Journal of Scientific & Industrial Research
Vol. 62, June 2003, pp 554-572
The Rules for Handling Transgenic Organisms and Biopharmaceuticals Produced
Therefrom in India *
P K Ghosh "
C2BISA, Janakpuri , New Delhi 110058
Received: 25 September 2002; accepted: 06 March 2003
The Indi an go vernment created and promulgated the rul es and procedures for dealing with genetic' ll y modified
organisms and products thereof in 1989 and these rules were enacted under the Indi an Environment (Protecti on) Act (EPA)
1986. In order to ease the enactmen t of the rules, the recombinant DNA (rONA) safety guidelines were made subsequ entl y by
the Indi an go vernment from the Department of Biotechnology in 1990, which were revised twice incorporating th e latest
revision up to September 1999. The rules al so defin e admini strative structures and procedures for handling ali kinds of rONA
products and substances. Within the rules, certain rONA mi croorgan isms have been authorized for use for the producti on of
therapeutic bi oactive proteins. Cu rrentl y, four rONA products are being produced locally and 12 in niJmber are consumed in
the market. Ind ian market is fed by local production as well as by imports. The first transgeni c plant, an insect resistant cotton,
was authori zed for co mmerciali zati on in 2002. The use of GMOs and products thereof require generation of environ mental
safety as well as food safety data under the Indi an EPA. Familiarity with the rules and guidelines enables entrepreneurs to
facilitate the market introduction process. The paper describes the infor mation needs, the administrati ve str ctures and the
step by step procedures required to be fo ll owed under the rules by entrepreneu rs for introduci ng GMOs and prod ucts thereof
in Indi a market.
Key words: Transgeni c organi sms, Biopharmaceuticals, rON A, GMOs, GMO handling
Introduction
Biotechnology encompasses techniques appli ed
to organisms or parts thereof to produce, identify or
design substances, o r to modify organisms for specific
applications. Cell fusion techniques, hybridomas,
recombinant DNA (rONA) technology for rONA
vaccin es and Bioactive prote ins, protein engineering
an d structure based molecular des ign are considered
as mod ern biotec hnology . Emanating from the above,
the s tru ~ tural and functi onal genomics complemented
with co mputer-aided informatics and biochips are
making fa st inroads into the frontiers of modern
biotechnology. Di fferentiated stem cell proliferation
and animal cloning techniques are developing very
fast. In addition , several technology platforms that
have emanated from increased understanding of
*
Views expressed in the paper are those of the author and they
do not necessaril y express the views of the organi zation to
which the author belongs.
.. Former Advi sor, Department of Biotechnology,
Go vernment of India
Presen.tly, Freel ance Consultant in Biotechnology
Email: [email protected]
signal tran sduction pathways of cells and ti ssues, ce ll
based immune rejections , t i sue eng ineerIng,
proteomics, bio-informatics inclu d ing bio-chips and
genomic sequencing of organisms ; a ll these areas ho ld
great promise for providing new biotech products that
wou ld have wide application s. Conventional
biotechnology includes fermentati n or conversion of
substrates into des ired products by bi o logical
processes; downstream process in g for reco very of
metabolites; use of microbes or enzy mes for
producing value added products; se ra , vaccines and
di agnostic s produced by conven ti onal meth ods;
re production, artificial inse mination and embryo
tran sfer technology for animal breeding ; meth ods for
fish spawning induction ; plant cel i oI' tissue culture;
plant breeding for producing better seeds or plant
cultivars; bio-fertilizers; bio-pest icides; plant growth
stimulants ; extraction, and iso lati o n of act i ve
principles from plants or animals or parts th ereof, etc.
Worldover, no sector of industrial activity was
specifically designated as biotechno logy industry per
se before the early 70s although co nventi onal
fermentation based industry was at its peak , especia ll y
GHOSH: HANDLING TRANSGENIC ORGANISMS & BIOPHARMACEUTICALS -
in the production of antibiotics, enzymes, fermented
beverages, certain organic chemicals, and biochem icals. Viral and bacteri al vaccines were also
produced by mUltiplying the target microorgani sms
(v iruses) in specific pathogen free eggs or in certain
safe cell lines or in defined biological media. The
products were used as such or after inactivation. This
scenari o fast a ltered with the rapid advances in
geneti cs, microbiol ogy and immunology at the
molecular leve l. The techniques of splicing and
reco mbin ation of nucl eotide sequences at specific
si tes, the discovery of vec tors for transporting transnucl eotide sequences to organisms and stabl y
integrat in g compos ite cassettes comprising promoters,
genes, enhancers, markers, and terminators into the
chromoso mes or through shuttle vectors, there by
enablin g the express ion of tran sgenes into unrelated
hosts brought revo luti on to the understanding of
biology of organisms. The Polymerase Chain
Reaction (PCR) and the acco mpanying knowledge
about the meth ods of magnification of nucl eotide
sequences enabled the production of large quantiti es
of hered itary materials at ease. The combined effects
of these deve lopments resu lted in the birth of modern
biotechno logy industry which started during the mid70s and spanned up to the late 90s at a hurricane
speed, as manifested by the production of a large
number of highl y potent bioactive prote ins including
hormones, cytokines, thrombolytic agents, growth
promoters, and regulatory molecules of several vital
physiological functions that revolutionized human
therapy. In agriculture, transgenic plants emerged that
were resistant to certain viral and so me other
microbi al diseases. Plants were produced that were
res istant to herbi c ides. Certain transgenic plants were
developed that had improved traits like enhancement
in vitamins or precursors, enrichment in certain amino
ac ids or reduction in unwanted metabolites. Thi s
situ ati on continues to unfold newer frontiers of
knowledge and technology in pharmaceutical s,
agriculture,
bio-chemicals,
foodstuffs,
and
environment management issues. Presently the
advancements in stem cell research, production of
biochips to unders tand genomics and proteomics,
adva nces in bio-informati cs, xeno-transplantation s
and organ/tissue revival method s in pharmaceutical s
are brin ging about newer hopes of prolonging
healthier lives with enh anced longev ity in humans.
Transgenic plants also hold enormous potential for
increasin g productivity and reduc ing production costs
in agriculture.
RULES
555
The upstream capabilities acquired by human
kind in modern biotechnology have brought in newer
questions of safety with reference to the environment
as well as human and animal hea lth . These have
resulted in the e mergence of newer regul atory laws
and closer scrutiny of the genetica lly modifi ed
organisms/substances on a case-by-case bas is. The
consequent requirement of tes ting for approval s from
regu latory authorities, market ing meth ods and
production techniques have also undergone a sea
change the worldover. Th e developing countri es are
often not able to keep pace with the ad vancements
and technol ogica l changes required to be acquired and
es tabli shed within their territory when co mpared with
the progress in the devel oped nat ions. Modern
biotechnology industry is thu s causi ng the creation of
a wide divergence in the techn ological ski ll s and
consequently economic disparity between the
developed and the developi ng nations. The
developing countries can bridge these gaps
considerably if they concentrate on generating and
updating
human
skill s,
in stall
sophi st icated
infrastructure for biotech research, and de liberate ly
channel efforts in specifi c need-based eco nomic
activities in their territori es . In additi on, training of
personnel abroad and creating hi gh culture of alli ance
with industries within and outside the ir territory along
with pronounc ing policies for promoting loca l
industrial development may he lp to sa lvage the
growlllg gap.
Consumption Scenario of Biotech Products in
India
India
has
been
practis ing
conventi onal
biotechnology
for several
decades . Products
manufactured by the use of genetic engineering,
immunological techniques, cell culture methods and
hybridoma technology are increas in gly be ing used
during the past 10 y and local research in these areas
has been intensified . Table I g ives th e curren t
consumption and future demand of biotech products
in India' .
Modern biotechnology is rather new to India.
Th e current consumption of modern biotech products
is about Rs 5 billion , of which only about Rs 1.4
billion worth of products are produced loca ll y. In
200 I , India was producing three recombinant DNA
products from basic stage using its own tec hn o logy ;
these were hepatitis B vaccine, erythropoi et in and GCSF (Granulocite Colony Stimulating Factor). In
SS6
J SCI IND RES VOL 62 J UNE 2003
Table 1-
Past co nsumption of biotech products in India and future consumpti on estimates
(Rupees in million )
Parti cul ars of biotech sub-sec tors
Ac tu al consump ti o n
Future cons um-ption
estimate
Future consumption
estimate
2000
2005
2010
I Human and animal hea lt h care prod ucts
32700
(37.5)
35320
(376)
93540
(40.0)
2 Agriculture (includin g traded seeds)
26200
(30.0)
28880
(30.7)
78720
(33. 7)
3 Indu stri al products
27320
(3 1.3)
28500
(30.3 )
53 590
(229)
1080
( 1.2 )
1300
(0 1.4 )
7940
(3.4)
87300
( 100)
94000
( 100)
2337 90
( 100 )
4 Other biotech product s
Total
In million US $
1850
2 186
4270
(Fi gures in brackets indicate contributions in percentage of th e total )
2002, Indi a has introd uced anoth e r o ne product,
namely inte rfe ro n a lph a 2 b and may introd uce a nothe r
one reco mbin ant DNA produ ct in the hea lth care
sector name ly human in sulin . In agriculture,
genetically modifi ed in sect resistant hybrid cotto n
seeds we re approved and in troduced in the country in
early 2002. More products would be introd uced in
nea r future. There is littl e doubt th at th e production
and consum pti o n of modern biotech products would
in c rease ve ry fast, as suc h produ cts ho ld great
pote nti a l for providing much better so luti o ns to
improve th e hea lth of people or th e quality of life,
improve agricultural productivity s igni fica nt ly along
w ith suppl y in g more nutrItI ous food, produce
industrial bio-products at much c heape r prices and
improve the quality of e nvi ro nme nt more effective ly
on a susta inab le basis .
Why Reglllations are Necessaryfor Using CMOs and
Products Th ereof?
It is expec ted that the Genetically M odifi ed
Orga ni sms (GMOs) a nd produ cts thereof will pl ay an
important role in the economi c uplift ing of th e
cou ntry in its va ri ous facets of app li cati ons inc ludin g
human and anima l hea lth care syste m, agriculture,
industrial products, and env iro nme nt management.
Concurre ntl y, it is a lso realized that there could be
unin tended haza rd s a nd risks from the use of GMOs
and produ cts the reof, if the new technology was not
2 J 1J
prope rl y assessed be fo re use . . • A gene co nstruct
compris in g a host-compatible-promoter, a gene of
interest
anJ
a
te rmin ator
seq ue nce
or
a
polyadeny lati on seq uence is integrated in a stab le
manner into th e genome of th e o rganism/ce ll line for
th e target ge ne to be exp ressed a nd stab ly inh erited. A
GM organism can be safe but this can be un safe too .
This will depend upo n the tran s-ge nes, the host
orga nism and the e nvironmen t wh e re the GMO is
be ing tested. GMOs ca n be microo rgani s ms, p lants ,
and anima ls. Products made from GMOs can have
contami na nts,
s uch
as
tran sge nic
nuc leo tid e
seq ue nces, tra nsgenic prote in s and lipid s, w hi ch may
come from the hos t orga ni sms and may rema in
e mbedded within th e fin is hed products. Post
translati o na l modifica ti o n of the protein wh ic h is host
dependant is a lso a factor to be eva lu ated for safety .
Finished products containin g unwanted material s may
create adverse reactions w he n used. This is
particularly the co ncern when the products are used as
pha rmaceuticals or food. Biopharmaceuti ca l products
prod uced by reco mbin ant DNA technology are
usually used by inj ectab le rout e where tran snu c leotide seque nces e ntering into ce ll s cou ld ex press
or get themselves integrated . C o nseque ntly th e
concern of safety becomes mo re pro noun ced.
Therefore. a case-by-case ana lys is of th e safety of
eac h GMOs a nd products th e reof need to be
conducted to assess the ir safety. Whe never GMOs a re
re leased into the o pe n e nviro nment they require to be
eva lu ated from enviro nmenta l safety as we ll as from
safety to human a nd animals . Keep ing th ese in view
th e Indi a n Government had enacted the Env ironment
(Protection) Act 2 in 1986 and th e reafter, notified
Rul es & Proced ures (R ul es) for hand lin g GMOs and
haza rdou s o rgani sms through a Gazette Noti ficat ion
No. G.S.R. 1037(E) dated S.12.19 89 fro m th e U ni on
Mini stry of Environme nt & Forests". T he Rul es cover
all kind s of GMOs and products th ereof, which are
GHOSH: HANDLING TRANSGENIC ORGANISMS & BIOPHARMACEUTICALS - RULES
co ntroll ed co mmoditi es for handlin g and use in the
country unde r the EPA. Guidelines have thereafte r
been framed in order to facilitate th e enactment of th e
Rul es 4. 6 . The Rul es and th e Guide lines apply
concurrently to genetically modified organisms and
the products deri ved o r produced the re from.
Indian EPA Implementation Structure for GMOs
The Indian EPA and Rules define Competent
Authorities and compos itions of suc h Authorities for
th e hand lin g of a ll aspects of GMOs and products
thereof. The GMOs inc lude microorgani sms, pl ants
and anima ls. The produc ts produced from GMOs are
also covered unde r the Rul es. Presentl y, the re are s ix
co mpetent authorities unde r the Rules . The Rul es
define the board responsibilities and auth oriti es of the
competent authorities 3. The re a re 20 para in the Rul es
1989. Different paras dea ls with differe nt as pec ts of
th e Rul es. Eac h para can a lso be des ig nated by
suffixing the number of the para and by prefixin g the
word Rule for ide ntifying the m. These rules need to
be familiar to peop le dealing with GMOs and
produc ts thereof. Their ap plicability as well as
implications has been di scussed by the author
e lsewhe re, es pec ia ll y in the co ntex t of transgenic
crops7.'). The Rul es, Authorities and Administrative
structures are dep icted in Figure I .
Indian Scenario on Transgenic Research in GMOs
and Products Thereof
Microorganisms
Efforts are being made to co nstruct transgeni c
microorganisms that code for bioactive therapeut ic
proteins. Transformed E.co li cod in g for severa l
recombinant
proteins,
suc h
as
interferons,
interleukin s, human growth ho rmo ne, bov ine gro wth
hormone, g ranul ocyte colony st imul atin g factors,
human pro-insulin, human ep iderma l growth factor,
streptok in ase, and reco mbinan t hepatiti s B vaccine are
bei ng experimen ted upo n in different laboratori es in
the country. Transformed Pichia pastoris has been
used for commercial production of recombin ant
inte rferon a lph a 2 b in the cou ntry. Other recombinant
yeast spec ies of Saccharomyces cerevisae a nd
Hanseneulla po lymorp ha have been modi fied to code
fo r spec ific th e rapeuti c proteins. Hepatitis B surface
ant igen gene has been coded in these yeast o rgani sms
and the anti gen has been iso lated and formulated into
dosages form for use as vacc ines to protect human
agai nst Hepatitis B. Such vaccines are being
557
commercially made in this country. These products
have been tested for safety unde r the Indi a n EPA and
these have bee n found to be safe. Genetically
modifi ed cholera microorgani sm is being eva lu ated
for its safety as we ll as efficacy; a non-virul e nt strain
of Vibrio c ho le ra was iso lated from natural sources
and an immunoge nic gene coding for a no n toxic
protein was incorporated into it to make th e stra in
responsibl e for e liciting immunogeni c res po nse and
for producin g antibodi es to it. Genetically modified
fungi be longing to Aspergillus spp. are being
experimented upo n fo r th e production of va lu e added
e nzymes. All suc h mi c roo rgani sms a re a lso tested for
e nvironmenta l safety (in case of acc ide nta l re lease in
to the open environment) as well as for human safety
unde r the Indi an EPA.
The re are severa l recombinant products th at are
being marketed'O in India includin g a few produced
loca lly as stated earli er. T ab le 2 provides the
necessary deta ils.
Plants
The first transgenic plant experime nt in the field
was started in 1995 when Brassica jUllcea plants
containing Bar gene regu lated wi th plant spec ific
constituti ve promoters a nd linked with Barnase and
Ba n tar genes regulated with floral ti ssue specific
promoters were planted at Gurgaon (Harya na), India,
under contained conditi o ns. These studi es were
conduc ted to assess the ex te nt of pollen escapes.'! .
Indian results of pollen escape stud ies were co ns istent
with earli er reported studi es". Subsequently, severa l
experime nts have been started in Indi a in the field in
different 10cati o'1 s u sing transgeni c mustard, cotton
and
to mato . Several
Indian
institutes
and
organizati o ns have claimed to develop transgenic
plants, which are ready for g reen house/ poly-house
eva lu ation a nd some are ready for field evaluat ion as
7
we ll .
Animals
Work o n transgenic anima ls ca rri ed out in India
is yet at rudimenta ry stage. Lab experiments have
been conducted to produce transgeni c mi ce conta in ing
different kinds of genes including growt h hormone
ge nes. Certain marker ge nes have a lso been utilized.
Genes th at code for subs tances that produce
luminescence were introduced in s il kworms and
glowi ng in sects were developed at lIS c Bangalore;
this work on ly reinstated that these orgalll sms are
VI
VI
00
Rules aulhorizing "ariou~
as perts of handling
GMO's & products
R:combinant DNA
Advisory COmmiuee
(RDAC) - Recommends on
Policy issues to GEAC and
Rul, 7. Approval for
individuals for handling
GMO's &: products thereof
RCGM
~
Led to the creation of Rules & Structures for handling GMO's
Ministry of Env & forests(GOI)
Notification no. 1037(E) dated 5-\2-\989 &
Guidelines of DBT amended upto 24-9-\999
AUlhorities &
Administrative
structure
Review Conunitt.. 00 Genetic
Manipulation (RCGM):
National Poltey making &
implementing body for GMOs.
Directs generation of environmental and food safety data for
GMOs and products tbere<>f.
Advises GEAC. Can set up
monitoring committees.
1
CIJ
o
z
"
;;0
tTl
CIJ
or<
0N
.....
C
Z
tTl
District Level Committee
(DLe): For tTlOIlitoring
distiicl IC'r'd exposure &
use of GMOs and
products thereof
Figure I -
State Bioteclmology
Coordination Committee
(SBCC). State le\'el
Commillee for
monilonng ertvuorunenl
safety & food «[ely
Genelic Engineering Approval
Committee (GEAC):
National Authority for Large
scale Reseat"h &
cOlnmercialization ofGMOs &
products ther<of
Advises & cOQ(dinates
Activities with S9CC
The rul es, aUlhoril ies and adminis trat ive st ru ctures for dealing with GMOs and prod uctgs th ereof
1
N
g
'-'
GHOSH : HANDLING TRANSGENIC ORGANISMS & BIOPHARM ACEUTI CALS -
559
RULES
Table 2- Recombinant DNA dru gs available in Indian market
Company
Strength
MRP
(in Rs)
I Human Insulin
(i)
Huminsulin -R
Eli Lil y & Co.
(ii )
Huminsulin-N
Eli Lily & Co.
(iii)
(iv)
(v)
Huminsu lin-L
Huminsulin-U
Huminsulin 30:70
Eli Lily & Co.
Eli Lily & Co
Eli Lily & Co
(vi)
(vi i)
Huminsulin 50: 50
Humalog
Eli Lily & Co
El i Lily & Co
(vi ii )
AClrapid HM Penfill
Novo Nordi sk
10 mL, 40 iu /mL
10 mL, 100 iu /mL
5X3mL cartridge, 100 iu/mL
10 mL, 40 iu/mL
5X 3mL cartridge, 100 iu/mL
10 mL, 40 iu/ mL
10 mL, 40 iu/ mL
10 mL, 40 iu/mL
10 mL, 100 iu/mL
5X 3ml cartridge, 100 iu/mL
10 mL, 40 iu/mL
10 mL, 40 iu/mL
5X3mL cartrid ge, 100 iu/mL
3mL cartridge, 100 iu/mL
206.00
398.00
990.00
209.00
990.00
206.00
206.00
209.00
398.00
990.00
206.00
375 .00
1920.00
993.69
(ix)
AClrapid Novo lel
Novo Nordisk
3X5 mL (pack of 5)
124.7 1
(x)
Human Actrapid
Novo No rdisk
(x i)
(x ii)
(x iii )
Human In sul alard
Insul alard HM Penfill
Human Monolard
Novo Nordi sk
Novo Nordisk
Novo Nordisk
(xiv)
I-Iuman Mixlard
Novo Nord isk
(xv)
Human Mi xlard 30
Novo Nordi sk
10 mL, 40 iu/mL
10 mL, 100 iu/ mL
10 mL, 40 iu/mL
5X3 mL cartrid ge
10 mL, 40 iu/mL
10 mL, 100 iu/mL
10 mL, 40 iu/mL
10 mL, 100 iu/mL
10 mL, 100 iu/mL
197.56
508. 15
195.69
99 1.50
197.52
508. 15
197 .52
508 . 15
508. 15
(xvi)
Human Mix lard 50
Novo Nordi sk
(xvii )
Mi xlard 30 HM Penfill
Novo Nordisk
10 mL, 40 iu/mL
10 mL, 100 iu/mL
3 mL penlill 100iu/mL
197.62
5 17. 35
834.00
(xvii i)
(x ix)
Mi xlard 50 HM Penfill
Novo Nordi sk
3 mL cartridge 100 iu/mL
993 .69
Human Faslacl
USV
10 mL, 100 iu/mL
199.24
(xx)
Human Longacl
USV
10 mL, 100 iu/mL
200.94
(xxi )
Huma- Mixacl
USV
10 mL. 100 iu/ mL
199.46
(xx ii )
Human Rap idica
Sarabhai
10 mL 40 iu/mL
189.84
(xx iii )
10 mL 40 iu/mL
19004
SI
No.
Brand
Human Prodica
Sarabhai
(xxiv)
Human Zi nul in
Sarabh ai
10 mL 40 iu/mL
189.84
(xxv)
Human Rapimix
Sarabhai
10 mL 40 iU/IllL
190.02
(xxvi)
Insuillan Rapid
HMR
10 mL 40 iU/IllL
2 17.68
(xxvii )
In suillan Bflsa l
HMR
10 mL 40 iU /IllL
2 17.6R
2 Erylh ropoietin
(i)
Ep rex
Ethnor
I IllL - 2000 iu/mL
I mL - 4000 iU/IllL
890.00
1690.00
(ii )
Heillax
Hi nd ustan Antibiotic
(iii)
Epox
Wock Hardl Mcrind
(iv)
Zyrop
Zydus B'iogen
( v)
Vintor
Eillcure
I mL - 2000 iu
I mL - 4000 iu
0.5 mL 2000 iU/IllL
I mL 4000 iu/MI
2 IllL - 2000 K
2 IllL - 4000 K
Vi al - 2000 iU/IllL
Vi al - 4000 iu/mL
882. 00
1750.00
98.00
1550.00
998. 91
1698 .00
550.00
1090.00
-
Coil ld
560
1 SCI IND RES VOL 621UNE 2003
Table 2- Recombinant DNA drugs availab le in Indian market SI
No
Brand
(Call/d)
Company
Strength
MRP
(in Rs )
0.5 mL inj. 10 mcg
I mL inj . 20 mcg
10 mL Multidose V
0 .5 mL inj. 10 mcg
I mL inj. 20 mcg
10 mL Multidose V
0.5 mL inj . 10 mcg
I mL inj. 20 mcg
10 mL Multidose V
0 .5 mL inj . 10 mcg
I mL inj. 20 mcg
10 mL Multidose V
0.5 mL Vial 10 mcg
I mL Vial 20 mcg
5 mL Vi al 20 mcglmL
10 mL Vial 20 mcglmL
0.5 mL inj. 10 mcg
I mL inj. 20 mcg
10mLV
0.5 mL Vial 10 meg
I mL Vial 20 mcg
5 mL Vi al 100 mcg
0.5 mL inj. 10 mcg
I mL inj. 20 mcg
10 mL V 20 mcglml
0.5 mL inj. 5 mcg
I mL inj. 10 mcg
I mL inj . 20 mcg
10 mL V 100 mcg
0 .5 mL inj.
I mLinj .
10mL V
0.5 mL inj .
I mLinj .
10 mL V (Multidose)
100.00
150.00
1500.00
140.00
190.00
1750.00
272.00
485.00
2880.00
100.00
170.00
1250.00
90.00
160.00
725 .00
1400.00
195 .00
295.00
2585 .00
97.00
178.00
834.00
150.00
222.00
1925.00
105 .00
148.00
198.00
834.00
160.00
230 .00
2 100.00
140.00
190 .00
1800.00
4 iu Vial
12 iuVial
4 iu Amp
10 iuAmp
1500.00
6000.00
7500.00
15000.00
1775.00
5415.00
2249.50
4750.00
N.A
N.A .
3 Recombinant hepatitis B vaccine
(i)
Bevac
Biological E
(ii )
Biovac B
Wockhard t
(iii )
Engerix B
SmithBeecham
(iv)
Enivac HB
Panacea
(v)
Genevac B
Serum Institu te
(vi)
HB Vac
Zydus Vaccine
(v ii)
Hepagen Plus
V H B Phanna
(v iii)
Hepashield
Pti zer
(ix)
Hepavax- B
V H B Phanna
(x)
Revac B
Intas
(xi)
Shanvac B
Shantha Biotech
4 Human growth hormone
(i)
Humatrope
Eli Lilly Ranbaxy
(ii)
Norditropin
Novo Nordisk
(iii)
Saizen
Serum International
5 Human Interleukin
(i)
Human interleukin
Ambala Sarabhai
Enterprises Ltd. ,
6 Granulocyte colony stimulating facto r
(i)
Neupogen
Pi ramal Health Care
30 million iu Vial
5 163.93
(ii)
Grastim
Dr .Reddy's Lab
30 mi llion iu Vial
2400.00
-Comd
GHOSH: HANDLING TRANSGENIC ORGANISMS & BIOPHARMACEUTICALS -
Table 2- Recombinant DNA dru gs avai lable in Indi an market SI
No
Brand
Company
561
RULES
(Comd)
Strength
MRP
(i n Rs)
Novartis
150 mcg
2650.00
7 Granulocyte macrophagecolony stimul ating factor
(i)
Leucomax
8 Interferon alpha
(i)
Alferon
Zydus
3 million iu Vi al
11 90.00
(i i)
Inron alpha Inj.
Biological E
3 million iu inj.
970.00
(iii )
Roferon-A
Piramal Health Care
3 million iu Vi al
136 1.00
(iv)
Shanferon
Shanta Biotech
3 million iu Vial
Nichol as Pi ramal
N.A.
N.A.
Hemophil a Federation of
Indi a
Kogenate
N.A.
.A .
9 Interferon gamma
(i)
y-Interferon
10 Bl ood factor VIII
(i)
Blood factor Vlll
II Foll olicl e stimulating hormone
(i)
Endogen
Uni Sankyo
Vi al 75 iu
760.00
(ii )
Endogen
Uni Sankyo
Vi al 150 iu
11 30.00
(iii)
Foliculin
Bharat Serum
Vi al 75 iu
700.00
(iv)
Foliculin
Bharat Serum
Vial 150 iu
1280.00
(v)
Follimon
Biochem
Vial 75 iu
850.50
(vi)
Fostine
Zydus Biogen
Vi al 75 iu
850.00
(vi i)
Gonal-F
Serum International
I Amp 37.5 iu
1011 .08
(viii)
Gonal-F
Serum International
I Amp 75 iu
2013.50
ix)
Gonotrop-F
Win-Medicare
I Amp 37.5 iu
850.00
(x)
Gonotrop-F
Win-Medicare
I Amp 75 iu
1445 .00
(x i)
Metrod in
Serum International
I Inj . 75 iu
11 90. 19
(x ii )
Ovimen
Life Medicare
Vi al 75 iu
800.00
(xiii )
Ovimen
Life Medicare
Vial 150 iu
1425 .00
(xiv)
Ovitrop
Sun Pharma
Vi al 75 iu
750.00
(xv)
Ovitrop
Sun Pharma
Vial150iu
1200.00
(xvi)
Puregon
Chemech Labs
I Amp
735.00
(xvii )
Recagon
Infar
I Amp 50 iu FSH
1462.50
(xvi ii )
Recago n
Infar
I Amp 100 iu FSH
2925.00
German Remedies
Vial 50 mL
43500.00
12 Ti ssue plasminogen acti vator
(i)
Actilyse
562
J SCI IND RES VOL 62 JUNE 2003
amenable to the strategy of genetic manipulation.
Efforts have also been made to produce transgenic
catfish, tilapia and Indian carps containing growth
hormone genes; the objectives were to obtain
tran sformants that mature fast. However, these
experiments have not provided any significant
success, and none of these works has yet come to the
stage of large- scale trials .
Conditions for Trials Using Transgenic Organisms
The RCGM monitors research on transgen ic
organisms in the laboratory and in the contained open
env ironment/fields. For transgenic plants, experiments are conducted in contained green house to
generate severa l vital safety information before
dec isions are taken to conduct contained open field
ex periments . In the field under contained co ndition s
bes ides designing ex periments for collecting data on
environmental
safety
aspects
the agronomic
advantages of the transgenic plants in small plots are
also assessed. The RCGM looks for information on
env ironmental safety including human and animal
food safety issues for all kinds of GMOs. Food safety
issues are linked with GMOs that may enter into
human or animal food chain directly. The info rmation
sought from the tri als of GMOs is summarized briefly
in Table 3.
The genetic materials can be allowed to be
imported or tran sferred within the country by the
RCGM for research use only, based on applications
subm itted through the IBSe.
For conducting experiments with transgenic
plants in contained Green House, designs have been
worked out for constructing low cost but substantially
contained environment where temperature, light and
humidity can be controlled to a considerable extent.
Nets ha ve been recommended that arrest the entry/exit
of insects below O.6-mm diam. Although s imilar
contained conditions for conducting experiments with
transgenic animals have not yet been published,
designs are available with the government authorities ,
and can be sent to the investigators on request.
There are severa l issues concerning the use of
transgenic plants in the country as mentioned above.
Entrepreneurs dealing with the introduction of
transgenic plants in Indian agriculture would have to
follow the above in a step-by-step manner. As the
emp hasi s of this paper is on the methods of
introduction of transgenic organisms or products
thereof which are to be used as pharmaceuticals and
which are to be introduced in Indi a within the frame
work of Indian laws, the following paragraphs
describes how this can be done.
Step by Step Methodsfor Introducing Recombinant
Bio-pha rmaceuticals
There are not many gen etically modified
organisms that are directly utilized as a drug in
therapy. One genetically modified vaccinia virus
coding for rabies antigen was introduced a couple of
years ago for imparting protection to domestic and
wild animals 12. Several defective viruses and
microorganisms are under testing that are targeted for
Several
recomb inant
biospecific
therapy .
ph armaceuticals have, however, been introduced the
worldover during the last two decad es and the number
presently exceeds thirty . These products ha ve been
produced in a wide ran ge of hosts that include
genetically engineered bacteri a, yeast and mammalian
cells. Plant and insect cell lines have also been
utilized ; however, products from such hosts have been
used mainly for diagnos tic purposes. The active
substances are usually lIsed as th erapeuti c or
prophylactic peptides and proteins. Concerns of safety
from the use of substances produced in transgenic
hosts arise from contaminants emanating from the
host cells as well as the special process ing steps
requiring use of toxic c hem ic als and substances.
Countries therefore have relied upon sc ientifically
valid and reproducible purification processes to
remove the contaminants, and establish acceptability
criteria for each bulk peptide o r prote in produced by
r-DNA technology . The goa ls that have been set for
new molecules produced for the fir t time in the world
are as under:
(a)
To evaluate the effects of host organi sm related
contaminants such as DNA, proteins, lipids,
nature
and
ex tent
of
ost-translational
modification .
(b)
To set standards of the limits of contaminants
into the final bulk material based on
scientifically sound evaluation criteria.
(c)
To set standards for the pr~ se nc e of toxic
processing chemicals used in the purification
processes of the bulk and fo rmulated r-DNA
bio-pharmaceuticals.
In addition, there has been extensive
requirement of documentation indicated in the
documentation details (Table 4).
GHOSH: HANDLING TRANSGENIC ORGANISMS & BIOPHARMACEUTICALS -
Table 3 -
RULES
Summary of the biosafety information sought from GMO trials
Particulars
Information sought
Rationale for the
development
•
Economic, agronomic and other benetits, and rational of development of the GMOs and/or
products therefrom
•
Description of the host organisms (micro-organisms, cell lines, pl ants , animals etc.)
•
Source and sequence of transgene/s
•
Sequential block diagram of all trans-nucleic acid stretches inserted
•
Cloning strategy
•
Characteristics of expression vectors
•
Characteristics of inserted genes with details of sequences
•
Characteristics of promoters
•
Genetic analysis including copy number of inserts, stability, level of expression of transgenes,
biochemistry of expressed gene products etc.
•
Transformation/cloning methods and propagation strategy
•
Back-crossing methods for plants
•
Seed setting characteristics of plants
•
Germination rates of seeds
•
Phenotypic characteristics of transgenics
•
Organism challenge tests where ever applicable
Details of the molecular
biology of GMOs and
genetically modi tied
products derived therefrom.
(Microorganisms, plants and
animals, as well as products
therefrom)
Laboratory, green house
trials (for plants) and
contained enclosure trials
(for animals)
Field trials in open
environment
563
•
Effects of chemical herbicides for all herbicide resistant plants
•
Growth characteristics and general health of animals, measured throu gh specitic scientific
parameters
•
Toxicity and allergenicity implications to human if any during handling of GMOs
•
Generation of foodlfeed safety information where applicable, through lab tests.
•
For GM plants, comparison of germination rates and phenotypic characteristics, usi ng nontransgenic as controls.
•
Study of gene flow of plants
•
Possibility of weed form ation for GM plants
•
Invasiveness studies of plants and animals compared to non-transgenes used as controls
•
Possibility of transfer of transgenes to near relatives through out crossing/ cross-fertilization
•
Implications of out crossing/cross-fertilization
•
Comparative evaluation of susceptibility to diseases and pests for plants and animals
•
For human foodl animal feed , elaborate determination of composition and assessment of quality
of transformed plants/ fruits/seeds as well as animals as the case may be, with appropriate
controls. Compositional analysis shall include near equivalence studies of all the major
ingredients in GMOs so as to assess substantial equivalence with reference to non-transgenics.
Change in the levels of allergens, toxicants if any, beyond acceptable limits is a matter of food
safety concern and such substances are unsuitable for commercial release.
•
Toxicity and allergenicity implications of transformed GMOs. This include microorganisms,
plants/fruits/seeds as well as animals; lab animal studies for food /feed safety evaluation is a
requisite.
•
Handling procedures for allergenic substances.
•
Agronomic evaluation for GM plants
•
Economic evaluation for GM animals and GM microorganisms
564
J SCI IND RES VOL 62 JUNE 2003
T ab le 4- E le ments of documentation for r-DNA bio-pharmaceuticals
2
C haracte ri zati on of the sys tem of production used
Details o f the transgen ic expression sys tem used
(i)
Description of the host cell line
(ii)
Identification of the genera and th e species
(iii )
The risks involved in handling th e cell lines I microorganisms
(iv)
The class ification of th e cell lin e I microorganisms in accordance with any accepted r-DNA cell line I
mi croorganisms us age guidelin e and their safety statu s if available.
(v)
The methodes) of maintenance and growth of the cell line I microorgani sms
(vi)
Th e nature and hazards of usin g sub strates, inducin g agents, e tc.
Characteri stics of the target transgenic DNA including promoters, genes, enhancers, markers. etc. and the vector
used
(i)
The full description of the source of the target transgenic DNA in cludin g pro mote rs, genes, enhancers ,
markers etc along wi th docume nt ation o f all th e trans - DNA seq uences
(ii)
The co mpositio n of th e vector used indi catin g th e promoter seq ue nce as well as the regulatory
mechanisms ut ilized in the express ion cassette
(i ii)
Schematic diagrams of the ex pression cassette to describe fully th e marker genes used
The res tri cti on si tes re lated to specilic endo nu cleases, and th e ce ll lines used fo r ' hutt ing and
(iv )
amp lifi cation into the host genome
(v)
The target gene should also contain, along with the nucl eo tide seq uence, th e description of the amino
ac ids below the codons.
Approac hes adopted for expression of the gene
(i)
The description of th e transc ript ed messenger RN A with its ana lysis of sequence ;1I1d ide ntitic ation
procedure
(ii )
The tran slat io n info rmat ion indi ca tin g wheth er th e protein product is C himaric, wheth e r the exp ress io n
is found as inclusio n bodies or as intracellul ar protein or whether the protein is ~ecre t ed out
(iii )
The extent of the target pro te in produced as percentage of the total cell dry-mass as we ll as its
percentage compared to the to tal proteins of th e ce ll
(iv)
The qua li ty of the target protein produced after the cell growth per lit er of the fermen ted broth
(v)
The full seq ue nce of the recombinant gene alon g with the promoters, the mark er genes and th e
terminator sequence
Description of the productio n process
(a)
Prodllclioll sel 1If!
(a)
(b)
(c)
(b)
(c)
(d)
(i)
The handling of the stock cell lin es
(ii)
The evalu atio n and uses of the ce ll lin es in pre-fermentation processes
(i ii )
The preparation o f the seed vesse l
The main production fermentation conditi o ns need to be described in bri ef
(iv)
Growth Kineti cs
(i)
Graphical plots of time versus substrate usage, opti cal density change biomass formation, protein
products formation , indu cing agents used and their effec ts on the target protein fo rmation
(i i)
The effect of change of standard parameters like Ph , dissolved oxygen, temperature, etc, in the main
production fermentation vessel
Fermentation parameters
(i)
The pH , temperat ure, aeration, and rpm of th e shaft
(ii )
Volume of seed to the volume, of main ferment ati on vesse l
(ii i)
Main sub strates used, inducing agents used if any
(iv)
The fermentation time and the concentrat io n of th e target protein in th e fermen ted broth
Approaches for the ex tract io n and purification of th e product
(i)
The methods of handli ng the cells
(ii )
The methods o f isolating the cell soup co ntai ning the target protein. The method o f enzyme treatment.
if any
(i ii )
The methods of concentration , precipitatio n (if app lied ), reconstituti o n, sal t separati on (if applicable).
absorpt ion and desorption meth ods, chro matographic methods used if any, and ultra-centrifugation
methods i I' used
(iv)
Sterilization and tinal dosage formulation
(v)
Processes to obtain the product in th e finished dosage form
-Conu/
GHOSH: HANDLING TRANSGENIC ORGANISMS & BIOPHARMACEUTICALS -
Table 4-- Elements of documentation for r-DNA bi o-pharmaceuti cals 3
4
5
6
7
(a)
565
RULES
(Call/d)
In-process control methods
All the analytical methods used for this in- process cont rol must be described to ensure the regulatory
(i)
autho rities that the chances of entry of unwanted products have been minimized
Description of the raw and processing materials used
(i)
Description of all raw materials and processing materials used starting from the stock cell s hand ling to
the fini shed dosage form must be in a tabular form
(i i)
Grades of materials used and their usual souring
Description of the plant and machinery used
(i)
List of all the equipment along with the indicative capacity
(ii )
Names of the manufacturers
(iii)
List of Main production equipment and the quality cont ro l equipment
Description of the building and facilities created for the manufacture
(i)
The manufacturing area
(ii)
The flow of the process/ operation starting from the stock culture area to the fini shing area,
(iii )
The plot plan of vari ous floors used in the manufacture and processing
(iv)
The building diagram in the plan and / or elevation in the outline indicating the cleanliness maintained
in different sections such as class 10,000, calls 1000 and class 100 areas, etc
(v)
Description of the air handling system, which is very important
(vi)
A separate write up indi cating the movement of operating personnel in the area
Quality Contro l and Quality Assurance
Bulk material:
(i)
Tests relating to the identification of the stock cells
(ii)
The plasmid constructs retention in the cell s durin g cell growth
(iii)
The media used
(iv)
The procedures adop ted for testi ng and the percentage retention of the target plasmid
(v)
Description of the contami nation test of the stock culture to assess and monitor the microbial
contamination including the media used and the procedures adopted
(vi)
The cri teria of acceptance of contamination free culture
(vii)
Characterization of the bio- active molecule produced by monitoring physical and chemical properti es
of the molecule by the following or more authentic method should be provided
(viii)
Microscopic examination including li ght, phase contract and electron microscopic studi es
(ix)
UV Spectroscopic analysis to show the absorption spectra of the product and comparison of thi s with
authentic material
Density gradient centrifugation to show si ngle peak
(x)
(xi)
Pattern in Hi gh performance liquid chromatograp hy to include how many peaks or if on ly one peak is
noticed in the produced and purified bulk
(xii)
Iso- electric focusing analysis to show the pi values
(xii i)
Western Blot and SDS-PAGE to map the peptide/ target protein
(xiv)
Existence of special bonds like disulfide bonds by breaking the protein / peptide with appropriate
reagents and subj ecting the products to SDS-PAGE mappi ng
(xv)
Immuno-diffusion Test to find out the absence of contaminants or the extent of the presence of
contaminants
(xvi)
Amino acid composition of the purified protein / peptide and its comparison with the aut hentic
material if available (gold standard)
(xvii)
N Terminal Amino Acid Sequence analysis and its compari son with the protein / peptide expressed by
the transgene
Determination of the biological activity in the appropriate animal model
(xviii)
(xix)
Determination of contami nant s per milligram or per any convenient unit of the manufactured bulk
purified protein is also to be carried out to indicate the extent of contaminated nucleic acid stretches,
proteins, carbohydrates , lipid, detergents, salts and other processing materials used in the purification
process, the impacts of the presence of these contaminants are also to be indicated with authentic
reference materials if any or by conducting animal toxicity / allergenicity tests in app ropriate animal
models to ensure the risk associated with their presence in quantities that are to be fixed , are minimal.
The limits of contaminants and Impurities are to be quantified for each foreign substance, and the
acceptability criteria for the bulk peptide / proteins with reference each of the contaminantslimpurities
need to be quantified
-
COIIU/
566
J SCI IND RES VOL 62 JUNE 2003
Table 4-- Elements of documentation for r-DNA bio-pharmaceuti cals -
(Co llld)
(b)
Formulated bio-pharmaceutical material s
(i)
The ingredi ents incorporated subsequently in the manufacture o f the formu lated material using
the bulk. The specification of the ti nal product needs to be detined in composition
(ii)
The qu ality co ntrol department of the organization would have to certify th at the tinal product
has the results within the specitications prescribed and accepted by the regulatory authoriti es
(iii )
The docu mentati on for the formul ated material should therefore specify at leas t th e fo llowing
parameters
Product present ation
Physical appearance of the product
(iv)
•
Preservative usage in percentage
•
pH of the solution! formul ated material when reco nstituted
•
Other extraneous materials used and their extent such as co ntent of DN A RNA
Carbohydrates, Lipids, processi ng materials like detergents, salts etc. wh~rever ;1p plicable
•
Total protein conten t and the acti ve protei n co ntent
•
Sterility status of the formul ation
•
Toxicity informatio n
•
Allergenicity info rmation
•
Pyrogen statu s.
•
Any other information relevant to the medical profession
Status o f certification i.e. if the formulated material co nforms to any accepted speci licati o n.
•
Every certificate wherever issued shall be accompani ed by other statuto ry inform ati on like
the manufacturing batch number like the date of manufacture, date of analys is o f the batch, of
the date of release of the certiticate, authorized signatory to the certiticate, etc
•
The final formulated product to be marketed I used needs to be certiti ed fo r accep tance by the
manufacturer indicating the date up to which the material can be used for th e intended
purpose! indication
Recombinant DNA bio-pharmaceuticals that are
new molecules are required to be fully investigated
for safety before they become eligible for marketing
and use. Such mol ecules produced and evaluated in
one cou ntry, and atlowed fo r marketing in the country
of origi n may require a fres h evaluation in another
country if the regulatory laws so require. In India,
depend ing upon the quality of evaluation of safety
and efficacy as also the sample size uSt?d for efficacy
studies, the Indian government may require the
applicants to conduct de-novo studies or may
authorize the conduct of phase III clinical studies in a
minimum number of human subjects. Often the phase
III trial s require a sample size of over 100 persons and
the studies should be conducted independently at least
in 4 centers under the supervi sion of qualified medical
practitioners. Even during such studies spec ial care is
required to be taken to generate data on safety issues
to human health that are part of the requ irement of the
Indian Environment (Protection) Act & Rul es.
Di rectly authorization for marketing can be prov ided
on ly in situation s where the recombinant DNA biopharmaceuticals have been extensive ly in use
elsewhere and have been evaluated! thoroughly over
very large number of human su bjects, and the
products are in use for a couple of years. In such cases
also the Indian Environment (Protection) Act & Rul es
require the generation of post market surveill ance data
where safety information on issues related to human
health are to be addressed and gathered . All protocols
used in phase I, II, III and IV require the approval of
the government.
GHOSH: HANDLING TRANSGENIC ORGANISMS & BIOPHARMACEUTICALS -
Use of all recombinant DNA products requires
pre-clinical safety evaluation in relevant animal
species. Evaluation carried out in non-relevant
animals is of no use. The testing in animals requires,
besides the selection of the relevant animal species a
statistically significant numbers, their · age with sex
distribution, the manner of delivery of the dose
including the routes of administration and the
treatment regimen (Schedule of treatment). The
material to be tested must be stable and must be
satisfied to be suitable for application/use. All toxicity
studies in animals are required to be consistent with a
protocol that requires the approval of the government.
Further, the use of the protocol as well as the test
animals also require the approval of the Animal
Ethics Committee of the institute. All studies should
follow Good Laboratory Practices . The safety
evaluation program should include at least two
re levant animals. Only in special cases, one relevant
animal species can be used ; however this requires the
approval of the regulatory authorities. Toxicity studies
should not be undertaken in non-relevant animal
species, as the results are misleading and irrelevant.
Transgenic animals can be used with the approval of
the regulatory authorities provided such animals have
the ability of expressing human receptors and that the
presence of receptors in the tissues of such animals
are comparable to human receptors. Data generated in
re levant animal models provide insight in determining
pharmacokinetics and pharmacological action besides
safety and the usefulness of the product. Some biopharmaceuticals produced by recombinant DNA
technology can be immunogenic in animals as well as
In
human
(when
repeatedly
applied).
The
immunogenic potential of such products requires to be
taken a note of and animal studies should document
such information at the start and at the end of the
animal experiments. Effect of hyper. sensitization on
the animals also requires to be recorded with effects
on vital organs. In thi s context, the safety of the host
cell needs to be taken cognizance to not only from its
potential for producing endo-toxins but also its
abilities for post translation modification of the
translated proteins . Toxic or immunogenic potential
of the non-protein components added to the transgenic
proteins need also to be evaluated before a decision
for approval is taken by the regulatory agencies. For
RULES
567
DNA vaccines, it is specifically required to study the
fate of the coding sequence in the target tissues /
animals and therefore the decay rate of the transgenic
sequences need to be studied. Further, data are to be
generated on whether the DNA coding sequences get
integrated into the tissues of the animal. It is also to
be determined whether the vaccine gets continuously
expressed and whether such a phenomenon hyperimmunizes the animal and whether such phenomenon
of hyper-immunization causes any toxic effect to the
blood or to any vital parts of the animals like kidney,
liver etc. The protocol has to address generating
information on these aspects. The new molecul es
require generations of pharmaco-kinetic and toxicokinetic data, evaluation of single doze toxicity as well
as
repeated
doze
toxicity ,
immuno-loxicity,
reproductive performance, geno-toxicity and carcinogenicity studies. A well-known molecul e does
however require de-novo generation of all these data
as there may be ex ten s ive published informati on
already available on these aspects. However, if the
known biopharmaceutical product is produced by a
different method where the host cell lines have been
modified or changed or where the construct has been
newly assembled using new method s or where the
product has been purified by applying down stream
processing methods that have not been eva luated, then
the product will be considered as new . However for
such a product only the sub-acute toxi c ity studies and
in some cases chronic toxicity studies in two rel evant
animals may be adequate. Effects of posttranslational
modification of the translated proteins must be kept in
sharp watch as indicated above. Post transpationally
modified product may behave quite differently as the
modification
depends
upon
the
tran s lational
machinery of the host organism. Carbohydrates, lipids
and other residues including Sialic Acid derivat ive
that get incorporated can impart different properties to
the resulting protein expressed by a set of well
characterized genes, as the latter is host dependent
while the former i.e. the production of the amino acid
sequence of the target protein is not host dependent,
but gene-dependent only.
After the conduct of animal studies and after
documenting the recombinant DNA biopharmaceutical product in the manner described above the
applicants can seek marketing permission for selling
568
J SCI IND RES VOL 62 JUNE 2003
such products . In India there can be two marketing
routes for introducing such biopharmaceuticals. In
one rou te, the biopharmaceutical can be developed in
a step by step manner in Indian laboratories/faciliti es
and the data generated on safety of the formulated
material can be submitted to the GEAC as well as to
the DC (G) I. T he former authorizes use and
marketing und er the Indian Environment (Protection)
Act whil e the later authorizes manufacture and
marketin g under the Dru g Act. In the other route, rDNA biopharmaceuticals can be directly imported
and marketed. In such cases also the applicants are
requires to provide data as above to the GEAC as well
as to the DC (G) I. The only difference is that such
data on safety of the products are not generated in
India.
The steps requ ired to be followed by applicants/
organizations/manufacturers for the introduction of
any r-DNA biopharmaceutical product in Indian
market based on loca l manufacture are elaborated in
Table 5.
For biopharmaceutical s to be directly imported
and marketed, the procedures to be followed by
applicants are given in Table 6.
The Kinds of r-DNA Biopharmaceuticals that
Require Clearance Under EPA
The information required to be generated, as
documented above, app lies to a.11 kinds of
biopharmaceutical s produced by recombinant DNA
tec hnol ogy. Th e usual products include growth
factors, fusion proteins, receptors, hormones,
cytokines, thrombolytic agents and plasma factors.
They al so include monoclonal antibodies produced by
the appl ication of phage display methods . Further,
they in clude proteins / peptide DNA vaccines, and
genetic material s / DNA material s used in ce llular and
gene therapies. They also apply to recombinant
primary and secondary metabolites used in therapy
such as enzymes, antibiotics, vitamins, blood
components, blood proteins, etc. However the rules
do not apply to conventional products like bacterial
and viral vaccines, antib iotics and other products
produced by rermentati on using non genetically
engineered microorganisms / cell lines . They also do
not apply to monoc lonal or polyclonal antibodies
produced by hybridoma tech no logy o r conventional
methods respectively .
Conclusions
The Indian Government created the rul es and
procedures for dea ling with GMOs in 1989 under th e
Indian Environment (Protection) Act 1986. Mis
T ransgene Vaccines Ltd , Hyderabad started the first
semi-commercial sca le transgenic e xperiments with
microorganisms in February 1995 Isin g geneti ca ll y
modified HWlseneula polymorpha th at codes for
Hepatitis B surface antigen. T he co mpan y procured
th e lab leve l technology from Mis Rhein Bi otec h
GmbH, Germany. The experiment were condu cted at
the Institute of Microbial Tec hnol ogy, Chandigarh .
The transgenic surface antigen of he patitis B kn own
as "S" antigen was isolated in bulk and it was proven
that the country could handl e geneti cal ly modi fied
organ isms under cGMP conditions. However, the
company could not subsequentl y set up its own
production facilities . Subseque ntl y, in August 1997,
Mis Shantha Biotechnics, Hyderabad introduced a
simi lar product, produced in recombinant Pichia
pastoris, developed by them. Later on after Shantha,
three more companies have come into production of
thi s
recombinant
vaccll1e
111
th e
country.
Subsequently, up
to end-2002, three more
recombinant
produ cts
namel y
erythropoi etin ,
granu locite colony st imu lating fact or and interferon
alpha 2b have been cleared by the Regul atory
Authorities
for
local
prod ucti on.
More
biopharmaceutical products woul d be authorized for
local production in the coming years. Presently (end2002), in all twelve biopharmaceuticals produced by
reco mbinant DNA technology have been authorized
for marketing in India of whi ch, as stated above, four
are authorized for local production . In GM plants, the
first transgenic plant experiment in the field was
started in 1995. Since then the cou ntry has acquired
substantial experience in understanding the issues
related to the handl in g of GMOs . In March 2002,
transgenic insect resistant Bt cotton was auth orized
for production in India. The Bt cotton was found to
reduce the consumption of chemical pesticides
considerably with increased yield o f cotton, provided
it rece ived the necessary quantities of fertili zers and
water, and the plants were sprayed fo r protection from
non-bollworm in sects, and where necessary to reduce
GHOSH: HANDLING TRA SGENIC ORGA ISMS & BIOPHARMACEUTICALS -
RULES
569
T abl e 5- Steps to be foll owed by applicants/in stitution s/organi zations for obtainin g the approval for thcir biotech drugs (reco mbinant
DNA drugs) developed and/o r produced in Indi a
The institution/organization makes an application for the approval of its Institutional Bio-Safety Committee (IBSC ) to
Department of Biotechnology (DBT), Govt. of India .
DBT approves the IBSC and nominates a govemment's nominee on the IBSC to oversee the activities to ensure that biosafety guidel ines are strictly followed .
IBSC is formed at the institution/organization and DBT takes composition of IBSC on record . Applicants including
institution/organization submit applications to IBSC for initiating experiments involving r-DNA products .
IBSC meetings are convened at the institute/organization at least twice a year. Every meeting shall have an agenda .
Minutes of all IBSC meetings are sent to Review Committee on Genetic Manipulation (RCGM). IBSC can approve
experiments of the Category I and Category II risk experiments. For experiments of Category III and above risks, IBSC
sends its recommendations to RCGM for approval. Applicants submit applications in prescribed formats to IBSC/RCGM .
RCGM grants the conduct of experiments of Category III and above risks , and asks fo r progress reports periodically from
the applicants/project-investigators through IBSC.
Experiments conceming drugs produced from Genetically Modified Organisms (GMOs) require generation of animal
toxicity data. Before such data are generated, results of at least five trial batches (including each bulk and formulation
batches) are evaluated first by the IBSC and then by RCGM to ascertain if a reasonably stable process has been
developed . Product acceptability criteria for both bulk and formulated materials are fixed .
Protocol must address risks emanating from host-related contaminants as well as residual toxic chemicals used in
processing . RCGM approves the conduct of animal toxicity studies; thereafter, Animal Ethics Committee of the
institution/organization has to approve the protocol.
Animal toxicity has to be carried out at least on two relevant species of animals (often , one rodent and one non-rodent
soecies are selected). Soecies of animal is to be indicated in the orotocol.
Animal toxicity studies are carried out and IBSC meeting is convened by institution/organization to discuss the report. If
satisfactory results are obtained , protocols for Phase I, Phase II and/or Phase III studies are prepared by the
institution/organization, and placed before IBSC members for approval. Phase I is needed for a new producUmolecule.
Phase II is needed when the dose is not known or when the dose finding has to be carried out. Straightaway Phase-III
stUdies can be carried out if the drug is well known and the host is assessed as safe.
,
.
Above documents, including protocols for human studies are submitted by the applicants to RCGM and Drug Controller
General of India (DCG (I» for approval. RCGM sends its recommendation to GEAC as it cannot approve human studies.
+
570
J SCI INO RES VOL 62 JUN E 2003
Ta bl e 5- Ste ps to be follow ed by app li cants/in st ituti ons/organizati ons for obta inin g the a pproval for their biotech d ru gs (recombin ant
o A drugs) developed and/o r produced in India - (COl/lei)
The protocol for the human studies and the phase for which the studies is conducted will have the approval of the Drugs
Controller General of India and the GEAC. GEAC requ ires generation of safety data while DCGI requires both safety and
efficacv data . GEAC consults RCGM .
Human Clinical Trial / studies are comp leted and data related to human safety including risks to human health are
generated and data subm itted to IBSC by the applicants.
ISSC meeting is convened by the institution / organization for approval of the human clinical trial report.
Clinical trial report is submitted to RCGM , GEAC and DCG(I). The applicant submits information in a format seeking
clearance under EPA For clearance under Drugs Act, a separate format is used .
•
GEAC sends the application to four or' five experts including DBT, and , on the basis of the recommendations of the
experts , GEAC members evaluate and approve the r-DNA drug under EPA for open environme ntal re lease , i.e., for
marketing .
+
The joint inspection of the production site may be required, and can be done by the representative of GEAC and DCG (I).
Thereafter, DCG (I) allows production of trial batches, and five such batches are sent to Central Research Institute (CRI) ,
Kasauli for testing under the Drugs Act.
•
After the receipt of Testing Reports from CRI , Kasauli, the DCG (I) grants the final approval to manufacture and market the
product. The DCG (I) has set up an expert committee for r-DNA drugs and the applicant is requ ired to make a presentation
before that committee. DCG (I) takes a decision on the basis of the recommendations of the expert committee.
Both DCG (I) and GEAC can impose conditions of surveillance on the product during marketing . Marketing under EPA can
be for a period of two to four years initially and this can be renewed on the basis of an application. Post-market
surveillance data may be required to be generated and submitted to DCG (I) and GEAC by the appl icants.
the load of bo ll worm too. In thi s co ntex t the ri c h
ex perie nce ga ined from hand s-o n ex pe rime nts in fi e ld
cond iti o ns for handlin g severa l other GM plants has
provided insights about the benefits as well as the
7
ri sks from th e use of such pl a nts . On overall
assessment it appears that the be nefits from the use of
suc h plants are substanti a l and they may outweigh the
ri sks , which in ma ny facets are presumpt ive and not
rea l. There a re issues on th e flow of tran s-genes into
the open e nvironment. Transgenic traits would spread
into th e natural e nvironment over the years, es pec ially
in si tuation s where GM plants set seeds by cross
pollination and whe re there are ma ny wild near
relatives o f suc h plants as is in the case of Brassica
species. Whil e approving suc h GM p lant s, g reat ca re
has to be taken to assess the potentia ls of spread of
transge nes into th e wild re latives throu g h suc h GM
plants, and the consequence o f such spread .
Obvious ly, in these cases, a close monitoring wou ld
be required for several years by th e auth or iti es.
Experime nts in tran sgeni c animal s including fish a re
yet at developme nta l stage and the cou ntry has to go a
long way befo re such products are deve loped for
comme rcial applications. A case by case assess men t
GHOSH : HANDLI NG TRA SGENIC ORGANISMS & BIOPHARMACEUTI CALS -
RULE S
57 1
Table 6 - Steps to be followed by applicamslinstitutions/organi zation s for obtaining the approval for th eir bi otech dru gs
(reco mbinant DNA drugs) to be imported and mark eted in India (S uch drugs cannot be co nsidered for import if th ey arc not
approved in the country of origin)
The applicant/institution/organization makes an application simultaneously to the Genetic Engineering Approval
Committee (GEAC) in a format (in about six copies), and to the Drugs Controller General of India (DCG (I» in
another format in five sets .
•
GEAC sends the application to four to five experts including the Department of Biotechnology (DBT) seeking their
evaluation/comments on the application . DCG (I) also examines the report in-house as well as consults experts .
...
After receipt of comments of all the experts, the GEAC examines the application along with the comments in its
meetings. At this stage, GEAC may ask for 21 sets of application from the applicants. GEAC may decide to direct the
applicant to conduct further Clinical Trials if the committee has any doubt about the product in terms of evaluation of
safety , lack of information on certain aspects of safety, the inadequacy of the sample size used in the evaluation etc.
Phase III clinical trials may be ordered incorporating in the protocol questions that address certain issues of human
risks/adversary actions .
•
•
•...
If the data are found to be satisfactory by the GEAC , the latter sends its recommendations to the DCG (I) incorporating
conditions that require the generation of post-market surveillance information (Phase IV data). If not approved , the
GEAC directs the applicant to apply afresh after generating the necessary data under Phase III clinical trial.
After receiving the authorization from the GEAC , if the DCG (I) is also satisfied with the data , it grants its initial
approval in accordance with 122A.
Applicants the n apply for form 10 along with 122A approval.
Authorization for imports is issued by the DCG (I) incorporating its conditions as well as those of the GEAC , and the '
applicant imports and markets the drug.
of ri sks o f all kinds of GMO s on a precautionary
principl e seems to be the wisest path to be followed at
thi s juncture. This will in still public confidence and
will enable the country to maximize the utilization of
thi s powerful technology to the benefit of India .
Authorities could also publish a list of GMOs and
produc ts thereof that are considered environmentally
safe and would not require any further scientific
scrutiny under the Indian EPA for handling and use,
based on published information or documentation or
data generated by the government. Al so, information
on the approved GMOs and products thereof as also
the bri ef basis of approval through an internet website
is lon g overdue.
References
Ghosh P K , Prospects for biotechnology in Indi a, In Business
opportunities in biotechnology (Confederation of Indian
Indu stry, New Delhi) March 200 I, pp 1-1 8.
2
3
4
5
Indian Environment (Protection ) Act, 1986 .
Rules Jo r the IlIanuJaclll re, lise, illlJiort, e),l'0 rt ami storage oj
hazardous
lIIicro
orgclllisllls/genetico/lv
engineaet/
organisms or cells, i ssued by the Un ion Ministry of
Environment and Forests, Government of Indi a, vide
Notification No. GSR 1037 (E) dated 05 December 1989.
Recombinant DNA Sa/ety Cllit/elin es, 1990, noti li ed by the
Department of Biotechnology, Un ion Mini stry of Sci ence
and Technology, Government of Indi a in 1990.
Revised Cuidelin es Jor Saj'ely in Biotechnolofn', notitl ed by
th e Department of Biotechnology, Union Mini stry of Science
and Technology, Government of Indi a in May 1994.
S72
Ii
7
J SCI IND RES VOL 62 JUNE 2003
Rl'l'ised GlIidelill es for Research ill Trall sgellic Plants &
GlIidl'lill es fo r Toxicitv alld A llergenicit )' Evaillatioll of
Trall sgellic Seeds, Plallts all d Plallt Pa rts, issued by the
Depart ment of Bi otechnology, Uni on Mini stry of Science
and Technology, Government o f Ind ia in Au gust 1998 and
partl y amended on 24. 09. 1999 .
Ghos h P K, Th e mles and procedllres fo r e vaLuatillg
trallsgellic crops ill Illdia in Hi-tech hort iculture, edi ted by K
L Chadh a, M L Choudhary and K V Prasad (Publi shed by
Hort icultural Society of India), 2002,428-443 .
R
Ghosh P K, ClIrr Sci, 72(3) 1997, 172- I 79
9
Ghos h P K , } Na tl Bot Soc, 51 ( 1997 ) I 1-32 .
10
Ghosh P K, M ark et Research co nducted by th e author
(Unpubli shed data)
II
Schae fll er 1 A & Dale P 1, Transgenic RI'.I', 3( 1994) 263-278.
12
Ghosh P K , Role of bi otechnoiogy i n heal thcare in India:
Present & future, ill Bioteclll lOlogy ami dl'l'e/o/JII /ell t reviell'
(RIS-BCIL Publi ca ti on), lune-December, 1995,1-22 .
13
Cartegana Protocol on Biosa/ety of th e COll vell tioll 0 11
BiologicaL Diversity of the United ations Enviro nmental
Program of the United Nati ons Indu strial Development
Organi zati on Montreal Canada, adopted on 29 January 2000
(hlip:!/ www. bi odiv. org/bi osaretyl).