Pure Albumin Research Articles

Development of an Online Isotope Dilution CE/ICP-MS Method for the Quantification of Sulfur in Biological Compounds.

We report an analytical methodology for the quantification of sulfur in biological molecules via a species-unspecific postcolumn isotope dilution (online ID) approach using capillary electrophoresis (CE) coupled online with inductively coupled plasma-mass spectrometry (online ID CE/ICP-MS). The method was optimized using a mixture of standard compounds including sulfate, methionine, cysteine, cystine, and albumin, yielding compound recoveries between 98 and 105%. The quantity of sulfur is further converted to the quantity of the compounds owing to the prior knowledge of the sulfur content in the molecules. The limit of detection and limit of quantification of sulfur in the compounds were 1.3-2.6 and 4.1-8.4 mg L-1, respectively, with a correlation coefficient of 0.99 within the concentration range of sulfur of 5-100 mg L-1. The capability of the method was extended to quantify albumin in its native matrix (i.e., in serum) using experimentally prepared serum spiked with a pure albumin standard for validation. The relative expanded uncertainty of the method for the quantification of albumin was 6.7% (k = 2). Finally, we tested the applicability of the method on real samples by the analysis of albumin in bovine and human sera. For automated data assessment, a software application (IsoCor)─which was developed by us in a previous work─was developed further for handling of online ID data. The method has several improvements compared to previously published setups: (i) reduced adsorption of proteins onto the capillary wall owing to a special capillary-coating procedure, (ii) baseline separation of the compounds in less than 30 min via CE, (iii) quantification of several sulfur species within one run by means of the online setup, (iv) SI traceability of the quantification results through online ID, and (v) facilitated data processing of the transient signals using the IsoCor application. Our method can be used as an accurate approach for quantification of proteins and other biological molecules via sulfur analysis in complex matrices for various fields, such as environmental, biological, and pharmaceutical studies as well as clinical diagnosis.

In vitro Validation Assessment of a Fecal Occult Blood Protein Test for Horses

A commercially available equine fecal blood test (FBT) claims to be able to detect the presence of blood proteins (albumin and hemoglobin) in manure. The purpose of this study was to determine the FBT test sensitivity, specificity, positive and negative predictive values (PPV, NPV), lower threshold of detection, time to obtain a visual positive result, effect of temperature and result stability in lab and field conditions. The FBT was assessed for its sensitivity and specificity for detecting pure albumin and hemoglobin at two temperatures over a range of concentrations. Time to result was measured for up to 60 minutes. PPV and NPV were assessed by measuring albumin and hemoglobin in manure from 13 pleasure horses over 25 days. Laboratory tests of hemoglobin alone, albumin alone, and hemoglobin and albumin combined were tested over a range of concentrations from 0.0125 ppm to 50 ppm. In the field study, fresh (within 30 minutes) manure was sampled and tested for proteins using the FBT. The FBT was both sensitive and specific to hemoglobin and albumin. The effect of cold temperature on time to a positive result at 15 minutes was not significant. Results were stable for up to 60 minutes. The field study showed evidence that the appearance of blood proteins in manure was intermittent, and that three tests on consecutive days provided a much better PPV and NPV. It is concluded that this FBT had high specificity, sensitivity, PPV, NPV, was equally functional at low and moderate temperatures, provided a rapid (within 15 minutes) and stable (for up to 60 minutes) reading. Its use in the field is simple and effective.

Dynamic Surface Properties of Solutions of Bovine Serum Albumin Complexes with Silica Nanoparticles

Surface properties of aqueous dispersions of bovine serum albumin complexes with silica nanoparticles have been studied using a set of methods that are sensitive to changes in the structure and composition of surface layers. In spite of the similar charges of the components, their interaction leads to changes in the surface properties of the dispersions; namely, the rates of variations in the dynamic surface elasticity and dynamic surface tension decrease due to the high electrostatic barrier of adsorption. The addition of an electrolyte decreases the electrostatic barrier. In this case, the adsorption of the complexes leads to a growth in the dynamic surface elasticity from the values characteristic of pure bovine serum albumin solutions (~80 mN/m) to 170 mN/m, which corresponds to the formation of macroscopically homogeneous adsorption film of nanoparticle–protein complexes.

Nanoparticle Tracking Analysis of Urine to Detect Exosomes Can Be Confounded by Albuminuria

In the article by Lv et al.1 published in the Journal of the American Society of Nephrology, the authors describe enhanced urinary exosomal release in experimental models of acute and chronic renal injury. They also describe increased exosomal release in patients with IgA nephropathy and a correlation between exosomal concentration and proteinuria. These findings are of clear interest, but we have reservations about some of the methodology used to ascertain this observation. They use nanoparticle tracking analysis (NTA) of urine and describe enhanced particle concentration in patients who are proteinuric. We have recently reported similar observations using NTA in 32 patients with varying levels of albuminuria.2 We found that albuminuria confounded particle analysis by NTA, because albumin-containing solutions contain particles of exosome-like size, and such particles can mimic exosomes in standard NTA. Immunodepletion of albumin from proteinuric urine resulted in a substantial reduction in the concentration of particles detected by NTA as did urine purification using iodixanol density gradients. More importantly, NTA measurements of pure albumin solutions yielded a substantial number of particles with a diameter of 105 nm. This reinforces the need for great caution in the interpretation of NTA results in fluids that have significant levels of protein. It also emphasizes the importance of stringent methods of purification, such as density gradients, in exosome analysis as recommended by the International Society for Extracellular Vesicles.3 Disclosures None.

Should total calcium be adjusted for albumin? A retrospective observational study of laboratory data from central Norway

ObjectivesAlbumin-adjusted total calcium is often used as a surrogate marker for free calcium to evaluate hypocalcaemia or hypercalcaemia. Many adjustment formulas based on simple linear regression models have been published,...

Changes of albumin secondary structure after palmitic acid binding. FT-IR spectroscopic study.

Albumin is an universal transport protein. Plasma pool of free fatty acids arising from triglyceride hydrolysis, critical in energy metabolism and etiology of metabolic disorders is transported by albumin. According to various studies albumin has from seven to nine binding sites with diverse affinity to long chain fatty acids. X-ray diffraction crystallography measurements have provided data only for pure human serum albumin or albumin with fully saturated binding sites. These results have shown that amount of -helices is higher after fatty acids binding. Molecular mechanics simulations suggest that binding of fatty acids in two high-affinity sites leads to major conformational changes in albumin structure. The aim of this research was to investigate albumin secondary structure upon gradually increasing fatty acids to protein mole ratio. Fourier transform infrared spectroscopy was applied to study changes of bovine serum albumin (as an analogue of human serum albumin) -helical structures after binding palmitic acid in a range of 0-20 palmitic acid: albumin molar ratios representing pure protein, partial, full saturation and excess binding sites capacity. Amount of -helices was increasing along with the amount of palmitic acid: bovine serum albumin molar ratio and reached maximum value around 2 mol/mol. Our studies confirmed molecular mechanics simulations and crystallographic studies. Palmitic acid binding in two high-affinity sites leads to major structural changes, filling another sites only slightly influenced bovine serum albumin secondary structure. The systematic study of fatty acids and albumin interactions, using an experimental model mimicking metabolic disorders, may results in new tools for personalized nanopharmacotherapy.

Influence of 1:4;3:6-dianhydro-d- mannitol-based polyamide as an additive on morphology, permeability and antifouling performance of PES ultrafiltration membrane

A novel hydrophilic polyamide, PA-OH containing 1:4;3:6-dianhydro-d-mannitol (isomannide) fragment was synthesized and used as an additive to prepare a series of polyethersulfone (PES)/PA-OH hybrid membranes with different content of PA-OH by phase separation method. And the addition of PA-OH was expected to improve the hydrophilicity, permeability, and antifouling performance of the PES ultrafiltration (UF) membrane. The scanning electron microscopic and atomic force microscopic images showed that the addition of PA-OH greatly changed the morphology of membranes by increasing the porosity, pore size and pore density, and the hydrophilicity was also improved with the increased PA-OH content. Based on the UF experiment, when the PA-OH content was 3%, the fluxes of pure water and bovine serum albumin solution reached 213.1 and 92.6 L m−2 h−1, which were close to 1.5 times and 2 times those of the bare PES membrane, respectively. When the PA-OH content was 5%, the FRR1st reached 85.2%, which was distinctly higher than that of the compared PES/polyethylene glycol5% hybrid membrane of 74.3%. And the FRR2st of the PES/PA-OH5% hybrid membrane was still over 80%, which indicated its long-term antifouling performance. Thus, the PA-OH can be a candidate for the hydrophilic additives.

On the importance of albumin binding for the flux of 7α-hydroxy-3-oxo-4-cholestenoic acid in the brain

We confirmed previous findings by a Japanese group that there is an accumulation of 7α-hydroxy-3-oxo-4-cholestenoic acid (7-Hoca) in human subdural hematomas. The accumulation correlated with the time from the bleeding to the sample collection. We present evidence that these accumulations are likely to be caused by the strong affinity of 7-Hoca to albumin and the marked difference between plasma and brain with respect to levels of albumin. In the circulation, 80-90% of 7-Hoca is bound to albumin with a ratio between the steroid acid and albumin of ∼1.4 ng/mg. In cerebrospinal fluid (CSF), the ratio between 7-Hoca and albumin is ∼30 ng/mg. When albumin or hemolyzed blood in a dialysis bag was exposed to CSF, there was a flux of 7-Hoca from CSF to the albumin. We suggest that the major explanation for accumulation of 7-Hoca in subdural hematoma is a flux from the brain into the hematoma due to the high affinity to albumin and the high capacity of 7-Hoca to pass biomembranes. We discuss the possibility that the markedly different ratios between 7-Hoca and albumin in circulation and brain can explain the flux of 7-Hoca from the brain into circulation against a concentration gradient.

The Physical and Morphological Characteristics of Mango Powder (Mangifera indica L. cv Tommy Atkins) Produced by Foam Mat Drying

The thermal behavior, moisture adsorption properties and structural and morphological characteristics of mango powders were evaluated. The powders were obtained by foam mat drying methodology using albumin (ALB), mixture (EB) of monoglycerides of fatty acids, sorbitan monostearate and polyoxyethylene sorbitan monostearate and a combination of the two (EB-ALB) as foaming agents. The evaluation was done by differential scanning calorimetry (DSC), X-ray diffraction (XRD) and scanning electron microscopy (SEM), and the moisture adsorption isotherms were also determined. The powder with EB had a glass transition temperature (Tg) of −4.2 °C. The denaturation temperature of pure albumin (82.2 °C) increased in the powders with ALB to 117 °C and in those with EB-ALB to 102 °C, due to the thermal stability provided by the pulp sugars. The moisture sorption isotherm of the EB-powder showed a higher water equilibrium content than the other powders. All the powders were in the amorphous state. The morphology of the powder with EB showed corrugated particles, whereas those with ALB and EB-ALB showed particles with a less porous aspect and more compact surfaces than the powders with EB.

CHEMICAL COMPOSITION STUDY OF VANADIUM PENTOXIDE XEROGELS DOPED BY BOVINE ALBUMIN

Metal–bioorganic compounds of vanadium pentoxide and bovine serum albumin (BSA) (Fraction V) were obtained by using sol–gel method. Series of the samples (BSA)xV2O[Formula: see text]H2O, where [Formula: see text], 0.01 and 0.001, were originally produced by the synthesis of vanadium pentoxide xerogels and subsequent blending with water-dissolved BSA in appropriate molar ratios. It was evident that the gelation process does not occur for [Formula: see text]. For the X-ray photoelectron spectroscopy (XPS) studies, the thin layers of these materials were prepared by drying the gel onto the glass and mica substrates. The surface morphology of the samples was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM) techniques. It follows from the analysis of experimental XPS spectra of (BSA)xV2O[Formula: see text]H2O that the nitrogen ions in pure albumin and in (BSA)[Formula: see text]V2O[Formula: see text]H2O are present in imine, amine and protonated amine groups. The additional protonated amine arises when the concentration of albumin in (BSA)xV2O[Formula: see text]H2O is low ([Formula: see text]). Increasing the amount of albumin results in decrease of the number of oxygen ions bonded to vanadium. At the same time (with increase of albumin), the component of oxygen bounded to carbon and nitrogen is increasing. In the samples with greater amount of albumin, the reduction of vanadium ions occurs. This means that the trivalent and tetravalent vanadium ions are present together with pentavalent ones.

Multivariate DoE Optimization of Asymmetric Flow Field Flow Fractionation Coupled to Quantitative LC-MS/MS for Analysis of Lipoprotein Subclasses

In this report we demonstrate a practical multivariate design of experiment (DoE) approach for asymmetric flow field-flow fractionation (AF4) method optimization using separation of lipoprotein subclasses as an example. First, with the aid of commercially available software, we built a full factorial screening design where the theoretical outcomes were calculated by applying established formulas that govern AF4 channel performance for a 5–35 nm particle size range of interest for lipid particles. Second, using the desirable ranges of instrumental parameters established from theoretical optimization, we performed fractional factorial DoE for AF4 separation of pure albumin and ferritin with UV detection to narrow the range of instrumental parameters and allow optimum size resolution while minimizing losses from membrane immobilization. Third, the optimal range of conditions were tested using response surface DoE for sub-fractionation of high and low density lipoproteins (HDL and LDL) in human serum, where the recovery of the analytes were monitored by fraction collection and isotope-dilution LC-MS/MS analysis of each individual fraction for cholesterol and apolipoproteins (ApoA-1 and ApoB-100). Our results show that DoE is an effective tool in combining AF4 theoretical knowledge and experimental data in finding the most optimal set of AF4 instrumental parameters for quantitative coupling with LC-MS/MS measurements.

Effects of protein on the detection of chlorobenzenes in fish and their elimination by modified QuEChERS method

Molecular fluorescence spectrometry, resonance Rayleigh scattering and gas chromatography-mass spectroscopy( GC-MS) were used to study the effect of pure fish serum albumin(FSA) as the model protein on the extraction of three chlorobenzenes(CBs: 1,3-DCB, 1,2-DCB and 1,2,4-TCB; DCB=dichlorobenzene; TCB=trichlorobenzene) in fish samples. The results show that there was a strong binding effect between CB and FSA. In an aqueous solution of 90%(volume fraction) acetone, a slow but full protein denaturation might take place, which would cause the unfolding of protein and the releasing of CBs. Based on these results, a QuEChERS(quick, easy, cheap, effective, rugged and safe) method was modified by replacing the traditional acetonitrile with acetone aqueous solution in the present work. This modified QuEChERS method was applied in the determination of CBs in fish samples. The spiked recoveries and the limits of detection were 80.4%–118.3% and 2.4–7.3 ng/g, respectively. This paper proposes a new strategy by slowing down the protein denaturation and releasing bound organic compounds to enhance the extraction efficiency of CBs in fish samples.

Abstract 492: Circulating Hydrogen Sulfide is Stored as Labile Sulfurs in Albumin

Despite several methods to measure H 2 S, physiological H 2 S serum concentrations remain highly variable and method-dependent. While modifying the classic methylene blue (MB) method to improve detection of serum H 2 S levels, we also probed potential sulfur-binding characteristics of serum albumin. To avoid loss of serum H 2 S and to trap sulfide ions effectively in the MB method serum was strongly alkalized to completely ionize gaseous H 2 S, followed by incubation with zinc acetate at 37°C, to trap sulfide ions effectively. After incubation, N,N-Diethyl-p-phenylenediamine and FeCl 3 were added to form MB product. Subsequently, trichloroacetic acid was added to remove matrix effects. Absorbance of MB product was at 670nm and compared to a dilution range of NaHS. Spectra (400-900nm) were taken to check quality of MB product and exclude matrix effects. Adaptations in experimental setup are mentioned below. Applying the modified MB method produced the unexpected finding that yield of serum H 2 S increases with higher pH and longer incubation time. H 2 S synthesizing enzymes cystathionine-β-synthase and cystathionine -lyase were both ruled out as sources of serum H 2 S by separately treating serum with their respective inhibitors hydroxylamine and DL-propargylglycine. Human serum protein standard containing only albumin and -globulin, or pure bovine serum albumin (BSA), also yielded H 2 S. Albumin was confirmed as source of H 2 S by absence of increasing H 2 S yield from analbuminemic rat serum, as compared to normal rat serum. That yield of H 2 S increased at least twofold after treating both BSA and human serum with dithiolthreitol (DTT), a disulphide reducer, and close correlation between albumin level and yield of H 2 S during DTT incubation (r 2 =0.99), both suggest H 2 S storage in albumin as “labile sulphurs”. The modified MB method allows measurement of release of H 2 S from albumin. Treating albumin with a disulphide reducer revealed H 2 S storage in albumin as sulfanes. Our work is consistent with suggestions that circulating 'pools' of biologically relevant sulfur/sulfide species exist. Thus, albumin may be a source of H 2 S, as has been observed for NO. How this is affected by disease states (e.g. chronic renal failure) remains to be seen.

P45 Circulating hydrogen sulfide is stored as labile sulfurs in albumin

Introduction Although several methods to measure hydrogen sulfide (H 2 S) are available, the physiological baseline H 2 S serum concentrations remain highly variable and method-dependent. While modifying the classic methylene blue (MB) method in order to improve the detection of serum H 2 S levels, we also probed the potential sulfur-binding characteristics of serum albumin. Methods The main modifications in the MB method in order to avoid loss of H 2 S were that serum was strongly alkalized such that all gaseous H 2 S was completely ionized and that alkalized serum was incubated with zinc acetate at 37 °C, in order to trap sulfide ions more effectively. After the incubation, N,N-Diethyl-p-phenylenediamine and FeCl 3 were added to form the MB product. Subsequently, trichloroacetic acid was added to remove matrix effects. Absorbance of MB product was at 670 nm and compared to a dilution range of NaHS. Spectra (400–900 nm) were taken from samples to check for quality of MB product and to exclude matrix effects. Adaptations in experimental setup are mentioned in the results. Results Initial observations from our modified MB method produced the unexpected finding that the yield of serum H 2 S increases with higher pH and with longer incubation times. The H 2 S synthesizing enzymes cystathionine- β -synthase and cystathionine γ -lyase were both ruled out as sources of serum H 2 S by separately treating serum with their respective inhibitors hydroxylamine and DL-propargylglycine. Applying the modified MB method on a human serum protein standard containing only albumin and gamma-globulin, or on pure bovine serum albumin (BSA), also yielded H 2 S. The absence of increasing H 2 S yield from analbuminemic rat serum, as compared to normal rat serum, further confirmed that albumin acts as a source of H 2 S. To investigate whether H 2 S is stored in albumin as “labile sulfurs”, both BSA and human serum were treated with dithiolthreitol, a disulfide reducer. The yield of H 2 S increased at least twofold during incubation. There was close correlation between albumin level and the yield of H 2 S during incubation ( r 2 = 0.99). Conclusion The modified MB method allows the measurement of the release of H 2 S from albumin. Treating albumin with a disulfide reducer revealed that H 2 S is stored in albumin as sulfanes. The current work is consistent with previous suggestions that various ’pools’ of biologically relevant sulfur/sulfide species exist in the circulation [1] . Thus, albumin may be a potential source of H 2 S in the circulation, as has also been observed for nitric oxide (NO) [2] . How this relationship is affected by disease states (e.g. chronic renal failure) remains to be seen.

Effect of SiC Nanoparticles on Thermal and Oxygen Barrier Properties of Albumin Bovine Protein

Albumin bovine/SiC nanobiocomposites were prepared by solution technique with variable percentage of silicon carbide loading. The interaction of SiC nanoparticle with albumin bovine nanobiocomposites was confirmed by Fourier transformed infrared (FTIR) spectroscopy. The structure of albumin/SiC composites were investigated by X-ray diffraction (XRD) and Transmission Electron Microscopy. Thermal stabilities of albumin bovine/SiC nanobiocomposites were studied by thermogravimetric analysis (TGA). It was noticed that albumin bovine/SiC nanobiocomposites were more thermally stable as compared to pure albumin bovine. A substantial reduction in oxygen permeability was obtained with increasing silicon carbide concentrations by which the synthesized nanobiocomposite may be applied as packaging material.

Fretting corrosion with proteins: The role of organic coating on the synergistic mechanisms

Fretting corrosion is one of the most deleterious mechanisms for the degradation of metallic biomaterials, especially in the orthopedic field. This work is dedicated to studying the synergistic effect of proteins and ionic concentration on the wear of 316L stainless steel against a polymer sample under fretting-corrosion conditions. A specialized device produces repeatable fretting corrosion conditions. In order to compare with previous investigations, the relative displacement was chosen to be ±40μm in a sinusoidal pattern. The duration of each test was 14,400s. 4 solutions of NaCl were selected in order to study the effect of the ionic strength: 10−3molL−1, 10−2molL−1, 10−1molL−1 and 1molL−1. In addition to NaCl, the following concentrations of proteins (in this case, pure albumin) were considered: 0, 1 and 20gL−1. Experiments were carried out at a temperature of 22±1°C. Measurements were obtained at open circuit potential, (OCP). During other experiments, the applied potential was equal to −400mV/SCE and the current density was measured. An integrated analytical approach was used in order to elucidate the synergy between mechanics and corrosion. The most interesting finding is that albumin changes the mode of synergy. At 0gL−1 of albumin, the main synergistic term is the influence of mechanics on corrosion. On the contrary, at 20gL−1, the main synergistic term is the influence of corrosion on mechanics.

Effect of nanoclay on morphological, thermal, and barrier properties of albumin bovine

The bionanocomposite of albumin bovine with nanoclay (albumin/clay) has been successfully prepared by solution technique using CuSO4/glycine chelate complex as the catalyst with various percentage of nanoclay loading. The uniform dispersion of nanoclay with the albumin bovine matrix was achieved by continuous sonication technique. The micro structural interactions of albumin bovine with nanoclay were evidenced by Fourier transform infrared spectroscopy. The structure of the albumin bovine/clay composites was investigated by X-ray diffraction and high resolution transmission electron microscopy. The thermal stability of the albumin/clay composites was more than that of the virgin albumin and remarkable increase in thermal property was obtained with increase in clay content. The oxygen barrier property of albumin/clay composites was measured and it was observed that the oxygen permeability was substantially reduced due to increase in nanoclay loading. The biodegradable property of albumin and its composites were measured. It was noticed that the pure albumin was degradable in 5 days whereas the albumin/clay bionanocomposites were stable up to 180 days. POLYM. COMPOS., 2012. © 2012 Society of Plastics Engineers

Study of antifouling modified ultrafiltration membrane based on the secondary treated water of urban sewage

Mixtures of polyvinylidene fluoride (PVDF) and polyvinyl alcohol (PVA) containing hydrophilic ultrafiltration membranes were prepared by adding PVA (5 to 30%) to PVDF by the phase inversion method. The hydrophilic contact angle (CA), equilibrium water content, pure water flux and bovine serum albumin retention were studied to assess the membrane performance. The anti-fouling performance of modified membrane to the secondary treated water was evaluated by flux decline, washing recovery rate and fouling resistance analysis. Scanning electron microscopy showed that the cross-section structure of the membranes had finger-like pores, which were well developed and uniformly distributed, and the sub-layer structure was looser and more porous with the increasing content of PVA. The CA gradually decreased. The steady flux was 800 L/m(2) h from P15 to P30, and the BSA retention sharply declined. The ultrafiltration tests for secondary treated water indicated that the main fouling source of the modified membrane was the concentration polarization and cake layer resistance. After physical flushing, the flux recovery ratio of the membrane could reach 100% when the PVA content was 5-15%, which shows excellent anti-pollution performance and good prospects for use in processing wastewater from urban sewage.

Isotherm Type Shift of Hydrophobic Interaction Adsorption and its Effect on Chromatographic Behavior

Adsorption isotherm type is a key factor in simulating chromatographic profiles. In this study, the adsorption isotherm type of pure bovine serum albumin (BSA) and immunoglobulin G (IgG) on hydrophobic interaction chromatography (HIC) media was found to shift from Freundlich to Langmuir with the increase of salt concentration. For BSA on butyl-, phenyl- and octyl-sepharose, the isotherm type shift occurred when the salt concentrations were higher than 1.8, 1.5 and 1.4 mol/L, respectively. For IgG, the turning points were 1.0 mol/L on phenyl-sepharose and 0.9 mol/L on octyl-sepharose. Circular dichroism spectra and high-performance size-exclusion chromatography found no obvious conformational change or oligomer formation for the proteins in the solutions with different salt concentrations. HIC profiles of BSA and IgG revealed that the isotherm type shift greatly affected the chromatographic behavior, because the original single peak was coincidentally split into two peaks at the salt concentrations over which the isotherm type shift occurred. Combining both the isotherm type shift and peak-splitting phenomena, it was possible that the change of protein-protein repulsion among adsorbed protein molecules under different salt concentrations caused the abnormal behavior of adsorption isotherm and chromatographic profiles.

Purification of IgG and albumin from human plasma by aqueous two phase system fractionation

The current shortages in human plasma products at global levels justify the development of new, cost effective plasma fractionation methods. We have developed a fractionation process to obtain immunoglobulin G (IgG) and albumin-enriched fractions based on polymer-salt aqueous two phase system (ATPS). A small-scale (0.02 L) ATPS composed of polyethyleneglycol 3350 (PEG), potassium phosphate and sodium chloride, at pH 6.1, was evaluated and subjected to 50-fold scale-up (1 L). Further purification of the fractions was performed using caprylic acid precipitation and ion exchange chromatography. Similar yield and purity were obtained at both small and large scales. IgG precipitated in the PEG rich upper phase at 83% recovery and 2.75-fold purification factor. An 81% pure albumin fraction was obtained in the salt rich bottom phase with a 91% yield. After polishing, IgG was obtained at a recovery of 70% and a purity of 92%. Corresponding values for albumin were 91% and 90%. This IgG and albumin fractionation technology deserves further evaluation as it may represent a potential alternative to conventional plasma fractionation methods.