TagedEnJournal of Pharmaceutical Sciences 112 (2023) 1492−1508

Contents lists available at ScienceDirect

TagedFiur TagedEn Journal of Pharmaceutical Sciences TagedFiur TagedEn

jo urn a l h om ep ag e : w ww.jp harms ci.o rg

Review

TagedH1Simulate SubQ: The Methods and the MediaTagedEn

TagedPDavid Li, Poh Yee Chow, Tzu Ping Lin, Celine Cheow, Zhuoxuan Li,
Matthias G. Wacker*TagedEn
TagedPNational University of Singapore, Faculty of Science, 4 Science Drive 2, 117544 Singapore
TagedEn
TAGEDPA R T I C L E I N F O TAGEDPA B S T R A C T

Article history: For decades, there has been a growing interest in injectable subcutaneous formulations to improve the
Received 16 August 2021 absorption of drugs into the systemic circulation and to prolong their release over a longer period. However,
Revised 25 October 2021 fluctuations in the blood plasma levels together with bioavailability issues often limit their clinical success.
Accepted 26 October 2021
This warrants a closer look at the performance of long-acting depots, for example, and their dependence on
Available online 30 October 2021TagedEn
the complex interplay between the dosage form and the physiological microenvironment. For this, biopredic-
TagedEnTagedPKeywords: tive performance testing is used for a thorough understanding of the biophysical processes affecting the
absorption of compounds from the injection site in vivo and their simulation in vitro. In the present work, we
Drug release
Dissolution testing
discuss in vitro methodologies including methods and media developed for the subcutaneous route of admin-
Performance testing istration on the background of the most relevant absorption mechanisms. Also, we highlight some important
Biorelevant knowledge gaps and shortcomings of the existing methodologies to provide the reader with a better under-
Biopredictive standing of the scientific evidence underlying these models.
Depot formulation © 2021 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.TagedEn
Long-acting injectables
Parenterals
Drug delivery
Drug absorption
ADME
In vitro-in vivo correlation (IVIVC)
Lymphatic uptake
Subcutaneous (SC)
Absorption
Non-oral complex drugs TagedEn

TagedH1IntroductionTagedEn agedEnTPSC delivery can be a convenient and effective alternative.1−3 Also,

TagedPIn recent years, drug formulations for subcutaneous (SC) injec-
tion have experienced a surge in demand due to a number of
advantages as compared to other dosage forms. From a drug
development perspective, drugs with poor aqueous solubility,
permeability, or stability issues in the gastrointestinal tract are
often limited by their peroral bioavailability. For these candidates,
TagedEnAbbreviations: DR, Dispersion Releaser; ECM, Extracellular matrix; FBS, Fetal bovine
serum; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IVIS, In vivo imag-
ing system; IVIVC, In vitro-in vivo correlation; MbCD, Methyl-b-cyclodextrin; NMP, N-
methyl-2-pyrrolidone; PBS, Phosphate buffered saline; PCL, Poly(e-caprolactone); PEG,
Polyethylene glycol; PLA, Poly(D,L-lactic acid); PLA-PCL, Poly(D,L-lactide-co-e-capro-
lactone); PLGA, Poly(D,L-lactic-co-glycolic acid); PLLA, Poly(L-lactic acid); PVA, Polyvi-
nyl alcohol; RES, Reticuloendothelial system; SBF, Simulated body fluid; SC,
subcutaneous(ly); SCISSOR, Subcutaneous Injection Site Simulator; SDS, Sodium
dodecyl sulphate; SSIF, Simulated subcutaneous interstitial fluid.
TagedEn* Corresponding author.
E-mail address: matthias.g.wacker@nus.edu.sg (M.G. Wacker).
the SC tissue often serves as a depot, leading to prolonged-release
intervals and reduced dosing frequency while avoiding extensive
first-pass metabolism.1 Furthermore, when combined with suit-
able devices, the formulation can be administered by the patient
and does not require healthcare professionals. They increase
patient compliance and reduce hospitalization costs.1,4 Although
the SC route of administration offers exciting opportunities in
drug delivery, pharmaceutical development is accompanied by
many challenges. So far, a widely recognized and validated pre-
clinical model to predict the absorption and bioavailability in
humans has yet to be established. Biopredictive in vitro perfor-
mance assays often fail to mimic the complexity of the in vivo
absorption process and safety characteristics.5,6 Also, many of the
known influences on drug absorption come with high uncertainty
due to the significant interspecies and interindividual differences
as only a small minority of literature studies are currently based
on larger animals or humans. Most information on the SC route
of administration is obtained from rodents (Fig. 1, left).TagedEn

https://doi.org/10.1016/j.xphs.2021.10.031
0022-3549/© 2021 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.
TagedEn TagedFiur TagedEnD. Li et al. / Journal of Pharmaceutical Sciences 112 (2023) 1492−1508
Figure 1. Of 295 articles listed on Pubmed (2011-2021) under the keywords “subcutaneous injection” and “formulation”, we evaluated a total of 136 randomly selected full-
research articles for the species used to investigate drug delivery of poorly soluble compounds (left), and the studied influences affecting this process in the SC tissue (right). The
number of review articles in this time interval was 36.TagedEn
TagedPThese knowledge gaps can put drug development but also the test
subjects at risk.7 They complicate translation to the clinical stages.5
Therefore, a better understanding of the various influences of the
dosage form on bioavailability and safety characteristics is required.
Improved prediction of events such as the initial burst release or the
lymphatic uptake of fine particles into the systemic circulation would
improve the translation from bench to bedside and ensure that more
candidates meet the regulatory and therapeutic requirements.8−11TagedEn
TagedPThe interspecies differences in the SC environment between
humans and animals result in conflicting outcomes and unpredict-
able pharmacokinetics.12,13 This diversity is reflected by the perfor-
mance assays as well. In the present work, we critically review the
current knowledge on absorption processes relevant for the SC route
of administrations. Emphasizing the physiological processes involved
in drug release and absorption, we highlight challenges and consider-
ations in the development of biopredictive performance assays. Fur-
ther, we lay open critical knowledge gaps and discuss open questions
in this research area.TagedEn
TagedH1Influences on Drug AbsorptionTagedEn
TagedPTo successfully develop drug products for SC injection, a careful
evaluation of all physicochemical characteristics involved in the
absorption process is required. Critical material attributes can have a
strong impact on the interactions of the dosage form with the physio-
logical microenvironment. They include parameters such as the
aqueous solubility in physiological fluids, the buffer capacity at the
injection site as well as physical or chemical stability. Also, the parti-
cle or molecular size of the formulation can have a strong influence
on the mobility of the drug in the interstitial space.14 To provide a
basis for our critical analysis, we evaluated articles from current liter-
ature dealing with SC delivery of poorly soluble compounds (Fig. 1,
right). Among all the influences on drug absorption, the rate or extent
of absorption, release kinetics of drugs from their delivery system,
the interaction of formulations with the lymphatic system as well as
the biocompatibility of the material were explored. For most biophar-
maceuticals, the chemical stability of the compound together with
the uptake by the reticuloendothelial system (RES) rather than the
aqueous solubility are limiting bioavailability. This includes larger
proteins such as antibodies but also nucleic acids and peptides. While
large proteins and nucleic acids have a higher risk of being recog-
nized by the immune system, peptides are sensitive to enzymes in
1493
TagedEnPthe SC tissue. Therefore, metabolic stability has to be considered as
one of the parameters of interest as well.TagedEn
TagedPPutting more focus on the administration site itself, the SC tissue
is characterized by the presence of mostly adipocytes, with access to
the blood capillaries and lymphatic vasculature,15 while the intersti-
tium, amongst other components, contains plasma proteins at a
lower concentration as compared to the vascular system.16 Being
more poorly perfused than the intramuscular environment, the SC
tissue enables drugs to be formulated into SC depots and sustained-
release formulations that are slowly absorbed into the systemic circu-
lation by passive diffusion.17 An illustration of the administration site
and the most important mechanisms involved in the in
vivo absorption process is presented in Fig. 2, providing the base for a
more thorough discussion in the following sections.TagedEn
TagedH2Lymphatic UptakeTagedEn
TagedPWhile many biopharmaceuticals such as large proteins or nucleic
acids are characterized by a high molecular weight, poorly soluble
compounds are often formulated into particles, vesicles, or micelles
to improve their solubility at the injection site. This includes the
encapsulation into liposomes,18 microsizing or nanosizing of the
crystalline drug,19 or the covalent modification of drugs by attaching
them to drug carriers such as dendrimers. The latter sometimes
includes the use of macromolecules.18 Generally speaking, macromo-
lecules with a molecular weight larger than 1 kDa have limited
mobility in the SC tissue. The absorption into blood vessels is much
slower as compared to smaller molecules. While for molecules with a
size below 100 nm, absorption into the lymphatic system is a prefera-
ble pathway, from where they eventually drain into the systemic cir-
culation at the thoracic lymph duct.16 In this section, we will look at
the factors that affect the lymphatic uptake and lymph node localiza-
tion, which may consequently influence the amount of drug that can
reach the systemic circulation. Firstly, a process that affects whether
the drug reaches the systemic circulation from the lymphatic system
is the susceptibility of these particles to phagocytosis by the macro-
phages from the RES. Foreign particles in the body are detected by
macrophages through their particle size, surface charge, and hydro-
phobicity when they differ largely from the endogenous compounds,
as part of the immune system to defend against pathogens. Thus, if
these signature recognition points can be concealed, phagocytosis
can be reduced. Using PEGylation, a process involving the attachment
 TagedEn TagedFiur
TagedEn1494 D. Li et al. / Journal of Pharmaceutical Sciences 112 (2023) 1492−1508
Figure 2. Illustration of the subcutaneous injection site including mechanisms involved in the absorption process. This illustration was created with BioRender (www.biorender.com).
TagedEn
TagedEnPof polyethylene glycol (PEG) molecules to the particle surface, lym-
phatic drainage can be improved as it can reduce the susceptibility of
nanoparticles, for example, to phagocytosis by macrophages. The
hydrophilic surface structure prevents opsonization by reducing the
binding of proteins that stimulate immune responses. It has been
demonstrated that the PEGylation of various drug carriers such as lip-
osomes, solid lipid nanoparticles, and dendrimers was able to reduce
phagocytic uptake.20−22 Besides PEGylation, a similar outcome was
also achieved through the incorporation of GM1 gangliosides into the
composition of liposomes,23 due to the presence of sialic acid which
was also able to impart hydrophilicity onto the nanoparticle surfaces
for RES evasion.24TagedEn
TagedPOther parameters that affect the surface structure and reduce
hydrophobic interactions are surfactants such as poloxamers and
poloxamines. They aid drug molecules in their movement through
the hydrophilic water channels in the interstitium.21,25,26 With
increased diffusion through the interstitium, more formulations are
thus likely to enter the lymphatic system, and subsequently, drain
into the systemic circulation.TagedEn
TagedPThe particle or molecular size of drugs and drug carriers affect
lymphatic uptake as well. Larger liposomes commonly lead to a low-
ered lymphatic uptake. This is likely due to the limited size of water
channels in the interstitium. They create a filter that particles have to
pass through to reach the lymphatic system.27 While molecules
smaller than 10 nm are more likely to be absorbed through blood
capillaries,28 the upper cut-off size for lymphatic uptake has been
reported differently in a few studies, with two review papers
referencing studies using rats and mice in their analysis concluding
the cut-off size to be 100 nm and 150 nm each,29,30 while primary lit-
erature using mice reported a cut-off size between 110 nm to
120nm.31 Overall, there seems to be a consensus that the cut-off is at
TagedEnPapproximately 100 nm,16,32−34 with another study specifying that
the optimal size for lymphatic uptake is between 10 to 50 nm.35 Pri-
marily rodent models were used to study lymphatic uptake, except
for one review34 which referenced a paper using a porcine model.36
The porcine skin is structurally more similar to the human skin as
compared to that of rodents.37 Since the studies using rodents largely
concur with the results of the study using a porcine model, the con-
clusion made with the porcine model can serve as supporting evi-
dence that the optimal size for lymphatic uptake may be between 10
and 100 nm. Though more research is still required to directly corre-
late this data to humans.TagedEn
TagedPWhile we can assume that an optimal size can lead to an increase in
lymphatic uptake, aforementioned modifications and exceptions need
to be remembered and its general impact on the overall absorption into
the blood circulations needs to be discussed. On one hand, a study in
mice highlighted that the size and surface modification of liposomes
with PEG altered drug absorption. Smaller liposomes and the presence
of PEG on the surface led to higher blood levels as compared to larger
liposomes and liposomes without PEG, respectively.31 On the other
hand, another study using rats concluded that lymphatic uptake is
unlikely to have a significant impact on systemic absorption,21 concur-
ring with Oussoren et al. that higher lymphatic uptake does not neces-
sarily correlate with an increase in blood concentration.38TagedEn
TagedPWith regards to liposomes, the drainage of drugs and their formu-
lations into the systemic circulation may furthermore be affected by
the exact injection site, liposomal bilayer fluidity, and the type of lip-
ids used in the formulation.27,39 Many of the abovementioned studies
used rodents to determine the impact of the lymphatic system on
drug absorption, some of them with conflicting outcomes. One reason
for this may be the aforementioned injection site. Allen et al. injected
liposomes into the neck or upper back just below the neck of the
 TagedEnD. Li et al. / Journal of Pharmaceutical Sciences 112 (2023) 1492−1508
TagedEnPanimal.31 In two other studies,21,31 the drug formulations were
injected into the hind legs and dorsal side of the foot, respectively.
Overall, the dorsolateral areas of the neck or back are among the pre-
ferred sites for SC injection in rats.40 As such, results from the earlier
study resulting in higher blood levels may be more reliable for our
interpretation.31TagedEn
TagedPIn conclusion, a carefully selected particle size, as well as surface
modifications with PEG, seem to increase absorption from the SC tis-
sue into the blood circulation, potentially through lymphatic uptake.
This is reflected by the most successful formulation strategies as well.
Surface PEGylation has become a common strategy to improve the
stability and absorption of large and medium-size proteins such as
interferon.41,42 Lymphatic drug exposure itself remains complex and
whether the abovementioned animal models are able to predict lym-
phatic distribution in humans is a topic that still requires more
research. Though, with the rise of lymphatic imaging techniques, pre-
viously invasive methods can be replaced to gain more insight and
enable more representative studies designed on humans in the near
future. In recent years, such techniques have already been used to
study the biodistribution of drugs from SC depots in animals43 as
well as in humans.44TagedEn
TagedH2Tissue Retention and Molecular MobilityTagedEn
TagedPIn addition to the lymphatic drainage of particles and mole-
cules, the bioavailability of therapeutics is also affected by the
retention of the drug in the SC tissue. Particles in the micrometer
range are known to remain at the injection site and exhibit poor
mobility while smaller particles undergo a distribution process
towards the systemic circulation. Diffusion properties are strongly
affected by the chemical structure and the excipients used in the
formulation. Introducing a negative charge to the carrier or the
molecule strengthens the repulsion forces between the formulation
and the negatively charged glycosaminoglycans or chondroitin
present in the SC tissue.45,46 Chondroitin has two ionizable groups
per repeated unit, comprising both carboxylate and sulfate moie-
ties (carboxylate pKa 3-5, sulfate pKa 1.5-2).47 Hyaluronic acid
exhibits a pKa of 2.9 at physiological pH. Although hyaluronan rep-
resents only 1% of the concentration of collagen in the skin, the
fluid exclusion volume potential is much higher as compared to
collagen.48,49 For negatively charged antibodies reduced endocyto-
sis and higher bioavailability have been reported.50 On the con-
trary, positively charged molecules often bind to the extracellular
matrix, leading to a delay in the absorption rate.51−53TagedEn
TagedPHydrophilic drugs tend to appear in the blood plasma more rapidly
due to their improved solubility and rapid diffusion through the SC
environment. For many biopharmaceuticals such as small peptides and
medium-size proteins, this increases absorption and bioavailability.54,55TagedEn
TagedPFor larger proteins such as antibodies, the molecular weight leads
to significant retention at the administration site. The three-dimen-
sional network formed by the extracellular matrix (ECM) reduces the
mobility of the molecule and, hence, allows enzymes to degrade the
drug before it appears in the blood plasma. The abovementioned
modification of proteins with PEG is a common strategy to improve
the stability of compounds. However, previous investigations
reported minor alterations in the molecular weight (PEG30 to PEG40)
having a strong impact on SC absorption.56 This change is due to the
altered tissue retention and diffusion rate and affects particles as
well.57 One major difference between the SC and the intramuscular
route of administration is the degree of vascularization. As a conse-
quence, the diffusion pathway of SC formulations is considerably lon-
ger resulting on average in lower bioavailability. A similar effect is
found for different injection sites.58TagedEn
TagedPHydrophobic interactions often lower the absorption.46 On one
hand, the poor aqueous solubility of these compounds decreases
1495
TagedEnPbioavailability. On the other hand, the presence of adipose tissue con-
tributes to a stronger partitioning effect.46,59 In summary, tissue
retention is a strong impact on absorption that needs to be consid-
ered.TagedEn
TagedH2Release KineticsTagedEn
TagedPFor many injectable formulations, the drug release from the car-
rier limits the bioavailability of the compound in systematic circula-
tion. The mechanism of release can be governed by various processes
such as the degradation or erosion of the carrier, the swelling, and
wetting of the matrix material, or the diffusion of excipients and the
drug through the SC tissue.8,60,61TagedEn
TagedPDrug diffusion is affected by the concentration gradient between
the injection site and the physiological microenvironment. Conse-
quently, the partitioning between adipocytes and the interstitial
space as well as the plasma protein binding can play an important
role. Critical changes in the degradation or diffusion processes will
affect the release rate. With regards to polymeric carriers and lipo-
somes, the degradation may occur through hydrolysis or enzymatic
degradation into smaller units of monomers or oligomers.60 The deg-
radation rate depends on the composition and structure of the matrix
in various types of drug carriers.33,62,63 For example, the use of chito-
san/poly(L-lactic acid) (PLLA) composite microspheres increased the
rate of degradation of the microspheres by hydrolysis. The positively
charged chitosan led to pore formation in the microparticle system.
This increased the surface area and thus the access of water mole-
cules to the polymeric matrix.62 In other cases, the degradation rate
was affected by the type of drug incorporated in the drug carriers as
well.64 The pore size of microparticles composed of poly(lactic-co-
glycolic acid) (PLGA) is commonly controlled by the ratio of lactide to
glycolide with a strong influence on water diffusion, hydrolysis, and
release behavior.65,66 When hydrophilic compounds were incorpo-
rated into these drug carriers, they increased the rate of hydrolysis
by facilitating the diffusion of water into the matrix. This has been
observed for small-molecular drugs aspirin and ibuprofen.64 Con-
versely, an increase in the ratio of hydrophobic lactide to glycolide in
a PLGA matrix reduces water diffusion, and thus hydrolytic degrada-
tion of the copolymer.64 Degradation of poly(D,L-lactic acid) (PLA)
and PLGA involves autocatalysis in a low pH environment within the
polymeric matrix following hydrolysis, due to the release of acidic
monomers upon hydrolysis of the polymers.67TagedEn
TagedPThe fluid volume and buffer capacity in the SC tissue are very lim-
ited. Therefore, the impact of the formulation on the pH is more pro-
nounced as compared to other administration routes. Other factors
that can contribute to a low pH environment would be the use of
acidic drugs or polymers in the formulation.8 A reduced pH has been
associated with a change in the release kinetics and is partially
affected by tissue changes. The effect was reported for implant for-
mulations composed of PLGA.68TagedEn
TagedPSo far, there is limited evidence for the autocatalysis of basic
drugs.65,69,70 However, porosity8 and dimensions of drug carrier71
have an influence on the diffusion of acidic degradation products out
of their matrices. The reduced diffusion causes an accumulation of
acidic compounds and enhances biodegradation. These effects have
been reported for a variety of PLGA and PLA polymers.72 As such,
autocatalysis contributes more significantly to the degradation of
larger and non-porous microparticles73 and implants72. Furthermore,
an evaluation of different mathematical models highlighted that
accounting for autocatalysis increased the predictability of drug
release kinetics.71TagedEn
TagedPThe use of chitosan in chitosan/PLLA composite microspheres
increased the buffer capacity, compensating for the hydrolysis of
PLLA to lactic acid. This led to a slower decrease in pH. Despite this
reduction in autocatalysis, the rate of degradation of composite
TagedEn1496 D. Li et al. / Journal of Pharmaceutical Sciences 112 (2023) 1492−1508
TagedEnPmicrospheres was still higher as compared to pure PLLA micro-
spheres. The availability of water due to the improved pore formation
outweighs the influence of pH in vitro. An in vivo experiment con-
ducted in rodents further confirmed the preferential dissolution of
chitosan over PLLA in a physiological environment.62TagedEn
TagedPThe next process that affects drug release is drug diffusion. It
can be delayed through stronger drug-drug interaction, drug-
matrix interactions, and larger particle sizes.65 Varying the
strength of the interactions between the drug and its carrier has
been demonstrated to vary the rate of release.63,74,75 However, the
parameter depends on multiple interactions of the drug delivery
system, and measuring the impact of the inherent binding forces is
challenging. A study comparing the drug release of beta-blockers
with similar molecular weights and pKa, but varying in their lipo-
philicities showed that despite the difference in drug release rate,
the difference in the rate of absorption was not statistically
significant.76TagedEn
TagedPOne major shortcoming of SC drug formulations is the occur-
rence of the “burst effect”. Sometimes, it is affected by the biodeg-
radation of the drug carrier and the diffusion behavior of the
drug. A very common explanation is the heterogeneous distribu-
tion of drug molecules in the matrix material, where each phase
comes with a distinct release and absorption behavior.77 Drug at
the surface of the carrier is released much more rapidly than
drug encapsulated into the core.63,78 The distribution widely
depends on the encapsulation process but may also be affected
by the storage conditions.79 This effect can be countered through
the creation of additional diffusion barriers.10 However, the burst
effect that is often held responsible for an initial increase in the
blood plasma concentration is only one puzzle piece of the initial
absorption and can overlap with lymphatic uptake or other
parameters discussed in this article.TagedEn
TagedH2Biocompatibility and Responses of the Immune SystemTagedEn
TagedPThe biocompatibility of formulations sometimes has a certain influ-
ence on drug absorption. Poor biocompatibility potentially induces
inflammation and may lead to drastic changes in the SC microenviron-
ment. These reactions are often caused by formulations with high con-
centrations of solubility enhancers. Co-solvents or complexation agents
are amongst the most critical excipients as common aims of formula-
tion development include the solubilization of poorly soluble com-
pounds.80 For those candidates, precipitation may occur when
supersaturated solutions are injected into the physiological environ-
ment. The diffusion of excipients such as water, buffer salts, solvents,
and co-solvents, from the injection site into the surrounding tissues
reduces the physical stability of the formulation14 and sometimes
causes rapid crystallization of the drug. As a consequence, inflammation
at the injection site may occur. However, supersaturated solutions do
not necessarily precipitate. The induction time, which is the time before
precipitate formation, is negatively correlated to the extent of supersat-
uration.80 Phospholipid formulations such as liposomes81 but also high
concentrations of colloid stabilizers with high affinity to the particle
surface are likely to reduce such precipitation effects.TagedEn
TagedPInflammation can also be caused by penetration trauma, pH
changes in the SC tissue, charge interactions, the high osmotic activ-
ity of the dosage form, or the released drug itself.82−85 The latter can
happen with biopharmaceuticals such as larger proteins or nucleic
acids in particular. Though in another example, the release of naltrex-
one from PLGA-based drug carriers in rats and rabbits has led to a sta-
tistically different inflammatory response as compared to placebo.
Naltrexone was the main contributor to the inflammatory response
rather than the degradation products of PLGA.83 The extent of inflam-
mation was reduced when the drug was administered at a lower
concentration.86TagedEn
TagedPInterestingly, the release of histamine and serotonin following the
injection trauma was found responsible for reduced absorption. This
phenomenon was observed in rats only.87 However, histamine indu-
ces vasodilation of small arteries88 and increases permeability89 in
the SC tissue of humans. Still, the effects of histamine on the vascular
tone vary between different species and can hardly be predicted
based on rodent studies alone.88 In some studies, antihistamines
have been used to reduce allergy-related side effects following the
injection of insulin.90 So far, there is limited information on the
effects of these treatments on drug absorption. However, future
research could consider monitoring the effect of antihistamines more
carefully. They are often used as a co-treatment and could provide
unexpected insights.TagedEn
TagedPOn another note, long-term exposure to foreign particles, in
general, can lead to long-lasting inflammation causing the forma-
tion of fibrotic tissue or a fibrous capsule. This is especially pro-
nounced for non-degradable materials.91 It is generally seen as an
indication of poor biocompatibility of the material that occurs as
part of an inflammatory response.92 This has been observed for
implanted microspheres,78 devices,93 but also proteins or nucleic
acids.94,95 The effect occurs two to three weeks after the adminis-
tration of implants96 and was found to pose a barrier to drug
absorption.96 Therefore, most implants and formulations change
their absorption behavior one to four weeks after implantation.97
The thickness of the capsule increases with the implant thickness
or diameter. This translates into a longer diffusion distance and
greater diffusion resistance respectively. Although the formation of
such a capsule may lead to prolonged-release intervals, local fibro-
sis must be considered a serious and widely unpredictable adverse
reaction. The expected interpatient variability and physiological
consequences of the fibrosis (e.g., formation of scars) do not allow
the use of this effect in drug delivery.TagedEn
TagedPAt last, PEGylation of biomolecules may also cause activation of
immune cells. The responses to such an adverse reaction are often
unpredictable as well and require additional immunogenicity assays
to elucidate the pharmacological responses to the delivery system.
These assays are not within the scope of this review article. Interested
readers are referred to summaries provided by Dingmann and Balu-
Iyer98 or Jarvi and Balu-Iyer.99TagedEn
TagedH2Metabolic StabilityTagedEn
TagedPWhile most chemical entities are characterized by high molecular
stability, biodegradable drugs such as peptides, proteins, or nucleic
acids undergo a rapid metabolism in the SC tissue. In this context, the
time required by the drug molecule to reach the nearest blood vessel
and drug degradation rate are interdependent parameters with a
strong impact on bioavailability. Metabolism can be widely driven by
the proteases secreted into the SC tissue.100 They include serine pro-
teases,101 cathepsin-B, and collagenase-like peptidase proteases,102
as well as aminopeptidases.103 The stability of glucagon-like peptide
1 analogs in the SC has been investigated in more detail.104 A direct
correlation between degradation of the drug and bioavailability was
found.104 Other drugs such as tacrolimus33 undergo non-enzymatic
chemical reactions including hydrolysis or oxidation.105,106 As men-
tioned in the previous sections, the metabolic stability of proteins
and peptides can be improved by conjugating the drug molecule
with PEG41,42 or hydroxyethyl starch.107,108 So far, PEGylation tech-
nology was most successfully used in the design of novel drug candi-
dates.109 Alternatively, the compound can be encapsulated into
delivery systems such as micro-/nanoparticles110 or liposomes.111
Predicting the impact of novel formulation strategies on the release,
stability, and bioavailability of drug molecules is one of the most
important goals of biopredictive performance testing.TagedEn
 TagedEnD. Li et al. / Journal of Pharmaceutical Sciences 112 (2023) 1492−1508
TagedH1Biopredictive Methods and MediaTagedEn
TagedPAt present, a wide variety of in vitro assays are being developed to
predict the in vivo performance of SC drug formulations.33,43,58,78,112
−115
Commonly, biorelevant methods provide an accurate reflection
of the physiological environment including the medium composition,
fluid volume, and hydrodynamics. They often contain proteins or
ions present at the injection site. For comparison, biopredictive meth-
ods demonstrate a partial ability to predict human in vivo perfor-
mance and may contain non-physiological ingredients such as
surfactants or stabilizers. More than an accurate reflection of the in
vivo situation, they trigger selected mechanisms of release such as
the dissolution of the drug or the degradation of the matrix material.
Most release assays for SC formulations do not achieve a high level of
biorelevance. Sometimes depot formulations with an in vivo release
over several days, weeks, or months are rather tested under acceler-
ated conditions.33,113,116,117 Quality control methods can use this to
discriminate quality differences between different batches of the
same dosage form. They reflect the rate-limiting release mechanism
but have a more limited predictive power for in vivo performances.118
Shen and Burgess present further information on accelerated in vitro
release testing methods for extended-release parenteral dosage
forms in general in their review article.119TagedEn
TagedPValidation of the in vitro assay can be accomplished by means of in
vitro-in vivo correlation (IVIVC). These mathematical relationships
between the in vitro and the in vivo data obtained from the same for-
mulation commonly use bioavailability as a surrogate parameter for
the expected in vivo response. The absorbed fraction of the drug can
be calculated by deconvolution from the plasma concentration-time
profile using model-dependent techniques such as the Wagner-Nel-
son or the Loo-Riegelmann method. They define a theoretical frame-
work (the mathematical model) to isolate the absorption process.
The evolution of computational methods in pharmaceutical develop-
ment has resulted in the integration of physiological parameters to
create more realistic simulations of the in vivo setting. Physiologi-
cally-based biopharmaceutics modeling (PBBM) is a discipline
involved with the explanation of the absorption phase.TagedEn
TagedPHowever, a significant number of influences on drug absorption
are not solely controlled by the formulation design. Therefore, new
standards must be defined for the application of computer models in
the development of IVIVCs.118TagedEn
TagedPIn the following, we will discuss the release media and methods
applied in the simulation of the SC tissue on the background of
parameters affecting drug absorption.TagedEn
TagedH2Release MediaTagedEn
TagedPMedium development often involves a compromise between the
existing knowledge on the microenvironment in vivo and the techni-
cal requirements of release testing. The most common parameters
are pH, buffer capacity, and ion concentration at the injection site.
They affect the ionization of compounds or excipients and can have a
strong impact on the release. Water, as a release medium, has no
buffer capacity and is thus, the most unreliable medium for the test-
ing of compounds and formulations affected by pH. However, even
for those formulations insensitive to such changes, there is a general
consensus to evaluate the drug release under conditions reflecting at
least some characteristics of the administration site.112TagedEn
TagedPA selection of the most common release media applied in release
testing of SC drug formulations is presented in Table 1. Most in vitro
studies were carried out using a buffer system at pH 7.4, reflecting
the physiological pH of the SC tissue. Commonly, phosphate buffers
with concentrations ranging from 10 mM120 to 100 mM75,121,122 are
used. With a rising number of poorly soluble compounds entering
the global healthcare market18 and their formulation into long-acting
1497
TagedEnPinjectables, more complex and biorelevant media reflecting the
microenvironment at the injection site may be required. Simple
buffer systems sometimes fail to mimic the most important processes
involved in the release and may not be suitable to predict the in vivo
performance.TagedEn
TagedPIon Species, Concentration, and Protein ContentTagedEn
TagedPFor some formulations, specific components of the medium such
as enzymes135 or certain ion species136,137 can have a strong impact
on the release of the drug product. Consequently, this influence
should be reflected by the in vitro assay. Simulated body fluid (SBF),
for example, comprises Ca2+ and Mg2+ cations in physiological con-
centrations. Hence, this fluid has been used for release and degrad-
ability studies of mesoporous silica microparticles where the cation
concentration plays a significant role.136,137 Although it does not spe-
cifically mimic the SC environment, SBF was used in a variety of bio-
activity, degradation, and dissolution studies focusing on these
applications.138−140 Kokubo et al. postulated that ion concentrations
and chemical composition reflect the physiological situation in
human plasma. This gives it a certain advantage as compared to other
fluids.141TagedEn
TagedPPhosphate buffered saline (PBS) is another common medium. As
compared to human plasma and SBF, it comprises a much higher
phosphate concentration and generally does not contain Ca2+ or
Mg2+.142 Nonetheless, PBS is widely applied in the simulation of
extracellular fluids. Compared to the other phosphate buffers, it
has the advantage that the composition is well-established. This
allows a direct comparison between different setups. Another
advantage of PBS is that the medium matches the osmolarity of
human body fluid. It contains sodium and chloride ions in physio-
logical concentrations.133 Another medium is Hanks’ balanced salts
solution (HBSS). It comprises ion species and concentrations more
comparable to SBF, while also containing glucose. For SBF and
HBSS, multiple versions have been published, depending on the
technical requirements of the assay. For example, Iyer et al. postu-
lated a modified HBSS for in vitro release testing of naltrexone
implants.133 Phenol red, a usual component of HBSS was removed
due to an interference with the analytical method. Also, he used 4-
(2-hydroxy ethyl) piperazine-1ethanesulfonic acid (HEPES) as a
buffer system, replacing the carbonate to reach a much higher
buffer capacity. This was to conduct the measurement under accel-
erated conditions. Primocin was used as a preservative. In sum-
mary, SBF, PBS, and HBSS exhibit some characteristics commonly
found at the injection site but, with regards to their biorelevance,
can be considered a simplified version of the physiology. SBF and
HBSS are closer to the composition of physiological fluids. This can
play an important role whenever the in vivo release is triggered by
a specific ion concentration or species.TagedEn
TagedPSerum proteins affect the release rate but, more often, have an
impact on chemical quantification methods and reproducibility of
the in vitro assay as well. Gao et al. explored the addition of pro-
teins, namely fetal bovine serum (FBS), to a release medium
intended for the simulation of the SC route of administration. This
simulated subcutaneous interstitial fluid (SSIF) leans on the com-
ponents and concentrations found in interstitial fluids instead of
human blood plasma, thus representing a biorelevant medium
more specific for the SC injection site.58 This is even more relevant
because, due to the limited permeability of the capillary mem-
brane, the protein concentration in the interstitial fluid is lower as
compared to the blood.143 Nonetheless, serum proteins can have a
stabilizing effect on particle dispersions, counteract agglomeration
and change the release rate. Table 2 summarizes the ion concentra-
tions and protein contents of blood plasma, interstitial fluid, and
the abovementioned media. Recently, the compositions of various
media simulating the SC route have been reviewed. The authors
TagedEn1498 D. Li et al. / Journal of Pharmaceutical Sciences 112 (2023) 1492−1508

TagedEnTable 1
Selected release media often used in vitro for the simulation of the SC environment.

Release media Notable modification Drug Formulation Refs.

75
Phosphate buffer (pH 7.4) 100 mM, Dexamethasone Core-crosslinked polymeric micelles
15 mM sodium chloride
75
100 mM, Paclitaxel Core-crosslinked polymeric micelles
15 mM sodium chloride,
Tween 80
121
100 mM, Nalmefene PLGA microspheres
sodium azide,
SDS
120
10 mM, Cytochrome c PLGA microspheres and implants
PVA,
sodium azide
122
100 mM, 4-benzoyloxy-2,2,6,6- In situ forming PLGA/NMP implants
tetramethylpiperidine-1-oxyl
123
50 mM, Buserelin PLGA implant
benzalkonium chloride,
sodium azide
117
67 mM Insulin Lipid implant
124
PBS (pH 7.4) Naproxen Nanosuspension
125
Bupivacaine Lipid-polymer hybrid nanoparticles
126
Tween 80 Oleanolic acid Liposomes
127
Carbonic anhydrase PLGA microspheres
127
Bovine serum albumin PLGA microspheres
128
Tween 20, Naltrexone PLGA microspheres
sodium ascorbate
10
Ibuprofen Composite PLGA microparticles
incorporating PCL nanoparticles
129
Dexamethasone PLGA microspheres mixed with
crosslinked hydrogel
130
Sodium azide Thymosin alpha 1 In situ forming PLGA implants
131
Tumor necrosis factor In situ forming PLGA implants
6
HEPES buffer (pH 7.4) 10 mM Dexamethasone Liposomes
113,132,133
Modified Hanks’ balanced HEPES buffer, Naltrexone PLA-PCL implant
salts solution (pH 7.4) primocin etc.
134
SBF (pH 7.4) SDS Toremifene citrate, Silica gel microspheres
dexmedetomidine HCl
58
SSIF FBS, Medroxyprogesterone acetate Microparticles
Pen-StrepÒ
33
FBS, Tacrolimus PLGA and PLA microspheres
MbCD
PBS = Phosphate buffered saline; SBF = Simulated body fluid; SSIF = Simulated subcutaneous interstitial fluid; HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
SDS = Sodium dodecyl sulfate; PVA = Polyvinyl alcohol; FBS = Fetal bovine serum; MbCD = Methyl-b-cyclodextrin; PLGA = Poly(D,L-lactic-acid); PCL = Poly(e-caprolactone);
NMP = N-methyl-2-pyrrolidone; PLA = Poly(D,L-lactic acid); PLA-PCL = Poly(D,L-lactide-co-e-caprolactone).

TagedEnPoutlined the importance of an actual measurement of the physio-
logical composition of the SC fluid.144 Although a review of the
data available has been timely, their analysis follows the common
narrative that today's biorelevant media offer an exact reflection of
the physiological environment. However, even for the well-estab-
lished media simulating the peroral route, a balance between bio-
prediction and biorelevance was necessary.145 In this context, with
regard to the SC route of administration, the poor standardization
of the administration site (e.g., upper arm, abdomen, thigh) and
difficulties in sample extraction must be taken into consider-
ation.144 Therefore, the capability of the medium to predict in vivo
TagedEnPperformances may be a more reliable benchmark than the varying
compositions measured in animals or humans.TagedEn
TagedPSurfactants, Solubilizers, and PreservativesTagedEn
TagedPFor many injectables, accelerated conditions are a key issue in the
development of new performance assays. An evaluation of the release
over several days, weeks, or months is affected by the chemical and
microbiological stability of the simulated microenvironment. This
weakness not only affects biorelevant media but all biologically
inspired in vitro systems and explains the relative insignificance of
three-dimensional cell models in the evaluation of depot

TagedEnTable 2
Relevant ion concentrations and protein content of blood plasma, interstitial fluid, and media used for in vitro release and degradation studies of SC formulations.

Cations [mM] Anions [mM] Proteins [g/L]

+ + 2+ 2+ 2 2
Medium Na K Ca Mg Cl HCO3 HPO4 SO4

Blood Plasma146 142 5 2.5 1.5 103 27 1 0.5 60-80147

Interstitial Fluid58 143 4 1.3 0.7 115 28 1 0.5 26
PBS146 157 4.5 - - 140 - 10 - -
SBF146 142 5 2.5 1.5 148.8 4.2 1 0.5 -
HBSS148 142.8 5.8 1.3 0.9 146.8 4.2 0.3 0.4 -
SSIF58 136 3.9 1.3 0.5 114.9 20.6 1 0.5 25 (FBS)
FBS = Fetal bovine serum; HBSS = Hanks’ balanced salts solution; PBS = Phosphate buffered saline; SBF= Simulated body fluid; SSIF = Simulated subcutaneous interstitial fluid.
 TagedEnD. Li et al. / Journal of Pharmaceutical Sciences 112 (2023) 1492−1508
TagedEnPformulations. Common additives include antioxidants such as sodium
ascorbate128 to prevent drug degradation, anti-adherents such as
polyvinyl alcohol120, and, more frequently, preservatives such as
sodium azide,120,121,123,130 benzalkonium chloride,123 penicillin-
streptomycin (Pen-StrepÒ ),33,58 and primocin113,132,133 when release
testing is done over an extended period.TagedEn
TagedPFurthermore, to assure a complete release process, many media
are supplemented with solubilizing agents.149 They comprise surfac-
tants or cosolvents to accelerate the dissolution process and enable
sink conditions.33,75,121,126,128,134 Surfactants do not reflect the physi-
ological conditions at the administration site. Still, they can allow a
more accurate prediction of the in vivo performance when absorption
is mainly limited by the dissolution.TagedEn
TagedPMedium Composition and CharacteristicsTagedEn
TagedPOther aspects to be considered are the medium composition, vol-
ume, and viscosity. The volume at the injection site is generally lower
and can be responsible for differences between the in vivo and the in
vitro performance. This has been observed for in situ implant formu-
lation, where degradation and drug release depend on the viscosity
of the matrix material.150 Testing at lower fluid volumes to accurately
mimic the SC environment leads to incomplete dissolution, especially
when poorly soluble drugs are tested. Also, the diffusion of the com-
pound into blood circulation is not sufficiently reflected. On one
hand, the use of higher fluid volumes and surfactants can improve
and accelerate the discrimination between drug formulations. On the
other hand, a change in hydrodynamics or medium composition may
alter the wetting, swelling, or dispersion properties of the drug for-
mulation. Therefore, the selection of the medium is widely based on
the mechanism of release. While the release from microcrystal for-
mulations is often driven by the poor aqueous solubility of the drug
in the SC tissue,58 other formulations such as oily liquids or disper-
sions may require their inherent structure to control the release in
the in vivo setting.151 Under these conditions, the effect of the release
medium on stability properties in vitro should be taken into consider-
ation. In this context, establishing IVIVCs for drug products exhibiting
similar formulation characteristics may support assay development.TagedEn
TagedPThe viscosity of SC injectables has been associated with an
increasing pain sensation during the administration of the drug.152
Surprisingly, high viscosity injections (up to 15−20 cP) were better
tolerated as compared to injectables with lower viscosity. Also, the
high drug and stabilizer concentrations required for the administra-
tion of depot formulations often lead to more viscous solutions. Con-
sequently, the viscosities of the formulation and the SC fluid likely
have a strong impact on drug diffusion as well.43 While most release
media are composed of buffer systems with low viscosity, this may
be an often-underestimated influence.TagedEn
TagedEnTable 3
Selection of release studies including the drug molecules, medium, and release set-up used for the assay.
Drug Medium
Cytochrome c Phosphate buffer, 10 mM (pH 7.4)
Carbonic anhydrase / bovine serum albumin PBS (pH 7.4)
Monoclonal antibody formulations Physiological, carbonate-based buffer solu-
tion (pH 7.4) and SCISSOR hydrogel
cartridge
Medroxyprogesterone acetate SSIF
Tacrolimus SSIF
Risperidone PBS (pH 7.4)
Naltrexone HBSS (pH 7.4)
Flurbiprofen / Lidocaine / Risperidone Hydrogel (agarose) in PBS (pH 7.4)
Interferon ß / Trypsinogen Hydrogel (agarose)
Recombinant human insulin Hydrogel (agarose)
HBSS = Hanks’ balanced salts solution; IVIS = In vivo imaging system; PBS = Phosphate buffered saline; SCISSOR = Subcutaneous Injection Site Simulator; SSIF = Simulated subcutane-
ous interstitial fluid.
1499
TagedPIn the past, hydrogel assays have been developed, to simulate the
resistance of the physiological environment.117,153 A certain intersti-
tial pressure resulting from constant muscle movement together
with the interconnected network of the ECM may affect the release
of SC injectables. To simulate this interstitial pressure, among others,
elastic agarose gels43 but also ECM components such as hyaluronic
acid and collagen have been used.117,153TagedEn
TagedPTemperatureTagedEn
TagedPAnother important parameter in the development of the release
assay is the temperature. Currently, there is no common standard for
the in vitro testing of SC drug formulations. Even the most recent USP
chapter <1001> In Vitro Release Test Methods for Parenteral Drug Prep-
arations provides no guidance in the area of SC drug formulations.
While 37 °C is often regarded as the core temperature of the human
body, temperatures in the SC tissue vary with an average of approxi-
mately 34 °C, depending on the administration site.154 In fact, a tem-
perature of 37 °C has been applied in every setup presented in
Table 1. Otte et al. specifically examined the influence of temperature
within a range of 35-37 °C on the in vitro release from naltrexone
PLGA microparticles with a significant impact on the release behav-
ior.128 At 37 °C, the release was considerably higher as compared to
the lower temperatures. This finding is strongly related to the poly-
mer properties and has been observed for many other PLGA and PLA
formulations. This is even more important when temperature
changes are used to accelerate the release.132TagedEn
TagedH2Instrumentation and Test MethodsTagedEn
TagedPAfter a long chain of discoveries, in 1970, the first dissolution
apparatus was adopted by the USP.155 It was a reflection of the most
recent efforts to provide a reproducible test system reflecting at least
some aspects of human physiology and, in particular, the human gas-
trointestinal tract. At present, there are no common standards for the
testing of SC drug formulations. This leads to a certain diversity in the
instrumentation and test conditions applied to these products. In the
following, we will provide an overview of some of the most relevant
methods. A selection of release studies including small-molecular
and biotechnological compounds is presented in Table 3.TagedEn
TagedPShake-Flask SetupsTagedEn
TagedPA very simple but popular setup uses tubes or vials with a defined
medium volume to evaluate the release. The vial is placed in shaking
water baths or incubators. In an attempt to simulate the microenvi-
ronment and hydrodynamics of the injection site, low agitation rates
of 30 rpm,122,128 80 rpm117 or 100 rpm,120,129 medium volumes of up
to 50 ml122 are selected. Some methods even further reduce the
Release setup Refs.
120
Shake-flask setup
127
Shake-flask setup with a dialysis bag
116
SCISSOR system
58
Dispersion releaser
33
Dispersion releaser
155
USP apparatus IV
113
Customized capillary cell device / modified
flow-through cells
156
Hydrogel in a shake-flask setup
Hydrogel assay in an IVISÒ system 43
117
UV imaging system
TagedEn TagedFiur
TagedEn1500 D. Li et al. / Journal of Pharmaceutical Sciences 112 (2023) 1492−1508
Figure 3. Illustration of the dialysis bag method used for the separation of dispersed drug formulations from the monomolecular drug. The illustrations were created with BioRen-
der (www.biorender.com).TagedEn
agedEnTPvolume to 300 ml.158 In this example, To reduce the aggregation,
agglomeration, and precipitation of the particles, surfactants, and sol-
ubilizers are sometimes added (e.g. sodium dodecyl sulfate, polysor-
bate). For small volumes, the sampling volume is commonly replaced
with fresh medium.117,129 Overall, this “shake-flask” technique can
be beneficial when compared to setups in complete stasis. Notewor-
thy, the sensitivity of the method to fluctuations in the release profile
is widely controlled by the stirring rate. Also, further separation steps
such as centrifugation or syringe filtration are often involved after
the sample collection. On one hand, the shake-flask method is highly
flexible and enables the testing at various stirring rates and fluid vol-
umes. On the other hand, the poor standardization, of this set-up
with regard to the size and agitation rate of magnetic stirrers and the
dimensions of the dissolution vessel may lead to more variability.
Also, evaporation has to be considered. However, their clinical rele-
vance will mostly depend on the mechanism of release of the formu-
lation.TagedEn
TagedPDialysis Membrane-Based SetupsTagedEn
TagedPFor comparison, when using dialysis-based techniques the mono-
molecular drug is continuously separated from the release medium
with the dialysis membrane forming a diffusion barrier. Depending
on the exact hydrodynamics of the system, the membrane can some-
times limit the release rate. Therefore, the sensitivity of the assay
involves an evaluation of the membrane permeation rate. In the area
of SC drug products, two different approaches are commonly found
in the literature. The membrane, sometimes in combination with
other barriers, is considered as a replacement for the physiological
diffusion barrier posed by the SC tissue. It is used to simulate tissue
retention and delays the availability of the drug in the acceptor
medium (Fig. 3).TagedEn
TagedPThe operator controls the diffusion rate by selecting different
membrane pore sizes, the fluid volume, temperature, and agitation
rate. Additionally, the liquid volume in the dialysis bag has a certain
influence on the release. It sometimes forms a static water layer that
further delays the release. On one hand, the methodology provides
hydrodynamics comparable to the SC tissue with a static liquid film
surrounding the formulation. On the other hand, an extremely slow
and variable release are major characteristics of the method.TagedEn
TagedPAs an alternative, the membrane can be used as an analytical tool
to separate the monomolecular drug from the medium. The influence
on release kinetics is considered an analytical error source and addi-
tional measures are required to validate the assay. Also, mathemati-
cal models have been established to estimate the release of the drug
from the formulation.159TagedEn
TagedPAs with other separation methods, the separation of the released
drug from the medium by dialysis comes with specific advantages
and disadvantages. These considerations are not specific to the SC
TagedEnProute of administration and will therefore not be discussed in more
detail. However, interested readers are referred to a recent stimuli
article published by the USP expert panel on New Advancements on
In-vitro Product Performance Testing.160 It describes the challenges
associated with a time-resolved separation of fine particles from the
liquid.TagedEn
TagedPSC Injection Site Simulator (SCISSOR)TagedEn. TagedPAn interesting alternative is the
SC Injection Site Simulator (“SCISSOR”) presented in Fig. 4.114 It com-
prises an injection cartridge immersed in a container filled with a
biorelevant medium. The dialysis-based cartridge is filled with hya-
luronic acid to mimic the influence of the ECM on SC drug formula-
tions. This design allows the simulation of release and diffusion
through the ECM, as well as the uptake through the blood capillaries
and/or the lymph vessels.TagedEn
TagedPThe system provides a simulated environment controlling tem-
perature, pH, and ionic strength. The interstitial pressure is simulated
by the donor cartridge due to the elastic deformation of the gel upon
injection. By increasing the fluid volume in the acceptor chamber,
sink conditions can be adjusted. The system is usually maintained at
34 °C and stabilized at pH 7.4 during measurements.114 To monitor
the current status of the injection chamber as well as injection events
with impact on the stability of the drug or the excipients a micro pH
probe, a thermometer, and a spectrophotometer are included. The
spectrophotometer measures the transmittance in the injection car-
tridge in real-time. Processes with impact on the UV/VIS spectrum
can be detected including the dispersion of particles as well as the
disintegration of the dosage form.161 Drug release can be determined
by sampling from the buffer system at selected time points.TagedEn
TagedPThe SCISSOR system has been used for characterizing
biopharmaceuticals114,116 and depot formulations of small mole-
cules162. Bown et al. studied the in vitro performance of a set of
monoclonal antibodies and obtained distinct profiles using the in
vitro setup.116 This investigation provided some evidence for the
capability of the system to predict the in vivo fate of SC injectables. A
recent study on diclofenac containing intra-articular injectables
highlighted the potential of the SCISSOR system for in vitro testing
and comparison of the release behavior of in situ depot formula-
tions.162 Major advantage of the SCISSOR is the simplified model of
the human ECM, limiting the availability of volume fluid and the
medium composition.161 However, it has limited sensitivity for com-
pound interactions. One disadvantage is the limited sensitivity of the
turbidity sensor. While macroscopic events (e.g., the disintegration of
the dosage form or dispersion of the particles) can be observed in the
UV/VIS spectrum, the background signal of the simulated ECM does
not allow the analysis of specific interactions of compounds with the
microenvironment. Further evaluation of this method may be
required before it can be recommended for broader application.161TagedEn
TagedEn TagedFiur TagedEnD. Li et al. / Journal of Pharmaceutical Sciences 112 (2023) 1492−1508 1501

Figure 4. Schematic (left) and photograph (right) of a SCISSOR system. The image was kindly provided by Karen Huffman, Pion Inc, Billerica, USA.TagedEn

TagedPDispersion ReleaserTagedEn. TagedPThe dispersion releaser (DR) is a dialysis-based
device and was developed at Goethe University in 2013164. The sys-
tem is mounted into a USP dissolution apparatus I/II equipped with
standard-vessel or mini-vessel configuration.164TagedEn
TagedPThe setup comprises a donor compartment surrounded by a dialy-
sis membrane, holding the dosage forms in the center position. The
dissolution vessel represents the acceptor compartment. The donor
and acceptor chamber are both agitated by a paddle stirrer (Fig. 5).
Overall, this design facilitates rapid and controlled membrane trans-
port, leading to highly reproducible release profiles. Additionally, the
molecular weight cut-off of the membrane can be optimized for
every release experiment. Evidently, the DR does not mimic the
hydrodynamics in the SC tissue. Still, it allows a highly selective sepa-
ration of different particle populations as well as sensitive detection
of release processes. It accelerates the medium exchange between
TagedEn TagedFiur
TagedEnPboth compartments and reduces the impact of membrane permeabil-
ity on the release.165 The DR has been used for the characterization of
SC depot formulations in presence of biorelevant media such as the
SSIF.33 A more recent investigation on long-acting tacrolimus micro-
spheres resulted in a good prediction in vivo fate of the microspheres.
In the context of the release mechanisms discussed previously, the
DR enables accurate detection of drug dissolution or release without
taking drug diffusion (through the ECM) into consideration. For the
drug tacrolimus, this diffusion step was simulated in silico.33 These
combinations of in vitro and in silico methods will become more com-
mon in the future.TagedEn
TagedPContinuous Flow-Through SetupsTagedEn
TagedPThe flow-through cell described by the USP (dissolution apparatus
IV) has been applied to the testing of a wide variety of parenteral

Figure 5. Schematic (left) and photograph (right) of the Pharma Test Dispersion Releaser. The images were kindly provided by Dirk Beilke, Pharma Test Apparatebau AG, Hainburg,
Germany.TagedEn
TagedEn TagedFiur
TagedEn1502 D. Li et al. / Journal of Pharmaceutical Sciences 112 (2023) 1492−1508

Figure 6. Flow-through cell setup described by the USP and one individual cell. The images were kindly provided by Michel Magnier, Sotax AG, Aesch, Switzerland.TagedEn

agedEnTPdrug formulations.156,165 It offers a wide range of commercially avail-
able flow-through cells and a flexible reservoir volume. Hence, USP
apparatus IV enables the testing of formulations where the release is
either limited by drug solubility or the detection limit.165 This allows
the development of IVIVCs for poorly soluble drugs.166 The system
comprises a pump, flow-through cells, a water bath, and medium res-
ervoirs. It can be operated with an open or a closed loop. Flow rates
and temperatures can be adjusted by the operator.167 Dissolution
apparatus IV was initially designed for in vitro dissolution testing of
controlled-release oral powders, granules, and solid dispersions
(Fig. 6).165TagedEn
TagedPMore often, adjustments are made to enable the testing of inject-
ables. For example, glass beads were added to a microsphere formu-
lation to avoid agglomeration in the release cell. Also, a filter can be
installed to prevent both glass beads and microspheres from entering
into the medium reservoir.156,165 Compared to USP apparatuses I and
II, advantages may arise from reduced agglomeration due to the
higher liquid volume. Further, a potential violation of sink conditions
can be avoided. Sampling procedures are comparable to other disso-
lution methods and come with the same advantages and disadvan-
tages. Burgess and co-workers developed a dissolution test based on
the flow-through cell using a dialysis membrane for the separation of
the drug from the carrier material (Fig. 6).6 The setup is comparable
to other dialysis-bag methods and comes with the same limita-
tions.163 However, the position of the dialysis adapter in the dissolu-
tion cell provides a certain standardization. Thus, the concept of
dissolution apparatus IV provides a good starting point for the devel-
opment of SC depot formulations. Continuous perfusion of tissues as
well as certain control of the release parameters such as flow rate
and temperature are the considerable advantages. Previous reports
made assumptions on the liquid flow in vivo, identifying a rate of
TagedEn TagedFiur
agedEnTPapproximately 1.06 ml/min.132 However, the robustness of the sys-
tem is often an issue. Filter membranes are affected by particles pres-
ent in the formulation and lead to variable flow rates between the
cells. Similarly, polymer migration limits the use of the continuous
flow-through setup to a certain degree.112TagedEn
TagedPHydrogel-Based SetupsTagedEn
TagedPAn increasing number of in vitro studies for injectables use hydro-
gels as the release medium to mimic the SC tissue.43,115,124,157,168−170
The formulations are entrapped into the hydrogel matrix represent-
ing the SC tissue. This environment controls the fluid volume
depending on the osmotic pressure of the formulation and tempera-
ture. Also, the hydrogel serves as a diffusion barrier.115,157,171 The
released drugs can be quantified using UV imaging or chemical quan-
tification methods. Agarose gels are most commonly used, due to the
ease of fabrication as well as their mechanical stability.115 Gietz et al.
described a release model based on an agarose hydrogel, simulating
the SC injection site. After injection of the formulation, PBS served as
the acceptor fluid.168 The released drug diffused through the agarose
gel into the liquid phase. Sink conditions were maintained by replen-
ishing the medium at defined time points. Another study used the in
vivo imaging system IVISÒ to measure the fluorescence emitted by
fluorescence-labeled interferon released from a solid implant in mice
and a hydrogel assay. The system is designed for analyzing the biodis-
tribution in animals and enables localization of fluorescent molecules
(Fig. 7).43TagedEn
TagedPThe hydrogel assay was used to characterize the in vitro behavior
of several parenteral formulations comprising either hirudin or insu-
lin. Leung et al. further explored the applications of the assay and
investigated the release of compounds with different molecular
weights.124 A UV-Vis spectrometer was used to measure the real-

Figure 7. Illustration of an agarose-based hydrogel assay using the IVISÒ system for measuring the drug release from solid implants. After the implantation of the solid implant into
animals and the hydrogel, the fluorescence was monitored over time. The illustration was created with BioRender (www.biorender.com).TagedEn
 TagedEnD. Li et al. / Journal of Pharmaceutical Sciences 112 (2023) 1492−1508
TagedEnPtime drug concentration in the buffer system. Biorelevance was dem-
onstrated by means of IVIVC.TagedEn
TagedPQuantification MethodsTagedEn
TagedPMost performance assays can be combined with various quantifi-
cation methods. Imaging methods have certain advantages. They
enable the observation of release processes in vitro and in vivo.43TagedEn
TagedPThis includes the stability of the injected components, swelling,
and erosion behavior of formulations, and drug transport. Impor-
tantly, every quantification method comes with its weaknesses and
strengths. While conventional high-performance liquid chromatog-
raphy setups detect the drug from various matrices, in situ methods
enable a continuous detection of more time points. These differences
are not specific to the SC route of administration and will therefore
not be discussed in more detail. With regards to imaging techniques,
diagnostic ultrasound imaging, fluorescence imaging, and UV-Vis
imaging are amongst the most popular non-destructive analytical
methods.122 One of the commercial set-ups that have been used for
the testing of drug release is the IVISÒ . The in vivo scanner was
designed for the analysis of biodistributions in small animals such as
rodents or mice (Fig. 7). After illumination of the inner chamber with
the light of a specific wavelength, the fluorescence emission from the
sample is detected resulting in a two-dimensional and, in the latest
versions of the instrument, three-dimensional image. After implanta-
tion into a hydrogel, the IVISÒ system has been used to detect the
release of a fluorescence-labeled protein from solid implant
formulations.43TagedEn
TagedPAt present, there are several methods under investigation, to con-
tinuously monitor the drug release. While instruments such as the
SCISSOR or UV imaging methods172 are more suitable for the detec-
tion in a controlled in vitro environment, fluorescence imaging, near-
infra-red, or magnetic resonance imaging are suitable to detect
changes in the performance in vitro and in vivo.173,174TagedEn
TagedH1Authors’ RecommendationsTagedEn
TagedPTo develop in vitro assays capable of predicting the in vivo
performance, the mechanisms involved in the release from the drug
product must be reflected by method and medium. Various influen-
ces on drug absorption were summarized in the previous sections.
Together with a dedicated investigation of the formulation properties
and responses, they are the basis for the selection of the release
conditions.TagedEn
TagedPOne of the critical influences on drug absorption is the amount of
the unbound drug present in the dosage form. Depending on the
exact manufacturing process, liquid dispersions have a higher risk for
safety-relevant changes in the encapsulation over time. Initial disper-
sion of this weakly bound fraction leads to “burst release” with a high
probability of reaching toxicologically relevant concentrations in the
blood plasma. In the in vivo setting, this “burst effect” often overlaps
with the initial distribution of the compound after injection and can
be hard to distinguish from the migration of nanosized particles into
the lymphatic capillaries. Therefore, both parameters, the fine parti-
cle fraction and the release behavior should be accurately moni-
tored.33 Both methods together, release testing and physical stability
testing, are potentially sensitive enough to detect these safety issues.
Noteworthy, the release assays utilized in the detection of “burst
effects” must be sensitive for rapid fluctuations in the release rate.162
Conventional dialysis approaches or hydrogels may not be able to
sufficiently discriminate these changes. The inherent barrier proper-
ties of the assay delay the release and reduce reproducibility. More
rapid separation techniques, even when using non-physiological
hydrodynamics and fluid volume, are more appropriate. In the pre-
clinical setting, most studies investigating the lymphatic transport of
drugs into systemic circulation are carried out in rodents. These
1503
TagedEnPpreclinical models may not be suitable to predict absorption from the
SC tissue. However, the size cut-off of 100 nm was also consistent
with porcine models suggesting that these animals may provide suffi-
cient information to study the burst effect related to lymphatic
uptake.16TagedEn
TagedPDepot formulations for SC administration are commonly adminis-
tered into a small volume present at the injection site. The protein
concentration in the interstitial space is lower as compared to the
blood plasma.143 Still, these proteins can have a stabilizing effect on
particle dispersions.58 Since agglomeration often reduces the fine
particle fraction and proteins counteract this process, the in vitro
studies should include physical stability in the presence and absence
of serum proteins.58 Currently, SSIF is the only release medium
including this essential component.33,58,144TagedEn
TagedPAmong other things, drug dissolution is affected by the fluid vol-
ume, temperature, aqueous solubility of the compound, and the par-
titioning into lipophilic tissues. While temperature usually varies in a
range of 3-5 °C and often remains without considerable impact on
pharmacokinetics, the fluid volume in the SC tissue is flexible and
depends on the osmotic pressure of the formulation. However, with
regard to dissolution, aqueous solubility and particle size remain the
most critical parameters. While temperature has only a minor influ-
ence on the solubility of microcrystal or nanocrystal formulations, it
sometimes represents an important trigger for the release from tem-
perature-sensitive polymer128 or lipid carriers.1 Therefore, whenever
the material properties indicate such a relationship, the temperature
should be monitored in the assay system over a wide temperature
range to ensure that there are no unexpected changes in the release
kinetics observed. For poorly soluble compounds, partitioning into
the SC fat layer affects bioavailability and dissolution behavior as
well.99 To study these interactions a biphasic release medium can be
used. Unfortunately, animal studies in mice are of limited value in
the prediction of these effects. The composition of the SC tissue dif-
fers from the human situation. For studies investigating drug distri-
bution in the skin layers, rats or non-human primates are of higher
value.33,46TagedEn
TagedPWith a growing number of biotechnological compounds entering
the global healthcare market, metabolic stability is becoming more
important. For insoluble compounds with poor metabolic stability,
chemical degradation may accelerate drug dissolution. The degrada-
tion step reduces the concentration of the free compound resulting in
more dissolution. Under these conditions, degradability is an impor-
tant release mechanism and requires an investigation in vitro and in
vivo. For example, hydrolysis of tacrolimus is not affected by the pres-
ence of serum33 while metabolic stability of other drugs depends on
the presence of enzymes and requires more effort in the simula-
tion.135 To predict and investigate the effects of the formulation on
the chemical stability of the compound in vivo, the preclinical model
plays an important role. Enzyme expression levels between different
species may vary and should therefore be considered when develop-
ing the drug formulations. With regard to the development of perfor-
mance assays, the expected degradation mechanisms can be
simulated by addition of enzymes to the release medium. Knowing
the processes affecting the chemical stability of the compound is,
therefore, is of utmost importance.TagedEn
TagedPFor many compounds with moderate or high aqueous solubility,
other processes such as the diffusion of the drug to the nearest blood
vessel or the unspecific interactions between the drug and the carrier
have a stronger impact on bioavailability. The differences in drug
absorption following SC and intramuscular injection are more pro-
nounced for biologics175 than for poorly soluble compounds.176 Diffu-
sion becomes the rate-limiting step in the absorption process. Here,
assay designs measuring this diffusion rate often provide a better
estimate of the drug absorption.43 The molecular size and protein
binding of a drug becomes a more important parameter. In this
TagedEn1504 D. Li et al. / Journal of Pharmaceutical Sciences 112 (2023) 1492−1508
TagedEnPcontext, breaking down the absorption into the individual processes
and simulating these steps either in vitro or in silico will enable a bet-
ter prediction than dissolution testing alone.TagedEn
TagedPMore advanced formulation designs control the release by using a
specific combination of excipients. With these ingredients controlling
the release behavior, their interaction with the physiological micro-
environment becomes more important. For example, changes in the
physiological fluid volume at the injection site over time may affect
the release behavior from in situ implant formulations.151 Consider-
ing that the influx of liquid varies over time, the release processes
would hardly be reflected by an in vitro setup with a static fluid vol-
ume. Hydrogel systems provide a flexible response to dissolution,
swelling, and osmotic pressure of the formulation and could poten-
tially lead to better outcomes.43 More than a representation of the
physiological environment, this biopredictive assay activates a physi-
ologically relevant trigger. Similarly, for some formulations, affinities
between the molecules in the formulation can be weakened by
endogenous binding partners. For liposomes, the exchange of phos-
pholipids between the vesicle and biomembranes has been observed
and potentially affects membrane permeability. For the SC formula-
tion InsulatardÒ , a complex of insulin, protamine, and zinc, insulin is
continuously replaced by negatively charged binding partners such
as heparin or chondroitin.177 Under these conditions, a release assay
not providing suitable binding partners may not be able to forecast
the in vivo performance.TagedEn
TagedPAs outlined by the previous sections, the biocompatibility of the
carrier is a key issue in its performance. Morphological changes of
the physiological environment can be a gamechanger in the release
and diffusion processes and make predictions more challenging.
With formulations remaining at the injection site over a considerable
time, the risk for long-term effects, inflammation, or immune
responses is higher as compared to other dosage forms. These
responses are not limited to the drug or excipients but may occur
due to the trauma caused by the injection. Also, the disassembly of
the formulation at the injection site may lead to inflammation reac-
tions.178 At present, the prediction of inflammation or immune reac-
tions still requires a significant number of preclinical studies. The
implications and models in vitro and in vivo have been discussed in
more detail by Jarvi and Balu-Iyer.99 Importantly, these responses are
difficult to predict using a release assay and are not within the scope
of this review article.TagedEn
TagedPOur recommendations provide some advice to the operator in the
selection of the in vitro conditions during the development of perfor-
mance assays. However, the abovementioned findings were the
result of a thorough understanding of the mechanisms of release.
We, therefore, caution the definition of general standards. The formu-
lation design must be reflected by the assay to a certain extent.TagedEn
TagedH1ConclusionTagedEn
agedPIT n conclusion, the SC route of administration provides an effective
delivery platform for many compounds by improving their bioavail-
ability or sustaining the release over a considerable period. Still, a
number of overlapping processes are involved in the absorption of
the drug, including lymphatic uptake, the release of the drug from
the delivery system, post-injection agglomeration, or inflammatory
responses. Knowledge gaps affect the predictive power of the assays
and still require a thorough investigation of the most important influ-
ences.TagedEn
TagedPBy gaining a better understanding of the mechanisms of release,
predictability of absorption can be improved and its impact on the
plasma drug concentration better simulated. This ensures that novel
drug formulations are safe and effective when entering clinical trials.
There is a common misunderstanding in dissolution science that a
more physiological assay automatically leads to better predictions. In
TagedEnPfact, the perfect balance between sensitivity, selectivity, and biorelev-
ance commonly leads to the best outcomes.TagedEn
TagedH1Declaration of InterestsTagedEn
TagedPThe authors declare that they have no known competing financial
interest.TagedEn
TagedH1AcknowledgmentsTagedEn
TagedPThe authors acknowledge Jennifer Dressman for her years of dedi-
cation to biorelevant release testing, her strong scientific and per-
sonal support, and for treating her staff, students, and co-workers as
a second family.TagedEn
TagedPD.L. acknowledges the National University of Singapore (NUS) and
A*STAR for providing him with a scholarship in the NUS Pharmacy
graduate research program. Also, all authors acknowledge Pion Inc.
(Billerica, USA), Pharma Test Apparatebau AG (Hainburg, Germany),
and Sotax AG (Aesch, Switzerland) for providing us with illustration
material as well as NUS and the Ministry of Education of Singapore
for financial support (grant no. R-148-000-282-133 and R-148-000-
297-114).TagedEn
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