Protein Stability and Aggregation

Since the AQS3™pro can directly measure protein secondary structure, it is a powerful tool for monitoring and understanding the mechanisms of protein stability and aggregation. As proteins are subjected to stress and the protein begins to change from its native state, details of the process can be easily followed.

Alpha Helix (⍺-helix) and Beta Sheet (β-Sheet)

Microfluidic Modulation Spectroscopy measurements are particularly sensitive to beta sheet structures, which dominate in protein antibody  based drugs. In addition, it is one of the only techniques which can directly monitor the formation of aggregates due to its ability to measure intermolecular beta sheet structures. Below we illustrate the use of the AQS3pro for two common types of stability studies, thermal and chemical.

Thermal Stability

A high beta sheet content protein at 1 mg/mL was incubated at an elevated temperature for differing periods of time. The protein series was measured using the AQS3pro and the second derivative spectra were overlaid and plotted to enhance the spectral changes. The data shown in Figure 1 clearly show the loss of intramolecular beta sheet content as a function of incubation time. Simultaneously, the amount of intermolecular beta sheet structure increases, which is associated with the formation of protein aggregates. Changes in other regions reflect the state of the protein  secondary structure and provide additional details of the denaturation process.

Figure 1. Incubation of a 1 mg/mL high beta sheet containing protein incubated at elevated temperature from 0 to 24 hours.  As the incubation time increases the (intramolecular) beta sheet content decreases and the intermolecular beta sheet increases, indicative of aggregate formation.
Figure 1. Incubation of a 1 mg/mL high beta sheet containing protein incubated at elevated temperature from 0 to 24 hours.  As the incubation time increases the (intramolecular) beta sheet content decreases and the intermolecular beta sheet increases, indicative of aggregate formation.

Protein Aggregation

A high beta sheet content protein at 1 mg/mL was incubated at an elevated temperature for differing periods of time. The protein series was measured using the AQS3pro and the second derivative spectra were overlaid and plotted to enhance the spectral changes. The data shown in Figure 1 clearly show the loss of intramolecular beta sheet content as a function of incubation time. Simultaneously, the amount of intermolecular beta sheet structure increases, which is associated with the formation of protein aggregates. Changes in other regions reflect the state of the protein  secondary structure and provide additional details of the denaturation process.

Figure 2.  As the protein was denatured insoluble aggregate formed and precipitated out of solution.  As only the supernatant of the sample was measured the intensity of the amide I band decreases, which is a direct indicator of soluble protein concentration.
Figure 2.  As the protein was denatured insoluble aggregate formed and precipitated out of solution.  As only the supernatant of the sample was measured the intensity of the amide I band decreases, which is a direct indicator of soluble protein concentration.

Chemical Stability: Comparing Microfluidic Modulation Spectroscopy with Circular Dichroism

Chemical stress studies are another method of studying protein stability. Alcohols are well known to denature the native state of proteins and also tend to stabilize the alpha-helical conformation in unfolded proteins and peptides . In this study a relatively high concentration of beta lactoglobulin was formulated in phosphate buffer at pH 7.4 at 0, 20, 40 and 60% isopropyl alcohol (IPA) concentration. The IPA/protein series were then measured by both Far UV-CD (circular dichroism) and MMS (microfluidic modulation spectroscopy)  to track the structural changes. In the UV-CD data, shown in Figure 3, a clear increase in the alpha helix structure occurs while a general decrease in beta sheet occurs.

Figure 3. Far UV-CD studies of the chemical denaturation of beta lactoglobulin in IPA show increasing alpha helix and decreasing beta sheet.
Figure 3. Far UV-CD studies of the chemical denaturation of beta lactoglobulin in IPA show increasing alpha helix and decreasing beta sheet.

In contrast, the RedShiftBio data of Figure 4 shows not only an increase in the alpha helix form at higher IPA concentration, it also shows a dramatic and clear shift in the beta sheet type as noted by the shift of the band from ~1630 cm-1 to 1620 cm-1, again indicating the formation of intermolecular beta sheet, something the Circular Dichroism data does not readily show. Not only does the AQS3pro provide greater insight into the denaturation process but it operates over a much wider range of concentrations.

Figure 4.  Protein characterization results obtained using RedShiftBio’s MMS analyzer not only show the expected increase in alpha helix with higher alcohol concentrations, but also shows a shift in beta sheet to the aggregate form of intermolecular beta sheet.
Figure 4.  Protein characterization results obtained using RedShiftBio’s MMS analyzer not only show the expected increase in alpha helix with higher alcohol concentrations, but also shows a shift in beta sheet to the aggregate form of intermolecular beta sheet.

1 Hirota N, Mizuno K, Goto Y, Cooperative alpha helix formation of beta lactoglobulin and melittin induce by hexafluoroisoprpanol.  Protein Science (1997) 6:416-421.

Related Resources:

Analyzing Protein Stability (immunoglobulins) in Formulation Development

  • Poster: Pressure-perturbation of protein secondary structure coupled with Microfluidic Modulation Spectroscopy – a powerful platform for biopharmaceutical formulations development.
  • Webinar: Pressure BioSciences and RedShiftBio Discuss Combining Microfluidic Modulation Spectroscopy and High hydrostatic Pressure to Study Protein Stability in Biopharmaceutical Formulations.

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