Introduction to Pharmacopoeial requirement, Diseases security and Biosecurity principals

Submitted by:Dr. Vijayata

B.V.Sc. & M.V.Sc

DEPARTMENT OF ANIMAL BIOTECHNOLOGY

Introduction to

pharmacopOeial requirement

• 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)

INTRODUCTION

Master Seed (MS) – • 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.

Master Cell Seed (MCS) – • 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.

Seed Lot System – • a system according to which successive batches of product are

prepared using the same Master Cell Seed or Master Seed.

Working Seed Lot –

• 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.

Working Cell Seed (WCS) –

• 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.

Primary Cell Culture – • 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.

Cell lines –

Cultures of cells that have a high capacity for multiplication

in vitro.

STARTING MATERIALS

A. Substances of animal origin

• 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.

.

Tests

• 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.

1. Freedom from extraneous viruses

• The suspension of the solid substance, or undiluted liquid substance should be tested for contaminants by suitably sensitive methods.

2. Sterility test

• Before use, substances shall be tested for sterility or properly sterilized to eliminate any bacterial, fungal or Mycoplasma contaminants.

B. Cell substrates

Requirements for Cell Lines :

• 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).

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.

Requirements for primary cells :

• 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.

Requirements for embryonated eggs :• Embryonated eggs must be obtained from an SPF flock.

Requirements for animals :• Animals must be free from specific pathogens, as appropriate

to the source species and the target animal.

C. Virus seed

General requirements• 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.

Propagation

• 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.

Identity

• 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.

Sterility

• The MSV shall pass the tests for sterility and freedom from mycoplasma.

Extraneous agents.

• 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.

D. Bacterial seed

General requirements

• 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.

Identity and purity

• 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.

Seed lot requirements

• 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.

E. Media for bacterial vaccines

• 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.

F. Antibiotics

• 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.

G. Other substances• 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.

H. Samples• Samples of all seed materials, reagents, in-process materials

and finished product shall be supplied to the competent authorities, on request.

Diseases security and

Biosecurity principals

What is Biosecurity

"Bio" means life.

"Security" means protection. 

Biosecurity is a set of preventive measures designed to reduce the

risk of transmission of infectious diseases and living modified

organisms.

FAO definition of ‘biosecurity’“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.”

• 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.

Biosafety or Disease security :-

Principles and practices employed to protect laboratory personnel and the environment from exposure or infection while working with living organisms, biological materials, or agents.

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.

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.

OBJECTIVE:

Components of Laboratory Biosecurity

• Physical security/protection

• Personnel security/reliability

• Material Control & Accountability (MC&A) / Pathogen accountability

• Transportation security

• Information security

•Defined perimeter•Access controls•Intrusion detection

Physical security(restricts access to authorized

individuals)

•Personnel screening (background checks)•Badges•Visitor control

Personnel security(determines who is authorized)

•Physical and procedural controls•Inventories•Accountable individuals

• Knowledge of recipient• Physical security of packages• Personnel screening for individuals who handle

packages• Chain of custody• Use of reliable carriers (Transport facility)

Transport security(MC&A for materials being

transferred between laboratories)

• Identification, marking, and control• Network and communication security

(Mail, email or fax security,Desktop security)

Information security

(protecting sensitive information from public

release)

Risk group

Containment

Bio-safety level

Vertebrate animal bio-safety level

Risk assessment

1. laboratory practice and technique2. safety equipment3. facility design.

Risk Group 1Risk Group 2Risk Group 3Risk Group 4

BSL 1 BSL 2BSL 3 BSL 4

A BSL 1 A BSL 2A BSL 3 A BSL 4

A. RISK GROUPS

• Agents that are not associated with disease in healthy adult humans.

Risk Group 1 (No or very low individual and

community risk)

• Agents that are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available.

Risk Group 2 (Moderate individual risk, low

community risk)

• Agents that are associated with serious or lethal human or animal disease for which preventive or therapeutic interventions may be available.

Risk Group 3 (High individual risk, low

community risk)

Risk Group 4 (High individual and

community risk)

•Agents that are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available.

Infectious agents are classified into risk groups based on their relative hazard.

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

B. CONTAINMENT

• 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 biosafety

• is the containment of potentially harmful biological agents.

The purpose of containment

• to reduce/eliminate exposure of lab workers, other persons and outside environment to biohazardous agent/

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.

Key elements of containment include:

Facility design and

construction (secondary barriers)

Safety equipment (primary barriers)

Laboratory practice

and technique

1. Laboratory Practice and Technique -

• 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. 

• 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. 

2. Safety Equipment (Primary Barriers) 

• 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.

Gloves

Goggles

Lab coat

• 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. 

3. Facility Design (Secondary Barriers) 

• 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. 

• 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.

C. BIOSAFETY LEVELS

• 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

Biological Safety Level 1 (BSL 1) 

• 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. 

• 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.

Biological Safety Level 2 (BSL 2) 

•  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. 

• 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.

Biological Safety Level 3 (BSL 3)  

• 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. 

• 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. 

Biological Safety Level 4 (BSL4) 

• 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

• Full-body Pressurized personnel suit. 

• 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.

A fully suited researcher in a BSL4 laboratory

View into the decontamination chamber of a BSL4 laboratory

Highlyrestricted area access door to BSL4 laboratory

D. VERTEBRATE ANIMAL BIOSAFETY LEVELS

Animal Biological Safety Level 1 (ABSL-1) -

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)

• 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. 

Animal Biological Safety Level 2 (ABSL-2) -• 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. 

Animal Biological Safety Level 3 (ABSL-3) -• 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.

Animal Biological Safety Level 4 (ABSL-4) -

• 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. 

E. RISK ASSESSMENT

• 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. 

1. Pathogenicity:• 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.

2. Route of transmission: 

• 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.

Factors to consider when evaluating risk include the following:

 3. Agent stability:

• 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.

4.  Infectious dose:

• 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.

5. Concentration: 

• 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.

6. Origin: 

• 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).

7. Availability of data from animal studies:

• 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.

8. Availability of an effective prophylaxis or therapeutic intervention :

• 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.

9. Medical surveillance:

• Medical surveillance programs may include monitoring employee health status, participating in post-exposure management, employee counseling prior to offering vaccination, and annual physicals.

10. Experience and skill level of at-risk personnel: 

• 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.

Reference

• 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

Thank you