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ProteoStat® Protein Aggregation Assay Flyer

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Aggregation is one of the most significant obstacles to the development of protein-based pharmaceuticals. During drug formulation, protein aggregation can lead to low yield, poor storage capacity and increased production costs. The ProteoStat® Protein Aggregation Assay is suitable for investigating the different environments to which a protein product might be exposed to:

Manufacture
Storage
Shipping
Freeze & thaw cycles
Oxygen exposure
Light & physical stress

The ProteoStat® assay can provide a better understanding of the overall impact that aggregates will have on drug product safety and quality.

Sources of Protein Aggregation

Protein Design	Aminoacid sequence Hydrophobicity, hydrogen bonding
Cell Culture	Expression system, disulfide scrambling Shear stress, pH, temperature
Purification	Membrane absorption, leachates, unfolding Exposure to various surfaces Exposure to buffers and reagents
Bulk DS	Freeze/Thaw bulk effects
Formulation	Excipient-protein interaction effects
Filtration UF/DF	Shear during mixing, filtration, filling Exposure and absorption to various surfaces Protein unfolding due to membrane interactions
DP Filling	Foaming during filling DP container interaction, light exposure Foreign particle, leachate, extractable
Lyophilization	Freeze/drying-independent stress Residual moisture level
Inspection	Exposure to light and shear Micro-bubbles formation
Label/Package	Temperature excursions Light exposure Long-term DP stability Interactions with primary container Silicone oil, glass interactions
Storage	Mechanical stress
Shipping	Pressure changes Temperature Stress
Delivery Device	Protein-device surface interaction Protein-device component interaction Shear during drug delivery

[1] Aggregates in Therapeutic Protein Products, U.S. Food and Drug Administration, Ruth Cordoba-Rodriguez, PhD

Identifying inhibitors of protein aggregation

Figure 1:Monitoring the ability of an excipient to inhibit protein aggregation. Lysozyme was incubated in the presence or absence of N,N’,N’’-triacetyl-chitotriose in 10 mM potassium phosphate, pH 7.3 for 16 hours. Aggregation was induced by 3.5 fold dilution into 50 mM potassium phosphate buffer, pH 12.2. Aggregation was monitored for several weeks at room temperature. The assay shows that chitotriose inhibits lysozyme aggregation.

Monitor Bulk Freezing and Freeze/Thaw Cycles

Figure 2: Bulk freezing presents a challenge to stable protein preparations because of the solute concentration effect that occurs during the process. Repeated freeze-thaw cycles can lead to further aggregation.aggregation.

Defining post-viral inactivation storage conditions

Importance of assessing the stability and aggregation tendency of antibodies at low pH and during titration from low to neutral pH values.Effects of Acid Exposure on the Stability and Aggregation Tendency of IgGs:

Exposure of antibodies to low pH is often unavoidable.
Antibodies are commonly purified by binding to a protein-A column and then eluting at low pH.
Based upon the premise that the eluted antibody is unstable at this pH, the antibody is often neutralized immediately.
It is unclear whether the antibodies would undergo slow conformational changes if stored in acidic solution following elution.
The most effective means of viral inactivation is acid treatment. The lower the pH the antibody preparation is exposed to, the more effectively the viruses are inactivated.
Thus, it is important to assess the stability and aggregation tendency of antibodies at low pH and during titration from low to neutral pH values.

Monitor pH Effects
Effects of Acid Exposure on the Stability and Aggregation Tendency of IgGs