intervals of time and checking the wet weight of
sample. The fermentation process is carried out until the desired wet weight is achieved (110130 g/L). This process generally takes around 16-17 hours.

STEP 5: Fermentation
When the desired wet weight has been achieved, the culture is transferred into the
production fermentor(600L) for production of desired protein. Before transferring the
inoculum the transfer line is sterilized. The working volume for this process is 350L. During
fermentation the wet weight analysis of the culture is carried out by taking samples at regular
intervals. After a desired wet weight is reached methanol is added at particular flow rate. This
is done because methanol acts as carbon source as well as inducer for gene (producing
required protein). Also as fermentation proceeds vitamins and trace elements along with yeast
extract are added at specific intervals of time through vitamin addition line. The fermentation
process is carried out for specific number of hours(130 hours) after which the batch is
harvested.

FERMENTOR
Basic Components of Fermentor: The fermentor basically consists of 4 components:

1)

Thermostatic

system:

The
fermentor
is
components
for
temperature during different processes:

equipped with following

controlling
the

25

a) Circulation Pump: its main purpose is to circulate water inside the jacket to maintain
temperature.
b) Steam injector:
c) Plate heat exchanger:
d) Pulsed valves for heating and cooling:
2) Agitator system:
a) Motor
b) 3 rushton turbines with adjustable height on shaft:
c) Double mechanical seal with condensate lubrication:
3) Aeration system:
The air inlet is equipped with:
a) Rota meter and needle valve:
b) Mass flow controller
c) Sterile filter
d) Valves for sterilization and operation
The exhaust gas line consists of :
a) Exhaust cooler
b) Thermal trap
c) Sterile filter
d) Pressure control valve
e) Valves for sterilization and operation
4) Addition ports:
a)
b)
c)
d)
e)
f)
g)
h)

Acid addition
Alkali addition
Antifoam addition
Methanol addition
Vitamin addition
Transfer from pre-fermentor
Transfer to harvest tank
Sampling port

Control System - There are 3 types of controllers to control various parameters:

1. Cascade controller: Cascade Controller uses the output of the primary controller
(PID controller) to manipulate the set point of the secondary controller as if it were
the final control element. Its typical application is DO control with airflow, stirrer and
pressure as secondary or servo controllers.
Reasons for cascade control:
Allow faster secondary controller to handle disturbances in the secondary loop.
26

Allow secondary controller to handle non-linear valve and other final control element
problems.
Allow operator to directly control secondary loop during certain modes of operation (such
as start-up).
2. Set point controller: In this type of controller a signal based on set point is sent to
external device to control a parameter. It also uses PID controller to reach set point
value. Its common application includes airflow control, stirrer control etc.
3. Timer controller: as the name suggests in it controller is triggered by a digital input
(which is generated after set time).
Depending upon type of controller used various parameters are controlled as follows:

Temperature control Loop:

It maintains temperature by controlling the steam valve or cooling water and tap water
valve (which are connected to heat exchanger). The temperature controller is
implemented as cascade control loop with vessel temperature as primary controller and
jacket temperature as secondary controller. The primary controller sends signals to the
secondary controller which in turn sends signals in form of pulses to the valves for either
steam or tap water and cold water. Depending upon whether heating or cooling is required
the valves are opened and hence temperature is achieved.
Po2 control loop:
Po2 is maintained by modulating the airflow via sparger and or stirrer speed and or
pressure. Po2 controller is a multi-stage cascade controller. Here po2 PID controller acts
as primary controller with output linked to set point of one of the 3 secondary controllers
for airflow, stirrer and pressure. The hierarchy can be set by the user. The default
hierarchy is airflow, stirrer and pressure in that order. The master controller tries to
achieve the set point by changing the set point of controller with first priority. Even after
reaching the maximum value for first controller if set point is not achieved then primary
controller shifts to controller with second in priority (with first controller running at
maximum value) and so on for third controller.

pH control loop:

27

pH is maintained by adding either acid or base. It is a setpoint controller. Ph probe senses

the ph and sends signal to ph controller which in turn generates signal in form of pulses.
Depending upon the signal sent by controller valves for either alkali or acid addition is
opened. It is also PID controller.
Stirrer control loop:
Stirrer control loop adjusts the speed of agitator via AC servo drive. The stirrer control
loop is a set point controller which sends continuous output signal to external servo drive.
The servo drive is equipped with resolver to measure the speed of rotor which sends the
signal back to the controller.
Pressure control loop:
The pressure control loop maintains the head pressure of the fermentor vessel by
regulating the back pressure control valve. It is a set point PID controller which sends
continuous output signal. The pressure controller can be operated as servo controller
when pO2 is in cascade mode.

Airflow control loop:

Airflow control loop maintains the flow of air by regulating the mass flow controller. It is
a set point PID controller which sends continuous output signal. The airflow controller
can be operated as servo controller when pO2 is in cascade mode.

STEP 5: Harvesting and Centrifugation

Before transferring the broth to the harvest tank the harvest line is steam sterilized. Then the
fermented broth is allowed to cool in the Harvest Tank. After a particular temperature is
reached the broth is fed to the Disc Stack Centrifuge. The concentrated fraction with cells is
collected in the Balance Tank and supernatant is discarded.

Disccentrifugation
Stack Centrifuge
The cells are washed using continuous
system by feeding Purified Water to the
harvest tank. After a series of washing when the desired conductivity is reached the pellet is
28

collected and supernatant is discarded. This step concentrates the fermentation broth to half
of its initial volume.

STEP 6: Cell Disruption

Since HBsAg is intracellular so to extract it, the cells need to be disrupted. The cell
disruption cream is prepared in the harvest tank by adding KSCN and Cell Disruption
Buffer consisting of Tris, EDTA, NaCl and Sucrose. A particular amount of each buffer is
added and a desired wet weight and pH are obtained. NaOH is used to adjust its pH. The
disruption cream is pumped to Feed Tank through stainless steel pipes and from here to the
Bead Mill Cell Disruption Dynomill.

Bead Mill Cell Disruption

Dynomill

This method uses small glass, ceramic, or steel beads along with a high level of agitation. The
method, often referred to as "beadbeating", works well for all types of cellular material - from
spores to animal and plant tissues. The glass beads are of 0.7-0.8 mm diameter. The cells get
disrupted because of the high mechanical pressure generated in the bead mill. The disrupted
cream is then collected in a Reception Tank. The disruption cream is again pumped back to
feed tank and then again to the dynomill. This process is carried out for 5 cycles to obtain the
final disrupted cream. The disruption cream is in highly concentrated form, so it is diluted
with buffer to maintain the osmolarity. The various buffers used for this purpose are Tris,
EDTA, KSCN, NaCl, and Sucrose. The diluted disruption cream is pumped to the Acid
Precipitation Room.

STEP 7: Acid Precipitation

29

For carrying out acid precipitation principle of iso-electric precipitation is employed which
precipitates all other proteins except the protein of our interest. The isoelectric point (pI) is
the pH of a solution at which the net primary charge of a protein becomes zero. At a solution
pH that is above the pI the surface of the protein is predominantly negatively charged and
therefore like-charged molecules will exhibit repulsive forces. Likewise, at a solution pH that
is below the pI, the surface of the protein is predominantly positively charged and repulsion
between proteins occurs. However, at the pI the negative and positive charges cancel,
repulsive electrostatic forces are reduced and the dispersive forces predominate. The
dispersive forces will cause aggregation and precipitation. The isoelectric point for HbsAg
is 4.6
Diluted disruption cream is collected in a 500L tank through stainless steel pipes. The
precipitation is carried out in a Precipation Reactor. The temperature of the system is
maintained at 0o C by circulation of chilled water in the jacket. pH is maintained using 1M
HCL.

STEP 8: Centrifugation
The precipitation cream is pumped to a feed tank which pumps it to three tubular
centrifuges.These centrifuges are used to centrifuge the solution and then remove the pellet
from the bowls. This process removes the unwanted precipated out protein. The centrifuges
are operated at 16000 rpm. The supernatant is collected in a collection tank and then is
pumped to two tubular centrifuges for second centrifugation and then the supernatant is
transferred to a Neutralization tank. In the tank the pH is again increased to 8.0 using 2M
NaOH and then the supernatant is pumped into a Supernatant Reception Tank. From this
reception tank the Supernatant of Acid Precipitation (SH) is then transferred to the
downstream processing area through sterile pipes. This is aerosol generating area. Hence it is
enclosed with PVC sheets so as to minimize addition of particulates in the room air.

Tubulur Centrifuge

30

9. Downstream processing
Downstream processing involves purification of that protein which was separated by acid
precipitation. The protein is purified here up to 98%. Purification of the process is very
critical since viability of the vaccine depends upon its purity. There are two steps involved in
the purification of the antigen: Primary purification and Secondary purification.

9.1 Primary Purification

The acidified supernatant from acid precipitation room is brought here to the Down Stream
processing area for primary purification. The supernatant is collected in a collection tank.
Further steps involves in purification are described below:

STEP 1: Adsorption
Adsorption is carried out in an Adsorption Reactor which contains celite as an adsorption
matrix. Celite is a naturally occurring, soft, siliceous sedimentary rock that is easily crumbled
into a fine white to off-white powder. It has a particle size ranging from less than
1 micrometre to more than 1 millimeter, but typically 10 to 200 micrometres. This powder
has an abrasive feel, similar to pumice powder, and is very light as a result of its
high porosity. The typical chemical composition of oven-dried diatomaceous earth is 80 to
90% silica, with 2 to 4% alumina (attributed mostly to clay minerals) and 0.5 to 2% iron
oxide. Prior to adsorption of the protein on the surface of celite, it needs to be activated and
regenerated. The Regeneration of celite is done by adding 60L of 2M NaOH, making the
volume to 600L by adding purified water and then leaving it for settlement for some time and
finally draining the solution. The Activation of celite is done by adding 1M HCl and purified
water and draining the solution after allowing settlement for some time. After activation,
celite is rinsed twice with purified water.
Now, 125L SH is added to each of the two adsorption reactors and is allowed to settle on the
surface of celite. During this process a low pH(4.4) and temperature(4C) is maintained for
efficient adsorption. Adsorbed protein settles down and the supernatant is discarded. Two
steps of washing are performed with purified water and 5M KSCN.

Adsorption Reactor
31

STEP 2: Desorption
Celite along with adsorbed protein is transferred to the Desorption Reactor containing Tris
HCl. In the desorption tank, protein of interest is separated from celite by increasing the
temperature (18-23o C) and pH (9.2). These conditions result in dissolution of celite leaving
behind pure protein in the upper liquid. Three elutions one after another are taken The
desorbed protein is held in a holding tank having pH 8.01.

STEP 3: Press and plate filtration

The desorbed protein is further purified by passing it through plate and frame filters.
Cellulose (0.1) pads fitted in the filter remove celite particles from the protein. Centrifugal
pump is used for pumping the protein through this filter. The filtered protein is transferred to
a feed tank.

STEP 4: Concentration
The feed tank transfers the eluates to Ultra Filteration System tank through a peristaltic
pump. Protein is now concentrated by ultra filtration. There are tangential flow cartridge
filters (0.1) through which impurities move out tangentially and the concentrate is retained.
The udesired protein is removed simultaneously A volume of 3600 L is concentrated to about
40 L by this system in less than 8 hours.

STEP 5: Centrifugation and Filteration

Further purification is achieved by centrifuging the protein in a tubular centrifuge(Reyna) at
16000 rpm. Any impurities present in the protein are pelleted out and supernatant is retained.
The supernatant is collected in a filteration vessel and from here it is filtered through a
filteration assembly having cartridge filter (0.8-3). The pH is maintained at 8.02 and
conductivity 26 +3 S/m.

8.2 Secondary Purification

Secondary purification is accomplished by a number of steps involves various types of
chromatography. This assures purity levels of 95-98% for the protein to be formulated. The
chromatographic techniques used here are summarized below:

STEP 1: Negative Ion Exchange Chromatography

Ion-exchange chromatography retains analyte molecules on the column based
on coulombic (ionic) interactions. The stationary phase surface displays ionic functional
32

groups (R-X) that interact with analyte ions of opposite charge. Negative ion exchange
chromatography retains positively charged cations because the stationary phase displays a
negatively charged functional group. The column is filled with cellulose gel DE-52. Before
addition of the protein, the column is sanitized with the help of 0.2M NaoH and is
equilibrated (the pH and conductivity of the buffer is set equal to that of the protein). The
elution is carried out using an elution buffer whose pH and conductivity are higher than that
of the protein in the column.

A typical Chromatographic
Column

STEP 2: Immunoaffinity Chromatography

Immunonaffinity chromatography is a powerful technique for rapid purification of proteins. A
specific antibody is covalently immobilised on a matrix, across which the sample to be
purified is passed. The protein adsorbs to the ligand and can then be selectively desorbed in
such a way as to retain the activity of the purified sample. The matrix used here is sepharose
gel. The protein from the previous step is filled into this column and is allowed to settle and
then elution is carried out by an elution buffer.

STEP 3: Incubation and Thermal Treatment

The protein purified from immunoaffinity chromatography is incubated for 7-21 days at a
temperature 7-21C. This step is done to bring back the protein to its tertiary structure. The
protein is then kept in water bath at a temperature 60 + 2C for 2 hours. It decreases the
chances of contamination by murein viruses and growth of other microbial flora.

33

STEP 4: Gel Filtration Chromatography

Gel filtration chromatography seprarates proteins, peptides, and oligonucleotides on the basis
of size. Molecules move through a bed of porous beads, diffusing into the beads to greater or
lesser degrees. Smaller molecules diffuse further into the pores of the beads and therefore
move through the bed more slowly, while larger molecules enter less or not at all and thus
move through the bed more quickly. Both molecular weight and three-dimensional shape
contribute to the degree of retention. The matrix used here is Sephadex G-25. The column is
sanitized in NaOH. The eluate collection from this column is UV based i.e., the eluate is
collected within certain O.D. limits.

STEP 5: Positive Ion-Exchange Chromatography

In positive ion exchange chromatography, negatively charged molecules are attracted to a
positively charged solid support. In contrast to negative ion exchange, here the protein is
absorbed while the impurities are eluted out. Prior to subjecting the protein to this stage, it is
filtered using a 0.2 filter. A total volume of 7 L protein is obtained at the end of this process.

Positive Ion Exchange

Chromatography

STEP 6: Concentration
The protein is highly diluted by this stage, so it is concentrated by ultra filtration or
difiltration by Millipore system(0.1). At this stage the protein concentrates to a volume of
300-500 ml. This protein concentrate is again passed through a 0.45 filter.

STEP 7: HPLC
High-performance liquid chromatography is a chromatographic technique used to separate
a mixture of compounds in analytical chemistry and biochemistry with the purpose of
identifying, quantifying and purifying the individual components of the mixture. It is
34

basically a highly improved form of column chromatography. Instead of a solvent being

allowed to drip through a column under gravity, it is forced through under high pressures of
up to 400 atmospheres. That makes it much faster. HPLC system purifies the protein up to
95%.
STEP 8: FGFE Chromatography
Final Gel Filteration Size Exclusion is used for buffer exchange form Tris buffer to PBS
(Phosphate Buffer Saline). The column is packed with sephadex G-25 matrix. This is the last
chromatographic exercise that is used.

STEP 9: Filtration
Finally, filtration is done using sterile filters (0.2). After this step, we obtain ARM (Active
Raw Material) which is ready for formulation.
ARM is sent to the formulation unit where it is mixed with thiomersal, buffer and alhydrogel.

10. Formulation
Formulation of bulk Hepatitis B vaccine is the most critical stage of fermentation and every
care is taken that the process is sterile. Components of the final product have to be mixed in
proper concentration. Aseptic Formulation System is used for the purpose in the plant. It is
manual as well as automatic. This area is also class 10,000 area and has a cold storage
maintained at 2o C. AFS has three jacketed tanks interconnected with SS pipes and very
much controlled by pneumatic valves. Software controls opening and closing of the valves.

35

Main elements of formulation: The main elements of formulation are

represented in the diagram shown below:

STEP 1: Thiomersal solution preparation

Thiomersal is used as preservative for the vaccine. It is prepared first of all and is transferred
to final formulation tank (V2).

STEP 2: Buffer preparation

PBS is used as the buffer and is prepared in the buffer preparation tank (V1).

STEP 3: Alhydrogel preparation

Aluminium hydroxide gel is the adjuvant used here. Concentrated hydrogel is placed in
alhydrogel preparation tank (V3). It is diluted with filtered sterile WFI. It is homogenized at
proper rpm. It transferred to V2.

STEP 4: Adsorption
Antigen is adsorbed on the adjuvant.

STEP 5: Homogenization
Then thiomersal and buffer are added to V2 and the solution is homogenized.

STEP 6: Filtration
The final product is then filtered and collected in 20 L sterile containers through sampling
ports.

36

References
Wenhui Zhang, Mehmet Inan, and Michael M. Meagher(2000)
Fermentation Strategies for Recombinant Protein Expression in the
Methylotrophic Yeast Pichia pastoris
Eugenio Hardy, Eduardo Martnez, David Diago, Rau l Daz, Daniel
Gonzalez, and Luis Herrera(1999) Large-scale production of
recombinant hepatitis B surface antigen from Pichia pastoris
www.analyticalventura.com/iex-hplc.shtml
www.sigmaaldrich.com/life-science/proteomics/proteinchromatography/gel-filtration-chromatography.html
World Health Organisation, Technical Report Series No. 786 (1989)
Requirements for Hepatitis B Vaccines made by recombinant DNA
Techniques
Lee YS, Kim BK, and Choi EC(1998) Physicochemical properties of
recombinant hepatitis B surface antigen expressed in mammalian cell

37