Application of fluorescence spectroscopy for the in-vial investigation of protein solutions

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ElKassas, Khaled
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University College Cork
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Therapeutic protein formulations are subjected to various stressors during manufacturing, transport, and storage, causing destabilisation, in turn leading to deleterious effects such as immunogenic reactions or inefficacy upon administration. An essential part of this production and supply process is establishing protein formulation stability at all stages. Spectroscopy is a common laboratory tool for the characterisation of therapeutic proteins. Conventional techniques for determining protein denaturation are lab-based and require sample removal from the sealed primary packaging vial and sample destruction. To overcome the limitations of traditional techniques, the development of a fluorescence-based spectroscopic technique for “in-vial” protein analysis was a key thesis aim. A fluorescence excitation wavelength of 365 nm was found and confirmed to be transmissible through borosilicate glass vial walls. The excitation resulted in an intrinsic emission at 462 nm for a model protein, bovine serum albumin (BSA). Using a bespoke apparatus, the change in fluorescence signal with time was monitored over the reconstitution period of lyophilised protein formulations, without breach of the vial seal. First, we demonstrated how analysis of changes in fluorescence signal with time during reconstitution, using principal component analysis, could be used to determine an instrumentally quantified reconstitution time. At high protein concentrations (10% w/v BSA) the variability of the reconstitution time measurements was reduced from 80.4% relative standard deviation obtained via the pharmacopeial visual method to 8.2% for the instrumental method. Spectroscopic measurements resulted in longer measured reconstitution time compared to the visual method, possibly owing to the detection of subvisible particulates undergoing dissolution. In the second part of this work, the bespoke spectroscopic apparatus was modified with polarising filters to monitor protein aggregation and denaturation in-vial. Detector measurement of the emission wavelength (365 nm) allowed measurement of protein aggregation via light scattering. Fluorescence anisotropy (a measurement of optical polarizability at different angles) at 462 nm was used to determine protein conformational changes. Fluorescence scattering and anisotropy measurements for freshly prepared, aggregated, and denatured BSA solutions were compared to reference circular dichroism (CD) and size-exclusion chromatography (SEC) measurements. Anisotropy analysis of thermal unfolding showed strong correlations with secondary structure as quantified through a CD-neural networking model. Scattering correlated with large molecular weight aggregate measurements via SEC. A study was conducted to find the relationship between the fluorescent amino acids and the overall protein fluorescence for 4 different proteins. Using a multivariate technique, multivariate curve resolution to resolve 3D emission excitation spectra, the fluorescence of individual amino acid solutions and the total fluorescence of the proteins was correlated. Finally, a partial least squares regression (PLSR) model relating the anisotropy and scattering measurements to reference CD and SEC measurements was established from a sample set of standardised and stored samples. A total of 72 spectra were collected for the batch standardised protein solutions and the stored stability study samples. Samples were stored under real-world-like stress conditions (temperature, shaking, ambient light exposure). The fluorescence anisotropy and scattering results were included as the predictors vs. CD and SEC measurements as the responses. The PLSR showed strong correlations with an average R2 of 0.87 and a root mean PRESS of 0.487 at 9 latent variables. In conclusion, the combination of bespoke apparatus and 365 nm excitation was capable of monitoring protein stability in-vial successfully. The spectroscopic measurement of reconstitution time resulted in more precise measurements. The method overcomes the challenges present in the current pharmacopeial standard measurement as it can detect subvisible particulates and is independent of analyst capabilities and subjectivity. The protein solution stability monitoring provided a holistic non-destructive alternative to multi-instrument analysis while maintaining reasonable correlations with established reference stability indicators. This research provides a platform for a cost-effective portable solution to provide a top-level overview of biopharmaceutical product stability in-vial, from manufacture to the point of administration.
Proteins , Spectroscopy , Fluorescence , Multivariate analysis , Analytical techniques
ElKassas, K. 2024. Application of fluorescence spectroscopy for the in-vial investigation of protein solutions. PhD Thesis, University College Cork.
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