April 15, 2012 (Vol. 32, No. 8)
Wayne F. Patton, Ph.D. Enzo Life Sciences
Quantifying Trace Subvisible Protein Aggregates Using a Protein Aggregation Assay and Standards
Aggregation represents one of the most significant obstacles to the development of protein-based pharmaceuticals. During drug formulation, protein aggregation can impact product quality in terms of biological activity and immunogenicity.
Protein aggregation can occur at all stages in the manufacturing process including cell culture, purification, formulation, storage, shipping, and handling. The pharmaceutical industry requires improved methods to detect, monitor, and quantify factors governing aggregation during manufacturing.
Lyophilized and excipient-stabilized protein aggregation reference standards have recently been introduced that are ideal for reliable and accurate quantification of protein aggregation in solution and are optimized for use with the ProteoStat® protein aggregation assay, a novel test that can be performed on a fluorescence microplate reader.
In this tutorial, the assay and standards are employed to determine levels of aggregated IgG by comparing the assay response of test samples to that of the standard curve—composed of reference material with known concentrations of aggregated IgG, focusing particularly on three common modes of aggregation encountered in the pharmaceutical industry:
- Thermally induced aggregation
- Mechanically induced aggregation
- Bulk freezing and freeze/thaw cycle-induced aggregation
Materials and Instrumentation
ProteoStat protein aggregation assay and ProteoStat protein aggregation standards were obtained from Enzo Life Sciences. The microplate-based assay employs a fluorescent molecular rotor dye specifically devised to detect aggregated proteins and peptides.
The probe is nonfluorescent in solution, but becomes brightly fluorescent when bound to the quaternary structure of aggregated proteins. Once the aggregation standards are reconstituted, they contain 0 to 12.5% aggregated protein, while the total protein concentration is maintained at 1 mg/mL.
Characterization of the reference material by imaging particle analysis demonstrated that 90% of the aggregates were 2–10 µm in length, while an additional 9% were in the 10–20 µm and 0.7% in the greater than 20 µm size ranges.
The Synergy™ Mx Multi-Mode Microplate Reader (BioTek Instruments) was used for all aggregation assays. The excitation monochromator was set to 500 nm and emission to 600 nm, with slit widths of 9 nm, for detection of the ProteoStat dye. Thioflavin T was detected with excitation set at 435 nm and emission set at 495 nm, both with a 9 nm slit-width.
ProteoStat and Thioflavin T dyes were compared to one another with respect to their ability to quantify protein aggregation in a 96-well microplate format. Purified rabbit anti-goat IgG (4.26 mg/mL) was incubated in aqueous HCl solution, pH 2.7 at 80° for 90 minutes to form protein aggregates.
The signal from the aggregate was determined after mixing aggregate with monomer at different ratios such that the total IgG concentration remained 60 µg/mL protein. The readings were taken in 50 mM potassium phosphate, pH 7, containing either 3 µM ProteoStat Detection Reagent or 5 µM Thioflavin T dye. These concentrations were previously determined to be optimal for each dye.
Protein was incubated with dye for 15 minutes prior to determining the fluorescence using a BioTek Synergy Mx Microplate Reader. Readings were taken at least in triplicate with samples deposited in Greiner µClear® black, clear bottom 96-well microplates (Greiner Bio One).
The signal from ProteoStat dye was more than 100 times stronger than the signal from Thioflavin T dye under these experimental conditions. For 1.2 µg/mL aggregate concentration, signal-to-noise ratio was calculated to be 12.8 for the ProteoStat dye, while only 1.2 for Thioflavin T, demonstrating that the former dye allows for more accurate and sensitive detection of protein aggregates.
Due to the highlighted performance advantages, ProteoStat protein aggregation assay, in conjunction with the ProteoStat protein aggregation standards, was employed to quantify the various common modes of protein aggregation.
Monitoring Thermally Induced Aggregation
The aggregation process can be accelerated by subjecting proteins to elevated temperature. Sheep IgG (4 mg/mL) in 100 mM sodium phosphate, pH 7.2, was maintained at 60oC with constant stirring of 400 rpm for various periods of time.
At each time point, 25 µL of IgG solution was retained for measurement. Sample was first diluted fourfold in 1x assay buffer to generate a final protein concentration of 1 mg/mL. 98 µL of test protein as well as 2 μL of the prepared ProteoStat Detection Reagent Loading Solution was added into each well. Each time point was performed in duplicate.
At the same time, the ProteoStat Protein Aggregation standards, which contain stabilized, high-quality reference samples, were used to generate a standard curve.
Figure 1 highlights thermally induced aggregation of sheep IgG, as determined using the ProteoStat dye and the Synergy Mx Multi-Mode Microplate reader. The generated curve suggests different stages associated with the formation of IgG protein aggregates.
First, a lag phase is observed during which minimal aggregation occurs. Then, a nucleation phase is observed, and aggregates start to form (growth phase). Eventually, the growth rate decreases and becomes zero, indicating exhaustion of the monomeric protein supply.
Monitoring Mechanically Induced Aggregation
Although mechanical stirring is recognized to enhance aggregate formation, it is not fully understood how the shear flow forces influence the kinetics of aggregate formation. Analogous mechanical stresses that a protein pharmaceutical might encounter can be generated by pumping and filtration.
Goat-Anti-Mouse IgG (10 mg/mL) was used as supplied, having a formulation of 10 mM Sodium phosphate, 150 mM NaCl, 0.05% sodium azide, pH 7.2. The mechanical agitation experiment was performed at room temperature by stirring 200 µL of IgG solution in a 4 mL amber glass vial with flat bottom at 990 rpm using a Variomag® Poly electronic stirrer (2Mag-USA). The stirring bar had dimensions of 1.0 x 0.4 x 0.2 cm.
At each time point, 10 µL of IgG solution was retained for measurement. Sample was first diluted 10 fold in 1x assay buffer to generate final protein concentration of 1 mg/mL. 98 µL of test protein as well as 2 μL of the prepared ProteoStat Detection Reagent Loading Solution was added into each well. Every time point was performed in duplicate.
Using ProteoStat Protein Aggregation Standards as reference material, mechanical agitation-induced aggregation was measured and quantified. With mechanically induced aggregation, using the described experimental conditions, protein aggregation appears to increase linearly with time (Figure 2).
Monitoring Bulk Freezing and Freeze/Thaw Cycle-Induced Aggregation
Bulk freezing presents a challenge to stable protein preparations because of the well-known solute concentration effect that occurs during the process. Goat-Anti-Mouse IgG (10 mg/mL) was used as supplied, with a formulation of 10 mM Sodium phosphate, 150 mM NaCl, 0.05% sodium azide, pH 7.2. The sample was stored at -20°C and then thawed at room temperature repeatedly.
At each cycle, 10 µL of IgG solution was retained for measurement. Sample was first diluted 10-fold in 1x assay buffer to generate a final protein concentration of 1 mg/mL. 98 µL of test protein as well as 2 μL of the prepared ProteoStat Detection Reagent Loading Solution was added into each well. Every time point was performed in duplicate.
Using ProteoStat Protein Aggregation Standards, freeze/thaw cycle-induced aggregation was measured and quantified. The aggregation data suggests that repeated freeze-thaw cycles also lead to aggregation. As with thermally induced aggregation, a sigmoidal response profile is observed, suggesting the possibility of nucleation, followed by elongation of the protein aggregates (Figure 3).
The ProteoStat protein aggregation assay, in combination with the Synergy Mx Multi-Mode Microplate Reader, was demonstrated to be suitable for monitoring protein aggregation generated by thermal-, mechanical agitation-, and freeze-thaw-induced stresses.
The stabilized subvisible protein particle reference standards enable rapid generation of a standard curve by rehydration, facilitating quantification of the aggregation response. The assay reliably detects less than 0.2% aggregated protein in a concentrated antibody formulation, with a linear dynamic range of two orders of magnitude.
The advantages of monitoring aggregation using the fluorescence microplate-based assay in combination with the aggregation reference standards include simple implementation, ability to quantify minute levels of aggregation, ~30 minute analysis time, and robust assay performance.