Loss of therapeutic proteins through nonspecific adsorption to surfaces during manufacture and storage can be problematic.
Nonspecific protein adsorption can lead to structural changes, denaturation, and inactivation of the protein because of irreversible
binding events and aggregation. Product loss as a result of these events can significantly decrease the concentration of the
active protein in solution, altering the efficacy of the drug. Nonspecific protein adsorption is a particular problem for
liquid product at low concentrations. Surfaces to which the protein product is exposed during the manufacturing and storage
process which can lead to adsorption include, but are not limited to, silicone tubing, glass and plastic containers, and drug
Protein adsorption is typically prevented or reduced with the use of a blocking agent through competitive binding of the agent
to the surface. Blocking agents commonly used in protein formulation include surfactants such as polysorbate 80 and Tween
20, which bind with higher affinity to surfaces than do proteins or protein-surfactant complexes. The suitability of these
surfactants as protein stabilizers raises certain other concerns because they are potential sources of peroxides, enhancing
the likelihood of oxidation of the protein product.
HSA has also been shown to be an effective blocking agent, allowing manufacturers to prevent thrombosis on plastic surfaces
and reduce pressure drop across device modules. The mechanism of action is not well understood, but albumin is thought to
bind to charged surfaces through opposite charged functional groups on the molecule. Hydrophobic interactions also occur but
at lower strength and are more easily reversed (10).
Transforming growth factor-β3 (TGF-β3), an active pharmaceutical ingredient (API) in scarless wound healing, is a hydrophobic
protein with a propensity to adsorb to container surfaces. To examine the ability of rAlbumin to act as a suitable blocking
agent, the percentage loss of TGF-β3 due to nonspecific binding to polypropylene or glass vial surfaces in the absence and
presence of rAlbumin was examined.
In this model, TGF-β3 (0.5–60 μg/ml) was added to a polypropylene or glass container containing a buffer solution. Each sample
was mixed and centrifuged then transferred to HPLC vials for analysis via reverse-phase HPLC. Percentage recoveries of TGF-β3
were calculated against the TGF-β3 reference standard. rAlbumin was then assessed for its ability to prevent the loss of TGF-β3
to the container surface. rAlbumin (0–0.5 mg/ml), then TGF-β3 (0.2 μg/ml) was added to the test container containing a buffer
solution. rAlbumin (0.1 mg/ml) was also compared with polysorbate 80 (0.1 mg/ml) for the ability to protect TGF-β3 (0.2–1.0
μg/ml) against nonspecific adsorption to glass surfaces using the method described above.
The results demonstrated rAlbumin significantly reduced protein loss that occurred due to adsorption of TGF-β3 to glass and
plastic surfaces. In the absence of rAlbumin, nonspecific binding of TGF-β3 increased as the concentration of TGF-β3 decreased
in solution below 60 μg/ml; the recovery of the protein was significantly reduced at lower concentrations (see Figure 5).
However, in the presence of rAlbumin the adsorption of TGF-β3 to vessel surfaces was minimal, with greater than 95% recovery
achieved using 0.05 mg/ml of rAlbumin (see Figure 6). rAlbumin was then compared to polysorbate 80 in preventing nonspecific
binding of proteins to plastic and glass surfaces. In this comparison, rAlbumin prevented the nonspecific binding of TGF-β3
at least as well as polysorbate 80 in plastic containers and significantly better in glass vials.