Biomedical Engineering Reference
In-Depth Information
Since more parameters (concentration, pH, ionic strength,
molecule type, and weight) contribute to the separation, this
technique has a high resolution power. ATPS is easily
scalable, has a high capacity, and can be used in a continuous
operation. ATPS could prove quite useful also for the
separation of fusion proteins if predictability and economi-
cal sustainability is verified [121].
administration as well. The protein itself can be available
either lyophilized (as dry substance) or as liquid formula-
tion, ready for injection. A self-administered therapeutic
such as insulin is usually available as prefilled syringe.
Noninvasive routes such as oral, pulmonary, nasal, or trans-
dermal delivery would be preferable but suffer from low
bioavailability [130]. Recently, a fusion protein approach
using transferrin as partner for granulocyte monocyte-
colony-stimulating factor (GM-CSF) was successfully
applied orally in mice [43]. Pulmonary delivery could be
achieved by using monomeric Fc fusions. Here, aerosolized
fusion proteins are carried across lung epithelial cells
utilizing the neonatal Fc receptor [131]. Part IIc of this
topic is dedicated to different
1.5.3 Formulation
Formulation covers the steps that are necessary to create a
drug product out of the drug substance. This means the
active pharmaceutical ingredient (API), the therapeutic
(fusion) protein, is combined with additives or excipients.
The proper selection of excipients is a challenging task
because only a limited range of molecules is available [122].
The goal of formulation is to prevent protein degradation
during storage and to optimize its delivery. To achieve that, a
number of buffer parameters such as pH, ionic strength, and
composition must be carefully assessed [123]. This can be
very demanding for a fusion protein that could be composed
of two proteins with different buffer preferences or different
sensitivity to degradation. Proteins can be degraded through
physical (denaturation, adsorption, aggregation, precipita-
tion) and chemical (hydrolysis, deamidation, oxidation,
isomerization, and disulfide exchange) instability. The pro-
tein can also be exposed to harmful conditions during
handling such as temperature, physical interfaces, and shear
forces [124].
Particularly, non-native aggregation of liquid dosage
forms can diminish the concentration of the active drug.
To limit the amount of aggregates formed, it is necessary to
understand the aggregation routes and to predict its forma-
tion rate [125].
From a safety point of view, the drug product has to be
sterile, free of aggregates that could cause immunogenicity
or reduce the concentration of the active substance. The final
product must be free of product or process related impurities
within a specified tolerance [126].
It was shown that a change of formulation of a well-
tolerated product can cause serious safety issues. For exam-
ple, a change of formulation and ingredients of epoetin- a
caused severe pure red cell aplasia [127]. The final step of
formulation, fill to finish, has to be done into vessels that
maintain sterility, but also do not contain inacceptable
extractables and leachables or cause immunogenicity
[128]. Both, leachables and extractables can be set free
during the filling procedures or later during storage; there-
fore these conditions must be defined carefully [129]. Sta-
bility can be tested in an accelerated approach, where long-
term effects are simulated by elevated stress in form of
intense temperature, light exposure, or pH.
Beyond physicochemical parameters also patient com-
pliance must be taken into account. This includes the
targeting and delivery
approaches with fusion proteins.
Due to the limited stability in the digestive tract thera-
peutic proteins are usually administered by injection. This
can be done through three different ways: intravenous (IV),
intramuscular (IM), or subcutaneously (SC). The adminis-
tration route has an impact on the concentration of the
protein. IV injections usually are in the concentration range
around 1mg/mL, IM injections are acceptable up to
100mg/mL, but only SC injections can have concentrations
up to 150mg/mL. It is very challenging to deliver a soluble,
aggregate-free formulation at these very high, viscous con-
centrations [132]. The concentration and dosage are obvi-
ously dependent on the potency and the clearance of the
drug. For example, highly active hormone or cytokine drugs
are administered at relatively low doses despite their rapid
clearance. Antibodies as large molecules on the other side
can be required at very high concentrations despite their long
half-life. This issue of manufacturability has to be consid-
ered from the start [133].
A lot of work has been done to diminish the aggregation.
Working with fusion proteins that consist of several
unrelated domains that did not evolve together is very
challenging. One technique is the selective domain stabili-
zation, where buffer conditions are evaluated that stabilize
the least stable domain, because from there aggregation is
initiated. Testing an hGH-HSA fusion, best results were
obtained when repulsion of protein-protein interaction was
increased [134].
1.5.4 Process Economies
As in all other manufacturing processes, cost of goods
(COG), processing time, and capital investment are major
determinants of fusion protein production. Another impor-
tant parameter is the usually high dose of therapeutic
proteins leading to high demands on capacity. In the last
years, a trend to improved processes delivering more output
from the same capacity and second generation products with
decreased doses and longer half-lives will change bio-
pharmaceutical manufacturing [135].
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