Development of a rapid polarized total synchronous fluorescence spectroscopy (pTSFS) method for protein quantification in a model bioreactor broth.

Protein quantification during bioprocess monitoring is essential for biopharmaceutical manufacturing and is complicated by the complex chemical composition of the bioreactor broth. Here we present the early-stage development and optimization of a polarized total synchronous fluorescence spectroscopy (pTSFS) method for protein quantification in a hydrolysate-protein model (mimics clarified bioreactor broth samples) using a standard benchtop laboratory fluorometer. We used UV transmitting polarizers to provide wider range pTSFS spectra for screening of the four different TSFS spectra generated by the measurement: parallel (||), perpendicular (⊥), unpolarized (T) intensity spectra and anisotropy maps. TSFS|| (parallel polarized) measurements were the best for protein quantification compared to standard unpolarized measurements and the Bradford assay. This was because TSFS|| spectra had a better analyte signal to noise ratio (SNR), due to the anisotropy of protein emission. This meant that protein signals were better resolved from the background emission of small molecule fluorophores in the cell culture media. SNR of >5000 was achieved for concentrations of bovine serum albumin/yeastolate 1.2/10 g L-1 with TSFS|| . Optimization using genetic algorithm and interval partial least squares based variable selection enabled reduction of spectral resolution and number of excitation wavelengths required without degrading performance. This enables fast (<3.5 min) online/at-line measurements, and the method had an LOD of 0.18 g L-1 and high accuracy with a predictive error of <9%.

Modelling Förster resonance energy transfer (FRET) using anisotropy resolved multi-dimensional emission spectroscopy (ARMES).

BACKGROUND: Förster Resonance Energy Transfer (FRET) is widely used to study the structure and dynamics of biomolecular systems and also causes the non-linear fluorescence response observed in multi-fluorophore proteins. Accurate FRET analysis, in terms of measuring changes in donor and acceptor spectra and energy transfer efficiency is therefore critical. METHODS: We demonstrate a novel quantitative FRET analysis using anisotropy resolved multidimensional emission spectroscopy (ARMES) in a Human Serum Albumin (HSA) and 1,8-anilinonaphathalene sulfonate (ANS) model. ARMES combines 4D measurement of polarized excitation emission matrices (pEEM) with multivariate data analysis to spectrally resolve contributing fluorophores. Multivariate analysis (Parallel Factor, PARAFAC and restricted Tucker3) was used to resolve fluorophore contributions and for modelling the quenching of HSA emission and the HSA-ANS interactions. RESULTS: pEEM spectra were modelled using Tucker3 which accommodates non-linearities introduced by FRET and a priori chemical knowledge was used to optimise the solution, thus resolving three components: HSA emission, ANS emission from indirect FRET excitation, and ANS emission from direct excitation. Perpendicular emission measurements were more sensitive to indirectly excited acceptor emission. PARAFAC modelling of HSA, donor emission, separated ANS FRET interacting (Tryptophan) and non-interacting (Tyrosine) components. This enabled a new way of calculating quenching constants using the multi-dimensional emission of individual donor fluorophores. CONCLUSIONS: FRET efficiency could be calculated using the multi-dimensional, resolved emission of the interacting donor fluorophores only which yielded higher ET efficiencies compared to conventional methods. GENERAL SIGNIFICANCE: Shows the potential of multidimensional fluorescence measurements and data analysis for more accurate FRET modelling in proteins.

Characterization of lysozyme PEGylation products using polarized excitation-emission matrix spectroscopy.

The growing use of therapeutic proteins requires accurate analytical techniques for measuring biophysical and structural changes during manufacturing. This is particularly true for the PEGylation of proteins, because characterization of PEGylation reactions and products can often be difficult due to the relatively small impact on protein structure, the lack of an accessible polyethylene glycol (PEG) chromophore, and the heterogeneous final product mixtures. Intrinsic fluorescence spectroscopy is one potential solution due to its relatively high sensitivity to small changes in protein structure and its suitability for online or atline measurements. In this study, we use the PEGylation of lysozyme as a model system to determine the efficacy of polarized excitation-emission matrix (pEEM) spectroscopy as a rapid tool for characterizing the structural variability of the lysozyme (LZ) starting materials and PEGylated products with varying PEG-to-protein ratios (PPR). Dynamic light scattering showed that as PPR increased from 0 to 2.8, the hydrodynamic radius increased from ∼2.2 to 4.8 nm. pEEM measurements provided several sources of information: Rayleigh scattering to identify size changes and aggregate/particle formation, and fluorescence emission to assess chemical and structural changes. PEGylation induced sufficient physicochemical changes in LZ, which produced changes in the pEEM spectra, largely due to variations in the hydrophobic environments of tryptophan residues close to a PEG attachment site. These significant spectral changes when modeled using conventional multivariate analysis methods were able to easily discriminate the raw product solutions according to the degree of PEGylation and were also able to predict PPR with reasonable accuracy (root mean square error for calibration ∼10%, relative error of prediction < 20%), considering the reference size exclusion chromatography method error of ∼7.2%. The variable selection of the pEEM data suggests that equivalent predictions could be obtained with faster and simpler two-dimensional spectra, making the method a more viable online measurement method.

Multi-attribute quality screening of immunoglobulin G using polarized Excitation Emission Matrix spectroscopy.

Immunoglobulin G (IgG) is often used as a starting material for the production of functionalised antibodies, like Antibody Drug Conjugates (ADCs), PEGlyated-conjugates, or radioimmunoconjugates. The gross structural quality of the protein starting material is, therefore, an important factor in determining final product composition, purity, and quality. In terms of structural quality, one needs to know both the aggregation content and the tertiary structure of the protein. The measurement of structural quality in solution can thus be difficult, but the use of intrinsic fluorescence measurements might offer a solution because of its high sensitivity, ease of use, and when implemented in via multi-dimensional techniques like polarized Excitation Emission Matrix (pEEM) spectroscopy, its high information content. Here we demonstrate how pEEM measurements can be used as a multi-attribute screening method for protein quality using a polyclonal rabbit immunoglobulin (rIgG) model system. By using both Rayleigh scatter and fluorescence emission in combination with simple chemometric data analysis methods like Principal Component analysis (PCA) and unfolded partial least squares (U-PLS) one can simultaneously measure protein concentration, structural variance, and particle/aggregate composition. Furthermore, one can generate quantitative prediction models for non-reversible aggregation content as described by size exclusion chromatography (SEC) and obtain qualitative information about reversible aggregate content, which cannot be obtained from SEC measurements. In conclusion, the pEEM measurement approach is a potentially useful Process Analytical Technology (PAT) method for downstream processing operations in biopharmaceutical manufacturing.

An excitation emission fluorescence lifetime spectrometer using a frequency doubled supercontinuum laser source.

The accurate fluorescence analysis of complex, multi-fluorophore containing proteins requires the use of multi-dimensional measurement techniques. For the measurement of intrinsic fluorescence from tyrosine (Tyr) and tryptophan (Trp) one needs tuneable UV excitation and for steady-state measurements like Excitation Emission Matrix (EEM) simple pulsed Xe lamps are commonly used. Unfortunately, simultaneous multi-dimensional wavelength and time resolved measurement of intrinsic protein fluorescence in the 260 to 400 nm spectral range are challenging and typically required the use of very complex tuneable laser systems or multiple single excitation wavelength sources. Here we have assembled and validated a novel Excitation Emission Fluorescence Lifetime Spectrometer (EEFLS) using a pulsed, frequency doubled, Super-Continuum Laser (SCL) source coupled with a 16 channel multi-anode Time Correlated Single Photon Counting (TCSPC) measurement system. This EEFLS enabled the collection of near complete lifetime and intensity maps over the most important intrinsic protein fluorescence spectral range (λ ex = 260-350/λ em = 300-500 nm). The 4-dimensional (λ ex/λ em/I(t)/τ) Excitation Emission Fluorescence Lifetime Matrix (EEFLM) data produced can be used to better characterize the complex intrinsic emission from proteins. The system was capable of measuring fluorescence emission data with high spectral (1-2 nm) resolution and had an Instrument Response Function (IRF) of ∼650 ps for accurate measurement of nanosecond lifetimes. UV power output was stable after a warm up period, with variations of <2% over 9 hours and reproducible (relative standard deviation RSD < 1.5%). This enabled the collection of accurate EEFLM data at low resolution (∼12 nm in excitation and emission) in 1-2 hours or high resolution (4 nm) in ∼17 hours. EEFLS performance in the UV was compared with a conventional commercial TCSPC system using pulsed LED excitation and validated using solutions of p-terphenyl and tryptophan.

Development and validation of a method for the determination of regulated fragrance allergens by High-Performance Liquid Chromatography and Parallel Factor Analysis 2.

This work presents the development and validation of a multivariate method for quantitation of 6 potentially allergenic substances (PAS) related to fragrances by ultrasound-assisted emulsification microextraction coupled with HPLC-DAD and PARAFAC2 in the presence of other 18 PAS. The objective is the extension of a previously proposed univariate method to be able to determine the 24 PAS currently considered as allergens. The suitability of the multivariate approach for the qualitative and quantitative analysis of the analytes is discussed through datasets of increasing complexity, comprising the assessment and validation of the method performance. PARAFAC2 showed to adequately model the data facing up different instrumental and chemical issues, such as co-elution profiles, overlapping spectra, unknown interfering compounds, retention time shifts and baseline drifts. Satisfactory quality parameters of the model performance were obtained (R2≥0.94), as well as meaningful chromatographic and spectral profiles (r≥0.97). Moreover, low errors of prediction in external validation standards (below 15% in most cases) as well as acceptable quantification errors in real spiked samples (recoveries from 82 to 119%) confirmed the suitability of PARAFAC2 for resolution and quantification of the PAS. The combination of the previously proposed univariate approach, for the well-resolved peaks, with the developed multivariate method allows the determination of the 24 regulated PAS.

Characterization and authentication of Spanish PDO wine vinegars using multidimensional fluorescence and chemometrics.

This work assesses the potential of multidimensional fluorescence spectroscopy combined with chemometrics for characterization and authentication of Spanish Protected Designation of Origin (PDO) wine vinegars. Seventy-nine vinegars of different categories (aged and sweet) belonging to the Spanish PDOs "Vinagre de Jerez", "Vinagre de Montilla-Moriles" and "Vinagre de Condado de Huelva", were analyzed by excitation-emission fluorescence spectroscopy. A visual assessment of fluorescence landscapes pointed out different trends with vinegar categories. PARAllel FACtor analysis (PARAFAC) extracted the potential fluorophores and their values in the PDO vinegars. This information, coupled with different classification methods (Partial Least Square Discrimination Analysis "PLS-DA" and Support Vectors Machines "SVM"), was able to discriminate the wine vinegar category within each PDO, for which SVM models obtained better results (>92% of classification). In each category, SVM also allows the differentiation between PDOs. The proposed methodology could be used as an analysis method for the authentication of Spanish PDO wine vinegars.

Fluorescence excitation-emission matrix spectroscopy as a tool for determining quality of sparkling wines.

Browning in sparkling wines was assessed by the use of excitation-emission fluorescence spectroscopy combined with PARAllel FACtor analysis (PARAFAC). Four different cava sparkling wines were monitored during an accelerated browning process and subsequently storage. Fluorescence changes observed during the accelerated browning process were monitored and compared with other conventional parameters: absorbance at 420nm (A420) and the content of 5-hydroxymethyl-2-furfural (5-HMF). A high similarity of the spectral profiles for all sparkling wines analyzed was observed, being explained by a four component PARAFAC model. A high correlation between the third PARAFAC factor (465/530nm) and the commonly used non-enzymatic browning indicators was observed. The fourth PARAFAC factor (280/380nm) gives us also information about the browning process following a first order kinetic reaction. Hence, excitation-emission fluorescence spectroscopy, together with PARAFAC, provides a faster alternative for browning monitoring to conventional methods, as well as useful key indicators for quality control.

Hyperspectral image analysis. A tutorial.

This tutorial aims at providing guidelines and practical tools to assist with the analysis of hyperspectral images. Topics like hyperspectral image acquisition, image pre-processing, multivariate exploratory analysis, hyperspectral image resolution, classification and final digital image processing will be exposed, and some guidelines given and discussed. Due to the broad character of current applications and the vast number of multivariate methods available, this paper has focused on an industrial chemical framework to explain, in a step-wise manner, how to develop a classification methodology to differentiate between several types of plastics by using Near infrared hyperspectral imaging and Partial Least Squares - Discriminant Analysis. Thus, the reader is guided through every single step and oriented in order to adapt those strategies to the user's case.