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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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S. R. Schmidt
while reducing volumetric productivity, leading to extended cultivation times. The
solution to this dilemma is a biphasic process where initially culture conditions are
established that support rapid expansion of cells followed by a second phase when
switching to parameters that reduce growth but improve productivity [87]. The disadvantage of incomplete sialylation at low temperatures could be compensated by
simultaneously reducing the pH [88]. A further benet of a biphasic cultivation
strategy is the reduction in EPO-Fc aggregates from 75% at standard conditions at
37°C and pH7.05 to less than 1% at 30°C and pH6.75. This synergistic effect even
led to reduced glucose and glutamine consumption while simultaneously lowering
lactate and ammonium generation [89].
The other signicant eukaryotic expression host is yeast. Either Saccharomyces
or Pichia pastoris cells are used to manufacture fusion proteins containing albumin
or transferrin [90]. For instance, a IL2-HSA fusion could be produced in P. pastoris
using glucose as carbon source. Besides the feeding strategy optimization, it was
found out that lower fermentation temperature reduced the presence of proteases
and a neutral pH of 7.0 was best for maximizing expression [91]. Surprisingly this
fusion protein did only achieve 50% of the expression levels compared to HSAalone.
Therefore, the co-expression of ve different secretion helper proteins, Er1, Kar2,
Pdi1, Sec1, and Sly1, was evaluated. All them could restore the initially observed
expression level of approximately 1g/L [92]. Expression yield in P. pastoris can
also be inuenced negatively by the structure of the fusion protein to be expressed.
For instance, a triple copy of somatostatin fused to HSA resulted in signicantly
lower expression than its tandem copy, indication that gene multiplication not necessarily is a suitable approach to improve fusion protein manufacturing [93].
11.4.2 Downstream
The downstream process (DSP) starts with the material containing the product of
interest. Depending on the expression system this can either be extracellular or
intracellular material.
Microbial expression utilizing Escherichia coli typically results in inclusion
bodies (IB) that contain denatured aggregated proteins. IBs can represent 10–50%
of the total cell protein containing up to 95% of a single protein species [94]. This
means during harvest the cells are collected and the later mechanically or chemically disrupted to set the IBs free. Typical impurities of that step are endotoxins that
are present in huge amounts as the cells wall is destroyed. The IBs are then washed
and solubilized by chaotropic substances such as guanidinium hydrochloride or
urea under reducing conditions to break all disulde bridges. After this denaturation, the so-called refolding process is initiated that essentially means gradual
removal of the denaturing agents while slowly establishing oxidizing conditions to
re-form disulde bridges, thus achieving the proper natural secondary and tertiary
structure [95]. Alternatively, recombinant proteins can be secreted to the
11 Fusion Proteins: Current Status andFuture Perspectives
311
periplasmatic space of E. coli, which avoids refolding, however, with lower expression yields.
Still some process and product related impurities are present that have to be
removed by typical downstream unit operations such as chromatography and
ltration.
In the case of extracellular production, the protein of interest is secreted to the
cell culture medium. Here the harvest process means the removal of cells either by
centrifugation or depth ltration or a combination of both to obtain a cell free uid
(CFF) for further processing. Hosts for secretion are typically eukaryotic cells such
as yeast or mammalian animal cells. Secretion is certainly preferred because in
modern serum free, chemically dened media only few host cell proteins are present together with the secreted product [96].
Fusion partners sometimes help in establishing platform purication processes.
In the case of elastin like peptides (ELP), the temperature-dependent aggregation
allows simple capturing by sedimentation as an initial enrichment [97]. The most
frequently used fusion partner IgG Fc can usually be treated just like regular antibodies. However, there are reports, where protein A resins exhibit a lower dynamic
binding capacity for Fc-fusion proteins primarily due to steric hindrance caused by
bulky fusion partners [98]. The rest of the capture process is comparable to an antibody process with binding at neutral pH, and elution at acidic pH below 3.7 that also
inactivates potential viruses. Unfortunately, sometimes aggregation during elution
at low pH might occur that is triggered either by high local protein concentrations
or the unnatural charges induced by low pH.Here the addition of a chaotropic agent
during elution can help maintaining monomeric species [99].
Other antibody derivatives like Fab fragments or scFv do not bind to a protein A
ligand. They require specic afnity matrices, for instance, protein L which is
kappa light chain specic [100, 101] or a synthetic ligand such as FabSorbent®
[102]. Recently a procedure to isolate Fab fragments with a series of protein G and
L chromatography was described, where some product-related impurities like fragmented light chains were co-puried on protein L resin [103].
Albumin from plasma used to be isolated by dye-afnity chromatography.
Nowadays advanced highly selective resins like Albupure™1 based on a synthetic
triazine or Captureselect™ with single domain antibody fragments are available to
capture recombinant albumin fusion proteins.
All other potentially more unorthodox fusion proteins without any of the well
understood fusion partners cannot be enriched from crude supernatants by afnity
chromatography but have to be collected by a more unspecic method with high
capacity but limited selectivity, ion exchange chromatography. Here the protein of
interest binds to immobilized charges of the stationary phase at a pH which is either
lower or higher than its isoelectric point to expose sufcient surface charges. Elution
is achieved by either changing the pH to modulate the charges of the protein or by
adding salt ions that disrupt the ionic binding. Besides bind and elute mode, proteins
1
https://www.astreabioseparations.com/resources/albumin-fusion-proteins/albupure-pc-3151
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S. R. Schmidt
can also be puried by a ow through method, that allows the protein of interest to
pass, while impurities are bound to the resin.
In the case of disulde containing proteins scrambling during purication might
occur [104] that requires careful monitoring. Obviously wrongly formed disulde
bonds have a negative impact on potency as described for Etanercept [105].
A high purity of the fusion protein is then achieved by combining different purication principles that selectively remove all kinds of impurities, while enriching
the protein of interest. Although it might seem preferable to execute a two-step
process from an economical perspective, a three-step approach could result in
improved purity [106]. So always cost must be balanced to a reasonable level.
Finally, the protein just has to be concentrated and mixed with suitable excipients to
be stabilized for its administration. Particularly the stabilization of fusion proteins
can be quite challenging as the two different fusion partners might have different
preferences for additives.
11.4.3 Glycosylation
Besides the proper formation of disulde bridges that determine the threedimensional structure, glycosylation is the other important post-translational modication of proteins. Glycosylation improves the solubility of proteins by adding
highly hydrophilic glycans. These large carbohydrate structures can also enhance
the stability by either covering protease sensitive regions or prohibiting aggregation
through hydrophobic patches. Another biological function is their recognition by
specic receptors that enable tissue targeting or the recruitment of immune cells.
Glycosylated proteins contain carbohydrates either N-linked to the nitrogen in
asparagine side chains or O-linked to the oxygen in serine or threonine. Interestingly
N-linked glycans require a recognition motif Asn-x-Ser/Thr, while O-linked glycosylation depends on the secondary structure and the accessibility of Ser or Thr. The
often observed heterogeneity of glycans is a result of the expression level of the
required enzymes and the availability of their substrates in the ER and Golgi [107].
The glycosylation can be controlled on several levels, starting at the protein sequence
level, the choice of host cells, the cultivation process parameters, and nally the
ability of the downstream process to select certain isoforms as described in Fig.11.5.
The CH2 part of the Fc-domain contains a canonical position for N-glycosylation
at Asparagine 297 (N297). N-glycans have important structural functions. They stabilize the CH2 domain of IgGs and the lack of glycans decreases the thermal stability of mAbs and make them more susceptible to unfolding. Furthermore,
deglycosylated mAbs are more prone to aggregation. Depending on the size of the
attached glycans, the structure of the CH2 domain can turn into either an open or
closed conformation which inuences the binding properties to Fc receptors. These
receptors transfer the antibody-dependent cell-mediated cytotoxicity (ADCC) or
complement-dependent cytotoxicity (CDC). The absence of fucose greatly enhances
ADCC, whereas the presence of terminal galactose improves the CDC activity.
11 Fusion Proteins: Current Status andFuture Perspectives
Fig. 11.5 Controlling glycosylation at different process steps. Glycosylation should be designed
into the protein from the start. The next important choice is the host cell or potential strain engineering approaches to improve homogeneity and intensity of glycosylation. During the upstream
process, the glycan pattern can be inuenced by the growth conditions or the presence of certain
substances in the culture medium. During downstream processing, the ideal glycoisoform must be
separated from wrong or incomplete forms to obtain a product with a glycosylation as homogeneous as possible
313
Capping of the terminal glycans with sialic acid positively inuences the pharmacokinetics, which is caused by prohibiting the clearance of desialylated proteins
through receptors the recognize mannose or galactose [108]. As glycosylation has
these wide-ranging effects, different approaches were undertaken to specically
address this functionality by either engineering the producer cells though eliminating fucosyltransferase or adding glycosyltransferases or optimizing the cultivation
conditions that support the enhancement of one or the other glycol-isoform. For
instance, the content of sialic acid could be increased through the addition of dexamethasone into the culture medium of CHO cells, expressing a CTLA4-Ig fusion
protein [109]. A similar effect was achieved by adding hydrocortisone to cell cultures of Fc-fusion protein which also improved the titer [110]. In one example, the
production level of a Fc-fusion protein was improved through a hyperosmotic culture medium, which unfortunately reduced the sialic acid content. This was due to
differential down-regulation of a series of genes, which could be restored through
the addition of betaine [111]. A more extensive recent study identied oxidative
stress resulting from gas transfer limitations in large bioreactors as source for low or
highly variable sialylation of a Fc-fusion protein. On a metabolic level, the hexosamine pathway became limited and thus the key building blocks for sialylation were
not sufciently available [112]. The obvious counteraction to thelack of educts is
their supplementation. Simply the addition of galactose enhanced the sialylation of
an Fc-fusion protein [113]. Beside the supply of precursors also extracellular factors
such as degradation via hydrolysis of α 2–3 sialic acids through sialidase activity
released from destroyed cells during cultivation can contribute to low levels of sialic
acid as seen during the cultivation of an Fc -fusion protein [114]. Modern tools
nowadays allow the monitoring of the complete N-glycosylation pathway on a
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S. R. Schmidt
molecular level. A recent study on CTLA4-Fc fusion protein revealed that, for
instance, at maximal cell density a bottleneck arises between endoplasmic reticulum (ER) and the cis-Golgi, leading to increased mannose 8 and 9 glycans, that
could be partly reduced by glucose deprivation. At later timepoints in the culture,
the transition between medial and trans-Golgi compartment becomes rate limiting,
resulting in decrease of sialylated species. This more detailed information will help
in better process understanding and control [115]. The situation gets more complicated if both fusion partners contain glycosylation sites and even vary between Nand O-linked species as in the case of EPO-Fc. With that molecule the inuence of
media composition on type and intensity of glycosylation was analyzed. Both fusion
protein partners maintained their original N-glycan characteristics. However,
depending on the media used for culture, either bi- and tri-antennary glycans were
generated, while the other medium delivered tetra-antennary glycans. With regard
to O-linked glycans, three species were preferred [116]. As O-glycans do not require
the presence of transferase recognition motifs, they can in principle occur at any
serine or threonine position. This unpredictability has recently been described on
the example of a CTLA4-Fc fusion protein that surprisingly contained a number of
unexpected new O-glycans. It could be attributed to the linker sequence derived
from the hinge region of IgG1. This specic post-translational modication resulted
in a complex formation of the hinge sequence and a glycan transferase. As the undesired O-glycans hindered proper inter-chain disulde bridge formation, O-glycanrich species tended to aggregation. Therefore, all potential O-glycosylation site
were eliminated by point mutations [33].
All examples above discuss experiments in CHO cells. But using a different host
such as Pichia Pastoris can have a dramatic effect on glycan pattern.
11.4.4 Aggregation
When investigating the causes of aggregation, it can be distinguished between
extrinsic and intrinsic factors. For instance, temperature changes during freezethawing can induce high molecular weight species. The same is true for physical
stress like stirring, ltration, or high ow rates. Finally, solution factors such as pH,
ionic strength, organic solvents or metal ions can lead to aggregation. Intrinsic factors are molecule derived and can be dependent on the presence of residues sensitive
to clipping, oxidation, deamidation, or isomerization. Furthermore, hydrophobic
patches or charges residues can trigger aggregation as well as the presence of
unpaired cysteines or scrambled disuldes [117].
As mentioned before, fusion proteins can be prone to aggregation due to domain
instability as in the case of HSA-hGH fusion. Modulating HSA stability with octanoic acid, it was revealed that colloidal instability but not conformational stability
of HSA contributes to aggregation of that molecule at low pH [118]. Orencia®, a
receptor trap consisting of several domains with different conformational stabilities
tends to aggregation due to partial unfolding of the CTLA-4 domain at pH6 [119].
11 Fusion Proteins: Current Status andFuture Perspectives
315
Aggregation can also be caused by disulde rearrangements resulting in intermolecular aggregation as demonstrated with a Fc-fusion protein. Here the presence of
N-linked glycosylation can reduce the tendency to form aggregates [120]. Proteins
containing a free thiol can generate also disulde crosslinking leading to massive
aggregation even under relatively inert conditions such as −30°C.Here during storage at elevated concentrations, salting out effects can occur that intensify aggregate
formation [121]. More detailed understanding of aggregation can be obtained by
hydrogen/deuterium exchange that identies surfaces with a tendency to aggregate [122].
Very often sensitivity to low pH has been observed for Fc fusion proteins. Adding
chaotropic substances such as urea during elution helps to prevent high molecular
weight species generation [123]. The so-called on-column disaggregation can
reduce aggregates by approximately 50% [124]. Interestingly aggregation can be
benecial, for instance, for the oral delivery. Here it prevents the degradation during
the stomach and intestine passage [125].
11.4.5 Analytics
Although many of the standardanalytical methods for antibodies can still be applied,
the level of complexity of fusion proteins often requires a more in-depth assessment. Particularly Fc-fusion proteins with a conserved N-linked glycosylation site
represent a difcult sample if the conjugated partner contains additional glycosylation sites. Therefore, site-specic glycan analysis is a key element of quality
control.
The typical assays to characterize any therapeutic protein comprised the assessment of identity, purity, and potency as displayed in Fig.11.6. Usually, platform
methods are established for the rst two assay, whereas potency determination often
requires highly specic bioassays. Identity determination nowadays relies primarily
on mass spectrometric methods. The content of variants is quantied with size
exclusion chromatography (SEC) or capillary electrophoresis (CE-SDS) with
respect to the content of high molecular weight species, monomers, or fragments or
capillary isoelectric focusing (cIEF) for charge variations that represent glycosylation isoforms. In case of high heterogeneity due to glycosylation, an enzymatic
pre-treatment for deglycosylation is done to establish the correct mass of the naked
polypeptide. Furthermore, large proteins can be digested by proteases into smaller
peptides to obtain either a peptide mass ngerprint or to identify modications that
prohibit a proper digest. Previously amino acid sequencing was applied to conrm
the correct N-terminus, but nowadays this method is replaced by liquidchromatography tandem mass spectrometry (LC-MS/MS). The advantage of
LC-MS/MS is the simultaneous detection of individual modications such as
methionine or tryptophan oxidation, asparagine deamidation, aspartic isomerization, and lysine glycation. In principle, fusion proteins can be seen as multispecic
molecules; therefore, a big part of the analytics developed for bispecic antibodies
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S. R. Schmidt
Fig. 11.6 Overview on typical analytical methods for fusion protein characterization
can be applied as well. For instance, it can be relevant to determine the thermal
stability by differential scanning calorimetry (DSC) and differential scanning uorimetry (DSF) that indicates correct pairing of polypeptide chains [126].
Particularly receptor traps require extensive analytics for S-S scrambling, incomplete folding, or aggregates. Another class of difcult molecules are enzyme fusions
that are sensitive to aberrant glycoisoforms and coagulation factors requiring carboxylation. For all these cases, specic detailed analytical methods must be established [127].
11.5 Therapeutic Concepts
11.5.1 Half-Life Extension
Therapeutic proteins have gained a wide acceptance in the last decades, but still
some of the most attractive biological substances in the class of hormones, cytokines, coagulation, and growth factors are small and suffer from a quick clearance
from the bloodstream. In the past, this small therapeutic window was compensated
for with frequent dosing, making treatment difcult for patients and causing
11 Fusion Proteins: Current Status andFuture Perspectives
Table 11.8 Half-life of approved fusion proteins
Brand [kDa] T½ range Average t½ [h]
Enbrel® 150 3–6 d 120
Ontak® 58 70–80min 1.2
Amevive® 92 11 d 264
Orencia® 92 8–25 d 400
Arcalyst® 251 9 d 216
Nplate® 60 1–34 d 408
Elonva® 47 59–82h 70
Nulojix® 90 8–10 d 220
Eylea®/Zaltrap® 115 5–6 d 130
Alprolix® 98 3.5 d 86
Tanzeum® 93 5–8 d 120
Eloctate® 220 10–23h 16
Trulicity® 63 5 d 120
Strensiq® 161 1.7–2.8 55
Idelvion® 125 4.3 d 104
Lumoxiti® 63 0.8–1.8h 1.4
Elzonris® 58 0.7–1h 1
Reblozyl® 76 13 d 312
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dose- limiting concentration peaks. Some of the currently approved fusion proteins
have signicantly increased half-life as described in Table11.8. The pharmacokinetic of drugs is inuenced by target-mediated drug disposition, antidrug antibodies, nonspecic catabolism, or kidney excretion [128].
Interestingly, receptor binding aids in recycling, keeping proteins in circulation
longer and protecting them from lysosomal degradation. This is actually a concept
utilized for some fusion protein types. Globular proteins smaller than 70kDa are
removed very rapidly by glomerular ltration in the kidneys. In addition to the 60Å
pore diameter of the glomerulus, another important parameter that regulates the
excretion of macromolecules is the negative charge on the cell surface that repels
anionic macromolecules. Multiple strategies to extend the plasma half-life have
now been feasible with fusion protein as summarized in Fig.11.7.
11.5.1.1 Half-Life Extension by Size andRecycling
Albumin Fusions
Human serum albumin (HSA), the most abundant serum protein with a size of
66kDa and a diameter of 90Å, exceeds the renal clearance threshold. However, its
exceptionally long half-life of 19days is more inuenced by recycling through the
neonatal Fc-receptor (FcRn). Its primary function during infancy is the capture protective maternal antibodies from milk by absorption through the intestinal
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Fig. 11.7 Fusion protein-based strategies for half-life extension (BSSL Bile salt stimulated-lipase,
CG chorionic gonadotropin)
S. R. Schmidt
epithelium. In adults, FcRn is downregulated on intestinal cells but remains present
on vascular cells. The mechanism of recycling relies on several conserved histidines
that can change their charge distribution at different pH.HSA, or IgG internalized
by pinocytosis bind strongly to the FcRn under acidic conditions in the endosome
thus being protected from degradation and remain bound to the FcRn until they
reach the cell surface again, where they are released at neutral pH.
Furthermore, the phenomenon of albumin accumulation in tumors, together with
the intrinsic ability to enhance the half-life of fusion or attachment proteins, could
be exploited for therapeutic approaches [129]. Another advantage is the ability of
HSA to stabilize proteins despite their own structural instability. For instance, the
fusion of HSA to granulocyte colony-stimulating factor (G-CSF) eliminated the
aggregation tendency of G-CSF [118]. As HSA can be attached either to the N-or
C- terminus, it gives design exibility even for fusion partners requiring an accessible terminus. Several HSA fusion proteins are now commercially available drugs
as can be seen in Table11.8. Designing adaptations of HSA fusions often involve
the modications of linkers. For instance, the blood clotting factor FIX not only
benetted from an increased plasma half-life but also from 10 to 30-fold higher
clotting activity when inserting a cleavable linker. This linker was split by proteases
simultaneously to FIX activation, thus triggering release from HSA when blood
clotting was needed [130].
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Fc Fusions
Probably the most successful class of fusion proteins are those containing an Fc
domain. The fragment crystallizable (Fc) region of an antibody consists of the second and third constant domains (CH2, CH3) of the heavy chain and forms a dimer
through two disulde bridges in the n-terminus of the hinge region. The rst ever
approved fusion protein, Enbrel (R), is a Fc-fusion protein. More specically it
belongs to the family of receptor traps that combine the extracellular domains of
receptors with the constant domain of antibodies.
The therapeutic success of Fc fusion proteins is primarily based on the wellunderstood concept of combining the high specic afnity of the extracellular
receptor domain and receptor-mediated antibody recycling through the Fc portion.
Although the addition of a 50kDaunits helps preventing rapid renal ltration, the
contribution of FcRn-mediated recycling has a much more profound effect.
Nevertheless, it was tried to correlate the contribution of FcRn-mediated recycling
to half-life with the afnity of antibodies and Fc fusion proteins for FcRn. As
expected, low afnity correlates to short half-life and vice versa. This was impressively demonstrated by optimizing Avastin® with a threefold longer half-life
through 11-fold higher afnity. The same effect was proven by modifying
Erbitux® [131].
Great efforts have been undertaken to identify critical residues in the Fc domain
that could be modied to increase FcRn binding and consequently extend the elimination half-life. Table11.9 summarizes the results [132].
But having a good FcRn afnity alone does not always help, as it could be shown
that some Fc-fusion proteins displayed shorter half-life than expected. The reduced
value could be attributed to sterical or conformational effects of the molecules fused
to the N-terminus [133]. For dimeric molecules, Fc fusion proteins are preferable
because the interaction between the antibody heavy chain and the disulde backbone of the hinge region automatically forces the formation of homodimers.
Sometimes the dimerization does not work due to exceptionally bulky fusion partners. Then a monomeric fusion might be advisable, where just one Fc-heavy chain
is combined with its fusion partner, while another Fc-only domain binds in an asymmetric way. Monomeric Fc-fusions might also be benecial in cases where
Table 11.9 Mutations in the Fc domain of IgG1 that inuence the plasma half-life
Company Half-life Mutation
Medimmune + M252Y, S254T, T256E
Protein Design Labs + T250Q, M428L
Genentech + N434A
Sally Ward + H433K, N434Y
Derry Roopenian + T307A, E380A, N434A
Xencor + M428L, N434S
Derry Roopenian − I253A
Eli Lilly − P257I, N434H or D376V, N434H