FUSION PROTEINS: APPLICATIONS AND CHALLENGES - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 14

Glycosylation is a key parameter of fusion proteins,
determining immunogenicity, solubility, and stability. Par-
ticularly, the terminal sugars of glycans in the CH 2 domain
of antibodies are responsible for influencing antibody-
dependent cellular cytotoxicity (ADCC) and complement-
dependent cytotoxicity (CDC) activity [149]. ADCC can be
significantly enhanced by the lack of core-fucose on the
Fc part as demonstrated with scFv-Fc fusion proteins [150].
An interesting example for the importance of glycosylation
is the half-life extension technology of Prolor that relies on a
carboxy-terminal peptide, originally derived from the cho-
rionic gonadotropin (CG) b -subunit, bearing four serine-
linked oligosaccharides. This short peptide can be fused to
the N- or C-terminus of other proteins, expanding the
hydrodynamic radius by extensive O-glycosylation, thus
preventing kidney filtration [151]. Obviously, glycosylation
in that case requires secreted expression in mammalian cells
to obtain the proper oligosaccharide pattern. Glycoengin-
eering was also used to optimize half-life and potency of an
erythropoietin (EPO) variant called Aranesp 1 . This novel
EPO has two additional N-linked carbohydrate chains,
resulting in threefold longer serum half-life [152].
Besides half-life extension, glycosylation also contrib-
utes to protein stability. The biggest effects are seen in
preventing proteolysis by shielding susceptible amino
acid sequences, inhibiting aggregation by the hydrophilic
nature of oligosaccharides and by protection of the proteins
against all kinds of physical denaturation, including freezing
[153].
The workhorses of antibody production, CHO cells,
deliver a slightly different glycosylation than original human
cells. Therefore, CHO cells were engineered to express
human glycosyltransferases to improve the ADCC and
CDC effects of antibodies [154].
Since cultivation of mammalian cells is rather costly
when compared to microbial cells, also attempts were under-
taken to obtain human glycoforms in yeasts. As suitable
starting point for humanization of yeast the a -1,6-mannose
extension must be eliminated. This was followed by the
introduction of a a -1,2-mannosidase for mannose trimming.
Finally, mannosidase II and N-acetylglucosaminyl transfer-
ase II was added to release uniform human-like glycans from
yeast [155]. As last step terminal sialysation had to be
implemented; this required the introduction of 14 foreign
genes into P. pastoris [156].
The biggest obstacle of yeast N-glycosylation is the high
mannose content that is potentially immunogenic and leads to
drastically reduced half lives by the removal of therapeutic
glycoproteins through mannose receptors on macrophages,
thus reducing the efficacy [157]. But in some cases manno-
sylation can be beneficial, for example, when an antibody-
enzyme fusion protein has to be quickly removed fromnormal
tissue to enable a highly specific antibody-directed enzyme
prodrug therapy (ADEPT) approach [158].

1.6 REGULATORY CHALLENGES

Despite the demonstrated success, fusion proteins consisting
of two molecules with different functions, still raises con-
cerns. Addressing these issues is of huge interest in a time
where biosimilars and biobetters enter clinical development
[159]. Several variants of approved drugs can be found
within the category of fusion proteins, having improved
half-life, potency, stability, or route of administration
[11]. In general, three aspects must be clarified satisfacto-
rily: quality, safety, and efficacy. The necessary information
is gathered through animal pharmacology and toxicology
studies. Here, the pharmacodynamics and pharmacokinetics
that cover clearance and absorption, distribution, metabo-
lism, and excretion (ADME), is assessed. The tightly regu-
lated release of proteins in the organisms has to be mimicked
by adapting dosing intervals or even activity of the protein
[160]. This is much more difficult for non-natural proteins
such as fusion proteins.
A separate document contains the chemistry, manufac-
turing and control (CMC) data. The manufacture must be
done under good manufacturing practice (GMP) conditions
and demonstrate the ability to consistently and reprodu-
cibly supply active batches of the drug. All that must be
included in an investigational new drug (IND) application
or a clinical trial authorization/exemption (CTA/CTX) for
the regulatory authorities in the Unitied States or Europe,
respectively.
Since protein therapeutics are produced in living orga-
nisms, they could contain intrinsic infectious agents and
other process- or product-related impurities. As biological
they could have a heterogeneous composition and require
extensive analysis. Expected and controllable parameters are
size, charge, activity, folding, but on the other hand
unexpected and undesired characteristics such as aggrega-
tion, amino acid modification or proteolysis could occur as
well [161]. Usually, viral clearance with two orthogonal
independent steps must be demonstrated. Before release the
drug substance's identity, purity, potency, and stability must
be verified according to predefined acceptance criteria in a
number of validated assays [162].
The key product attributes that determine efficacy, in
other words potency and activity, under the conditions of the
intended use must be characterized. This includes nonclin-
ical pharmacokinetic, toxicology, and safety studies [163].
Manufacturing conditions have to be chosen that can gene-
rate the desired attributes, or product quality, in a reproduc-
ible and cost-efficient manner. In the preclinical studies
often imperfect replicas of human disease conditions in
animal models are used [164]. A further hurdle for human
therapeutic (fusion) proteins is that sometimes nonclinical
assays must be performed with the respective animal homo-
log because the human version is nonfunctional

in the

selected animal species [165].