Calibration, standardization, and quantitative analysis of multidimensional fluorescence (MDF) measurements on complex mixtures (IUPAC Technical Report)

Ryder, Alan G.; Stedmon, Colin A.; Harrit, Niels; Bro, Rasmus

doi:10.1515/pac-2017-0610

Cited by 18 publications

(6 citation statements)

References 110 publications

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“…IFE is usually considered a problem because it causes a nonlinear dependence between intensity and concentration. However, it is a valuable source of information about protein‐based samples because of the high sensitivity to changes in sample composition (Panigrahi & Mishra, 2019 ; Ryder et al, 2017 ) and can be incorporated into variance assays once identified and taken into account.…”

Section: Resultsmentioning

confidence: 99%

Analyzing protein conjugation reactions for antibody‐drug conjugate synthesis using polarized excitation emission matrix spectroscopy

Silva

Ryder

2022

Biotech & Bioengineering

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Antibody‐drug conjugates (ADCs) are promising anticancer therapeutics, which offer important advantages compared to more classical therapies. There are a variety of ADC critical quality attributes (CQAs) such as the protein structure, aggregation, and drug‐to‐antibody ratio (DAR), which all impact on potency, stability, and toxicity. Production processes can destabilize antibodies via a variety of physical and chemical stresses, and or by increased aggregation after conjugation of hydrophobic drugs. Thus, a proper control strategy for handling, production, and storage is necessary to maintain CQA levels, which requires the use of in‐process quality measurements to first identify, then understand, and control the variables which adversely affect ADC CQAs during manufacturing. Here, we show how polarized excitation emission matrix (pEEM) spectroscopy, a sensitive, nondestructive, and potentially fast technique, can be used for rapidly assessing aggregation and DAR in a single measurement. pEEM provides several sources of information for protein analysis: Rayleigh scatter for identifying aggregate/particle formation and fluorescence emission to assess chemical and structural changes induced by attachment of a linker and/or a small molecule drug payload. Here, we used a nontoxic ADC mimic (monoclonal antibody with linker molecule) to demonstrate efficacy of the measurement method. Emission changes caused via light absorption by the attached linker, allowed us to predict DAR with good accuracy using fluorescence signal from the final purified products (6% relative error of prediction [REP]) and also from unpurified alkylation intermediates (11% REP). pEEM changes could also be correlated with size (hydrodynamic radius, Rh) and aggregate content parameters obtained from dynamic light scattering and size exclusion chromatography (SEC). For the starting material and purified product samples, pEEM correlated better with Rh (R2 = 0.99, 6% REP) than SEC determined aggregate content (18% REP). Combining both fluorescence and light scatter signals also enabled in‐process size quantification (6% REP). Overall, combining polarized measurements with EEM and Rayleigh scatter provides a single measurement, multi‐attribute test method for ADC manufacturing.

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Section: Resultsmentioning

confidence: 99%

Analyzing protein conjugation reactions for antibody‐drug conjugate synthesis using polarized excitation emission matrix spectroscopy

Silva

Ryder

2022

Biotech & Bioengineering

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“…This effectively prevents the use of absorbance spectroscopy as a quantitative method for in‐process protein analysis. This high absorbance also causes very strong IFE, which has a significant impact on UV fluorescence (Ryder et al, 2017).…”

Section: Resultsmentioning

confidence: 99%

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

Boateng

Elcoroaristizabal

Ryder

2021

Biotech & Bioengineering

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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%.

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“…Inner-filtering is an artifact caused by the absorption of excitation and emission light and is greatest at the shortest EEM wavelengths . Inner-filtering effects could either be (1) intentionally removed by sample dilution or algorithmic correction methods or (2) utilized as the intrinsic characteristic of the sample …”

Section: Resultsmentioning

confidence: 99%

In Situ and Ex Situ 2D Infrared/Fluorescence Correlation Monitoring of Surface Functionality and Electron Density of Biochars

Uchimiya

Noda

Orlov

et al. 2018

ACS Sustainable Chem. Eng.

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Carboxyl, hydroxyl, and other oxygen-containing functional groups play key roles in the interfacial reactions of soil surfaces including biochar (solid-phase slow pyrolysis product) soil amendment. Intensity and directionality in both real (synchronous) and imaginary (asynchronous) coordinates of 2D infrared correlation spectra were confirmed by the time courses of pyrolysis reaction (temperature × wavenumber × absorbance; 10 °C min −1 , 1 h residence time at 500 °C) utilizing high-density (74 total spectra) in situ diffuse reflectance Fourier transform (DRIFTs) monitoring. Similar primary trends were observed for four different lignocellulosic biomass feedstocks: cottonseed hull, cotton ginning waste, flax shive, and pecan shell. In the OH stretch region (3100−3750 cm −1 ), free OH was most sensitive to pyrolysis temperature and reacted before H-bonded OH indicating the evaporation of water, followed by the cleavage of interchain H-bonds. Aromatic CH (R=CH n ) was the primary CH functionality (within 2700−3100 cm −1 ) impacted by the pyrolysis temperature perturbation and formed as the aliphatic CH x was removed. Of C=O/C=C groups, electron-deficient C=O (1740 cm −1 ) was most sensitive to pyrolysis, reacted synchronously (in the same direction) with the aromatic C=C (1510 cm −1 ), and was formed after the most electron-rich C=O (1620 cm −1 ). This electron-density trend in the C=O/C=C (1400−1800 cm −1 ) region of infrared coincided with the formation of aromatic extractable carbon before aliphatic structures in 2D fluorescence emission−emission correlation spectra using 340 nm excitation wavelength. Results could be used to drive biomass pyrolysis toward desirable solid-(carboxyl-enriched biochar) and liquid-phase (less hydrophilic bio-oil) products.

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Calibration, standardization, and quantitative analysis of multidimensional fluorescence (MDF) measurements on complex mixtures (IUPAC Technical Report)

Cited by 18 publications

References 110 publications

Analyzing protein conjugation reactions for antibody‐drug conjugate synthesis using polarized excitation emission matrix spectroscopy

Analyzing protein conjugation reactions for antibody‐drug conjugate synthesis using polarized excitation emission matrix spectroscopy

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

In Situ and Ex Situ 2D Infrared/Fluorescence Correlation Monitoring of Surface Functionality and Electron Density of Biochars

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