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
The details required in
response to each parameters is 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 of bodies responsible for carrying out the studies.
d. A 1-2 page summary of the proposal shall be appended with
Name of the Applicant
Name of organization/ firm
4. Quantity per year of the product to be manufactured/ imported/
Name of the product :
d. Others (please specify)
(pathological, Metabolic and Immune Response)
SAFETY CONCERN :
Nucleic acid segments from sources)
Vector (DNA) molecules to
which heterologous nucleic acid segments are joined for transfer to hosts)
c. Hosts (living cells or organisms into which rDNA molecules are
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).
Characterization of the
system of production used
Details of the expression
Description of the host
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 agents, etc.
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 cassette.
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 cassette.
The method of constructing
the target gene along with all the sequences added or deleted.
The extent of target gene
amplification into the host genome.
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 identification
information indicating whether the protein product is Chimeric, 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 along with the promoters the marker genes and the
Description of the
The handling of the stock
The evaluation and uses of
the cell lines in pre-fermentor processes
The preparation of the
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
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.
In-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
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
Description of the Plant
and Machinery used
List of all the equipment
along with the indicative capacity.
Names of the
List of Main production equipment
and the quality control equipment.
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 processing.
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 methods.
The methods of handling
The methods of isolating
the cell soup containing the target protein the methods of enzyme treatment
The methods of
concentration, precipitation (if applied) reconstitution, salt separation
(if applicable), absorption and desorption methods, chromatographic methods
used if any, ultra-centrifugation methods if used.
Sterilization and final
e. Processes to obtain the product in the finished dosage form.
Quality Control and
Bulk material :
Test relating to the
identification of the stock cells.
The plasmid construct
retention, in the cells during cell growth.
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
Characterization of the
bio-active molecule produced by monitoring physical & chemical
properties of the molecules by the following or more authentic method
should be provided.
including light, phase contract and electron microscopic studies.
UV Spectroscopic analysis
to show the absorption spectra of the product and comparison of this with
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 purified bulk.
analysis to show the pI values.
Western Blot and SDS-PAGE
to map the peptide/ target protein.
Existence of special bonds
like disulfide bonds by breaking the protein and subjecting to SDS-PAGE
Immuno-diffusion Test to
find out the absence of contaminants or the extent of the presence of
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 model.
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.
Formulated Materials :
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
The documentation should
therefore indicate the following specifically :
literature and label claim
Volume/ Quantity per pack/
Potency of the product.
Particulate matter limits
Other Extraneous materials
used, their extent such as contents of DNA, RNA, Carbohydrates, Lipids,
processing materials like detergents, salts, etc.
Protein content in the
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
Stability and Shelf Life
Method of disposal of
vials of syringes/ wastes matter
Regulatory status India/
abroad (enclose 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.
Applicants' Signature with Seal