Optimizing the Bioavailability of Subcutaneously Administered Biotherapeutics Through Mechanochemical Drivers
Optimizing the Bioavailability of Subcutaneously Administered Biotherapeutics Through Mechanochemical Drivers
D. S. Collins 0 1 2
L. C. Kourtis 0 1 2
N. R. Thyagarajapuram 0 1 2
R. Sirkar 0 1 2
S. Kapur 0 1 2
M. W. Harrison 0 1 2
D. J. Bryan 0 1 2
G. B. Jones 0 1 2
J. M. Wright 0 1 2
0 Clinical & Translational Science Institute, Tufts University Medical Center , 800 Washington St, Boston, Massachusetts 02111 , USA
1 Division of Plastic and Reconstructive Surgery, Lahey Hospital and Medical Center , Burlington, Massachusetts 01805 , USA
2 Eli Lilly Innovation Center , 450 Kendall Street, Cambridge, Massachusetts 02142 , USA
The subcutaneous route offers myriad benefits for the administration of biotherapeutics in both acute and chronic diseases, including convenience, cost effectiveness and the potential for automation through closed-loop systems. Recent advances in parenteral administration devices and the use of additives which enhance drug dispersion have generated substantial additional interest in IV to SQ switching studies. Designing pre-clinical and clinical studies using SQ mediated delivery however requires deep understanding of complex inter-related physiologies and transport pathways governing the interstitial matrix, vascular system and lymphatic channels. This expert review will highlight key structural features which contribute to transport and biodistribution in the subcutaneous space and also assess the impact of drug formulations. Based on the rapidly growing interest in the SQ delivery route, a number of potential areas for future development are highlighted, which are likely to allow continued evolution and innovation in this important area.
biodistribution; biologics; drug delivery; lymphatics; subcutaneous
ABC Accelerated blood clearance
AUC Analytical ultracentrifugation
The subcutaneous (SQ) route of parenteral drug
administration offers numerous benefits, including reduced
administration costs, increased patient compliance and preference, and
the potential to ultimately integrate drug demand and delivery
through closed loop systems, where drug infusion rates are
controlled by automated sensors. Challenges in switching
from intravenous (IV) to SQ delivery include physically
accommodating the large volumes of formulated drugs needed
in the SQ space, often lengthy mechanical administration
times, and achieving bioequivalence through altered
pharmacokinetics (and potentially pharmacodynamics). The latter is a
consequence of the SQ environment and variations in rates of
blood flow and lymphatic drainage among patients’
physiology and according to injection location. In order to
fully realize the potential for SQ drug delivery, thorough
understanding of the architecture of the SQ region is required,
coupled with comprehension of the myriad parameters
influencing drug transport. An ultimate objective would be
to establish a set of guidelines pertaining to a given drug,
which can be used to model SQ mediated delivery with
precise metrics on optimal physical (injection location, depth, and
rate) and chemical (drug concentration and formulation)
). Agnostic of therapeutic area, this review provides
a holistic overview of the challenges and opportunities, and
outlines potential areas for innovation in this rapidly
developing field (
DEFINING THE STRUCTURE
OF THE SUBCUTANEOUS REGION
The SQ region in humans is a complex, variable domain
located between the dermis and muscular layers. Its sequence
is comprised of superficial adipose tissue, a fibrous layer of
connective tissue (often referred to as the membranous layer),
and deep adipose tissue with a boundary to a fascia and the
muscle walls (Figure 1). Thickness of the SQ region is
dependent on location, personal characteristics, and gender. It
increases with body mass index (BMI), decreases with age, and is
typically greater in females of comparable BMI These factors
are necessarily incorporated into SQ drug delivery regimens.
For example, one of the preferred sites for SQ administration
of insulin is the abdominal region, which is particularly
impacted by patient BMI. This necessitates patient training and
rotation of injection sites to maximize biodistribution, and
reduce the potential for induration and lipohypertrophy.
The use of animal models to mimic SQ drug uptake and
distribution has only limited relevance as a major difference
to humans lies in the fact that their SQ connective tissue is
typically much less rich in fibrous components, presenting a
less rigid structure with flexibility to accommodate relatively
large volumes of injected solutions with comparative ease (
Additionally the SQ region in animals presents a pronounced
sub-dermal striated muscle known as the panniculus carnosus,
which can impact studies on injection mechanics, as it is
essentially absent in humans (
). An outlier to these differences
are pigs and mini-pig varieties (
). Having fibrous connective
tissue similar to humans, their panniculus is not located at the
boundary to the dermis, instead separating adipose tissue
layers. However, although the subcutis of the porcine
model shares many anatomical similarities to human,
lymphatic and vascular uptake and subsequent biodistribution
can often proceed at markedly different rates (
Accordingly, precise modeling of the human SQ
environment is needed (10).
Chemical components of the interstitial matrix
Injection to the SQ region requires a degree of accuracy to
penetrate the epidermal, dermal and pannicular layers while
not transitioning to the skeletal muscle below (Figure 1) (
humans, 4 mm is an approximate injection depth but ranges
from 1–39 mm (average 16 mm) in abdomen, 1–34 mm
(average 6 mm) in the arm and 1–32 mm (average 8 mm) in the
leg have been reported (
). The subcutis is composed of
adipose tissue bound by an extracellular matrix, through which
the venous system and lymphatic channels are interspersed,
and is enveloped in interstitial fluid derived from plasma. The
extracellular matrix is composed of several key
macromolecules with unique chemical properties. For example, type I &
III collagen is represented by fibrils composed of individual
collagen units which are in fact three polypeptide strands
(known as tropocollagen) whose triple helices form a
quaternary structure stabilized by multiple hydrogen bonds. The
high glycine content (every third amino acid) contributes
substantively to collagen’s ability to establish key hydrogen bonds
and cross links which enhance its mechanical strength. Of
significance, the isoelectric point of one of the collagen
components (type I) has been calculated to be ~10, thus rendering
the fibers net cationic at physiological pH and establishing the
potential for attractive interactions with negatively charged
matrix components (
). Elastin fibers, formed from multiple
66kD tropoelastin proteins are rich in amino acids which form
hydrophobic regions bridged by lysine cross links, which may
also foster attractive through-space interactions. Other key
macromolecules present in the matrix include proteoglycans
and appended glycosoaminoglycans (GAG’s). Examples of
GAG’s include heparin, an oligosaccharide (average MW
~12-14kD) composed of sulfated α 1,4 linked units of iduronic
acid and glucosamine, which bears a net negative charge as a
consequence of the ionized sulfate moieties. Chondroitin is
another key GAG, exemplified by chondroitin sulfate B
(sometimes referred to as dermatan sulfate) a variable mass
oligosaccharide composed of GalNAc or GlcA linked via
β1,4 or 1,3 linkages, which is known to engage in multiple
binding interactions with cytokines, matrix components and
growth factors, and plays a key role in wound healing and
tissue damage (
). Per building block, chondroitin bears
two ionizable sites per repeated unit, comprising both
carboxylate and sulfate moieties and rendering the oligomer highly
anionic in physiologic pH (carboxylate pKa ~3–5, sulfate pKa
). Hyaluronic acid (often referred to a hyaluronan)
is another negatively charged component with a pKa of 2.9 in
physiologic pH (
), and is of renewed significance, as its
enzymatic degradation [typical MW = 6–8 x 106] via injected
hyaluronidases is a method used to enhance uptake and
trafficking of SQ injected drugs [vide infra] (
). Interestingly and
significantly, though hyaluronan represents approximately
1% of the concentration of collagen in skin, its fluid exclusion
volume potential is ten times that of collagen (
). Solutions of
hyaluronic acid can be highly viscous, and as molecular weight
increases it adopts a spherical conformation with a
hydrodynamic volume of ~600 nm for a 106 MW oligomer (
Hyaluronic acid is also hygroscopic and contributes to the
viscoelasticity of skin. Its properties are related to molecular
weight, the native polymer having defined structural
properties whereas oligomers can vary and are responsible for
numerous biologic events including proliferation of endothelial
cells and cell migration (
). Interactions between GAG’s is
also possible, and it has been suggested that the viscosity of
hyaluronic acid is enhanced by chondroitin sulfate (
Subcutaneous adipose tissue
Surrounding the complex labyrinth of fibers, proteoglycans and
GAG’s are variable deposits of adipose [fat] tissue, and the
entire region is bathed in interstitial fluid. Adipose layers can
be characterized as either deep, membranous or superficial
layers and thickness is dependent on patient BMI and location
). Of significance for drug delivery, an immunostaining study
showed the presence of critical lymphatic vessels is highest in the
dermis and fascia regions but low in superficial and deep
adipose layers independent of patient BMI (
). Models have been
developed to interrogate injection physiology into adipose
tissue. On injection, hydraulic fracturing of the tissue results in
micro-cracks, which can increase permeability to injected fluids.
X-ray imaging methods have been used to visualize the injected
plume, which initially adopts a conical formation then begins to
). Adipose tissue is classified into white adipose and
less abundant but mitochondria-rich brown adipose tissue
(BAT), which is becoming of renewed importance in diabetes
). White fat cells are 90–99% triglyceride, 2–3%
protein and 5–30% water (20), and in obese patients,
remodeling of the extracellular matrix is common to accommodate
growth of adipocytes which may have the potential to
contribute to patient variability in SQ drug uptake as evidenced by
studies on insulin (
The vascular and lymphatic systems
Interspersed within the SQ tissue is the inter-related network
of arteries, veins and lymphatic channels. Transport of ISM
solutes into the vascular system is influenced by oncotic
pressure exerted by dissolved proteins in the surrounding
interstitial fluid and the uptake of lower molecular weight injected
drugs via endothelial cells is now well understood (
). In the
case of larger biomolecules however, absorption through the
lymphatic network is the predominant pathway and involves a
complex interplay of mechanical and chemical processes
). Lymphatic capillaries (with high surface area)
are abundant in dermal layers, and transdermal delivery has
indeed been exploited (26), but in the case of large molecular
weight biomolecules and antibodies, the volumes required to
mirror doses delivered by IV methods are sufficiently large to
render transdermal delivery impractical. Lymphatic vessels
however are abundant in the SQ layers, albeit present a lower
surface area (relative to capillaries) for drug uptake,
necessitating additional considerations for large volume capture and
transport. The lymphatic system originates as a network of
capillaries which transport fluid from dermal layers and the
SQ interstitium (
). The capillary networks (or initial
lymphatics as they are sometimes referred to) drain into
lymphatic collecting vessels, several of which feed into afferent
trunks which in turn connect to lymph nodes (Figure 3).
Nodes are often interconnected in regional groups. From the
nodes, fluid is transported via efferent trunks towards the
thoracic duct, entering venous circulation at the intersect of the
left subclavian and jugular veins (
Injection site remodeling events
An additional consideration for SQ drug administration in
chronic diseases is the potential for tissue induration and
scarring at the injection site (
). Similarly, for the SQ
administration of large formulated drug volumes in short time
periods, pressure build up at the injection site can cause
complications including leakage and tissue scarring as a consequence
of hydraulic forces (
). Future developments of ‘closed loop’
SQ drug delivery systems using implanted devices will also
have to address an additional injection site event known as
the ‘foreign body reaction’. In this situation a fibrous network
develops as a consequence of injection site inflammatory
response, limiting drug perfusion (
TRANSPORT OF BIOMOLECULES
IN THE SUBCUTANEOUS REGION
On injection to the SQ region low molecular weight drugs
and proteins (<16kD) can be absorbed through capillaries
and then enter systemic circulation (
). Since their size
precludes entry however, higher molecular weight proteins and
antibodies must traffic through the lymphatic system where
they enter general circulation at the interface of the thoracic
lymph duct with the subclavian vein (
). As lymphatic flow
rates can vary between 0.2% and 2% to that of blood,
understanding lymphatic uptake is of key importance (
Drug uptake through the lymphatic system
Movement within the interstitial matrix involves a
combination of diffusion and convection, and transport models
between components of the interstitium have been developed
based on the Starling equation and the Brinkman and Darcy
). Interstitial fluid traffics through the
lymphatic system at a rate of 0.2-1 μm/s, the movement being
dependent on pressure gradients between the lymphatic
system and interstitium. A commonly accepted model suggests
cleft like junctions open between lymphatic endothelial cells in
the capillaries to allow passage of macromolecules into the
lymph system (Figure 2), with expansion from 10 nm to over
1000 nm based on chemical and physical gradients,
influenced by attached collagen and elastin fibers (
picture may underestimate the complexity of the process
), and the possibility of active transport
through numerous endothelial cell surface proteins (e.g.
cadherins, catenins) in both the vascular and lymphatic
systems are an under explored strategy, as are charge-based
gating pathways (
). Another interesting option could be to
actively target and exploit the FcRn neonatal receptor, (which is
known to play a significant role in the SQ uptake of
monoclonal antibodies) through chimeric affinity constructs (
Mechanical stimulation of the lymphatic system
Interstitial fluid dynamics and lymphatic flow are known to be
both temperature and exercise dependent which has been
attributed to filtration forces acting across capillary walls
). Tissue compression [e.g. when walking] increases
interstitial flow whereas stretching impedes this in a process that
may be governed by fibroblasts (
). The impact of these
mechanical phenomena on SQ drug dispersion and uptake
has not been extensively studied though models have been
). In an effort to promote lymphatic flow [and
thus drug uptake] it is also possible to employ other
mechanical methods. Clinical studies have been conducted on the
impact of manual lymph drainage (MLD) in fibromyalgia
). The methods, which include use of soft tissue
massage regimens, targeted physical exercise [e.g. yoga], use of
graduated compression bandages and pneumatic devices (e.g.
the Lympha Press system) have shown a dramatic increase in
lymph flow, reducing edema and other clinical markers
(Figure 3) (
). Studies have also shown synergistic impact of
ultrasound with manual drainage to increase lymph flow (
which may afford additional benefits. The connective tissues
are not fully hydrated at physiological state, and compression/
stretching cycles results in substantial flux as GAG’s attract
water based on their net negative charges.
Phoretic stimulation of lymphatic uptake
Given the impact of hydration of GAG’s on their function and
properties, sonophoresis, which is known to impact intra and
inter-molecular hydrogen bonding networks, is likely to have a
pronounced impact on drug dynamics. Indeed, in addition to
disrupting hydrogen bonding networks of oligosaccharides
), sonophoresis has been shown to reduce solution viscosity
), and promote localized thermally induced massaging
effects which may also improve flow (
). Elsewhere, the
impact of sonophoresis on SQ and adipose tissue is routinely
exploited during abdominoplasty surgery, where it is used to
aid mechanical dissolution of adipose tissue via lipolysis (47).
Given that (degraded) lipids are actively up taken by the
lymphatic system it may be insightful to study post-sonophoretic
lymphatic activity in control subjects not undergoing
subsequent adipose aspiration surgery. Additional possibilities
include the use of radio-frequency induced thermal effects (
which are routinely used in cosmetic dermatology procedures
to effect skin-tightening and re-sculpting, assumed to occur via
FM based interaction with dermal collagen (
is another method that can enhance the transport of charged
molecules through the skin and the subcutaneous space,
demonstrated most notably with anti-inflammatory agents (
This can be achieved by applying a continuous low-voltage
current of appropriate polarity. Highly charged drugs can be
transported by means of electrophoresis, whereas uncharged
compounds transport can be enhanced by the electroosmotic
flow of water assisted by the dissolved mobile cations (e.g.
Na + or K+) movement (51). However, there are limitations
to the size of the molecules that can be transported. One of the
benefits of iontophoresis, is that the rate of drug transport can
be controlled at will by adjusting the electrical current which is
correlated to the drug flux almost linearly.
Modulating interstitial pressure
Interstitial pressure increases rapidly when solutions of drugs
are injected SQ, and a variety of methods can be used to
). Elevated pressures in the interstitium and at
Fig. 3 Lymphatic ducts and nodes
(left) and lymphatic drainage points
the injection site can increase pain nociception and thus
means to dissipate large injected boluses are of interest.
Administration of the enzyme hyaluronidase (e.g. rHuPH20)
reduces this pressure substantially and with rapid onset, by
enzymatic degradation of hyaluronan, thereby reducing
viscosity in the interstitium (
). The co-administration of this
enzyme in clinical studies has been successfully demonstrated
through IV to SQ non-inferiority trials with various biologics,
a function of its ability to act as a volume expander (
Though initial concerns regarding the potential for
immunogenic response had been raised (
), clinical application in
immunoglobulin therapy (
), and oncology (57) has been
demonstrated and additional applications can be anticipated
). Use of hyaluronidase to enhance perfusion and
distribution of injected solutions dates several decades (
initial studies [using non recombinant sources of enzyme], repeat
administration resulted in hypersensitivity towards the agent
). In addition to application in drug delivery however, it has
also been employed under off-label use in cosmetic surgery to
modulate the physical effects of injected hyaluronic acid based
). This is potentially significant, as the enzymatic
reaction is considered reversible, tempting speculation that the
degraded components may in theory subsequently reform high
molecular weight oligomers and polymers of hyaluronic acid
(60). Additionally, it has been suggested that the presence of
very high molecular weight hyaluronic acids confers cancer
chemoprotective properties in some species (
). The impact
of altering the mass distributions of hyaluronic acids through
enzymatic processes and homeostatic processes (baseline
turnover of HA is around 1/3 per day in humans) (
) may thus
become an area of importance for long term clinical studies.
Movement through the ISM
The movement of biomolecules through the interstitial matrix
is impacted by electrostatic interactions, and the pI of the
protein though important, is not always a predictor for
transport dynamics. This is because in the ISM, the hyaluronic acid
[and other GAG’s] functions as a polyelectrolyte, and even
close to the protein pI, local charges on the protein can foster
associations with the ISM components (
). In terms of the
interstitial volume, it is known that the β1 integrin receptor
causes contraction of collagen fibers resulting in compaction
of collagen gels. The process is governed by fibroblasts, and
during inflammation, inhibition of the integrins allows tissue
expansion and concomitant fluid influx, resulting in swelling
and edema (66). Deliberate administration of an anti
βintegrin IgG likewise promotes influx and edema suggesting
this may be a druggable target (
). Interstitial flow is a
complex process with multiple components, and as a result
algebraic approaches to modeling [akin to principal component
analysis] need to be continually developed and refined to help
establish criteria that can be used to predict drug transport at
the level of the individual patient (
Injection site considerations
The actual site of SQ administration is of relevance in
addition to the depth of injection, and may have marked,
patientspecific impact in terms of drug bioavailability in comparison
to alternate delivery modes (
). Rotation protocols employed
to reduce induration and edema (an example being studies on
) also need to be influenced by optimal access
to the lymphatic system itself as shown on studies of human
growth hormone (
), erythropoietin (
) and insulin (
Systems level mapping of the human lymphatic system has
been conducted providing insight to lymph collectors and
connecting nodes (
). For example, in the anterior
abdominal wall, the lymphatic channels are in the midline
watershed and periumbilical regions in the subdermal plane
). However, in the lower abdomen the lymph
collectors lie deeper in the SQ tissue as they travel inferiorly,
penetrating Scarpa’s fascia approximately 2–3 cm superior to the
inguinal ligament before emptying into the inguinal lymph
nodes (Figure 3) (
). Accordingly, based on collector
location, bioavailability would be predicted to be higher in the
mid-abdominal periumbilical region compared to when
injected into the lower abdomen (
). This can be expected
to be a fertile area for future investigation, as studies to date
have been inconclusive (2), or focused solely on clinical
). Imaging at the tissue level of lymphatic
duct density will also be useful in order to prioritize injection
sites. Lymphoscintigraphy, commonly used to assess
lymphedema and lymph associated metastases by assessing gross
transport to the lymphatic nodes, has obvious potential in
mapping the lymphatic microenvironment and the impact of
long term SQ drug administration in chronic conditions
). Another useful tool will be to establish anatomical
maps of soft tissue massage points which can be used to
mechanically stimulate lymphatic flow to aid in drug uptake
(Figure 3). Developing appropriate methods to image the
distribution of drug & vehicle boluses from the SQ tissue into
lymph are also important. Aside from scintigraphy, basic
echography has recently proven useful and is commonly
accessible in healthcare settings (
Age related anatomical changes
Given that transport in the SQ environment involves
mechanical forces, it is expected that the fidelity and efficiency of the
processes will naturally alter with patient age. For example,
reduction of both SQ adipose tissue (
) and hyaluronic acid
) have been associated with aging. Hyaluronic acid
levels in the epidermis begin to diminish in the 60–80’s which
impacts the function of fibroblasts, eventually reducing tissue
elasticity as collagen level production falls (
collagen has a half life of approximately 15 years, hyaluronan is
under 24 h (
), its age-associated reduction, MW distribution
and composition linked to catabolism and degradation via the
hyaluronan synthase family of enzymes (
). Injected native
hyaluronic acid [exogenous] is degraded rapidly, thus to
increase and maintain levels (
), regulation of dermal fibroblast
function is instead necessary or else cross-linked hyaluronic
acid introduced (which when used in cosmetic indications
typically has persistence of 9–12 months). Another concern
relates to drug catabolism at the SQ delivery site, which can
diminish bioavailability (
). Adipose redistribution takes place
in old age and has many potential implications, including
metabolic dysfunction and inflammatory processes which
might impact the fidelity of SQ delivered drugs (
), and also
hyaluronan related repair at the injection site (
Accordingly, the administration of SQ drugs in chronic
diseases afflicting the elderly [Alzheimer’s disease being a prime
candidate] needs to be informed by these anatomic and
physiologic alterations in terms of drug potency, formulation and
injection site rotation (
THE IMPACT OF FORMULATIONS
Charge, molecular weight, formulants, pH, temperature,
viscosity and tonicity all play a role in the PK and PD of injected
). Given the impact of hydration
and charge on ECM and interstitium components and their
transport properties, it is imperative that formulation of SQ
delivered drugs is considered appropriately. In addition to the
active drug substances, the nature of buffers used and their
relative tonicity (
) impacts biodistribution and lymphatic
uptake of biopharmaceuticals (
). Numerous studies have
examined the role of buffer and substrate charge, pI and
tonicity and their impact on uptake and pK, with variable results
). In one example, the use of a charged buffer,
Ophosphoserine, enhanced lymphatic uptake of an antibody
). Anecdotally, the hyaluronic acid GAG bears negative
charge, thus positively charged species may associate (thereby
reducing lympatic uptake) whereas negatively charged species
could traffic more rapidly to the lymph based on repulsive
Coulombic forces. The role of additives have been explored,
including use of the protein albumin to enhance drug uptake,
by acting as a volume expander in the ISM in a similar
manner to hyaluronidases (
). Active albumin mediated uptake
into the lymphatic system can also be exploited, by derivitizing
the drug in question with an albumin binding glyceride motif,
effecting what has been dubbed ‘albumin hitchiking’ (
Potentially relevant, association of serum albumin with
immunoglobulins (through non specific binding events) is known to
impact certain immunoassays, and should be examined in
formulated drugs using relevant methods (
A primary objective will be to predict the interaction of
candidate biopharmaceuticals with various ISM components and
incorporate this methodology into drug screening assays and
selection. This will allow modeling of how they can be
expected to transform in the subcutaneous milieu (
). To achieve this
requires deep understanding of fundamental properties of the
drug and how these impact its bioavailability. For example, a
useful tool for drug development would be to correlate zeta
potential and isoelectric point with viscosity of the drug (
This might be used for the re-engineering of antibodies with
more desirous properties (
), including solubility (
). It is
also important to assess the nature of any charged variants
of proteins which might impact affinity and transport in the
ISM using appropriate techniques (89). In this regard, the
assessment of charge remains a critical yet ill-defined
parameter. Electrophoretic methods have been described to
quantitate the Debye-Huckel-Henry charge, and studies of the
colloidal properties of proteins reveal that proximity energies,
which are electrostatic in nature, play a dominant role (
This is important, as the typically highly concentrated
formulations used can lead to the formation of aggregates, gels and
). Interactions between [charged] protein drugs
and cellular macromolecules is also important. It has been
revealed that despite charge variances and regional
permutations, anions selectively and preferentially accumulate on the
surface of proteins in salt solutions (
). Accurate modeling of
the electrostatic surface of proteins might then be used to
assess potential interactions with matrix components
(Figure 4). Though charge based interactions between
proteins can be attractive or repulsive [important for trafficking
to the lymphatic from the interstitial matrix], so-called
‘excluded volume’ interactions are always repulsive in nature.
Of available methods, electrophoresis can be used to calculate
protein valence using the equation, where μ represents
electrophoretic mobility, zeff corresponds to the effective valence, f
signifies translational friction coefficient, and Qp fundamental
proton charge (
Collectively, we anticipate that these design considerations
will pave the way for what will eventually become known as
quantitative subcutaneous targeting (QUEST) strategies.
μ ¼ zeff = f Q p
Considerations for monoclonal antibodies
In the case of monoclonal antibodies, interactions of
formulated drugs with buffers and excipients are vastly different in
serum. Methods to study this include analytical
ultracentrifugation [AUC] using fluorescence detection (
). At high
antibody concentrations, both the nature and magnitude of
intermolecular interactions is a key parameter impacting its
viscosity, and charge distribution plays a pivotal role (
primary concern with antibodies is the potential for
aggregate formation, as this can have immunogenic consequence
). At low concentration, changes in conformational
stability and weak protein-protein interactions can induce
aggregation, and various techniques including dynamic light
scattering can be employed to monitor these events (
has also been demonstrated that SQ delivery of highly
concentrated formulations of mAb’s can be achieved using
crystalline suspensions (
), and more recent investigations have
been reported on mAb based gel beads (
). An increasingly
common strategy is the derivitization of proteins as poly
ethylene glycol conjugates (PEGylation). This can have the
impact of increasing the Stokes radius of a given protein,
and methods have been developed to assess the impact of
this modification on properties, including hydrodynamic
). Though PEGylation of certain proteins
has been shown to increase SQ mediate lymphatic uptake,
in the case of antibody derivatives the impact is less
pronounced, but has been shown to enhance plasma clearance
in rodent models (
). It has also been postulated that
PEGylation of certain SQ delivered nano-encapsulated
drugs results in accelerated blood clearance through
intravenous pathways, suggesting potential for synergistic
enhancement of drug delivery (
Ultimately it may be possible to model potential
interactions in silico, prior to investing in costly preclinical
programs. Aggregation of proteins at high salt concentration,
a known problem in drug development, can be measured
in vitro but may eventually be predictable based on charge
density mapping (
), as might viscosity (
protein-protein interactions can be modeled using light
scattering techniques and can give insight to potential
limitations of a drug candidate (
). Finally, molecular
dynamics simulations, previously unimaginable studies on
antibodies, are now proving insightful, and with the advent of
ever increasing computational methods and processing
power, can be expected to play a role making meaningful
assessments of drug candidates (
). A major concern for
the injection of high molecular weight antibodies in
concentrated form is precipitation at the injection site, which can
occur through steric exclusion processes involving GAG’s
and formulants (
). One means to assess potential for this
is to inject constituted drugs ex vivo into tissue, then section
the tissue using MALDI-MS, scanning for aggregates (
Also to be considered is the potential to impact the local
environment with co-administered adjuvants. For example
lipolysis in fat cells can occur via stimulation of
βadrenoceptors, which might be used to alter lipid levels
during drug administration e.g. using isoprenaline (
degradation of adipose tissue (similarly enhanced by
sonophoretic lipolysis during abdominoplasty) may
enhance bulk distribution of injected drugs at the site of
lipolysis as the degraded products [glycerol] may have a
stimulatory impact on lymphatic flow. Likewise, addition of anti
β-integrin IgG to induce edema/influx and promote uptake
of the drug may become a viable strategy, allied to injection
site modeling. Conversely, as trafficking to the lymphatic
system from the ISM relies on aqueous transport,
hydrophobic components of the formulated drug are likely to
accumulate in the matrix, may impact dispersion of the
injected bolus and contribute to aggregation (
STRATEGY AND OUTLOOK
The SQ drug administration route is becoming of increased
significance in the delivery of biopharmaceuticals. Several
compounds administered by the route are now in clinical
), and long term studies are beginning to
validate the merit of hyaluronidase based co-formulations (
Realizing the full potential of subcutaneous delivery methods
is likely to have a pronounced impact on patient care and
disease management (
) and it is incumbent on the
pharmaceutical industry to devise strategies which fully exploit this
). When incorporated into closed-loop
automated delivery systems, this may open the possibility of achieving
homeostasis by dosing (through pulsatile processes) from a
reservoir to the interstitium. The lymphatic system, the main
route for uptake of biologic drugs, flows at a much lower rate
than via capillary uptake however. Algorithms used to predict,
monitor, and analyze kinematics of drug flow will become
necessary and may be informed by other systems – e.g. the
Riemann method for modeling merged traffic flow (
biologic filtration bed processes (
) and porous drain
engineering principles (
), which rely on the Darcy and
Brinkman equations (
). Ultimately systems may emerge that
allow us to stress test candidate biomolecules using simulated
subcutaneous environments and closed loop drug delivery
Based on findings in this rapidly unfolding field, it seems
likely that certain areas will prove fertile in the search for long
term solutions, as outlined in Table I.
Equally important will be parallel long term studies on the
impact of rHuPH20 and volume expanders on interstitial and
lymphatic integrity. It has been suggested that enzymatic
degradation of the interstitium leads to collapse of the lymphatic
). Designing in vivo lymphatic imaging experiments,
and high resolution analysis of basement membranes e.g.
using SEM techniques may reveal the impact of the enzyme
in appropriate detail. There may be additional avenues to
enhance drug uptake and permeation, including the use of
encapsulated vectors which possess charges (or can be induced
on demand) to exploit Coulombic forces in the interstitium
). Another strategy may be via induction of accelerated
blood clearance (ABC) pathways through derivatization of the
drug substance itself (
). We look forward to the
incorporation of these and related quantitative subcutaneous targeting
(QUEST) strategies in mainstream drug development. Given
the evident commercial opportunities in this space (
innovations are likely to proceed at a rapid pace and will form
the cornerstone of a new era in patient engaged drug delivery.
1. accurate determination of net charges on candidate proteins and formulants
2. the impact of mechanical forces (massage, stretching) on lymphatic flow and
3. the impact of hydration, and disruption of hydrogen bonding (ultrasound) in
drug dispersion and uptake
4. the potential for electrical stimulation of drug uptake via pulsed and linear
5. thermal processes that improve drug uptake and patient nociception (e.g.
6. processes which enhance dissipation of injected plumes to lymphatic
7. the impact of adjuvants to enhance drug uptake including
-integrin receptor inhibitors to induce edema / drug influx
-lipolysis inducers for drug dispersion and uptake
-endothelial targeting agents which promote lymphatic cleft opening and
-analgesics which increase patient pain thresholds for the introduction of
large injected volumes (
-co-administration of protease inhibitors to enhance bioavailability (
8.Exploring the utility of mAb-FcRn targeting chimeras to enhance lymphatic
ACKNOWLEDGMENTS AND DISCLOSURES
D. S. Collins, L. C. Kourtis, N. R Thyagarajapuram, R.
Sirkar, S. Kapur, M. W. Harrison, and J. M. Wright are
employees of Eli Lilly Corporation. G. B. Jones acknowledges
funding from the NIH National Center for Advancing
Translational Sciences through grant UL1 TR001064.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License
permits unrestricted use, distribution, and reproduction in any
medium, provided you give appropriate credit to the original
author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made.
1. Kinnunen HM , Sharma V , Contreras-Rojas LR , Yu Y , Alleman C , Sreedhara A , et al. A novel in vitro method to model the fate of subcutaneously administered biopharmaceuticals and associated formulation components . J Control Release . 2015 ; 214 : 94 - 102 .
2. Richter WR , Bhansali SG , Morris ME . Mechanistic determinants of biotherapeutics absorption following SQ administration . APPS J . 2012 ; 14 : 559 - 70 .
3. McLennan DN , Porter CJH , Charman SA . Subcutaneous drug delivery and the role of the lymphatics . Drug Discov Today Technol . 2005 ; 2 : 89 - 96 .
4. Akkus O , Oguz A , Uzunlulu M , Kizilgul M . Evaluation of Skin and Subcutaneous Adipose Tissue Thickness for Optimal Insulin Injection . J Diabetes Metab . 2012 ; 3 : 216 .
5. Kinnunen HM , Mrsny RJ . Improving the outcomes of biopharmaceutical delivery via the subcutaneous route by understanding the chemical, physical and physiological properties of the subcutaneous injection site . J Control Release . 2014 ; 182 : 22 - 32 .
6. Lancerotto L , Stecco C , Macchi V , Porzionato A , Stecco A , De Caro R . Layers of the abdominal wall: anatomical investigation of subcutaneous tissue and superficial fascia . Surg Radiol Anat . 2011 ; 33 ( 10 ): 835 - 42 .
7. Martinez MN . Factors Influencing the Use and Interpretation of Animal Models in the Development of Parenteral Drug Delivery Systems . AAPS J . 2011 ; 13 ( 4 ): 632 - 49 .
8. Dolgin E . Minipig, minipig, let me in. Nat Med . 2010 ; 16 : 1349 .
9. Zheng Y , Tesar DB , Benincosa L , Birnböck H , Boswell CA , Bumbaca D , et al. Minipig as a potential translatable model for monoclonal antibody pharmacokinetics after intravenous and subcutaneous administration . MAbs . 2012 ; 4 ( 2 ): 243 - 55 .
10. Friedman T , Coon D , Kanbour-Shakir A , Michaels J , Rubin JP . Defining the lymphatic system of the anterior abdominal wall: an anatomical study . Plast Reconstr Surg . 2015 ; 135 ( 4 ): 1027 - 32 .
11. Trowbridge JM , Gallo RL . Dermatan sulfate: new functions from an old glycosaminoglycan . Glycobiology . 2002 ; 12 ( 9 ): 117R - 25R .
12. Chandran PL , Horkay F . Aggrecan, an Unusual Polyelectrolyte: Review of Solution Behavior and Physiological Implications . Acta Biomater . 2012 ; 8 ( 1 ): 3 - 12 .
13. Lapcik L , Lapcik L . Hyaluronan: preparation, structure, properties and applications . Chem Rev . 1998 ; 98 ( 8 ): 2663 - 84 .
14. Cowman MK , Schmidt TA , Raghavan P , Stecco A . Viscoelastic Properties of Hyaluronan in Physiological Conditions . F1000Res . 2015 ; 4 : 622 .
15. Bookbinder LH , Hofera A , Hallera MF , Zepedab ML , Kellera GA , Lima JE , et al. A recombinant human enzyme for enhanced interstitial transport of therapeutics . J Control Release . 2006 ; 114 : 230 - 41 .
16. Gall Y. Hyaluronic acid: structure, metabolism and implication in cicatrisation . Ann Dermatol Venereol. 2010 ; 137 ( Suppl 1 ): S30 - 9 .
17. Nishimura M , Yan W , Mukudai Y , Nakamura S , Nakamasu K , Kawata M , et al. Role of chondroitin sulfate-hyaluronan interactions in the viscoelastic properties of extracellular matrices and fluids . Biochim Biophys Acta Gen Subj . 1998 ; 1380 : 1 - 9 .
18. Comley K , Fleck NA . Deep penetration and liquid injection into adipose tissue . J Mech Mater Struct . 2011 ; 6 : 127 - 40 .
19. Sacks H , Symonds ME . Anatomical Locations of Human Brown Adipose Tissue Functional Relevance and Implications in Obesity and Type 2 Diabetes . Diabetes. 2013 ; 62 : 1783 - 90 .
20. Geerligs M , Peters GWM , Ackermans PAJ , Oomens CWJ , Baaijens FPT . Linear viscoelastic behavior of subcutaneous adipose tissue . Biorheology . 2008 ; 45 : 677 - 88 .
21. Alkhouli N , Mansfield J , Green E , Bell J , Knight B , Liversedge N , et al. The mechanical properties of human adipose tissues and their relationships to the structure and composition of the extracellular matrix . Am J Physiol Endocrinol Metab . 2013 ; 305 ( 12 ): E1427 - 35 .
22. Gagnon-Auger M , du Souich P , Baillargeon JP , Martin E , Brassard P , Ménard J , et al. Dose-dependent delay of the hypoglycemic effect of short-acting insulin analogs in obese subjects with type 2 diabetes: a pharmacokinetic and pharmacodynamic study . Diabetes Care . 2010 ; 33 : 2502 - 7 .
23. Fathallah AM , Turner MR , Mager DE , Balu-Iyer SV . Effects of Hypertonic Buffer Composition on Lymph Node Uptake and B i o a v a i l a b i l i t y o f R i t u x i m a b , A f t e r S u b c u t a n e o u s Administration . Biopharm Drug Dispos . 2015 ; 36 : 115 - 25 .
24. Negrini D , Moriondo A . Lymphatic anatomy and biomechanics . J Physiol . 2011 ; 589 ( 12 ): 2927 - 34 .
25. Aukland K , Reed R . Interstitial-lymphatic mechanisms in the control of extracellular fluid volume . Physiol Rev . 1993 ; 73 : 1 - 78 .
26. Harvey AJ , Kaestner SA , Sutter DE , Harvey NG , Mikszta JA , Pettis RJ . Microneedle-Based Intradermal Delivery Enables Rapid Lymphatic Uptake and Distribution of Protein Drugs . Pharm Res . 2011 ; 28 : 107 - 16 .
27. Kang DW , Jadin LM , Nekoroski TA , Zepeda ML . Recombinant Human Hyaluronidase (rHuPH20) Facilitates Subcutaneous Infusion of Immunoglobulin, Increases Local Fluid Dispersion, and Reduces Induration in a Porcine Model . J Allergy Clin Immunol . 2012 ; 129 ( 2 ): AB85 .
28. Thomsen M , Hernandez-Garcia A , Mathiesen J , Poulsen M , Sørensen DN , Tarnow L , et al. Model Study of the Pressure Build-Up during Subcutaneous Injection . PLoS One . 2014 ; 9 ( 8 ): e104054 .
29. Klopfleisch R , Jung F. The pathology of the foreign body reaction against biomaterials . J Biomed Mater Res Part A . 2017 ;105A: 927 - 40 .
30. Swartz MA , Fleury ME . Interstitial Flow and Its Effects in Soft Tissues . Annu Rev Biomed Eng . 2007 ; 9 : 229 - 56 .
31. Levick JR , Michel CC . Microvascular fluid exchange and the revised Starling principle . Cardiovasc Res . 2010 ; 87 ( 2 ): 198 - 210 .
32. Skobe M , Detmar M. Structure , Function, and Molecular Control of the Skin Lymphatic System . J Investig Dermatol Symp Proc . 2000 ; 5 : 14 - 9 .
33. Leak LV , Burke JF . Ultrastructural studies on the lymphatic anchoring filaments . J Cell Biol . 1968 ; 36 ( 1 ): 129 - 49 .
34. Michel CC , Curry FE. Microvascular Permeability . Physiol Rev . 1999 ; 79 ( 3 ): 703 - 61 .
35. Trevaskis NL , Kaminskas LM , Porter CJ . From Sewer to Saviour -Targeting the Lymphatic System to Promote Drug Exposure and Activity . Nat Rev Drug Discov . 2015 ; 14 : 781 .
36. Renkin EM , Tucker VL . Measurement of microvascular transport parameters of macromolecules in tissues and organs of intact animals . Microcirculation . 1998 ; 5 : 139 - 52 .
37. Ko S , Pegu A , Rudicell RS , Yang Z , Joyce MG , Chen X , et al. Enhanced neonatal fc receptor function improves protection against primate SHIV infection . Nature . 2014 ; 514 : 642 - 5 .
38. Davis HA , Jooste PL . Subcutaneous interstitial pressure in man and dogs exposed to heat and exercise stress . Eur J Appl Physiol Occup Physiol . 1980 ; 44 ( 2 ): 117 - 22 .
39. Langevin HM , Nedergaard M , Howe A . Cellular Control of Connective Tissue Matrix Tension . J Cell Biochem . 2013 ; 114 ( 8 ): 1714 - 9 .
40. Lu Y , Wang W. Interaction between the interstitial fluid and the extracellular matrix in confined indentation . J Biomech Eng . 2008 ; 130 ( 4 ): 041011 .
41. Asplund R . Manual lymph drainage therapy using light massage for fibromyalgia sufferers: a pilot study . J Orthop Nurs . 2003 ; 7 : 192 - 6 .
42. Ekici G , Bakar Y , Akbayrak T , Yuksei I . Comparison of manual lymph drainage therapy and connective tissue massage in women with fibromyalgia: A randomized controlled trial . J Manip Physiol Ther . 2009 ; 32 : 127 - 33 .
43. Citak-Karakaya I , Akbayrak T , Demirturk F , Ekici G , Bakar Y. Short and long-term results of connective tissue manipulation and combined ultrasound therapy in patients with fibromyalgia . J Manip Physiol Ther . 2006 ; 29 ( 7 ): 524 - 8 .
44. Venegas-Sanchez JA , Motohiro T , Takaomi K. Ultrasound effect used as external stimulus for viscosity change of aqueous carrageenans . Ultrason Sonochem . 2013 ; 20 : 1081 - 91 .
45. Venegas-Sanchez JA , Tagaya M , Kobayashi T . Effect of ultrasound on the aqueous viscosity of several water-soluble polymers . Polym J . 2013 ; 45 : 1224 - 33 .
46. Rao R , Nanda S. Sonophoresis : recent advancements and future trends . J Pharm Pharmacol . 2009 ; 61 ( 6 ): 689 - 705 .
47. Sklar LR , El Tal AK , Kerwin LY . Use of transcutaneous ultrasound for lipolysis and skin tightening: a review . Aesthet Plast Surg . 2014 ; 38 ( 2 ): 429 - 41 .
48. Gold MH . The Increasing Use of Nonablative Radiofrequency in the Rejuvenation of the Skin . Expert Rev Dermatol . 2011 ; 6 ( 2 ): 139 - 43 .
49. Elsaie ML , Choudhary S , Leiva A , Nouri K. Nonablative radiofrequency for skin rejuvenation . Dermatol Surg . 2010 ; 36 : 577 - 89 .
50. Kalia YN , Naik A , Garrison J , Guy RH . Iontophoretic drug delivery . Adv Drug Deliv Rev . 2004 ; 56 ( 5 ): 619 - 58 .
51. Prausnitz MR , Langer R . Transdermal drug delivery . Nat Biotechnol . 2008 ; 26 ( 11 ): 1261 - 8 .
52. Kang DW , Oh DA , Fu GY , Anderson JM , Zepeda ML . Porcine model to evaluate local tissue tolerability associated with subcutaneous delivery of protein . J Pharmacol Toxicol Methods . 2013 ; 67 : 140 - 7 .
53. Ismael G , Hegg R , Muehlbauer S , Heinzmann D , Lum B , Kim SB , et al. Subcutaneous Versus Intravenous Administration of (neo) Adjuvant Trastuzumab in Patients with HER2-Positive, Clinical Stage I-III Breast Cancer (HannaH study): A Phase 3 , Open-Label , Multicentre, Randomised Trial . Lancet Oncol . 2012 ; 13 : 869 - 78 .
54. Rosengren S , Dychter SS , Printz MA , Huang L , Schiff RI , Schwarz H-P , et al. Clinical Immunogenicity of rHuPH20, a Hyaluronidase Enabling Subcutaneous Drug Administration . AAPS J . 2015 ; 17 ( 5 ): 1144 - 56 .
55. Wasserman RL , Melamed I , Stein MR , Engl W , Sharkhawy M , Leibl H , et al. Long-Term Tolerability , Safety, and Efficacy of Recombinant Human Hyaluronidase-Facilitated Subcutaneous I n f u s i o n o f H u m a n I m m u n o g l o b u l i n f o r P r i m a r y Immunodeficiency . J Clin Immunol . 2016 ; 36 : 571 - 82 .
56. Danieli MG , Pulvirenti F , Rocchi V , Morariu R , Quinti I. Selfadministered hyaluronidase-facilitated subcutaneous immunoglobulin therapy in complicated primary antibody deficiencies . Immunotherapy . 2016 ; 8 ( 9 ): 995 - 1002 .
57. Jackisch C , Müller V , Maintz C , Hell S , Ataseven B . Subcutaneous Administration of Monoclonal Antibodies in Oncology . Geburtshilfe Frauenheilkd . 2014 ; 74 ( 4 ): 343 - 9 .
58. Dychter SS , Harrigan R , Bahn JD , Printz MA , Sugarman BJ , DeNoia E , et al. Tolerability and pharmacokinetic properties of ondansetron administered subcutaneously with recombinant human hyaluronidase in minipigs and healthy volunteers . Clin Ther . 2014 ; 36 : 211 - 24 .
59. Holborow EJ , Keech MK . Hyaluronidase skin spreading effect. An analysis of repeated measurements . Br Med J. 1951 ; 2 : 1173 - 8 .
60. Rzany B , Becker-Wegerich P , Bachmann F , Erdmann R , Wollina U. Hyaluronidase in the correction of hyaluronic acid-based fillers: a review and a recommendation for use . J Cosmet Dermatol . 2009 ; 8 ( 4 ): 317 - 23 .
61. Landau M. Hyaluronidase Caveats in Treating Filler Complications . Dermatol Surg . 2015 ; 41 ( Suppl 1 ): S347 - 53 .
62. Tian X , Azpurua J , Hine C , Vaidya A , Myakishev-Rempel M , Ablaeva J , et al. High molecular weight hyaluronan mediates the cancer resistance of the naked mole-rat . Nature . 2013 ; 499 : 346 - 9 .
63. Stern R . Hyaluronan catabolism: a new metabolic pathway . Eur J Cell Biol . 2004 ; 83 ( 7 ): 317 - 25 .
64. Mach H , Gregory SM , Mackiewicz A , Mittal S , Lalloo A , Kirchmeier M , et al. Electrostatic interactions of monoclonal antibodies with subcutaneous tissue . TherDeliv . 2011 ; 2 : 727 - 36 .
65. Boswell CA , Tesar DB , Mukhyala K , Theil F-P , Fielder PJ , Khawli LA . Effects of charge on antibody tissue distribution and pharmacokinetics . Bioconjug Chem . 2010 ; 21 : 2153 - 63 .
66. Reed RK , Woie K , Rubin K. Integrins and Control of Interstitial Fluid Pressure News . J Physiol Sci . 1997 ; 12 : 42 - 8 .
67. Dongaonkar RM , Laine GA , Stewart RH , Quick CM . Balance point characterization of interstitial fluid volume regulation . Am J Phys Regul Integr Comp Phys . 2009 ; 297 ( 1 ): R6 - R16 .
68. Jin J-F , Zhu LL , Chen M , Xu H-M , Wang H-F, Feng X-Q , et al. The Optimal Choice of Medication Administration Route Regarding Intravenous , Intramuscular, and Subcutaneous Injection . Patient Pref Adh . 2015 ; 9 : 923 - 42 .
69. Martin JR , Beegle NL , Zhu Y , Hanisch EM . Subcutaneous Administration of Bortezomib: A Pilot Survey of Oncology Nurses . J Adv Pract Oncol . 2015 ; 6 : 308 - 18 .
70. Beshyah SA , Anyaoku V , Niththyananthan R , Sharp P , Johnston DG . The effect of subcutaneous injection site on absorption of human growth hormone: abdomen versus thigh . Clin Endocrinol . 1991 ; 35 : 409 - 12 .
71. Macdougall IC , Jones JM , Robinson MI , Miles JB , Coles GA , Williams JD . Subcutaneous erythropoietin therapy: comparison of three different sites of injection . Contrib Nephrol . 1991 ; 88 : 152 - 6 .
72. ter Braak EW , Woodworth JR , Bianchi R , Cerimele B , Erkelens DW , Thijssen JH , et al. Injection site effects on the pharmacokinetics and glucodynamics of insulin lispro and regular insulin . Diabetes Care . 1996 ; 19 : 1437 - 40 .
73. Yuan Z , Chen L , Luo Q , Zhu J , Lu H , Zhu R . The role of radionuclide lymphoscintigraphy in extremity lymphedema . Ann Nucl Med . 2006 ; 20 ( 5 ): 341 - 4 .
74. Tourani SS , Taylor GI , Ashton MW . Scarpa Fascia Preservation in Abdominoplasty: Does it Preserve the Lymphatics? PRS. 2015 ; 136 ( 2 ): 258 - 62 .
75. Kalawat TC , Chittoria RK , Reddy PK , Suneetha B , Narayan R , Ravi P. Role of lymphoscintigraphy in diagnosis and management of patients with leg swelling of unclear etiology . Indian J Nucl Med . 2012 ; 27 ( 4 ): 226 - 30 .
76. Berteau C , Filipe-Santos O , Wang T , Rojas HE , Granger C , Schwarzenbach F. Evaluation of the Impact of Viscosity, Injection Volume and Injection Flow Rate on Subcutaneous Injection Tolerance . Med Devices . 2015 ; 8 : 473 - 84 .
77. Palmer AK , Kirkland JL . Aging and adipose tissue: potential interventions for diabetes and regenerative medicine . Exp Gerontol . 2016 ; 86 : 97 - 105 .
78. Matuoka K , Hasegawa N , Namba M , Smith GJ , Mitsui Y. A decrease in hyaluronic acid synthesis by aging human fibroblasts leading to heparan sulfate enrichment and growth reduction . Aging . 1989 ; 1 ( 1 ): 47 - 54 .
79. Papakonstantinou E , Roth M , Karakiulakis G . Hyaluronic acid: A key molecule in skin aging . Dermato Endocrinol . 2012 ; 4 ( 3 ): 253 - 8 .
80. Garg SK , Delaney C , Shi H , Yung R . Changes in adipose tissue macrophages and T cells during aging . Crit Rev Immunol . 2014 ; 34 : 1 - 14 .
81. Mangoni AA , Jackson SHD . Age-related changes in pharmacokinetics and pharmacodynamics: basic principles and practical applications . Br J Clin Pharmacol . 2003 ; 57 ( 1 ): 6 - 14 .
82. Fathallah AM , Balu-Iyer SV . Anatomical, Physiological, and Experimental Factors Affecting the Bioavailability of scAdministered Large Biotherapeutics . J Pharm Sci . 2015 ; 104 : 301 - 6 .
83. Bumbaca D , Boswell CA , Fielder P , Khawli L . Physiochemical and biochemical factors influencing the pharmacokinetics of antibody therapeutics . AAPS J . 2012 ; 14 : 554 - 8 .
84. Bocci V , Muscettola M , Grasso G , Magyar Z , Naldini A , Szabo G. The lymphatic route. 1 Albumin and hyaluronidase modify the normal distribution of interferon in lymph and plasma . Experientia . 1986 ; 42 ( 4 ): 432 - 3 .
85. Hilger C , Grigioni F , De Beaufort C , Michel G , Freilinger J , Hentges F. Differential binding of IgG and IgA antibodies to antigenic determinants of bovine serum albumin . Clin Exp Immunol . 2001 ; 123 ( 3 ): 387 - 94 .
86. Yadav S , Shire SJ , Kalonia DS . Viscosity behavior of highconcentration monoclonal antibody solutions: correlation with interaction parameter and electroviscous effects . J Pharm Sci . 2012 ; 101 ( 3 ): 998 - 1011 .
87. Chow C-K , Allan BW , Chai Q , Atwell S , Lu J . Therapeutic Antibody Engineering To Improve Viscosity and Phase Separation Guided by Crystal Structure . Mol Pharm . 2016 ; 13 ( 3 ): 915 - 23 .
88. Pindrus M , Shire SJ , Kelley RF , Demeule B , Won R , Xu Y , et al. Solubility Challenges in High Concentration Monoclonal Antibody Formulations: Relationship with Amino Acid Sequence and Intermolecular Interactions . Mol Pharm . 2015 ; 12 ( 11 ): 3896 - 907 .
8 9 . Du Y , W a ls h A , E h r i ck R , X u W , M ay K , L i u H. Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies . MAbs . 2012 ; 4 ( 5 ): 578 - 85 .
90. Filoti DI , Shire SJ , Yadav S , Laue TM . Comparative Study of Analytical Techniques for Determining Protein Charge . J Pharm Sci . 2015 ; 104 : 2123 - 31 .
91. Gokarn YR , Fesinmeyer RM , Saluja A , Razinkov V , Chase SF , Laue TM , et al. Effective charge measurements reveal selective and preferential accumulation of anions, but not cations, at the protein surface in dilute salt solutions . Protein Sci . 2011 ; 20 ( 3 ): 580 - 7 .
92. Scapin G , Yang X , Prosise WW , McCoy M , Reichert P , Johnston JM , et al. Structure of full-length human anti-PD1 therapeutic IgG4 antibody pembrolizumab . Nat Struct Mol Biol . 2015 ; 22 : 953 - 8 .
93. Laue T. Charge matters . Biophys Rev . 2016 ; 8 : 287 - 9 .
94. Hill JJ , Laue TM . Protein Assembly in Serum and the Differences from Assembly in Buffer . Methods Enzymol . 2015 ; 562 : 501 - 27 .
95. Yadav S , Laue TM , Kalonia DS , Singh SN , Shire SJ . The Influence of Charge Distribution on Self-Association and Viscosity Behavior of Monoclonal Antibody Solutions . Mol Pharm . 2012 ; 9 : 791 - 802 .
96. Roberts CJ . Therapeutic protein aggregation: mechanisms, design, and control . Trends Biotechnol . 2014 ; 7 : 372 - 80 .
97. Ghosh R , Calero-Rubio C , Saluja A , Roberts CJ . Relating Protein-Protein Interactions and Aggregation Rates From Low to High Concentrations . J Pharm Sci . 2016 ; 105 ( 3 ): 1086 - 96 .
98. Yang MX , Shenoy B , Disttler M , Patel R , McGrath M , Pechenov S , et al. Crystalline Monoclonal Antibodies for Subcutaneous Delivery . Proc Natl Acad Sci U S A . 2003 ; 100 : 6934 - 9 .
99. Johnson HR , Lenhoff AM . Characterization and Suitability of Therapeutic Antibody Dense Phases for Subcutaneous Delivery . Mol Pharm . 2013 ; 10 : 3582 - 91 .
100. Gokarn YR , McLean M , Laue TM . Effect of PEGylation on protein hydrodynamics . Mol Pharm . 2012 ; 9 : 762 - 73 .
101. Chan LJ , Bulitta JB , Ascher DB , Haynes JM , McLeod VM , Porter CJH , et al. PEGylation Does Not Significantly Change the Initial Intravenous or Subcutaneous Pharmacokinetics or Lymphatic Exposure of Trastuzumab in Rats but Increases Plasma Clearance after Subcutaneous Administration . Mol Pharm . 2015 ; 12 ( 3 ): 794 - 809 .
102. Zhao Y , Wang C , Wang L , Yang Q , Tang W , She Z , et al. A frustrating problem: accelerated blood clearance of PEGylated solid lipid nanoparticles following subcutaneous injection in rats . Eur J Pharm Biopharm . 2012 ; 81 ( 3 ): 506 - 13 .
103. Weidenhaupt M , BenKhalifa M , Hugo N , Choulier L , Altschuh D , Vernet T. Functional mapping of conserved, surface-exposed charges of antibody variable domains . J Mol Recognit . 2002 ; 15 : 94 - 103 .
104. Li L , Kumar S , Buck PM , Burns C , Lavoie J , Singh SK , et al. Concentration Dependent Viscosity of Monoclonal Antibody Solutions: Explaining Experimental Behavior in Terms of Molecular Properties. Pharm Res . 2014 ; 31 : 3161 - 78 .
105. Roberts D , Keeling R , Tracka M , VanderWalle CF , Uddin S , Warwicker J , et al. The Role of Electrostatics in Protein−Protein Interactions of a Monoclonal Antibody . Mol Pharm . 2014 ; 11 : 2475 - 89 .
106. Brandt JP , Patapoff TW , Aragon SR . Construction, MD Simulation, and Hydrodynamic Validation of an All-Atom Model of a Monoclonal IgG Antibody . Biophys J . 2010 ; 99 ( 3 ): 905 - 13 .
107. Wang W , Singh S , Zeng DL , King K , Nema S . Antibody structure, instability, and formulation . J Pharm Sci . 2007 ; 96 : 1 - 26 .
108. Eberlin LS , Mulcahy JV , Tzabazis A , Zhang J , Liu H , Logan MM , et al. Visualizing Dermal Permeation of Sodium Channel Modulators by Mass Spectrometric Imaging . J Am Chem Soc . 2014 ; 136 : 6401 - 5 .
109. Barbe P , Millet L , Galitzky J , Lafontan M , Berlan M. In situ assessment of the role of the β1, β2 and β3 adrenoceptors in the control of lipolysis and nutritive blood flow in human subcutaneous adipose tissue . Br J Pharmacol . 1996 ; 117 : 907 - 13 .
110. Pivot X , Gligorov J , Müller V , Barrett-Lee P , Verma S , Knoop A , et al. Preference for Subcutaneous or Intravenous Administration of Trastuzumab in Patients with HER2-Positive Early breast cancer (PrefHer): An Open-Label Randomised Study . Lancet Oncol . 2013 ; 14 ( 10 ): 962 - 70 .
111. Salar A , Avivi I , Bittner B , Bouabdallah R , Brewster M , Catalani O , et al. Comparison of Subcutaneous Versus Intravenous Administration of Rituximab As Maintenance Treatment for Follicular Lymphoma: Results From a Two-Stage, Phase IB Study . J Clin Oncol . 2014 ; 32 : 1782 - 91 .
112. Perraudin C , Bourdin A , Spertini F , Berger J , Bugnon O. S w i t c h i n g P a t i e n t s t o H o m e - B a s e d S u b c u t a n e o u s I m m u n o g l o b u l i n : a n E c o n o m i c E v a l u a t i o n o f a n Interprofessional Drug Therapy Management Program . J Clin Immunol . 2016 ; 36 ( 5 ): 502 - 10 .
113. Richter WF , Jacobsen B . Subcutaneous Absorption of Biotherapeutics: Knowns and Unknowns . Drug Metab Dispos . 2014 ; 42 : 1881 - 9 .
114. Jin W-L. Continuous kinematic wave models of merging traffic flow . Transp Res B Methodol . 2010 ; 44 : 1084 - 103 .
115. Lian X , Liu Z , Wang Z. A Modified T-S Model Fuzzy Adaptive Control System Based on Genetic Algorithm . Int J Inf Tech Comput Sci . 2011 ; 3 : 8 - 14 .
116. Skaggs RW , Breve MA , Gilliam JW . Hydrologic and water quality impacts of agricultural drainage . Crit Rev Environ Sci Technol . 1994 ; 24 ( 1 ): 1 - 32 .
117. Wilinska ME , Chassin LJ , Acerini CL , Allen JM , Dunger DB , Hovorka R. Simulation Environment to Evaluate Closed-Loop Insulin Delivery Systems in Type 1 Diabetes . J Diabetes Sci Technol . 2010 ; 4 ( 1 ): 132 - 44 .
118. Dias C , Abosaleem B , Crispino C , Gao B , Shaywitz A . Tolerability of High-Volume Subcutaneous Injections of a Viscous Placebo Buffer: A Randomized, Crossover Study in Healthy Subjects . AAPS Pharm Sci Tech . 2015 ; 16 : 1101 - 7 .
119. Takeyama M , Ishida T , Kokubu N , Komada F , Iwakawa S , Okumura K , et al. Enhanced bioavailability of subcutaneously injected insulin by pretreatment with ointment containing protease inhibitors . Pharm Res . 1991 ; 8 ( 1 ): 60 - 4 .
120. Jayant RD , McShane MJ , Srivastava R . Polyelectrolyte-coated alginate microspheres as drug delivery carriers for dexamethasone release . Drug Deliv . 2009 ; 16 ( 6 ): 331 - 40 .
121. Mullard A. Robust Biotech Sector Increases R&D Spend . Nat Rev Drug Discov . 2015 ; 14 : 449 .