Regulatory Toxicology and Pharmacology 73 (2015) 595e606

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Regulatory Toxicology and Pharmacology

journal homepage: www.elsevier.com/locate/yrtph

Proof of concept for a banding scheme to support risk assessments

related to multi-product biologics manufacturing
Jeffrey W. Card a, *, Hana Fikree a, Lois A. Haighton a, James Blackwell b, Brian Felice c,
Teresa L. Wright d
a
Intertek Scientific & Regulatory Consultancy, Mississauga, ON, Canada
b
The Quantic Group, Livingston, NJ, USA
c
Shire, Lexington, MA, USA
d
Dimension Therapeutics, Inc., Cambridge, MA, USA

a r t i c l e i n f o a b s t r a c t

Article history: A banding scheme theory has been proposed to assess the potency/toxicity of biologics and assist with
Received 26 June 2015 decisions regarding the introduction of new biologic products into existing manufacturing facilities. The
Received in revised form current work was conducted to provide a practical example of how this scheme could be applied. Infor-
31 August 2015
mation was identified for representatives from the following four proposed bands: Band A (lethal toxins);
Accepted 2 September 2015
Band B (toxins and apoptosis signals); Band C (cytokines and growth factors); and Band D (antibodies,
Available online 8 September 2015
antibody fragments, scaffold molecules, and insulins). The potency/toxicity of the representative sub-
stances was confirmed as follows: Band A, low nanogram quantities exert lethal effects; Band B, repeated
Keywords:
Biologics
administration of microgram quantities is tolerated in humans; Band C, endogenous substances and re-
Manufacturing combinant versions administered to patients in low (interferons), intermediate (growth factors), and high
Cross-contamination (interleukins) microgram doses, often on a chronic basis; and Band D, endogenous substances present or
Cleaning validation produced in the body in milligram quantities per day (insulin, collagen) or protein therapeutics adminis-
Impurities tered in milligram quantities per dose (mAbs). This work confirms that substances in Bands A, B, C, and D
Risk assessment represent very high, high, medium, and low concern with regard to risk of cross-contamination in
manufacturing facilities, thus supporting the proposed banding scheme.
© 2015 Elsevier Inc. All rights reserved.

1. Introduction pharmacological effects, since a desired pharmacological effect in

It is widely recognized that manufacturing multiple pharma-
ceutical products in a single facility is associated with risks related
to cross-contamination. Carryover of a given drug substance [or
active pharmaceutical ingredient (API)] into another drug product
represents a potential risk for cross-contamination that can be of
concern to a patient who is subsequently administered the
contaminated drug product. This risk can be due to known toxi-
cological effects of the contaminating substance or to its recognized
Abbreviations: ADE, acceptable daily exposure; API, active pharmaceutical
ingredient; FDA, Food and Drug Administration; G-CSF, granulocyte colony stimu-
lating factor; GM-CSF, granulocyte-macrophage colony stimulating factor; IFN,
interferon; IL, interleukin; IM, intramuscular; IU, international unit; IV, intravenous;
LD50, median lethal dose; mAbs, monoclonal antibodies; MTD, maximum tolerated
dose; PDE, permitted daily exposure; SC, subcutaneous; TNF, tumor necrosis factor.
* Corresponding author. 2233 Argentia Road, Suite 201, Mississauga, ON, L5N 2X7,
Canada.
E-mail address: jeffrey.card@intertek.com (J.W. Card).
an intended recipient may be undesirable or dangerous in an un-
intended recipient.
Several guidance documents and thought papers have been
published on the topics of cross-contamination of APIs and rec-
ommended methods to establish safe exposure limits that can be
used to assist with cleaning validation efforts (Dolan et al., 2005;
ISPE, 2010; Walsh, 2011a,b,c; Bercu et al., 2013; Sargent et al.,
2013). The derived exposure limit for a given substance is typi-
cally referred to as a permitted daily exposure (PDE) value or an
acceptable daily exposure (ADE) value that is applicable for lifetime
daily exposure in all patient populations and by all routes of
exposure. From a regulatory perspective, it is noted that the Euro-
pean Medicines Agency (EMA) has issued a guideline on estab-
lishing health based exposure limits (PDE values) related to
manufacturing different medicinal products in shared facilities
which came into effect in June 2015 (EMA, 2014).
The existing literature and guidance documents focus on small

http://dx.doi.org/10.1016/j.yrtph.2015.09.003
0273-2300/© 2015 Elsevier Inc. All rights reserved.
596 J.W. Card et al. / Regulatory Toxicology and Pharmacology 73 (2015) 595e606
molecule APIs, and very little attention appears to have been given
to large molecule biologics. One notable exception is the article by
Mott et al. (2013) which describes several methods that can be used
to evaluate the extent of biopharmaceutical product inactivation as
it relates to cleaning validation and potential carryover on
manufacturing equipment. In this regard, the EMA guideline notes
that most cleaning processes employ extremes of pH and/or heat
and that therapeutic macromolecules and peptides are “known to
degrade and denature when exposed to pH extremes and/or heat,
and may become pharmacologically inactive” (EMA, 2014). As such,
the guideline concludes that it may not be necessary to use a PDE
approach to determine health based exposure limits for biologics.
This language is somewhat ambiguous, and concerns may remain in
cases where cleaning efforts are insufficient. In addition, knowledge
of potential exposure limits for biologics or of the potency/toxicity of
one type of biologic relative to that of another would assist in early
decisions regarding the suitability of introducing a new biologic into
a facility in which other biologics are already manufactured.
A banding scheme for assessing the potency/toxicity of biologics
was proposed in a recent article by Carver (2013). This concept
would involve categorizing biologics into different “bands” based
on their potency and toxicological potential. This banding scheme is
intended to help identify more potent/toxic biologics from those
that are more innocuous, and is anticipated to assist with decisions
regarding bringing new products into an existing facility or
whether to outsource manufacturing efforts to a dedicated facility
and therefore eliminate the potential for cross-contamination. As
outlined by Carver (2013), the proposed banding scheme and ex-
amples of substances within each band are as follows (potency/
toxicity decreases from Band A to Band D):

Band A: Lethal toxins (e.g., botulinum toxin);

Band B: Toxins and apoptosis signals [e.g., tumor necrosis factor-
a (TNF-a)];

Band C: Cytokines, growth factors, etc. (e.g., interferons, in-
terleukins, growth factors); and

Band D: Antibodies, antibody fragments, scaffold molecules, and
insulins [e.g., insulins, monoclonal antibodies (mAbs), antibody
fragments, scaffold proteins].
While the proposed banding scheme appears to make sense
intuitively, quantitative or qualitative information are not provided
to support the proposed bands or the example substances that are
indicated within each band. The purpose of the work summarized
here was to determine whether there is quantitative and/or quali-
tative information to support the proposed bands from a scientific
perspective. Such information would be useful to consider when
conducting risk assessments of biologics pertaining to cleaning
validation and cross-contamination.
2. Methods
To determine whether the proposed banding scheme could be
affirmed with scientific data, representative substances were
selected from each of the four proposed bands and searches for
information were performed. A summary of the representative
substances and the resources that were searched for potency/
toxicity information is presented in Table 1.
3. Results
3.1. Information on representative substances from Band A
3.1.1. Botulinum toxin
Botulism is a rare disease caused by the preformed toxins of
Clostridium botulinum. There are four naturally occurring botulism
syndromes, namely foodborne botulism, wound botulism, infant
botulism (caused by intestinal colonization and toxin production),
and adult intestinal toxemia (caused by intestinal colonization and
toxin production). All forms of botulism have the same clinical
syndrome comprised of cranial nerve paralysis followed by
descending symmetrical flaccid paralysis of voluntary muscles; in
the worst cases this can progress to respiratory compromise and
death (Sobel, 2005).
There are seven immunologically distinct botulinum toxins,
designated by the letters A to G, which are responsible for causing
botulism. Botulinum toxins A, B, E, and F cause human forms of
botulism while toxins C and D cause botulism in animals; botuli-
num toxin G has not been reported to cause botulism in humans
(Kotsonis and Burdock, 2008). All of the botulinum toxins are
neurotoxic. Their mechanism of action involves blockade of
acetylcholine release from presynaptic motor-neuron terminals
thereby preventing stimulation of motor fibers. Recovery from
botulism often takes weeks to months, and respiratory distress and
paralysis may persist for six to eight months (Kotsonis and Burdock,
2008).
Botulinum toxins are some of the most potent toxins known.
Data summarized by Gill (1982) demonstrate the potency of bot-
ulinum toxins in mice, with reported median lethal dose (LD50)
values ranging from 0.4 ng/kg body weight for botulinum toxin D
(intraperitoneal) to 2.5 ng/kg body weight for botulinum toxin F
[intravenous (IV)]. As noted by Sobel (2005), commonly cited
estimated lethal doses for botulinum toxin A for a 70 kg human are
90e150 ng when introduced intravenously (approximately
1.3e2.1 ng/kg body weight), 800e900 ng when introduced via
inhalation, and 70 mg (70,000 ng) when introduced orally. Others
have estimated that the lethal dose of botulinum toxin in humans
may be as low as 1 ng (Kotsonis and Burdock, 2008).
3.1.2. Diphtheria toxin
Diphtheria is an acute, communicable disease caused by the
exotoxin-producing bacteria Corynebacterium diphtheria (Hadfield
et al., 2000). It is normally transmitted by sneezing, coughing, or
direct contact. The bacteria usually localizes in the upper respira-
tory tract where it induces the formation of an inflammatory
pseudomembrane. From here, released diphtheria toxin is absor-
bed into the systemic circulation and causes severe systemic pa-
thology. Diphtheria toxin does not have a specific target organ
although peripheral nerves and myocardium appear to be most
often affected (Hadfield et al., 2000). Diphtheria toxin enters cells
via binding to heparin binding epidermal growth factor precursor
that is present on cell membranes. Once inside the cell, diphtheria
toxin acts to inhibit protein synthesis and ultimately leads to pro-
grammed cell death (Shapira and Benhar, 2010).
Diphtheria toxin is extremely potent. It has been demonstrated
that introduction of one molecule of diphtheria toxin fragment A
(the catalytic domain) into the cytosol of a cell is sufficient to induce
cell death (Yamaizumi et al., 1978). Barksdale (1970) reported that
diphtheria toxin is lethal to humans and animals at a dose level of
130 ng/kg body weight; further details were not reported. A
comparably low IV LD50 value of 10 ng/kg body weight (0.01 mg/kg
body weight) for diphtheria toxin in mice is presented in a publicly-
available database maintained by the University of Florida (avail-
able at: http://www.ehs.ufl.edu/programs/bio/toxins/toxin-table/;
last accessed April 21, 2015).
3.2. Information on representative substances from Band B
3.2.1. Crotoxin
Phospholipase A2 catalyzes the hydrolysis of phospholipids to
 J.W. Card et al. / Regulatory Toxicology and Pharmacology 73 (2015) 595e606
Table 1
Representative substances and sources of information.
Banda Representative substancea
A Botulinum toxin (lethal toxin)
Diphtheria toxin (lethal toxin)
B Crotoxin (a less potent toxin) and TNF-a
(a pro-apoptotic factor)
C Interferons, interleukins, and growth
factors (representative examples from each)
D Insulin
Collagen
mAbs (representative examples)
a
As outlined by Carver (2013).
form free arachidonic acid and lysophospholipids. Numerous
phospholipase A2 enzymes have been identified in various snake
venoms and appear to contribute to their effect on the host
(Watkins, 2008). Crotoxin is found in the venom of the South
American snake, Crotalus durissus terrificus. It is a non-covalent
complex composed of two subunits; an acidic subunit A and a
basic subunit B (Cardoso et al., 2001; Cura et al., 2002). Subunit B is
a cytotoxic and neurotoxic phospholipase A2 that is released from
the crotoxin complex upon binding to the cell surface.
A limited number of toxic effects are induced by crotoxin, and it
is believed that the epidermal growth factor receptor plays a role in
its cell targeting (Cura et al., 2002). Neurotoxicity manifested as
peripheral blockade of neuromuscular transmission is the principal
effect mediated by crotoxin, while intramuscular (IM) administra-
tion of crotoxin has been demonstrated to cause myotoxicity (Cura
et al., 2002). As reported in a publicly-available database main-
tained by the University of Florida (available at: http://www.ehs.ufl.
edu/programs/bio/toxins/toxin-table/; last accessed April 21, 2015),
the IV LD50 value for crotoxin in mice is 82 mg/kg body weight.
Crotoxin has been evaluated in a Phase 1 study in patients with
advanced cancer (Cura et al., 2002). In this study, crotoxin was
administered by IM injection for 30 consecutive days at a dose level
of 0.03, 0.06, 0.12, 0.18, 0.21, or 0.22 mg/m2/day (equivalent to 0.8,
1.6, 3.2, 4.9, 5.7, or 5.9 mg/kg body weight/day). Pharmacokinetic
data reported for the 0.21 mg/m2 (5.7 mg/kg body weight) dose level
on Study Days 1 and 15 indicated rapid absorption of crotoxin into
the bloodstream, with maximum plasma concentrations observed
within 20 min of dosing. No neurological toxicity was observed at
dose levels 0.12 mg/m2 (3.2 mg/kg body weight). The most
common neurological adverse effect at dose levels 0.18 mg/m2
(4.9 mg/kg body weight) was diplopia (double vision). This was
attributed to paresis of the external ocular muscles and was not
observed in any patient beyond Day 21. The only other neurological
toxicities observed at the highest dose level (0.22 mg/m2, or 5.9 mg/
kg body weight) were occasional incidences of ptosis, nystagmus,
and anxiety. No effects of crotoxin were observed on hematopoietic,
hepatic, or renal function. Diarrhea and excessive salivation were
occasionally observed, and one patient had an anaphylactic reac-
tion on the last day of dosing. Based on the available data, the
maximum tolerated dose (MTD) was determined to be 0.21 mg/m2
(5.7 mg/kg body weight) (Cura et al., 2002).
3.2.2. Tumor necrosis factor-a
TNF-a is a member of the TNF family of cytokines which in-
cludes TNF-b, CD40 ligand, Fas ligand (FasL), TNF-related apoptosis
inducing ligand (TRAIL), and others (Chu, 2013). TNF-a and other
members of the TNF family have important functions in various
physiological processes. TNF-a is an endogenous pyrogen that is
best known as an inducer of necrosis and apoptosis of tumor cells.
597
Information source
Reference textbooks and the published scientific literature.
Published scientific literature.
Published scientific literature.
Product monographs obtained via the Drugs@FDA
database (http://www.accessdata.fda.gov/scripts/cder/drugsatfda/).
Reference textbooks, published scientific literature, and product monographs obtained
via the Drugs@FDA database (http://www.accessdata.fda.gov/scripts/cder/drugsatfda/).
Published scientific literature.
Reference textbooks, published scientific literature, and product monographs obtained
via the Drugs@FDA database (http://www.accessdata.fda.gov/scripts/cder/drugsatfda/).
Acute and chronic systemic inflammatory reactions are modulated
by TNF-a, which induces its own secretion and the secretion of
several other inflammatory cytokines and chemokines. Evidence is
available to support a central role for TNF-a in several autoimmune
diseases such as rheumatoid arthritis, Crohn's disease, ulcerative
colitis, multiple sclerosis, and others, and TNF-a appears to be an
important risk factor for tumorigenesis, tumor progression, and
metastasis (Chu, 2013).
TNF-a is produced and secreted mainly by macrophages
although it can be generated by other cell types. Based on data
presented in 18 different human studies and summarized in the
Plasma Proteome Database, endogenous plasma/serum concen-
trations of TNF-a are generally very low, specifically in the low pg/
mL range (Nanjappa et al., 2014).
A single IV dose of recombinant human TNF-a to mice was not
lethal at a dose level of up to 0.4 mg/kg body weight (400 mg/kg
body weight) (Myers et al., 1990).
A recent review by Roberts et al. (2011) summarized data from
18 Phase 1 studies and ten Phase 2 studies in cancer patients in
which recombinant human TNF-a was administered as a single
agent. All of the identified clinical trials were conducted in the
1980s and 1990s and all but one of the studies utilized the IV route
of administration (one Phase 1 study utilized IM injection). The
design of the Phase 1 studies varied in terms of dosing posology;
single dose administration, multiple dosing (daily to every three
weeks), and continuous IV infusion (one to five days in duration)
were evaluated in various studies. As summarized by Roberts et al.
(2011), a dose level of 150e200 mg/m2 administered as a 30-
min infusion was identified as the MTD in several of the Phase 1
studies. This is equivalent to a dose of 4e5.4 mg/kg body weight for
a 50 kg adult. Dose-limiting toxicities appeared to be generally
well-tolerated and reversible; these included hypotension, throm-
bocytopenia, leucopenia, neurotoxicity, fever, nausea/vomiting, and
general symptoms of malaise and weakness. A total of ten Phase 2
studies were identified in which recombinant human TNF-a was
administered by the IV route to patients with advanced/metastatic
cancers of various types. The most common dosing posology
involved a 30-min infusion of a dose of 150e200 mg/m2 (equivalent
to a dose of 4e5.4 mg/kg body weight for a 50 kg adult) adminis-
tered daily for three to five days and repeated every one to four
weeks. The most serious toxicities included respiratory failure and
coagulopathies, while more common adverse events included hy-
potension, thrombocytopenia, leucopenia, fever/chills, headache,
and nausea/vomiting. Notably, tumor responses were rare and
when they occurred, only partial responses were observed.
The collective data from the available clinical studies indicates
that systemic administration of recombinant human TNF-a has
been evaluated in several studies in cancer patients at dose levels
generally in the range of 4e5.4 mg/kg body weight. A common
598 J.W. Card et al. / Regulatory Toxicology and Pharmacology 73 (2015) 595e606
profile of adverse events was noted in these studies and included
hypotension, thrombocytopenia, leucopenia, fever, and nausea/
vomiting.
3.3. Information on representative substances from Band C
3.3.1. Interferons
3.3.1.1. Interferon alpha-2b. Interferon alpha-2b (IFNa-2b) is an
endogenous cytokine that induces the innate anti-viral immune
response. Its biological effects include inhibition of viral replication
in virus-infected cells, the suppression of cell cycle progression/cell
proliferation, induction of apoptosis, anti-angiogenic activities, and
numerous immunomodulating activities, such as enhancement of
the phagocytic activity of macrophages, activation of NK cells,
stimulation of cytotoxic T-lymphocytes, and the upregulation of the
Th1 T-helper cell subset.
PegIntron® (Merck & Co., Inc.) is a covalent conjugate of re-
combinant human IFNa-2b and polyethylene glycol. The pegylation
of IFNa-2b allows for a longer circulation time, permitting a
decreased dosing frequency relative to free IFNa-2b. PegIntron® is
indicated for the treatment of chronic Hepatitis C in patients with
compensated liver disease. The recommended dose of PegIntron® is
1.5 mg/kg body weight/week by subcutaneous (SC) injection for one
year.
According to the product label, the most common adverse re-
actions (>40%) in adult patients receiving PegIntron® are injection
site inflammation/reaction, fatigue/asthenia, headache, rigors, fe-
vers, nausea, myalgia and anxiety/emotional lability/irritability. The
most common adverse reactions (>25%) in pediatric patients
receiving PegIntron® are pyrexia, headache, neutropenia, fatigue,
anorexia, injection-site erythema, and vomiting.
3.3.1.2. Interferon beta-1a. Interferon beta-1a (IFNb-1a) is an
endogenous cytokine that is produced by eukaryotic cells in
response to viral infection and other biologic agents. IFNb-1a pos-
sesses immunomodulatory, anti-viral, and anti-proliferative bio-
logical activities.
Avonex® (Biogen Idec Inc.) and Rebif® (EMD Serono, Inc.) are
products containing recombinant IFNb-1a that are indicated for the
treatment of relapsing forms of multiple sclerosis to slow the
accumulation of physical disability and decrease the frequency of
clinical exacerbations. The recommended dose of Avonex® is 30 mg
once per week administered by IM injection, while the recom-
mended dose of Rebif® is 22 or 44 mg three times per week
administered by SC injection.
According to the product labels for Avonex® and Rebif®, the
most common adverse reactions are flu-like symptoms including
chills, fever, myalgia, and asthenia. Other, more severe adverse re-
actions include depression, suicidal thoughts, and psychotic dis-
orders, hepatic injury, anaphylaxis or other allergic reactions,
congestive heart failure, decreased peripheral blood counts, sei-
zures, and autoimmune disorders.
3.3.1.3. Interferon gamma-1b. Interferon gamma-1b (IFNg-1b) is an
endogenous cytokine that is produced by various immune cells. It is
an activator of macrophages and has anti-viral, immunoregulatory,
and anti-tumor properties.
ACTIMMUNE® (InterMune, Inc.) is recombinant human IFNg-1b
that is indicated for reducing the frequency and severity of serious
infections associated with Chronic Granulomatous Disease and for
delaying time to disease progression in patients with severe, ma-
lignant osteoporosis. The recommended dose of ACTIMMUNE® for
patients with a body surface area >0.5 m2 is 50 mg/m2 (equivalent to
approximately 1.35 mg/kg body weight) three times per week
administered by SC injection. For patients with a body surface area
0.5 m2 the recommended dose of ACTIMMUNE® is 1.5 mg/kg body
weight three times per week administered by SC injection.
According to the product label, the most common adverse re-
actions associated with ACTIMMUNE® include fever, headache,
rash, chills, injection site reactions, fatigue, and others. Other
adverse reactions include decreased mental status, gait distur-
bance, dizziness, bone marrow toxicity (reversible neutropenia and
thrombocytopenia), and hepatic toxicity.
3.3.2. Interleukins
3.3.2.1. Interleukin-2. Interleukin-2 (IL-2) is an endogenous cyto-
kine that has a number of immunoregulatory properties, including
the following:

Enhancement of lymphocyte mitogenesis and stimulation of
long-term growth of human IL-2-dependent cell lines;

Enhancement of lymphocyte cytotoxicity;

Induction of killer cell [lymphokine-activated (LAK) and natural
(NK)] activity; and

Induction of IFNg production.
Proleukin® (Prometheus Laboratories Inc.) is a recombinant
human IL-2 that differs from native IL-2 in the following ways: i) it
is not glycosylated because it is derived from Escherichia coli; ii) it
has no N-terminal alanine as the codon for this amino acid was
deleted during the genetic engineering procedure; and iii) it has
serine substituted for cysteine at amino acid position 125 (accom-
plished by site-specific manipulation during the genetic engineer-
ing procedure). Proleukin® is indicated for treatment of adults with
renal cell carcinoma or metastatic melanoma. The recommended
dose of Proleukin® is 0.037 mg/kg body weight (37 mg/kg body
weight) every eight hours by 15-min IV infusion for a total of 14
doses. This is repeated after nine days of rest for a total of 28 doses
per course, as tolerated.
A boxed warning on the product label for Proleukin® indicates
that it has been associated with “capillary leak syndrome, which is
characterized by a loss of vascular tone and extravasation of plasma
proteins and fluid into the extravascular space”. Several other
adverse effects are listed in the product label.
3.3.2.2. Interleukin-11. Interleukin-11 (IL-11) is an endogenous
cytokine that stimulates proliferation of hematopoietic stem cells
and megakaryocyte progenitor cells and induces megakaryocyte
maturation resulting in increased platelet production.
Neumega® (Wyeth Pharmaceuticals, Inc.) is a recombinant hu-
man IL-11 that differs from native IL-11 only in that it lacks the
amino-terminal proline residue. Neumega® is indicated for the
prevention of severe thrombocytopenia and the reduction of the
need for platelet transfusions following myelosuppressive chemo-
therapy in adult patients with non-myeloid malignancies who are
at high risk of severe thrombocytopenia. The recommended dose of
Neumega® is 0.05 mg/kg body weight/day (50 mg/kg body weight/
day) administered as a SC injection for a maximum of 21 days per
treatment course. Treatment should be initiated six to 24 h after
completion of chemotherapy and continued until the post-nadir
blood platelet count is >50,000/mL.
A number of adverse effects are listed in the Neumega® product
label, including allergic reactions and anaphylaxis.
3.3.3. Growth factors
3.3.3.1. Granulocyte colony stimulating factor. Granulocyte colony
stimulating factor (G-CSF) is an endogenous glycoprotein that is
produced by monocytes, fibroblasts, and endothelial cells. It stim-
ulates proliferation, differentiation commitment, and some end-
cell functional activation of neutrophils.
 J.W. Card et al. / Regulatory Toxicology and Pharmacology 73 (2015) 595e606
NEUPOGEN® (Amgen Inc.) is recombinant human G-CSF that
differs from native G-CSF only in that it has an additional N-ter-
minal methionine (necessary for expression in E. coli) and it is not
glycosylated (because it is produced in E. coli). NEUPOGEN® is
indicated for the following:

To decrease the incidence of infection, as manifested by febrile
neutropenia, in patients with non-myeloid malignancies
receiving myelosuppressive anti-cancer drugs associated with a
significant incidence of severe neutropenia with fever;

To reduce the time to neutrophil recovery and the duration of
fever following induction or consolidation chemotherapy
treatment of adults with Acute Myeloid Leukemia;

To reduce the duration of neutropenia and neutropenia-related
clinical sequelae (e.g., febrile neutropenia) in patients with non-
myeloid malignancies undergoing myeloablative chemotherapy
followed by marrow transplantation;

To mobilize hematopoietic progenitor cells into the peripheral
blood for collection by leukapheresis; and

To reduce the incidence and duration of sequelae of neutropenia
(e.g., fever, infections, oropharyngeal ulcers) in symptomatic
patients with congenital neutropenia, cyclic neutropenia, or
idiopathic neutropenia.
The recommended dose of NEUPOGEN® varies depending on
the indication. For cancer patients receiving myelosuppressive
chemotherapy, the recommended dose is 5 mg/kg body weight/day
for up to two weeks, administered by SC injection, short IV infusion,
or continuous SC or IV infusion. For patients receiving a bone
marrow transplant, the recommended dose is 10 mg/kg body
weight/day administered by IV or SC infusion at least 24 h after
receipt of bone marrow infusion; treatment should continue until
neutrophil recovery is achieved. For blood progenitor cell collec-
tion, the recommended dose is 10 mg/kg body weight/day by SC
bolus injection or continuous infusion for at least four days prior to
collection. For patients with cyclic or idiopathic neutropenia, the
recommended dose is 5 mg/kg body weight/day by SC injection.
According to the product label, severe adverse reactions asso-
ciated with NEUPOGEN® include allergic reactions, splenic rupture,
acute respiratory distress syndrome, and alveolar hemorrhage and
hemoptysis (expectoration of blood). Other common adverse re-
actions include medullary bone pain, nausea/vomiting, alopecia,
and diarrhea.
3.3.3.2. Granulocyte-macrophage colony stimulating factor.
Granulocyte-macrophage colony stimulating factor (GM-CSF) is an
endogenous growth factor that is secreted by various cell types. It
promotes the number and function of white blood cells, including
neutrophils, monocytes/macrophages, and dendritic cells.
LEUKINE® (Bayer HealthCare Pharmaceuticals, Inc.) is recombi-
nant human GM-CSF that differs from native GM-CSF only by the
substitution of leucine at position 23. LEUKINE® is indicated for the
following:

To shorten time to neutrophil recovery and to reduce the inci-
dence of severe and life-threatening infections and infections
resulting in death after induction chemotherapy in older adults
with acute myelogenous leukemia;

To mobilize hematopoietic progenitor cells into peripheral
blood for collection by leukapheresis;

To accelerate myeloid recovery in patients with non-Hodgkin's
lymphoma, acute lymphoblastic leukemia, and Hodgkin's dis-
ease undergoing autologous bone marrow transplantation;

To accelerate myeloid recovery in patients undergoing alloge-
neic bone marrow transplantation; and
599

To prolong survival of patients who are experiencing graft fail-
ure or engraftment delay, in the presence or absence of infec-
tion, following autologous or allogeneic bone marrow
transplantation.
The recommended dose of LEUKINE® is 250 mg/m2/day
(approximately 6.75 mg/kg body weight/day) administered by IV
infusion, although the duration of treatment varies depending on
the indication. For the treatment of bone marrow transplantation
failure or engraftment delay, the recommended duration of treat-
ment is 14 days.
According to the product label, severe adverse reactions that
have been associated with LEUKINE® include fluid retention
(edema, capillary leak syndrome, and pleural and/or pericardial
effusion), respiratory symptoms (dyspnea), and cardiovascular
symptoms (transient supraventricular arrhythmia). Other adverse
reactions include fever, headache, bone pain, chills, myalgia,
dizziness, hypotension, and injection site reactions.
3.4. Information on representative substances from Band D
3.4.1. Insulin
The processes whereby insulin is synthesized, stored, and
secreted by b-cells of the pancreas are well understood. Insulin
secretion is a tightly regulated process that provides for stable
glucose levels in the bloodstream during periods of fasting and
feeding, with glucose serving as the primary stimulus for insulin
secretion. The half-life of insulin in the blood is approximately five
to six minutes, with degradation occurring mainly in the liver,
kidney, and muscle via the enzyme insulinase.
Under normal fasting conditions, insulin secretion from the
pancreas occurs at a rate of approximately 25 ng/kg body weight/
minute (Hall, 2011a). This is equivalent to “background” rates of
approximately 0.25 mg/min for a 10 kg child and 1.25 mg/min for a
50 kg adult, or 360 and 1800 mg/day, respectively. It should be noted
that these “background” rates are based on insulin secretion only
during fasting and do not account for the higher insulin secretion
that occurs after meals.
Several human insulin drug products are available, including
Humulin® R (Eli Lilly and Company) and Novolin® R (Novo Nordisk
A/S). These products are intended to be administered by SC injec-
tion prior to meals. Based on information provided in the product
labels, the total daily insulin requirement varies but usually ranges
from 0.5 to 1.0 units/kg body weight/day. According to the National
Institute for Biological Standards and Control (NIBSC, 2010), one
international unit (IU) of human insulin corresponds to the activity
contained in 0.03846 mg of insulin (i.e., there are 26.0 IU/mg of
human insulin). As such, the total daily insulin requirement of
0.5e1.0 units/kg body weight/day is equivalent to approximately
0.019e0.038 mg/kg body weight/day. For a 10 kg child this is
equivalent to approximately 190e380 mg/day, while for a 50 kg
adult this is equivalent to approximately 950 to1900 mg/day. These
values are comparable to the “background” rates of insulin secre-
tion that were calculated in the preceding paragraph.
According to the product labels for Humulin® R and Novolin® R,
hypoglycemia is the most common adverse reaction associated
with all insulin therapies. Excess insulin may cause hypoglycemia
and hypokalemia, particularly after IV administration. Mild epi-
sodes of hypoglycemia can usually be treated with oral glucose.
Severe hypoglycemia may lead to unconsciousness and/or convul-
sions and may result in temporary or permanent impairment of
brain function, or death. More severe episodes with coma, seizure,
or neurologic impairment may be treated with IM/SC glucagon or
concentrated IV glucose.
600 J.W. Card et al. / Regulatory Toxicology and Pharmacology 73 (2015) 595e606
3.4.2. Collagen
Collagen is an endogenous compound produced primarily by
fibroblasts. It is insoluble in water and is composed of a triple helix
which contains a high content of hydroxyproline. Collagen is a
major constituent of the extracellular matrix in mammals and is the
most abundant protein in the human body (Rennie, 1999; Di Lullo
et al., 2002; Smith and Rennie, 2007). By one estimate, collagen
comprises 3.5 kg of the weight of a 70 kg human, or approximately
30% of total body protein (Smith and Rennie, 2007). Approximately
28 types of collagen have been identified to date (Smith and Rennie,
2007). Current evidence suggests that there are fast-turnover
(soluble or immature) and slower-turnover (insoluble or mature)
pools of collagen, with the proportion of soluble collagen converted
to insoluble collagen being dependent on the tissue and on the age
and nutritional status of the animal (Smith and Rennie, 2007). In-
formation was not identified on the presence of collagen in blood,
although markers of collagen degradation (e.g., crosslinked telo-
peptides of type I collagen) can be measured in blood and urine
(Smith and Rennie, 2007).
Collagen is a major constituent of intercellular connective tissue,
including that of meats that are ingested by humans. Digestion of
ingested collagen is initiated in the stomach via the action of the
enzyme pepsin, with subsequent digestion to smaller polypeptides
and ultimately amino acids occurring due to other enzymatic ac-
tivity (e.g., trypsin, peptidases, and others) (Hall, 2011b).
No drug products that contain collagen as an active ingredient
were identified in the Drugs@FDA database (search conducted April
23, 2015). A search of the U.S. Food and Drug Administration (FDA)
inactive ingredient database (http://www.accessdata.fda.gov/
scripts/cder/iig/index.cfm; search conducted April 23, 2015) iden-
tified three approved drug products in which collagen is present as
an inactive ingredient; these approved drug products include a
topical gel, a topical lotion, and a shampoo. The maximum reported
potency of collagen in these products is 1%.
Hydrolyzed collagen (also referred to as collagen hydrolysate,
gelatin, gelatine, gelatine hydrolysate, and hydrolyzed gelatine)
refers to collagen that has been hydrolyzed from its triple helix
conformation into its monomeric components. Safety data for hy-
drolyzed collagen are available, although they relate mainly to the
oral and dermal routes of administration (CIR, 1985). The oral LD50
for hydrolyzed collagen in rats was reported to be >10,000 mg/kg
body weight while subchronic dermal studies with two cosmetic
formulations containing 2% hydrolyzed collagen were negative for
systemic toxicity (likely due to lack of absorption). Hydrolyzed
collagen was found to be non-sensitizing in dermal studies in
guinea pigs (CIR, 1985). More recent evidence from a study in mice
suggests the potential for oral absorption of small amounts of hy-
drolyzed collagen (Oesser et al., 1999), while food-derived collagen
peptides have been identified in human blood after ingestion of
gelatin hydrolysates (Iwai et al., 2005). A recent health claims
petition to support the use of collagen hydrolysate in the mainte-
nance of joint health in active individuals was denied by the Eu-
ropean Food Safety Authority on the basis that a cause-and effect
relationship had not been established between the consumption of
collagen hydrolysate and maintenance of joint health (EFSA, 2011).
In summary, collagen is an endogenous substance that is the
most abundant protein in the human body.
3.4.3. Monoclonal antibodies
Therapeutic mAbs have been in clinical use for over 20 years
(Hansel et al., 2010; Reichert, 2012). As outlined by Hansel et al.
(2010), mAbs have high target specificities which facilitate precise
action, and long half-lives which permit infrequent dosing. More-
over, advances in molecular engineering have enabled fine-tuning
of mAb structure for specific therapeutic actions and to minimize
immunogenicity, thus improving their overall risk-benefit ratio. In
general, mAbs are considered to be well-tolerated in humans
despite containing elements that may be recognized by the re-
cipient's immune system as being foreign (Hansel et al., 2010).
A search of the Drugs@FDA database (conducted April 22, 2015)
using the term “mab” identified a total of 40 therapeutic mAb drug
products that have been approved by the FDA, although not all of
them are currently marketed in the U.S. Moreover, three of these
products are actually antibodyedrug conjugates [gemtuzumab
ozogamicin (Mylotarg®; Wyeth Pharmaceuticals Inc.), brentuximab
vedotin (Adcetris®; Seattle Genetics), and ado-trastuzumab
emtansine (Kadcyla™; Genentech, Inc.)]. A brief summary of
these 40 therapeutic mAb drug products, including indications,
dose routes, and dose levels, is provided in Table 2.
As noted above in Table 2, the majority of approved therapeutic
mAb drug products are indicated for the treatment of various types
of cancer and arthritis. With the exception of palivizumab (Syna-
gis®; MedImmune, LLC) and ranibizumab (Lucentis®; Genentech,
Inc.), which are administered by the IM and intravitreal routes,
respectively, all of the therapeutic mAbs that have been approved
by the FDA are administered by the IV or SC route. A review of the
available information indicates that mAbs are generally adminis-
tered to patients in milligram amounts per kilogram of body weight
(i.e., mg/kg body weight dose levels), supportive of the targeted
nature of these therapeutics. Despite their targeted nature, toxic-
ities due to mAbs can occur as a result, for example, of their
recognized ability to cross the placental barrier and potentially
cause fetal toxicity. Therefore, although relatively high (milligram)
quantities are administered therapeutically, an evaluation of all of
the pharmacology and toxicology data for a given mAb would be
required to fully appreciate its target effects and potential off-target
effects, all of which would impact the derivation of an ADE value.
One exception to the relatively high doses of mAbs that are typically
administered is blinatumomab (Blincyto™; Amgen, Inc.), which is a
newly-developed bi-specific T cell engager (BiTE®; Amgen, Inc.)
antibody construct that simultaneously targets CD19 on cancer
cells and CD3 on T cells and is administered in low microgram
quantities per day. Other emerging technologies are likely to lead to
the development of additional novel mAb-based therapeutics that,
like Blincyto™, are more potent than traditional mAbs and are
likely to be administered in much lower amounts. The metabolism
of mAbs and other biotechnology-derived products is well-known
and involves degradation to small peptides and individual amino
acids (ICH, 2011).
4. Discussion
The information on representative substances that is summa-
rized above in Section 3 supports the categorization of biologics
into the four proposed potency bands as suggested by Carver
(2013). This is outlined in Table 3.
A summary of the identified information for the representative
substances in each band is as follows.
Band A: Only low nanogram quantities of these toxins are
required to exert lethal effects. As such, based on information
identified for the representative substances, this band is considered
to represent a very high hazard for cross-contamination.
Band B: Repeated administration of microgram quantities of
crotoxin (285 mg/day) and TNF-a (270 mg/day) have been demon-
strated to be tolerated in humans. However, there are inherent risks
given the foreign nature and potential lethality (crotoxin) or the
recognized pro-inflammatory nature (TNF-a) of these substances.
As such, based on information identified for the representative
substances, this band is considered to represent a high hazard for
cross-contamination.
 J.W. Card et al. / Regulatory Toxicology and Pharmacology 73 (2015) 595e606 601

Table 2
Therapeutic mAbs approved by the U.S. Food and Drug Administration.a

International non-proprietary name First U.S. Indication Dose route Dosing posology
(trade name) approvalb

Rituximab (Rituxan®; MabThera) 1997 Non-Hodgkin's lymphoma IV 375 mg/m2 (~10 mg/kg bw) once
(Biogen Idec Inc.) weekly for 4 or 8 doses.
Chronic lymphocytic leukemia IV 375 mg/m2 (~10 mg/kg bw) in first cycle
and 500 mg/m2 (~13.5 mg/kg bw) in
Cycles 2 to 6.
Rheumatoid arthritis IV 1000 mg on day 0 and 14, repeated
every 24 weeks.
Granulomatosis with Polyangiitis and IV 375 mg/m2 (~10 mg/kg bw) once
Microscopic Polyangiitis weekly for 4 doses.
Daclizumab (Zenapax®) (Hoffmann-La 1997 Prophylaxis of acute organ rejection in IV 1 mg/kg bw every 2 weeks for 5 total
Roche Inc.) renal transplantation doses.
Basiliximab (Simulect®) (Novartis 1998 Prophylaxis of acute organ rejection in IV 20 mg prior to surgery and again 4 days
Pharmaceuticals Corporation) renal transplantation after surgery.
Palivizumab (Synagis®) (MedImmune, 1998 Prevention of RSV disease in children IM 15 mg/kg bw every month during RSV
LLC) season.
Infliximab (Remicade®) (Janssen 1998 Crohn's disease IV 5 mg/kg bw at 0, 2, and 6 weeks, then
Biotech, Inc.) every 8 weeks.
Pediatric Crohn's disease IV 5 mg/kg bw at 0, 2, and 6 weeks, then
every 8 weeks.
Ulcerative colitis IV 5 mg/kg bw at 0, 2, and 6 weeks, then
every 8 weeks.
Pediatric ulcerative colitis IV 5 mg/kg bw at 0, 2, and 6 weeks, then
every 8 weeks.
Rheumatoid arthritis IV 3 mg/kg bw at 0, 2, and 6 weeks, then
every 8 weeks.
Ankylosing spondylitis IV 5 mg/kg bw at 0, 2, and 6 weeks, then
every 6 weeks.
Psoriatic arthritis IV 5 mg/kg bw at 0, 2, and 6 weeks, then
every 8 weeks.
Plaque psoriasis IV 5 mg/kg bw at 0, 2, and 6 weeks, then
every 8 weeks.
®
Trastuzumab (Herceptin ) (Genentech, 1998 HER2 overexpressing breast cancer IV 4 mg/kg bw initially, then 2 mg/kg bw
Inc.) weekly for 52 weeks.
HER2 overexpressing metastatic gastric IV 8 mg/kg bw initially, then 6 mg/kg bw
or gastroesophageal junction every 3 weeks.
adenocarcinoma
Gemtuzumab ozogamicin (Mylotarg®) 2000 CD33 positive acute myeloid leukemia IV 9 mg/m2 (~0.24 mg/kg bw) every 2
(Wyeth Pharmaceuticals Inc.) weeks for 2 total doses.
Alemtuzumab (Lemtrada™; Campath®) 2001 Relapsing multiple sclerosis IV 12 mg/day for 5 days, then 12 mg/day
(Genzyme Corporation) for 3 days (12 months later).
Adalimumab (Humira®) (AbbVie Inc.) 2002 Rheumatoid arthritis SC 40 mg every 2 weeks.
Juvenile idiopathic arthritis SC 10 to 40 mg every 2 weeks.
Psoriatic arthritis SC 40 mg every 2 weeks.
Ankylosing spondylitis SC 40 mg every 2 weeks.
Adult Crohn's disease SC 160 mg on day 1, 80 mg on Day 15, then
40 mg every other week.
Pediatric Crohn's disease SC 80 mg on day 1, 40 mg on Day 15, then
20 mg every other week.
Ulcerative colitis SC 160 mg on day 1, 80 mg on Day 15, then
40 mg every other week.
Plaque psoriasis SC 80 mg on Day 1, then 40 mg every other
week.
®
Ibritumomab tiuxetan (Zevalin ) 2002 Non-Hodgkin's lymphoma IV 250 mg/m2 (~6.8 mg/kg bw) on Day 1
(Spectrum Pharmaceuticals, Inc.) and Day 7, 8, or 9.
Efalizumab (Raptiva®) (Genentech, Inc.) 2003 Plaque psoriasis SC 0.7 mg/kg bw initial dose, then 1 mg/kg
every week.
Tositumomab-I131 (Bexxar®) 2003 Non-Hodgkin's lymphoma IV 450 mg once weekly for 2 doses.
(GlaxoSmithKline)
Omalizumab (Xolair®) (Novartis AG) 2003 Moderate to severe persistent asthma SC 150 to 375 mg every 2 or 4 weeks.
Chronic idiopathic urticaria SC 150 or 300 mg every 4 weeks.
®
Cetuximab (Erbitux ) (ImClone LLC) 2004 Head and neck cancer IV 400 mg/m2 (~10.8 mg/kg bw) followed
by weekly doses of 250 mg/m2
(~6.8 mg/kg bw).
Colorectal cancer IV 400 mg/m2 (~10.8 mg/kg bw) followed
by weekly doses of 250 mg/m2
(~6.8 mg/kg bw).
Bevacizumab (Avastin®) (Genentech, 2004 Metastatic colorectal cancer IV 5 mg/kg bw every 2 weeks.
Inc.) Non-squamous non-small cell lung IV 15 mg/kg bw every 3 weeks.
cancer
Glioblastoma IV 10 mg/kg bw every 2 weeks.
Metastatic renal cell carcinoma IV 10 mg/kg bw every 2 weeks.
Cervical cancer IV 15 mg/kg bw every 3 weeks.
(continued on next page)
602
Table 2 (continued )
International non-proprietary name
(trade name)
Natalizumab (Tysabri®) (Biogen Idec
Inc.)
Ranibizumab (Lucentis®) (Genentech,
Inc.)
Panitumumab (Vectibix®) (Amgen Inc.)
Eculizumab (Soliris®) (Alexion
Pharmaceuticals, Inc.)
Golimumab (Simponi®; Simponi®
ARIA™) (Janssen Biotech, Inc.)
Canakinumab (Ilaris®) (Novartis
Pharmaceuticals Corporation)
Ofatumumab (Arzerra®)
(GlaxoSmithKline)
Ustekinumab (Stelara®) (Janssen
Biotech, Inc.)
Tocilizumab (Actemra®) (Genentech,
Inc.)
Denosumab (Prolia®; Xgeva®) (Amgen,
Inc.)
Belimumab (Benlysta®)
(GlaxoSmithKline)
Ipilimumab (Yervoy®) (Briston-Myers
Squibb Company)
Brentuximab vedotin (Adcetris®)
(Seattle Genetics)
Raxibacumab (RAXIBACUMAB)
(GlaxoSmithKline)
Pertuzumab (Perjeta®) (Genentech,
Inc.)
Obinutuzumab (Gazyva™) (Genentech,
Inc.)
Ado-Trastuzumab Emtansine
(Kadcyla™) (Genentech, Inc.)
Blinatumomab (Blincyto™) (Amgen,
Inc.)
Nivolumab (Opdivo®) (Bristol-Myers
Squibb Company)
Pembrolizumab (Keytruda®) (Merck &
Co., Inc.)
J.W. Card et al. / Regulatory Toxicology and Pharmacology 73 (2015) 595e606
First U.S. Indication Dose route Dosing posology
approvalb
Platinum-resistant recurrent epithelial IV 10 mg/kg bw every 2 weeks or 15 mg/
ovarian, fallopian tube or primary kg bw every 3 weeks.
peritoneal cancer
2004 Multiple sclerosis IV 300 mg every 4 weeks.
Crohn's disease IV 300 mg every 4 weeks.
2006 Neovascular (wet) age-related macular Intravitreal 0.5 mg every month.
degeneration
Macular edema following retinal vein Intravitreal 0.5 mg every month.
occlusion
Diabetic macular edema Intravitreal 0.3 mg every month.
Diabetic retinopathy in patients with Intravitreal 0.3 mg every month.
diabetic macular edema
2006 Metastatic colorectal cancer IV 6 mg/kg bw every 2 weeks.
2007 Paroxysmal nocturnal hemoglobinuria IV 600 mg every week for 4 weeks, then
900 mg for Week 5, then 900 mg every
2 weeks.
Atypical hemolytic uremic syndrome IV 900 mg every week for 4 weeks, then
1200 mg for Week 5, then 1200 mg
every 2 weeks.
2009 Rheumatoid arthritis IV 2 mg/kg bw at Weeks 0 and 4, then
2 mg/kg bw every 8 weeks.
Rheumatoid arthritis SC 50 mg every month.
Active psoriatic arthritis SC 50 mg every month.
Active ankylosing spondylitis SC 50 mg every month.
Ulcerative colitis SC 200 mg initial dose, 100 mg at week 2,
then 100 mg every 4 weeks.
2009 Cryopyrin-associated periodic SC 150 mg every 8 weeks.
syndrome
Systemic juvenile idiopathic arthritis SC 4 mg/kg bw every 4 weeks.
2009 Chronic lymphocytic leukemia IV 300 mg first dose, followed by 2000 mg
weekly for 7 doses, then 2000 mg every
4 weeks for 4 doses.
2009 Moderate to severe plaque psoriasis SC 45 mg initial, 45 mg at Week 4, then
45 mg every 12 weeks.
Active plaque arthritis SC 45 mg initial, 45 mg at Week 4, then
45 mg every 12 weeks.
2010 Rheumatoid Arthritis in adults IV 4 mg/kg bw every 4 weeks.
Rheumatoid Arthritis in adults SC 162 mg bw every 2 weeks.
Polyarticular Juvenile Idiopathic IV 8 mg/kg bw every 4 weeks.
Arthritis
Systemic Juvenile Idiopathic Arthritis IV 8 mg/kg bw every 2 weeks.
2010 Postmenopausal women with SC 60 mg once every 6 months.
osteoporosis at high risk for fracture
Increase bone mass in men with SC 60 mg once every 6 months.
osteoporosis
Bone loss in men receiving androgen SC 60 mg once every 6 months.
deprivation therapy for prostate cancer
Bone loss in women receiving adjuvant SC 60 mg once every 6 months.
aromatase therapy for breast cancer
2011 Systemic lupus erythmatosus IV 10 mg/kg bw every 2 weeks for 3 doses,
then once every 4 weeks.
2011 Unresectable or metastatic melanoma IV 3 mg/kg bw every 3 weeks for total of 4
doses.
2011 Hodgkin lymphoma IV 1.8 mg/kg bw every 3 weeks.
Anaplastic large cell lymphoma IV 1.8 mg/kg bw every 3 weeks.
2012 Inhalational anthrax IV 40 mg/kg bw single dose.
2012 HER2-positive breast cancer IV 840 mg initial dose, then 420 mg every
3 weeks.
2013 Chronic lymphocytic leukemia IV Cycle 1: 100 mg on Day 1, 900 mg on
Day 2, 1000 mg on Day 8 and 15; Cycles
2 to 6: 1000 mg on Day 1.
2013 HER2-positive metastatic breast cancer IV 3.6 mg every 3 weeks.
2014 Philadelphia chromosome-negative IV Cycle 1: 9 mg/day on Days 1 to 7, 28 mg/
relapsed or refractory B-cell precursor day on Days 8 to 28; Subsequent cycles:
acute lymphoblastic leukemia 28 mg/day for 28 days.
2014 Unresectable or metastatic melanoma IV 3 mg/kg bw every 2 weeks.
Metastatic squamous non-small cell IV 3 mg/kg bw every 2 weeks.
lung cancer
2014 Unresectable or metastatic melanoma IV 2 mg/kg bw every 3 weeks.
 J.W. Card et al. / Regulatory Toxicology and Pharmacology 73 (2015) 595e606 603

Table 2 (continued )

International non-proprietary name First U.S. Indication Dose route Dosing posology
(trade name) approvalb

Ramucirumab (Cyramza®) (Eli Lilly and
Company)
Siltuximab (Sylvant™) (Janssen Biotech,
Inc.)
Vedolizumab (Entyvio™) (Takeda
Pharmaceuticals America, Inc.)
Secukinumab (Cosentyx™) (Novartis
Pharmaceuticals Corporation)
Dinutuximab (Unituxin™) (United
Therapeutics Corp.)
2014 Advanced or metastatic gastric or IV 8 mg/kg bw every 2 weeks.
gastro-esophageal junction
adenocarcinoma
Metastatic non-small cell lung cancer IV 10 mg/kg bw on Day 1 of a 21-day cycle.
(in combination with docetaxel)
2014 Multicentric Castleman's disease IV 11 mg/kg bw every 3 weeks.
2014 Adult ulcerative colitis IV 300 mg at 0, 2, and 6 weeks and every 8
weeks thereafter.
Adult Crohn's disease IV 300 mg at 0, 2, and 6 weeks and every 8
weeks thereafter.
2015 Moderate to severe plaque psoriasis SC 300 mg at 0, 1, 2, 3, and 4 weeks and
every 4 weeks thereafter.
2015 Pediatric high-risk neuroblastoma IV 17.5 mg/m2/day for 4 consecutive days
(over 10 to 20 h/day) for a maximum of
5 cycles (cycle days and durations are
variable).

bw ¼ body weight; HER2 ¼ human epidermal growth factor receptor 2; IM ¼ intramuscular; IV ¼ intravenous; RSV ¼ respiratory syncytial virus; SC ¼ subcutaneous.
a
Only therapeutic monoclonal antibodies and monoclonal antibodyedrug conjugates are included. Not included are diagnostic products or products that are based on
fragment antigen-binding (Fab) regions of antibodies [e.g., abciximab (ReoPro®) and certolizumab (Cimzia®)].
b
Listed chronologically.

Band C: These cytokines and growth factors are endogenous
substances. Recombinant human versions of these substances are
administered to patients in low (interferons), intermediate (growth
factors), and high (interleukins) microgram quantities for the
treatment of various disease conditions, sometimes on a chronic
basis. As such, based on information identified for the representa-
tive substances, this band is considered to represent a medium
hazard for cross-contamination.
Band D: These substances are endogenous and are present or
produced in the human body in milligram quantities per day (in-
sulin, collagen), or are protein therapeutics that are administered in
milligram quantities but are targeted to specific receptors within
the body and therefore are associated with minimal off-target ef-
fects (mAbs). It is anticipated that calculated ADE values for mAb
products would still be higher than those for substances in other
potency bands, even after adjusting for the intermittent dosing
schedules and prolonged half-lives that are typical of therapeutic
mAb products. As such, based on information identified for the
representative substances, this band is considered to represent a
low hazard for cross-contamination.
It is recognized that there is some overlap for some of the
substances in Bands B and C with regard to the estimated exposure
(i.e., daily dose) values that are presented in Table 3. Specifically, the
calculated maximum daily exposure values for crotoxin and TNF-a,
which are representative substances for Band B, are somewhat
higher than the calculated maximum daily exposure values for the
interferons (IFNa-2b, IFNb-1a, and IFNg-1b) that are representative
substances in Band C. It should be noted, however, that these in-
terferons are administered to patients once or several times per
week on a continual basis for the treatment of chronic diseases (e.g.,
IFNa-2b for the treatment of chronic Hepatitis C), and single or
shorter-term exposures to higher levels likely would be tolerable.
Conversely, the representative substances for Band B (crotoxin and
TNF-a) have been evaluated only in a limited number of clinical
trials of relatively short duration, and it is possible (and perhaps
likely) that longer exposure periods would only be tolerable if the
exposure level were to be decreased. Given this information, it is
considered appropriate to classify crotoxin and TNF-a as Band B
substances and the interferons as Band C substances despite the
apparent similarity in numerical exposure values (as presented in
Table 3).
It is not known how the representative substances for each of
the proposed potency bands were identified by Carver (2013), but
the potency/toxicity data that have been identified in the current
assessment would seem to support the overall approach, at least in
general terms. The results of this evaluation indicate that the ulti-
mate designation of a given substance into any of the proposed
potency bands will require a combination of quantitative and
qualitative data and information related to its source (endogenous
vs. foreign), potency, toxicity, allergenic potential, and other factors.
It should be noted that the calculated “maximum daily expo-
sure” values that are presented in Table 3 represent established
dose/exposure levels for the representative substances (e.g., known
toxic or therapeutic doses) and are not synonymous with ADE
values (or PDE values as they are more commonly known). While
ADE values for the four proposed potency bands are presented in
Figs. 2 and 3 of the recent publication by Carver (2013), it is stated
in the publication that these ADE values are “indicative and are only
presented for illustrative purposes”. Indeed, as outlined by Carver
(2013), an ADE value for a given biologic is a value that is consid-
ered safe for any and all potential “recipients” of the biologic as an
unintended impurity in another product. As such, in addition to a
lack of toxicological potential, there shouldn't be any meaningful
pharmacological activity at the ADE because what is efficacious (i.e.,
desired) for a biologic in an intended patient may be detrimental in
someone else who receives a small quantity of the biologic as an
impurity.
To derive an ADE value for any given substance would require a
complete dataset for that substance (pharmacology data, pharma-
cokinetics information, full toxicology profile, etc.); this would
represent a significant effort which is beyond the scope of the
current assessment. In addition, much of this information is usually
lacking in early development programs.
Based on the information identified in this proof-of-concept
exercise, it is anticipated that if sufficient information was avail-
able to calculate ADE values for each of the representative sub-
stances from each potency band, the ADE values would align in a
similar manner as the maximum daily exposure values that are
presented above in Table 3. In other words, it is expected that the
ADE values for Band A substances would be lowest (most potent/
toxic substances) and the ADE values for Band D substances would
be highest (least potent/toxic substances). This would be of prac-
tical benefit since maximum daily exposure values would be easier
to estimate for early development programs and more amenable
604 J.W. Card et al. / Regulatory Toxicology and Pharmacology 73 (2015) 595e606

Table 3
Summary of information to support the 4 proposed potency bands.

Potency Representative Noteworthy information on potency/ Effect level/dose level Maximum daily exposure as a toxin
band substance toxicity (Band A) or as an endogenous substance
or therapeutic agent (Bands B, C, and D)
for a 50 kg patienta

A Botulinum toxin Potent bacterial toxin. Low ng/kg body weight range Low nanogram amount
(estimated IV or IP lethal dose).
Diphtheria toxin Potent bacterial toxin. Low ng/kg body weight range Low nanogram amount
(estimated IV lethal dose).
B Crotoxin Snake venom toxin. Low mg/kg body weight range Low microgram amount
Evaluated in a Phase 1 clinical trial in (estimated IV lethal dose).
cancer patients. Up to 5.7 mg/kg body weight/day for 30 285 mg
days evaluated in patients (IM
injection).
TNF-a Endogenous pyrogen with multiple 4 to 5.4 mg/kg body weight range 270 mg
physiological functions and a central generally considered to be the MTD on
role in several autoimmune diseases. various dosing schedules (IV infusion).
Evaluated in multiple Phase 1 and 2
clinical trials in cancer patients with
expected dose-limiting toxicities
(hypotension, fever, etc.).
C IFNa-2b Endogenous cytokine with anti-viral 1.5 mg/kg body weight/week (SC 75 mg
properties. injection).
Recombinant version is used to treat
chronic Hepatitis C.
IFNb-1a Endogenous cytokine with Up to 44 mg three times per week (SC 44 mg
immunomodulatory, anti-viral, and injection).
anti-proliferative properties.
Recombinant version is used to treat
relapsing forms of multiple sclerosis.
IFNg-1b Endogenous cytokine that activates 1.35 mg/kg body weight three times per 67.5 mg
macrophages and has anti-viral, week (SC injection).
immunoregulatory, and anti-tumor
properties.
Recombinant version is used to reduce
frequency and severity of serious
infections associated with Chronic
Granulomatous Disease and for
delaying time to disease progression in
patients with severe, malignant
osteoporosis.
IL-2 Endogenous cytokine with numerous 37 mg/kg body weight every 8 h by 15- 5550 mg
immunoregulatory properties. min IV infusion for a total of 14 doses;
Recombinant version is used to treat repeated after 9 days of rest for a total of
adults with renal cell carcinoma or 28 doses per course, as tolerated.
metastatic melanoma.
IL-11 Endogenous cytokine that stimulates 50 mg/kg body weight/day for a 2500 mg
proliferation of hematopoietic stem maximum of 21 days per treatment
cells and megakaryocyte progenitor course (SC injection).
cells and induces megakaryocyte
maturation resulting in increased
platelet production.
Recombinant version is used to prevent
severe thrombocytopenia and reduce
the need for platelet transfusions
following myelosuppressive
chemotherapy in adult patients with
non-myeloid malignancies who are at
high risk of severe thrombocytopenia.
G-CSF Endogenous growth factor that Dose level depends on indication. 250 mg
stimulates proliferation, differentiation For patients with cyclic or idiopathic
commitment, and some end-cell neutropenia, the recommended dose is
functional activation of neutrophils. 5 mg/kg body weight/day (SC injection)
Recombinant version is used to
stimulate neutrophil production in
various indications.
GM-CSF Endogenous growth factor that 6.75 mg/kg body weight/day (IV 337.5 mg
promotes the number and function of infusion; duration varies depending on
white blood cells, including the indication).
neutrophils, monocytes/macrophages,
and dendritic cells.
Recombinant version is used to
stimulate white blood cell production in
various indications.
D Insulin Endogenous peptide hormone that 360 to 1800 mg/day in children (10 kg) 1800 mg (normal background)
regulates blood glucose levels. and adults (50 kg), respectively 1900 mg (therapeutic in diabetics)
 J.W. Card et al. / Regulatory Toxicology and Pharmacology 73 (2015) 595e606
Table 3 (continued )
Potency Representative Noteworthy information on potency/
band substance toxicity
Several human insulin drug products
are available.
Collagen Endogenous compound produced
primarily by fibroblasts.
It is the most abundant protein in the
human body.
Information was not identified on the
presence of collagen in blood, although
markers of collagen degradation can be
measured in blood and urine.
mAbs Targeted protein therapeutics used to
treat various diseases.
G-CSF ¼ granulocyte colony stimulating factor; GM-CSF ¼ granulocyte-macrophage colony stimulating factor; IFN ¼ interferon; IL ¼ interleukin; IM ¼ intramuscular;
IP ¼ intraperitoneal; IV ¼ intravenous; mAbs ¼ monoclonal antibodies; MTD ¼ maximum tolerated dose; SC ¼ subcutaneous; TNF ¼ tumor necrosis factor.
a
Refers to the dose level on any given day and is not intended to imply daily dosing.
for use in their risk assessments. Moreover, this would be funda-
mentally similar to the banding scheme and corresponding ADE
values that were derived by Dolan et al. (2005) for small molecule
drug substances, although it is presently unknown whether ADE
values for different categories of biologics and small molecules
might overlap.
5. Conclusions
Based on an evaluation of information that was identified for the
representative substances, it is considered that the banding scheme
proposed by Carver (2013) is a feasible approach to provide an
initial evaluation of risks associated with inadvertent exposure to
biologics as potential carryover impurities in other products. Sub-
stances in Bands A, B, C, and D are considered to represent very
high, high, medium, and low risk, respectively, with regard to po-
tential cross-contamination in manufacturing facilities. The ulti-
mate designation of a given substance into any of the proposed
potency bands will require consideration of a combination of
quantitative and qualitative data and information related to a
substance's source (endogenous vs. foreign), potency, toxicity,
allergenic potential, and other factors.
The approach outlined by Carver (2013) can be utilized to help
establish safe exposure limits that in turn can be used to assist with
cleaning validation efforts, similar to what has been proposed for
APIs (ISPE, 2010; Walsh, 2011a,b,c; Bercu et al., 2013; Sargent et al.,
2013). It should be noted, however, that the purpose of this eval-
uation was not to derive ADE values for any of the representative
substances or to establish absolute upper and lower exposure limits
for each of the proposed bands. Rather, the purpose was to deter-
mine whether information was available to support the proposed
banding scheme from a scientific perspective, based on a limited
assessment of representative substances from each band. Given the
vast number of substances that may fall into each of the proposed
bands, additional detailed work beyond this initial assessment
would be required to allow for derivation of ADE values for the
representative substances and, if possible, delineation of absolute
upper and lower exposure limits for each of the proposed bands.
Acknowledgments
The authors appreciate discussion with and input from Dr. M.
605
Effect level/dose level Maximum daily exposure as a toxin
(Band A) or as an endogenous substance
or therapeutic agent (Bands B, C, and D)
for a 50 kg patienta
(“background” rates of secretion from
the pancreas).
190 to 380 mg/day (10 kg child) and
950e1900 mg/day (50 kg adult) (total
daily insulin requirement for diabetics).
N/A N/A
Dose levels in the mg/kg body weight Several milligrams
range are typically utilized on various
dosing schedules (usually SC or IV
route).
Carver on his work in this area.
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.yrtph.2015.09.003.
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