Assessment of biotechnology products for therapeutic use

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Toxicology Letters 120 (2001) 59±66
Assessment of biotechnology products for therapeutic use
Jennifer Sims *
No6artis Pharma AG,WSH2881,Auhafen,4132Muttenz,Basel,Switzerland
Abstract
Biotechnology products for therapeutic use include a very diverse range of products,including growth factors,
cytokines,hormones,receptors,enzymes,clotting factors,monoclonal antibodies,vaccines,DNA vaccines,gene
transfer products,cell therapies and tissue:organ grafts.While some of these products are regulated as medicinal
products,the regulatory status of others such as some cell therapies and tissue:organ-based products differs globally
and falls within the borderline between the practice of medicine,medical devices and medicinal products.The unclear
regulatory status of some products can add to the complexity of the safety assessment of such products.Conventional
non-clinical testing paradigms and guidelines for small molecule development are often not relevant for biotechnology
products.Guidelines relating to the non-clinical safety evaluation of biotechnology products,gene transfer products
and cell therapy products are available and represent a set of general guiding principals to be applied on a
case-by-case basis.The quality,safety and ef®cacy of biotechnology products for therapeutic use are intricately
linked,far more so than for conventional medicinal products,leading to the need for increased communication
between those responsible for ensuring product quality and those responsible for non-clinical safety testing.Safety
issues include microbiological safety (due to the use of biological materials either during the manufacturing process
or as an integral part of the products),pharmacological:biological toxicity (due to excessive primary pharmacology
or undesirable secondary pharmacology),immunogenicity and potential tumourigenicity (for example,for growth
factors,immunosuppressive monoclonal antibodies and cell therapy products).Genotoxicity and intrinsic chemical
toxicity are less of a problem for biotechnology products.© 2001 Published by Elsevier Science Ireland Ltd.
Keywords:Biotechnology;Pharmaceutical;Safety assessment
www.elsevier.com:locate:toxlet
1.Spectrum of biotechnology-derived
pharmaceutical products
Biotechnology products for therapeutic use in-
clude a very diverse range of products,as outlined
in Tables 1 and 2.Some products are intended to
mimic the human counterpart,whereas others are
intended to differ from the human counterpart
and may be analogues,chemically modi®ed (e.g.
pegylated) or novel products (e.g.single chain or
Abbre6iations:LH,luteinising hormone;PDGF,platelet-
derived growth factor;NGF,nerve growth factor;IGF-1,
insulin growth factor-1;HSV,Herpes simplex virius;TSE,
transmissable spongiform encephalopathies.
* Tel.:41-61-3241523.
E-mail address:jennifer.sims@pharma.novartis.com (J.
Sims).
0378-4274:01:$ - see front matter © 2001 Published by Elsevier Science Ireland Ltd.
PII:S0378- 4274( 01) 00310- 1
J.Sims:Toxicology Letters 120(2001)59±6660
Table 1
Range of biotechnology-derived pharmaceuticals
a
Hormones FSH,LH,growth hormone,insulins,
insulin analogues
Growth factors PDGF,NGF,IGF-1
Interferons,interleukins,colonyCytokines
stimulating factor,erythropoietin
Others Albumin,clotting factors,enzymes
Murine,chimeric,humanised,rDNAMonoclonal
antibodies technology
Antibody-related Single chain,fragments,fusion products
products
Conventional,recombinant proteinVaccines
antigen,modi®ed bacteria or viruses,
anti-idiotype,mimotopes
Nucleic Gene therapy,DNA vaccines,ribozymes
acid-based
products
Autologous,allogeneic,xenogeneicCells,tissues
and organs
a
FSH,Follicle stimulating hormone;LH,luteinising hor-
mone;PDGF,platelet-derived growth factor;NGF,nerve
growth factor;IGF-1,insulin growth factor-1.
The expressed protein or gene may have the iden-
tical amino acid or nucleotide sequence as the
human endogenous form,or may be intentionally
different in sequence to confer some technical
advantage such as an optimised pharmacokinetic
or pharmacodynamic pro®le.The glycosylation
pattern of protein products is likely to differ from
the endogenous human form due to the different
glycosylation preferences of the expression system
used.Furthermore,intentional post-translation
modi®cations or alterations may be made such as
pegylation.It is important for the toxicologist to
be aware of the nature of the product to be tested
in terms of primary,secondary and tertiary struc-
ture,and any post-translational modi®cations
such as glycosylation status,particularly as these
may be altered if the manufacturing system is
modi®ed.
The diversity in the range of products and the
uncertainty about the regulatory status of some
cell:tissue-based products create a challenge for
toxicologists to design relevant safety evaluation
programmes to ensure patient safety,and which
provide useful information to the clinician respon-
sible for clinical trials,the prescribing physician
and the patient.The ICH harmonised tripartite
guideline on the preclinical safety evaluation of
biotechnology-derived pharmaceuticals (Interna-
tional Conference on Harmonisation of Technical
Requirements for Registration of Pharmaceuticals
for Human Use,1997) emphasises the need for a
science-driven rational approach using relevant
animal models to assess the safety of these
products.
2.Safety concerns
There are a number of safety issues relating to
biotechnology products that differ from those
raised by low molecular weight products and need
to be taken into account when designing the
safety evaluation programme for a biotechnology-
derived pharmaceutical product (Table 3).The
quality and consistency of the product requires
careful control in terms of product identity,po-
tency and purity because of concerns about mi-
crobiological safety,impurities arising from the
fragment antibody products,gene transfer vec-
tors,tissue-engineered products).Most of these
products are regulated as medicinal products;
however,the regulatory status of others such as
some cell therapies and tissue:organ-based prod-
ucts differs globally and falls within the borderline
between the practice of medicine,medical devices
and medicinal products.
Biotechnology-derived pharmaceuticals may be
derived from a variety of expression systems such
as Escherichia coli,yeast,mammalian,insect or
plant cells,transgenic animals or other organisms.
Table 2
Potential gene therapy products
Plasmid DNA
Non viral vectors as Cationic lipids,polymers,
fusogenic peptide,gene gundelivery system
Adenovirus,adeno-associatedViral vectors as
virus,retrovirus,vaccinia,delivery system
lentivirus,HSV,phage,
pseudotyping
Other novel nucleic DNA:RNA chimeras
acid products
Genetically modi®ed
somatic cells
J.Sims:Toxicology Letters 120(2001)59±66 61
Table 3
What are the safety issues?
Microbiological safety
Adventitious agents:bacterial,mycoplasma,fungal,viral,
TSEs,etc.
Potential for reconstitution of replication competent viral
vectors for gene transfer products
Cells harbour homotropic and xenotropic viruses,e.g.
retroviruses,Epstein±Barr virus,cytomegalovirus
Potential for environmental spread,e.g.vector
dissemination and viral shedding
Immunological
Anti-product antibodies
Host cell proteins or other process impurities
Viral vectors for gene transfer products
Cell impurities in cell therapy products
`Foreign'epitopes on cell:tissue:organ-based products
Development of immune tolerance to DNA vaccines
Pharmacological
Excessive pharmacology or unintentional receptor binding
Distribution to and exposure of non-target tissues
Altered cell phenotype,cell products,function and
localisation for cell therapies
Biodistribution
Distribution and persistence of gene and cell therapies
Genetic transfer to germ cells for gene transfer products
Insertional mutagenesis for gene transfer products
Vector dissemination and viral shedding
Tumourigenicity
Pharmacology or product,e.g.growth factor activity,
immunomodulatory activity
Oncogenic residual DNA in product
Insertional mutagenesis for gene transfer products
Donor-derived malignant or premalignant cells in cell
therapy products
Cell culture process leading to
immortalisation:trnasformation to malignant
phenotype:growth factor independence
Cellular impurities in cell therapy products
General safety concerns
Physical charactersitics,e.g.of the protein,viral vector
Covalently bound ligand molecule,e.g.toxin
Product formulation and excipients
Local tolerance
ogy products span line functions within the phar-
maceutical industry and require a cross-functional
approach in generating appropriate data.There-
fore,communication between functions such as
manufacturing,quality control,preclinical safety
and clinical functions must be optimal to ensure
the overall safety of the product.The toxicologist
must understand the nature of the material under
evaluation and the nature of changes conferred by
process improvements and scale-ups.
The immunogenic nature of heterologous
proteins,vectors,cells,tissues and process con-
taminants must also be considered in the design of
the safety evaluation programme and appropriate
monitoring for anti-product antibodies,particu-
larly neutralising antibodies included in toxicity
studies to aid interpretation of the ®ndings.
For gene transfer products,there are concerns
about the distribution and persistence of vector
sequences,the potential for expression of vector
sequences in non-target cells:tissues and,in partic-
ular,the potential for inadvertent gonadal distri-
bution and germ-line integration.In 1997,the
Food and Drug Administration (FDA) became
aware that preclinical studies from multiple clini-
cal trial applications indicated evidence of vector
DNA in animal gonadal tissues following extrago-
nadal administration.These positive polymerase
chain reaction (PCR) signals were for DNA ex-
tracts from whole gonads subsequent to vector
administration.The observations involved multi-
ple classes of vectors,formulations and routes of
administration.There was no information at the
time on whether these sequences were intracellular
or integrated and,furthermore,no information on
whether the sequences were in gametes or somatic
cells.These ®ndings led to concerns over inadver-
tent germ-line integration.FDA recommenda-
tions regarding biodistribution studies for gene
transfer products were discussed at the Depart-
ment of Health and Human Services Recombi-
nant DNA Advisory Committee,National
Institutes of Health in June 1999 and reported in
the Regulatory Issues section of Human Gene
Therapy (Anonymous,2000).Biodistribution
studies should normally be conducted prior to
clinical trials unless the product falls within the
class where such studies can be postponed.It is
manufacturing process (e.g.host-cell contami-
nants,endotoxin,residual DNA levels and pro-
cess chemicals),and the ®delity of the protein
sequence and post-translational modi®cations
during process improvements and scale-up.
Many of the safety issues raised by biotechnol-
J.Sims:Toxicology Letters 120(2001)59±6662
recommended that biodistribution studies should
use the clinical route of administration,should
include early and late times of killing to evaluate
the kinetics of vector transduction and the persis-
tence of the effect,and should use a direct DNA
PCR assay with a sensitivity level of detection
B100 copies:mg DNA per tissue sampled.
3.Designing relevant toxicity studies to evaluate
safety
Ideally,non-clinical toxicity studies of a poten-
tial therapeutic biotechnology-derived pharma-
ceutical should be performed in species that have
the relevant target receptor or epitope.The selec-
tion of the a test species is usually accomplished
by in vitro comparison of binding af®nity or
functional activity of the product to animal and
human cells,followed by demonstration of the
expected pharmacological activity in the test spe-
cies in vivo.In addition,in order to provide useful
information to the clinician,the design of the
toxicity studies must take account of the dose and
dosing frequency,exposure levels,and the end-
points to be monitored.In some cases,the use of
homologous proteins and:or transgenic animal
models may be considered.
Animal models of disease have also been used
to generate useful information to support clinical
trials and can predict some adverse effects ob-
served in patients but not seen in normal animals.
For example,recombinant human erythropoietin
was associated with hypertension in patients with
chronic renal failure and also in uremic dogs but
not in normal dogs (Dempster,1998).Therefore,
if an animal model of disease is available and used
to pro®le pharmacodynamic activity or ef®cacy
prior to initial clinical trials,it may be useful to
evaluate certain safety endpoints in addition,such
as effects on vital functions,haematology,clinical
pathology and targeted histopathology.However,
while animal models of disease may be useful to
evaluate the potential for exacerbations of disease,
safety pharmacology and short-term toxic effects,
they should not be regarded as a routine model
for use in toxicity testing to support extended
clinical trials or marketing,particular as the
model disease process may cause dif®culty in in-
terpreting any toxicity ®nding observed in such
animals.
Often,a relevant species is not available for
toxicity testing,particularly for humanised mono-
clonal antibodies that frequently have limited spe-
cies cross-reactivity (humans and chimpanzees).
Evaluating the safety of products that are not
biologically active in any species available for
toxicity testing presents a challenge to the toxicol-
ogist and clinician.This is particularly so when
the potential patient population will include males
and females of child-bearing age and treatment
will be administered chronically.Such products
are often biologically active in chimpanzees and
some companies have conducted limited`in-life'
studies in two to three chimpanzees,studies that
mimic Phase I clinical studies and that include
collection of in-life pharmacokinetic,pharmaco-
dynamic and tolerance (e.g.clinical signs,vital
functions,haematology,clinical chemistry) end-
points only.However,other companies have a
policy of not using chimpanzees as this is an
endangered species,and have to consider alterna-
tive approaches to ensure patient safety.These
alternative approaches have involved a combina-
tion of in vitro approaches and clinical studies
starting with doses at least an order of magnitude
below estimated therapeutic clinical doses and
escalating gradually to therapeutic doses.
Other approaches that have been taken are to
evaluate an homologous protein in a rodent
model or to develop a transgenic mouse model
expressing the human receptor:epitope (for a
monoclonal antibody).These two approaches
have been used to conduct chronic toxicity stud-
ies,reproductive toxicity studies and even a car-
cinogenicity study for the homologous protein
approach.The approach of using a homologous
monoclonal antibody to evaluate the reproductive
and chronic toxicity potential was adopted for a
humanised anti-tumour necrosis factor (TNF)a
monoclonal antibody,in¯iximab,which is used
for the treatment of several chronic autoimmune
diseases,such as Crohn's disease and rheumatoid
arthritis.(The homologous anti-TNFa mono-
clonal antibody selectively inhibited the functional
activity of mouse TNFa (Treacy,2000).) There
J.Sims:Toxicology Letters 120(2001)59±66 63
was no effect on foetal development in mice and
no effects on male or female reproductive
function.
A human CD4 transgenic mouse model (murine
CD4 knock-out,human CD4 knock-in) was used
to assess the reproductive and chronic toxicity
and genotoxicity of keliximab,a human-cynomol-
gus monkey chimeric monoclonal antibody with
speci®city for human and chimpanzee CD4
(Bugelski et al.,2000).This model was also used
to evaluate changes in the production cell line and
formulation,and for a variety of special studies to
evaluate immune function and host defence,in-
cluding experimental metastases following chal-
lenge with B16 melanoma cells.Prior to the use of
this transgenic mouse model,extensive work was
conducted to characterise the transgenic mouse
and assess its suitability for safety evaluation
studies.This included demonstrating that human
CD4 is functionally active in mice and that the
animals do not have background pathologies that
could confound the interpretation of any toxicity
®ndings.
For biotechnology-derived pharmaceuticals,it
is recommended to include safety pharmacology
endpoints such as monitoring for vital functions
in the toxicity studies,wherever possible,rather
than conduct separate safety pharmacology stud-
ies.The predictivity of non-clinical studies for
patients may be improved by incorporating devel-
opments derived form the applied and basic sci-
ences to identify and follow effects in
pharmacodynamic and:or toxicity studies and in
vitro,and applying these methods to patients.
However,when applying new developments to
non-clinical safety evaluation,there are concerns
over assay standardisation,reproducibility,the
mechanistic basis for assay outcomes and the
interpretation of the results in a regulatory
environment.
If the product has immunomodulatory proper-
ties,a programme of focused immunotoxicity test-
ing may be required.However,even when all
efforts are made to design relevant non-clinical
safety evaluation programmes,the information
generated may not be predictive of long-term
immunological effects in the patient,particularly
for immunomodulatory products.For example,
one-third of multiple sclerosis patients treated
with a short-term treatment (5-day pulse) of the
humanised anti-CD52 monoclonal antibody
(Campath-1H),which depleted 95% of circulating
lymphocytes,developed antibodies against the
thyroptopin receptor and carbimazole-responsive
autoimmune hyperthyroidism (Coles et al.,1999).
The depleted peripheral lymphocyte pool was re-
constituted with cells that had decreased mitogen-
induced proliferation and interferon gamma
secretion in vitro.The autoimmune hyperthy-
roidism was noted in multiple sclerosis patients
between 9 and 18 months after the 5-day pulse
treatment,but has not been observed in any other
clinical trial in other diseases.So,non-clinical
studies may be able to model the depletion of
circulating lymphocytes and the characteristics of
the reconstituted cells in the recovery phase,but it
is probably unrealistic to expect non-clinical stud-
ies to be able to predict the consequences of
altered characteristics of the reconstituted cells in
a particular clinical population who may be genet-
ically predisposed to autoimmune diseases.Such
®ndings indicate the need for long-term follow-up
of patients to evaluate the potential for long-term
immunological effects.
4.Non-clinical studies prior to ®rst-dose-in-man
studies
The range of non-clinical studies to support the
®rst clinical study include in vitro and in vivo
pharmacodynamic data from an animal model,
possibly an animal model of disease.If the latter
is used,it can be useful to include monitoring for
safety parameters (e.g.vital function and other
clinical signs,haematology,clinical chemistry,
limited pathology) in the study,as toxicity may be
evident only in the disease state Very important is
a review of the literature to obtain a good under-
standing of the physiological and biochemical
processes relating to the target of the product
because this may give some clues as to the ex-
pected effects of the product and suggest appro-
priate endpoints for monitoring during the study.
Assays for pharmacokinetic analysis,pharma-
codynamic monitoring and anti-product antibod-
J.Sims:Toxicology Letters 120(2001)59±6664
ies will need to be developed to aid in the inter-
pretation of non-clinical studies.There is evidence
that allometric scaling of clearance and volume of
distribution is possible for protein therapeutics
(Mordenti et al.,1991) and pharmacokinetic
pro®ling,combined with comparative in vitro
pharmacodynamic data in animal and human
cells:tissues,and in vivo pharmacodynamic data
in an animal model,allowing pharmacokinetic:
pharmacodynamic modelling of estimated clinical
therapeutic doses for the initial clinical study.
Based on this estimated therapeutic clinical dose,
toxicity studies can be designed (single dose,2±4
weeks duration) in one,or possibly two,relevant
animal species.Evaluation in two species may be
required if two relevant species are available and
if little is known about the pharmacodynamic
properties of the product.
Information on the disposition of the product is
useful to understand the mode of clearance.For
monoclonal antibodies,an in vitro tissue cross
reactivity screen is required and also provides
useful information on unexpected tissue binding.
For products that do not contain inorganic
linker molecules or toxin conjugates,genotoxicity
studies are not considered to provide meaningful
information and are not generally required.
5.Non-clinical studies to support full development
programmes
Following the initial clinical study,the pharma-
codynamic:pharmacokinetic model should be re-
evaluated and re®ned,and new estimates made of
likely therapeutic clinical doses based on real or
surrogate endpoints for ef®cacy if possible.The
adequacy of the initial toxicity study should be
re-evaluated based on new estimates of the clinical
dose and exposure.Further toxicity studies,in-
cluding an assessment of chronic toxicity,if neces-
sary based on the proposed clinical indication,
can then be designed,with doses up to at least
tenfold the pharmacologically equivalent clinical
exposures,taking account of both pharmacoki-
netic exposure ratios and differences in pharmaco-
dynamic potency.An assessment of local
tolerance of the clinical formulation may be re-
quired if not available from the toxicity studies.
The most problematical aspects of the safety
evaluation of biotechnology products to support
extended clinical trials and marketing are what to
do if the product is not biologically active in any
species available for toxicity testing,how to assess
toxicity to reproduction for products active only
in primate species,and the assessment of carcino-
genic potential.For the assessment of both toxic-
ity to reproduction and carcinogenic potential,it
is important to identify the questions to be an-
swered and to de®ne what is feasible and useful
for human risk assessment and product labelling.
6.Assessment of reproductive toxicity
For products that are biologically active only in
primate species,the assessment of toxicity to re-
production can be problematical as the standard
reproduction,developmental toxicity studies in
rats and rabbits are considered inappropriate to
provide relevant information on the potential to
produce reproductive,developmental effects in
humans.The extent of the evaluation will depend
on the clinical indication,the clinical populations
to be treated,the disease severity,the availability
of alternative therapies,the availability of a rele-
vant animal model,the pharmacodynamic activity
of the products (level of perceived risk),and the
immunogenicity issues in the animal model.
Again,a variety of approaches has been
adopted including conducting fertility,embryo-
foetal development and peri-postnatal studies in
primates,testing homologous proteins in mice
and:or rats,and the use of a transgenic mouse
model for the human receptor:epitope.
The advantages of using a primate species for
evaluation of toxicity to reproduction are the
similarity to humans in terms of the endocrinol-
ogy of the menstrual cycle and early pregnancy,
placental morphology and physiology,rates of
embryonic developments and the similarity in re-
sponse to known human teratogens such as
thalidomide and retinoids (Hendrickx et al.,
2000).Current teratology protocols in these mod-
els may require slight modi®cations to assess ade-
quately the safety of biotechnology-derived
products,particularly immunomodulatory prod-
J.Sims:Toxicology Letters 120(2001)59±66 65
ucts.However,the disadvantages of using pri-
mates include the low number of animals that can
be used leading to statistical issues,the lack of
historical data,cost,the length of study if postna-
tal evaluations are required and animal welfare
considerations.
Where it is not possible to use primates,ho-
mologous proteins or transgenic mouse models
have been used,but these models probably
provide more useful data when positive signals for
adverse effects on reproduction are evident lead-
ing to label warnings,rather than in the event of
negative ®ndings where there are doubts about
the validity and predictivity of the model em-
ployed.This is particularly the case where the
concern relates to potential immunotoxicity in the
offspring.
7.Assessment of tumourigenic potential
As with the assessment of toxicity to reproduc-
tion,the assessment of carcinogenic potential pre-
sents a challenge to toxicologists.Conventional
approaches using rodent carcinogenicity bioassays
are often not feasible because of the lack of
biological activity in rodents or the immunogenic
nature of the product,and probably not meaning-
ful for products where the desired pharmacologi-
cal activity represents the basis for the concern
about a tumourigenic hazard (e.g.growth factors
or immunosuppressant products).The challenge is
to evaluate tumourigenic risk for patients for
products that are considered to represent a tu-
mourigenic hazard as a result of their biological
activity.
A variety of approaches have been taken,in-
cluding:conducting a conventional rodent
bioassay in one species with the product itself;
conducting a conventional rodent bioassay with a
homologous protein;evaluation of the impact of a
growth factor on tumour growth and metastasis
in a mouse human tumour xenograft model where
the human tumour cells have been shown to
express the receptor for the growth factor;and
evaluation of the impact of an immunosuppres-
sive product on host resistance to neoplasia in a
B16 mouse melanoma model in comparison with
other immunosuppressive agents for which there
is extensive clinical experience (Bugelski et al.,
2000).The usefulness of the alternative ap-
proaches of testing for human risk assessment is
debatable,but probably provides as good infor-
mation as would a conventional carcinogenicity
bioassay,if feasible.Information on risk relative
to other commonly used therapeutics in similar
indications may provide useful information but
there is a need to understand whether relative risk
in a rodent model translates into equivalent rela-
tive risk for patients.
8.Conclusion
Safety evaluation programmes need to be de-
signed to answer speci®c questions rather than
designed merely to comply with regulatory guide-
lines.There are a number of examples of unex-
pected effects that have arisen due to the safety
evaluation programme not taking full account of
the possibility of unintentional receptor binding
and:or cross reactivity with other receptors.In
some cases,this cross-reactivity with another re-
ceptor(s) could have been predicted from knowl-
edge of the nature or biological activity of the
product.In some cases,it would appear that
adverse effects observed in patients would have
been dif®cult to predict from the non-clinical
studies,which indicates the need for long-term
clinical follow-up of patients and tailor-made clin-
ical monitoring.
The challenge posed by the non-clinical safety
evaluation of biotechnology-derived pharmaceuti-
cals is one that needs to be taken up by industry,
regulatory and academic toxicologists working in
partnership to develop new and relevant ap-
proaches to improve human risk assessment for
novel products and to ensure the safety of
patients.
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