AGGREGATION
Aggregation of protein products is a major concern during the manufacturing and delivery of protein therapeutics. This concern
is primarily because of the increased potential for aggregates to lead to an immunogenic reaction, and possible problems with
drug administration (6). There are various mechanisms by which aggregation of proteins occur, including a formation of polymeric-like
structures, misfolded proteins, and covalently linked proteins either in a native or denatured state.
Significant product losses during manufacture and storage from aggregation are also a concern, influencing product recovery
and effective dosage forms. Protein aggregation can occur during numerous stages of the manufacturing and storage process,
such as refolding, purification, mixing, freeze-thawing, freeze-drying, and reconstitution. Formation of these associated
species is generally concentration dependent, which is a particular challenge for protein therapeutics formulated at high
concentrations.
Exposure of protein therapeutics to bulk freeze-thaw processes is a stress that protein drug substances can be exposed to
during the manufacturing process to enhance operational flexibility while maintaining product stability. In this study, a
range of rAlbumin concentrations was evaluated for the ability to suppress amyloid-fibril formation of the malarial vaccine
antigen, merozoite surface protein 2 (MSP-2), after a single freeze-thaw cycle. MSP-2 was chosen as the model to investigate
aggregation because of its tendency to form amyloid-like fibril aggregates (7, 8).
Briefly, rAlbumin at various concentrations was dissolved in a buffer solution; the MSP-2 protein (3.5 mg/ml) was then added
to all samples followed by a single freeze-thaw cycle. Samples were then plated in a 96-well plate and stored at 2–8 °C. Amyloid-like
fibrils are known to affect light scattering when measured at λ 320 nm. Therefore, absorbance readings were taken at λ 320
nm at multiple time intervals over a five-day period to test for the formation of aggregation products.
Figure 3
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Excipients commonly used to improve protein stability were also compared against rAlbumin for their ability to inhibit protein
aggregation. rAlbumin (15.0 mg/ml), glycine (20.0 mg/ml), PEG 400 (1.0 mg/ml), polysorbate 80 (0.82 mg/ml), or polysorbate
80 (8.2 mg/ml) were tested in the same model as described above. Absorbance readings were taken at multiple time intervals
at λ 320 nm.
Figure 4
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Results showed aggregation was suppressed by 50% at a 1:1 molar ratio of the MSP-2 antigen to rAlbumin and at the highest
concentration of rAlbumin, aggregation of the MSP-2 protein was reduced by 80% (see Figure 3). These results also demonstrated
that rAlbumin suppressed aggregation of the MSP-2 antigen to a greater extent when compared with other commercially available
excipients (see Figure 4).
The mechanism by which albumin inhibits aggregation is not well understood. HSA is known to sequester and transport metal
ions such as Cu and therefore has the potential to reduce the complexes between Cu and amyloid peptides that are involved
in the formation of amyloid-like fibrils (9).
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