MONOMERIC Fc-FUSION PROTEINS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 113

FIGURE 7.6 Expression of monomeric fusion proteins. Two proteins, effector Fc fusion and Fc
alone are expressed in the expression system. After disulfide bond formation, three products are
produced, including Fc fusion dimer, Fc fusion monomer, and Fc dimer.

Protein G and the fact that Protein A withstands harsher
conditions used in cleaning procedures, Protein A is pre-
ferred for Fc-fusion protein and antibody purifications.
Both Protein A and Protein G chromatography columns
employ binding around neutral pH and elution at acidic
conditions. When the effector protein is sensitive to the low
pH elution, an alternative purification strategy is necessary.
In addition to binding to Protein A and Protein G, human
IgG and Fc fragment bind to Fc receptors such as neonatal
Fc receptor (FcRn) [96] and FcRn affinity chromatography
can be a good alternative for purification of Fc-fusion
proteins. The Fc fragment binds to FcRn with high affinity
at slightly acidic conditions (below pH 6.5) and dissociates
at physiological pH or higher (at or above pH 7.4) [97].
This feature can be conveniently employed in FcRn affinity
chromatography. The interaction of FcRn and Fc fragment
hasbeenfoundtobesimilaracrossspecies,withdifferent
affinity and binding kinetics [98], which provides options
to select appropriate FcRn for affinity resin depending on
the characteristics of the Fc fusion monomer.
The presence of Fc dimer fusion and Fc dimer during
monomer Fc fusion expression requires appropriate chro-
matography processes to separate them during downstream
purification. Protein A or FcRn affinity chromatography are
not able to separate monomer from homodimer Fc fusion or
Fc dimer. Depending on the characteristics of the effector
proteins fused to Fc fragment, Fc monomer fusion and Fc
dimer alone can have similar behaviors on the some chro-
matography columns and cause challenges in purification.
Separation of Fc monomer fusion from Fc dimer fusion and
Fc dimer can be achieved based on charge, hydrophobicity,
and

hydrophobic interaction chromatography, and size exclusion
chromatography [12,61]. Immunoaffinity chromatography
specific to the effector part of the Fc-fusion protein has also
been successfully used for separation of monomer Fc-fusion
protein [99]. During the early research phase, including a tag
to either the effector protein-Fc fusion or Fc alone can
facilitate the separation of monomer from homodimer Fc
fusion [12]. The formulation of Fc monomer is usually
dependent on the characteristics of effector protein fused
to Fc fragment. In general, fusion of Fc fragment could
potentially increase the stability of the effector proteins.
The monomeric Fc-fusion protein provides several
advantages over traditional dimeric Fc fusion technology.
For example, many of the Fc monomer fusion proteins have
enhanced biological activity in comparison to Fc dimer
fusion proteins, presumably in part due to reduction or
elimination of interference between the effector proteins
which could occur in dimeric Fc-fusion proteins [12,32].
However, like any other technologies, monomeric Fc fusion
has its challenges and limitations. One of them is that Fc
monomer fusion proteins are co-expressed with Fc homo-
dimer (Fc dimer fusion protein) and Fc dimer in the expres-
sion system. Because a monomeric Fc-fusion protein is a
heterodimer of effector protein Fc fusion and Fc alone, two
expression cassettes need to be introduced into the expres-
sion system during the construction of recombinant cell
lines. Appropriate ratio of these two proteins can be impor-
tant for the productivity of the monomeric Fc-fusion pro-
teins. The ratio of the three products (monomer Fc fusion,
dimer Fc fusion, and Fc dimers) in the expression system is
affected by multiple factors such as the ratio of effector Fc
fusion and Fc alone at a given time, the condition and size by using ion exchange chromatography,