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The protein cloud-point temperature (T Cloud ) is a known representative of protein-protein interaction strength and provides valuable information during the development and characterization of protein-based products, such as biopharmaceutics. A high-throughput low volume T Cloud detection method was introduced in preceding work, where it was concluded that the extracted value is an apparent T Cloud (T Cloud,app ). As an understanding of the apparent nature is imperative to facilitate inter-study data comparability, the current work was performed to systematically evaluate the influence of 3 image analysis strategies and 2 experimental parameters (sample volume and cooling rate) on T Cloud,app detection of lysozyme. Different image analysis strategies showed that T Cloud,app is detectable by means of total pixel intensity difference and the total number of white pixels, but the latter is also able to extract the ice nucleation temperature. Experimental parameter variation showed a T Cloud,app depression for increasing cooling rates (0.1-0.5 °C/min), and larger sample volumes (5-24 μL). Exploratory thermographic data indicated this resulted from a temperature discrepancy between the measured temperature by the cryogenic device and the actual sample temperature. Literature validation confirmed that the discrepancy does not affect the relative inter-study comparability of the samples, regardless of the image analysis strategy or experimental parameters. Additionally, high measurement precision was demonstrated, as T Cloud,app changes were detectable down to a sample volume of only 5 μL and for 0.1 °C/min cooling rate increments. This work explains the apparent nature of the T Cloud detection method, showcases its detection precision, and broadens the applicability of the experimental setup.

Short-term parameters correlating to long-term protein stability, such as the protein cloud point temperature (T cloud ), are of interest to improve efficiency during protein product development. Such efficiency is reached if short-term parameters are obtained in a low volume and high-throughput (HT) manner. This study presents a low volume HT detection method for (sub-zero) T cloud determination of lysozyme, as such an experimental method is not available yet. The setup consists of a cryogenic device with an automated imaging system. Measurement reproducibility (median absolute deviation of 0.2 °C) and literature-based parameter validation (Pearson correlation coefficient of 0.996) were shown by a robustness and validation study. The subsequent case study demonstrated a partial correlation between the obtained apparent T cloud parameter and long-term protein stability as a function of lysozyme concentration, ion type, ionic strength, and freeze/thaw stress. The presented experimental setup demonstrates its ability to advance short-term strategies for efficient protein formulation development.

Identification of long-term stable biopharmaceutical formulations is essential for biopharmaceutical product development. Reduction of the number of long-term storage experiments and a well-defined formulation search space requires knowledge-based formulation screenings and a detailed protein phase behavior understanding. To achieve this, short-term analytical techniques can serve as predictors for long-term protein phase behavior. Protein phase behavior studies that investigate this concept commonly display shortcomings such as limited and small datasets, sample adjustments, or simplistic data analysis. To overcome these shortcomings, 150 unique lysozyme solutions were analyzed using six different short-term analytical techniques. Lysozyme's structural properties, conformational stability, colloidal stability, surface charge, and surface hydrophobicity were obtained directly after formulation preparation. Employing the empirical phase diagram method, this short-term data was correlated to long-term physical stability data obtained during 40 days of storage. Short-term protein properties showed partial correlation to long-term phase behavior. Structural differences, changing surface properties, colloidal stability, and conformation stability as a function of formulation conditions were observed. This study contributes to long-term protein phase behavior research by presenting a systematic, data-dependent, and multidimensional data evaluation workflow to create a comprehensive overview of short-term protein analytics in relation to long-term protein phase behavior., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Knowledge of protein behavior/stability during freeze/thaw (FT) operations is essential for storage and production processes in the biopharmaceutical industry. FT stress involves freeze concentration, cold denaturation, and ice crystals formation which can result in protein aggregation. Therefore, it is important to understand the ongoing FT processes, and the influence of different solution parameters. In order to evaluate the ongoing processes during FT (up to -80°C), phase diagrams with lysozyme from chicken egg white and sodium chloride were generated. Thereby, three different buffer systems with varying buffer substances and ionic strengths at pH 3 and pH 5 were investigated. As indicators for the ongoing FT processes, the phase behavior, crystal morphology and solubility were used. An increased number of cycles led, for example, to the formation of micro crystals, sea urchin crystals - indicating LLPS and/or high supersaturation - and precipitate. Furthermore, the buffer substances had a more distinct influence on the phase behavior and morphology compared to the ionic strength differences. The solubility line itself was only shifted when distinct changes in the phase behavior could be observed. In summary, a tool was developed for using the phase behavior and especially the crystal morphology as indicator for underlying processes during FT operations., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Covalent attachment of synthetic polymers to proteins, known as protein-polymer conjugation, is currently one of the main approaches for improving the physicochemical properties of these biomolecules. The most commonly employed polymer is polyethylene glycol (PEG), as evidenced by extensive research and clinical track records for its use in biopharmaceuticals. However, the occurrence of allergic reactions or hypersensitivity and the discovery of PEG antibodies, on the one hand, and the rise of controlled polymerization techniques and novel monomers, on the other hand, have been driving the search for alternative polymers for bioconjugation. The present study describes the synthesis, purification, and properties of conjugates of lysozyme with poly( N-acryloylmorpholine) (PNAM) and poly(oligoethylene glycol methyl ether methacrylate) (POEGMA). Particularly, conjugate species with distinct conjugation degrees are investigated for their residual activity, aggregation behavior, and solubility, by using a high-throughput screening approach. Our study showcases the importance of evaluating conjugates obtained by nonsite-specific modification through isolated species with discrete degrees of conjugation rather than on the batch level. Monovalent conjugates with relatively low molar mass polymers displayed equal or even higher activity than the native protein, while all conjugates showed an improved protein solubility. To achieve a comparable effect on solubility as with PEG, PNAM and POEGMA of higher molar masses were required.

Protein phase diagrams are a tool to investigate the cause and consequence of solution conditions on protein phase behavior. The effects are scored according to aggregation morphologies such as crystals or amorphous precipitates. Solution conditions affect morphologic features, such as crystal size, as well as kinetic features, such as crystal growth time. Commonly used data visualization techniques include individual line graphs or phase diagrams based on symbols. These techniques have limitations in terms of handling large data sets, comprehensiveness or completeness. To eliminate these limitations, morphologic and kinetic features obtained from crystallization images generated with high throughput microbatch experiments have been visualized with radar charts in combination with the empirical phase diagram method. Morphologic features (crystal size, shape, and number, as well as precipitate size) and kinetic features (crystal and precipitate onset and growth time) are extracted for 768 solutions with varying chicken egg white lysozyme concentration, salt type, ionic strength, and pH. Image-based aggregation morphology and kinetic features were compiled into a single and easily interpretable figure, thereby showing that the empirical phase diagram method can support high-throughput crystallization experiments in its data amount as well as its data complexity., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Recent studies highlighted the potential of PEGylated proteins to improve stabilities and pharmacokinetics of protein drugs. Ion-exchange chromatography (IEX) is among the most frequently used purification methods for PEGylated proteins. However, the underlying physical mechanisms allowing for a separation of different PEGamers (proteins with a varying number of attached PEG molecules) are not yet fully understood. In this work, mechanistic chromatography modeling is applied to gain a deeper understanding of the mass transfer and adsorption/desorption mechanisms of mono-PEGylated proteins in IEX. Using a combination of the general rate model (GRM) and the steric mass action (SMA) isotherm, simulation results in good agreement with the experimental data are achieved. During linear gradient elution of proteins attached with PEG of different molecular weight, similar peak heights, and peak shapes at constant gradient length are observed. A superimposed effect of increased desorption rate and reduced diffusion rate as a function of the hydrodynamic radius of PEGylated proteins is identified to be the reason of this anomaly. That is why the concept of the diffusion-desorption-compensation effect is proposed. In addition to the altered elution orders, PEGylation results in a considerable decrease of maximum binding capacity. By using the SMA model in a kinetic formulation, the adsorption behavior of PEGylated proteins in the highly concentrated state is described mechanistically. An exponential increase in the steric hindrance effect with increasing PEG molecular weight is observed. This suggests the formation of multiple PEG layers in the interstitial space between bound proteins and an associated shielding of ligands on the adsorber surface to be the cause of the reduced maximum binding capacity. The presented in silico approach thus complements the hitherto proposed theories on the binding mechanisms of PEGylated proteins in IEX., (© 2017 The Authors Biotechnology Journal Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

During production, purification, formulation, and storage proteins for pharmaceutical or biotechnological applications face solution conditions that are unfavorable for their stability. Such harmful conditions include extreme pH changes, high ionic strengths or elevated temperatures. The characterization of the main influencing factors promoting undesired changes of protein conformation and aggregation, as well as the manipulation and selective control of protein stabilities are crucially important to biopharmaceutical research and process development. In this context PEGylation, i.e. the covalent attachment of polyethylene glycol (PEG) to proteins, represents a valuable strategy to improve the physico-chemical properties of proteins. In this work, the influence of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme is investigated. Specifically, conformational and colloidal properties were studied by means of high-throughput melting point determination and automated generation of protein phase diagrams, respectively. Lysozyme from chicken egg-white as a model protein was randomly conjugated to 2kDa, 5kDa and 10kDa mPEG-aldehyde and resulting PEGamer species were purified by chromatographic separation. Besides protein stability assessment, residual enzyme activities were evaluated employing a Micrococcus lysodeikticus based activity assay. PEG molecules with lower molecular weights and lower PEGylation degrees resulted in higher residual activities. Changes in enzyme activities upon PEGylation have shown to result from a combination of steric hindrance and molecular flexibility. In contrast, higher PEG molecular weights and PEGylation degrees enhanced conformational and colloidal stability. By PEGylating lysozyme an increase of the protein solubility by more than 11-fold was achieved., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In concentrated protein solutions attractive protein interactions may not only cause the formation of undesired aggregates but also of gel-like networks with elevated viscosity. To guarantee stable biopharmaceutical processes and safe formulations, both phenomenons have to be avoided as these may hinder regular processing steps. This work screens the impact of additives on both phase behavior and viscosity of concentrated protein solutions. For this purpose, additives known for stabilizing proteins in solution or modulating the dynamic viscosity were selected. These additives were PEG 300, PEG 1000, glycerol, glycine, NaCl and ArgHCl. Concentrated lysozyme and glucose oxidase solutions at pH 3 and 9 served as model systems. Fourier-transformed-infrared spectroscopy was chosen to determine the conformational stability of selected protein samples. Influencing protein interactions, the impact of additives was strongly dependent on pH. Of all additives investigated, glycine was the only one that maintained protein conformational and colloidal stability while decreasing the dynamic viscosity. Low concentrations of NaCl showed the same effect, but increasing concentrations resulted in visible protein aggregation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The colloidal stability of a protein solution during downstream processing, formulation, and storage is a key issue for the biopharmaceutical production process. Thus, knowledge about colloidal solution characteristics, such as the tendency to form aggregates or high viscosity, at various processing conditions is of interest. This work correlates changes in the apparent diffusion coefficient as a parameter of protein interactions with observed protein aggregation and dynamic viscosity of the respective protein samples. For this purpose, the diffusion coefficient, the protein phase behavior, and the dynamic viscosity in various systems containing the model proteins α-lactalbumin, lysozyme, and glucose oxidase were studied. Each of these experiments revealed a wide range of variations in protein interactions depending on protein type, protein concentration, pH, and the NaCl concentration. All these variations showed to be mirrored by changes in the apparent diffusion coefficient in the respective samples. Whereas stable samples with relatively low viscosity showed an almost linear dependence, the deviation from the concentration-dependent linearity indicated both an increase in the sample viscosity and probability of protein aggregation. This deviation of the apparent diffusion coefficient from concentration-dependent linearity was independent of protein type and solution properties for this study. Thus, this single parameter shows the potential to act as a prognostic tool for colloidal stability of protein solutions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

PEGylation, i.e. the covalent attachment of chemically activated polyethylene glycol (PEG) to proteins, is a technique commonly used in biopharmaceutical industry to improve protein stability, pharmacokinetics and resistance to proteolytic degradation. Therefore, PEGylation represents a valuable strategy to reduce autocatalysis of biopharmaceutical relevant proteases during production, purification and storage. In case of non-specific random conjugation the existence of more than one accessible binding site results in conjugates which vary in position and number of attached PEG molecules. These conjugates may differ considerably in their physicochemical properties. Optimizing the reaction conditions with respect to the degree of PEGylation (number of linked PEG molecules) using high-throughput screening (HTS) technologies requires a fast and reliable analytical method which allows stopping the reaction at defined times. In this study an analytical protocol for PEGylated proteases is proposed combining preservation of sample composition by trichloroacetic acid (TCA) precipitation with high-throughput capillary gel electrophoresis (HT-CGE). The well-studied protein hen egg-white lysozyme served as a model system for validating the newly developed analytical protocol for 10kDa mPEG-aldehyde conjugates. PEGamer species were purified by chromatographic separation for calibrating the HT-CGE system. In a case study, the serine protease Savinase(®) which is highly sensitive to autocatalysis was randomly modified with 5kDa and 10kDa mPEG-aldehyde and analyzed. Using the presented TCA protocol baseline separation between PEGamer species was achieved allowing for the analysis of heterogeneous PEGamer mixtures while preventing protease autocatalysis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Microrheological measurements prove to be suitable to identify rheological parameters of biopharmaceutical solutions. These give information about the flow characteristics but also about the interactions and network structures in protein solutions. For the microrheological measurement tracer particles are required. Due to their specific surface characteristic not all are suitable for reliable measurement results in biopharmaceutical systems. In the present work a screening of melamine, PMMA, polystyrene and surface modified polystyrene as tracer particles were investigated at various protein solution conditions. The surface characteristics of the screened tracer particles were evaluated by zeta potential measurements. Furthermore each tracer particle was used to determine the dynamic viscosity of lysozyme solutions by microrheology and compared to a standard. The results indicate that the selection of the tracer particle had a strong impact on the quality of the microrheological measurement dependent on pH and additive type. Surface modified polystyrene was the only tracer particle that yielded good microrheological results for all tested conditions. The study indicated that the electrostatic surface charge of the tracer particle had a minor impact than its hydrophobicity. This characteristic was the crucial surface property that needs to be considered for the selection of a suitable tracer particle to achieve high measurement accuracy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This study aims at defining rheological parameters for the characterization of highly concentrated protein solutions. As a basis for comparing rheological behavior with protein solution characteristics the protein phase behavior of Lysozyme from chicken egg white with concentrations up to 225 mg/mL, changing pH values and additive concentrations was studied in a microbatch scale format. The prepared phase diagrams, scored after 40 days (t40) give insights into the kind and kinetics of the phase transitions that occur. Oscillatory frequency sweep measurements of samples with exactly the same conditions were conducted immediately after preparation (t0). The protein solutions behave viscoelastic and show a characteristic curve shape of the storage modulus (G') and the loss modulus (G″). The graphs provide information about the cross-linking degree of the respective sample. The measured rheological parameters were sensitive concerning solution composition, protein concentration and solution inner structure. The rheological moduli G' and G″ and especially the ratio of these parameters over a frequency range from 100 to 40000 rad/sec give information about the aggregation tendency of the protein under tested conditions. We succeeded to correlate protein phase behavior with the defined rheological key parameter ωCO. This point represents the frequency value of the intersection point from G' and G″. In our study Lysozyme expressed a ωCO threshold value of 20000 rad/sec as a lower limit for stable protein solutions. The predictability of lysozyme aggregation tendency and crystallization by means of squeeze flow rheometry is shown., (© 2015 Wiley Periodicals, Inc.)

Undesired protein aggregation in general and non-native protein aggregation in particular need to be inhibited during bio-pharmaceutical processing to ensure patient safety and to maintain product activity. In this work the potency of different additives, namely glycerol, PEG 1000, and glycine, to prevent lysozyme aggregation and selectively manipulate lysozyme phase behavior was investigated. The results revealed a strong pH dependency of the additive impact on lysozyme phase behavior, lysozyme solubility, crystal size and morphology. This work aims to link this pH dependent impact to a protein-specific parameter, the conformational stability of lysozyme. At pH 3 the addition of 10% (w/v) glycerol, 10% (w/v) PEG 1000, and 1 M glycine stabilized or destabilized lysozymes' native conformation resulting in a modified size of the crystallization area without influencing lysozyme solubility, crystal size and morphology. Addition of 1 M glycine even promoted non-native aggregation at pH 3 whereas addition of PEG 1000 completely inhibited non-native aggregation. At pH 5 the addition of 10% (w/v) glycerol, 10% (w/v) PEG 1000, and 1 M glycine did not influence lysozymes' native conformation, but strongly influenced the position of the crystallization area, lysozyme solubility, crystal size and morphology. The observed pH dependent impact of the additives could be linked to a differing lysozyme conformational stability in the binary systems without additives at pH 3 and pH 5. However, in any case lysozyme phase behavior could selectively be manipulated by addition of glycerol, PEG 1000 and glycine. Furthermore, at pH 5 crystal size and morphology could selectively be manipulated., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The combination of PEG-protein conjugation and chromatographic separation is generally known as solid-phase or on-column PEGylation and can provide advantages compared to commonly applied batch PEGylation. Even though the concept was already applied by several authors, changes in the isoform distribution compared to liquid-phase PEGylation due to sterically hindered PEGylation sites could not be confirmed. In this manuscript, a method for solid-phase PEGylation experiments in a 96-well plate format, using an automated liquid handling station is described. Applying size exclusion chromatography (SEC) and highly sensitive isoform analytics for mono-PEGylated lysozyme, we were able to investigate the differences in reaction kinetics and isoform distribution between adsorber-based PEGylation and modifications in free solution. Accordingly, solid-phase PEGylation with SP Sepharose FF and XL generally showed a reduced PEGylation reaction. In contrast to the predominant N-terminal PEGylation of lysozyme in liquid phase, a main modification of lys 97 and lys 116 was found for solid-phase experiments, which could be explained by binding orientations on corresponding adsorbent materials. Further experiments with varying amounts of bound protein additionally showed an influence on the isoform distribution of mono-PEGylated lysozyme., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Knowledge of protein phase behavior is essential for downstream process design in the biopharmaceutical industry. Proteins can either be soluble, crystalline or precipitated. Additionally liquid-liquid phase separation, gelation and skin formation can occur. A method to generate phase diagrams in high throughput on an automated liquid handling station in microbatch scale was developed. For lysozyme from chicken egg white, human lysozyme, glucose oxidase and glucose isomerase phase diagrams were generated at four different pH values – pH 3, 5, 7 and 9. Sodium chloride, ammonium sulfate, polyethylene glycol 300 and polyethylene glycol 1000 were used as precipitants. Crystallizing conditions could be found for lysozyme from chicken egg white using sodium chloride, for human lysozyme using sodium chloride or ammonium sulfate and glucose isomerase using ammonium sulfate. PEG caused destabilization of human lysozyme and glucose oxidase solutions or a balance of stabilizing and destabilizing effects for glucose isomerase near the isoelectric point. This work presents a systematic generation and extensive study of phase diagrams of proteins. Thus, it adds to the general understanding of protein behavior in liquid formulation and presents a convenient methodology applicable to any protein solution., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Covalent attachment of PEG to proteins, known as PEGylation, is currently one of the main approaches for improving the pharmacokinetics of biopharmaceuticals. However, the separation and characterization especially of positional isoforms of PEGylated proteins are still challenging tasks. A common purification strategy uses ion exchange chromatography with increasing ionic strength by shallow salt gradients. This paper presents a method which applies a linear pH gradient chromatography to separate five of six possible isoforms of mono-PEGylated lysozyme, modified with 5 kDa and 10 kDa mPEG-aldehyde. To identify the corresponding PEGylation sites a comparison of elution pH values and calculated isoelectric points of each isoform, was used. The resulting correlation showed an R(2)>0.99. Fractionation, tryptic digestion and subsequent MALDI-MS analysis of each peak, verified the predicted elution order. Based on UV areas the N-terminal amine at lysine 1 exhibited the highest reactivity, followed by the lysine 33 residue., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Optimization of protein refolding parameters by automated, miniaturized, and parallelized high throughput screening is a powerful approach to meet the demand for fast process development with low material consumption. In this study, we validated methods applicable on a standard liquid handling robot for screening of refolding process parameters by dilution of denatured lysozyme in refolding buffer systems. Different approaches for the estimation of protein solubility and folding were validated concerning resolution and compatibility with the robotic system and with the complex buffer and protein structure composition. We established an indirect method to assess soluble lysozyme concentration independent of matrix effects and protein structure varieties by automated separation of aggregated protein, resolubilization, and measurement of absorption at 280 nm. Using this nonspecific solubility assays the correlation between favorable parameters for high active and soluble lysozyme yields were evaluated. An overlap of good refolding buffer compositions was found provided that the redox environment was controlled with redox reagents. In addition, the need to control unfolding conditions like time, temperature, lysozyme, and dithiothreitol concentration was pointed out as different feedstocks resulted in different refolding yields., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chromatography is the most widely used technique for the purification of biopharmaceuticals in the biotech industry. Surprisingly, process development is often still based on empirical studies or experience; recently high-throughput screening stations are employed to minimize development time and to improve screening quality. Still, experimental effort remains high and a more detailed understanding of adsorption mechanisms on a molecular level underlying chromatographic separation could help in the future to select and design chromatography steps in silico. In this study, we focused on the elucidation of protein orientation upon adsorption onto a chromatographic resin. We identified two characteristic binding sites of lysozyme on SP Sepharose Fast Flow and one multipoint interaction of lysozyme with SP Sepharose XL. Increasing ionic strength did not significantly influence the binding, whereas changes in the mobile phase pH led to a re-orientation on SP Sepharose FF. This phenomenon agrees well with theoretical considerations, including a detailed description of the surface charge distribution with changing pH and linear elution experiments, giving an idea why proteins are often retained on ion-exchange materials beyond their isoelectric point.

Confocal laser scanning microscopy (CLSM) is a method allowing in situ visualization of protein transport in porous chromatography resins. CLSM requires labeling a protein with a fluorescent probe. Recent work has shown that conjugation of the protein with fluorescent probes can lead to significant changes in the retention time of the protein-dye conjugate with respect to the unlabeled protein. In this study, we show that common labeling procedures result in a heterogeneous mixture of different variants and that attachment location of the fluorescent probe on the protein surface can have a strong effect on the retention of protein-dye conjugate. Lysozyme was labeled with Cy5 and BODIPY-FL succinimidyl esters, followed by chromatographic separation of the different lysozyme-dye conjugates and subsequent determination of the label position using MALDI-TOF-MS. Finally, homogenously labeled lysozyme-dye conjugates were used in CLSM experimentation and compared to published results arising from heterogeneously labeled feedstocks. The results confirm that the attachment location of the fluorescent probe has a strong effect on chromatographic retention behavior. When addressing the binding affinities of the different labeled protein fractions, it was found that native lysozyme was able to displace lysozyme-dye conjugates when the fluorescent label was attached to lysine-33, but not when attached to lysine-97. Finally, it could be shown that when superimposing the single profiles of the three major fractions obtained during a labeling procedure a qualitative picture of the net profile is obtained.

Much work has been done to qualify and quantify chromatographic adsorption and transportation mechanisms in different adsorber materials. An important aspect in all studies is the understanding of the binding mechanism between protein and resin on a molecular level in order to optimize processes on the level of adsorber design. We established a method to determine the binding orientation of lysozyme for different materials under various experimental conditions enabling us to observe changes in the mode of adsorption. We varied the protein load of two different adsorber types, Source 15S, a conventional cation exchange resin and EMD Fractogel SO(3), a tentacle-type cation exchanger. We found similar preferential binding sites for the interaction between lysozyme and the surface of these adsorbers at low surface coverage, however, the tentacle adsorber exhibited multi-point binding whereas the binding on Source was limited to one binding site only. With increasing protein density on the surface, lysozyme rotates from a space-consuming side-on to a space-saving end-on orientation on Fractogel, explaining a higher maximum binding capacity for Fractogel. This re-orientation could not be observed for Source.

Confocal laser scanning microscopy has been previously applied to the study of protein uptake in porous chromatography resins. This method requires labeling the protein with a fluorescent probe. The labeled protein is then diluted with a large quantity of native protein so that the fluorescence intensity is a linear function of the labeled protein concentration. Ideally, the attachment of a fluorescent probe should not affect the affinity of the protein for the stationary phase; however, recent experimental work has shown that this assumption is difficult to satisfy. In the present study, we present a mathematical model of protein diffusion and adsorption in a single adsorbent particle. The differences in adsorption behavior of labeled and native protein are accounted for by treating the system as a two-component system (labeled and native protein) described by the steric mass action isotherm (SMA). SMA parameters are regressed from experimental linear gradient elution data for lysozyme and lysozyme-dye conjugates (for the fluorescent dyes Cy3, Cy5, Bodipy FL, and Atto635). When the regressed parameters are employed in the model, an overshoot in the labeled lysozyme concentration is predicted for Cy5- and Bodipy-labeled lysozyme, but not for Atto635-labeled lysozyme. The model predictions agree qualitatively well with recent work showing the dependence of the concentration overshoot on the identity of the attached dye and provide further evidence that the overshoot is likely caused by the change of binding characteristics due to the fluorescent label., ((c) 2006 Wiley Periodicals, Inc.)