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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5981_Библиотеки_им_академика_М_И_Перельмана
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Homologous proteins have been developed and used successfully for mouse, rat, dog, and
nonhuman primate targets (Bussiere et al., 2009). When developing these molecules there is
an expectation by health authorities that they are fully characterized (i.e., pharmacological
mechanism, production process, impurities/contaminants, pharmacokinetics, and immuno-
genicity) and produce pharmacologically relevant effects in the toxicity studies similar to
those expected to be produced by the clinical candidate in humans. The same questions posed
in Table 3.2 need to be addressed for these homologous molecules. In addition, it is expected
that the toxicity studies utilizing homologous proteins will be conducted under Good Labo-
ratory Practices.
Other alternative models that can be considered include transgenic animal expressing the
human target, where the target has been overexpressed or knocked out (to simulate agonist
or antagonist activity), and animal models of disease. In all cases, these models need to be
carefully characterized to understand the pharmacologic mechanisms and relationship to
pharmacologic activity in humans. Health authorities will typically want to review this infor-
mation in a dossier to understand the validity of the model. For animal models of disease,
additional information around background lesions and the ability to distinguish adverse ef-
fects from the background findings will need to be documented. Collection of concurrent con-
trol and baseline data from the disease model may be critical in optimizing study design and
interpretation of data.
In cases where the biopharmaceutical is directed at a foreign target such as a bacterial or
viral antigen, in accordance with the ICH S6(R1), these products could be tested in a short-
term toxicity study in one species to be justified by the sponsor. In most cases, this single
study would be conducted in a rodent. No additional studies, such as developmental and
reproductive toxicity studies, would need to be conducted. Alternatively, the product could
be tested in an animal model of disease being used to evaluate efficacy and could include
appropriate safety assessments.
Study design considerations for repeat-dose studies
In general, the clinical strategy should be taken into consideration when designing the
nonclinical safety strategy to support entry into the clinic. With regard to study timing
needed to support the stages of clinical development, the guidance in ICH M3(R2) is appli-
cable to biopharmaceuticals. In addition, nonclinical study duration and dosing regimen
need to be taken into consideration. With the exception of late-stage oncology products,
the nonclinical study duration should be the same length or longer than the proposed length
of the clinical trial the studies are designed to support ( DeGeorge et al., 1999). In accordance
with ICH S6(R1), repeat-dose chronic studies of 6 months duration are generally appropriate
to support product registration for chronic use, including lifetime administration. Using a
retrospective analysis of completed biopharmaceutical programs consisting of predominantly
recombinant proteins, Clarke et al. showed that little to no additional important safety data
were collected in repeat-dose studies greater than 6 months in duration (Clarke et al., 2008). A
more recent survey across multiple pharmaceutical companies and government agencies
covering 142 monoclonal antibodies (mAbs) showed that in the majority of cases (71%) no
3. Regulatory guidelines and their application in the nonclinical evaluation of biological medicines66
I. Development of biopharmaceuticals
new toxicity was observed in the chronic (6 months) studies (Chien et al., 2023). Conversa-
tions with Regulators around scientifically justified waivers for chronic studies (6 months)
is strongly encouraged and an increasingly utilized approach to both 3Rs considerations and
NHP scarcity (Sewell et al., 2016; Chien et al., 2023).
Studies 3 months in durat ion are generally appropriate to support product registration for
late-stage cancer indications- (see Chapter 11 Biological Therapies for Cancer and ICH 2009).
The dosing interval for nonclinical studies, in most cases, should be the same or more
frequent and the exposure greater in the nonclinical studies as compared to the clinical trial
dosing interval and predicted exposure. In general, animals are dosed once every half-life for
repeat-dose studies (e.g., weekly or twice weekly for a typical monoclonal antibody). Some
health authorities consider recovery to start immediately after the last dose of a biopharma-
ceutical is administered, even if exposure remains high for some time postdose; thus consid-
eration should be given as to when necropsy will occur after the last dose. One strategy is to
perform necropsies on the animals in the first few days after the last dose rega rdless of drug
exposure and to use animals assigned to recovery groups to fully characterize the pharmaco-
kinetic pro file of the drug.
The use of one species is justified when the biopharmaceutical is pha rmacologically active
in only one species. In cases where there are two relevant species (a rodent and a nonrodent)
and short-term to xicology studies (generally <3 months) have shown similar toxicities,
chronic studies (>3 months) with a single species (preferably rodent) may be justified
(ICH, 2010; Prior et al., 2022). Studies in a second species with a homologous product do
not add further value to the toxicity evaluation and are not recommended. This is because,
unlike small-molecule drugs, toxicity associated with biopharmaceuticals is generally due
to exaggerated pharmacology and not intrinsic toxicity. The number and gender of animals
used in the nonclinical program is the same as used for small-molecule pharmaceuticals.
Although here again 3Rs considerations have prompted the proposal of single sex studies
(where a single sex may be the patient population), reduction of animals per dosing group,
and eliminating control groups (Ackley et al., 2023). Careful consideration should be given as
to whether study designs like these are scientifically justified for a the rapeutic area and/or
patient population.
As described above, an observat ion of immunogenicity is not a scientifically valid reason
to eliminate a species for inclusion in the nonclinical safety program. If immunogenicity is not
manageable (e.g., most or all animals lose exposure in all dose groups), or the immunoge-
nicity results in deleterious effects (e.g., anaphylaxis causing severe moribundity or mortality)
at any stage of the nonclinical program, then discontinuing testing in that species can be
considered. For progra ms where the first-in-human (FIH)-enabling studies were conducted
in two species, there are circumstances where only one species may need to be evaluated
in postFIH repeat-dose toxicity studies. If the toxicity findings are similar in the FIH-
enabling studies in both species, or the findings are understood based on the mechanism
of action of the drug, then one species may be considered sufficient for the postFIH
repeat-dose studies. If the rodent is the most relevant species based upon the above criteria,
in accordance with ICH S6(R1), the postFIH repeat-dose studies should be conducted in the
rodent unless there is a specific scientific rationale for using the nonrodent species (Prior et al.,
2023; Ackley et al., 2023).
Study design considerations for repeat-dose studies 67
I. Development of biopharmaceuticals
Safety pharmacology endpoints
In addition to ICH S6(R1), ICH S7B gives supplementary guidance on the conduct of safety
pharmacology studies. Cardiovascular, central nervous system (CNS), and pulmonary func-
tion should be assessed before FIH studies are initiated. If rodents are considered a relevant
species, then standard safety pharmacology studies can be cond ucted in these species or
incorporated into the repeat-dose studies (e.g., adding an additional dose group to collect
CNS data from the functional observational battery). Cardiovascular, respiratory, and CNS
endpoints can be incorporated directly into the repeat-dose toxicity studies in the case of
nonhuman primate (NHP)-only programs, although these endpoints can be challenging to
assess in the NHP. Typically, the following endpoints are collected from NHP studies to sup-
port safety pharmacology assessments: heart rate, electrocardiogram measurements, respira-
tion rate and clinical observations (to assess for neurological endpoints). Additional studies or
endpoints that are sometimes warranted, depending on a cause for concern, include use of
telemetry or jackets for collecting robust cardiovascular data or more detailed neurological
evaluations. Stand-alone safety pharmacology studies are not required unless a concern
has been identified in previous studies or there is a theoretical cause for concern based on
the target biology or mechanism of action of the biopharmaceutical. In addition, for proteins,
including monoclonal antibodies, in vitro hERG testing is generally not warranted as their
physical characteristics and specificity prevent interaction with the central cavity or binding
to the outer portion of the hERG channel, respectively (Vargas et al., 2008).
According to the guidelines for immunotoxicology evaluation, immunotoxicity parameters
do not need to be added to the repeat-dose toxicity studies for biopharmaceuticals unless
there is a specific cause for concern (ICH, 2008; FDA, 2020).
Dose selection
The rationale for dose selection for the repeat-dose studies should take into account the
exposureeresponse (pharmacologic and/or toxicologic response) relationships. Pharma-
cology and pharmacokinetics/pharmacodynamics (PK/PD) information can be used to assist
in dose selection. Dose selection for in vivo studies should be based on exposure rather than
dose (mg/kg) whenever possible, because the exposure and/or pharmacologic response at a
specific dose in one species may not directly translate to a similar exposure/response at the
same dose in another nonclinical species or humans.
High-dose selection is assisted by identification of (1) a dose (exposure) which provides the
maximum intended pharmacological effect and (2) a dose which provides an approximate 10-
fold exposure multiple over the maximum exposure to be achieved at the highest dose in the
clinic. It is important to note that this exposure multiple is not based on the minimum effica-
cious dose or the clinical starting dose. In accordance with ICH S6(R1) the higher of these two
doses (exposures) should be chosen for the high-dose group unless there is justification for
using a lower dose (e.g., a maximum tolerated dose has been identified in previous studies).
In practical terms, the clinical program would be limited by exposure obtained from the
repeat-dose toxicity studies depending on the type of toxicity (e.g., monitorable, reversible,
or species specific) observed. Because of the uncertainty around clinical dose escalation to
higher exposure levels as a program progresses, it is not uncommon to increase the high
3. Regulatory guidelines and their application in the nonclinical evaluation of biological medicines68
I. Development of biopharmaceuticals
dose in the repeat-dose toxicity studies if a maximum tolerated dose has not been reached to
achieve the highest possible NOAEL. Doing this achieves two objectives: first, this allows the
greatest flexibility for the clinical program as dose escalation proceeds, and second, it gives
health authorities greater confidence that the potential toxicity of the biopharmaceutical
has been adequately assessed. One misconception is that the amount of protein administered
to animals must be limited due to potential toxicity from “protein overload” and that the high
dose must be capped at some arbitrary limit such as 100 or 300 mg/kg. There is no scientific
data to support that high doses of protein cannot be administered to animals for long periods
of time. Similarly, the concept of a maximum feasible dose must be approached with caution
and in many cases is not necessary if a maximal pharmacologic effect has been achieved.
There are several ways to increase exposure to a biopharmaceutical if necessary, including
more intensive dosing regimens (i.e., giving twice weekly instead of once weekly doses),
administering more than one dose in a short period of time (i.e., giving two IV bolus doses
1 h apart or IV infusion), or increasing the number of injection sites in the case of subcutane-
ously administered drugs. The expectation from health authorities is that the high dose will
be scientifically justified and supported with exposure data. In some cases, it is difficult to
obtain robust exposure data and, in these cases, PK/PD modeling can be used to justify
dose selection. The low dose is generally selected to be similar in exposure to a previously
identified efficacious dose and the mid-dose is selected to further characterize the range of
toxicity between the high and low doses (usually a geometric mean approach is used).
Adjustments to dose selection should be made based upon known differences in target
binding affinity and/or pharmacological activity between different nonclinical species and
humans. For example, a large relative difference in binding affinity (i.e., a 10-fold difference
or greater) or in vitro/in vivo potency (e.g., human >> nonclinical species) would suggest
that testing higher doses in the toxicity studies would be appropriate.
Route of administration
In general, one route of administration is sufficient and should be selected based on the
proposed clinical route of administration that will be used for the commercialized drug. If
the final clinical route has not yet been determined, an educated guess as to the commercial
clinical route is appropriate (i.e., many biopharmaceuticals for chronic diseases are adminis-
tered by the subcutaneous route; many biopharmaceuticals used in late-stage cancer are
administered by the intravenous route). Once the clinical route of administration has been
confirmed, all subsequent toxicity studies should incorporate that route of administration.
A common approach for FIH-enabling repeat-dose toxicity studies if the final clinical route
has not been established, is to add an additional dose group to the study. The primary
dose groups should receive the biopharmaceutical by the route most likely to be the final
commercial route of administration. The additional dose group using an altern ative route
of administration is typically a high-dose group (i.e., the study will have two high-dose
groups). A workshop between Industry and the US FDA reported that FDA rarely saw dif-
ferences in systemic toxicity based on route of administration (Ackley et al., 2023). Thus,
designs where different dosing groups have different routes of administration is generally
acceptable (Ackley et al., 2023). It is possible to use a singl e control group with individual
animals receiving the vehicle by all routes being tested (i.e., administered both intravenous
Study design considerations for repeat-dose studies 69
I. Development of biopharmaceuticals
(IV) and subcutaneous doses). Further, a toxicology program that is primarily IV-based may
be able to support subcutaneous dosing in the clinic via a nonterminal pharmacokinetics sub-
cutaneous “bridging” study in the relevant toxicology species (with doses at parity with the
clinic) and a rabbit and/or rodent subcutaneous study for local tolerability assessment (Ack-
ley et al., 2023). However, some divisions may request a bridging toxicology study with the
new route; alignment with Health Authorities prio r to study conduct is recommended.
When feasible, the route of administration and formulation matching the planned com-
mercial product should be used in the post-FIH-toxicity studies, and any developmental
and reproductive toxicity (DART) and chronic studies. If the intravenous route is being pro-
posed, and particularly if novel excipients have been incorporated into the formulation,
in vitro blood compatibility testing in all relevant nonclinical species and humans can be
considered prior to the first in vivo study using the formulation.
Similarly, consider nonclinical comparability studies (e.g., pharmacokinetic and/or toxicity
studies) if a manufacturing change occurs after toxicity studies have already been conducted
and bioanalytical comparability has not been demonstrated; the need will also depend upon
the stage of the development program and the extent of the manufacturing change. A full dis-
cussion of comparability is beyond the scope of this chapter. In brief, comparability refers to
an evaluation undertaken to compare a biopharmaceutical product before and after a
manufacturing change. Comparability testing is covered by ICH Q5E and various country-
specific guidelines (ICH, 2004). It is worth noting that “the demonstration of comparability
does not necessarily mean that the quality attributes of the prechange and postchange prod-
ucts are identical, but that they are highly similar and that the existing knowledge is suffi-
ciently predictive to ensure that any differences in quality attributes have no adverse
impact on safety and efficacy of the drug product” (ICH, 2004). In vivo nonclinical studies
may or may not be needed. Conducting nonclinical comparability studies may be warranted
to assess PK/PD in an appropriate animal model or to assess novel excipients, new or
increased levels of impurities, or altered potency of the product. Nonclinical studies, if deemed
necessary, should be discussed and agreed upon in advance with health authorities.
Recovery periods for repeat-dose studies
Recovery from pharmacological and toxicological effects with potential clinical impact
should be understood. The purpose of a recovery period is to understand reversibility of
these effects, not to assess delayed toxicity or potential immunogenicity. The demonstration
of complete recovery may not be needed in all cases (e.g., the recovery process of a micro-
scopic lesion in a specific tissue is understood). In accordance with regulatory guidelines,
reversibility of clinically relevant effects should usually be demonstrated in at least one
repeat-dose toxicity study and evaluated within at least one dose level. Recovery groups
may not be warranted on every study when no toxicity is suspected or observed, there are
no significant known or theoretical concerns with the target, and/or therapy is for an unmet
medical need where the risk/benefit may warrant a more limited nonclinical program. In
other cases, recovery groups may be included on more than one study and at more than
one dose level. A decision on whether to include recovery groups, and on which study or
studies, should be determined based on program needs and existing data, on a case-by-
case basis. The ICH M3, S6, and S9 guidelines do allow for scientifically justified reduction
or omission of recovery groups (ICH, 2010; ICH, 2011; ICH, 2018). With the increased concern
3. Regulatory guidelines and their application in the nonclinical evaluation of biological medicines70
I. Development of biopharmaceuticals
around the use of animals, careful consideration for the need for recovery groups is becoming
an accepted, and perhaps expected, practice (Ackley et al., 2023 ; Prior et al., 2023). If a recov-
ery is included, its duration is typically determined by the half-life of the biopharmaceutical
and the expected duration of pharmacodynamic effects. Drugs with longer half-lives or those
with longer lasting pharmacodynamic effects may require longer recovery periods. If the re-
covery period is based on the half-life of the drug, a duration of approximately five half-lives
is generall y applied.
Immunogenicity
It is not uncommon for the nonclinical species to generate an immune response to a bio-
pharmaceutical because in most cases the drug is of human origin or has been humanized
and appears as foreign. The most common immune response to protein-based molecules is
the development of antibodies. The formation of antibodies against biologic drugs or anti-
drug antibodies (ADAs) is assessed using specialized immunogenicity assays in many
in vivo toxicity studies including routine toxicity, DART, and carcinogenicity studies. For a
comprehensive overview of immunogenicity assessment, including historical data from mar-
keted products and case studies, see Ponce et al. (Ponce et al., 2009).
Immunogenicity assessments are used to aid in the interpretation of nonclinical study re-
sults and may also be instrumental in the design of subsequent studies. The development of
ADAs can have a range of effects, the most common being decreased exposure to the admin-
istered biologic drug and associated PD effects. Other effects include increased exposure and
prolonged PD. In some cases no secondary effects are observed despite ADA detection in a
study. Conversely, in some instances, ADA formation can induce off-target toxicity resulting
from immune complex (ADAs complexed with the biopharmaceutical) deposition (e.g., im-
mune complex deposition in kidney). Measurements of ADAs can aid in inter pretation of
changes in exposure and are important to consider when assessing the toxicity findings. It
is critically important to note that the development of ADAs and the incidence or other char-
acteristics (e.g., affinity, isotype, etc.) of the ADAs in the nonclinical species is not predictive
for, and thus not relevant to, the development or incidence of ADAs in humans. Neverthe-
less, it is valuable to assess immunogenicity and use the collected data to aid in the interpre-
tation of both noncl inical and clinical study results (exposure, efficacy, and/or toxicity).
In accordance with ICH S6(R1), immunogenicity (measureme nt of ADAs) “should be eval-
uated when there is 1) evidence of altered pharmacodynamic activity; 2) unexpected changes
in expos ure in the absence of a pharmacodynamic marker; or 3) evidence of immune-
mediated reactions (immune complex disease, vasculitis, anaphylaxis, etc.).” Based on the
above criteria, it can be difficult to predict in advance of study completion whether immuno-
genicity assessments will be necessary. Therefore, it is common practice to collect samples for
ADA measurement during the study and analyze the samples if warranted to aid in study
interpretation. To better characterize the ADA response it may be necessary to collect samples
during the recovery period; this is particularly useful if high levels of drug cause interference
with detection of ADAs in the assay format being utilized. However, ICH S6(R1) makes it
clear that addition of a recovery group or extending the duration of the recovery period is
not required solely to augment immunogenicity assessments.
Immunogenicity 71
I. Development of biopharmaceuticals
The most common assay formats for detecting ADAs measure binding antibodies. Data
from these assays in conjunction with pharmacokinetic data and any measured PD effect
are generally sufficient to interpret study results. In some cases, a pharmacodynamic
endpoint may not be measurable in toxicity studies for a particular drug candidat e. It may
be unclear if any detected ADAs neutralized the PD activity of the biopharmaceutical if
PD endpoints are not measurable, no toxicity is seen on study, and the pharmacokinetic assay
cannot measure differences in PD effect. In these circumstances, there may be a regulatory
expectation to develop an assay to determine if neutralizing ADAs were formed and contrib-
uted to the lack of observed toxicity. Any ADAs that decrease or fully ablate the PD activity
of a biopharmaceutical are defined as neutralizing antibodies. These should not be confused
with clearing ADAs, which decrease expos ure by increasing clearance of a biopharmaceutical;
clearing ADAs may also be neutralizing. Since it is generally not necessary to characterize
whether a decrease in any PD activity is due to clearing or neutralizing antibodies, a pharma-
cokinetic target binding assay is usually sufficient to characterize the ADA response and any
correlations to reduced exposure or toxicity. If the potential for neutralizing ADAs needs to
be assessed there are several assay formats available, including pharmacokinetic assays that
can also measure a disruption of pharmacologic activity, ex vivo bioactivity assays or other
specialized formats to specifically detect neutralizing antibodies.
To further aid in study interpretation there are practical measures for sample collection
and analysis that can be employed and are also helpful in justifying results to health author-
ities. When feasible, it is recommended that pharmacokinetic and ADA samples be collected
from the same animal since the interpretation of any changes in exposure is one of the key
uses of ADA data. To help minimize drug interference in the assays, ADA samples should
be collected before each dose when drug levels are lowest (trough level). It is not uncommon
to detect preexisting antibodies in nonclinical species, particularly NHPs, and therefore it is
recommended to collect samples prior to the first administration (prestudy) of the biophar-
maceutical. These samples can help determine if potential mis-dosing occurred when
ADAs are detected in control animals or if the animals have preexisting levels of ADAs.
Generally, it takes approximately 14 days for ADAs to develop after dosing commences
and sample collection should begin around this time period to best understand the onset
of the ADA response. Samples are typically collected every 15e30 days, although for chronic
studies the interval between sampling may be lengthened (e.g., every 45e60 days). Samples
are also generally collected after administration of the last dose, at each necropsy and at the
time of any unscheduled euthanasia. Samples are also usually taken from developmental and
reproductive studies including embryo-fetal development and pre/postnatal studies. For
these studies the assays will need to be validated for use with special matrices including preg-
nant animal and fetal serum. ADA assessments in milk are generally not recommended.
There may be cases when immunogenicity causes a loss of drug exposure in most or all of
the animals in a dose group or on an entire study due to a high incidence of clearing ADAs.
This is most commonly observed in the low-dose group where drug exposure is already low
and the effect of clearing ADAs is most pronounced. Inclusion of similar dose levels in sub-
sequent studies may not be practical in these cases. However, as discussed previously, it is
not acceptable to health authorities that a relevant nonclinical species be eliminated because
ADAs develop in this species. There are several approaches that can be used to manage loss
of exposure due to clearing ADAs in rodents and nonrodents (Ponce et al., 2009). For most
3. Regulatory guidelines and their application in the nonclinical evaluation of biological medicines72
I. Development of biopharmaceuticals
biopharmaceuticals tested in toxicology studies, exposure to a drug is impacted differently at
different dose levels and typically a few animals in one or more dose groups maintain reason-
able exposure. In this instance, additional animals can be added to each dose group to in-
crease the numbers of animals with adequate exposure at the end of the study. Increasing
the absolute dose or using a loading dose approach where higher levels of the biopharmaceu-
tical are given initially, are other ways to potentially manage imm unogenicity. It is hypoth-
esized that these methods either overwhelm the antibody clearance mechanism by saturating
the FcRn receptor or may induce a state of tolerance and decrease ADA production. In either
case, higher levels of exposure are maintained in more animals on the study.
One of the more severe consequences of ADA development is toxicity due to various types of
hypersensitivity reactions. These reactions can be acute or delayed and include anaphylaxis/
anaphylactoid reactions and the development of immune complexes that can deposit in tissues
and cause damage (Sethu et al., 2012). Hypersensitivity reactions can occur after multiple doses
of a biopharmaceutical or if preexisting antibodies exist can be observed after the first dose.
Acute hypersensitivity (i.e., occurring within minutes of drug administration) reactions may
arise after the initial dose of a biopharmaceutical without the presence of pre-existing antibodies
and may be due to the pharmacologic activity of the drug or excipients and/or impurities in the
drug product. It is well documented, for example, that dogs can have acute hypersensitivity re-
actions to some excipients including serum albumins and polysorbates (Cohn et al., 2007). When
ADAs bind to a biopharmaceutical, immune complexes can be formed that under the right stoi-
chiometric conditions can deposit into tissues, activate complement and cause mild to severe
inflammation, and lead to tissue damage. This type of hypersensitivity is called immune com-
plex disease (ICD) and its sequelae can range from mild histopathological changes in one to
several tissues to severe toxicity including moribundity or death. Hypersensitivity reactions
do not have to be dose dependent and in many cases occur with greater incidence/severity
in low- and mid-dose versus high-dose groups where, for example, the stoichiometry of the
ADAs and biopharmaceutical favor complex formation. Acute, type I hypersensitivity reactions
may be managed with antihistamines, but this must always be weighed against the ability to
interpret results from the toxicity study. In some cases, acute hypersensitivity will wane during
the course of the study with appropriate management.
When an acute hypersensitivity reaction or ICD is believed to be the underlying cause of
an observed toxicity it is the expectation of hea lth authorities that the appropriate weight of
evidence data are presented to support this interpretation. Hypersensitivity can be confirmed
by assessing one or more endpoints. Clinical signs, clinical pathology parameters (coagula-
tion, renal, and inflammatory), and histopathology to look for immune complexes are
standard parameters that are typically measured on most general toxicity studies. In addi-
tion, several other specialized endpoints/assays can be added once immunogenicity is a
suspected cause of an observed toxicity. These include immunohistochemical analysis of le-
sions for complement and immunoglobulin in association with drug in immune complexes.
In this case, species-specific reagents are used to qualitatively assess complement and ADAs
and human reagents are used to stain for drug detection. Other methods include electron mi-
croscopy of the lesion to further characterize putative immune complexes, and m easurement
of cytokine, complement and complement split product serum levels. A combination of these
endpoints in addition to ADA titers and exposure data can help to build a convincing
scientific argument for ADA-mediated toxicity.
Immunogenicity 73
I. Development of biopharmaceuticals
Reproductive and developmental toxicity
The need to evaluate potential developmental and reproductive toxicity (DART) depends
on the therapeutic indication, intended patient population, and predicted biological activity
of a new drug and should be conducted in accordance with the principles of ICH S5(R3)
(ICH, 2020). With the third revision of ICH S5, there is been an emphasis on expanding
the scope of the guidance to include vaccines and biologics. As mentioned throughout this
chapter, an increased emphasis on the 3Rs has been embraced with the inclusion of study de-
signs and overall DART strategies that allow for: combining typical study types, clinical
stage-specific conduct of studies, and alternative assays (where scientifically justified).
Another important feature of the revised guidance is consideration for severely debili-
tating/life-threatening (SDLT) or late life onset (LLO) diseases. ICHS5 (R3) cautiously
encourages discussion with Health Authorities around the use of weight of evidence and/
or alternative assays to support a DART assessment in these types of diseases.
ICHS5 (R3) suggests that investigators shoul d evaluate effects on reproduction and devel-
opment as part of a holistic view of the entire toxicology program. Investigators should be
mindful of information around patient population (e.g., SDLT and LLO disease, pediatric,
primarily women of child-bearing potential (WoCBP), duration of treatment, route of admin -
istration, toxicokinetics, and hazards identified in repeat-dose toxicology studies. For
biologics, where adverse effects are often the product of excessive on-target pharmacology,
a deep understanding of target biology, genetic disease data (human and transgenic animals),
and clinical data from drugs with similar pharmacology or targeting similar pathways is key
to DART risk assessment. All of the above are factors in determining timing (e.g., earlier, if
signals of concern are identified) and extent (e.g., would alternative assays or weight of ev-
idence approaches be appropriate versus standard studies) of DART studies and study
design. The recommendation for stage-specific timing of studies is conducive to a 3Rs
approach as larger DART studies can potentially be deferred until after hurdles such as a suc-
cessful Phase 2 are completed while still allowing enrollment of a limited number of WoCBP.
Species selection considerations
The size, species specificity, and potential immunogenicity of biopharmaceuticals may
require different approaches to species selection, study types, and study designs. Similar to
other toxicity studies, DART studies should be conducted only in pharmacologically rele vant
species. The type of molecule and the expected placental transfer should also be considered.
Large molecular weight therapeutic proteins (20,000e30,000 Da) exhibit minimal if any trans-
fer across the placenta by simple diffusion (Martin et al., 2009; Skaggs et al., 2019), whereas
for monoclonal antibodies, which can have molecular weights as high or higher than
150 kDa, there exists a specific transport mechanism (FcRn), which determines fetal exposure
but varies across species. In human and nonhuman primates, very little maternal IgG crosses
the placenta during the first trimester; this increases early in the second trimester through
term, with most of the IgG being transferred during the third trimester (Simister, 2003).
Following birth, maternal IgG is only excreted in the milk initially (i.e., in the colostrum)
and not later during the lactation and nursing phases. Therefore, very little fetal exposure
is expected to occur during organogenesis and postnatally (Pent
suk and and van der Laan,
3. Regulatory guidelines and their application in the nonclinical evaluation of biological medicines74
I. Development of biopharmaceuticals
2009; Fujimoto et al., 1983; Skaggs et al., 2019). In contrast to human and nonhuman primates,
transfer of IgG in rodents occurs early in pregnan cy via the yolk sac and then again postna-
tally through breast milk, which can be absorbed by the fetal gut using FcRn pathway (Mar-
tin et al., 2009; Pent
suk and and van der Laan, 2009; Skaggs et al., 2019). Placental transfer in
rabbits is similar to that in rodents and occurs within initial gestation days (GD) via the yolk
sac (until about GD8) and, as in human and nonhuman primates, it increases during the latter
part of pregnancy (Simister, 2003; Martin et al., 2009; Skaggs et al., 2019). In mini-pigs, there is
no transfer of IgG during pregnancy and therefore the mini-pig is unlikely to be a useful spe-
cies for reproductive testing for monoclonal antibodies (Bode et al., 2010; Skaggs et al., 2019).
Alternative approaches to reproductive/developmental toxicity evaluation should be
considered for proteins and monoclonal antibodies due to their differences in placental trans-
fer. Although only minimal exposure to the fetus is expected with proteins, as long as the an-
imal species is pharmacologically relevant DART studies can provide useful data with
respect to indirect fetal effects resulting from maternal toxicity and/or effects on placental
function (development and maintenance or pregnancy). In addition to indirect effects, mono-
clonal antibodies can also produce direct fetal effects because of the FcRn transport pathway.
Although there are differences in placental transfer among different species, it is recommen-
ded by ICH S6(R1) that rodent species and rabbits, if pharmacologically relevant should be
considered prior to using nonhuman primates. The advantage of rodent/rabbit reproductive
toxicity studies is that larger numbers of animals/fetuses can be evaluated, gestation periods
are relatively short compared to higher species, and, if necessary, all periods of reproductive
performance can be assessed (Martin et al., 2009; Skaggs et al., 2019). However, since IgG
crosses the yolk sac in rodents, the relevance of an adverse finding may make hazard assess-
ment difficult given that, as discussed above, monoclonal antibodies do not cross the placenta
during organogenesis in humans. Therefore, additional mechanistic studies may be required
to determine relevance of an adverse finding to humans.
The species specificity and potential immunogenicity of a number of biopharmaceuticals
have led to an increased use of nonhuman primates, in particular, cynomolgus macaques.
Although nonhuman primates share a number of similarities such as reproductive physi-
ology, placental transfer of monoclonal antibodies, and similar response to human teratogens
with humans there are disadvantages associated with primates, such as expense and genetic
diversity of the animals, low fertility rates, high rate of fetal wastage, and limited historical
background data, group size, and offspring (Henck et al., 1996). Therefore, study design
and types of studies need to be carefully considered.
ICH S5(R3) contains useful information around the selection of species for DART studies
(ICH, 2020). In Table 1 of the R3 Annex 1, a comparison of the principal advantages and dis-
advantages of various species for DART evaluation is presented.
When no pharmacologically relevant animal model is available for testing the clinical candi-
date, alternative models such as transgenic mice that express the human target or homologous
protein in a species expressing an ortholog of the human target should be considered. Howev-
er, these alternative models need to be properly characterized (see above in Species Selection
Considerations). When the nonhuman primate is the only pharmacologically relevant species,
it is preferred to use them with the clinical candidate versus using an alternative model
(including homologous proteins). Alternate models can be applied when scientifically justified
by the sponsor particularly given the NHP supply and 3Rs considerations. Consultation with
the applicable health authorities early in planning is recommended.
Species selection considerations 75
I. Development of biopharmaceuticals
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