October 1, 2010 (Vol. 30, No. 17)
Recombinant Albumin Characterization as a Robust Formulation Pharmaceutical Excipient
Protein therapeutics can be susceptible to a range of stresses during manufacturing and storage that can trigger protein instability and degradation. Protein degradation can take the form of aggregation, denaturation, or absorption and can arise as a result of changes in temperature, shear stresses, and lyophilization.
Oxidation of the protein during processing and storage is also common. With the potential to increase immunogenicity and decrease efficiency and shelf life of the protein drug product, protein stability is a key issue.
Protein therapeutics are formulated to protect against degradation and provide a suitable shelf life for transportation and storage. Human serum albumin (HSA) has been commonly used as an excipient in therapeutic protein formulations. As the most abundant protein in human plasma, the potential for HSA to elicit an immunogenic response is minimal.
Formulation scientists, however, have moved away from its use due to concerns that blood-borne contaminants such as prions or viruses that may contaminate the final drug product.
rAlbumin’s Stabilization Effect
Aggregation can cause numerous issues during the therapeutic protein manufacturing process. This association of mis-folded proteins can result in significant product losses, potentially increasing the immunogenicity of the drug product. Aggregation can occur as a consequence of process operations like refolding, purification, mixing, freeze-thawing, freeze drying, and reconstitution as well as during transportation and storage.
In a study conducted at Novozymes Biopharma, recombinant albumin was evaluated for its ability to suppress amyloid-fibril formation by the merozoite surface protein, MSP-2 protein, after a single freeze–thaw cycle.
A range of rAlbumin concentrations was evaluated for their ability to suppress aggregation, where fibril formation was measured by the effect on light scattering at 320 nm.
rAlbumin at various concentrations was dissolved in a solution of PBS buffer; the MSP-2 protein (3.5 mg/mL) was then added to all samples followed by a single freeze-thaw cycle. Samples were then aliquoted into 96-well plates and stored at 2–8ºC. Absorbance readings were taken at multiple time intervals over a five-day period.
Excipients, which are widely applied across the industry to enhance protein stability, were also assessed against rAlbumin for their ability to reduce protein aggregation. rAlbumin (15 mg/mL), glycine (20 mg/mL), PEG 400 (1 mg/mL), polysorbate 80 (0.82 mg/mL), or polysorbate 80 (8.2 mg/mL) were tested in the same model as described above.
Aggregation was suppressed by 50% at a 1:1 molar ratio of the antigen to rAlbumin and reduced by 80% in the presence of the highest concentration of rAlbumin. rAlbumin also suppressed aggregation of the MSP-2 antigen to a greater extent compared to other commercially available excipients (Figure 1).
Oxidation of the protein therapeutic can also present challenges for the biomanufacturer where it can lead to a range of functional issues such as altered binding activities, increased susceptibility to aggregation and proteolysis, increased or decreased uptake by cells, and altered immunogenicity. The rate of oxidation can be affected by a number of factors (particularly during storage) including oxygen (head space), light, the physical state of the product, and temperature.
Insulin-like growth factor-I (IGF-I), an important anabolic growth factor, has been shown to be susceptible to oxidation. To test the functionality of rAlbumin as an antioxidant, pharmaceutically relevant conditions in protein oxidation were modeled using trace amounts of hydrogen peroxide (H2O2). rAlbumin or L-methionine were dissolved in a solution of PBS buffer. The IGF-I protein was then added to all samples followed by H2O2 to a final concentration of 0.0005%.
The reaction was terminated and degree of oxidation analyzed. Oxidation of IGF-I was shown to be significantly reduced by the presence of increasing concentrations of rAlbumin and the highest concentration reduced oxidation of IGF-I by 93%.
The study also assessed the ability of rAlbumin to act as an antioxidant following exposure to hydrogen peroxide when compared to the commonly used antioxidant L-methionine (Figure 2). The oxidative protection of IGF-I by rAlbumin was achieved at molar concentrations ~13-fold less than that of L-methionine. The antioxidant function of HSA is primarily due to the single free-thiol at position Cys 34, where HSA-SH acts as a potential scavenger for reactive oxygen and nitrogen species.
The loss of drug product due to non-specific adsorption to surfaces can decrease the concentration in solution, significantly altering the efficacy of the drug. Structural change, denaturation, and inactivation are also results of nonspecific adsorption and present a particular problem for products administered at low concentrations. Many surfaces that a drug product may come in contact with during manufacturing processes such as delivery pumps, silicone tubing, and glass and plastic containers, can lead to product losses.
HSA has been utilized as a blocking agent to prevent therapeutic proteins binding to various surfaces. Though the mechanism is not well understood, 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 reversible. Transforming growth factor-β3 (TGF-β3), an active pharmaceutical ingredient, is a hydrophobic protein with a propensity to adsorb to container surfaces. The percentage loss of TGF-β3 due to nonspecific binding to polypropylene or glass vial surfaces in the absence and presence of rAlbumin was also examined.
TGF-β3 was added to a polypropylene or glass container containing citrate buffer (pH 3.6). Samples were analyzed and percentage recovery of TGF-β3 was 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.
The study demonstrated that rAlbumin significantly reduced protein loss due to nonspecific binding of TGF-β3 to glass and plastic. In the absence of rAlbumin, nonspecific binding increased progressively at concentrations less than 60 µg/mL, and the recovery of the protein was significantly reduced at lower concentrations.
However, in the presence of rAlbumin the nonspecific binding of TGF-b3 to vessel surfaces was minimal with >95% recovery achieved using just 0.05 mg/mL of rAlbumin.
It is vital that therapeutic proteins be formulated to provide optimal stability during manufacturing processes, transportation, and storage. Protein degradation occurs through both physical and chemical pathways and a range of excipients are used throughout the industry to reduce its occurrence.
This study showed rAlbumin to be an effective multipurpose excipient with the ability to protect proteins against aggregation, act as an antioxidant in preventing protein oxidation and as a blocking agent to prevent nonspecific adsorption to surfaces. The use of a single or reduced number of excipients not only simplifies the formulation strategy for biopharmaceutical manufacturers but also accelerates development time.
Phuong Tran (PHUT@novozymes.com) is senior scientist in the development and discovery group, Geoffrey Francis is chief scientist, Larissa Chirkova is discovery and development manager, and Sally Grosvenor is scientific communications manager, all at Novozymes Biopharma.