FORM � II
APPLICATION FOR ENVIRONMENTAL APPROVAL OF� CLINICAL TRIALS/ PHARMACETICALS /
VETERINARY� DRUGS DERIVED FROM
LIVING� MODIFIED ORGANISMS /� HAZARDOUS MICRO-ORGANISMS.
Not all the points included will apply to every case. It is
to be expected, therefore, that individual applicant will address only the
particular parameters that are appropriate to individual situations. In each
case where it is not technically possible or it does not appear necessary to
give the information, the reasons shall be stated.
The details required in response to each parameter are also
likely to vary according to the nature and scale of the proposed release.
The description of the methods used or the reference to the
standardized or internationally recognized methods shall also be mentioned in
the form together with the name of the body or bodies responsible for carrying
out the studies.
A 1-2 page summary of the proposal shall be appended with
Name of the Applicant
Name of organization/firm
Quantity per year of the product to be
Name of the product:
Others (please specify)
Intended use (Pathological, Metabolic and Immune Response)
Donor (heterologous Nucleic acid segments from sources)
Vector (DNA) molecules to which heterologous nucleic acid
segments are joined for transfer to hosts)
Hosts (living cells or organisms into which rDNA molecules
Production method (the production method should give the
details of the cell lines used, the information on the production of the
recombinant proteins within or outside the cells, the concentration of the
products in units per litre of the fermented broth as well as the concentration
in physical weights. The description should also include in brief the methods
applied for reducing the genomic particles, proteins and living contaminants
including viruses, bacteria, fungi, parasites. Etc.)
(a)�� Characterisation of the system of
Details of the expression system:
Description of the host cell line
Identification of the genera and the species.
The risks involved in handling the cell line.
The classification of the cell line according to the
Government of India�s Biosafety guidelines or any other accepted Recombinant
cell line usage guideline.
The method(s) of maintenance and growth of the cell line.
The nature and hazards of using substrates, inducing
Characteristics of the target gene and vector
The full description of the source of the target gene.
The composition of the vector used indicating the promoter
sequence as well as the regulatory mechanisms utilized in the expression
Schematic diagrams of the expression cassette to describe
fully the marker genes used.
The restriction sites related to specific endo nucleases,
and the cell lines used for shuttling and amplification of the expression
The method of constructing the target gene along with all
the sequences added or deleted.
The extent of target gene amplification into the host
The target gene should also contain, along with the
nucleotide sequence, the description of the amino acids below the codons.
Approaches adopted for expression of the gene.
The description of the transcripted messenger RNA� with its analysis of sequence and
The translation information indicating whether the protein
product is Chimaric, whether the expression is found as inclusion bodies or as
intracellular protein or whether the protein is secreted out.
The extent of the target protein produced as the percentage
of the total cell dry mass as well as its percentage compared to the total
proteins of the cell.
The quantity of the target protein produced after the cell
growth per litre of the fermented broth.
The full sequence of the recombinant gene alongwith the
promoters the marker genes and the terminator se sequence.
(b)����� Description of the production process
�� ��(i)� Production set up
The handling of the stock cell lines.
The evaluation and uses of the cell lines in pre-fermentor
The preparation of the seed vessel.
The main production fermentation conditions need to be
described in brief.
Graphical plots of the versus substrate usage, optical
density change, biomass formation, protein products formation, inducing agents
used and their effects on the target protein formation.
The effects of change of standard parameters like pH dissolved
oxygen, temperature etc. in the main production fermentor.
The pH, temperature, aeration, and rpm of the shaft.
Volume of seed to the volume of main fermentor.
Main substrates used inducing agents used if any.
The fermentation time and the concentration of the target
protein in the fermented broth.
process Control Methods
All the analytical methods used for this in-process control
must be described to ensure the regulatory authorities that the chances of
entry of unwanted products have been minimized.
�Description of the
raw and processing materials used
Description of all the raw materials and processing
materials used starting from the stock cells handling to the finished dosage
form must be in a tabular form.
Grades of materials used and their usual sourcing.
(e)����� Description of the
plant and Machinery used
List of all the equipment alongwith the indicative
Names of the manufacturers.
List of main production equipment and the quality control
Description of the building and facilities created for the manufacture
The manufacturing area.
The flow of the process/operation starting from the stock
culture area to the finishing area.
The plot plan of various floors used in the manufacture and
The building diagram in the plan and/or elevation in the
outline indicating the cleanliness maintained in different sections such as
class 10,000, class 1000 and class 100 areas etc.
Description of the air handling system which is very important.
A separate write-up indicating the movement of operating
personnel in the area.
Approaches for the extraction and purification of the
product. The purification methods should particularly indicate the modes of
elimination of viruses, bacteria, fungi, parasites, etc., if the cell lines are
mammalian origin. It should also include methods for the removal of genomic
particles, and proteins irrespective of cell lines/hosts used for production,
removal of adjuvants, reagents chemicals including vectors, donor and recipient
organisms foreign DNA and adventitious materials associated with production
The methods of handling the cells.
The methods of isolating the cell soup containing the
target protein the methods of enzyme treatment, if any.
The methods of concentration, precipitation (if applied),
reconstitution, salt separation ( if applicable), absorption and desorption
methods, chromatographic methods used if any, ultracentrifugation methods if
Sterilization and final dosage formulation.
Processes to obtain the product in the finished dosage
Quality Control and Quality Assurance
(a)������������������ Bulk material:
Test relating to the identification of the stock cells.
The plasmid construct retention in the cells during cell
The media used.
The procedures adopted for testing and the percentage
retention of the target plasmid.
Description of the contamination test of the stock culture
to assess and monitor the microbial contamination including the media used and
the procedures adopted.
The criteria of acceptance of contamination free culture.
Characterisation of the bio-active molecule produced by
monitoring physical & chemical properties of the molecules by the following
or more authentic method should be provided.
Microscopic examination including light, phase contract and
electron microscopic studies.
UV Spectroscopic analysis to show the absorption spectra of
the product and comparison of this with authentic material.
Density gradient centrifugation to show single peak.
Pattern in High performance liquid chromatography to
indicate how many peaks or if only one peak is noticed in the produced and
Iso-electric focussing analysis to show the pl values.
Western Blot and SDS-PAGE to map the peptide/target
Existence of special bonds like disulfide bonds by breaking
the protein and subjecting to SDS-PAGE mapping.
Immunodiffusion Test to find out the absence of
contaminants or the extent of the presence of contaminants.
Amino acid composition of the purified protein and its
comparison with the authentic material.
N-Terminal Amino Acid Sequence analysis and its comparison
with the gene construct used.
Determination of the biological activity in the animal
Determination of Contaminants per milligram or any
convenient unit of the manufacturing bulk purified protein is also to be
carried out to indicate the extent of contaminant nucleic acid stretches,
proteins, carbohydrates, lipids, detergents, salts and other processing
chemicals used in the purification. The impacts of the presence of these
contaminants are also to be indicated with authentic references if any to
ensure that the risks associated with their presence are minimal. The limits of
contaminants and the acceptance criteria need to be quantified for each contaminant.
(b)����� Formulated Material:
The ingredients incorporated subsequently in the
manufacture. The specification of the final product.
The Quality Control Department would have to certify that
the final product has the results within the specifications prescribed and
accepted by the Regulatory Authorities.
The documentation should therefore indicate the following
Product inserts, literature and label claim
Volume/Quantity per pack/dose.
Potency of the product.
Particulate matter limits for liquid.
Preservative usage percentage.
Other extraneous materials used their extent such as
contents of DNA, RNA, Carbohydrates, Lipids, processing materials like
detergents, salts, etc.
Protein content in the product.
Possible Hazards to environment from release of GMO/nucleic
acid/micro-organisms or products.
Clinical field trials done in India/abroad (whether
permission of Institutional Ethics Committee obtained)
Stability and Shelf Life of product.
Method of disposal of vials of syringes/wastes matter.
Regulatory status in India/abroad (enclosed certificates
like free sale certificate/GMP certificate/certificates granted by Health &
Environment Authorities of the country of origins signed by applicant. In case
the certificate is issued by the concerned authority of country of origin, the
certificate should be endorsed/ authenticated by Indian Embassy/High
Commission/Consulate in that country.
Every certificate shall be accompanied by other statutory
information like manufacturing batch, no. date of manufacture, date of
analysis, date of release of the certificate, signatory to the certificate etc.
The final formulated product needs to be certified for acceptance
by the manufacturer.
signature with seal.