DOI: 10.1002/cmdc.

201600374 Reviews

Half-Life Extension of Biopharmaceuticals using Chemical

Methods: Alternatives to PEGylation
Søren B. van Witteloostuijn,[a, b] Søren L. Pedersen,[b] and Knud J. Jensen*[a]

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Peptides and proteins constitute a vast pool of excellent drug
candidates. Evolution has equipped these molecules with su-
perior drug-like properties such as high specificity and poten-
cy. However, native peptides and proteins suffer from an inade-
quate pharmacokinetic profile, and their outstanding pharma-
cological potential can only be realized if this issue is ad-
dressed during drug development. To overcome this challenge,
a variety of half-life extension techniques relying on covalent
chemical modification have been developed. These methods
1. Introduction
Since the approval of recombinant human insulin in 1982 by
the US Food and Drug Administration (FDA), biopharmaceuti-
cals have become increasingly important from both a medical
and an economic perspective. Currently, approximately 250 bi-
opharmaceutical drug products are approved and available on
the US and European markets for the treatment of a variety of
diseases, including several cancer forms, inflammatory disease,
diabetes, obesity and heart disease.[1] The total annual revenue
generated by biopharmaceuticals has been steadily increasing,
reaching USD 140 billion in 2013[1] and predicted to reach USD
239 billion in 2015.[2]
The vast majority of biopharmaceuticals are based on en-
dogenous peptides and proteins, such as hormones, enzymes,
growth factors, interferons, and antibodies. Inherently, these
natural ligands possess superior drug properties such as highly
selective receptor binding and potent receptor activation. This
minimizes off-target side effects and allows administration of
very low drug doses, thereby improving the drug safety profile
and increasing patient compliance. Nevertheless, direct use of
most native polypeptides as drugs is hampered by their poor
pharmacokinetic (PK) properties, owing to their rapid metabo-
lism, proteolytic degradation and, especially in the case of pep-
tides and smaller proteins, susceptibility to renal clearance.
These obstacles result in very short systemic half-lives, typically
in the range of minutes to few hours.[3] This challenge has
prompted the development of several half-life extension strat-
egies to improve the PK properties of native peptides and pro-
teins.
Chemical modification of peptides and proteins has become
an essential route to enhance the PK profile of biopharmaceut-
icals. Arguably, the most common and so far most commercial-
ly successful approach has been PEGylation, that is, the cova-
lent attachment of polyethylene glycol (PEG) chains. However,
there is growing concern about the safety of PEGylation, as
[a] S. B. van Witteloostuijn, Prof. K. J. Jensen
Department of Chemistry, University of Copenhagen, Thorvaldsensvej 40,
1871 Frederiksberg C (Denmark)
E-mail: kjj@chem.ku.dk
[b] S. B. van Witteloostuijn, Dr. S. L. Pedersen
Gubra ApS, Hørsholm Kongevej 11B, 2970 Hørsholm (Denmark)
The ORCID identification number(s) for the author(s) of this article can
be found under http://dx.doi.org/10.1002/cmdc.201600374.
& ChemMedChem 2016, 11, 1 – 23 www.chemmedchem.org
Reviews
include PEGylation, fusion to unstructured polypeptide-based
PEG mimetics, conjugation of large polysaccharides, native-like
glycosylation, lipidation, fusion to albumin or the Fc domain of
IgG, and derivatization with bio-orthogonal moieties that
direct self-assembly. This review provides an overview of avail-
able conjugation chemistries, biophysical properties, and
safety data associated with these concepts. Moreover, the ef-
fects of these modifications on peptide and protein pharmaco-
kinetics are demonstrated through key examples.
this modification may lead to hypersensitivity reactions,[4] anti-
body formation,[5] and PEG bioaccumulation[6] (see below).
Herein we provide an overview of the most important chem-
ical modifications to provide half-life extension of biopharma-
ceuticals, which are alternatives to PEGylation. The primary
focus is on the conjugation of large carbohydrate polymers,
native O- and N-glycosylation, lipidation, and experimental
methods that direct nano-scale self-assembly. The chemical at-
tachment of unstructured polypeptides, albumin conjugation,
and Fc fusion is also mentioned (Table 1). The emphasis is on
chemical moieties, the biophysical properties they direct, and
their biological compatibility. The impact of each half-life ex-
tension method is illustrated through selected examples.
The focus of this review is on alternatives to PEGylation;
chemical modification by PEGylation has previously been ex-
tensively reviewed.[7] Noncovalent drug formulation is not cov-
ered in this review.
2. PEGylation
The attachment of poly(ethylene glycol) (PEG) moieties to pep-
tides and proteins is a well-established and efficient method
for improving their pharmacokinetic properties. PEG polymers
are highly hydrophilic and provide with their associated water
molecules a hydration shell, which expands the hydrodynamic
volume of a polypeptide. This makes the PEGylated molecule
less susceptible to renal clearance as well as to protease degra-
dation, and can decrease the immunogenicity of native pep-
tides and proteins.[7a] The concept of PEGylation was intro-
duced in the 1970s by Davis and co-workers,[8] and today 12
PEGylated peptide- and protein-based biopharmaceuticals are
available on the American and European markets.[9]
PEG polymers are produced synthetically and can be either
linear or branched, and the end group(s) may be either the
standard hydroxy group or a methoxy group (denoted mPEG)
(Figure 1). Typically, commercially available PEG polymers used
for chemical modification of biopharmaceuticals range up to
50 kDa. PEGylation of polypeptides may be obtained with dif-
ferent strategies.[7b, c, 10] Usually, the N-terminal amino group or
the Ne-amine of a Lys residue is used as nucleophile in either
alkylation or acylation reactions with an activated PEG species.
An example of an alkylating reagent is mPEG-propionaldehyde,
which reacts with the amino groups of polypeptides to form
an imine (Figure 2 A). Subsequent reduction with NaBH3CN
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Søren B. van Witteloostuijn obtained
his MSc degree in Biology–Biotech-
nology from the University of Copen-
hagen in 2013. He currently holds a po-
sition as research scientist and indus-
trial PhD student with Gubra ApS and
the University of Copenhagen under
the supervision of Dr. Søren L. Peder-
sen and Prof. Knud J. Jensen. His re-
search interests include the design and
synthesis of peptide-based drugs, bio-
physical characterization of peptide
oligomers, and development of novel principles for half-life exten-
sion of biopharmaceuticals.
Dr. Søren L. Pedersen obtained his PhD
in bioorganic chemistry in 2010 with
Prof. Knud J. Jensen, University of Co-
penhagen, in collaboration with Rheo-
science. Subsequently, he was a post-
doctoral researcher with Prof. Knud J.
Jensen at the Lundbeck Foundation
Center for Biomembranes in Nanome-
dicine at the University of Copenha-
gen. In 2011, he joined Gubra ApS,
where he is now a senior scientist and
group leader. His research group is en-
gaged in peptide drug discovery within diabetes and obesity, and
target discovery in specialized regions of the gut as well as the
brain.
Knud J. Jensen obtained a PhD degree
in synthetic bioorganic chemistry from
the University of Copenhagen in 1992
with Prof. Morten Meldal, after which
he did postdoctoral research with Prof.
George Barany, University of Minneso-
ta. He became assistant professor at
the Technical University of Denmark in
1997 and associate professor at KVL in
Copenhagen in 2001, which in 2007
became part of the University of Co-
penhagen. The same year he was pro-
moted to full professor in nanobioscience. He is now a full profes-
sor at the Department of Chemistry, University of Copenhagen and
Head of the section for Chemical Biology. He is the co-author of
more than 130 peer-reviewed publications, has edited two books
on the design and synthesis of peptides, and has co-authored nu-
merous book chapters and proceedings. He was awarded the
Zervas Award of the European Peptide Society in 2012. His re-
search covers a broad range of topics at the interface between
synthetic chemistry, medicinal chemistry, biology, biophysics, and
nanobioscience.
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Figure 1. Different types of PEG structures; mPEG: methoxy-PEG. Adapted
from Harris & Chess 2003.[7a]
provides the secondary amine.[7b, 10] Overall, this process is a re-
ductive amination.
Active succinimidyl esters of PEG-carboxylic acids (PEG-NHS)
are the most widely used reagents for acylation reactions.[10]
Acylation of amines with PEG-NHS yield a polypeptide-PEG
conjugate held together by an amide bond (Figure 2 B). PEGy-
lation of amines is generally feasible and occur under mild
conditions. However, a potential problem with targeting Lys is
the high prevalence of accessible Lys residues in native pep-
tides and proteins, possibly leading to a mixture of products
and batch variation.
An alternative is to use the nucleophilic thiol functionality of
a free Cys. If no free Cys is present, it may be introduced
chemically or recombinantly to enable site-specific PEGylation.
Typical and widely used reagents for this purpose are PEG-mal-
eimides.[7c] Under neutral to slightly basic conditions, Michael
addition of the thiol to the maleimide results in the formation
of a thioether (Figure 2 C). The resulting thioether is, however,
not completely stable under aqueous conditions or in vivo, po-
tentially leading to deconjugation of a thiol-reactive linker.[11]
Maleimide exchange and succinimide ring hydrolysis in the
conjugated linker have been reported for several conjuga-
tes.[11b, 12] PEG-maleimide can exchange with albumin, cysteine,
or glutathione in vivo, giving the non-modified biopharma-
ceutical, or undergo succinimide ring hydrolysis. To avoid
these challenges, PEG-haloacetamides may be employed for
Cys modification (Figure 2 D).
The PEGylation technology has been highly successful, as re-
flected by the large number of PEGylated drugs available on
the market and in development. However, because PEGs are
synthetic polymers, many commercially available reagents are
provided as polydisperse mixtures, a feature that inevitably is
reflected in the PEGylated drug product. PEGs with narrow and
well-characterized dispersities are thus preferred. Moreover,
the safety of PEG is still a matter of debate.[2, 13] Data suggests
that administration of PEGylated drugs may cause both hyper-
sensitivity reactions as well as antibody formation.[4, 5] Other
concerns include bioaccumulation of PEG and the fate of this
synthetic polymer after injection.[6] These issues have led to
the investigation and development of alternatives to PEGyla-
tion for extending the half-life of biopharmaceuticals.
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Table 1. Currently approved peptide- and protein-based biopharmaceuticals that are PEGylated, lipidated, or fused to albumin or the Fc domain of IgG.[a]

INN[b] Brand name t1/2 extension Indication Approval

method year[c]
Pegadamase Adagen PEGylation Severe combined immunodeficiency disease (SCID) 1990
Pegaspargase Oncaspar PEGylation Leukemia 1994
Peginterferon-a2b PegIntron PEGylation Hepatitis C 2000
Peginterferon-a2a Pegasys PEGylation Hepatitis C 2001
Pegvisomant Somavert PEGylation Acromegaly 2002
Pegfilgrastim Neulasta PEGylation Neutropenia 2002
Methoxy polyethylene glycol-epoetin b Mircera PEGylation Anemia associated with chronic kidney disease 2007
Certolizumab pegol Cimzia PEGylation Rheumatoid arthritis, Crohn’s disease 2008
Pegloticase Krystexxa PEGylation Chronic gout 2010
Lipegfilgrastim Lonquex PEGylation Neutropenia 2013
Peginterferon-a2a Plegridy PEGylation Relapsing multiple sclerosis 2014
Antihemophilic factor (recombinant) Adynovate PEGylation Hemophilia A 2016
Insulin detemir Levemir Lipidation Type 1 diabetes 2004
Liraglutide Victoza Lipidation Type 2 diabetes 2009
Insulin degludec Tresiba Lipidation Type 1 diabetes 2013
Liraglutide Saxenda Lipidation Obesity 2014
Albiglutide Tanzeum, Eperzan Albumin fusion Type 2 diabetes 2014
Etanercept Enbrel Fc fusion Rheumatoid arthritis, psoriasis 1998
Abatacept Orencia Fc fusion Rheumatoid arthritis 2005
Rilonacept Arcalyst Fc fusion Cryopyrin-associated periodic syndromes (CAPS) 2008
Romiplostim NPlate Fc fusion Immune thrombocytopenic purpura (ITC) 2008
Aflibercept Eylea Fc fusion Wet macular degeneration 2011
Belatacept Nujolix Fc fusion Kidney transplant rejection 2011
Ziv-aflibercept Zaltrap Fc fusion Colorectal cancer 2012
Antihemophilic factor (recombinant) Eloctate Fc fusion Hemophilia A 2014
Coagulation Factor IX (recombinant) Alprolix Fc fusion Hemophilia B 2014
Dulaglutide Trulicity Fc fusion Type 2 diabetes 2014

[a] All fusion proteins listed are obtained by recombinant expression. [b] International nonproprietary name. [c] Approval year reflects first approval in
either USA or Europe.

Figure 2. Examples of PEGylation chemistry. A) mPEG-propionaldehyde reacts with an amino group to form an imine, which is reduced to form a secondary
amine. B) PEG-NHS reacts with an amino group to form an amide bond. C) PEG-maleimides react with free Cys to provide a thioether linkage. D) PEG iodo-
acetamides also react with free Cys to form a thioether bond. R: peptide or protein.

3. Recombinant PEG Mimetics

As an alternative to the synthetic PEG polymers, a number of
recombinant ‘PEG mimetics’ for half-life extension has been re-
ported. These polypeptides are specific amino acid sequences
that are attached to biopharmaceuticals as fusion proteins ob-
tained by recombinant DNA technology. In general, these poly-
mers are hydrophilic or even charged, and they adopt an un-
structured conformation when attached to proteins.[14] The hy-
drophilic properties ensure solvation of the polymers, which
decreases the risk of aggregation during storage and allows
high-dose formulation of the fusion protein. The absence of
hydrophobic residues also minimizes unspecific interactions
with biological membranes and hydrophobic cell components.
Moreover, the unstructured nature of these polypeptides pro-

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vides conformationally flexible polymers with an expanded hy- 3.2 Other PEG mimetics
drodynamic volume, which retards glomerular filtration and
renal clearance of the active fusion protein.[14] As such, these In addition to XTEN, a variety of other unstructured polypep-
polymers may be regarded as biocompatible alternative to tides have been reported for optimizing the PK profile of bio-
PEG. pharmaceuticals. However, these technologies all rely on re-
combinant expression to provide a fusion protein with extend-
ed half-life. Examples include elastin-like polypeptide (Phase-
3.1 XTEN
Bio, www.phasebio.com),[19] gelatin-like protein,[20] and Pro-Ala-
XTEN is an 83.5 kDa recombinant polypeptide developed by Ser (PASylation; XL-Protein, www.xl-protein.com).[21] Several
Amunix Inc. The XTEN sequence is a nonrepetitive polymer drug candidates using these technologies are currently in clini-
consisting only of the amino acids Ala, Asp, Gly, Pro, Ser, and cal development. For a detailed account, the reader is referred
Thr. This amino acid composition provides a biopolymer that is to the overview by Binder & Skerra.[14]
unstructured, nonimmunogenic, highly soluble, and chemically
stable.[15] XTENylated biopharmaceuticals were originally pro-
duced as fusion proteins of the XTEN sequence and the phar- 4. Carbohydrate Polymers
maceutically active peptide or protein using recombinant DNA
4.1 Dextran conjugation
technology. The most advanced drug candidate using the
XTEN technology is somavaratan (VRS-317, Amunix/Versatis), Conjugation of dextran was one of the first alternatives to
which is currently in phase III clinical development for pediatric PEGylation, with early efforts dating back to the 1970s.[22] Dex-
growth hormone deficiency (GHD).[16] tran polymers are obtained from bacteria such as L. mesenter-
In a proof-of-concept study, the PK properties of a recombi- oides and are d-glucose polymers linked by a(1–6) glycosidic
nant fusion protein consisting of XTEN and the GLP-1 analogue linkages and a small extent of a(1–3) bonds (~ 95 % a(1–6) and
exenatide (named E-XTEN) were evaluated in cynomolgus 5 % a(1–3) in the case of L. mesenteroides) (Figure 3). The a(1–
monkeys. Fusion to XTEN prolonged the terminal (biological) 3) linkages serve as attachment points for primarily mono- or
half-life of exenatide from 0.48 to 60 h following subcutaneous disaccharide units, giving rise to a low degree of molecular
(s.c.) administration, corresponding to a 125-fold increase, em- branching.[23] Dextran preparations have been used as plasma
phasizing the potential of the technology.[15] volume expanders since the 1950s, and today two dextran so-
Although construction of XTEN fusion proteins initially was lutions (containing 10 % of 40 kDa dextran and 6 % of 70 kDa
an entirely recombinant process, it is now also possible to dextran, respectively) are approved in the US for this pur-
make XTEN conjugates by chemical means. Chemical XTENyla- pose.[24] For research purposes, dextrans with mean molecular
tion uses special versions of the XTEN polymer containing for weight (MMW) in the range of 4 to 200 kDa and low
example, amines, thiols, or dibenzocyclooctyne (DBCO) groups, polydispersity ( 2) are commercially available.[24]
which react selectively with succinimidyl esters, maleimides, or
azides, respectively. These reactive groups may then be intro-
duced into peptides by chemical methods to allow subsequent
conjugation. Examples include the glucagon-like peptide 2
(GLP-2) analogue GLP2-2G-XTEN developed by Amunix, which
consists of an XTEN variant and the GLP-2 analogue teduglu-
tide assembled by maleimide–thiol chemistry. The resulting
conjugate has a markedly decreased in vitro potency, as re-
flected in a > 50-fold decrease in relative in vitro activity rela-
tive to unmodified teduglutide. However, s.c. administration to
Sprague–Dawley rats revealed a half-life extension to 38.5 h,
resulting in a largely increased drug exposure.[17]
Chemical XTENylation has also been used to produce multi-
valent peptide-XTEN conjugates otherwise inaccessible by re-
combinant methods. Ding et al. have published conjugates of
XTEN and the HIV fusion inhibitor T-20 (enfuvirtide, Fuzeon) as-
Figure 3. Example of dextran architecture. Degree of cross-linking varies
sembled by maleimide–thiol chemistry. The conjugates contain among different dextran preparations.
multiple copies of T-20 attached to a single XTEN polymer, but
instead of traditional end-to-end fusion, the T-20 molecules are
attached at various positions along the XTEN sequence
through the side-chains of Cys residues. Investigation of the PK In addition to unmodified dextran, various synthetic dextran
properties of the most active conjugate (named T-20-XTEN-4) derivatives such as carboxymethyl-dextran (CMD), diethylami-
following s.c. administration to Sprague–Dawley rats revealed noethyl-dextran (DEAED), glycosylated versions of CMD such
an elimination half-life of 55.7 h, corresponding to a 20-fold in- as galactose-CMD (Gal-CMD) and mannose-CMD (Man-CMD),
crease relative to the reported half-life of unmodified T-20.[18] carboxymethyl benzylamide dextran (DCMB), carboxymethyl

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sulfate dextran (DCMSu), and carboxymethyl benzylamide sul-
fate dextran (DCMBSu) have also been reported.[25]
Historically, methods for coupling proteins to dextran have
focused on obtaining dextran conjugates with multiple copies
per dextran moiety rather than site-specific incorporation. First
attempts involved cyanogen bromide (BrCN) activation of dex-
tran hydroxy groups with subsequent aminolysis by protein
amino groups.[26] However, large product diversity combined
with the apparent instability of the protein–dextran linkage as
well as the toxicity of the BrCN reagent made this method un-
attractive.[23, 27]
Periodate oxidation to form dextran dialdehydes[28] followed
by reductive amination to yield a secondary amine linkage has
been used successfully for synthesizing protein–dextran conju-
gates (Figure 4 A).[25b, 29] Takakura et al. prepared conjugates of
dextran and soybean trypsin inhibitor (STI) by six different
chemical methods, including periodate oxidation and cyano-
gen bromide activation. Of all the tested methods, periodate
oxidation provided both high reaction yield, high degree of
molar substitution and high activity of the modified enzyme,
indicating that periodate oxidation is the preferred method for
synthesizing protein–dextran conjugates.[30] A similar conclu-
sion was made by Yasuda et al. from studies of dextran-conju-
gated uricase.[31] However, although periodate oxidation of
dextran may be controlled to some extent,[28a] inherent draw-
backs associated with this method include instability of the di-
aldehyde intermediate, product heterogeneity due to the pres-
ence of multiple ring-opening sites, and cross-linking of the
target protein if this contains multiple amino groups.[2, 23]
Dextran conjugation via a spacer has been extensively ex-
plored for small molecules,[23] but has also been applied to
peptides[22] and enzymes.[32] Introduction of spacers such as e-
aminocaproic acid at the reducing end has enabled site-specif-
ic conjugation of dextran to the N-terminus of catalase sub-
units (Figure 4 B).[32c] Lastly, dextran conjugation to proteins has
also been obtained by an enzymatic approach using microbial
transglutaminase to attach dextran with hexylenediamine at
the reducing end to surface Gln residues of catalase.[32d]
Similar to PEGylation, half-life extension by dextran conjuga-
tion is driven by the increase in hydrodynamic radius, which
decreases the renal clearance of the target compound. The
Figure 4. Dextran conjugation by A) periodate oxidation and subsequent reductive amination, and B) attachment of e-aminocaproic acid at the reducing end
followed by amide bond formation. Only monoconjugated product is shown; R: peptide or protein.
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threshold for renal clearance of dextran is ~ 40 kDa, as indicat-
ed by studies of both unconjugated dextran as well as fluores-
cein–dextran conjugates.[33] Studies of carboxypeptidase–dex-
tran conjugates in tumor-bearing Balc/c mice showed that
elimination of dextran is size-dependent, with conjugates con-
taining large dextran moieties exhibiting the longest half-
life.[34] This notion was further supported by PK studies of fluo-
rescein–dextran conjugates in rats.[24, 33b] Gel filtration studies of
the fluorescein–dextran conjugates demonstrated a linear rela-
tionship between dextran size and column retention time,
with larger dextrans displaying shorter retention times.[24, 33b] In
addition, conjugation of dextran has been shown to decrease
in vitro enzymatic degradation of catalase[32c] and to reduce
the immunogenicity of xenogeneic antibodies.[35]
Since 1975, dextran has been labeled as ‘Generally Regarded
As Safe’ (GRAS) by the FDA,[36] testifying to the overall safety of
these polymers. Dextran is metabolized in vivo by dextranases,
which are found predominantly in the liver and spleen.[23]
The ability of dextran and other colloid volume substitutes
to induce anaphylactoid reactions was investigated by Ring &
Messmer and the incidence of severe reactions following dex-
tran administration was found to be 0.008 %.[37] This was similar
to the incidence observed following administration of hydroxy-
ethyl starch (HES, see below) (0.006 %) and slightly raised rela-
tive to plasma protein solutions (0.003 %).[37] In a similar study
conducted by Laxenaire et al., the incidence of anaphylactoid
reactions following dextran administration was found to be
0.273 %, corresponding to a slightly increased incidence rela-
tive to HES (0.0058 %) and albumin (0.099 %).[38] However, the
very low incidence rates observed in both studies emphasize
the low risk of developing anaphylactoid reactions in response
to dextran infusion.
Although dextran-reactive antibodies have been implicated
in dextran-induced anaphylactoid reactions,[39] there are, to the
best of our knowledge, no published reports of antibodies
toward dextran conjugates in human subjects. However, Sep-
pl et al. were able to generate antibodies against a conjugate
of 10 kDa dextran and chicken serum albumin (CSA) after im-
munization of mice, and the immune response of the conju-
gate was stronger than the response obtained after immuniza-
tion with plain dextran.[40] Importantly, immunogenicity was
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most pronounced for CSA–dextran conjugates with small dex-
trans (1 and 4 kDa, respectively), but was lower for a conjugate
containing a large 40 kDa dextran moiety.[41]
A long-acting somatostatin–dextran conjugate has been pre-
pared for cancer treatment. Using reductive amination, soma-
tostatin was coupled to a 70 kDa dextran moiety activated
using sodium periodate to yield a conjugate with a 4:1 soma-
tostatin/dextran ratio. The somatostatin–dextran conjugate
showed receptor binding affinity in the low nanomolar range,
which was similar to octreotide (a somatostatin analogue). Fol-
lowing s.c. administration to mice, the conjugate displayed an
extended PK profile with delayed absorption (maximal concen-
tration being reached 24 h post-injection) and prolonged half-
life relative to octreotide (27 h for conjugate,[42] compared to
less than 2 h in man for octreotide).[43] The conjugate has been
evaluated in phase I clinical trials and was found to be well-tol-
erated.[44]
Villalonga and co-workers have reported the synthesis of
catalase–dextran conjugates. Catalase is a tetrameric enzyme
important for neutralization of reactive oxygen species that
have been associated with a variety of diseases. First, a cata-
lase–dextran conjugate was prepared using a 5 kDa dextran
with a hexyleneamine installed at the reducing end. Coupling
of dextran to Asp and Glu residues situated on the surface of
catalase was performed at pH 6 under aqueous conditions and
resulted in a product containing an average of four dextrans
per catalase molecule, corresponding to one dextran chain per
catalase subunit. The specific activity of the catalase–dextran
conjugate was slightly increased over unmodified catalase
(from 14.6 U g 1 to 15.5 U g 1). Moreover, intravenous (i.v.) ad-
ministration of the conjugate to lean rats illustrated a markedly
improved PK profile, with a 20-fold longer elimination half-life
(from 0.8 to 16 h), a 171-fold increased exposure (from 1.8 to
307 U h mL 1), and a 176-fold decreased clearance (from 120 to
0.68 mL min 1).[32a]
Second, Villalonga and co-workers prepared a dextran–cata-
lase conjugate using a dextran variety activated with an e-ami-
nocaproic acid spacer. By using this spacer, site-specific inser-
tion of dextran at the N-terminus of each catalase subunit was
achieved. Although catalase activity was decreased by 23 %,
this catalase–dextran variant displayed improved thermostabili-
ty as well as decreased susceptibility to trypsin digestion (func-
tional half-life extension from 40 min to 2 h as compared with
native catalase). Intravenous administration of the dextran–cat-
alase conjugate to rats revealed a seven-fold extension of half-
life relative to unmodified catalase (from 0.7 to 5.1 h) as well
as a 104-fold increase in exposure (from 1.9 to 197 U h mL 1),
and a 100-fold decrease in clearance (from 110 to
1.1 mL min 1).[32c]
Villalonga and co-workers have also reported the synthesis
of a conjugate of superoxide dismutase (SOD) and dextran.
Like catalase, SOD is an enzymatic scavenger of reactive
oxygen species, specifically the superoxide anion. The dextran
conjugate was prepared using hexyleneamine chemistry and
provided a SOD–dextran conjugate containing an average of
4.4 dextran units per SOD subunit. The conjugate retained
81 % of initial enzymatic activity, but showed a 10.8-fold in-
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creased half-life (from ~ 20 min to 3.6 h) in a H2O2 inactivation
assay. As in the case of catalase, the PK profile of the SOD–dex-
tran conjugate was markedly improved over wild-type SOD.
The conjugate showed a 150-fold longer elimination half-life
(from 4.8 min to 12 h), a 9.7-fold increase in exposure, and
a 9.8-fold decrease in clearance (from 62.5 to 6.4 U h 1) follow-
ing i.v. administration to rats. The improved PK profile and the
increased resistance toward H2O2 inactivation further translated
into improved pharmacological properties of the SOD–dextran
conjugate, as visualized by a carrageenan-induced paw edema
test.[32b]
Other examples include dextran conjugates of asparagi-
nase,[45] carboxypeptidase G2,[34, 46] and hemoglobin,[47] respec-
tively, as well as CMD-insulin conjugates.[48]
4.2 HESylation
Hydroxyethyl starch (HES) is a modified natural polymer ob-
tained by controlled hydroxyethylation of the plant polysac-
charide amylopectin. Amylopectin is a polymer of d-glucose
containing primarily a-1,4 glycosidic bonds, but also a lower
abundance of a-1,6 linkages, leading to a naturally branched
carbohydrate (Figure 5).[49] Hydroxyethylation of the starch pre-
cursor serves two purposes: first, to increase the water solubili-
ty by increasing water-binding capacity and decreasing viscosi-
ty, and second, to prevent immediate degradation by plasma
a-amylase and subsequent renal excretion.[49] Hydroxyethyla-
tion predominantly occurs at C2 and C6, and to a lesser extent
at C3.[50]
Figure 5. Example of HES architecture. Degree of cross-linking as well as the
amount and location of hydroxyethyl substituents vary among different HES
preparations.
The first use of HES for pharmacological purposes dates
back to the 1970s, when this modified polysaccharide was in-
troduced by Fresenius Kabi as a plasma volume substitute.
Commercially available HES products are characterized by
three parameters: mean molecular weight (MMW); molar sub-
stitution (MS), calculated as the average number of hydroxy-
ethyl groups per glucose unit; and the C2/C6 ratio, which de-
scribes the substitution pattern at C2 relative to C6 (Table 2).
HES preparations are labeled according to mean molecular
weight and molar substitution (e.g., HES 450/0.7, which has
MMW = 450 kDa and MS = 0.7) (Table 2).[50]
7  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim &
 Reviews
The conjugation of HES moieties increases the hydrodynam-
Table 2. Common HES preparations and their parameters.
ic volume of the modified molecule, thereby making it less
Name MMW [kDa][a] MS[b] C2/C6 ratio susceptible to renal clearance.[49] The most relevant parameters
HES 450/0.7[156] 450 0.7 5:1 for describing the PK properties of the various HES polymers,
HES 200/0.62[49] 200 0.62 9:1 in addition to size, are molar substitution and C2/C6 ratio.
HES 200/0.5[157] 200 0.5 5:1 High molar substitution, reflecting a high degree of hydroxy-
HES 70/0.5[157] 70 0.5 3:1
ethylation, decreases enzymatic degradation by a-amylase via
HES 130/0.4[156] 130 0.4 9:1
HES 130/0.4[156] 130 0.42 6:1 steric factors, resulting in prolonged elimination half-life after
both single and multiple doses.[50] In addition, a high C2/C6
[a] MMW: mean molecular weight. [b] MS: molar substitution.
ratio further decreases susceptibility to enzymatic degradation.
This is especially pronounced for HES preparations of similar
Interestingly, correct choice of analytical method is essential molar substitution.[50, 57] However, it should be noted that HES
for accurate size determination of HES polymers. Using size ex- polymers with high MMW are more effective providers of steric
clusion chromatography coupled to low-angle laser light scat- shielding, as indicated by studies on HES-shielded polyplex-
tering (SEC-LALLS), Lederer et al. showed that commercially es.[54] HESylation has also been shown to increase protein sta-
available HES 450/0.7 has a MMW of ~ 740 kDa, corresponding bility by increasing the melting temperature and the enthalpy
to a 64 % increase from the nominal value determined by con- of melting as well as decreasing stress-induced aggregation.[56]
ventional light scattering.[51] HES is ultimately metabolized in vivo by a-amylase in
Chemical coupling of HES to proteins was originally explored plasma and excreted by the kidneys as smaller fragments. In-
in the 1970s, typically as a random conjugation approach in- tracellular degradation of HES by lysosomal enzymes such as
volving BrCN activation of available alcohols followed by ami- a-glucosidase has also been suggested,[58] but the exact mech-
nolysis to yield an isourea or carbamate linkage.[49, 52] However, anism remains unknown. Because of the close resemblance be-
the latter approach often results in cross-linked products, and tween the semisynthetic HES polymer and the natural glucose
random conjugation has now been superseded by site-specific polymers known to the human body (e.g., glycogen and
incorporation. This approach utilizes that the HES polymer only starch), HES is generally considered nonimmunogenic. This
contains one available hemiacetal (i.e., the reducing end), notion was emphasized by Dieterich et al., who investigated
which may be oxidized using iodine,[49] KMnO4,[53] or NaClO[53] antibody formation in 1004 patients in response to infusion of
to yield the terminal aldonic acid. The aldonic acid can be acti- various HES preparations.[59] Only one patient (corresponding
vated either by dehydration to produce the corresponding lac- to 0.1 %) developed low-titer IgM antibodies, corresponding to
tone or by formation of an active NHS-ester (Figure 6 A).[49] In a very low incidence. Importantly, the antibodies were specifi-
both cases, subsequent aminolysis results in the formation of cally directed toward the hydroxyethyl groups, with no ob-
a polypeptide–HES conjugate linked by an amide bond. served cross-reactivity toward amylose, a natural component
An alternative to oxidation exploits that hemiacetals are in of starch.[59] In rabbits, however, Richter & de Belder were able
equilibrium with the corresponding open-chain aldehyde, to generate high-titer anti-HES antibodies after immunization
which enables HESylation via reductive amination using with a bovine serum albumin–HES (BSA–HES) conjugate. No
NaBH3CN (Figure 6 B).[49, 54] The aldehyde may also be obtained cross-reactivity toward amylopectin or glycogen was observed
by oxidation of the hemiacetal with NaIO4.[55] Finally, the alde- for the anti-HES antibodies.[52]
hyde functionality may be introduced via a spacer at the re- As indicated by the previously mentioned studies by Ring &
ducing end.[56] Messmer and Laxenaire et al., the risk of developing anaphy-

Figure 6. Examples of HESylation chemistry. A) The hemiacetal of the reducing end of the polymer chain may be oxidized to yield the corresponding aldonic
acid, which then can be activated by dehydration to form the lactone or by formation of the NHS ester. B) The hemiacetal exists in an equilibrium with the
open-chain aldehyde, which enables HESylation via reductive amination. R: peptide or protein; TSTU: N,N,N’,N’-tetramethyl-O-(N-succinimidyl)uronium tetra-
fluoroborate; DIEA: N,N-diisopropylethylamine.

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lactoid reactions following HES infusion is very low (< 0.1 %)
and does not exceed the risk associated with dextran infu-
sion.[37, 38]
Despite the effective enzymatic degradation of infused HES,
transient bioaccumulation of the polymer is known to occur. In
a recent meta-analysis of HES bioaccumulation conducted by
Wiedermann & Joannidis, several safety concerns were raised.
Based on data from 37 human studies (635 patients in total),
the authors documented accumulation in a wide range of
tissue types, including skin, kidney, liver, bone marrow, lymph
nodes, spleen, lung, pancreas, intestine, muscle, trophoblasts,
as well as placental stroma. Tissue uptake was very rapid (as
early as 30 min after infusion), and storage increased in a dose-
dependent manner. HES deposits could be detected in the
skin and in the kidneys for as long as eight and ten years, re-
spectively, after administration. Other adverse events were pru-
ritus and renal failure, and cases of liver dysfunction and bone
marrow suppression were also associated with HES administra-
tion.[60]
HES has also been associated with negative effects on the
blood coagulation system. In a study of 30 patients with cere-
brovascular disease, administration of three different HES prep-
arations (HES 200/0.62, HES 200/0.5, HES 40/0.5, n = 10 for
each group) for ten days resulted in a decrease in platelet
volume, with the more substituted HES 200/0.62 causing the
largest decrease.[61] A further comparison of HES 200/0.62 and
HES 200/0.5 in a ten-day hemodilution study revealed that in-
fusion of HES200/0.62 led to acquired von Willebrand syn-
drome as well as large decreases in FVIII function (72.2 %), von
Willebrand ristocetin cofactor (61.3 %), and von Willebrand
factor antigen (64 %). In contrast, no adverse effects were ob-
served for HES 200/0.5. The discrepancy was attributed to the
slower degradation of HES 200/0.62, which upon repeated ad-
ministration leads to an in vivo build-up of high molecular
weight HES fragments and causes negative effects on blood
coagulation.[62]
The first attempt to use HES for improving PK properties
dates back to 1989, when the polymer was used to prolong
the half-life of deferoxamine, a siderophore isolated from S. pi-
losus. The deferoxamine–HES conjugate was prepared by initial
activation of HES (obtained from HES 450/0.7) with sodium pe-
riodate to provide the aldehyde, followed by reductive amina-
tion with NaBH3CN to yield a substituted HES polymer contain-
ing 10–20 % (w/w) deferoxamine.[55] Intravenous administration
of the deferoxamine–HES conjugate to male Swiss-Webster
mice showed that the half-life of the conjugate was increased
15-fold relative to unmodified deferoxamine (from 5.5 to
84 min), while the ability to induce urinary excretion of excess
iron was fully maintained.[55]
HES was also used to prepare a conjugate of AGE M400,
a novel dimeric erythropoietin mimetic peptide (EMP) with op-
timized in vitro performance. The conjugate, named
AGEM400(HES), was assembled by reacting the free thiol of the
C-terminal Cys of the AGEM400 sequence with a maleimide-
functionalized HES polymer (MW = 130  20 kDa, obtained
from HES 200/0.5) under aqueous conditions to provide conju-
gates containing an average of five peptides per HES poly-
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mer.[63] The conjugate was deemed equipotent to erythropoie-
tin (EPO), as reflected by similar half-maximal effective concen-
tration (EC50) values in survival assays conducted in EPO-re-
sponsive cell lines.[63] Notably, the conjugate was also found to
be equipotent to both EPO and Aranesp in stimulating eryth-
ropoiesis from bone marrow cells in vitro.[63]
A long-acting factor VIII (FVIII) for prophylactic treatment of
hemophilia A was prepared by site-specific covalent attach-
ment of HES to B-domain deleted rFVIII. Addition of up to
700 kDa HES provided FVIII–HES conjugates with fully pre-
served in vitro activity and retained ability to bind von Wille-
brand factor with high affinity.[64] Moreover, the FVIII–HES con-
jugate exhibited acute efficacy comparable to rFVIII in a hemo-
philia A mouse-tail bleeding model, while also demonstrating
prolonged exposure as a result of extended half-life.[64]
More recently, a long-acting HESylated version of anakinra
(recombinant human interleukin 1 receptor antagonist, rhIL-
1ra) was reported. Unmodified anakinra is approved for the
treatment of rheumatoid arthritis, but has a short-acting PK
profile. A HESylated analogue, anakinra–HES, was obtained by
conjugation of 85 kDa HES at the N-terminus. The HES moiety
carried a propionaldehyde spacer at the reducing end, en-
abling conjugation by reductive amination. Biophysical charac-
terization of the anakinra–HES conjugate confirmed the ex-
pected increase in molar mass (from 16.6 to 105.5 kDa) and hy-
drodynamic radius (from 4.36 to 14.73 nm). The secondary
structure of anakinra was unaffected by HESylation, and the
conjugate retained a dissociation constant (KD) in the nanomo-
lar affinity range toward the interleukin-1 receptor, as shown
by surface plasmon resonance (SPR) and microscale thermo-
phoresis (MST) experiments. In addition, differential scanning
calorimetry (DSC) revealed that HESylation increased protein
stability, as illustrated by an increase in melting point (from
58.0 to 62.8 8C) and in enthalpy of melting (from 58.4 to
100.8 kcal mol 1). The conjugate exhibited no stress-induced
aggregation during a seven day shaking test, whereas aggre-
gation of unmodified anakinra was observed already after
30 min. Intravenous injection of anakinra–HES to rats revealed
a markedly improved PK profile of the conjugate, with a 45-
fold decrease in clearance (from 403.3 to 9.1 mL kg 1 h 1),
a 6.5-fold increase in half-life (from 1.7 to 10.8 h) and a 45-fold
increase in exposure (from 12.3 to 548.8 ng mL 1).[56]
4.3 Polysialylation
Conjugation of polysialic acid (PSA) to drugs in order to im-
prove their pharmacokinetic properties (polysialylation) was
first suggested and explored by Gregoriadis and co-workers in
1993.[65] Strictly speaking, the term ‘sialic acid’ refers not to
a single chemical entity, but rather to an entire group of nine-
carbon monosaccharides, with the most important examples
being 5-N-acetylneuraminic acid (Neu5Ac), 5-N-glycolylneura-
minic acid (Neu5Gc), and 2-keto-3-deoxynonulosonic acid
(Kdn).[66] However, the only observed PSA variant in humans is
colominic acid (CA), the linear a-2,8-linked homopolymer of
Neu5Ac (Figure 7).[67]
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Figure 7. Structure of colominic acid, the linear polymer of Neu5Ac used for
polysialylation.
CA is a naturally occurring polysaccharide found mainly as
an extracellular glycan of the neural cell adhesion molecule
(NCAM) protein, which is known to be very important in cell–
cell adhesion and synaptic plasticity. NMR studies have shown
that the ‘basal conformational unit’ of CA is an extended heli-
cal structure with n = 9.[66, 68] For pharmaceutical purposes, CA
is used almost exclusively. The technology is currently being
commercialized as PolyXen by Xenetic Biosciences (formerly
Lipoxen PLC, www.xeneticbio.com), and several polysialylated
drug candidates are now in development, including EreproXen
(polysialylated EPO) in phase II/III clinical trials.[69]
For the production of PSA-drug conjugates, an activated
form of CA containing a terminal aldehyde moiety is used. This
species may be obtained by mild NaIO4 oxidation of the C7–C8
diol system found in the non-reducing end of the polysaccha-
ride to yield an aldehyde functionality. Conjugation may then
be performed by nucleophilic attack of an amino group to
yield a Schiff base, followed by reductive amination under
aqueous conditions using excess of oxidized colominic acid in
the presence of NaBH3CN (Figure 8 A).[70] The reductive amina-
tion strategy has so far been the most widely used method for
producing polysialylated drugs.[71] However, because peptides
and proteins often contain multiple amino groups in the form
of N-terminal amino group(s) and Lys Ne-amines, this approach
may lead to a mixture of products.
Alternatively, polysialylation may also be obtained by the
chemoselective maleimide–thiol conjugation reaction. A malei-
mide-activated PSA species can be prepared by reacting the al-
dehyde functionality of oxidized PSA with N-[b-maleimidopro-
pionic acid] hydrazide to produce a maleimide-functionalized
PSA hydrazone. Subsequent conjugation to the thiol function-
ality of an available Cys may then be performed under slightly
basic aqueous conditions (Figure 8 B).[71e] This approach is
Figure 8. Chemical introduction of PSA. A) The non-reducing end of CA may be oxidized to yield an aldehyde, which may then undergo reductive amination,
enabling conjugation via the N-terminal amino group(s) and Lys Ne amines. B) CA aldehyde may also be further modified by incorporation of a maleimide,
which may then be selectively attached to the thiol functionality of a free Cys. R: peptide or protein.
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often site-specific, as free Cys residues occur at a low frequen-
cy in natural polypeptides or have been specifically engineered
into the peptide or protein.
In addition to these chemical methods, a chemo-enzymatic
method using the bacterial enzymes C. jejuni Cst-II sialyltrans-
ferase and N. meningitides a2,8-polysialyltransferase has also
been described for the production of polysialylated proteins.[72]
Similar to PEG and the PEG mimetics dextran and HES, the
driving force behind the improved PK properties of polysialy-
lated compounds is thought to be an increase in hydrodynam-
ic volume, resulting in decreased renal clearance as well as
shielding from enzymatic degradation and antibody recogni-
tion.[65, 71e] A notable physicochemical feature of PSA is the per-
manent negative charges. It may be speculated that this fea-
ture decreases glomerular filtration even further because of
electric repulsion by the negatively charged surfaces of glo-
merular capillaries.[73]
It has been proposed that the increase in proteolytic stabili-
ty occurs by a different mechanism than observed with PEGyla-
tion.[71a] Catalase is a very large protein with a molecular
weight of 240 kDa. It was observed that a catalase–PSA conju-
gate containing four 10 kDa CA units was less prone to degra-
dation by trypsin and chymotrypsin than native catalase. The
negatively charged CA polymer may interact with positively
charged amino acids residues of the enzyme, thereby forming
an extensive hydrophilic layer able to shield against protea-
ses.[71a]
Interestingly, PSA has been shown to act as a ‘lyo-protec-
tant’. A catalase–PSA conjugate was shown to retain 35–40 %
of its initial activity after lyophilization, compared with only
20 % for the corresponding native enzyme.[71a] The exact mech-
anism behind this observation is unknown, but it was speculat-
ed that during the freeze-drying process, the extreme hydro-
philicity of the PSA polymer is able to maintain an aqueous mi-
croenvironment around the enzyme necessary for maintaining
the active conformation.[71a]
In contrast to the synthetic PEG and semi-synthetic HES
polymers, PSAs are truly natural polymers. Colominic acid is
biodegradable and the ultimate degradation product, Neu5Ac,
is not known to be toxic.[65] Moreover, PSAs are T-cell inde-
pendent antigens, and immunization of mice with colominic
acid resulted in no detectable antibody formation, suggesting
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that this PSA variant is nonimmunogenic.[65, 74] Immunization
with PSA-protein conjugates, however, have been shown to
elicit an immune response, but in the case of colominic acid,
this only leads to generation of low-affinity antibodies that cir-
culate at low concentration.[65] These circumstances all contrib-
ute to create an attractive safety profile for PSA.
Early proof-of-concept for polysialylation was obtained when
Gregoriadis and co-workers showed that conjugation of
a 24 kDa CA (82 Neu5Ac units) to fluorescein increased the
half-life of this otherwise rapidly excreted compound. The fluo-
rescein–PSA conjugate exhibited a half-life of 20 h following
i.v. administration to mice, corresponding to the half-life of the
PSA polymer itself.[65]
The concept was later expanded to also include modulation
of protein pharmacokinetics. In 1996, Fernandes & Gregoriadis
presented a modified version of catalase containing an average
3.8 molecules of 10 kDa CA per molecule enzyme produced by
the reductive amination approach. Compared with the native
enzyme, the catalase–PSA conjugate exhibited increased stabil-
ity toward degradation by the endopeptidases trypsin and chy-
motrypsin (illustrated by 95 % retained activity of the conju-
gate compared with only 15 % retained activity of the native
enzyme after incubation with chymotrypsin for 3 h at 37 8C).[71a]
In addition, polysialylated versions of asparaginase were re-
ported with ~ 85 % retained enzymatic functionality and im-
proved plasma stability relative to the native enzyme. The
lowest degree of polysialylation provided the largest increase
in stability, with 87 % retained activity being observed for of
a conjugate with an average of 4.2 CA units per asparaginase
molecule compared with only 18 % retained activity of the
native enzyme after 6 h of incubation at 37 8C in diluted
mouse plasma.[71b] Intravenous injection of asparaginase–PSA
to mice revealed an improvement in functional and terminal
half-life proportional to the degree of PSA conjugation, with
a conjugate containing an average of 8.1 CA units per aspara-
ginase molecule exhibiting a half-life of almost 40 h.[71b, 75]
Moreover, measurement of antibody titers following immuniza-
tion of mice with asparaginase–PSA conjugate suggested
a lower immunogenicity compared to the native enzyme.[75]
Two polysialylated versions of insulin, modified with 22 and
39 kDa CA chains, respectively, have also been described.[71c]
The insulin–PSA conjugates were assembled by the reductive
amination approach using excess of CA aldehyde, yielding
compounds with PSA/insulin molar ratios of 1.60–1.74 and
2.37–2.45 for the 22 kDa and 39 kDa CA variant, respectively.
Subcutaneous administration of insulin–PSA to mice showed
that polysialylated insulin had a prolonged efficacy relative to
unmodified insulin, with the 22 kDa CA variant having a glu-
cose lowering effect for 9 h, compared with 6 h for the 39 kDa
CA variant and 3 h for native insulin.[71c]
Conjugation of PSA has also been used to modulate anti-
body pharmacokinetics. Polysialylation of a Fab antibody frag-
ment specific for the placental-like alkaline phosphatase (PLAP)
antigen was performed by reductive amination using either
11 kDa or 22 kDa periodate-oxidized CA, providing Fab–PSA
immunoconjugates with a varying degree of molar substitu-
tion.[71d] Antigen affinity of the obtained immunoconjugates
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was only slightly decreased relative to unmodified Fab (2.7- to
3.7-fold or 1.3- to 3.4-fold, depending on the antibody used for
ELISA detection).[71d] Intravenous administration of the Fab–
PSA immunoconjugates to mice showed a 4.2- to 5.2-fold in-
crease in bioavailability and a 1.8- to 3.3-fold increase in total
tumor exposure compared to native Fab, with a higher degree
of polysialylation providing the biggest improvements.[71d]
Site-specific polysialylation of the single-chain variable frag-
ment (scFv) MFE-23, a well-described anti-carcinoembryonic
antigen, was obtained using the chemoselective maleimide–
thiol conjugation reaction. An engineered version of MFE-23
containing a terminal Cys residue was produced by recombi-
nant expression and polysialylated using a maleimide-function-
alized PSA hydrazine, which was produced from 11 kDa CA al-
dehyde and N-[b-maleimidopropionic acid] hydrazide.[71e] The
MFE-23-Cys–PSA immunoconjugate retained its antigen affinity
compared with both native MFE-23 and MFE-23-Cys, and s.c.
administration of MFE-23-Cys–PSA to mice revealed a 3.6-fold
prolongation of elimination half-life (from 4.3 to 15.6 h) and an
9.8-fold increase in bioavailability compared to native MFE-23,
which was accompanied by an increase in tumor exposure and
uptake.[71e]
Recently, a polysialylated version of the anorectic gut pep-
tide oxyntomodulin (Oxm) with improved protease resistance
was published.[71f] The Na-substituted Oxm–PSA conjugate was
produced by reductive amination using a commercially avail-
able 14.1 kDa oxidized PSA.[71f] Oxm is a well-known substrate
for the enzyme dipeptidyl peptidase IV (DPP-4), which partly
explains the very short in vivo half-life of native Oxm. Incuba-
tion of the Oxm–PSA conjugate with DPP-4 at a concentration
of 2.9 U mL 1 over a period of 20 h revealed no enzymatic deg-
radation, suggesting that N-terminal polysialylation of Oxm
confers complete resistance toward DPP-4.[71f]
Also recently, conjugates of PSA and butyrylcholinesterase
(BChE), a bioscavenger useful for treatment of organophospho-
rus poisoning, was reported.[71g] A commercially available
27 kDa mono-oxidized CA polymer containing a terminal
Neu5Ac unit with a C7-keto group (named CAO27) was conju-
gated to recombinant human BChE (rhBChE) by reductive ami-
nation, resulting in a polysialylated product (rhBChE-CAO27)
with a protein/PSA ratio of 1:6 and full catalytic activity relative
to unmodified rhBChE.[71g] Intravenous administration of
rhBChE-CAO27 to mice revealed a 6.3-fold increase in mean
residence time (from 3.7 to 23.3 h) and a 5.6-fold increase in
elimination half-life (from 3 to 16.6 h) in comparison with un-
modified rhBChE.[71g]
A polysialylated version of the 52 kDa glycoprotein a-1-anti-
trypsin (A1AT), a serine protease inhibitor targeting the
enzyme neutrophil elastase, has also been described.[72] Native
A1AT is currently used as a therapeutic protein useful in treat-
ment of patients with congenital A1AT deficiency, a disorder
associated with lung emphysema, chronic obstructive lung dis-
ease (COPD), as well as other disorders.[76] Using their chemo-
enzymatic method (see above), Lindhout et al. attached six
PSA undecamers to the three available biantennary sialylation
sites of A1AT. The bioactivity of the resulting product (PSA-
A1AT) was examined using an enzyme activity assay measuring
11  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim &

the inhibition of neutrophil elastase, with PSA-A1AT exhibiting
retained bioactivity compared to native A1AT.[72] Intravenous
administration of PSA-A1AT to mice revealed an 18-fold greater
bioavailability (measured as area under the curve) and a 5.4-
fold increase in elimination half-life (from 5 h to 27 h).[72] Inter-
estingly, a version of A1AT containing very short PSA oligomers
with only 1–3 repeats of Neu5Ac (named diSA-A1AT) exhibited
an approximate 2-fold increase in bioactivity, a 6.5-fold in-
crease in bioavailability as well as a 2.6-fold increase in elimina-
tion half-life (from 5 to 13 h) relative to unmodified A1AT.[72]
These data indicate that introduction of only very few sialic
acid residues may have dramatic effects on the pharmacokinet-
ic properties of biopharmaceuticals.
4.4 HAylation
As in the case of PSA, hyaluronic acid (HA) is a negatively
charged linear polysaccharide. HA consists of alternating d-glu-
curonic acid (GlcA) and N-acetyl-d-glucosamine (GlcNAc) units
(repeating disaccharide: [b(1-4)-GlcA-b(1-3)-GlcNAc]n (Figure 9)).
It is a naturally occurring biopolymer found primarily in the ex-
tracellular matrix of connective tissues, with highest abun-
dance in the synovial fluid of joints, the dermis of the skin, and
the vitreous humor of the eye.[77] The HA polymer serves multi-
ple physiological purposes, including tissue viscoelasticity and
repair, control of tissue hydration, as well as cell proliferation,
differentiation, and migration.[78] These features have prompted
the use of HA for various medical applications such as visco-
supplementation for treating joint disease, wound healing,
ophthalmic surgery, and cosmetic applications.[79]
Figure 9. Chemical structure of the repeating GlcA-GalNAc disaccharide that
makes up HA.
Under normal physiological conditions, a HA polymer con-
sists of 20 000–25 000 disaccharide units, corresponding to
a molar weight of up to 10 MDa and a length of 2–25 mm.[80]
For conjugation chemistry, however, polymers of maximal 100–
300 kDa are used to avoid viscosity problems.[2] HA variants of
appropriate size and quality for medical applications may be
obtained by fermentation using S. equi as expression host,[81]
or alternatively by degradation of high-MW HA polymers[82] or
by chemoenzymatic synthesis using P. multocida hyaluronic
acid synthase (PmHAS).[83]
Conjugation of large HA polymers increases the hydrody-
namic radius of the modified therapeutic, thereby decreasing
renal clearance and prolonging plasma half-life. In addition,
the hydrodynamic nature of HA combined with its ability to
retain water can shield conjugated proteins from enzymatic
degradation.[80, 84] Due to its presence in the human body and
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long-term use in various medical applications, HA is regarded
as biocompatible, nontoxic, and nonimmunogenic.[85] The poly-
mer is degraded in vivo by enzymes known as hyaluronidases
in a highly controlled manner, which also involves receptor-
mediated cellular uptake.[79] This controlled degradation pat-
tern has suggested a potential use of HA for drug delivery, pro-
viding sustained release of the active component upon HA
degradation in vivo.[86]
Numerous options exist for chemical modification of HA,
and these have been used for preparing both cross-linked HA
derivatives as well as several drug-HA conjugates.[79, 85] For the
purpose of preparing protein-HA conjugates, preferred meth-
ods have been either direct coupling of protein amino groups
to the COOH of GlcA or partial periodate oxidation of 2-OH
and 3-OH of GlcA to yield the dialdehyde followed by reduc-
tive amination.[78] However, both of these strategies are prone
to result in cross-linking of HA and the target protein and may
also lead to significant product heterogeneity.[78, 86a]
To address this limitation, strategies to avoid cross-linking
and produce multivalent HA conjugates have been developed
(Figure 10). Pasut and co-workers attached a 4-aminobutyral-
dehyde diethyl acetal as spacer to the COOH groups of GlcA
(Figure 10 A), creating 200 kDa HA polymers with varying
degree of acetal derivatization. The acetal may be hydrolyzed
under mildly acidic conditions to yield the corresponding alde-
hyde, which may then be used for conjugation of protein
amino groups by reductive amination.[86a]
Initially, the HA-acetal was used to prepare multivalent HA
conjugates of trypsin, RNase A, and insulin.[86a] By varying the
pH value, the conjugation process could be directed toward
Lys residues or be selective for the N-terminus. HA conjugation
increased the hydrodynamic radius of both trypsin and
RNase A, but also decreased the enzymatic activity relative to
the unmodified enzymes, possibly due to steric hindrance.
However, thermal stability was increased in the case of the N-
terminal trypsin-HA conjugate.[86a] HAylation of insulin provided
a conjugate with a prolonged and enhanced glucose-lowering
effect after administration to diabetic rats, suggesting that
HAylation of insulin increases the half-life and facilitates forma-
tion of a soluble depot.[86a]
Pasut and co-workers have subsequently employed the HA-
acetal for synthesizing a conjugate of HA and salmon calcito-
nin (sCT-HA). The HA polymer was site-selectively attached to
the N-terminus of sCT by reductive amination, and the result-
ing conjugate displayed an extended pharmacokinetic profile
and chondro-protective effects in the ALCT rabbit model of os-
teoarthritis.[87] Also using the HA-acetal, the same authors pre-
pared a conjugate of the interferon a2a (INFa2a) and HA with
increased bioavailability due to a HAylation-induced depot
effect and improved antitumor activity in an ovarian cancer
xenograft mouse model.[88]
In another approach, Hahn and co-workers used a methacry-
lated version of 200 kDa HA to obtain multivalent conjugates
of HA and CWRYMVm, a peptide agonist of the formyl peptide
receptor like 1 (FPRL1) receptor.[89] Aminoethyl methacrylate
HA (AMEA-HA) was synthesized by coupling AEMA to the
COOH functionality of the GlcA residues of HA (Figure 10 B).
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Figure 10. Representative GlcA-GlcNAc disaccharides from HA derivatives used for controlled preparation of multivalent HA conjugates: A) HA acetal, B) HA
methacrylate, C) HA vinyl sulfone, and D) HA dialdehyde.
AMEA-HA was subsequently coupled to the Cys residue of
CWRYMVm by Michael addition of the thiol to the methacry-
late double bond. By changing the amount of AMEA used for
preparing the AMEA-HA polymers, the average number of pep-
tides per HA conjugate could be controlled in the range from
5 to 23. Compared to unmodified CWRYMVm, the multivalent
HA conjugates had increased hydrodynamic volume and ex-
hibited increased stability in diluted serum, with no degrada-
tion observed after 96 h of incubation. The multivalent
CWRYMVm-HA conjugates displayed partially decreased bio-
logical activity in terms of FPR L1 receptor activation, but full
activity was recovered upon treatment with hyaluronidases.[89]
Using a related strategy, Hahn and co-workers have also pre-
pared multivalent conjugates of exendin-4 (Ex4) and HA using
a 100 kDa HA variant containing a vinyl sulfone functionality at
the COOH position (VS-HA) (Figure 10 C).[90] Ex4 was prepared
with an additional C-terminal Cys residues and efficiently con-
jugated to VS-HA by Michael addition under slightly alkaline
conditions to yield multivalent Ex4-HA conjugates with a vary-
ing number of peptides per HA polymer. The conjugates have
an increased hydrodynamic volume and a markedly prolonged
half-life when incubated with human serum (half-life of > 96 h,
corresponding to a 20-fold increase over unconjugated Ex4).
The in vivo efficacy of the Ex4-HA conjugates was evaluated in
diabetic db/db mice. Compared to unmodified Ex4, the hypo-
glycemic effect of Ex4-HA was similar in an intraperitoneal glu-
cose tolerance test (IPGTT) but prolonged following adminis-
tration of a single s.c. dose, with the effect being maintained
for up to three days.[90]
Although the periodate oxidation strategy is associated with
product heterogeneity, it has been used to prepare multivalent
conjugates of HA and interferon a (INFa) in a controlled fash-
ion.[91] Using NaIO4, Kahn and co-workers obtained a partly oxi-
dized 100 kDa HA polymer (Figure 10 D) and used it to prepare
INFa-HA conjugates with varying INFa content (2–9 copies per
HA polymer). Conjugation by reductive amination was per-
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formed under mildly acidic conditions to target only the N-ter-
minus of INFa. HAylation increased the hydrodynamic volume
while retaining the secondary structure of INFa, as indicated
by gel permeation chromatography (GPC) and circular dichro-
ism (CD) spectroscopy, respectively. Compared with unconju-
gated INFa, a HA conjugate containing six INFa molecules ex-
hibited decreased biological activity in an anti-proliferation
Daudi cell assay, but displayed improved serum stability (half-
life of > 120 h, corresponding to a 3-fold increase). Following
i.v. administration to rats, INFa-HA exhibited prolonged circula-
tion and could be detected for 50–110 h (depending on the al-
dehyde content of the polymer), whereas native INFa was
cleared within 24 h.[91]
5. N- and O-Glycosylation
Protein glycosylation is one of the most important and most
complex post-translational modifications.[92] It has been esti-
mated that more than 50 % of all proteins may in fact be gly-
coproteins, emphasizing the ubiquity of glycosylation.[93] Glyco-
sylation of peptides and proteins has many functions, includ-
ing modulation of protein physicochemical properties, control
of protein folding, stabilization of protein conformation, pro-
tein interactions and cellular recognition, protection from pro-
teolytic degradation, regulation of protein transport and locali-
zation, modification of immunological properties, and media-
tion of protein-protein and cell–cell interactions.[94] Many of
these effects directly provide half-life extension of a glycosyla-
ted peptide or protein.
In N-glycosylation, a glycan is attached by a glycosidic
amide from GlcNAc to the side-chain of an Asn residue in an
Asn-X-Ser/Thr consensus sequence, with X being any amino
acid except Pro.[95] N-linked glycans share a common pentame-
ric core motif (Man3GlcNAc2, where Man is mannose) and are
derived from a common 14-mer oligosaccharide precursor
(Glc3Man9GlcNAc2, where Glc is glucose), which is further trim-
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med to provide three main architectures known as the high-
mannose, complex, and hybrid type oligosaccharide structu-
res.[95a, 96]
In O-glycosylation, a glycan is linked by an O-glycosidic
bond to side-chain hydroxy groups, most commonly Ser or
Thr, but apparently all types of side-chain hydroxy groups can
be found glycosylated. The glycans in the widely occurring
mucin-type O-glycosylation are linked by an a-glycosidic bond
from N-acetylgalactosamine (GalNAc) to the hydroxy group of
either Ser or Thr. Mucin-type glycans can have a wide variety
of structures.[97]
In nature, glycoproteins are obtained by enzymatic co- or
post-translational modification of a polypeptide precursor.
Many glycosylated biopharmaceuticals are produced by re-
combinant techniques using expression hosts that to some
extend can mimic the human glycosylation pattern.[94b] Howev-
er, glycosylated biopharmaceuticals are often obtained as a het-
erogeneous mixture of several glycoforms. More recently,
chemically well-defined glycosylated biopharmaceuticals have
also been obtained by synthetic or semisynthetic methods.
Kent and co-workers have prepared a monodisperse, polymer-
modified, synthetic erythropoiesis protein (SEP) as a homogene-
ous alternative to the multiple glycoforms obtained by re-
combinant EPO production. To mimic the physicochemical
properties of wild-type EPO glycosylation, SEP contains two
PEG-like, branched, negatively charged polymers installed at
engineered Lys(Ne-levulinyl) residues using oxime ligation. SEP
was constructed from four peptide fragments synthesized by
solid-phase peptide synthesis (SPPS). The full SEP sequence
was assembled using native chemical ligation (NCL) and exhib-
ited full hematopoietic activity in mice. Compared with re-
combinant EPO, the half-life of SEP was increased approxi-
mately 2-fold (from 5.1 to 9.5 h) following i.v. administration to
rats. This benefit was attributed to the use of PEG-like poly-
mers instead of native glycosylation.[98]
Danishefsky and co-workers synthesized human EPO as
a single, native-like glycoform. The glycoform of choice had
three N-linked chitobiose disaccharides at Asn24, Asn38, and
Asn83, as well as an O-linked glycan at Ser126. The 166-residue
EPO sequence was prepared as four glycosylated fragments
synthesized by solid-phase methods and assembled by itera-
tive ligations. Following protein folding, the fully glycosylated
EPO exhibited clear erythropoietic activity in a cell proliferation
assay using cord blood CD34 + cells.[99]
The semisynthesis of the immunomodulatory glycoprotein
interleukin-6 (IL6) was recently reported by Unverzagt and co-
workers. The 183-residue IL6 protein was prepared from three
peptide fragments: a short glycopeptide thioester (correspond-
ing to AAs 43–48) prepared by chemical synthesis, and a thio-
ester fragment (AAs 1–42) and a Cys-peptide fragment (AAs
49–183) obtained by recombinant methods using intein splic-
ing and cleavage of a SUMO tag, respectively. Two N-glycosy-
lated versions of IL6 (containing either GalNAc or a biantennary
nonasaccharide at Asn44) were assembled by NCL-based
methods using a hydrazide as a latent thioester for fragment
43–48. Relative to recombinant IL6, both semisynthetic IL6 ver-
sions displayed full in vitro bioactivity in a proliferation assay
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using Ba/F3 cells, indicating correct disulfide bond formation
and protein folding.[100]
6. Lipidation
Lipidation of peptides and proteins is a well-known and impor-
tant posttranslational modification, with examples including S-
prenylation (either farnesylation or geranylgeranylation), N-
myristoylation, S-palmitoylation, N-palmitoylation, O-octanoyla-
tion, and cholesteroylation.[101] A special case is the glycosyl-
phosphatidylinositol (GPI) anchor, a glycolipid which besides
a phospholipid tail also contains a carbohydrate core struc-
ture.[102] In nature, covalent attachment of lipids to peptides
and proteins is mediated by enzymes and serves to increase
the hydrophobicity of the modified protein, often with the
purpose of aiding anchoring to the cell membrane. Lipidation
as post-translational modification has been described else-
where and is not discussed further.[103]
Acylation of peptides and proteins with long-chain fatty
acids has become a well-described and validated approach to
improve the half-life of several native peptides and small pro-
teins. The concept was originally explored by scientists at
Novo Nordisk and Eli Lilly in the 1990s for the generation of
novel insulin analogues with a protracted mode of action,[104]
and the approach has since been adapted by others. For an
overview of selected lipid constructs used for half-life exten-
sion of biopharmaceuticals, please see Figure 11.
Lipidation of biopharmaceuticals is typically obtained by N-
acylation with a fatty acid, either directly or via a spacer, to
produce a stable amide bond. While S-prenylation and S-palmi-
toylation of peptides and proteins are abundant in nature and
may also be obtained by means of chemical synthesis,[101b]
these lipidation forms are rarely used in the context of bio-
pharmaceuticals. S-prenylation is not known to facilitate half-
life extension, and the thioester linkage obtained by S-palmi-
toylation is less stable under physiological conditions.
Importantly, the conditions for lipidation by N-acylation
depend on whether it is a peptide or protein that is being
modified. If the target is a protein, lipidation can almost exclu-
sively be performed in aqueous solution because of the insta-
bility of most proteins toward organic solvents. However, the
patent literature describes methods for lipidation of coagula-
tion factor FVIIa (406 AA, 50 kDa) using hydroxypropyl-b-cyclo-
dextrin (HPCD) as a lipid solubilizer under aqueous condi-
tions,[105] as well as lipidation of human growth hormone (hGH,
190 AA, 22 kDa) by enzymatic approaches.[106]
If the target is a peptide or a small protein like insulin
(51 AA, 5.8 kDa), lipidation may take place in solution using
polar aprotic solvents, as peptides are often stable to organic
conditions. A successful approach has been to employ NHS-ac-
tivated fatty acid constructs for acylation of available amino
groups.[107] However, this approach often requires a two-step
HPLC purification process, which decreases the overall yield of
reaction.[107b]
For peptides synthesized by SPPS, lipidation can also be per-
formed in organic solvents while the peptide is attached to
the solid support. Typically, the point of lipidation will be
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Figure 11. Examples of lipid moieties used for half-life extension of biopharmaceuticals. A) Myristic acid, which is used for acylation of the LysB29 position of
insulin detemir. B) Palmitic acid linked to a g-glutamic acid (gGlu) spacer, which is inserted at the e-amine of Lys26 position of liraglutide. C) Hexadecanoic
diacid with a gGlu spacer, used for modification of the LysB29 position of insulin degludec. D) Octadecanoic diacid coupled to a spacer consisting of gGlu and
two 8-amino-3,6-dioxaoctanoic acid (OEG) units, used for acylating position Lys26 of semaglutide.
either the N-terminus or a Lys residue carrying an orthogonal
protecting group. In Fmoc-SPPS, this could be for example, al-
lyloxycarbonyl (Alloc),[107b] 2-acetyl-5,5-dimethyl-1,3-cyclohexa-
nedionyl (Dde),[108] or (4-methoxyphenyl)diphenylmethyl
(Mmt).[109] During assembly of the peptide chain, the orthogo-
nal protecting group may be selectively removed to reveal an
attachment point for anchoring of the lipid moiety. By per-
forming the lipidation on-resin, purification in one step is usu-
ally sufficient to achieve a pure product.[107b]
The ability of fatty acid conjugation to improve the half-life
of biopharmaceuticals relies on noncovalent binding to albu-
min. Human serum albumin (HSA) is the most abundant pro-
tein of the bloodstream and contains nine different fatty acid
binding sites, which may bind free fatty acids as well as fatty
acids conjugated to larger molecules.[110] When bound to HSA,
lipidated peptides are sterically shielded from proteolytic deg-
radation, and because of the large size of HSA (MW of
~ 66 kDa), this protein, along with any molecule associated
with it, is protected from rapid glomerular filtration.
Following binding to serum albumin, lipidated biopharma-
ceuticals are released gradually into the circulation, thereby
obtaining a protracted half-life and an extended mode of
action. Early studies on fatty acid-modified insulin analogues,
which include insulin detemir, revealed that half-life was corre-
lated with the affinity for HSA, such that strong albumin affini-
ty resulted in prolonged action of these analogues.[111]
Conjugation with fatty acids has in several cases been
shown to elicit in vivo self-assembly of drug monomers into
stable oligomeric structures.[112] Such oligomers are less suscep-
tible to proteolytic degradation and may also form a depot
from which the active monomer is gradually released. In some
cases, lipidation also increases the chemical stability of the
modified protein. For instance, a study of guanidinium hydro-
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chloride-mediated denaturation of insulin detemir (Levemir,
Novo Nordisk) showed that myristoylation increases the free
energy of unfolding by 30 %.[113]
Lipidation with dietary fatty acids such as myristic acid and
palmitic acid is generally perceived as a safe approach to half-
life extension, as these compounds are known to be complete-
ly metabolized by the human body. Whether this is also true
for the structurally similar fatty diacids remains unknown. Gen-
eration of antibodies toward lipidated biopharmaceuticals has
been reported,[114] but so far the level of antibody formation is
low and without clinical relevance. The long-term safety profile
associated with more elaborate lipid motifs containing PEG-
based spacers is currently unknown.
The first lipidated polypeptide to obtain regulatory approval
was insulin detemir in 2004. Insulin detemir is a basal insulin
for treatment of type 1 diabetes (T1D). It is based on desB30
human insulin and is derivatized with myristic acid (C14) at the
Ne-amine of LysB29 (Figure 11 A). It has a half-life of 5–7 h fol-
lowing s.c. administration to healthy human subjects.[115] The
mechanism underlying the half-life extension involves both al-
bumin binding (more than 95 % of circulating insulin detemir
is albumin-bound)[111, 112] as well as self-association. Insulin dete-
mir is co-formulated with phenol and Zn2 + to induce forma-
tion of insulin hexamers, but size exclusion chromatography
(SEC) studies have revealed that insulin detemir exists in a sta-
bilizing hexamer–dihexamer equilibrium in the s.c. depot,
which delays the absorption. Once in the bloodstream, the
hexamers dissociate into monomers, which are then able to
bind to albumin.[112a] The X-ray crystal structure indicates that
dihexamer formation is mediated by hydrophobic interactions
of the myristic acid groups.[113]
In 2009, the once-daily GLP-1 analogue liraglutide (Victoza,
Novo Nordisk) was marketed for the treatment of type 2 diabe-
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tes (T2D). In 2014, it was also approved for treatment of obesi-
ty under the brand name Saxenda. The peptide backbone of
liraglutide is similar to native GLP-1 with the exception of the
Arg residue at position 34 (Lys in native GLP-1). Liraglutide is
a full agonist of the GLP-1 receptor (GLP-1R) and equipotent to
native GLP-1, as shown by in vitro functional assays using baby
hamster kidney (BHK) cells.[107a, 116] Lipidation with palmitic acid
(C16) is obtained by acylation of the Ly s26 position via a short
g-glutamic acid (gGlu) spacer, which is inserted to assist albu-
min binding and to increase compound solubility (Fig-
ure 11 B).[107a] This results in a very high degree of albumin
binding (~ 99 %), as indicated by equilibrium dialysis studies.[117]
Analytical ultracentrifugation studies have revealed that liraglu-
tide forms a heptamer under aqueous conditions. The ability
of liraglutide to undergo self-assembly is thought to delay ab-
sorption following s.c. injection and thereby contribute to the
markedly prolonged half-life of liraglutide (11–15 h)[118] com-
pared to native GLP-1 (1–1.5 h).[119]
Insulin degludec (Tresiba, Novo Nordisk) is a long-acting
once-daily basal insulin approved by the European Medicines
Agency (EMA) in 2013 and FDA in 2015 for treatment of T1D.
It contains a gGlu-spaced hexadecanoic diacid at position
LysB29 (Figure 11 C). In addition to albumin binding, the ex-
tended half-life of insulin degludec relies on in vivo oligomeri-
zation. Insulin degludec is formulated as a dihexamer using
Zn2 + and phenol as excipients. Following injection, rapid diffu-
sion of phenol away from the injection site results in a confor-
mational change of the dihexamer (from intermediate T3R3
conformation to the exposed T6 conformation; T ‘tense’, R ‘re-
laxed’), leading to the formation of a s.c. depot containing
large chain-like multihexamers (MW > 5 MDa), as indicated by
SEC and dynamic light scattering (DLS).[112b] Interestingly, the
presence of a fatty diacid seems to be critical for multihexamer
formation. Using transmission electron microscopy (TEM), it
was shown that the architecture of insulin degludec multihex-
amers resembles ‘pearls on a string’,[120] with the individual
hexamers being anchored to each other through the interac-
tion between Zn2 + and the terminal carboxylic acid of hexade-
canoic diacid.[112b] Subsequent passive diffusion of Zn2 + releas-
es the bioactive monomer, which is then absorbed into the
bloodstream.[112b] As a result of both albumin affinity and multi-
hexamer formation, insulin degludec exhibits a half-life of 25 h
in humans, with the glucose-lowering effect being preserved
for more than 42 h after administration, as indicated by eugly-
cemic clamp studies in patients with diabetes.[120, 121]
Semaglutide (Novo Nordisk) is a once-weekly GLP-1 analogue
in phase III clinical development for treatment of T2D and obe-
sity. It is based on the same peptide backbone as liraglutide,
with the exception of the Ala residue at position 8, which has
been changed to the nonproteinogenic amino acid 2-aminoi-
sobutyric acid (Aib) in order to confer resistance toward enzy-
matic degradation by DPP-4. Semaglutide is lipidated at Lys26
with octadecanoic diacid using a spacer consisting of gGlu and
two 8-amino-3,6-dioxaoctanoic acid (OEG) units, representing
one of the most optimized lipid moieties currently used for bi-
opharmaceuticals (Figure 11 D).[109] Semaglutide is equipotent
to native GLP-1 in terms of GLP-1R activation as shown by in
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vitro functional assays using BHK cells.[109] In humans, semaglu-
tide has a half-life of ~ 160 h following s.c. injection.[122] This re-
sults from very high albumin affinity (5.6-fold greater than lira-
glutide as assessed by analytical ultracentrifugation under
near-physiological conditions) as well as resistance toward
DPP-4.[109] Whether semaglutide also forms oligomers under
physiological conditions has not yet been reported.
Somapacitan (NNC0195-0092, Novo Nordisk), a long-acting
lipidated version of human growth hormone (hGH), is in late-
stage clinical development for the treatment of GHD. Plasma
protein binding of somapacitan was investigated using SPR
biosensing and revealed that > 99 % of somapacitan was
bound to plasma proteins, with albumin as the primary binder
in human plasma.[123] The chemical structure of somapacitan
has not been publicly disclosed at the time of writing.
The lipidation motif used for liraglutide (C16-gGlu-) has been
used successfully for half-life extension of other biopharma-
ceuticals in preclinical development. Bellmann-Sickert et al.
have reported that lipidation of radiolabelled pancreatic poly-
peptide with gGlu-spaced palmitic acid at Lys13 provided a 7-
fold increase in half residence time after i.v. infusion to Wistar
rats.[108] Also, Dalbøge et al. reported that lipidation of neuro-
medin U (NMU) with C16-gGlu- at either the N-terminus or an
engineered Lys at position nine resulted in a 15-fold prolonga-
tion of in vitro plasma stability at 37 8C.[107b]
Other examples of lipidated biopharmaceuticals in various
stages of development include a N-palmitoylated GLP-1/glu-
cose-dependent insulinotropic polypeptide co-agonist,[124] N-
palmitoylated analogues of prolactin-releasing peptide, a long-
acting N-acylated (OEG-gGlu-spaced octadecanoic diacid) ana-
logue of calcitonin,[125] a cholesteroylated analogue of oxynto-
modulin obtained by S-alkylation of a free Cys with cholesterol
via a short PEG-based spacer,[126] and insulin-327, a liver-specific
insulin analogue obtained by N-acylation at LysA22 with
a OEG-gGlu-spaced icosanoic diacid.[127]
Very recently, van Witteloostuijn et al. combined lipids and
carbohydrates in a series of novel neoglycolipid conjugates.
They consist of an open-chain carbohydrate, mucic acid, and
fatty acids of varying length. The impact of neoglycolipidation
on peptide properties was evaluated from a library of neogly-
colipidated GLP-1 analogues. Biophysical studies revealed that
neoglycolipidation mediated albumin binding and directed for-
mation of soluble peptide oligomers of variable size, depend-
ing on the chemical composition of the neoglycolipid. More-
over, the neoglycolipidated GLP-1 analogues had maintained
or even improved in vitro potency relative to native GLP-1.
This translated into potent in vivo efficacy, manifested as de-
creased food intake and improved glucose tolerance following
s.c. administration to lean mice.[128]
Besides lipidation, a variety of other techniques also use
noncovalent albumin binding for half-life extension. Examples
include albumin binding domains (ABDs),[129] albumin binding
peptides,[130] albumin-binding domain antibodies (Albu-
dAbs),[131] Nanobodies,[132] and small-molecule albumin binders
such as dicoumarol.[133] An overview is provided by Sleep
et al.[134]
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7. Fusion Proteins Using FcRn-Mediated
Recycling
Compared with other plasma proteins, albumin and immuno-
globulin G (IgG) exhibit a remarkably long half-life of approxi-
mately three weeks.[135] This is due in part to their large size
(66 kDa and 150 kDa, respectively), which inhibits renal clear-
ance, but also due to a unique receptor-mediated pH-con-
trolled recycling process, which makes these proteins less sus-
ceptible to intracellular degradation. The receptor responsible
is known as the neonatal Fc receptor (FcRn). Its presence was
first suggested by Brambell[136] and later confirmed upon isola-
tion by Simister & Rees.[137] FcRn was first recognized for its
role in IgG recycling, but it was later discovered that FcRn also
salvages albumin.[138] FcRn is situated in the cell membrane of
immune cells and binds and internalizes IgG (via the Fc
domain) as well as albumin under acidic conditions. Binding to
FcRn ensures that IgG and albumin escapes lysosomal degra-
dation and are redirected to the cell surface, where the pro-
teins dissociate from FcRn at physiological pH and are released
back into circulation.[135]
The FcRn-mediated recycling mechanism has been used to
create several half-life extended conjugates of various thera-
peutic proteins with albumin or the Fc portion of IgG.[139] Ex-
amples include etanercept (Enbrel, TNFR75-Fc, Wyeth/
Amgen),[140] dulaglutide (Trulicity, GLP-1-Fc, Eli Lilly),[141] and al-
biglutide (Tanzeum/Eperzan, GLP-1-albumin, GlaxoSmithK-
line).[142] For the development of albiglutide, an engineered al-
bumin variant with improved FcRn binding (Veltis, Albumedix/
Novozymes) was used.[143]
Although all approved albumin and Fc conjugates are ob-
tained as recombinant fusion proteins, there are examples of
albumin conjugates that have been obtained by chemical con-
jugation. Using maleimide–thiol chemistry, a NMU-HSA conju-
gate was prepared by coupling of a iodoacetamide-functional-
ized NMU variant to the free Cys34 residue on the surface of
albumin. The conjugate exhibited prolonged in vivo efficacy in
diet-induced obese (DIO) mice and had a predicted half-life in
humans of approximately 3–4 days.[11c]
In humans, the half-life extension obtained by conjugation/
fusion to albumin and Fc typically amounts to several days, ex-
emplified by for example, etanercept (2–4 days),[144] dulaglutide
(3–4 days),[145] and albiglutide (6–8 days).[146]
Despite albumin’s human origin, antibodies may develop
toward albumin and Fc fusion proteins. In a phase II study of
albiglutide in T2D patients, 5 out of 356 patients (1.4 %) devel-
oped anti-drug antibodies, although these were mainly transi-
ent, low titer, and non-neutralizing.[147] In a population of pa-
tients with rheumatoid arthritis, 16 % (corresponding to 80 pa-
tients) developed antibodies toward etanercept at some point
during a five year follow-up period.[148] However, in a phase I
study of dulaglutide containing 20 healthy subjects, no anti-
drug antibodies were observed.[145]
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8. Other Chemical Modifications—Insulin as
Example
Numerous other chemical entities have also been covalently
anchored to peptides and proteins. Most have seen limited if
any use in biopharmaceutical development or have only been
reported recently. Instead of providing a full overview, we have
selected a few examples of chemically modified insulin ana-
logues.
As mentioned, chemical lipidation of insulin at LysB29 has
been a very successful strategy for developing long-acting in-
sulin variants such as insulin detemir and insulin degludec.
Upon complexation with two Zn2 + ions, insulin readily forms
well-defined hexamers, which can occur in two states. The oli-
gomeric state of new insulin variants, that is, whether they are
preferably monomers/dimers, hexamers or multimers of hex-
amers, determines whether they are fast-acting or slow-acting
in the treatment of diabetes. Insulin has only one Lys (B29),
and hence only one free amine besides the two N-terminals.
Ligands may be attached by pH-controlled regioselective acyla-
tion, as the pKa of the Ne-amine of LysB29 is ~ 11.2 whereas it is
8.4 and 7.1 for the Na-amine of the A and B-chain, respective-
ly.[149]
8.1 Bile acid derivatives
In a collaboration between Jonassen et al. at Novo Nordisk and
Dodson and co-workers, several different bile acids (cholic acid
and derivatives) were anchored to the LysB29 position of
desB30 human insulin and studied.[150] Insulin desB30 acylated
in position LysB29 with derivatives of cholic acid displayed
a wide range of affinities for HSA, but serum albumin affinity
made only a minor contribution to the long elimination times
observed for these analogues. The most promising analogue,
LysB29-(N-(lithocholyl-g-Glu)) des(B30) human insulin (NN344),
showed glucose-lowering activity lasting more than 24 h in
pigs. NN344 forms a stable dodecameric zinc complex in the
presence of phenol, but becomes a high-MW structure com-
posed of insulin zinc hexamers in the absence of phenol. With-
out phenol, the apparent molecular mass was > 5000 kDa. The
formation of a large aggregate after injection and depot-for-
mation may have contributed to the long duration of action of
NN334. A low but measurable affinity for albumin of the litho-
cholic acid ligand may also have contributed to the prolonged
action. The crystal structure revealed that the cholic acid
moiety served as a hook, connecting the insulin hexamers.[150b]
However, NN344 appears not to have been further developed
in clinical trials.
8.2 Perfluoroalkylation
Jensen and co-workers used a bioorthogonal strategy to ex-
plore the self-segregating behavior of perfluoroalkyl moieties
for intermolecular protein self-assembly.[151] Highly fluorinated
molecules tend to segregate into a fluorous phase, a phenom-
enon, which is often referred to as the fluorous effect.[152] At-
taching a perfluoroalkyl group on the surface of a protein
17  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim &

could potentially direct intermolecular fluorous self-assembly,
representing a bio-orthogonal approach to control the oligo-
meric state of proteins in solution. Six new insulin variants car-
rying perfluoroalkyl moieties were prepared by regioselective
chemical acylation of LysB29 in desB30 human insulin at high
pH.[151] The perfluoroalkyl chain length was varied systematical-
ly to control the level of fluorous interactions between the per-
fluoroalkyl insulins. The six new insulin variants maintained sat-
isfactory levels of binding to the insulin receptor. Relatively
short perfluoroalkyl chains did not influence the overall quater-
nary structure of insulin, whereas the longer perfluoroalkyl
chains provided increased degrees of self-association of insulin
hexamers. With even longer perfluoroalkyl chain length,
a breakdown of the hexameric substructure was observed in
favor of a new structure, apparently with local cylindrical ge-
ometry. This new cylindrical geometry was observed for insu-
lins with C7F15, and C8F17 chains, and it was observed that the
increased molecular attraction introduced by the longer per-
fluoroalkyl chains also gave rise to a significant increase in the
overall length of the cylindrical structures.[151] Thus, it seems
that with short perfluoroalkyl appendices the hexameric subu-
nit core is still conserved as the dominant structure, whereas
with longer perfluoroalkyl appendices it breaks down and the
fluorous interactions start to override and promote intermolec-
ular self-assembly leading to larger structures.
8.3 Bipyridyl
Jensen and co-workers attached the metal ion binding ligand
2,2’-bipyridyl (bipy) to LysB29 on insulins. Bipy has a high affin-
ity for FeII and forms a chiral tris-bipy FeII complex, which is
colored. FeII forms a six-coordinated octahedral complex with
three bipyridine units and gives rise to a charge-transfer band
at 550 nm, which enabled monitoring of the coordination reac-
tion. Besides inducing self-assembly, the combination of bipyri-
dine and FeII also acts as a molecular probe, as the color
change from colorless to magenta is specific for formation of
the trimeric compound. Bipy was first attached to an insulin
variant, insulin X2, which has a low propensity to form the
hexamer and is mainly in the monomer/dimer form.[153] The
self-assembly through FeII chelation was reversible and provid-
ed the first insulin trimer reported in the literature as well as
the first insulin variant where assembly/disassembly could be
followed by visual inspection. This study gave the first indica-
tion that the use of a metal ion binding ligand was possible.
Next, bipy was anchored to human insulin. This conjugate is
able to lower blood glucose upon i.v. injection. It forms higher-
ordered structures, combining native Zn2 + complexation with
abiotic Fe2 + binding. Insulin 18-mers were observed by small
angle X-ray scattering in solution, while 54-mers were ob-
served on surfaces by atomic force microscopy (AFM).[154] This
is a significant step toward construction of novel peptide and
protein drugs, which change their oligomeric state in response
to metal ion concentration. While the half-life extension of the
two latter strategies was not tested, it can be speculated that
they have the potential for this.
& ChemMedChem 2016, 11, 1 – 23 www.chemmedchem.org
Reviews
9. Summary and Outlook
Today, 40 years after the first work on protein PEGylation was
published, the attachment of PEG remains the most successful
method for chemically modifying peptides and proteins to
protract their half-life. As of today, 12 PEGylated peptide- and
protein-based biopharmaceuticals are approved in the USA
and Europe, which clearly outnumbers any other chemical
technique for half-life prolongation. However, the bioincom-
patibility of the large PEG polymers remains an issue. No cellu-
lar machinery is able to fully degrade PEG into metabolically
useful constituents and, although PEG polymers can be excret-
ed by the kidneys and in feces, there are indications of PEG im-
munogenicity and bioaccumulation. This is especially concern-
ing given that many PEGylated biopharmaceuticals are ap-
proved for chronic treatment and thus are administered over
long periods of time. The current concerns associated with
chronic use should not be neglected, and the search for bio-
compatible PEG mimetics is an important effort toward mini-
mizing the risks of long-term toxicity of biopharmaceuticals.
Of the current carbohydrate-based alternatives to PEGyla-
tion, dextran conjugation and HESylation resembles PEGylation
the most. Both techniques decreases renal excretion by in-
creasing the hydrodynamic radius of the target molecule.
Moreover, dextran and HES carry no charge, making them
structurally similar to PEG. In contrast, the PSA and HA poly-
mers carry multiple negative charges, which confer increased
water solubility to the biopharmaceuticals and further decrease
their renal clearance by electric repulsion in addition to steric
factors. This makes PSA and HA attractive for modification of
for example, hydrophobic peptides with low intrinsic solubility.
A similar advantage could be expected from CMD and other
charged dextran derivatives.
The glucose or glucose-based constituents of dextran, HES,
and HA exhibit greater chemical stability than the Neu5Ac
units of CA chains, and the options for conjugation chemistry
is also more diverse for these three polymers than for PSA. The
apparent lack of reported methods for obtaining monovalent
protein-HA conjugates (i.e., with a 1:1 molar ratio of protein
and polymer) is a potential drawback for the use of this tech-
nology in a biopharmaceutical context. However, the degree of
HA substitution can be regulated by controlled reaction condi-
tions to yield multivalent protein-HA conjugates with a well-
defined degree of substitution.[86a, 89, 91] Also, as in the case of
dextran and HES, it should be possible to selectively modify
the reducing end of HA, thereby providing an opportunity for
‘true’ (i.e., 1:1) site-specific HA conjugation.
One advantage of PEG over the carbohydrate-based poly-
mers is that molecular properties of synthetic polymers are
more easily controlled than those of polymers obtained from
natural sources. Despite optimized production methods, poly-
mer heterogeneity is a challenge for dextran, HES, PSA, and
HA. From a drug development perspective, this is a potential
problem because the molecular structure of drug products
must be very well-defined. Efforts to further optimize produc-
tion of carbohydrate polymers may still be needed to ensure
18  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

maximal homogeneity, thereby strengthening the case for
these biocompatible polymers.
While carbohydrate-based PEG mimetics are often claimed
as nontoxic alternatives to PEG, the safety of these molecules
may be questioned. Generation of antibodies is only rarely ob-
served in the clinic, but bioaccumulation of dextran and HES is
known to occur, and this has been associated with unwanted
side effects (see above). Bioaccumulation of PSA and HA is not
known to occur, emphasizing the safety of these molecules.
Despite substantial effort and strong preclinical evidence, no
biopharmaceutical containing a carbohydrate-based PEG mim-
etic has yet made it to the market. In contrast, several lipidated
peptide-based biopharmaceuticals have obtained regulatory
approval. Early examples used dietary fatty acids, which are in-
expensive and highly biocompatible. The use of fatty diacids
has provided drug products with optimized properties, but
this often requires extra synthetic effort, and the long-term
safety of this kind of lipids is yet unknown. Lipidation increases
the overall hydrophobicity of the target biopharmaceutical,
and this may result in solubility problems. This issue may be
addressed by inserting a hydrophilic spacer between the lipid
and the target biopharmaceutical, as in the case of the gGlu
moiety used in liraglutide, insulin degludec, and semaglutide.
As indicated by a growing number of publications, the use
of lipidation for protraction of peptides is increasing within
drug discovery, and the methodology is also expanding into
the protein field. Lipidation is a safe and efficient way of pro-
longing the half-life of biopharmaceuticals, and it may super-
sede PEGylation as the preferred method for half-life extension.
Lipid constructs are becoming more diverse in terms of both
composition and function, illustrated by the development
from insulin detemir to semaglutide. Moreover, lipidation may
also hold potential for targeted delivery, exemplified by the
central delivery of liraglutide following peripheral administra-
tion[155] and the liver-specificity of insulin-327.[127]
Designing and developing lipidated peptides is not
a straightforward task. In the development of semaglutide,
many related analogues exhibited an extended PK profile, but
only a few withheld the high potency of native GLP-1.[109] Also,
solubility problems may arise when introducing hydrophobic
lipid constructs, as illustrated by the lipidated NMU ana-
logues.[107b] Consequently, there is no ‘one size fits all’ solution
for lipidation of biopharmaceuticals. For each target peptide or
protein, a significant effort is required to confirm the optimal
position for lipidation and to develop the optimal lipid con-
struct in terms of composition and length of both the spacer
and the lipid moiety.
Anchoring chemical groups that enable controlled self-as-
sembly of peptides and proteins by bioorthogonal principles
hold great promises for precise control of the oligomeric state
of biopharmaceutical drug candidates. However, the biocom-
patibility of these moieties needs to be considered.
Keywords: biopharmaceuticals · drug delivery · half-life
extension · peptides · proteins
ChemMedChem 2016, 11, 1 – 23 www.chemmedchem.org
These are not the final page numbers! ÞÞ
Reviews
[1] G. Walsh, Nat. Biotechnol. 2014, 32, 992 – 1000.
[2] G. Pasut, Polymers 2014, 6, 160 – 178.
[3] R. Kontermann in Therapeutic Peptides: Strategies to Modulate Their
Plasma Half-Lives (Ed.: R. Kontermann), Wiley-Blackwell, Weinheim,
2012, pp. 3 – 21.
[4] A. Chanan-Khan, J. Szebeni, S. Savay, L. Liebes, N. M. Rafique, C. R.
Alving, F. M. Muggia, Ann. Oncol. 2003, 14, 1430 – 1437.
[5] a) R. P. Garay, R. El-Gewely, J. K. Armstrong, G. Garratty, P. Richette,
Expert Opin. Drug Delivery 2012, 9, 1319 – 1323; b) N. J. Ganson, S. J.
Kelly, E. Scarlett, J. S. Sundy, M. S. Hershfield, Arthritis Res. Ther. 2006, 8,
R12; c) M. S. Hershfield, N. J. Ganson, S. J. Kelly, E. L. Scarlett, D. A. Jag-
gers, J. S. Sundy, Arthritis Res. Ther. 2014, 16, R63.
[6] T. Yamaoka, Y. Tabata, Y. Ikada, J. Pharm. Sci. 1994, 83, 601 – 606.
[7] a) J. M. Harris, R. B. Chess, Nat. Rev. Drug Discovery 2003, 2, 214 – 221;
b) F. M. Veronese, Biomaterials 2001, 22, 405 – 417; c) G. Pasut, F. M. Ver-
onese, J. Controlled Release 2012, 161, 461 – 472; d) S. Jevševar, M. Kun-
stelj, V. G. Porekar, Biotechnol. J. 2010, 5, 113 – 128.
[8] a) A. Abuchowski, T. van Es, N. C. Palczuk, F. F. Davis, J. Biol. Chem.
1977, 252, 3578 – 3581; b) A. Abuchowski, J. R. McCoy, N. C. Palczuk, T.
Van Es, F. F. Davis, J. Biol. Chem. 1977, 252, 3582 – 3586.
[9] a) A. Grigoletto, K. Maso, A. Mero, A. Rosato, O. Schiavon, G. Pasut, J.
Drug Delivery Sci. Technol. 2016, 32, 132 – 141; b) FDA approves modi-
fied antihemophilic factor for hemophilia A, FDA News Release:
www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/
ucm472643.htm (accessed October 12, 2016).
[10] M. J. Roberts, M. D. Bentley, J. M. Harris, Adv. Drug Delivery Rev. 2002,
54, 459 – 476.
[11] a) A. D. Baldwin, K. L. Kiick, Bioconjugate Chem. 2011, 22, 1946 – 1953;
b) B. Q. Shen, K. Y. Xu, L. N. Liu, H. Raab, S. Bhakta, M. Kenrick, K. L. Par-
sons-Reponte, J. Tien, S. F. Yu, E. Mai, D. W. Li, J. Tibbitts, J. Baudys,
O. M. Saadi, S. J. Scales, P. J. McDonald, P. E. Hass, C. Eigenbrot, T.
Nguyen, W. A. Solis, R. N. Fuji, K. M. Flagella, D. Patel, S. D. Spencer,
L. A. Khawlil, A. Ebens, W. L. Wong, R. Vandlen, S. Kaur, M. X. Sliwkow-
ski, R. H. Scheller, P. Polakis, J. R. Junutula, Nat. Biotechnol. 2012, 30,
184 – 189; c) P. Neuner, A. M. Peier, F. Talamo, P. Ingallinella, A. Lahm, G.
Barbato, A. Di Marco, K. Desai, K. Zytko, Y. Qian, X. Du, D. Ricci, E. Mon-
teagudo, R. Laufer, A. Pocai, E. Bianchi, D. J. Marsh, A. Pessi, J. Pept. Sci.
2014, 20, 7 – 19.
[12] a) S. C. Alley, D. R. Benjamin, S. C. Jeffrey, N. M. Okeley, D. L. Meyer, R. J.
Sanderson, P. D. Senter, Bioconjugate Chem. 2008, 19, 759 – 765; b) D.
Lin, S. Saleh, D. C. Liebler, Chem. Res. Toxicol. 2008, 21, 2361 – 2369.
[13] a) K. Knop, R. Hoogenboom, D. Fischer, U. S. Schubert, Angew. Chem.
Int. Ed. 2010, 49, 6288 – 6308; Angew. Chem. 2010, 122, 6430 – 6452;
b) H. Schellekens, W. E. Hennink, V. Brinks, Pharm. Res. 2013, 30, 1729 –
1734.
[14] U. Binder, A. Skerra in Therapeutic Proteins: Strategies to Modulate Their
Plasma Half-Lives (Ed.: R. Kontermann), Wiley-Blackwell, Weinheim,
2012, pp. 63 – 80.
[15] V. Schellenberger, C. W. Wang, N. C. Geething, B. J. Spink, A. Campbell,
W. To, M. D. Scholle, Y. Yin, Y. Yao, O. Bogin, J. L. Cleland, J. Silverman,
W. P. Stemmer, Nat. Biotechnol. 2009, 27, 1186 – 1190.
[16] a) J. L. Cleland, N. C. Geething, J. A. Moore, B. C. Rogers, B. J. Spink,
C. W. Wang, S. E. Alters, W. P. C. Stemmer, V. Schellenberger, J. Pharm.
Sci. 2012, 101, 2744 – 2754; b) K. C. J. Yuen, G. S. Conway, V. Popovic,
G. R. Merriam, T. Bailey, A. H. Hamrahian, B. M. K. Biller, M. Kipnes, J. A.
Moore, E. Humphriss, G. M. Bright, J. L. Cleland, J. Clin. Endocrinol.
Metab. 2013, 98, 2595 – 2603.
[17] V. N. Podust, B. C. Sim, D. Kothari, L. Henthorn, C. Gu, C. W. Wang, B.
McLaughlin, V. Schellenberger, Protein Eng. Des. Sel. 2013, 26, 743 –
753.
[18] S. Ding, M. Song, B. C. Sim, C. Gu, V. N. Podust, C. W. Wang, B.
McLaughlin, T. P. Shah, R. Lax, R. Gast, R. Sharan, A. Vasek, M. A. Hart-
man, C. Deniston, P. Srinivas, V. Schellenberger, Bioconjugate Chem.
2014, 25, 1351 – 1359.
[19] a) H. Betre, W. Liu, M. R. Zalutsky, A. Chilkoti, V. B. Kraus, L. A. Setton, J.
Controlled Release 2006, 115, 175 – 182; b) M. F. Shamji, H. Betre, V. B.
Kraus, J. Chen, A. Chilkoti, R. Pichika, K. Masuda, L. A. Setton, Arthritis
Rheum. 2007, 56, 3650 – 3661; c) M. Amiram, K. M. Luginbuhl, X. Li,
M. N. Feinglos, A. Chilkoti, J. Controlled Release 2013, 172, 144 – 151;
d) M. Amiram, K. M. Luginbuhl, X. H. Li, M. N. Feinglos, A. Chilkoti, Proc.
Natl. Acad. Sci. USA 2013, 110, 2792 – 2797.
19  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim &

[20] Y. S. Huang, X. F. Wen, Y. L. Wu, Y. F. Wang, M. Fan, Z. Y. Yang, W. Liu,
L. F. Zhou, Eur. J. Pharm. Biopharm. 2010, 74, 435 – 441.
[21] a) M. Schlapschy, U. Binder, C. Borger, I. Theobald, K. Wachinger, S. Kis-
ling, D. Haller, A. Skerra, Protein Eng. Des. Sel. 2013, 26, 489 – 501; b) V.
Morath, F. Bolze, M. Schlapschy, S. Schneider, F. Sedlmayer, K. Seyfarth,
M. Klingenspor, A. Skerra, Mol. Pharm. 2015, 12, 1431 – 1442.
[22] A. E. Vasilev, G. F. Zhukova, G. A. Ravdel, R. G. L. Shchukin, Zh. Obshch.
Khim. 1973, 43, 2529 – 2532.
[23] C. Larsen, Adv. Drug Delivery Rev. 1989, 3, 103 – 154.
[24] R. Mehvar, J. Controlled Release 2000, 69, 1 – 25.
[25] a) Y. Takakura, T. Fujita, M. Hashida, H. Sezaki, Pharm. Res. 1990, 7,
339 – 346; b) T. Fujita, M. Nishikawa, C. Tamaki, Y. Takakura, M. Hashida,
H. Sezaki, J. Pharmacol. Exp. Ther. 1992, 263, 971 – 978; c) M. Nishikawa,
A. Kamijo, T. Fujita, Y. Takakura, H. Sezaki, M. Hashida, Pharm. Res.
1993, 10, 1253 – 1261; d) M. C. Degat, G. Dubreucq, A. Meunier, L.
Dahri-Correia, L. Sedel, H. Petite, D. Logeart-Avramoglou, J. Biomed.
Mater. Res. Part A 2009, 88A, 174 – 183; e) D. Logeart-Avramoglou, R.
Huynh, F. Chaubet, L. Sedel, A. Meunier, Biochem. Pharmacol. 2002, 63,
129 – 137.
[26] R. Axn, J. Porath, S. Ernback, Nature 1967, 214, 1302 – 1304.
[27] J. F. Kennedy, J. A. Barnes, J. B. Matthews, Br. Polym. J. 1983, 15, 133 –
138.
[28] a) G. M. Lindenbaum, O. A. Mirgorodskaya, B. V. Moskvichev, Khim.-
Farm. Zh. 1977, 11, 80 – 83; b) O. A. Mirgorodskaya, L. V. Poletaeva,
Khim.-Farm. Zh. 1985, 19, 594 – 599.
[29] a) V. P. Torchilin, E. G. Tischenko, V. N. Smirnov, E. I. Chazov, J. Biomed.
Mater. Res. 1977, 11, 223 – 235; b) V. P. Torchilin, E. V. Il’lna, A. V. Mazaev,
B. S. Lebedev, V. N. Smirnov, E. I. Chazov, J. Solid-Phase Biochem. 1977,
2, 187 – 193.
[30] Y. Takakura, Y. Kaneko, T. Fujita, M. Hashida, H. Maeda, H. Sezaki, J.
Pharm. Sci. 1989, 78, 117 – 121.
[31] Y. Yasuda, T. Fujita, Y. Takakura, M. Hashida, H. Sezaki, Chem. Pharm.
Bull. 1990, 38, 2053 – 2056.
[32] a) Y. Prez, A. Valdivia, H. L. Ramrez, R. Villalonga, Macromol. Rapid
Commun. 2005, 26, 1304 – 1308; b) Y. Perez, A. Valdivia, L. Gomez, B. K.
Simpson, R. Villalonga, Macromol. Biosci. 2005, 5, 1220 – 1225; c) R. Vil-
lalonga, A. Valdivia, Y. Perez, B. Chongo, J. Appl. Polym. Sci. 2006, 102,
4573 – 4576; d) A. Valdivia, R. Villalonga, P. Di Pierro, Y. Perez, L. Mari-
niello, L. Gomez, R. Porta, J. Biotechnol. 2006, 122, 326 – 333.
[33] a) R. L. S. Chang, I. F. Ueki, J. L. Troy, W. M. Deen, C. R. Robertson, B. M.
Brenner, Biophys. J. 1975, 15, 887 – 906; b) R. Mehvar, T. L. Shepard, J.
Pharm. Sci. 1992, 81, 908 – 912.
[34] R. G. Melton, C. N. Wiblin, R. L. Foster, R. F. Sherwood, Biochem. Phar-
macol. 1987, 36, 105 – 112.
[35] R. Fagnani, S. Halpern, M. Hagan, Nucl. Med. Commun. 1995, 16, 362 –
369.
[36] Select Committee on GRAS Substances (SCOGS) Opinion: Dextran, US
Food and Drug Administration: www.fda.gov/Food/IngredientsPacka-
gingLabeling/GRAS/SCOGS/ucm261271.htm (accesssed October 12,
2016).
[37] J. Ring, K. Messmer, Lancet 1977, 309, 466 – 469.
[38] M. C. Laxenaire, C. Charpentier, L. Feldman, Ann. Fr. Anesth. Reanim.
1994, 13, 301 – 310.
[39] a) H. Hedin, W. Richter, J. Ring, Int. Arch. Allergy Appl. Immunol. 1976,
52, 145 – 159; b) D. Kraft, H. Hedin, W. Richter, O. Scheiner, H. Rumpold,
M. E. Devey, Allergy 1982, 37, 481 – 489.
[40] I. Seppl, J. Pelkonen, O. Mkel, Eur. J. Immunol. 1985, 15, 827 – 833.
[41] I. Seppl, O. Mkel, J. Immunol. 1989, 143, 1259 – 1264.
[42] M. Behe, J. Du, W. Becker, T. Behr, C. Angerstein, M. Mrquez, J. Hiltu-
nen, S. Nilsson, A. R. Holmberg, Med. Oncol. 2001, 18, 59 – 64.
[43] I. Lancranjan, C. Bruns, P. Grass, P. Jaquet, J. Jervell, P. Kendall-Taylor,
S. W. J. Lamberts, P. Marbach, H. Ørskov, G. Pagani, M. Sheppard, L.
Simionescu, Metabolism 1995, 44, 18 – 26.
[44] T. K. Joensuu, S. Nilsson, A. R. Holmberg, M. Marquez, M. Tenhunen, T.
Saarto, H. Joensuu in Signal Transduction and Communication in
Cancer Cells, Vol. 1028 (Eds.: H. L. Bradlow, L. Castagnetta, L. Massimo,
K. Zaenker), New York Academy of Sciences, New York, 2004, pp. 361 –
374.
[45] a) T. E. Wileman, R. L. Foster, P. N. C. Elliott, J. Pharm. Pharmacol. 1986,
38, 264 – 271; b) T. E. Wileman, Adv. Drug Delivery Rev. 1991, 6, 167 –
180.
& ChemMedChem 2016, 11, 1 – 23 www.chemmedchem.org
Reviews
[46] R. G. Melton, C. N. Wiblin, A. Baskerville, R. L. Foster, R. F. Sherwood,
Biochem. Pharmacol. 1987, 36, 113 – 121.
[47] S. C. Tam, J. Blumenstein, J. Tzefeiwong, Proc. Natl. Acad. Sci. USA
1976, 73, 2128 – 2131.
[48] M. Baudyš, D. Letourneur, F. Liu, D. Mix, J. Jozefonvicz, S. W. Kim, Bio-
conjugate Chem. 1998, 9, 176 – 183.
[49] T. Hey, H. Knoller, P. Vorstheim in Therapeutic Proteins: Strategies to
Modulate Their Plasma Half-Lives (Ed.: R. Kontermann), Wiley-Blackwell,
Weinheim, 2012, pp. 117 – 140.
[50] C. Jungheinrich, T. A. Neff, Clin. Pharmacokinet. 2005, 44, 681 – 699.
[51] K. Lederer, C. Huber, M. Dunky, J. K. Fink, H. P. Ferber, E. Nitsch, Arz-
neim.-Forsch. 1985, 35, 610 – 614.
[52] A. W. Richter, A. B. de Belder, Int. Arch. Allergy Appl. Immunol. 1976, 52,
307 – 314.
[53] M. A. Abbas, S. Hameed, J. Kressler, Starch/Staerke 2013, 65, 264 – 272.
[54] M. Noga, D. Edinger, R. Klager, S. V. Wegner, J. P. Spatz, E. Wagner, G.
Winter, A. Besheer, Biomaterials 2013, 34, 2530 – 2538.
[55] P. E. Hallaway, J. W. Eaton, S. S. Panter, B. E. Hedlund, Proc. Natl. Acad.
Sci. USA 1989, 86, 10108 – 10112.
[56] R. Liebner, R. Mathaes, M. Meyer, T. Hey, G. Winter, A. Besheer, Eur. J.
Pharm. Biopharm. 2014, 87, 378 – 385.
[57] S. Schramm, C. Thyes, P. Frascarolo, T. Buclin, M. Burki, A. Fisch, M.-A.
Burmeister, L. Asmis, D. R. Spahn, Anesthesiology 2007, 107, 442 – 451.
[58] P. Kiehl, D. Metze, H. Kresse, S. Reimann, D. Kraft, A. Kapp, Br. J. Derma-
tol. 1998, 138, 672 – 677.
[59] H.-J. Dieterich, D. Kraft, C. Sirtl, H. Laubenthal, W. Schimetta, W. Pçlz, E.
Gerlach, K. Peter, Anesth. Analg. 1998, 86, 1123 – 1126.
[60] C. J. Wiedermann, M. Joannidis, Intensive Care Med. 2014, 40, 160 – 170.
[61] J. Treib, A. Haass, G. Pindur, W. Treib, E. Wenzel, K. Schimrigk, Eur. J.
Haematol. 1996, 56, 168 – 172.
[62] J. Treib, A. Haass, G. Pindur, M. T. Grauer, E. Wenzel, K. Schimrigk, Trans-
fusion 1996, 36, 450 – 455.
[63] A. Greindl, C. Kessler, B. Breuer, U. Haberl, A. Rybka, M. Emgenbroich,
A. J. G. Pçtgens, H.-G. Frank, Open Hematol. J. 2010, 4, 1 – 14.
[64] J. Sommer, D. Sim, L. Leong, F. Hacket, B. Subramanyan, J. Haaning,
Haemophilia 2010, 16, 39.
[65] G. Gregoriadis, B. McCormack, Z. Wang, R. Lifely, FEBS Lett. 1993, 315,
271 – 276.
[66] M. Mhlenhoff, M. Eckhardt, R. Gerardy-Schahn, Curr. Opin. Struct. Biol.
1998, 8, 558 – 564.
[67] A. Constantinou, C. Chen, M. P. Deonarain in Therapeutic Peptides:
Strategies to Modulate Their Plasma Half-Lives (Ed.: R. Kontermann),
Wiley-Blackwell, Weinheim, 2012, pp. 95 – 115.
[68] H. Baumann, J.-R. Brisson, F. Michon, R. Pon, H. J. Jennings, Biochem-
istry 1993, 32, 4007 – 4013.
[69] PolyXen Technology-Based Candidates, Xenetic Biosciences: www.xene-
ticbio.com/product-pipeline/polyxen (accessed October 12, 2016).
[70] H. J. Jennings, C. Lugowski, J. Immunol. 1981, 127, 1012 – 1018.
[71] a) A. Fernandes, G. Gregoriadis, Biochim. Biophys. Acta Protein Struct.
Mol. Enzymol. 1996, 1293, 90 – 96; b) A. Fernandes, G. Gregoriadis, Bio-
chim. Biophys. Acta Protein Struct. Mol. Enzymol. 1997, 1341, 26 – 34;
c) S. Jain, D. H. Hreczuk-Hirst, B. McCormack, M. Mital, A. Epenetos, P.
Laing, G. Gregoriadis, Biochim. Biophys. Acta Gen. Subj. 2003, 1622, 42 –
49; d) A. Constantinou, A. A. Epenetos, D. H. Hreczuk-Hirst, S. Jain, M. P.
Deonarain, Bioconjugate Chem. 2008, 19, 643 – 650; e) A. Constantinou,
A. A. Epenetos, D. H. Hreczuk-Hirst, S. Jain, M. Wright, K. A. Chester,
M. P. Deonarain, Bioconjugate Chem. 2009, 20, 924 – 931; f) I. Vorobiev,
V. Matskevich, S. Kovnir, N. Orlova, V. Knorre, S. Jain, D. Genkin, A. Ga-
bibov, A. Miroshnikov, Biochimie 2013, 95, 264 – 270; g) D. G. Ilyushin,
I. V. Smirnov, A. A. Belogurov, Jr., I. A. Dyachenko, T. Zharmukhamedo-
va, T. I. Novozhilova, E. A. Bychikhin, M. V. Serebryakova, O. N. Kharybin,
A. N. Murashev, K. A. Anikienko, E. N. Nikolaev, N. A. Ponomarenko,
D. D. Genkin, G. M. Blackburn, P. Masson, A. G. Gabibov, Proc. Natl.
Acad. Sci. USA 2013, 110, 1243 – 1248.
[72] T. Lindhout, U. Iqbal, L. M. Willis, A. N. Reid, J. Li, X. Liu, M. Moreno,
W. W. Wakarchuk, Proc. Natl. Acad. Sci. USA 2011, 108, 7397 – 7402.
[73] a) G. Gregoriadis, A. Fernandes, M. Mital, B. McCormack, Cell. Mol. Life
Sci. 2000, 57, 1964 – 1969; b) B. M. Brenner, C. Baylis, W. M. Deen, Physi-
ol. Rev. 1976, 56, 502 – 534.
[74] C. Moreno, M. R. Lifely, J. Esdaile, Infect. Immun. 1985, 47, 527 – 533.
[75] A. Fernandes, G. Gregoriadis, Int. J. Pharm. 2001, 217, 215 – 224.
20  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

[76] E. Kelly, C. M. Greene, T. P. Carroll, N. G. McElvaney, S. J. O’Neill, Respir.
Med. 2010, 104, 763 – 772.
[77] T. C. Laurent, J. R. Fraser, Faseb J. 1992, 6, 2397 – 2404.
[78] A. Mero, M. Campisi, Polymers 2014, 6, 346 – 369.
[79] C. E. Schant, G. Zuber, C. Herlin, T. F. Vandamme, Carbohydr. Polym.
2011, 85, 469 – 489.
[80] B. P. Toole, Nat. Rev. Cancer 2004, 4, 528 – 539.
[81] V. Rangaswamy, D. Jain, Biotechnol. Lett. 2008, 30, 493 – 496.
[82] R. Stern, G. Kogan, M. J. Jedrzejas, L. Soltes, Biotechnol. Adv. 2007, 25,
537 – 557.
[83] P. L. DeAngelis, Curr. Pharm. Biotechnol. 2008, 9, 246 – 248.
[84] A. J. Day, J. K. Sheehan, Curr. Opin. Struct. Biol. 2001, 11, 617 – 622.
[85] E. J. Oh, K. Park, K. S. Kim, J. Kim, J. A. Yang, J. H. Kong, M. Y. Lee, A. S.
Hoffman, S. K. Hahn, J. Controlled Release 2010, 141, 2 – 12.
[86] a) A. Mero, M. Pasqualin, M. Campisi, D. Renier, G. Pasut, Carbohydr.
Polym. 2013, 92, 2163 – 2170; b) S. J. Kim, S. K. Hahn, M. J. Kim, D. H.
Kim, Y. P. Lee, J. Controlled Release 2005, 104, 323 – 335.
[87] A. Mero, M. Campisi, M. Favero, C. Barbera, C. Secchieri, J. M. Dayer,
M. B. Goldring, S. R. Goldring, G. Pasut, J. Controlled Release 2014, 187,
30 – 38.
[88] I. M. Montagner, A. Merlo, D. Carpanese, A. Dalla Piet, A. Mero, A. Gri-
goletto, A. Loregian, D. Renier, M. Campisi, P. Zanovello, G. Pasut, A.
Rosato, J. Controlled Release 2016, 236, 79 – 89.
[89] E. J. Oh, J. W. Kim, J. H. Kong, S. H. Ryu, S. K. Hahn, Bioconjugate Chem.
2008, 19, 2401 – 2408.
[90] J. H. Kong, E. J. Oh, S. Y. Chae, K. C. Lee, S. K. Hahn, Biomaterials 2010,
31, 4121 – 4128.
[91] J. A. Yang, K. Park, H. Jung, H. Kim, S. W. Hong, S. K. Yoon, S. K. Hahn,
Biomaterials 2011, 32, 8722 – 8729.
[92] F. Fares in Therapeutic Proteins: Strategies to Modulate Their Plasma
Half-Lives (Ed.: R. Kontermann), Wiley-Blackwell, Weinheim, 2012,
pp. 81 – 94.
[93] R. Apweiler, H. Hermjakob, N. Sharon, Biochim. Biophys. Acta Gen. Subj.
1999, 1473, 4 – 8.
[94] a) H. Lis, N. Sharon, Eur. J. Biochem. 1993, 218, 1 – 27; b) H. J. Li, M.
d’Anjou, Curr. Opin. Biotechnol. 2009, 20, 678 – 684.
[95] a) B. Imperiali, S. E. O’Connor, Curr. Opin. Chem. Biol. 1999, 3, 643 – 649;
b) R. Kornfeld, S. Kornfeld, Annu. Rev. Biochem. 1985, 54, 631 – 664.
[96] G. Opdenakker, P. M. Rudd, C. P. Ponting, R. A. Dwek, Faseb J. 1993, 7,
1330 – 1337.
[97] P. Van den Steen, P. M. Rudd, R. A. Dwek, G. Opdenakker, Crit. Rev. Bio-
chem. Mol. Biol. 1998, 33, 151 – 208.
[98] a) G. G. Kochendoerfer, S. Y. Chen, F. Mao, S. Cressman, S. Traviglia, H. Y.
Shao, C. L. Hunter, D. W. Low, E. N. Cagle, M. Carnevali, V. Gueriguian,
P. J. Keogh, H. Porter, S. M. Stratton, M. C. Wiedeke, J. Wilken, J. Tang,
J. J. Levy, L. P. Miranda, M. M. Crnogorac, S. Kalbag, P. Botti, J. Schin-
dler-Horvat, L. Savatski, J. W. Adamson, A. Kung, S. B. H. Kent, J. A.
Bradburne, Science 2003, 299, 884 – 887; b) S. B. H. Kent, Angew. Chem.
Int. Ed. 2013, 52, 11988 – 11996; Angew. Chem. 2013, 125, 12208 –
12217.
[99] P. Wang, S. W. Dong, J. A. Brailsford, K. Iyer, S. D. Townsend, Q. Zhang,
R. C. Hendrickson, J. Shieh, M. A. S. Moore, S. J. Danishefsky, Angew.
Chem. Int. Ed. 2012, 51, 11576 – 11584; Angew. Chem. 2012, 124,
11744 – 11752.
[100] A. Reif, S. Siebenhaar, A. Troster, M. Schmalzlein, C. Lechner, P. Velisetty,
K. Gottwald, C. Pohner, I. Boos, V. Schubert, S. Rose-John, C. Unverzagt,
Angew. Chem. Int. Ed. 2014, 53, 12125 – 12131; Angew. Chem. 2014,
126, 12321 – 12327.
[101] a) R. K. Mann, P. A. Beachy, Annu. Rev. Biochem. 2004, 73, 891 – 923;
b) D. Kadereit, J. Kuhlmann, H. Waldmann, ChemBioChem 2000, 1,
144 – 169.
[102] M. G. Paulick, C. R. Bertozzi, Biochemistry 2008, 47, 6991 – 7000.
[103] a) F. L. Zhang, P. J. Casey, Annu. Rev. Biochem. 1996, 65, 241 – 269; b) P.
Orlean, A. K. Menon, J. Lipid Res. 2007, 48, 993 – 1011; c) M. D. Resh,
Biochim. Biophys. Acta Mol. Cell Res. 1999, 1451, 1 – 16.
[104] a) P. Kurtzhals, S. Havelund, I. Jonassen, B. Kiehr, U. D. Larsen, U. Ribel,
J. Markussen, Biochem. J. 1995, 312, 725 – 731; b) S. R. Myers, F. E.
Yakubu-Madus, W. T. Johnson, J. E. Baker, T. S. Cusick, V. K. Williams,
F. C. Tinsley, A. Kriauciunas, J. Manetta, V. J. Chen, Diabetes 1997, 46,
637 – 642.
ChemMedChem 2016, 11, 1 – 23 www.chemmedchem.org
These are not the final page numbers! ÞÞ
Reviews
[105] C. Behrens, P. W. Garibay, P. W. Garibay, H. S. Andersen, N. L. Johansen,
B. Peschke, S. Bak (Novo Nordisk A/S), Int. PCT Pub. No.
WO2010015668 A1, 2010.
[106] N. L. Johansen, H. S. Andersen, J. Buchardt, C. Behrens, L. Norskov-
Lauritsen, J. Su (Novo Nordisk Health Care AG), Int. PCT Pub. No.
WO2011015649 A1, 2011.
[107] a) L. B. Knudsen, P. F. Nielsen, P. O. Huusfeldt, N. L. Johansen, K.
Madsen, F. Z. Pedersen, H. Thøgersen, M. Wilken, H. Agersøe, J. Med.
Chem. 2000, 43, 1664 – 1669; b) L. S. Dalbøge, S. L. Pedersen, S. B. van
Witteloostuijn, J. E. Rasmussen, K. T. Rigbolt, K. J. Jensen, B. Holst, N.
Vrang, J. Jelsing, J. Pept. Sci. 2015, 21, 85 – 94; c) C. Christensen, R. Se-
verinsen, A. K. Petersen, S. Kidal, C. U. Jessen, P. Madsen, H. Valore,
T. M. Tagmose, J. L. Sorensen (Novo Nordisk A/S), Int. PCT Pub. No.
WO2010029159 A1, 2010.
[108] K. Bellmann-Sickert, C. E. Elling, A. N. Madsen, P. B. Little, K. Lundgren,
L. O. Gerlach, R. Bergmann, B. Holst, T. W. Schwartz, A. G. Beck-Sicking-
er, J. Med. Chem. 2011, 54, 2658 – 2667.
[109] J. Lau, P. Bloch, L. Schaffer, I. Pettersson, J. Spetzler, J. Kofoed, K.
Madsen, L. B. Knudsen, J. McGuire, D. B. Steensgaard, H. M. Strauss,
D. X. Gram, S. M. Knudsen, F. S. Nielsen, P. Thygesen, S. Reedtz-Runge,
T. Kruse, J. Med. Chem. 2015, 58, 7370 – 7380.
[110] G. Fanali, A. di Masi, V. Trezza, M. Marino, M. Fasano, P. Ascenzi, Mol.
Aspects Med. 2012, 33, 209 – 290.
[111] J. Markussen, S. Havelund, P. Kurtzhals, A. S. Andersen, J. Halstrom, E.
Hasselager, U. D. Larsen, U. Ribel, L. Schaffer, K. Vad, I. Jonassen, Diabe-
tologia 1996, 39, 281 – 288.
[112] a) S. Havelund, A. Plum, U. Ribel, I. Jonassen, A. Vølund, J. Markussen,
P. Kurtzhals, Pharm. Res. 2004, 21, 1498 – 1504; b) I. Jonassen, S. Have-
lund, T. Hoeg-Jensen, D. B. Steensgaard, P. O. Wahlund, U. Ribel, Pharm.
Res. 2012, 29, 2104 – 2014; c) D. B. Steensgaard, J. K. Thomsen, H. B.
Olsen, L. B. Knudsen, Diabetes 2008, 57, A164.
[113] H. B. Olsen, N. C. Kaarsholm, Biochemistry 2000, 39, 11893 – 11900.
[114] a) J. B. Buse, A. Garber, J. Rosenstock, W. E. Schmidt, J. H. Brett, N. Vide-
baek, J. Holst, M. Nauck, J. Clin. Endocrinol. Metab. 2011, 96, 1695 –
1702; b) J. Vora, P. Hollander, S. C. Tamer, T. Johansen, R. Bergenstal, Di-
abetologia 2012, 55, S53 – S54.
[115] G. A. Brunner, G. Sendlhofer, A. Wutte, M. Ellmerer, B. Søgaard, A. Sie-
benhofer, S. Hirschberger, G. J. Krejs, T. R. Pieber, Exp. Clin. Endocrinol.
Diabetes 2000, 108, 100 – 105.
[116] K. Madsen, L. B. Knudsen, H. Agersoe, P. F. Nielsen, H. Thøgersen, M.
Wilken, N. L. Johansen, J. Med. Chem. 2007, 50, 6126 – 6132.
[117] A. Plum, L. B. Jensen, J. B. Kristensen, J. Pharm. Sci. 2013, 102, 2882 –
2888.
[118] B. Elbrønd, G. Jakobsen, S. Larsen, H. Agersø, L. B. Jensen, P. Rolan, J.
Sturis, V. Hatorp, M. Zdravkovic, Diabetes Care 2002, 25, 1398 – 1404.
[119] M. K. Gutniak, B. Linde, J. J. Holst, S. Efendic, Diabetes Care 1994, 17,
1039 – 1044.
[120] P. Kurtzhals, T. Heise, H. M. Strauss, S. G. Bottcher, C. Granhall, H. Haahr,
I. Jonassen, Diabetologia 2011, 54, S426.
[121] a) T. Heise, U. Hovelmann, L. Nosek, S. G. Bottcher, C. Granhall, H.
Haahr, Diabetologia 2011, 54, S425; b) T. Heise, L. Nosek, S. G. Bottcher,
H. Hastrup, H. Haahr, Diabetes Obes. Metab. 2012, 14, 944 – 950.
[122] a) Abstracts of the 48th EASD Annual Meeting of the European Associa-
tion for the Study of Diabetes: M. A. Nauck, J. R. Petrie, G. Sesti, E. Man-
nucci, J. P. Courreges, S. Atkin, M. During, C. B. Jensen, S. Heller, Diabe-
tologia 2012, 55 (Suppl. 1), S7; b) C. Kapitza, L. Nosek, L. Jensen, H.
Hartvig, C. B. Jensen, A. Flint, J. Clin. Pharmacol. 2015, 55, 497 – 504.
[123] P. Thygesen, G. Konradsen, C. B. Schjødt, P. F. Nielsen, Endocr. Rev.
2016, SAT-034.
[124] B. Finan, T. Ma, N. Ottaway, T. D. Muller, K. M. Habegger, K. M. Heppner,
H. Kirchner, J. Holland, J. Hembree, C. Raver, S. H. Lockie, D. L. Smiley,
V. Gelfanov, B. Yang, S. Hofmann, D. Bruemmer, D. J. Drucker, P. T.
Pfluger, D. Perez-Tilve, J. Gidda, L. Vignati, L. Zhang, J. B. Hauptman, M.
Lau, M. Brecheisen, S. Uhles, W. Riboulet, E. Hainaut, E. Sebokova, K.
Conde-Knape, A. Konkar, R. D. DiMarchi, M. H. Tschçp, Sci. Transl. Med.
2013, 5, 209ra151.
[125] C. Nilsson, T. K. Hansen, C. Rosenquist, B. Hartmann, J. T. Kodra, J. F.
Lau, T. R. Clausen, K. Raun, A. Sams, Eur. J. Pharmacol. 2016, 773, 24 –
31.
[126] A. Pocai, P. E. Carrington, J. R. Adams, M. Wright, G. Eiermann, L. Zhu,
X. Du, A. Petrov, M. E. Lassman, G. Jiang, F. Liu, C. Miller, L. M. Tota, G.
21  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim &

Zhou, X. Zhang, M. M. Sountis, A. Santoprete, E. Capito, G. G. Chicchi,
N. Thornberry, E. Bianchi, A. Pessi, D. J. Marsh, R. SinhaRoy, Diabetes
2009, 58, 2258 – 2266.
[127] a) D. S. Edgerton, M. C. Moore, J. J. Winnick, M. Scott, B. Farmer, H.
Naver, C. B. Jeppesen, P. Madsen, T. B. Kjeldsen, E. Nishimura, C. L.
Brand, A. D. Cherrington, Diabetes 2014, 63, 3946 – 3954; b) A. N.
Zaykov, J. P. Mayer, R. D. DiMarchi, Nat. Rev. Drug Discovery 2016, 15,
425 – 439.
[128] S. B. van Witteloostuijn, K. Mannerstedt, P. Wismann, E. M. Bech, M. B.
Thygesen, N. Vrang, J. Jelsing, K. J. Jensen, S. L. Pedersen, accepted for
publication in Mol. Pharm.
[129] A. Jonsson, J. Dogan, N. Herne, L. Abrahmsen, P. A. Nygren, Protein
Eng. Des. Sel. 2008, 21, 515 – 527.
[130] M. S. Dennis, M. Zhang, Y. G. Meng, M. Kadkhodayan, D. Kirchhofer, D.
Combs, L. A. Damico, J. Biol. Chem. 2002, 277, 35035 – 35043.
[131] a) C. Herring, O. Schon in Therapeutic Proteins: Strategies to Modulate
Their Plasma Half-Lives (Ed.: R. Kontermann), Wiley-Blackwell, Wein-
heim, 2012, pp. 249 – 268; b) L. J. Holt, A. Basran, K. Jones, J. Chorlton,
L. S. Jespers, N. D. Brewis, I. M. Tomlinson, Protein Eng. Des. Sel. 2008,
21, 283 – 288.
[132] B. M. Tijink, T. Laeremans, M. Budde, M. S. V. Walsum, T. Dreier, H. J. de
Haard, C. R. Leemans, G. van Dongen, Mol. Cancer Ther. 2008, 7, 2288 –
2297.
[133] J. Han, L. D. Sun, Y. Y. Chu, Z. Li, D. D. Huang, X. Y. Zhu, H. Qian, W. L.
Huang, J. Med. Chem. 2013, 56, 9955 – 9968.
[134] D. Sleep, J. Cameron, L. R. Evans, Biochim. Biophys. Acta Gen. Subj.
2013, 1830, 5526 – 5534.
[135] J. Kim in Therapeutic Proteins: Strategies to Modulate Their Plasma Half-
Lives (Ed.: R. Kontermann), Wiley-Blackwell, Weinheim, 2012, pp. 143 –
155.
[136] a) F. W. Brambell, W. A. Hemmings, I. G. Morris, Nature 1964, 203, 1352;
b) F. W. Brambell, Lancet 1966, 288, 1087.
[137] N. E. Simister, A. R. Rees, Eur. J. Immunol. 1985, 15, 733 – 738.
[138] C. Chaudhury, S. Mehnaz, J. M. Robinson, W. L. Hayton, D. K. Pearl,
D. C. Roopenian, C. L. Anderson, J. Exp. Med. 2003, 197, 315 – 322.
[139] W. R. Strohl, Biodrugs 2015, 29, 215 – 239.
[140] B. Jarvis, D. Faulds, Drugs 1999, 57, 945 – 966.
[141] M. Sanford, Drugs 2014, 74, 2097 – 2103.
[142] R. M. Poole, M. L. Nowlan, Drugs 2014, 74, 929 – 938.
[143] a) J. T. Andersen, B. Dalhus, D. Viuff, B. T. Ravn, K. S. Gunnarsen, A.
Plumridge, K. Bunting, F. Antunes, R. Williamson, S. Athwal, E. Allan, L.
Evans, M. Bjoras, S. Kjaerulff, D. Sleep, I. Sandlie, J. Cameron, J. Biol.
Chem. 2014, 289, 13492 – 13502; b) Veltis Optimized Drug Dosing, Albu-
& ChemMedChem 2016, 11, 1 – 23 www.chemmedchem.org
Reviews
medix: albumedix.com/drug-delivery/veltis/ (accessed October 12,
2016).
[144] J. M. Korth-Bradley, A. S. Rubin, R. K. Hanna, D. K. Simcoe, M. E. Leb-
sack, Ann. Pharmacother. 2000, 34, 161 – 164.
[145] P. Barrington, J. Y. Chien, F. Tibaldi, H. D. Showalter, K. Schneck, B. Ellis,
Diabetes Obes. Metab. 2011, 13, 434 – 438.
[146] M. A. Bush, J. E. Matthews, E. H. De Boever, R. L. Dobbins, R. J. Hodge,
S. E. Walker, M. C. Holland, M. Gutierrez, M. W. Stewart, Diabetes Obes.
Metab. 2009, 11, 498 – 505.
[147] J. Rosenstock, J. Reusch, M. Bush, F. Yang, M. Stewart, Albiglutide
Study Group, Diabetes Care 2009, 32, 1880 – 1886.
[148] B. Haraoui, J. P. Pelletier, J. Martel-Pelletier, Curr. Rheumatol. Rep. 2007,
9, 265 – 267.
[149] J. M. Gao, M. Mrksich, F. A. Gomez, G. M. Whitesides, Anal. Chem. 1995,
67, 3093 – 3100.
[150] a) I. Jonassen, S. Havelund, U. Ribel, A. Plum, M. Loftager, T. Hoeg-
Jensen, A. Volund, J. Markussen, Pharm. Res. 2006, 23, 49 – 55; b) J. L.
Whittingham, I. Jonassen, S. Havelund, S. M. Roberts, E. J. Dodson, C. S.
Verma, A. J. Wilkinson, G. G. Dodson, Biochemistry 2004, 43, 5987 –
5995.
[151] L. Malik, J. Nygaard, R. Hoiberg-Nielsen, L. Arleth, T. Hoeg-Jensen, K. J.
Jensen, Langmuir 2012, 28, 593 – 603.
[152] a) E. Neil, G. Marsh, Chem. Biol. 2000, 7, R153 – R157; b) M. Cametti, B.
Crousse, P. Metrangolo, R. Milani, G. Resnati, Chem. Soc. Rev. 2012, 41,
31 – 42.
[153] H. K. Munch, S. T. Heide, N. J. Christensen, T. Hoeg-Jensen, P. W. Thulstr-
up, K. J. Jensen, Chem. Eur. J. 2011, 17, 7198 – 7204.
[154] H. K. Munch, J. Nygaard, N. J. Christensen, C. Engelbrekt, M. Oster-
gaard, T. Porsgaard, T. Hoeg-Jensen, J. D. Zhang, L. Arleth, P. W. Thulstr-
up, K. J. Jensen, Angew. Chem. Int. Ed. 2016, 55, 2378 – 2381; Angew.
Chem. 2016, 128, 2424 – 2427.
[155] A. Secher, J. Jelsing, A. F. Baquero, J. Hecksher-Sorensen, M. A. Cowley,
L. S. Dalbøge, G. Hansen, K. L. Grove, C. Pyke, K. Raun, L. Schaffer, M.
Tang-Christensen, S. Verma, B. M. Witgen, N. Vrang, L. Bjerre Knudsen,
J. Clin. Invest. 2014, 124, 4473 – 4488.
[156] R. Bellmann, C. Feistritzer, C. J. Wiedermann, Clin. Pharmacokinet. 2012,
51, 225 – 236.
[157] M. Jamnicki, T. Bombeli, B. Seifert, A. Zollinger, V. Camenzind, T. Pasch,
D. R. Spahn, Anesthesiology 2000, 93, 1231 – 1237.
Received: July 21, 2016
Revised: September 24, 2016
Published online on && &&, 0000
22  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!
REVIEWS
An alternative direction: Despite the S. B. van Witteloostuijn, S. L. Pedersen,
commercial success of PEGylation, new K. J. Jensen*
strategies for extending the half-lives of
&& – &&
biopharmaceuticals are warranted. In
this review, we describe the available al- Half-Life Extension of
ternatives with an emphasis on conju- Biopharmaceuticals using Chemical
gation chemistry, biophysical properties, Methods: Alternatives to PEGylation
and biological compatibility.

ChemMedChem 2016, 11, 1 – 23 www.chemmedchem.org 23  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim &

These are not the final page numbers! ÞÞ