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size generally less than w60 kDa will be filtered by the kidney (Meibohm and Braeckman,
2008). Antibody fragments or constructs that retain the Fc portion of the molecule may still
be able to bind to FcRn and have slow clearance and limited distribut ion. However, Fc con-
structs have a wide range of clearances and half-lives, suggesting that the physical and chem-
ical characteristics of the protein fused to the Fc, as well as the target interactions, have
considerable impact (Chen et al., 2012).
Therapeutic proteins and peptides
Physical/chemical characteristics of therapeutic proteins including size, molecular weight
and charge are the primary determinants of nonspecific distribution and clearance (Table 4.1)
(Meibohm and Braeckman, 2008). Many therapeutic proteins (growth hormone (GH), IGF-I,
dornase alpha, VEGF) and peptides also have specific binding proteins or shed receptors
(VEGF), which serve to modulate the exposure and receptor interactions of these molecules.
These binding proteins may serve as sinks or depots or control cellular uptake. Peptides can
also interact nonspecifically with plasma proteins (albumin, lipoproteins), which can impact
both distribution and clearance of the peptides (Meibohm and Braeckman, 2008).
The main organs for nonspecifi c clearance of therapeutic proteins and peptides are the kid-
ney and liver (Table 4.1). Molecular weight or “effective hydrodynamic size” is the key deter-
minant for kidney clearance (Fig. 5.1). The kidney is the major organ of clearance for
molecules smaller than the glomerular filtration cut-off (w60 kDa). Filtration is most ef ficient
for peptides and proteins < 30 kDa (Edwards et al., 1999; Maack et al., 1979). Molecules are
filtered by the glome rulus, metabolized within the proximal tubule or extracted from postglo-
merular capillaries and metabolized (Maack et al., 1979). The resultant peptide fragments and
amino acids are then released into systemic circulation where they become part of the
FIGURE 5.1 Schematic molecule structures, sizes and half-lives in rodents. Molecules smaller than the kidney
filtration cut-off (w60 kDa) generally have shorter half-lives than larger molecules. Molecules that retain an antibody
Fc have longer half-lives than non-Fc-containing molecules of similar size. ADC, antibodyedrug conjugate; TP,
therapeutic protein. Adapted from Holliger and Hudson (2005). Data from unpublished Merck studies and published literature
(Meibohm and Braeckman, 2008; Beals et al., 2008; Bazin-Redureau et al., 1997; Kim et al., 1995; Chapman et al., 1999).
5. Novel biopharmaceuticals: Pharmacokinetics, pharmacodynamics, and bioanalytics136
I. Development of biopharmaceuticals
TABLE 5.2 Example clearance mechanisms and human pharmacokinetic parameters for selected biotherapeutics.
Molecule description
Type
(MW kDa) PK type
Clearance
mechanism
Clearance
(L/day) #1 Half-life (days) #1
Bioavailability
(%) References
Rituximab;
anti-CD20 mu/hu IgG1
mAb (145) Nonlinear IG, TM RA: 0.34 NHL: 22 (6.1 - 52);
CLL: 32 (14 - 62);
RA: 18 (5.2 - 78) #2
Rituxan US
prescribing
information, 2012
Rituximab; anti-CD20 mu/hu
IgG1 and human recombinant
hyaluronidase
FL: 0.18
(34%); CLL:
0.20 (31%)
FL: 34 (27%);
CLL: 32 (24%)
FL: 63 - 66;
CLL: 60 - 67 #4
Rituxan hycela US
prescribing
information, 2017
Trastuzumab; anti-HER2
IgG1 and human recombinant
hyaluronidase
mAb (148) Nonlinear IG, TM 0.11 (30%) NR 77 (13%) Herceptin hylecta US
prescribing
information, 2019
Daratumumab; anti-CD38
human IgG1
mAb (148) Nonlinear IG, TM MM: 0.12
(59%); AL
0.21 (42%)
MM: 20 (22%);
AL: 28 (74%)
Darzalex US
prescribing
information, 2021
Daratumumab; anti-CD38
human IgG1 and human
recombinant hyaluronidase
69 Darzalex Faspro US
prescribing
information, 2021
Herceptin; anti-HER2
humanized IgG1
mAb Nonlinear IG, TM NR NR Herceptin US
prescribing
information, 2021
Filgastrim; recombinant human
G-CSF
Therapeutic
protein
(18.8)
Linear TM, RM 0.72 - 0.10
(/kg)
0.15 NR Neupogen US
prescribing
information, 2013
Epoetin alfa; recombinant
human erythropoietin
Protein
(30.4)
Nonlinear TM, RM 0.22 - 0.57
(/kg)
IV: 0.17 - 0.54; SC:
0.21 - 1
NR Epogen US
prescribing
information, 2017
Blinatumomab; bispecific
CD19-directed CD3 T-cell
engager
mAb
fragment
bispecific
(54)
Linear NR NR 0.092 Blincyto US
prescribng
information, 2023
Epcoritamab; bispecific CD20-
directed CD3 T-cell engager
bsAb (149) Nonlinear IG, TM 0.53 (40%) 22 (58%) NR Epkinly US
prescribing
information, 2023
(Continued)
Absorption, distribution and elimination of biopharmaceuticals 137
I. Development of biopharmaceuticals
TABLE 5.2 Example clearance mechanisms and human pharmacokinetic parameters for selected biotherapeutics.dcont’d
Molecule description
Type
(MW kDa) PK type
Clearance
mechanism
Clearance
(L/day) #1 Half-life (days) #1
Bioavailability
(%) References
Talquetamab; bispecific
GPRC5D-directed CD3 T-cell
engager
bsAB (147) Nonlinear IG, TM 0.90 (63%) First dose: 8.4 (41%);
16 weeks: 12 (46%)
59 (22%) Talvey US prescribing
information, 2023
Etanercept; TNF receptor 2
extracellular domain and
IgG1 Fc fusion protein
Dimeric
fusion
protein
(150)
Linear NR 3.8 (1.9) #3 4.3 (1.3) #3 NR Enbrel US prescribing
information, 2023
Abatacept; IgG1 Fc and CTLA4
extracellular domain fusion
protein
Dimeric
fusion
protein
(92)
Linear NR RA: 0.0067
(/kg)
RA: 14 79 Orencia US
prescribing
information, 2021
Pembrolizumab, anti-PD1
humanized IgG4 mAb
mAb (149) Linear IG 0.20 (40%) 22 (32%) Keytruda US
prescribing
information, 2021
Values represent means and %CV unless otherwise specified; #1 Values may have been converted and/or rounded from reported units; #2 median and range; #3 mean
and standard deviation; #4 95% confidence interval; AL, light chain amyloidosis; bsAb, bispecific antibody; CLL, chonic lymphocytic leukemia; FL, follicular lymphoma; IG,
Fc-mediated immunoglobulin clearance; mAb, monoclonal antibody; MM, multiple myeloma; NR, not reported; RA, reumatoid arthritis; RM, nonspecific receptor medi-
ated elimination; TM, target-mediated elimination.
5. Novel biopharmaceuticals: Pharmacokinetics, pharmacodynamics, and bioanalytics138
I. Development of biopharmaceuticals
endogenous amino acid pool and are likely re-incorporated into newly formed peptides and
proteins. Hepatic metabolism is largely determined by the amino acid sequence of the pep-
tide or therapeutic protein, and exposed carbohydrates. Uptake into hepatocytes is mediated
via a variety of mechanisms, including specific glycoprotein receptor-mediated endocytosis
and uptake by the low-density lipoprotein (LDL), transferrin, or insulin receptors ( Meibohm
and Braeckman, 2008; Cumming, 1991; Marks et al., 1995). After uptake, the endocytotic ves-
icles fuse with lysosomes or bile canalicular membranes and the proteins and peptides are
metabolized by peptidases into fragments and amino acids. The fragments and amino acids
may be released into the blood or excreted by exocytosis into the bile.
Distribution and elimination: Target-mediated processes
Monoclonal antibodies
Specific target-mediated distribution and clearance is common with mAbs, especially
mAbs, which target high concentration soluble ligands or mAbs, which bind to cell-based tar-
gets. Target-mediated drug disposition (TMDD) is frequently observed initially as nonlinear
PK behavior, where observed plasma or serum concentrations, clearance and half-lives
change disproportionally with increasing doses, suggesting capacity-limited, saturable pro-
cesses (Fig. 5.2)(Levy, 1994; Mager, 2006). However, the effects of target-mediated disposi-
tion can be complex, impacting not only drug profiles, but response profiles, timing, and
duration of responses.
Factors that impact the extent and magnitude of TMDD include affinity of antibody for
target, target levels and distribution, target kinetics, and the fate of the antibody when bound
to the target. If mAb binding results in internalization of both targe t and mAb, this mecha-
nism becomes a new clearance pathway for the mAb. That pathway can be the major elim-
ination mechanism if the target is highly abundant and/or recycles rapidly after
internalization (Wang et al., 2008; Tabrizi et al., 2006). The impact of target-mediated clear-
ance may change with time dep ending upon the fate of the target or target-bearing cells after
drug dosing. Rituximab is an example of time-dependent changes due to elimination of target
(CD20) by cell death (Dayde et al., 2009; Scheidhauer et al., 2002). The terminal half-life is
0 1 2 3 4 5 6 7
0.1
1
10
100
1000
LLOQ
Time (days)
FIGURE 5.2 Pharmacokinetic profiles for an
anti-receptor mab showing nonlinear, dose- and target-
mediated pk in wild-type mice and linear,
dose-independent pk in knockout (ko) mice. Open
symbols ¼ wild-type mice (target receptor present).
Solid symbols ¼ ko mice (target receptor absent). Anti-
body is specific for the mouse receptor. N ¼ three to
four mice/time point. IV doses: Squares, 25 mg/kg;
triangles, 4.3 mg/kg; circles, 1.6 mg/kg; diamonds,
0.8 mg/kg.
Absorption, distribution and elimination of biopharmaceuticals 139
I. Development of biopharmaceuticals
approximately 2.5 days for the first dose of rituximab when large numbers of CD20þ cells are
present (Table 5.2)(Rituxan US prescribing information, 2012). The terminal half-life after the
fourth dose increases to approximately 7.25 days, reflecting depletion of many CD20þ target
cells. Cellular targets can also exist in circulating soluble or “shed” forms (e.g., HER2, CD25),
which can also serve as a periphera l sink for the mAb (Mould et al., 1999 ; Herceptin US pre-
scribing information, 2021). TMDD and dose-dependent clearance may also be seen when
mAbs bind to high concentration soluble targets (e.g., IgE). Binding can result in formation
of large amounts of mAb: target complexes. The complexes may not recycle via the FcRn
pathway and may traffic through Fcg or phagocytotic clearance pathways in liver or spleen
with resulting faster clearance (Ward and Ober, 2009; Alvares et al., 2012).
In addition to target binding, antibodies also interact with cells and other molecules via the
Fc portion of the antibody, and these effector function interactions can induce antibody-
dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP)
or complement-dependent cytotoxicity (CDC) (Anand et al., 2008). Fc effector function can
be impacted by IgG framework types and target cell characteristics. ADCC, ADCP or CDC
can be an important part of the mechanism of action for the antibody but can also contribute
to unwanted cell death. ADCC, ADCP or CDC can also have an indirect effect on antibody
clearance by depleting the target-bearing cell populations (Scheidhauer et al., 2002).
Therapeutic proteins and peptides
Target-mediated drug disposition can have a large impact on distribution and clearance
of therapeutic proteins. Specific receptor-mediated uptake is the dominant factor controlling
both drug exposure and pharmacodynamic responses for the hematopoietic cytokines, gran-
ulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF),
and erythropoietin (EPO) (Foote, 2008; Yang and Kido, 2011; Agoram et al., 2006; Ramak-
rishnan et al., 2004). Interactions of G-CSF, M-CSF, or EPO with their specific receptors
on target cells (bone marrow progenitors, circulating cells) serve as a direct elimination
pathway, but also a stimulus for production of more target cells resulting in time-
dependent changes in exposure and responses (Fig. 5.3)(Bauer et al., 1999; Roskos et al.,
2006).
Modifications and impact on PK and biodistribution
Biopharmaceuticals are often modified to optimize their properties and in vivo behavior.
Examples include modifications to reduce or increase systemic clearance, selectively target
tissues with potent small molecule drugs, bind multiple targets, or enhance effector
mechanisms.
Multiple methods have been used to reduce systemic clearance or maintain systemic
exposure including: (1) increasing hydrodynamic size, (2) altering glycosylation, (3) fusion
to long-lived serum proteins, (4) improvement of FcRn binding (mAbs, Fc constructs), and
(5) modification of absorption properties (Beals et al., 2008; Kontermann, 2011). Increases
in hydrodynamic size are usually introduced to block rapid kidney filtration of smaller pro-
teins, peptides, and mAb fragments, mostly via conjugation to a variety of polymers (PEG,
5. Novel biopharmaceuticals: Pharmacokinetics, pharmacodynamics, and bioanalytics140
I. Development of biopharmaceuticals
PSA, HES, HPMA, dextrans) or by recombinant glycosylation or polysialylation (Table 5.2)
(Kontermann, 2011; C Chen et al., 2011)(Fig. 5.4). Modification of N-glycans has also reduced
clearance for therapeutic proteins (darbopoetin) and mAb fragments (Mould et al., 1999; Mac-
dougall et al., 1999). Higher efficiency antibody recycling should theoretically result from
tighter binding to FcRn at acidic pH . Modifications to improve FcRn binding have shown
reduced clearance with some mAbs (Zalevsky et al., 2010; Dall’Acqua et al., 2006). Constructs
that fuse mAb fragments, therapeutic proteins, or peptides to the Fc portion of an antibody
take advantage of the Fc framework’s ability to use the FcRn-mediated recycling process to
maintain serum exposure (Chen et al., 2012)(Fig. 5.4). Fc fusion constructs have also been
designed to add Fc effector functionality (e.g., ADCC) to the mechanism of action of the mol-
ecules. Fusions of therapeutic proteins and mAb fragments with other long-lived serum pro-
teins, notably albumin, have also been developed (Kratz, 2008; Smith et al., 2001; Shen et al.,
2012). GLP-1 receptor agonists (liragl utide, exenatide) and long-acting insulins (insulin glar-
gine, insulin detemir) extend their systemic exposure by using a variety of controlled-release
strategies, which delay absorption from SC injection sites (Beals et al., 2008; Evans et al., 2011;
Davies et al., 2011). Strategies include adding zinc or protamine to formulations, changing
isoelectric point, creating precipitates, and binding to serum albumin.
Serum Pegfilgrastim (ng/mL)
1000
100
10
1
0246810
Study Day
12 14 16
0.1
60
50
40
0
Hours Postdose
0
5
10
15
20
25
2468
Absolute Neutrophil Count (cells × 10
9
/mL)
30
0246810
Study Day
12 14 16
10
0
20
30 mcg/kg (n=8)
100 mcg/kg (n=8)
60 mcg/kg (n=8)
300 mcg/kg (n=8)
FIGURE 5.3 Nonlinear, dose- and time-dependent pharmacokinetics (PK) in humans. Target (G-CSF receptor)-
mediated clearance (left panel) and induced proliferation of target-bearing cells (right panel) following subcutane-
ous administration of pegfilgastrim (peg G-CSF). Pegfilgastrim concentrations drop rapidly as target (G-CSF
receptor)-bearing cell (neutrophil) numbers increase. Target cell numbers increase due to pegfilgastrim stimulation
of neutrophil proliferation and accelerated release of mature neutrophils from bone marrow. Left panel shows mean
serum pegfilgastrim concentrations. Right panel shows absolute neutrophil counts. Lines are model fits of both drug
levels and target-bearing cell numbers. Lines for both panels: solid line, 30 mg/kg; long dash, 60 mg/kg; medium
dash, 100 mg/kg; short dash, 300 mg/kg. Reprinted with permission from Roskos et al. (2006).
Modifications and impact on PK and biodistribution 141
I. Development of biopharmaceuticals
Molecules have also been modified to increase clearance and reduce systemic exposure,
especially with toxic molecules, imaging agents or drugs with intended rapid onset of action.
Fab fragments that target various tumor types have been conjugated with imaging agents or
chemotoxics to take advantage of the specificity of the Ab-binding region and the rapid clear-
ance of Fab fragments (Holliger and Hudson, 2005). Utilization of Fab fragments reduces sys-
temic exposure to chemotoxics and reduces background levels of drug in circulation for
imaging (Dennis et al., 2007). Specific antiidiotype antidrug antibodies (ADAs) have also
been developed for experimental use to facilitate rapid systemic clearance of toxic or
radiolabeled drugs via formation of drug: ADA complexes (Johansson et al., 2002;
Montero-Julian et al., 1995). Rapid-acting insulin analogs (insulin lispro, insulin aspart, insu-
lin glulisine) have also been developed, which avoid multimer formation, resulting in rapid
absorption (Evans et al., 2011).
Antibodyedrug conjugates (ADCs) utilize the specific targeting of mAbs to deliver highly
potent small molecule drugs to specific tissues, notably tumors. ADC disposition is a
FIGURE 5.4 Schematic structures for selected types of modified biopharmaceutical molecules. Fc fusion mole-
cules are schematic representations of two marketed products: Etanercept, a TNFReFc fusion (left) and romiplostim,
a TPO-mimetic peptideeFc fusion (right). The bispecific IgG diagram is a generic representation where two different
binding regions have been engineered into a common IgG framework. The second hybrid bispecific IgG diagram
represents molecules generated by hybrid hybridoma (quadroma) methods, which produce different heavy and light
chains for each target. The PEGylated Fab molecule is a generalized representation of certolizumab pegol, a marketed
PEGylated anti-TNF Fab product. The scFv2 and scFv2ealbumin fusion diagrams are generalized representations of
molecule types, which are currently in development (scFv2 ¼ blinatumomab BiTE technology from Amgen
(Nagorsen et al., 2012); scFv2-albumin ¼ MM-111 Her2:HSA:Her3 molecule from Merrimack (Kontermann, 2011)).
5. Novel biopharmaceuticals: Pharmacokinetics, pharmacodynamics, and bioanalytics142
I. Development of biopharmaceuticals
combination of both mAb and small molecule distribution and clearance processes. Clearance
and distribution of ADCs is dependent upon the modifications to the basic antibody struc-
ture, including the number of nonantibody molecules attached, sites of attachment, and sta-
bility of the conjugated molecule and linker (Shen et al., 2012; Pasqu etto et al., 2011; Boswell
et al., 2011). PK characteristics and tissue distribution are generally similar to unmodified
antibody molecules, if a limited number of conjugate molecules are present (Fig. 5.1)(Boswell
et al., 2011). When liberated from the mAbs, the small molecule and linker components of the
ADC will distribute and clear via processes, which are typical for the compound class, usu-
ally hepatic elimination.
Disposition of dual and multispecific antibodies may be heavily influenced by the mAb
targets and molecule design (IgG, fragments, other constructs) (Kontermann, 2012; Thakur
and Lum, 2010; Chames and Baty, 2009; Muller and Komtermann, 2010). Most multispecific
mAbs are in development for cancer immunotherapy (see Chapter 12, Biological Therapies
for Cancer), designed for redire cting effector cells to tumor cells, specific immunomodulation
in the tumor microenvironment or combined engagement of multiple immunomodulatory
pathways (Dahlén et al., 2018 ). Target-mediated distribution and elimination appear to domi-
nate over generic immunoglobulin processes for many dual and multispecific molecules and
nonlinear PK is common (Ruf et al., 2010). Blinatu momab, a 55 kDa anti-CD19, anti-CD3
fusion protein composed of two single-chain antibodies (ScFvs) has a half-life of w2h in
humans (Nagorsen et al., 2012). Epcoritamab, a bispecific antibody targeting CD3 and
CD20, showed a half-life of 22 days at the therapeutic dose of 48 mg, which can be considered
typical for IgG1 in absence of TMDD. Exposures increased more than proportionally with
dose at lower dose levels (Epkinly US prescribing information, 2023). The bispecific
GPRC5D-directed CD3 T-cell engager talquetamab showed time-dependent PK, with a
mean half-life increasing from 8.4 days after the first administration to 12.2 days at 16 weeks
after the first dose (Talvey US prescribing information, 2023). Preclinical studies with other
IgG-like molecule formats have shown half-lives (average w13 days) that were typical of un-
modified mAbs (Dong et al., 2011a). Multispecific molecules constructed of various types of
mAb fragments are rapidly eliminated because of their small size. Modifications to extend
exposure include PEGylation and fusion with proteins such as HSA (Muller and Konter-
mann, 2007; Stork et al., 2008; Strohl, 2015; Mahmood et al., 2023). For instance, etanercept
and abatacept consist of therapeutic proteins e
TNFR and the CTLA-4 extracellular domain,
respectively e fused with an IgG1 Fc fragment, extending their respective half-lives to around
4 and 13 days, respectively (Strohl, 2015; Enbrel US prescribing information, 2023; Orencia US
prescribing information, 2021 ).
Several antib ody modifications have been developed to enhance or control Fc effector
functions of mAbs (Anand et al., 2008; Kang and Jung, 2019; van der Horst et al., 2020).
ADCC and/or ADCP have been enhanced by glycoengineering (modification of the glycan
structure) of the FcgR binding domain, site directed mutations to increase Fc gR binding af-
finity, and Fc multimerization. Mutations can also be introduced to silence undesired FcgR
interactions. Enhanced CDC has been achieved through glycoengineering of the binding
site of C1q e the first component of the classical complement pathway, by site-directed mu-
tations to improve C1q affinity or to increase Fc-Fc interactions that promote clustering of
antibody-receptor complexes on the cell surface in hexamers, the ideal C1q docking confor-
mation (Diebolder et al., 2014; De Jong et al., 2016). Such modifications generally aim to
Modifications and impact on PK and biodistribution 143
I. Development of biopharmaceuticals
control effector functions without compromising FcRn interactions, so as to maintain favor-
able PK properties. Whether Fc effector functions are desired for the intended clinical use
should be an importan t part of mAb design considerations.
“Metabolism” and biodistribution for biopharmaceuticals
Toxic metabolites are not a potential risk for unmodified biopharmaceuticals. All native
mAbs, therapeutic proteins, and peptides are ultimately catabolized to their natural amino
acid constituents. However, mAbs, therapeutic proteins, and peptides can have a wide vari-
ety of interactions in vivo with other proteins, carbohydrates, lipids, and cells as part of their
normal physiologic functions. mAb, therapeutic protein, or peptide in vivo interacti ons with
other proteins or cells may be specific and desirable, or nonspecific, exposure- or potency-
damaging, or a potential significant safety risk. “Metabolism” studies for biopharmaceuticals
should evaluate those interactions with potential binding partners in vivo, characterize the
stability of the molecules and complexes, and map the fate of various forms of the molecules
in tissues and cells (Hamuro and Kishnani, 2012; Prueksaritanont and Tang, 2012).
The fate of the molecule upon target engagement can also dictate additional characteriza-
tion studies. Demonstration of internalization of a therapeutic protein or mAb by a cell sur-
face receptor may be important in defining the clearance pathways and understanding the
kinetics of the target, or demonstrating delivery of “payload” for ADCs. Formation of circu-
lating complexes between mAbs and ligands or therapeutic protein and binding proteins
should be characterized to understand the amounts and sizes of the circulating complexes
and assess any safety risks or unexpected PD from these complexes.
Research mAbs, surrogate mAbs, and reference/benchmark molecules can also be used
early in nonclinical development programs to help map the distribution of the target
in vitro or in vivo, especially with new targets where biology is less well understood. This
early target mapping can help de fine “on-target” versus “off-target” distribution for later
screening of drug candidate molecules.
Characterization of tissue distribution for the drug candidate molecules in disease or activ-
ity mo dels can be used to demonstrate distribution to disease-relevant target tissues and to
quantitate target tissue drug levels. However, distribution studies with the human drug
candidate are often limited by species specificity, and studies usually need to be performed
with a surrogate molecule. When use of a surrogate is required, full quantitative comparisons
of the surrogate and candidate molecule characteristics, as well as a thorough understanding
of target distribution and target kinetics across species, is required for proper interpretation of
the biodistribution data.
Metabolism and biodistribution evaluations can be conducted both in vitro, ex vivo and
in vivo. In vitro evaluations include assessment of stability and interactions in biomatrices
and assessment of interactions with blood or tissue cells. In vivo evaluations include tissue
distribution and imaging studies.
Characterization of in vitro stability of mAbs, therapeutic proteins, or peptides in bio-
matrices (serum, plasma, blood, target fluids) and in vitro protein array screening for protein:
protein interactions are examples of early assessments to help identify unexpected interac-
tions or molecule stability problems. Stability and interaction evaluations typically use
5. Novel biopharmaceuticals: Pharmacokinetics, pharmacodynamics, and bioanalytics144
I. Development of biopharmaceuticals
radiolabeled or fluorescently labeled versions of the drug molecules, which are incubated un-
der physiologic conditions in biomatrices of interest followed by physical/chemical analysis
(SEC HPLC, LC-MS, gels). Protein array screening involves application of the drug molecules
of interest to chip-based microarrays, which are preprinted with a wide variety of potential
target or interacting molecules. The arrays can be used to screen large numbers of molecules
for potential interactions, but these must be carefully benchmarked with appropriate controls.
Screening for interactions with blood cells typically utilizes fluorescently labeled mAbs, ther-
apeutic proteins, or peptides followed by flow cytometry. Evaluation of the cellular fate (e.g.,
internalization followed by metabolism, no internalization, internalization followed by recy-
cling to cell surface) of a mAb, therapeutic protein, or peptide can also be assessed in vitro.
Appropriate cells bearing receptors or other targeted cell surface molecules are incubated
with labeled molecules and the fate of those molecules is tracked by a variety of methods
including flow cytometry, immunohistochemical analysis, and physical/chemical analysis
of cell lysates.
Classic tissue distribution studies are the most common type of in vivo study and usually
include harvesting and analysis of individual tissues (Fox et al., 1996; Vugmeyster et al., 2010;
Xiang et al., 2004). Harvested tissues can be further analyzed using immunohistochemical
(IHC) methods to localize the biopharmaceutical to specific cell types (Alvares et al., 2012;
Nguyen et al., 2000). Tissue lysates can be analyzed to determine if the tissue/cells are the
site of catabolism (Alvares et al., 2012; Xiang et al., 2004; Coffey et al., 2005). Nondestructive
in vivo imaging (fluorescent, PET) can also provide useful tissue localization information,
especially with smaller therapeutic proteins and peptides (Hoppin et al., 2011).
Tissue distribution and in vivo imaging studies have historically relied upon isotopic tags
(125I, 123I, 111In, 99Tc), which have limited the types of analyses and turnaround times due
to health and safety concerns or short half-lives of the isotopes (Vugmeyster et al., 2010).
However, long wavelength and near infrared fluorophores are being used for both in vitro
and in vivo work and should facilitate rapid analyses on multiple platform s (Alvares et al.,
2012; Cilliers et al., 2017; Ji et al., 2020).
Immunogenicity and impact on PK and biodistribution
Development of ADAs is common in nonclinical development of biopharmaceuticals,
especially with chronic administration of therapeutic proteins or peptides or even with single
administration of long half-life mAbs (Meibohm and Braeckman, 2008; Schellekens and Jis-
koot, 2008; Chirmule et al., 2012). ADAs bind to the drug molecule and form immune com-
plexes, which are then present in the bloodstream and tissues. The impact of ADAs on
exposure and observed PK and PD responses can vary from total removal of all active
drug with concomitant loss of responses (Fig. 5.5)(Wagner et al., 2003; Anderson, 2005), to
increased drug exposure and persistence of response (Rehlaender and Cho, 1998; Rosenblum
et al., 1985; Working, 1992). The development of ADAs can cause serious or fatal responses
due to acute or delayed hypersensitivity reactions, immune complex-mediated pathologies,
or neutralization of critical endogenous protein counterparts to the administere d therapeutic
protein (US Department of Health and Human Services, 2014).
Immunogenicity and impact on PK and biodistribution 145
I. Development of biopharmaceuticals
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