In-Vial Detection of Protein Denaturation Using Intrinsic Fluorescence Anisotropy

Abstract

The conventional quality control techniques for identifying the denaturation of biopharmaceuticals includes sodium dodecyl sulfate-polyacrylamide gel electrophoresis for identifying fragmentation, ion exchange chromatography and isoelectric focusing for identifying deamidation, reverse-phase high-performance liquid chromatography (HPLC) for identifying oxidation, and size-exclusion HPLC for identifying aggregation. These stability assessments require essential processes that are destructive to the product tested. All these techniques are lab based and require sample removal from a sealed storage vial, which can breach the sterility. In this work, we investigate the heat- and surfactant-induced denaturation of an in-vial-stored model protein, bovine serum albumin (BSA), by analyzing its intrinsic fluorescence without removing the sample from the vial. A lab-based bespoke setup which can do the measurement in vial is used to demonstrate the change in fluorescence polarization of the protein to determine the denaturation level. The results obtained are compared to circular dichroism and size-exclusion HPLC measurements. The results prove that in-vial fluorescence measurements can be performed to monitor protein denaturation. A cost-effective portable solution to provide a top-level overview of biopharmaceutical product stability from manufacture to the point of patient administration can be further developed using the same technique.

Figure 1

Principle of intrinsic fluorescence polarization: a significant difference in the total intensity is detected between the orthogonal polarization directions if the luminescence emitted from the molecule retains the polarization.

Figure 2

Schematic comparison of in-vial UV excited intrinsic fluorescence polarization to traditional lab-based methods. For conventional methods, sample removal is a must, and an ideal method should be developed and identified; in addition, lab downtime should be considered to identify if the vial is denatured. The exact process is to be repeated for multiple vials. If in-vial direct measurements are employed, this downtime can be reduced to a minimum.

Figure 3

Simple schematic of the bespoke setup used for measurement: light from the 365 nm UV LED is focused onto the sample via a collimator-refocusing unit. The light passing through the polarizer produces fluorescence isotropically, and the fluorescence is detected via the bottom of the vial through an analyzer. The light collected is fed to the spectrometer via a fiber.

Figure 4

Transmission spectra of the closed quartz vial, polarizer, analyzer, and the emission spectrum of the source LED at 365. The LED spectra in orthogonal polarization directions shown are obtained using the setup by measuring the empty vial and recording the corresponding reflection and Rayleigh scattering. The absorption spectra and emission spectra upon excitation at 365 nm for BSA are also indicated in dotted lines. All spectra are normalized or scaled to enable a comparison between them. Further details are provided in the Supporting Information, Section S1(a), regarding the choice of excitation wavelength.

Figure 5

Raw data. (a) Reference spectrum measured for the sample set 1. (b) Fluorescence spectra for orthogonal polarization directions for the thermally stressed sample set 1. The dotted lines are horizontal polarization directions, and the solid lines are vertical polarization directions. The inset shows the same intensities zoomed. All sample sets are at 100 mg/mL concentration. The samples are kept at 75 °C for periods of 1 to 4 h.

Figure 6

(a) SEC peak areas for 100 mg/mL BSA under different temperature-induced stress levels. Each sample is kept at 75 °C for periods of 1, 2, and 4 h. The decrease in the monomer peak area is accompanied by the appearance and increase of an aggregation peak that elutes slightly earlier than the main peak conglomerate. (b) The area under the curve for Rayleigh scattering was observed for the same samples. All the measurements are done in triplicate (n = 3).

Figure 7

(a) Molar ellipticity values for 100 mg/mL BSA samples thermally stressed at 75 °C for 1, 2, and 4 h. (b) Comparison between CD secondary structure analysis and fluorescence analysis. Although there is a sharp spike at 1 h, the BSA structure refolds for the remaining observation period. Fluorescence measurements show a similar trend to beta-sheet content as measured using CD (n = 3 for all).

Figure 8

Raw data. (a) Reference spectrum measured for the sample set 1. (b) Fluorescence spectra for orthogonal polarization directions for the chemically stressed sample set 1. The dotted lines are horizontal polarization directions, and the solid lines are vertical polarization directions. The inset shows the same intensities zoomed. All sample sets are at 100 mg/mL concentration.

Figure 9

(a) Molar ellipticity values for 100 mg/mL BSA samples chemically stressed at 25, 50, and 100 mM SDS concentrations. (b) Comparison between CD secondary structure analysis and fluorescence: the primary structure of BSA 100 mg/mL samples obtained by CD secondary structure analysis vs the conformational changes detected by fluorescence polarization. SDS unfolding proceeds via electrostatic and hydrophobic interactions on multiple states on the protein structure, leading to various unfolded states (n = 3 for all).

Figure 10

Degree of anisotropy measured for (a) thermally stressed and (b) chemically stressed samples. The data shown for (a) is for 6 sample sets, and that for (b) is for 9 sample sets. The dotted black curve shows the average response from all the samples. The error bars show the variations occurring for the repetition of the measurements for individual samples.