Introduction to Pharmacopoeial requirement, Diseases security and Biosecurity principals

1. DEPARTMENT OF ANIMAL BIOTECHNOLOGY
Submitted by:
Dr. Vijayata
B.V.Sc. & M.V.Sc

3. • The basic requirement for manufacture and control of vaccine
shall Comply with the specifications of the relevant monograph of
the European pharmacopoeia.
• In order to assure the safety and efficacy of vaccine, ingredients
and the finished product must be manufactured to an acceptable
quality standard.
• The role of the default standard(s) is to define the minimum
quality standard with which vaccines must comply.
• A comprehensive testing strategy is therefore required in order to
address the issues of product origin, target species and market
place of the test material which could encompass:
- Master and working cell seeds
- Master virus seeds ,Bacterial seeds
- Materials of animal origin (e.g. trypsin, serum, plasma etc)

4. • A culture of a micro-organism distributed from a single bulk
into containers and processed together in a single operation in
such a manner as to ensure uniformity and stability and to
prevent contamination.
• A culture of cells distributed into containers in a single
operation, processed together and stored in such a manner as
to ensure uniformity and stability and to prevent
contamination.
• a system according to which successive batches of product are
prepared using the same Master Cell Seed or Master Seed.

5. • A culture of a micro-organism derived from the master
seed lot and intended for use in production, distributed into
containers and processed together in a single operation in such
a manner as to ensure uniformity and stability and to prevent
contamination.
• A culture of cells derived from the master cell seed and
intended for use in the preparation of production cell
cultures, processed together and stored in such a manner as to
ensure uniformity and stability and to prevent contamination.

6. • Culture of cells, essentially identical to those of the tissue of
origin and being no more than 5 in vitro passages to production
level from the initial preparation from the animal tissue.
Cultures of cells that have a high capacity for multiplication
in vitro.

7. • Substances of animal origin (e.g. serum trypsin and serum albumin)
may be used during the manufacture of veterinary immunological
products, as ingredients of culture media or as added constituents of
vaccines or diluents.
• Substances of animal origin shall be prepared from homogeneous
bulk, designated with a batch number.
• All batches of substances shall be shown to be free from
contaminants or shall be subject to a suitable validated inactivation
procedure.
• It shall also contain details of the inactivation procedure to which
the substance has been subjected and details of tests performed on
the substance and results obtained.

8. • For examination of the substance for freedom from
contaminants, any solid substance should be suspended in a
suitable medium in such a way as to create a suspension that is at
least 30% substances (w/v) or less.
• Any batch of substance that is found to contain living organisms
of any kind is not satisfactory and must be discarded.
• The suspension of the solid substance, or undiluted liquid
substance should be tested for contaminants by suitably sensitive
methods.
• Before use, substances shall be tested for sterility or properly
sterilized to eliminate any bacterial, fungal or Mycoplasma
contaminants.

9. • Cell substrates used in manufacture shall normally be
produced according to a Seed Lot System.
• Each MCS shall be assigned a specific code for identification
purposes.
• The MCS shall be stored in aliquots at -70 C or lower.
• Production of vaccine shall not normally be undertaken on
cells further than 20 passages from the MCS.
• The history of the cell line must be known in detail and
recorded in writing (e.g. origin, number of passages and media
used for their multiplication, storage conditions).

10. Extraneous contaminants
• The cells must be checked for their appearance under the
microscope, for their rate of growth and for other factors
which will provide information on the state of health of the
cells.
• The cells must be checked for contamination with
bacteria, virus, fungi, mycoplasma.
• Contaminated cells must be discarded.

11. • For most of the mammalian vaccines the use of primary cells is
not acceptable for the manufacture of vaccines.
• If a vaccine has to be produced on primary cells, they should be
obtained from a specific pathogen free herd or flock with
complete protection from introduction of diseases (e.g. disease
barriers, filters on air inlets, no new animals introduced without
appropriate quarantine).
• In the case of chicken flocks these should comply with the
requirements of the European Pharmacopoeia monograph for
SPF chickens.
• For all other animals and species of birds, the herd or flock must
be shown to be free from appropriate pathogens.

12. • Embryonated eggs must be obtained from an SPF flock.
• Animals must be free from specific pathogens, as appropriate
to the source species and the target animal.

13. • Viruses used in manufacture shall be derived from a Seed Lot
System.
• A record of the origin, passage history (including purification
and characterisation procedures) and storage conditions shall
be maintained for each Seed Lot.
• Each Master Seed Virus (MSV) shall be assigned a specific
code for identification purposes.
• The MSV shall normally be stored in Aliquots at -70 C or
lower if it is in liquid form or at -20 C or lower if in a
lyophilised form.
• Production of vaccine shall not normally be undertaken using
virus more than 5 passages from the MSV.

14. • The MSV and all subsequent passages shall be propagated on
cells, on embryonated eggs or in animals which have been
shown to be suitable for vaccine production, and all such
propagations shall only involve substance of animal origin.
• The MSV shall be shown to contain only the virus stated.
• A suitable method shall be provided to identify the vaccine
strain and to distinguish it as far as possible from related
strains.
• If the MSV is shown to contain living organisms of any
kind, other than virus of the species and strain stated, then it is
unsuitable for vaccine production.

15. • The MSV shall pass the tests for sterility and freedom from
mycoplasma.
.
• Each batch shall be shown to be free of any non-specific
inhibition effects on the ability of viruses to infect and
propagate within cells (or eggs – if applicable).
• Each batch shall be treated at 56 C for 30 minutes to
inactivate complement.

16. • The bacteria used in the vaccine shall be stated by genus and
species and varieties.
• The origin, date of isolation and designation of the bacterial
strains used shall be given, and details provided, where
possible, of the passage history, including details of the media
used at each stage.
• Bacteria used in manufacture shall be derived from a Seed Lot
System wherever possible.
• A record of the origin, passage history (including purification
and characterisation procedures) and storage conditions shall
be maintained for each Seed Lot.

17. • Each Seed Lot shall be shown to contain only the species and
strain of bacterium stated.
• A brief description of the method of identifying each strain by
biochemical, serological and morphological characteristics and
distinguishing it as far as possible from related strains shall be
provided, as shall also the methods of determining the purity
of the strain.
• If the Seed Lot is shown to contain living organisms of any
kind other than the species and strain stated, then it is
unsuitable for vaccine production.

18. • The minimum and maximum number of subcultures of each
Seed Lot prior to the production stage shall be specified.
• The methods used for the preparation of seed
cultures, preparation of suspensions for seeding, techniques for
inoculation of seeds, titre and concentration of inocula and the
media used shall be described.
• It shall be demonstrated that the characteristics of the seed
material (e.g. dissociation or antigenicity) are not changed by
these subcultures.
• The conditions under which each seed lot is stored shall be
described.

19. • At least the qualitative composition should be given of media
used for seed culture preparation and for production.
• Named ingredients should be specified as to grade.
• Where ingredients are claimed as proprietary, this should be
indicated and an appropriate description given.
• Ingredients which are derived from animals should be
specified as to the species source and country of origin.
• Preparation processes for media used, including sterilisation
procedures shall be described.

20. • The addition of antibiotics in the process of the manufacture of
the product shall normally be restricted to cell culture fluids
and other media, egg inoculum and material from skin or other
tissues.
• Not more than three antibiotics shall be permitted for
simultaneous use for these purposes.
• If the antibiotics used are not recommended for use in the
target species, they shall be shown to have no harmful effect
on the vaccinated animals.

21. • All other substances used in vaccine production shall be
prepared in such a way as to prevent contamination of the
vaccine with any living organism or toxin.
• Samples of all seed materials, reagents, in-process materials
and finished product shall be supplied to the competent
authorities, on request.

23. is a set of preventive measures designed to reduce the
risk of transmission of infectious diseases and living modified
organisms.
“A strategic and integrated approach that encompasses the policy
and regulatory frameworks for analysing and managing relevant
risks to human, animal and plant life and health and associated
risks to the environment.”

24. • While biosecurity does encompass the prevention of the
intentional removal (theft) of biological materials from research
laboratories.
• These preventative measures are a combination of systems and
practices put into its place at legitimate bioscience laboratories to
prevent the use of dangerous pathogens and toxins for malicious
use, as well as by customs agents and agricultural and natural
resource managers to prevent the spread of these biological agents
in natural and managed.
Principles and practices employed to protect laboratory personnel
and the environment from exposure or infection while working with
living organisms, biological materials, or agents.

25. The relationship between biosafety and biosecurity :
• Biosafety and biosecurity are both different approaches to ensure
containment, and they both share an end goal of minimizing the risk of
accidental or intentional exposures and releases of pathogens or toxins.
• They also share a number of elements such as inventory control, access
restriction, accountability and compliance with biosecurity
procedures, incident reporting, evaluation and revision, and education and
training.

26. To protect biological agents against the theft or diversion
of high-consequence microbial agents, which could be
used by someone who maliciously intends to conduct
bioterrorism or pursue biological weapons proliferation.

27. Components
of
Laboratory
Biosecurity
• Physical security/protection
• Personnel security/reliability
• Material Control & Accountability
(MC&A) / Pathogen accountability
• Transportation security
• Information security

28. Physical security • Defined perimeter
• Access controls
(restricts access to authorized
individuals) • Intrusion detection
• Personnel screening (background checks)
Personnel security • Badges
(determines who is authorized)
• Visitor control
Material Control & • Physical and procedural controls
Accountability • Inventories
(MC&A) • Accountable individuals
(Provides awareness of what
materials exist where and who is
responsible)

29. • Knowledge of recipient
Transport security • Physical security of packages
(MC&A for materials • Personnel screening for individuals who handle
being transferred between packages
laboratories) • Chain of custody
• Use of reliable carriers (Transport facility)
Information
security • Identification, marking, and control
(protecting sensitive • Network and communication security
information from public (Mail, email or fax security,Desktop
release) security)

30. Risk Group 1
Risk Group 2
Risk group Risk Group 3
Risk Group 4
1. laboratory practice
and technique
Containment 2. safety equipment
3. facility design.
BSL 1
BSL 2
Bio-safety level BSL 3
BSL 4
A BSL 1
Vertebrate animal A BSL 2
bio-safety level A BSL 3
A BSL 4
Risk assessment

32. Infectious agents are classified into risk groups based on their
relative hazard.
Risk Group 1 • Agents that are not associated with disease in
(No or very low individual and healthy adult humans.
community risk)
Risk Group 2 • Agents that are associated with human disease
(Moderate individual risk, low which is rarely serious and for which preventive
community risk) or therapeutic interventions are often available.
Risk Group 3 • Agents that are associated with serious or lethal
(High individual risk, low human or animal disease for which preventive or
community risk) therapeutic interventions may be available.
Risk Group 4 •Agents that are likely to cause serious or lethal
(High individual and human disease for which preventive or therapeutic
community risk) interventions are not usually available.

33. E.coli K-12, Transgenic
Plants, Plasmids, Fungi, Mold, Yeast
Human or Primate Cells, Salmonella ,
Herpes Simplex Virus,
Patient specimens
Human Immunodeficiency
Virus, Mycobacterium tuberculosis,
Coxiella burnetii
Lassa Fever Virus, Ebola Hemmorrhagic
Fever Virus, Marburg Virus, Herpes B
Virus

35. • The term "containment" is used in describing safe methods for
managing infectious agents in the laboratory environment where
they are being handled or maintained.
The objective of • is the containment of potentially
biosafety harmful biological agents.
• to reduce/eliminate exposure of lab
The purpose of workers, other persons and outside
containment environment to biohazardous agent/

36. Primary containment
• The protection of personnel and the immediate laboratory
environment from exposure to infectious agents.
• Provided by
– Good microbiological technique and the use of appropriate safety
equipment.
– The use of vaccines may provide an increased level of personal
protection
Secondary containment
• The protection of the environment, external to the laboratory from
exposure to infectious materials.
• Provided by
- Combination of facility design
- Operational practices.

37. Laboratory
practice and
technique
Safety
equipment
(primary
barriers)
Facility design
and construction
(secondary
barriers)

38. • The most important element of containment is strict adherence
to standard microbiological practices and techniques.
• Persons working with infectious agents or infected materials
must be aware of potential hazards, and must be trained and
proficient in the practices and techniques required for handling
such material safely.
• The laboratory supervisor is responsible for providing or
arranging for appropriate training of personnel.
• Each laboratory should develop an operational manual which
identifies specific hazards that will or may be encountered, and
which specifies practices and procedures designed to minimize or
eliminate risks.

39. • Personnel should be advised of special hazards and should be
required to read and to follow the required practices and
procedures.
• The laboratory supervisor is responsible for selecting
additional safety practices, which must be in keeping with the
hazard associated with the agent or procedure.
• Laboratory personnel, safety practices and techniques must be
supplemented by appropriate facility design and engineering
features, safety equipment and management practices.

40. • Safety equipment includes biological safety cabinets, enclosed
containers (i.e., safety centrifuge cups) and other engineering
controls designed to remove or minimize exposures to hazardous
biological materials.
• The biological safety cabinet (BSC) is the principal device used to
provide containment of infectious splashes or aerosols generated
by many microbiological procedures.

41. • Safety equipment such as personal protective clothing
(gown), respirators, face shields, safety glasses or goggles used
for personal protection.
• In some situations, personal protective clothing may form the
primary barrier between personnel and the infectious materials.
Goggles
Gloves
Lab coat

42. • The design of a facility is important in providing a barrier to
protect people working inside and outside the laboratory, and to
protect people or animals in the community from infectious
agents which may be accidentally released from the laboratory.
Secondary barriers in laboratories include:
-separation of the laboratory work area from
public access,
-availability of a decontamination facility
(e.g., autoclave) and
-hand washing facilities,
-waste decontamination facilities.

43. • As the risk for aerosol transmission increases, higher levels of
primary containment and multiple secondary barriers may
become necessary to prevent infectious agents from escaping
into the environment.
• Such design features could include:
- specialized ventilation systems to assure directional airflow,
- air treatment systems to decontaminate or remove agents from
exhaust air,
- controlled access zones,
- airlocks at laboratory entrances,
- or separate buildings or modules for isolation of the laboratory.

45. • A biosafety level is the level of biocontainment precautions
required to isolate dangerous biological agents in an enclosed
facility.
• Each level represents a combination of
– laboratory practices,
– laboratory techniques,
– safety equipment,
– Laboratory facilities
designed to minimize release and exposure.
• Different than the Risk Groups!!
– Risk groups used in risk assessment
– BSL are used in risk management

46. • is appropriate for work done with defined and
characterized strains of viable microorganisms not
known to cause disease in healthy adult humans.(
non-pathogenic organisms or agents)
• Minimal hazard to lab personnel and the
environment
• Example:
– E coli K-12 strains
• It represents a basic level of containment that relies
on standard microbiological practices with no
special primary or secondary barriers
recommended, other than a sink for hand washing.

47. • Mouth pipetting is prohibited- MUST USE
MECHANICAL PIPETTING DEVICES
• Work surfaces are decontaminated with
disinfectant on a daily basis.
• All cultures, stocks and other infectious materials
are decontaminated by an approved method
(usually autoclaving)
• Biohazard signs are posted at lab entrance
• Washing of hands after handling viable
organisms, after removing gloves and before
leaving the lab.
• Eating, drinking, smoking, brushing teeth, storing
food is absolutely forbidden.

48. • is applicable to work done with indigenous moderate-risk agents
present in the community and associated with human disease of varying
severity. (Agents of moderate hazard to personnel or environment)
Examples:
– Hepatitis A & B , Salmonella
– Human derived blood and blood products
– Cell culture work
• Not spread by aerosol.
• Agents can be used safely on the open bench.
• Primary hazards to personnel working with these agents relate to
accidental Percutaneous exposure (Scratch, Puncture, Needle stick)
,Mucus membrane exposure (Eyes, Mouth, open cut) or ingestion of
infectious materials.

49. • Procedures with high aerosol or splash potential
must be conducted in primary containment
equipment such as biosafety cabinets.
• Primary barriers such as splash shields, face
protection, labcoat and gloves should be used as
appropriate.
• Secondary barriers such as hand washing and waste
decontamination facilities must be available.
• Extreme precautions taken with contaminated
sharps.

50. • is applicable to work done with indigenous or exotic agents with
a potential for respiratory transmission and which may cause
serious and potentially lethal infection.
(Agents of high hazard to personnel or environment)
• Aerosol transmission
• Examples:
– Mycobacterium tuberculosis,
– B. anthracis
– Coxiella burnetii
– SARS and West Nile viruses
• Primary hazards to personnel working with these agents include
auto-inoculation, ingestion and exposure to infectious aerosols.

51. • Greater emphasis is placed on primary and secondary barriers to
protect personnel in adjoining areas, the community and the
environment from exposure to infectious aerosols.
• For example, all laboratory manipulations should be performed
in a biological safety cabinet.
• Secondary barriers include controlled access to the laboratory
and a specialized ventilation system that minimizes the release of
infectious aerosols from the laboratory.

52. • is applicable for work with dangerous and exotic agents that pose
a high individual risk of life-threatening disease, which may be
transmitted via the aerosol route and for which there is no
available vaccine or therapy.
• Examples: Ebola, Marburg and Lassa Fever viruses
• Primary hazards to workers include respiratory exposure to
infectious aerosols, mucous membrane exposure to infectious
droplets and auto-inoculation.
• Levels of personal protection increases
• Lab access is restricted
• Lab design becomes more critical

53. A fully suited researcher
• Full-body Pressurized personnel suit. in a BSL4 laboratory
• The facility is generally a separate building or a
completely isolated zone within a complex with
specialized ventilation and waste management
systems to prevent release of viable agents to the
environment.
• Total containment, airtight labs, “submarine”
doors, air pumps, water treatment, HEPA filtration.
Highly
restricted area access
door to BSL4 laboratory
View into the
decontamination chamber
of a BSL4 laboratory

55. • There are four animal biosafety levels (ABSLs) for work with
infectious agents in mammals.
• The levels are combinations of practices, safety equipment and
facilities for experiments on animals infected with agents that
produce or may produce human infection.
• In general, the biosafety level recommended for working with
an infectious agent in vivo and in vitro is comparable.
is suitable for work with animals infected with agents that are not
known to cause disease in healthy adult humans, and that are of
minimal potential hazard to laboratory personnel and the
environment. ( non-pathogenic organisms or agents)

56. • is suitable for work with animals infected with those agents associated
with human disease. (Agents of moderate hazard to personnel or
environment)
• It addresses hazards from ingestion as well as from percutaneous and
mucous membrane exposure.
is suitable for work with animals infected with indigenous or exotic
agents that present the potential of aerosol transmission and of causing
serious or potentially lethal disease.
• is suitable for work with animals infected with dangerous and exotic
agents that pose high risk of like threatening disease, aerosol
transmission, or related agents with unknown risk of transmission.

58. • A risk assessment will determine the degree of correlation
between an agent’s risk group classification and biosafety
level. (Risk assessment can evaluate the threat posed by any
laboratory activity.)
• It is the responsibility of the principal investigator or laboratory
director to conduct a risk assessment to determine the proper
work practices and containment requirements for work with
biohazardous material.
• The risk assessment process should identify features of
microorganisms as well as host and environmental factors that
influence the potential for workers to have a biohazard exposure.
• The principal investigator or laboratory director should consult
with a Biosafety Officer to ensure that the laboratory is in
compliance with established guidelines and regulations.

59. Factors to consider when evaluating risk include the following:
• The more severe the potentially acquired disease, the higher the
risk.
• Salmonella (Risk Group 2 agent), can cause diarrhea, septicemia if
ingested. Treatment is available.
• Viruses such as Ebola, Marburg, and Lassa fever (Risk Group 4
Agents) cause diseases with high mortality rates. There are no
vaccines or treatment available.
• Agents that can be transmitted by the aerosol route have been known
to cause the most laboratory-acquired infections.
• The greater the aerosol potential, the higher the risk of infection.
• Work with Mycobacterium tuberculosis is performed at Biosafety
Level 3 because disease is acquired via the aerosol route.

60. • The greater the potential for an agent to survive in the
environment, the higher the risk.
• Consider factors such as desiccation, exposure to sunlight or
ultraviolet light, or exposure to chemical disinfections when
looking at the stability of an agent.
• Consider the amount of an infectious agent needed to cause
infection in a normal person.
• An infectious dose can vary from one to hundreds of thousands
of organisms or infectious units.
• An individual’s immune status can also influence the infectious
dose.

61. • Consider whether the organisms are in solid tissue, viscous
blood, sputum, etc., the volume of the material and the
laboratory work planned (amplification of the
material, sonication, centrifugation, etc.).
• In most instances, the risk increases as the concentration of
microorganisms increases.
• This may refer to the geographic location (domestic or
foreign), host (infected or uninfected human or animal), or
nature of the source (potential zoonotic or associated with a
disease outbreak).

62. • If human data is not available, information on the
pathogenicity, infectivity, and route of exposure from animal
studies may be valuable.
• Use caution when translating infectivity data from one species
to another.
• Effective vaccines, if available, should be offered to laboratory
personnel in advance of their handling of infectious material.
• However, immunization does not replace engineering
controls, proper practices and procedures and the use of personal
protective equipment (PPE).
• The availability of post-exposure prophylaxis should also be
considered.

63. • Medical surveillance programs may include monitoring
employee health status, participating in post-exposure
management, employee counseling prior to offering
vaccination, and annual physicals.
• Laboratory workers must become proficient in specific tasks
prior to working with microorganisms.
• Laboratory workers may have to work with non-infectious
materials to ensure they have the appropriate skill level prior to
working with biohazardous materials.
• Laboratory workers may have to go through additional training
(e.g., HIV training, BSL-3 training, etc.) before they are
allowed to work with materials or in a designated facility.

64. • EUROPEAN PHARMACOPOEIA 5.0
• OIE Terrestrial Manual 2008 17 (Biosafety and biosecurity in
the veterinary microbiology laboratory and animal facilities)
• Handbook of applied biosecurity for life science laboratories -
peter clevestig

65. Thank you