Polyacrylic acid based plasma fractionation for the production of albumin and IgG: Compatibility with existing commercial downstream processes.

Commercial fractionation of human plasma into immunoglobulin- and albumin-rich fractions is often initiated with sequential cold ethanol-based precipitation methods, which have changed little over the past 70 years. The required low temperature (-4 to -8°C) and high concentrations of ethanol 8-40%) necessitate large-scale fixed processing lines, and major capital investment and operating costs. The resulting fractions are then further purified by ethanol based precipitation or chromatographic procedures to obtain the purified final product. Aqueous polyacrylic acid (PAA) based precipitation, which readily interfaces with existing downstream processing, could offer advantages with respect to cost, safety, environmental impact, and flexibility. Sequential precipitation with 7%, 12%, and 20% (w/v) solutions of PAA 8000 in the presence of a kosmotropic salt (sodium citrate) gave fibrinogen-, immunoglobulin-, and albumin-rich fractions with 80-90% yield and 64%, 55%, and 82% purity, respectively. Further purification of the IgG-rich precipitate by caprylic acid precipitation and anion exchange chromatography, achieved a target purity of >99%. This was also achieved for the downstream processing of the albumin-rich precipitate using a two-step ion exchange chromatographic procedure. This work shows that PAA precipitation can be used in place of cold ethanol precipitation to generate crude IgG and albumin fractions which can be purified to final products of acceptable purity.

A Near-infrared Turn-on Fluorescent Sensor for Sensitive and Specific Detection of Albumin from Urine Samples.

A readily synthesizable fluorescent probe DMAT-π-CAP was evaluated for sensitive and selective detection of human serum albumin (HSA). DMAT-π-CAP showed selective turn-on fluorescence at 730 nm in the presence of HSA with more than 720-fold enhancement in emission intensity ([DMAT-π-CAP] = 10 µM), and rapid detection of HSA was accomplished in 3 seconds. The fluorescence intensity of DMAT-π-CAP was shown to increase in HSA concentration-dependent manner (Kd = 15.4 ± 3.3 µM), and the limit of detection of DMAT-π-CAP was determined to be 10.9 nM (0.72 mg/L). The 1:1 stoichiometry between DMAT-π-CAP and HSA was determined, and the displacement assay revealed that DMAT-π-CAP competes with hemin for the unique binding site, which rarely accommodates drugs and endogenous compounds. Based on the HSA-selective turn-on NIR fluorescence property as well as the unique binding site, DMAT-π-CAP was anticipated to serve as a fluorescence sensor for quantitative detection of the HSA level in biological samples with minimized background interference. Thus, urine samples were directly analyzed by DMAT-π-CAP to assess albumin levels, and the results were comparable to those obtained from immunoassay. The similar sensitivity and specificity to the immunoassay along with the simple, cost-effective, and fast detection of HSA warrants practical application of the NIR fluorescent albumin sensor, DMAT-π-CAP, in the analysis of albumin levels in various biological environments.

MS-Based Proteomic Analysis of Serum and Plasma: Problem of High Abundant Components and Lights and Shadows of Albumin Removal.

Blood serum or plasma proteome is a gold mine of disease biomarkers. However, complexity and a huge dynamic range of their components, combined with multiple mechanisms of degradation and posttranslational modifications, further complicated by the presence of lipids, salts, and other metabolites, represent a real challenge for analytical sensitivity, resolution, and reproducibility. This problem exists particularly in the case of potential disease-specific markers, most typically represented by low-abundant proteins (LAPs), whose detection is usually impaired by the dominance of albumins, immunoglobulins, and other high-abundant serum/plasma proteins (HAPs). Hence, analysis of biomarker candidates in serum/plasma samples frequently requires separation of their components, usually including depletion of albumin in a fraction of interest. Such "preprocessing" of serum/plasma specimens is critical in proteomic analysis based on mass spectrometry. This approach is very potent; nevertheless a wide range of protein concentrations in serum/plasma represents a particular challenge, since high-abundant proteins (mostly albumin) dominate in a sample subjected to mass spectrometry and suppress peptide ions originating from low-abundant proteins, thus limiting probability and reliability of their detection. An emerging approach in serum-/plasma-based biomarker-oriented studies is the proteome component of exosomes - nanovesicles secreted by cells and involved in multiple aspects of intercellular communication. However, the presence of albumin, frequent contaminant of exosomes isolated from human serum/plasma, represents a real challenge also in this type of study. A similar problem is encountered in proteomic studies based on exosomes obtained in in vitro experiments where culture media are normally supplemented with fetal bovine serum containing growth factors and hormones. In this case exosomes are frequently contaminated with bovine serum albumin and other bovine serum proteins which should be removed before proteomic analysis of exosome cargo.

A comparison of albumin removal procedures for proteomic analysis of blood plasma.

Blood biomarkers are usually present in low concentration and can be masked by the high-abundance proteins, of which albumin is the predominant one. The purpose of this study was to compare four different albumin removal methods compatible with in-gel based proteomics, applicable for plasma, without requiring specific techniques and high financial input. Plasma underwent albumin depletion with ultrafiltration device Amicon Ultra, commercial ProteoPrep Blue Albumin and IgG Depletion Kit, acetonitrile precipitation method and precipitation with acetonitrile-methanol protocol. All samples were evaluated by 1-D and 2-D gel electrophoresis with subsequent mass spectrometry protein identification. Two of the tested methods (ProteoPrep BlueKit and acetonitrile-methanol precipitation) maintained sufficient protein content for further in-gel analyses. Their 2-D protein profiles were distinctively separated and overlapped with protein profile of crude plasma. Protein spot count showed significant increase in protein spots, compared to crude plasma, only with acetonitrile-methanol precipitation method. Precipitation with acetonitrile-methanol method significantly increased number of protein spots on 2-D protein profile and improved score of mass spectrometry identification. However, albumin was still present and found in number of protein spots.

Effects of Ultrasonic-Assisted Extraction on the Physicochemical Properties of Different Walnut Proteins.

The effects of ultrasonic-assisted extraction (UAE, 200 W, 20 min) on the yield and physicochemical properties of different walnut proteins (WNPs, including albumin, globulin, and glutelin) were investigated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that UAE could result in protein molecular fragmentation of albumin, but did not affect the major bands of globulin and glutelin. The CD spectra demonstrated that different WNPs obtained by UAE had different changes in their secondary structure. Under UAE, there was an increase in surface hydrophobicity (H0) of albumin and gluten and no change in the fluorescence intensity, while decreases were observed in the H0 and fluorescence intensity of globulin; and the contents of total and surface free sulfhydryl in albumin dramatically decreased. UAE reduced the size of the particles and the dimension of the microstructures in albumin and gluten, indicating that ultrasound could unfold protein aggregates. In addition, UAE increased the solubility, emulsifying activity (EA), foaming capacity (FC), and foam stability (FS) of the obtained proteins. The above results indicate that ultrasound extraction is a promising approach to improve the extraction yield and properties of walnut proteins.

Assessing the Sensitizing and Allergenic Potential of the Albumin and Globulin Fractions from Amaranth (Amaranthus hypochondriacus) Grains before and after an Extrusion Process.

Background: The first cases of food allergy to amaranth grain have recently been published. This pseudocereal is considered hypoallergenic, and there is scarce information about the allergenic potential of amaranth proteins, either before or after food processing. Objective: To evaluate, in a mouse model of food allergy, the sensitizing and allergenic potential of extruded and non-extruded albumin and globulin fractions from amaranth grains. Materials and Methods: Amaranth (Amaranthus hypochondriacus) flour was obtained and the albumin and globulin fractions isolated. These protein fractions were also obtained after flour extrusion. An intraperitoneal 28-day protocol was carried out to evaluate the sensitizing and allergenic potential of the proteins. The common and rarely allergenic proteins ovalbumin and potato acidic phosphatase were utilized as reference. Specific IgE and IgG antibodies were evaluated for all the proteins tested. Mast cell protease-1 (mMCP-1) responses were evaluated in serum samples collected after intragastric challenges with the proteins of interest. All serological evaluations were carried out using ELISA. Results: Mice were sensitized to the non-extruded albumin fraction from amaranth grains and to ovalbumin (p = 0.0045). The extrusion process of amaranth proteins abrogated the IgE responses triggered under non-extruded conditions (p = 0.0147). mMCP-1 responses were significantly detected in the group of mice sensitized to ovalbumin (p = 0.0138), but not in others. Conclusions: The non-extruded albumin fraction from amaranth has the potential to sensitize BALB/c mice, but this sensitizing potential fails to induce detectable serum levels of the mast cell degranulation marker mMCP-1 after intragastric challenges. Furthermore, the extrusion process abolished the sensitization potential of the amaranth albumins.

Molecular and Functional Properties of Protein Fractions and Isolate from Cashew Nut (Anacardium occidentale L.).

Some molecular and functional properties of albumin (83.6% protein), globulin (95.5% protein), glutelin (81.3% protein) as well as protein isolate (80.7% protein) from cashew nut were investigated. These proteins were subjected to molecular (circular dichroism, gel electrophoresis, scanning electron microscopy) and functional (solubility, emulsification, foaming, water/oil holding capacity) tests. Cashew nut proteins represent an abundant nutrient with well-balanced amino acid composition and could meet the requirements recommended by FAO/WHO. SDS-PAGE pattern indicated cashew nut proteins were mainly composed of a polypeptide with molecular weight (MW) of 53 kDa, which presented two bands with MW of 32 and 21 kDa under reducing conditions. The far-UV CD spectra indicated that cashew proteins were rich in ß-sheets. The surface hydrophobicity of the protein isolate was higher than that of the protein fractions. In pH 7.0, the solubility of protein fractions was above 70%, which was higher than protein isolate at any pH. Glutelin had the highest water/oil holding capacity and foaming properties. Protein isolate displayed better emulsifying properties than protein fractions. In summary, cashew nut kernel proteins have potential as valuable nutrition sources and could be used effectively in the food industry.

Report- Immunological study of different fraction of wheat proteins.

Wheat allergy specifically refers to the adverse reaction involving IgE antibody to one or more protein fraction of wheat such as albumin, globulin, gliadin and glutenin (gluten). The majority of IgE-mediated reactions to wheat involve albumin and globulin fraction while gluten (gliadin & glutenin) also cause allergy (Celiac disease). Allergic reactions to wheat may be caused by ingestion of wheat containing foods or inhalation of flour (Bakers asthma). The present study was an effort to explore the antibody response of different proteins present in wheat. ELISA results revealed that the antibody response for albumin varied from 0.92-1.78, whereas, for globulin ranged from 1.39-1.60. Antibody response against glutenin and gliadin ranged from 0.57-1.05 and 0.98-1.95 respectively, among the different varieties of wheat. All the tested wheat varieties showed the significant difference antibody response against the different fractions of protein.

Purification of Biotinylated Proteins Using Single Walled Carbon Nanotube-Streptavidin Complexes.

In this study, Single walled carbon nanotube (SWNT)-streptavidin complexes were used to capture and purify biotinylated proteins, including bio-GFP and bio-DBS using a pull-down method. The purification conditions were systematically studied, including surface blocking of SWNT using chicken egg albumin (CEA), the ratio of SWNT-streptavidin complexes to the cell lysate, as well as the centrifugation speed. Optimization of the protein purification using SWNT-streptavidin complexes shows the possibility of carbon nanotubes as a promising candidate for protein purification applications. The SWNT-streptavidin could be used as a scaffold to analyze protein structure directly by cryo-transmission electron microscopy, which provides better understanding in protein­protein interactions and biological processes.

Capillary electrophoresis in a fused-silica capillary with surface roughness gradient.

The electro-osmotic flow, a significant factor in capillary electrophoretic separations, is very sensitive to small changes in structure and surface roughness of the inner surface of fused silica capillary. Besides a number of negative effects, the electro-osmotic flow can also have a positive effect on the separation. An example could be fused silica capillaries with homogenous surface roughness along their entire separation length as produced by etching with supercritical water. Different strains of methicillin-resistant and methicillin-susceptible Staphylococcus aureus were separated on that type of capillaries. In the present study, fused-silica capillaries with a gradient of surface roughness were prepared and their basic behavior was studied in capillary zone electrophoresis with UV-visible detection. First the influence of the electro-osmotic flow on the peak shape of a marker of electro-osmotic flow, thiourea, has been discussed. An antifungal agent, hydrophobic amphotericin B, and a protein marker, albumin, have been used as model analytes. A significant narrowing of the detected zones of the examined analytes was achieved in supercritical-water-treated capillaries as compared to the electrophoretic separation in smooth capillaries. Minimum detectable amounts of 5 ng/mL amphotericin B and 5 µg/mL albumin were reached with this method.

Amorphous-Amorphous Phase Separation of Freeze-Concentrated Protein and Amino Acid Excipients for Lyophilized Formulations.

The objective of this study was to elucidate the mixing state of proteins and amino acid excipients concentrated in the amorphous non-ice region of frozen solutions. Thermal analysis of frozen aqueous solutions was performed in heating scans before and after a heat treatment. Frozen aqueous solutions containing a protein (e.g., recombinant human albumin, gelatin) or a polysaccharide (dextran) and an amino acid excipient (e.g., L-arginine, L-arginine hydrochloride, L-arginine monophosphate, sodium L-glutamate) at varied mass ratios showed single or double Tg' (glass transition temperature of maximally freeze-concentrated solutes). Some mixture frozen solutions rich in the polymers maintained the single Tg' of the freeze-concentrated amorphous solute-mixture phase. In contrast, amino acid-rich mixture frozen solutions revealed two Tg's that suggested transition of concentrated non-crystalline solute-mixture phase and excipient-dominant phase. Post-freeze heat treatment induced splitting of the Tg' in some intermediate mass ratio mixture solutions. The mixing state of proteins and amino acids varied depending on their structure, salt types, mass ratio, composition of co-solutes (e.g., NaCl) and thermal history. Information on the varied mixing states should be valuable for the rational use of amino acid excipients in lyophilized protein pharmaceuticals.

Treatment of Alzheimer disease using combination therapy with plasma exchange and haemapheresis with albumin and intravenous immunoglobulin: Rationale and treatment approach of the AMBAR (Alzheimer Management By Albumin Replacement) study. / Tratamiento de la enfermedad de Alzheimer mediante terapia combinada de aféresis terapéutica y hemoféresis con albúmina e inmunoglobulina intravenosa: fundamentos y aproximación terapéutica al estudio AMBAR (Alzheimer Management By Albumin Replacement).

INTRODUCTION: There is a growing interest in new therapeutic strategies for the treatment of Alzheimer disease (AD) which focus on reducing the beta-amyloid peptide (Aß) burden in the brain by sequestering plasma Aß, a large proportion of which is bound to albumin and other proteins. This review discusses the concepts of interaction between Aß and albumin that have given rise to AMBAR (Alzheimer's Disease Management by Albumin Replacement) project, a new multicentre, randomised, controlled clinical trial for the treatment of AD. DEVELOPMENT: Results from preliminary research suggest that Albutein(®) (therapeutic albumin, Grifols) contains no quantifiable levels of Aß. Studies also show that Albutein(®) has Aß binding capacity. On the other hand, AD entails a high level of nitro-oxidative stress associated with fibrillar aggregates of Aß that can induce albumin modification, thus affecting its biological functions. Results from the phase ii study confirm that using therapeutic apheresis to replace endogenous albumin with Albutein(®) 5% is feasible and safe in patients with AD. This process resulted in mobilisation of Aß and cognitive improvement in treated patients. The AMBAR study will test combination therapy with therapeutic apheresis and haemopheresis with the possible leverage effect of Albutein(®) with intravenous immunoglobulin replacement (Flebogamma(®) DIF). Cognitive, functional, and behavioural changes in patients with mild to moderate AD will be assessed. CONCLUSIONS: the AMBAR study represents a new therapeutic perspective for AD.

Tunable electrophoretic separations using a scalable, fabric-based platform.

There is a rising need for low-cost and scalable platforms for sensitive medical diagnostic testing. Fabric weaving is a mature, scalable manufacturing technology and can be used as a platform to manufacture microfluidic diagnostic tests with controlled, tunable flow. Given its scalability, low manufacturing cost (<$0.25 per device), and potential for patterning multiplexed channel geometries, fabric is a viable platform for the development of analytical devices. In this paper, we describe a fabric-based electrophoretic platform for protein separation. Appropriate yarns were selected for each region of the device and weaved into straight channel electrophoretic chips in a single step. A wide dynamic range of analyte molecules ranging from small molecule dyes (<1 kDa) to macromolecule proteins (67-150 kDa) were separated in the device. Individual yarns behave as a chromatographic medium for electrophoresis. We therefore explored the effect of yarn and fabric parameters on separation resolution. Separation speed and resolution were enhanced by increasing the number of yarns per unit area of fabric and decreasing yarn hydrophilicity. However, for protein analytes that often require hydrophilic, passivated surfaces, these effects need to be properly tuned to achieve well-resolved separations. A fabric device tuned for protein separations was built and demonstrated. As an analytical output parameter for this device, the electrophoretic mobility of a sedimentation marker, Naphthol Blue Black bovine albumin in glycine-NaOH buffer, pH 8.58 was estimated and found to be -2.7 × 10(-8) m(2) V(-1) s(-1). The ability to tune separation may be used to predefine regions in the fabric for successive preconcentrations and separations. The device may then be applied for the multiplexed detection of low abundance proteins from complex biological samples such as serum and cell lysate.

Microscale depletion of high abundance proteins in human biofluids using IgY14 immunoaffinity resin: analysis of human plasma and cerebrospinal fluid.

Removal of highly abundant proteins in plasma is often carried out using immunoaffinity depletion to extend the dynamic range of measurements to lower abundance species. While commercial depletion columns are available for this purpose, they generally are not applicable to limited sample quantities (<20 µL) due to low yields stemming from losses caused by nonspecific binding to the column matrix and concentration of large eluent volumes. Additionally, the cost of the depletion media can be prohibitive for larger-scale studies. Modern LC-MS instrumentation provides the sensitivity necessary to scale-down depletion methods with minimal sacrifice to proteome coverage, which makes smaller volume depletion columns desirable for maximizing sample recovery when samples are limited, as well as for reducing the expense of large-scale studies. We characterized the performance of a 346 µL column volume microscale depletion system, using four different flow rates to determine the most effective depletion conditions for ∼6-µL injections of human plasma proteins and then evaluated depletion reproducibility at the optimum flow rate condition. Depletion of plasma using a commercial 10-mL depletion column served as the control. Results showed depletion efficiency of the microscale column increased as flow rate decreased, and that our microdepletion was reproducible. In an initial application, a 600-µL sample of human cerebrospinal fluid (CSF) pooled from multiple sclerosis patients was depleted and then analyzed using reversed phase liquid chromatography-mass spectrometry to demonstrate the utility of the system for this important biofluid where sample quantities are more commonly limited.

[Electrophoretic Analysis of Abnormal Data on Serum Protein Electrophoresis].

We recently demonstrated glycation of monoclonal IgA and the presence of IgA-albumin complexes, but the significance of the complexes was not clear. We describe a non-diabetic patient with IgA type M-protein whose serum fructosamine and glycoalbumin levels were elevated. On electrophoresis of the serum protein of the patient, the albumin band shifted to the cathode side. The abnormal precipitin arc of IgA-albumin complexes was detected by immunoelectrophoresis. To elucidate the mechanism of IgA-albumin complexes, we analyzed their properties using immunoelectrophoresis, Western blotting, and two-dimensional gel electrophoresis. The macromolecularized albumin spots were demonstrated by two-dimensional Western blotting with antiserum to human albumin of the patient's serum. Moreover, the IgA-albumin complexes were dissociated on treatment with 2-mercaptoethanol. It can be considered that albumin is bound to the monoclonal IgA molecule by covalent disulfide bonds, and that the albumin binding site is located near the hinge region (311Cys) of the IgA molecule and involves the free SH group, thought to be present in the α-chain.

A fast and reproducible method for albumin isolation and depletion from serum and cerebrospinal fluid.

Analysis of serum and plasma proteomes is a common approach for biomarker discovery, and the removal of high-abundant proteins, such as albumin and immunoglobins, is usually the first step in the analysis. However, albumin binds peptides and proteins, which raises concerns as to how the removal of albumin could impact the outcome of the biomarker study while ignoring the possibility that this could be a biomarker subproteome itself. The first goal of this study was to test a new commercially available affinity capture reagent from Protea Biosciences and to compare the efficiency and reproducibility to four other commercially available albumin depletion methods. The second goal of this study was to determine if there is a highly efficient albumin depletion/isolation system that minimizes sample handling and would be suitable for large numbers of samples. Two of the methods tested (Sigma and ProteaPrep) showed an albumin depletion efficiency of 97% or greater for both serum and cerebrospinal fluid (CSF). Isolated serum and CSF albuminomes from ProteaPrep spin columns were analyzed directly by LC-MS/MS, identifying 128 serum (45 not previously reported) and 94 CSF albuminome proteins (17 unique to the CSF albuminome). Serum albuminome was also isolated using Vivapure anti-HSA columns for comparison, identifying 105 proteins, 81 of which overlapped with the ProteaPrep method.

Long-circulating poly(ethylene glycol)-coated poly(lactid-co-glycolid) microcapsules as potential carriers for intravenously administered drugs.

The intrinsic advantages of microcapsules with regard to nanocapsules as intravenous drug carrier systems are still not fully exploited. Especially, in clinical situations where a long-term drug release within the vascular system is desired, if large amounts of drug have to be administered or if capillary leakage occurs, long-circulating microparticles may display a superior alternative to nanoparticles. Here, microcapsules were synthesised and parameters such as in vitro tendency of agglomeration, protein adsorption and in vivo performance were investigated. Biocompatible poly(ethylene glycol) (PEG)-coated poly(DL-lactide-co-glycolide) (PLGA) as wall material, solid and perfluorodecalin (PFD)-filled PEG-PLGA microcapsules (1.5 µm diameter) were manufactured by using a modified solvent evaporation method with either 1% poly(vinyl alcohol) (PVA) or 1.5% cholate as emulsifying agents. Compared to microcapsules manufactured with cholate, the protein adsorption (albumin and IgG) was clearly decreased and agglomeration of capsules was prevented, when PVA was used. The intravenous administration of these microcapsules, both solid and PFD-filled, in rats was successful and exhibited a circulatory half-life of about 1 h. Our data clearly demonstrate that PEG-PLGA microcapsules, manufactured by using PVA, are suitable biocompatible, long-circulating drug carriers, applicable for intravenous administration.

Amino acid profiles and quality from lotus seed proteins.

BACKGROUND: Protein composition, amino acid profile and nutritional value of the lotus seed and its Osborne fractions were investigated. The seed was rich in protein with 19.85%, and showed well balanced amino acid composition compared with FAO/WHO pattern, Its nutritive properties were similar to those observed in the reference soybean protein. Phenylalanine, tyrosine, leucine and lysine were the limiting amino acids in the seed proteins. The albumin and globulin were the main protein fraction, the amino acid profile and nutritional value were close to the seed protein. RESULTS: Changes in transition temperature and thermal stability were observed through different solvent extractions. Albumin possessed the predominant thermal stability (81.4 °C) followed by globulin (74.49 °C), prolamin (69 °C) and glutelin (65.6 °C). So, solvent compositions influence the profile of AAs and their nutritive value, and aqueous solvent with 0.1 mol L⁻¹ NaCl was an efficient protein solubiliser. CONCLUSION: The results indicated that the extraction processes influenced the lotus seed protein quality and thermal stability. Overall, the study revealed that the lotus seed protein was nutritionally well-balanced protein and might be of significant importance in the formulation of diets for humans.

Molecularly imprinted composite cryogel for albumin depletion from human serum.

A new composite protein-imprinted macroporous cryogel was prepared for depletion of albumin from human serum prior to use in proteom applications. Polyhydroxyethyl-methacylate-based molecularly imprinted polymer (MIP) composite cryogel was prepared with high gel fraction yields up to 83%, and its morphology and porosity were characterized by Fourier transform infrared, scanning electron microscopy, swelling studies, flow dynamics, and surface area measurements. Selective binding experiments were performed in the presence of competitive proteins human transferrin (HTR) and myoglobin (MYB). MIP composite cryogel exhibited a high binding capacity and selectivity for human serum albumin (HSA) in the presence of HTR and MYB. The competitive adsorption amount for HSA in MIP composite cryogel is 722.1 mg/dL in the presence of competitive proteins (HTR and MYB). MIP composite cryogel column was successfully applied in the fast protein liquid chromatography system for selective depletion of albumin in human serum. The depletion ratio was highly increased by embedding beads into cryogel (85%). Finally, MIP composite cryogel can be reused many times with no apparent decrease in HSA adsorption capacity.

Albumin handling by renal tubular epithelial cells in a microfluidic bioreactor.

Epithelial cells in the proximal tubule of the kidney reclaim and metabolize protein from the glomerular filtrate. Proteinuria, an overabundance of protein in the urine, affects tubular cell function and is a major factor in the progression of chronic kidney disease. By developing experimental systems to study tubular protein handling in a setting that simulates some of the environmental conditions of the kidney tubule in vivo, we can better understand how microenviromental conditions affect cellular protein handling to determine if these conditions are relevant in disease. To this end, we used two in vitro microfluidic models to evaluate albumin handling by renal proximal tubule cells. For the first system, cells were grown in a microfluidic channel and perfused with physiological levels of shear stress to evaluate the effect of mechanical stress on protein uptake. In the second system, a porous membrane was used to separate an apical and basolateral compartment to evaluate the fate of protein following cellular metabolism. Opossum kidney (OK) epithelial cells were exposed to fluorescently labeled albumin, and cellular uptake was determined by measuring the fluorescence of cell lysates. Confocal fluorescence microscopy was used to compare uptake in cells grown under flow and static conditions. Albumin processed by the cells was examined by size exclusion chromatography (SEC) and SDS-PAGE. Results showed that cellular uptake and/or degradation was significantly increased in cells exposed to flow compared to static conditions. This was confirmed by confocal microscopy. Size exclusion chromatography and SDS-PAGE showed that albumin was broken down into small molecular weight fragments and excreted by the cells. No trace of intact albumin was detectable by either SEC or SDS-PAGE. These results indicate that fluid shear stress is an important factor mediating cellular protein handling, and the microfluidic bioreactor provides a novel tool to investigate this process.