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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5608_Библиотеки_им_академика_М_И_Перельмана.pdf
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Table 11.4 Overview on point mutations to enable heterodimerization of Fc domains
Company Technology Mutations HC1 Mutations HC2
Genentech Knobs-into-
holes
Genmab DuoBody F405L K409R
Zymeworks Azymetric T350V, L351Y,
Amgen Charge Pair K392D, K409D E356K, D399K
Rinat-Pzer Charge Pair D221E, P228E, L368E D221R, P228R, K409R
Xencor HA-TF S364H, F405A Y349T, T394F
T366W T366S,L368A, Y407V
T350V, T366L, K392L,
F405A,Y407V
T394W
S. R. Schmidt
11.3.3.3 Trimer
Trimeric formats can be generated by utilizing naturally occurring proteins based on
trimers such as collagen. Some years ago, the combination of scFV with the
collagen- like peptide scaffold (GPP)10 was evaluated. Fusions at the N-terminus of
the peptide resulted in stable homotrimers. Due to the increased avidity, the trimer
bound 20 and 1000-fold stronger than the bivalent or monovalent versions of the
same scFv, respectively. When expressed in NS0 cells, the murine prolyl
4- hydroxylase converted approximately 61% of the 10 available prolines into
hydroxyproline, thus giving a thermo-stability comparable to the naturally occurring collagen. The so-called Collabody is currently evaluated as a CD3-specic
T-cell engager for therapeutic applications [38].
11.3.3.4 Tetramer
The next oligomeric level are tetramers. A naturally available building block is the
self-assembling tetramerization domain from p53 (p53TD) that can surpass the
naturally occurring afnity threshold of antibodies through higher avidity. In multiple examples, modules such as dAbs, scFv, Fabs, and extracellular protein domains
were fused to p53TD either at its N- or C-terminus. Due to its self-assembling capabilities active tetramers could be successfully expressed, secreted, and puried from
a single poly-peptide chain. As the p53 domain is deeply embedded in the overall
structure, no detrimental immunogenic effects are expected. Involving a dimerization unit such as the Fc-domain, even an octamer could be obtained. Octameric
versions of Humira were approximately 50-fold more potent than the original divalent Humira. Due to the higher potency the so-called Quads could either be used at
lower doses than the original antibody or allow a switch from intravenous to subcutaneous application [39].
Tetrameric antibodies have been described since many years. One recent example is the evaluation of tetrameric formats to ght COVID infections. Here the variable domain of heavy chain is either duplicated at the N-terminus or fused to the
C-terminus of the Fc-domain, while maintaining the light chain. This antibody with
11 Fusion Proteins: Current Status andFuture Perspectives
301
four binding domains neutralizes the virus with enhanced potency so limiting its
ability to achieve resistance though escape mutations [40].
11.3.3.5 Pentamer
Some receptor ligand interaction happens at low afnity and therefore requires multiple ligands to bind simultaneously. For instance, CD200 binds to its corresponding
receptor at approximately 1μM.When generating a pentamer by fusing CD200
with an eleven amino acid linker to cartilage oligomeric matrix protein (COMP), the
receptor binding was highly improved [41].
11.3.3.6 Hexamer
Larger multimers have also been described in the form of tandem repeats of Fc
domains to increase the antibody-dependent cellular cytotoxicity (ADCC) and
complement- dependent cytotoxicity (CDC) [42]. Even larger hexameric complexes
have been designed by adding small 18-mer peptides to theC-terminus. These tailpieces connect six Fc-dimers to form a large multi-subunit molecule with the ability
to crosslink low afnity FcγR binding. The hexameric format exhibits stronger
avidity with 12 potential binding sites or fusion partners [43].
Looking at other fusion partners beyond Fc-domains, a dimerization can be
enforced by leucine zippers, naturally occurring peptide sequences. For instance, a
noncovalently bound heterodimeric Fab fragment, a so-called “Zipbody” was
designed by fusing leucine zipper pairs LZA and LZB or c-Jun and c-Fos to the
C-terminus of heavy and light chain thus enhancing correct pairing and resulting in
a properly folded active Fab [44]. A similar example was described earlier by creating bispecic homodimers based on two different designed ankyrin repeat proteins
(DARPins) and a leucine zipper linker [45].
More complicated trivalent molecules based on tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) utilized leucine zippers forming trimers. One
case incorporated the yeast GCN4-pII leucine zipper in fusion to the N-terminus of
TRAIL.The Ile substitutions in the “a” and “d’ positions stabilizes the trimer formation. Replacing the yeast protein GCN4-pII by the human ATF7-pII greatly
reduced the immunogenicity while maintaining high antitumor potency [46]. To
enable an even more oligomeric molecule, the building blocks human IgG4
Fc-domain, the TNF receptor-associated factor 2 (TRAF2) coiled coil domain, and
the extracellular receptor binding domain (RBD) of human OX40 ligand (OX40L)
were fused together to obtain a 300kDa large hexameric fusion protein [47]. A
functional OX40L multimer can also be obtained by using similar building blocks,
but using trimerizing isoleucine zipper domain GCN4 instead of TRAF2 and IgG1
Fc instead of IgG4 Fc. The nal hexameric molecule consists of three Fc-induced
disulde linked dimers that forming two connected trimers [48]. A more elegant and
less complicated solution to generating trimeric fusion proteins was described in the
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S. R. Schmidt
hexavalent receptor agonist (HERA) Technology. These hexameric fusion proteins
comprise three receptor binding domains in a single chain arrangement, connected
to an Fc-silenced human IgG1 not requiring FcγR-mediated crosslinking for tumor
killing activity [49]. Other approaches with the class of homo-multimeric proteins
focused on nding the most active version of CD40L as adjuvant for DNA vaccines.
Three different self-assembling versions were compared. The starting point was a
leucine zipper-based trimer. The next variant contained two trimers connected
through fusion to the N-terminal part of adiponectin (Acrp30). The third molecule
was a tetra-trimer containing 12 individual CD40L arranged through fusion to surfactant protein D.Their immunostimulatory activity increased in direct proportion
to the valency of the trimers [50]. Naturally occurring trimeric proteins such as the
noncollagenous domain (NC1) of human collagen XVIII NC1 can be utilized as
well in fusion protein applications. NC1 is composed of a trimerization domain
(TD), a hinge region, and the endostatin domain. Proteolytic cleavage on the hinge
region releases the endostatin domains to establish a fully functional TRAIL-TD
trimer [51].
11.3.3.7 Octamer
So far, the highest described order of fusion proteins are octamers. Here the naturally occurring afnity threshold can be surpassed by higher avidity. The base element in that context is the self-assembling tetramerization domain from p53.
11.3.4 Orientation
On the rst glance, the orientation of both fusion partners seems to be irrelevant as
long as a single polypeptide chain can be obtained. However, as soon as a free N- or
C- terminus is required for activity this parameter needs to be considered in the
design. An overview with current examples for an orientation preference can be
seen in Table11.5.
Sometimes simply following the natural conguration is giving the best results.
For instance, this is true for all receptor traps consisting of the extracellular domain
of membrane bound receptors fused at their C-terminus to an IgG Fc domain.
Peptibodies are another class of Fc fusions but with opposite orientation. Here small
peptides or concatemers are fused to the C-terminus of Fc domains. One underlying
reason for this conguration is the protection from peptidases that attack free
N-termini. Furthermore, for so far unknown reasons, a C-terminally fused NGF
peptibody was 1500 times more potent than its N-terminal counterpart [52].
A wide range of other examples can be found in the literature about human serum
albumin (HSA) fusions. Interestingly in few cases N-terminal fusion of HSA was
preferable. Although the real reason for that has rarely been properly identied, but
in the here mentioned examples the N-terminal fusion typically resulted in better
11 Fusion Proteins: Current Status andFuture Perspectives
Table 11.5 Examples of fusion proteins with a clear preference of the orientation of their fusion
partners
N-Term C-Term Effect (better) Reason
TNF-R Fc Functionality Natural conguration
Fc FGF21 Activity Unknown
Fc EPO Half-life and activity Endosomal stability
X ELP Activity, expression Mis-folding in N-Term fusion
scFv ANG Functionality Free C-Term required
ANG END Potency Unknown
IFN-a2b HSA Reduced aggregation Improved S-S formation
TNF-RI HSA Activity Improved S-S formation
HSA IL28B Activity and expression Unknown
HSA BNP
HM3 HSA Half-life, stability, activity Unknown
hLF HSA Stability, low proteolysis Steric hindrance
HSA TMP
SS28
2
2
2
HSA Activity and expression Lower degradation
Activity and expression Lower degradation
Expression Unknown
303
expression levels and higher activity. For instance, only IL28B fused to HSA at its
N-terminus was active and exhibited better stability than its C-terminal counterpart [53].
On the contrary for IFNα2b, lower heterogeneity and instability were observed
for this orientation but could be signicantly enhanced by changing to a C-terminal
fusion [54]. The underlying reason for that effect seems to be the improved ability
to form di-sulde bridges. The same cause was identied for the better TNF-α neutralizing activity of a receptor trap consisting of TNF-RI fused to HSA on its
C-terminus [55].
Sometimes the benet of one version is different in the other version. For
instance, evaluating the ideal orientation for a HSA lactoferrin fusion, it was found
out that placing HSA at the N-terminus of the construct results in sixfold increased
half-life, but for the cost of reduced thermal and proteolytic stability [56]. Peptides
typically suffer from a short half-life that can be extended by HSA-fusion. On the
one hand, there are examples such as the integrin antagonistic peptide HM-3, where
the best result in terms of higher stability and activity was achieved with connecting
HSA to the C-terminus of the peptide [57]. On the other hand, cases such as the
human brain natriuretic peptide (BNP) obtained best biological potency when two
copies are fused to the C-terminus of HSA [58]. Similarly TPO mimetic peptide
(TMP) in tandem could signicantly better expressed having HSA at its N-terminus
[59]. Interestingly another example utilizing tandem peptides showed lower degradation and higher activity for the variant with somastatin at the N-terminus [60].
The orientation of bifunctional molecules not relying on the classical building
blocks Fc domain or albumin is even more difcult to predict. When connecting
angiostatin (ANG) and endostatin (ENG) to inhibit vascularization a clear preference of the arrangement is not obvious. However, in biological assays the
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S. R. Schmidt
ANG-ENG version was signicantly more potent than its ENG-ANG counterpart
[61]. Another special case is the fusion to elastin like peptides (ELP). When testing
the position of ELP in fusion to four different proteins, it was found out that the ELP
at the C-terminus resulted in higher expression and better activity than the N-terminal
fusion of ELP.The underlying reason for that effect is most probably an increase in
misfolded protein when the translation starts with ELP [62]. The class of immunotoxins consisting of a targeting ligand such as scFv and a toxin was also evaluated
for the optimal orientation of both elements. Chimeric toxins containing the scFv at
the C-terminus of restrictocin were shown to be more active than those havinga
ligand at the N-terminus of the toxin, most probably due to folding differences [12].
The multitude of examples here clearly demonstrates the fact that it is very difcult to predict the ideal orientation upfront, but rather requires empirical results
testing both variations. Typical parameters to differentiate between the options are
expression levels, activity, stability, and improved pharmacokinetics.
11.3.5 Protein Engineering
The paragraph above highlighted design details on linker sequences. But protein
engineering can also help in solving other issues such as efcacy, production yield
that impact the therapeutic effect of fusion proteins.
11.3.5.1 Modications ofFc asBuilding Block
When designing Fc-fusion proteins, it is worthwhile to have a closer look on the
inbuilt features of the constant region. The Fc domain has multiple functions such
as dening half-life through recycling via the FcRn receptor, triggering effects such
as ADCC or CDC via FcyR, and enabling oligomerization from asymmetrical
monomer and homo- or heteromeric dimer. Over the last decade, the underlying
amino acid interactions were revealed. In some cases, when bivalency is not possible due to steric hindrance of too large fusion partners or not desirable to avoid
receptor crosslinking, the availability of a truly monomeric Fc would be benecial.
Unfortunately, most attempts were unsuccessful and resulted in instable molecules
or suffered from decreased half-life. The rst stable Fc monomer was obtained by
introducing two additional N-glycosylation sites at the CH3-CH3 interface with the
double replacement Ser364Asn and Phe405Asn [63]. The contemporary approaches
on Fc optimizations to address receptor binding have been summarized in a comprehensive review [64]. Details can be found in Table11.6.
Fc receptor functions have often been used to optimize therapeutic effects [65].
For instance, an improved version of Enbrel® had higher ADCC and CDC activity
than the original fusion protein that could extend its use to other chronic disease
such as ulcerative colitis or Morbus Crohn. Additionally, higher afnity to the target
TNF-α was observed. Taken together, these modications can lead to a better
11 Fusion Proteins: Current Status andFuture Perspectives
Table 11.6 Point mutations in the Fc domain modulating FcyR-mediated functions
Company ADCC ADCP CDC IgG Mutation
Genentech + + 1 S298A, E333A, K334A
Xencor + + 1 S239D, I332E
Xencor + + − 1 S239D, A330L, I332E
Applied Molecular
Evolution
Applied Molecular
Evolution
Macrogenics + + 1 F243L, R292P, Y300L +/− P396L or
Xencor + 1 G236A, S239D, I332E
Abgenix/Genentech = + 1 K326A, E333A
Abgenix/Genentech 0 + 1 K326W, E333S
GlaxoSmithKline/Tolerx − − 1 N297A
Ortho Biotech − − 1 L234A, L235A
Protein Design labs − − 2 V234A, G237A
Welcome Labs 4 L235A, G237A, E318A
Merck − − − 2 H268Q, V309L, A330S, A331S
Bristol-Myers Squibb − − 1 C220S, C226S, C229S,
Seattle Genetics − − − 1 C226S, C229S, E233P, L234V,
Medimmune − − − 1 L234F, L235E, P331S
+ + 1 P247I, A339D/Q
+ + 1 D280H, K290S +/− S298D/V
V305I
L235A
305
efcacy and a wider applications [66]. A different approach to improve Enbrel®
against its primary indication rheumatoid arthritis was taken with a hybrid Fc
domain that consists of a immunological silent IgG4 domain connected to the highly
exible hinge region of IgD, hiding the junction site inside the structure to avoid
unwanted immunogenicity. The molecule showed a 1.5-fold higher neutralizing
activity than Enbrel® [67]. The three-dimensional structure of the Fc domain is
governed by S-S bridges [68].
11.3.5.2 Modications ofHuman Serum Albumin asBuilding Block
Protein engineering has been applied to HSA fusion proteins as well. An obvious
target is the free Cys in HSA that causes aggregation and reduces stability. Cys34
was replaced by Ser by site directed mutagenesis in a IFNα2b-HSA fusion protein.
After incubation at 60 °C or prolonged agitation still 90% of the fusion protein was
in a monomeric state [69]. Interestingly additional free Cys residues could be useful
for chemical conjugation of drugs. Therefore, two more thiol groups were introduced at position 93 and 294 resulting in the replacements Lys93Cys and
Glu294Cys [70].
As the primary reason for HSA fusion is the extension of half-life, several
attempts were undertaken to optimize its binding to FcRn which is the main
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S. R. Schmidt
contributor. Typically, albumin has a serum half-life of 3weeks. The rst described
site directed exchange of Lys573Pro at the C-terminus in domain DIIIb improved
afnity to FcRn by a factor of 12, leading to a 1.5-fold prolonged half-life [71]. As
the binding to FcRn happens at the C-terminus of HSA, it is obvious that fusions to
the N-terminus of HSA will have less impact on recycling through FcRn and are
therefore preferable.
Combining yeast display and co-crystallization of FcRn with HSA variants, a
double-mutant Glu505Gly and Val547Ala was identied that exhibited a ten-fold
higher afnity to FcRn leading to a 1.3-fold improved half-life in cynomolgus monkeys [72].
Although the DIII domain is the main contributor for FcRn binding, it was found
out that two loops in DI at the N-terminus modulate the interaction [73]. It will be
worthwhile to utilize that region as well to optimize FcRn afnity at different pH
values together with further mutations in DIII to ne tune new designer HSA molecules as fusion partners.
Another group selected different amino acid residues to not only improve halflife but also enabling transcellular delivery. The new albumin variant QMP contains
three substitutions: Glu505Gln, Thr527Met and the known mutation Lys573Pro.
Compared to the wild-type, QMP was translocated twofold more efcient over
mucosal barriers at intranasal application. Fusing the triple mutant HSA to coagulation factor VII, the half-life improved 3.6-fold [74]. Table 11.7 summarizes the
mutagenesis approaches based on albumin.
11.3.5.3 Modication ofOther Functions
Serum half-life is typically inuenced either by the diameter of the molecule that
prevents kidney ltration, charge density that would cause repulsion or the ability of
recycling through FcRn or transferrin receptors. On the example of immunocytokines (fusions between antibodies and cytokines), it was demonstrated that the
faster than expected clearance from the bloodstream can be inuenced by altering
the afnity to other FcRn either by changing the Fc isotype or mutagenesis of the
FcRn binding site on the Fc domain [75]. In a second study, the half-life was further
improved by optimizing the junction between IL-2 and the antibody to prevent
intracellular degradation during the FcRn-mediated recycling [76].
Stability issues cannot only be attributed to aggregation but can also be the result
of degradation. Peptidbodies with N-terminal fusion of short amino acid sequences
Table 11.7 Point mutations in human serum albumin to optimize certain functionalities
Additional functions Half-life extension Translocation
Cys34Ser
Improved stability
Lys93Cys, Glu294Cys
Additional conjugation sites
Lys573Pro
1.5-fold longer half-life
Glu505Gly, Val547Ala
1.3-fold longer half-life
Glu505Gln, Thr527Met, Lys573Pro
Twofold better translocation
3.6-fold longer half-life
11 Fusion Proteins: Current Status andFuture Perspectives
307
can suffer from enzymatic proteolysis. This can be avoided if a glutamate residue is
introduced at the rst position of the polypeptide which forms pyroglutamate after
cyclization, protecting against protease attacks. Studying the functionality of this
peptidbody, an unexpected O-glycosylation site was removed by exchanging Thr to
Ala, thus signicantly increasing potency [77]. Similarly, the negative impact of
proteolysis and aggregation on an FGF21-Fc fusion protein could be successfully
abolished through the replacements Pro171Gly and Leu98Ar, respectively [78].
The next level of engineering targets three-dimensional alterations. One example
in that context is the introduction of four point-mutations into the EPO domain
within an Fc-EPO fusion protein to improve multiple features. Overall, the amino
acid exchanges were His32Gly, Cys33Pro, Trp88Cys and Pro90Ala that resulted in
a disulde bridge rearrangement from Cys29-Cys33 to Cys29-Cys88. The modied
fusion protein was secreted as monomer with better pharmacokinetic and stability
after the removal of N-linked glycosylation [79].
11.3.6 Immunogenicity
Some unwanted effects like glycosylation at novel positions introduced through
linker sequences, nonhuman glycosylation or degradation at cryptic protease recognition motifs have been described in the paragraphs above. However, another even
more deleterious biologic consequence of a novel, non-natural protein design is the
eliciting immunogenicity, particularly at novel epitopes created at the junction
between both fusion partners, even if only proteins of human origin are being used.
Furthermore, fusion proteins might be taken up or processed differently than their
native counterparts which could lead to an increased immune response. Therefore,
it is benecial to apply rational design of fusion proteins to minimize immunogenicity of the new construct. The current approach to identify amino acid positions for
targeted mutagenesis is in silico analysis to predict T- and B-cell epitopes [80].
Particularly Fc-fusion proteins could show increased immunogenicity as they
bind via Fcy receptors (FcyR) to antigen presenting cells (APC) thus targeting antigens to these cells. If that results in enhanced uptake to APCs, or if the fusion protein stimulates these cells, then a stronger immune response could be triggered.
Current deimmunization strategies based on prediction of particularly immunogenic
epitopes focus on modifying epitope sequences to disrupt HLA binding to eliminate
T-cell recognition [81].
The effect of the Fc domain in fusion proteins has also been studied as means to
modulate immunogenicity. Primarily CD4 T-cells and B-cells are involved with an
important participation of APCs. In case of crosslinking activating FcyR on APCs
the antigen presentation to T-cells increases, strengthening the immune response.
The other case of signaling through inhibitory FcyR can also occur and will downregulate immune responses. Interestingly the nature of the fusion partner can inuence the afnity of the corresponding Fc- domain to the FcyR [82]. The hypothesis
of lowered immunogenicity of Fc-fusion proteins by binding to inhibitory Fc
308
receptors on B lymphocytes, the FcyRIIb was experimentally proven with the injection of DNA coding for a Exendin-4 Fc-fusion protein that did not result in the
generation of neutralizing antibodies, which was the case for Exendin-4 alone [83].
Just recently a Fc receptor trap fusion protein consisting of fully human sequences
only was risk assessed for immunogenicity. Although fully human sequences reduce
the risk of antidrug antibodies (ADA), the effects of ADA might be higher if they
occur, as they target the corresponding component on cells with all potential negative consequences [84].
Ideally B- and T-cell epitopes are removed, as both contribute to immunogenicity. It is easier to remove B-cell epitopes, as they are not restricted to major histocompatibility complex (MHC). In contrast to that T-cell epitopes rely on the high
polymorphism of MHC [85].
S. R. Schmidt
11.4 Manufacturing
On the rst glance, the production of fusion proteins cannot be distinguished from
that of regular therapeutic proteins. Manufacturing typically comprises the steps of
upstream processing including all aspects of cell culture, downstream processing
covering steps to capture and purify the protein of interest from impurities and
nally formulation steps that convert the protein into a storable and administrable
form. One of the many advantages of fusion proteins is the uninterrupted manufacturing process of a single but multifunctional molecule. However, fusion proteins
suffer from a lot of issues that can complicate the production. The combination of
two different proteins with different properties might not always be compatible with
respect to their pH preferences, hydrophobicity, native cellular localization, and
glycol-isoforms. Furthermore, they are sometimes sensitive to low pH which then
does not allow conventional virus inactivation. Fusion proteins are often larger than
well-studied molecules like antibodies and therefore might have lower expression
levels. Figure 11.4 gives a comprehensive overview on current manufacturing
practices.
11.4.1 Upstream
Due to their size and complexity, most modern fusion proteins are expressed in
eukaryotic cells. This allows the proper formation of post-translational modications such as glycosylation or disulde bridges. The major host cell is derived from
Chinese hamster ovaries (CHO) which is frequently used for Fc-fusions. Yeast cells
are applied primarily for albumin fusions. Prokaryotic expression is favored either
for relatively small proteins with a molecular mass below 30kDa or proteins that
form inclusion bodies to enable the production of toxic proteins like
immunotoxins.
11 Fusion Proteins: Current Status andFuture Perspectives
Fig. 11.4 Comparison of mammalian and microbial bioprocesses for the manufacturing of fusion
proteins. (a) The upstream part of cultivation and expanding the cells takes typically several weeks
in a mammalian bioprocess. Here the protein of interest is secreted to the medium; therefore, cells
are separated from the supernatant by either depth ltration or centrifugation. Protein from the cell
free harvest is then captured on the rst column and potential virus impurities are inactivated by
typical low pH exposure. The protein is further puried in several polishing steps to remove impurities before any remaining viruses are removed by virus ltration. In the last step, the pure protein
is formulated by the addition of excipients and lled as bulk drug substance. (b) Microbial cultivation processes are usually nished in days. Here the protein of interest often accumulates in the
form of inclusion bodies in the host cell. Therefore, these cells are collected by centrifugation,
disrupted, and the inclusion bodies are washed and solubilized by denaturing agents. Then the
unfolded protein has to be refolded before the regular purication process is started. With the
exception of the absence of virus removal steps, the downstream procedure is quite similar. (WCB
working cell bank, DF depth ltration, BDS bulk drug substance, IB inclusion body)
309
In principle, the upstream process contains three elements: the DNA construct
coding for the gene of interest plus regulating sequences, the host cell, and the cultivation conditions. All three elements contribute to the expression level, or titer that
can be achieved during upstream processing. Prokaryotic expression can directly
work by introducing a plasmid that codes for the fusion protein, accompanied by at
least a promoter sequence, a replication origin, and typically a selection marker
such as an antibiotic resistance gene into the microbial cell. However, in mammalian cells the expression construct is integrated into the cell genome. Often this
integration happens at multiple sites simultaneously increasing the expression level.
To generate the producer cell line, a selection process is frontloaded that identies
the best cell clone based on expression yield, growth characteristics, and genomic
stability.
Temperature inuences cell growth and product titer. It was observed that in the
case of Etanercept growth at 30 °C resulted in a threefold higher titer than at
37°C.However, increasing the viable cell density at 30°C decreased the productivity. So, essentially both effects have to be systematically assessed to nd optimal
expression conditions [86]. A more detailed analysis of cultivation parameters pH,
temperature (T), and dissolved oxygen (DO) has been undertaken for EPO-Fc
expressed in CHO.Lowering T and pH reduce cell growth and metabolism but
increase the viable cell integral and consequently enhance the specic productivity