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

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.

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%.

Effects of Viscosity and Refractive Index on the Emission and Diffusion Properties of Alexa Fluor 405 Using Fluorescence Correlation and Lifetime Spectroscopies.

Fluorescence Correlation Spectroscopy (FCS) studies of the interaction of polymers or proteins in solution are strongly affected by the viscosity and refractive index of the medium, and the effects are likely to be more significant with the use of short wavelength excitation (e.g., 405 nm diode lasers). Failing to account for these issues can lead to incorrect measurement of average size, conformational changes, and dynamic behaviour of polymers and proteins. Steady-state, time-resolved, and FCS measurements of Alexa 405 in glycerol:water mixtures were performed to determine its suitability for FCS measurements with 405 nm excitation. The effects of the refractive index and viscosity on the diffusion coefficient and photophysical parameters (lifetime and relative quantum yield) of the fluorophore were determined. Alexa 405 lifetime decreased from 3.55 ns in water to 3.25 ns in a 50:50 glycerol:water mixture, while its diffusion coefficient dropped from 333 ± 16 to 44 ± 1 µm2s- 1. Lifetime data collected from micromolar solutions of Alexa 405 did however also suggest that as solvent polarity decreased, aggregates (excimers) were formed as evidenced by the appearance of a rising edge in the decay plots. The interdependence between lifetime, refractive index, and diffusion coefficient could be accurately fitted by a simple polynomial function indicating that the probe is well behaved and predictable in the glycerol:water model system. Overall, Alexa 405 is a most promising and reliable probe for FCS measurement using violet laser diode excitation sources.

Advanced Spectroscopy and APBS Modeling for Determination of the Role of His190 and Trp103 in Mouse Thymidylate Synthase Interaction with Selected dUMP Analogues.

A homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. In these mutants, histidine at position 190 and tryptophan at position 103 are substituted with alanine and glycine, respectively. Several emission-based spectroscopy methods used in the paper demonstrate an especially important role for Trp 103 in TS ligands binding. In addition, the Advanced Poisson-Boltzmann Solver (APBS) results show considerable differences in the distribution of electrostatic potential around Trp 103, as compared to distributions observed for all remaining Trp residues in the mTS family of structures. Together, spectroscopic and APBS results reveal a possible interplay between Trp 103 and His190, which contributes to a reduction in enzymatic activity in the case of H190A mutation. Comparison of electrostatic potential for mTS complexes, and their mutants, with the substrate, dUMP, and inhibitors, FdUMP and N4-OH-dCMP, suggests its weaker influence on the enzyme-ligand interactions in N4OH-dCMP-mTS compared to dUMP-mTS and FdUMP-mTS complexes. This difference may be crucial for the explanation of the "abortive reaction" inhibitory mechanism of N4OH-dCMP towards TS. In addition, based on structural analyses and the H190A mutant capacity to form a denaturation-resistant complex with N4-OH-dCMP in the mTHF-dependent reaction, His190 is apparently responsible for a strong preference of the enzyme active center for the anti rotamer of the imino inhibitor form.

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.

Super Stable Fluorescein Isothiocyanate Isomer I Monolayer for Total Internal Reflection Fluorescence Microscopy.

Total internal reflection fluorescence microscopy (TIRFM) is an important method in surface science and for the analysis of surface-bound macromolecules. Here, we developed and explored the use of a novel fluorescein isothiocyanate isomer I (FITC)-adsorbed monolayer for alignment and validation of TIRFM measurements and configurations. Aqueous solutions of FITC exist as several different protolytic forms (dianionic, anionic, neutral, and cationic) with each form having different emission characteristics. However, the emission behavior of FITC adsorbed on hydrophilic, hydrophobic, and unmodified glass surfaces at different pH was unknown. TIRFM imaging and spectroscopy were used to study FITC and FITC-labeled bovine serum albumin (BSA-FITC) monolayers generated on three different glass surfaces. Monolayer emission intensity, spectra, and the photobleaching profiles were all dependent on pH and the surface properties of the glass. Very strangely, however, at pH 5.0 on hydrophobic surfaces, the FITC monolayers produced were both bright and apparently unbleachable over ∼20 min of imaging (60 s total exposure). During monolayer formation at pH 5.0, we saw clear evidence for concentration-based quenching, indicating high surface coverage. When the monolayer had been rinsed with buffer to remove unbound FITC, we observed an increase in emission intensity during illumination indicative of some form of photoactivated species being present. Eventually, the fluorescence emission stabilized and remained constant for extended periods of time with no evidence of photobleaching. We hypothesize that during the adsorption process (a hydrophobic-hydrophobic interaction) there was conversion to the fluorescent quinoid form of FITC. In contrast, at pH 7.4 and 9.6 on hydrophobic surfaces, FITC monolayers had well-defined, fast photobleaching kinetics (decay to ∼50% intensity in 5-10 s). The equivalent BSA-FITC monolayers were slightly brighter, with similar photobleaching kinetics. While the precise mechanism for this unusual behavior is still unknown, all these low-cost monolayers were easily prepared, were reproducible, and can serve as convenient test samples for TIRFM alignment, calibration, and validation prior to undertaking measurements with more sensitive biogenic or biological specimens.

Low-content quantification in powders using Raman spectroscopy: a facile chemometric approach to sub 0.1% limits of detection.

A robust and accurate analytical methodology for low-content (<0.1%) quantification in the solid-state using Raman spectroscopy, subsampling, and chemometrics was demonstrated using a piracetam-proline model. The method involved a 5-step process: collection of a relatively large number of spectra (8410) from each sample by Raman mapping, meticulous data pretreatment to remove spectral artifacts, use of a 0-100% concentration range partial least-squares (PLS) regression model to estimate concentration at each pixel, use of a more accurate, reduced concentration range PLS model to calculate analyte concentration at each pixel, and finally statistical analysis of all 8000+ concentration predictions to produce an accurate overall sample concentration. The relative prediction accuracy was ∼2.4% for a 0.05-1.0% concentration range, and the limit of detection was comparable to high performance liquid chromatography (0.03% versus 0.041%). For data pretreatment, we developed a unique cosmic ray removal method and used an automated baseline correction method, neither of which required subjective user intervention and thus were fully automatable. The method is applicable to systems which cannot be easily analyzed chromatographically, such as hydrate, polymorph, or solvate contamination.

Comprehensive, quantitative bioprocess productivity monitoring using fluorescence EEM spectroscopy and chemometrics.

This study demonstrates the application of fluorescence excitation-emission matrix (EEM) spectroscopy to the quantitative predictive analysis of recombinant glycoprotein production cultured in a Chinese hamster ovary (CHO) cell fed-batch process. The method relies on the fact that EEM spectra of complex solutions are very sensitive to compositional change. As the cultivation progressed, changes in the emission properties of various key fluorophores (e.g., tyrosine, tryptophan, and the glycoprotein product) showed significant differences, and this was used to follow culture progress via multiple curve resolution alternating least squares (MCR-ALS). MCR-ALS clearly showed the increase in the unique dityrosine emission from the product glycoprotein as the process progressed, thus provided a qualitative tool for process monitoring. For the quantitative predictive modelling of process performance, the EEM data was first subjected to variable selection and then using the most informative variables, partial least-squares (PLS) regression was implemented for glycoprotein yield prediction. Accurate predictions with relative errors of between 2.3 and 4.6% were obtained for samples extracted from the 100 to 5000 L scale bioreactors. This study shows that the combination of EEM spectroscopy and chemometric methods of evaluation provides a convenient method for monitoring at-line or off-line the productivity of industrial fed-batch mammalian cell culture processes from the small to large scale. This method has applicability to the advancement of process consistency, early problem detection, and quality-by-design (QbD) practices.

Size exclusion chromatography for screening yeastolate used in cell culture media.

Yeastolate is often used as a media supplement in industrial mammalian cell culture or as a major media component for microbial fermentations. Yeastolate variability can significantly affect process performance, but analysis is technically challenging because of its compositional complexity. However, what may be adequate for manufacturing purposes is a fast, inexpensive screening method to identify molecular variance and provide sufficient information for quality control purposes, without characterizing all the molecular components. Here we used Size Exclusion Chromatography (SEC) and chemometrics as a relatively fast screening method for identifying lot-to-lot variance (with Principal Component Analysis, PCA) and investigated if Partial Least Squares, PLS, predictive models which correlated SEC data with process titer could be obtained. SEC provided a relatively fast measure of gross molecular size hydrolysate variability with minimal sample preparation and relatively simple data analysis. The sample set comprised of 18 samples from 12 unique source lots of an ultra-filtered yeastolate (10 kDa molecular weight cut-off) used in a mammalian cell culture process. SEC showed significant lot-to-lot variation, at 214 and 280 nm detection, with the most significant variation, that correlated with process performance, occurring at a retention time of ∼6 min. PCA and PLS regression correlation models provided fast identification of yeastolate variance and its process impact. The primary drawback is the limited column lifetime (<300 injections) caused by the complex nature of yeastolate and the presence of zinc. This limited long term reproducibility because these age-related, non-linear changes in chromatogram peak positions and shapes were very significant.

Evaluating the interaction of human serum albumin (HSA) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes in different aqueous environments using anisotropy resolved multi-dimensional emission spectroscopy (ARMES).

Studying the interaction between plasma proteins and liposomes is critical, particularly for their use as drug delivery systems. Here, the efficacy of anisotropy resolved multidimensional emission spectroscopy (ARMES) for investigating the interaction of human serum albumin (HSA) with liposomes was explored and compared to conventional spectroscopic techniques. Dynamic Light Scattering (DLS) and absorbance spectroscopy (with Multivariate Curve Resolution (MCR) modeling) indicated that the highest degree of liposome rupturing, and aggregation occurred in water, with less in ammonium bicarbonate buffer (ABC) and phosphate buffered saline (PBS). Fluorescence emission spectra of HSA-liposome mixtures revealed significant hypsochromic shifts for water and ABC, but much less in PBS, where the data suggests a non-penetrating protein layer was formed. Average fluorescence lifetimes decreased upon liposome interaction in water (6.2→5.2 ns) and ABC buffer (6.3→5.6 ns) but increased slightly for PBS (5.6→5.8 ns). ARMES using polarized Total Synchronous Fluorescence Scan measurements with parallel factor (PARAFAC) analysis resolved intrinsic HSA fluorescence into two components for interactions in water and ABC buffer, but only one component for PBS. These components, in water and ABC buffer, corresponded to two different HSA populations, one blue-shifted and penetrating the liposomes (λex/em = ~ 280/320 nm) and a second, similar to free HSA in solution (λex/em = ~ 282/356 nm). PARAFAC scores for water and ABC buffer suggested that a large proportion of HSA interacted in an end on configuration. ARMES provides a new way for investigating protein-liposome interactions that exploits the full intrinsic emission space of the protein and thus avoids the use of extrinsic labels. The use of multivariate data analysis provided a comprehensive and structured framework to extract a variety of useful information (resolving different fluorescent species, quantifying their signal contribution, and extracting light scatter signals) all of which can be used to discriminate between interaction mechanisms.

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.

Prediction of cell culture media performance using fluorescence spectroscopy.

Cell culture media used in industrial mammalian cell culture are complex aqueous solutions that are inherently difficult to analyze comprehensively. The analysis of media quality and variance is of utmost importance in efficient manufacturing. We are exploring the use of rapid "holistic" analytical methods that can be used for routine screening of cell culture media used in industrial biotechnology. The application of rapid fluorescence spectroscopic techniques to the routine analysis of cell culture media (Chinese hamster ovary cell-based manufacture) was investigated. We have developed robust methods which can be used to identify compositional changes and ultimately predict the efficacy of individual fed batch media in terms of downstream protein product yield with an accuracy of +/-0.13 g/L. This is achieved through the implementation of chemometric methods such as multiway robust principal component analysis (MROBPCA), and n-way partial least-squares-discriminant analysis and regression (NPLS-DA and NPLS). This ability to observe compositional changes and predict product yield before media use has enormous potential and should permit the effective elimination of one of the major process variables leading to more consistent product quality and improved yield. These robust and reliable methods have the potential to become an important part of upstream biopharmaceutical quality control and analysis.

Rapid characterization and quality control of complex cell culture media solutions using raman spectroscopy and chemometrics.

The use of Raman spectroscopy coupled with chemometrics for the rapid identification, characterization, and quality assessment of complex cell culture media components used for industrial mammalian cell culture was investigated. Raman spectroscopy offers significant advantages for the analysis of complex, aqueous-based materials used in biotechnology because there is no need for sample preparation and water is a weak Raman scatterer. We demonstrate the efficacy of the method for the routine analysis of dilute aqueous solution of five different chemically defined (CD) commercial media components used in a Chinese Hamster Ovary (CHO) cell manufacturing process for recombinant proteins.The chemometric processing of the Raman spectral data is the key factor in developing robust methods. Here, we discuss the optimum methods for eliminating baseline drift, background fluctuations, and other instrumentation artifacts to generate reproducible spectral data. Principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA) were then employed in the development of a robust routine for both identification and quality evaluation of the five different media components. These methods have the potential to be extremely useful in an industrial context for "in-house" sample handling, tracking, and quality control.

Monitoring local unfolding of bovine serum albumin during denaturation using steady-state and time-resolved fluorescence spectroscopy.

In a previous report (J. Fluoresc. 16, 153, 2006) we studied the chaotropically induced denaturation of Bovine Serum Albumin (BSA) using the fluorescence decay kinetics at different stages in the denaturation of BSA by guanidinium hydrochloride (GuHCl). In this work, we gain a more detailed insight into the BSA denaturation process by investigating the thermodynamics of the process. Structural changes were monitored spectrophotometrically via the intrinsic protein fluorescence from tryptophan residues, and the extrinsic fluorescence from 1,8-anilinonaphthalene sulphonate (ANS). ANS tends to locate in a variety of binding sites in BSA which are located in different domains, and these can be selectively populated using different, 1:1 and 1:10 molar ratios of BSA to ANS. The data from steady-state and time-resolved fluorescence spectroscopy were analyzed using thermodynamic two-state and three-state models and the lifetime data clearly indicated the presence of an intermediate state during denaturation. A global analysis using non-linear regression gave a DeltaG(H(2)O,D)(0) = 6.7 kcal x mol(-1) for the complete unfolding of the BSA-ANS complexes, and a DeltaG(H(2)O,I) = 0.9 kcal x mol(-1) for the first step to the intermediate. Therefore, the unfolding energy of the intermediate, which appears mostly at intermediate GuHCl concentrations (1.0 to 1.5 M), to the denatured state, is 5.8 kcal x mol(-1). The lifetime analysis of the BSA-ANS complexes also shows clearly that there are differences in stability of the BSA domains, with domain III unfolding first at low GuHCl concentrations (<1.5 M).

Polarity assessment of thermoresponsive poly(NIPAM-co-NtBA) copolymer films using fluorescence methods.

The in-situ, non-contact, and non-destructive measurement of the physicochemical properties such as the polarity of thin, hydrophilic polymer films is desirable in many areas of polymer science. Polarity is a complex factor and encompasses a range of non-covalent interactions including dipolarity/polarizability and hydrogen bonding. A polarity measurement method based on fluorescence would be ideal, but the key challenge is to identify suitable probes which can accurately measure specific polarity related parameters. In this manuscript we assess a variety of fluorophores for measuring the polarity of a series of relatively hydrophilic, thermoresponsive N-isopropylacrylamide/N-tert-butylacrylamide (NIPAM/NtBA) copolymers. The emission properties of both pyrene and 3-Hydroxyflavone (3-HF) based fluorophores were measured in dry polymer films. In the case of pyrene, a relatively weak, linear relationship between polymer composition and the ratio of the first to the third vibronic band of the emission spectrum (I(1)/I(3)) is observed, but pyrene emission is very sensitive to temperature and thus not suitable for robust polarity measurements. The 3-HF fluorophores which can undergo an excited-state intramolecular proton transfer (ESIPT) reaction have a dual band fluorescence emission that exhibits strong solvatochromism. Here we used 4'-diethylamino-3-hydroxyflavone (FE), 5,6-benzo-4'-diethylamino-3-hydroxyflavone (BFE), and 4 -diethylamino-3-hydroxy-7-methoxyflavone (MFE). The log ratio of the dual band fluorescence emission (log (I(N*)/I(T*))) of 3-HF doped, dry, NIPAM-NtBA copolymer films were found to depend linearly on copolymer composition, with increasing hydrophobicity (greater NtBA fraction) leading to a decrease in the value of log (I(N*)/I(T*)). However, the ESIPT process in the polymer matrix was found to be irreversible, non-equilibrated and occurs over a much longer timescale in comparison to the results previously reported for liquid solvents.

Quantitative analysis of weakly bound insulin oligomers in solution using polarized multidimensional fluorescence spectroscopy.

Being able to measure the size and distribution of oligomers in solution is a critical issue in the manufacture and stability of insulin and other protein formulations. Measuring oligomers reliably can however be complicated, due to their fragile self-assembled structures, which are held together by weak forces. This can cause issues in chromatographic based methods, where dissociation or re-equilibration of oligomer populations can occur e.g. upon dilution in a different eluting buffer, but also for light scattering based methods like dynamic light scattering (DLS) where the size difference involved (often less than a factor 3) does not allow mixtures of oligomers to be resolved. Intrinsic fluorescence offers an attractive alternative as it is non-invasive, sensitive but also because it contains scattered light when implemented via excitation emission matrix (EEM) measurements, that is sensitive to changes in particle size. Here, using insulin at formulation level concentrations, we show for the first time how EEM can both discriminate and quantify the proportion of oligomeric states in solution. This was achieved by using the Rayleigh scatter (RS) band and the fluorescence signal contained in EEM. After validating size changes with DLS, we show in particular how the volume under the RS band correlated linearly with protein/oligomer molecular weight, in agreement with the Debye-Zimm relationship. This was true for the RS data from both EEM and polarized EEM (pEEM) measurements, the latter providing a stronger scatter signal, more sensitive to particle size changes. The fluorescence signal was then used with multivariate curve resolution (MCR) to quantify more precisely the soluble oligomer composition of insulin solutions. In conditions that promoted the formation of mainly one type of oligomer (monomer, dimer, or hexamer), pEEM-MCR helped identify the presence of small amounts of other oligomeric forms, while in conditions that were previously said to favour the insulin tetramer, we show that in the presence of zinc, these insulin samples were instead a heterogenous mixture composed of mostly dimers and hexamers. These MCR results correlated in all cases with the observed discrimination by principal component analysis (PCA), and deviations observed in the RS data. In conclusion, using pEEM scatter and emission components with chemometric data analysis provides a unique analytical method for characterising and monitoring changes in the soluble oligomeric state of proteins.

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.

Trigger factor from the psychrophilic bacterium Psychrobacter frigidicola is a monomeric chaperone.

In eubacteria, trigger factor (TF) is the first chaperone to interact with newly synthesized polypeptides and assist their folding as they emerge from the ribosome. We report the first characterization of a TF from a psychrophilic organism. TF from Psychrobacter frigidicola (TF(Pf)) was cloned, produced in Escherichia coli, and purified. Strikingly, cross-linking and fluorescence anisotropy analyses revealed it to exist in solution as a monomer, unlike the well-characterized, dimeric E. coli TF (TF(Ec)). Moreover, TF(Pf) did not exhibit the downturn in reactivation of unfolded GAPDH (glyceraldehyde-3-phosphate dehydrogenase) that is observed with its E. coli counterpart, even at high TF/GAPDH molar ratios and revealed dramatically reduced retardation of membrane translocation by a model recombinant protein compared to the E. coli chaperone. TF(Pf) was also significantly more effective than TF(Ec) at increasing the yield of soluble and functional recombinant protein in a cell-free protein synthesis system, indicating that it is not dependent on downstream systems for its chaperoning activity. We propose that TF(Pf) differs from TF(Ec) in its quaternary structure and chaperone activity, and we discuss the potential significance of these differences in its native environment.

Investigating tryptophan quenching of fluorescein fluorescence under protolytic equilibrium.

Fluorescein is one of most used fluorescent labels for characterizing biological systems, such as proteins, and is used in fluorescence microscopy. However, if fluorescein is to be used for quantitative measurements involving proteins then one must account for the fact that the fluorescence of fluorescein-labeled protein can be affected by the presence of intrinsic amino acids residues, such as tryptophan (Trp). There is a lack of quantitative information to explain in detail the specific processes that are involved, and this makes it difficult to evaluate quantitatively the photophysics of fluorescein-labeled proteins. To address this, we have explored the fluorescence of fluorescein in buffered solutions, in different acidic and basic conditions, and at varied concentrations of tryptophan derivatives, using steady-state absorption and fluorescence spectroscopy, combined with fluorescence lifetime measurements. Stern-Volmer analyses show the presence of static and dynamic quenching processes between fluorescein and tryptophan derivatives. Nonfluorescent complexes with low association constants (5.0-24.1 M(-1)) are observed at all pH values studied. At low pH values, however, an additional static quenching contribution by a sphere-of-action (SOA) mechanism was found. The possibility of a proton transfer mechanism being involved in the SOA static quenching, at low pH, is discussed based on the presence of the different fluorescein prototropic species. For the dynamic quenching process, the bimolecular rate constants obtained (2.5-5.3 x 10(9) M(-1)s(-1)) were close to the Debye-Smoluchowski diffusion rate constants. In the encounter controlled reaction mechanism, a photoinduced electron transfer process was applied using the reduction potentials and charges of the fluorophore and quencher, in addition to the ionic strength of the environment. The electron transfer rate constants (2.3-6.7 x 10(9) s(-1)) and the electronic coupling values (5.7-25.1 cm(-1)) for fluorescein fluorescence quenching by tryptophan derivatives in the encounter complex were then obtained and analyzed.

Measuring the micro-polarity and hydrogen-bond donor/acceptor ability of thermoresponsive N-isopropylacrylamide/N-tert-butylacrylamide copolymer films using solvatochromic indicators.

Thin polymer films are important in many areas of biomaterials research, biomedical devices, and biological sensors. The accurate in situ measurement of multiple physicochemical properties of thin polymer films is critical in understanding biocompatibility, polymer function, and performance. In this work we demonstrate a facile spectroscopic methodology for accurately measuring the micro-polarity and hydrogen-bond donor/acceptor ability for a series of relatively hydrophilic thermoresponsive copolymers. The micro-polarity of the N-isopropylacrylamide (NIPAM) and N-tert-butylacrylamide (NtBA) co-polymers was evaluated by means of the E(T)(30), alpha, beta, and pi empirical solvatochromic polarity parameters. The data shows that increasing the NtBA fraction in the dry copolymer film reduces polarity and hydrogen-bonding ability. Within the Kamlet-Taft polarity framework, the NIPAM/NtBA copolymer films are strong hydrogen-bond acceptors, strongly dipolar/polarizable, and rather moderate hydrogen-bond donors. This characterization provides a more comprehensive physicochemical description of polymers, which aids the interpretation of film performance. Comparison of the measured E(T)(30) values with literature data for other water-soluble polymers show that dry NIPAM/NtBA copolymers are slightly more polar than poly(ethylene oxide), less polar than polyvinylalcohol, and approximately the same polarity as poly(N-vinyl-2-pyrrolidone). These findings indicate that this spectroscopic method is a facile, rapid, and nondestructive methodology for measuring polymer properties in situ, suitable for most biomaterials research laboratories.