Overview of Albumin and Its Purification Methods

2016 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers. Adv Pharm Bull, 2016, 6(4), 495-507 doi: 10.15171/apb.2016.063 http://apb.tbzmed.ac.ir Advanced Pharmaceutical Bulletin


Introduction
The name of albumin protein is taken from Albumen (etymologically goes back to Albus). 1 There exist different types of albumin, including ovalbumin, human serum albumin (HSA), and bovine serum albumin (BSA) which are described briefly in Table 1.Albumin is the most predominant circulating protein in healthy adults (normal physiological concentration is 0.6 Mm).It is synthesized in the liver and it causes 80% of plasma colloid osmotic pressure (COP).Since last century, due to critical physiological and biopharmaceutical function of albumin, efforts have been made to achieve high pure and qualified albumin in order to be utilized in therapeutic and research approaches.Human albumin has the highest demand among other biopharmaceutical solutions.Currently, the annual request of albumin is guessed approximately 500 metric tons in the world. 2 Till now various researchers and teams have tried to innovate new albumin production methods.Traditional techniques such as fractionation have been developed and currently diverse chromatographic techniques are used to attain albumin with high yield and purity.So, in the following review, different methods of albumin purification, production and cons and pros of each of them will be discussed.

Albumin Structure
Albumin is a single chain protein with low molecular weight (66/5 kDa) which containing 585 amino acids.It is a simple protein, non-glycosylated polypeptide, hydrophobic patches/cavities, and it lacks prosthetic groups.Human albumin gene is located on chromosome 4 q (11-22) and mutations of this gene will end in anomalous protein.4][5][6] Albumin structure is composed of three domains which are homologous in structural features (this has been elucidated by using Xray crystallography) (Figure 1).The structure includes domains I in residues 1 to 195, domain II in residues 196 to 383 and domain III residues 384 to 585.Each domain consists of 2 identical subunits (A and B) and is composed of 4 and 6 α-helices consecutively.In X-ray crystallography, albumin was displayed as a heart-shaped tertiary in the human body and is containing 17 pairs of disulfide bridges.In the structure of albumin, there exists only one free cysteine (Cys34 is unpaired).Its approximate dimensions are of 80× 80 × 30 Å and about 68% α-helix (any β-sheet). 1,7zymatic properties Human albumin has interesting enzymatic properties including: esterase activity, enolase activity, effects on eicosanoids, aryl acylamidase activity, stereospecificity, condensation reactions, binding and activation of drug conjugates.Also, some of enzymatic properties of albumin-ligand complexes are as follows: heme-and hemin-human albumin, human albumin and Buckminster fullerene, inactivation of reactive oxygen and nitrogen species, metalloenzymes constructed using albumin, lipid peroxide peroxidase activity and nanoparticles.

Albumin synthesis
Albumin synthesis occurs in hepatocyte cells, but isn't stored by the liver.Once produced, it is secreted into the portal circulation. 9The normal concentration of albumin is 3.5-5 g/dl in healthy adults and 2.9-5.5 g/dl in children.Nearly 35 percent of the total body albumin is exist in the intravascular compartment.The rate of synthesis is approximately 12-25 grams per day.Its biological half-life is approximately 19 days.Routinely albumin turnover occurred around 14 grams in a normal 70 kg adult which is approximately 50 percent in the muscles and skin. 10,11Among factors that modify albumin metabolism and leads to its reduced synthesis, can be noted to decreased gene transcription (such as trauma, sepsis, hepatic diseases, diabetes, decreased growth hormone, decreased corticosteroids) and ribosome disaggregation (like protein depletion, fasting). 12Hypoalbuminemia, which is described as insufficient low levels of HSA in the blood is a common symptom of ill patients.This symptom is not only a result of reduction in albumin synthesis, but related to the breakdown, protein uptake and leakage to the extravascular space. 13,14These conditions are created in sub-conditions such as liver and kidney, infections, AIDS, lymphoma, cancer, poverty nutrition, surgery, burn damage, chemotherapy, spontaneous bacterial peritonitis, taking medication, prematurity infant, acute respiratory distress syndrome, chronic respiratory diseases, acute necrotizing, infant brain hemorrhage, hydrops fetalis, systemic inflammatory response syndrome, sepsis and infant edema.[17][18][19][20]

Functions
Albumin is known as a multifunctional plasma protein.It is the main regulator of plasma oncotic pressure.Beside this, any alteration in the oncotic pressure can lead to the stimulation of HSA synthesis.It can bind to different molecules due to its tertiary structure.Such molecules can include metabolites, gases or exogenous substances such as drugs.There has been no evidence about presence of HSA receptor besides only a receptor-mediated endocytosis mechanism is assumed for loaded HSA.It also acts as a chaperone molecule which helps promoting the folding of various proteins and prevents the formation of aggregated proteins related to diseases. 8,21,22lasma, as a place which is exposed to different types of oxidative stresses, encompasses the HSA.This protein is well-known for its anti-oxidant activity that encounters varieties of reactive oxygen species (ROS) and reactive nitrogen species (RNS).It also reduces the production chance of reactive species by binding to the major cause of free radical production, free Cu (II).4][25] There exist other less-known properties of HSA such as anti-coagulant and antithrombotic functions.It also leads to delayed aggregation of platelets by binding to Nitric oxide (NO) and inhibiting its inactivation.Like other plasma proteins, HSA, due to its long half-life, is prone to glycation. 26Glycated HSA will cause irremeable damages in different diseases.Studies also reported that HSA protects other proteins from glycation.HSA is also supposed to be responsible for membrane permeability.The hypothesis of mechanism is due to binding of HSA to subendothelium and interstitial layers and then altering their permeability.HSA is reported to have neuroprotective function on neuronal and glial cells and regulates brain circulation.Experimental ischemic and Alzheimer's disease model indicates HSA administration, which can play a neuroprotective and anti-oxidant role.This function is mainly related to anti-oxidant properties of HSA.In vitro studies on hepatocytes or liver microsomes of rats indicated that HSA is increased with aging only in male rats.In human, sex-related HSA amount is doubted but there is a fact that before age 14, plasma protein levels are sex-independent and the variation is only occur between ages 18-24.[29][30][31][32][33][34]

Applications
Serum albumin is utilized under various clinical conditions.Restoration of blood volume, emergency treatment of shock, acute management of burns, and other situations associated with hypovolemia are some of the clinical applications of albumin.6][37][38][39] In addition, albumin has different applications in the related research field, including cryopreservation, stabilizer of some of the proteins and as a supplement in cell culture.Novel application of HSA is consisted of a fusion of peptides, a drug nanocarrier and an oxygen transporter. 2,11,40erapeutic formats Clinical indication of albumin is done in two forms.The first one is albumin 4-5% (near iso-oncotic solution) which leads to intravascular volume expansion, and the second one is albumin 20-25% (hyper oncotic solution) which is applied in the restoration of COP and fluid balance preservation among compartments. 41Injection of these 5%, 20%, or 25% albumins is used for hypoalbuminemia treatment which is sterilized under 60 ± 0.5°C for 10-11 h to viral inactivation.[44][45] Indications Clinical indications of albumin 4-5% solution includes emergency treatment of hypovolemic shock, cardiopulmonary bypass, acute liver failure, sequestration of protein rich fluids. 46,47Clinical indications of albumin 20-25% solution may include hypovolemia, hypoproteinemia, adult respiratory distress syndrome (ARDS), hemolytic disease of the newborn (HDN), acute nephrosis, acute liver failure, renal dialysis, hypovolemic shock, burn therapy, cardiopulmonary bypass, sequestration of protein rich fluids, and erythrocyte resuspention. 48

Contraindications
Although albumin is a valuable therapeutic product in a broad range of disorders, but its administration in some situations may be hazardous.Precede the prescription of albumin, its circulation amount should be checked, the level of more than 2.5 g/dl (hyperalbuminemia) cause contraindication.Cardiac and renal failures, acute or chronic pancreatitis, pulmonary edema or severe anemia because of the risk of acute circulatory overload are the other situations in which albumin is forbidden.In addition, utilization of albumin in patients with nervous system disorders such as cerebral ischemia and traumatic brain injury (TBI) may be dangerous and life threatening.[51][52]

Side effects
Along with the beneficial effects of albumin, it can have a variety of side effects when indicated intravenously.List of these side effects are noted in Table 2. 53,54

Plasma fractionation method with ethyl alcohol
Approximately 60 years ago, Cohn and colleagues developed a protein purification method by using plasma fractionation technique for the first time.This approach is based on albumin solubility difference with other plasma proteins.Comparing to other plasma proteins, albumin sediments in lower pH and higher ethanol concentration.In this method there are five available systems for separation of plasma protein.6][57] Protein separation is a result of the increase in ethyl alcohol concentration occurring in each fraction, so albumin will be seen in the last fraction.This method which is done under bacteriostatic condition, can be used as therapeutic agents.The method is suitable for large-scale and industrial production.But this method has some disadvantages, too; It is not enable to produce high purity product, protein denaturation may occur, and this method needs cold areas. 57

The Cohn method combined with chromatography
The Cohn method was modified by Oncley et al. in 1949 and Kistler-Nitschrnan et al. in 1962 in order to improve albumin purification. 58It is shown in Figure 2.

The Cohn method combined with liquid chromatography
This method at first was described by Tanaka et al.
Large volumes of plasma with low price is fractionated in Chon method, so Victoria and colleagues to raise the purity and quality of the product, added liquid chromatography technique.Due to the benefits of both techniques, this method was considered as an integrated method for the purification of plasma proteins.Among advantages of this method one can point to the low cost and high purified albumin which leads to the promoted product quality.The purified product using the present method is yielded approximately 99% which is higher than Cohn's method.In this method albumin is a byproduct and other important therapeutic proteins including factor VIII, intravenous immunoglobulin (IVIG) and some other proteins are isolated from plasma.This method is used for industrial production of plasma proteins in the world. 59

Purification from placenta
Another method which has been developed for production of albumin is purification from human placenta.Joaquin Cabrera-Crespo and colleagues described this method which was performed using solvent precipitation (with ethanol) and ion exchange chromatography.Since human placenta collection is available, purification from placenta is one of the most efficient ways of purification of albumin.Among the advantages of the mentioned technique, one can point to the reduction in the volume of resin used in the chromatographic process in comparison with the isolation processes.They have acquired albumin of placenta with 97.1 percent purity. 60,61finity precipitation (using a thermo-response polymer attach with an L-thyroxin ligand) Currently, Cao and Ding reported affinity precipitation process for purification of plasma proteins (such as albumin).It is performed by using reversibly soluble-insoluble polymers combined with an affinity ligand which depends on the polymer features such as temperature, pH and light response.With using N-methylol acrylamide, butyl acrylate and N-isopropyl acrylamide as monomers, a thermoresponse copolymer was synthesized which was called P NBN .Then, L-thyroxin ligand, as an affinity ligand, was coupled to P NBN and was used to purify albumin from human serum. 62One of the advantages of this new production method is tolerable purity HSA production in a single step.

Heat shock method
Since the albumin is stable in thermal fluctuation comparing to the other proteins of the plasma, it can be purified in heat shock purification method.So, albumin protein resists to increasing the temperature up to 60 °C in which potential pathogen may be inactivated.To obtain albumin from serum, 0.04 Ϻ caprylic acid was used to stabilize in pH 5 at 60 °C.In this state other proteins of the serum are denatured and precipitated in solution.Then albumin concentrated by precipitation and ultrafiltration with purity around 98 %. 57

Ammonium sulfate precipitation combined with liquid chromatography
Another method for albumin purification is ammonium sulfate precipitation combined with liquid chromatography which yields approximately more than 90 percent purified albumin.In this method albumin is separated from immunoglobulins by 50% ammonium sulfate.Then lipids are removed using icecold acetone (in 4 centigrade).In next stage for separating albumin from other proteins (especially transferrin which is the main contaminant), ion exchange and size exclusion chromatography are utilized.DNase and immune detection assays proved that the purified albumin was free of any DNases and immunoglobulins.Eventually, purified albumin was evaluated using western blotting and chemiluminescence. 63Steps of the aforementioned method are shown in Figure 3.

TCA/Acetone precipitation method
Although many low-abundant serum proteins play an important role in disease detection as a biomarker but some of them such as albumin which exists in high amount is an obstacle for detection.For better detection, we can eliminate albumin and enrich the serum for low-abundant proteins.In order to remove albumin from plasma for disease detection, Chen et al. developed a precipitation method using trichloroacetic acid (TCA)/acetone in 2005.They claim that their method can be a rapid and large scale albumin purification method.The albumin isolated by this method seemed to maintain its native structure beside other characteristics such as solubility, temperature stability, electrophoretic mobility, dye-binging properties, crystallization characteristics, and immunologic behavior. 64lumn chromatography for the purification of HSA Although the ethyl alcohol fractionation method is used in industry for blood protein`s fractionation, the purity is not sufficient enough. 57So, chromatography might be the best choice for albumin and other protein`s purification.To achieve this goal, there have been variable chromatography techniques which some of their most predominant ones include. 57

Ion exchange chromatography (IEC)
IEC is widely used for albumin 65,66 and other protein production. 67,68Among them the anion exchange method has the most usage. 66Three steps of conventional method for IEC consisting pre-treatment, purification and polishing was shown in Figure 4. Some of the limitations of conventional methods for chromatography such as delayed processing time, low flow rates and etc. which are apparently known for everyone.To cope with these limitations, the new method for ion exchange membrane chromatography (IEM) using ameliorated adsorbers was developed.The simulation studies which compare the IEM with IEC were done by Frerick, et al. 69,70 Comparing to columnbased separations, the membrane chromatography has some advantages such as high separation efficiencies due to shorter diffusion times, [71][72][73] reduction in buffer usage, small floor space requirements, 74 simple method without complex hardware or packing necessities. 75In spite of this advantages, expensive membrane and low efficiency of this technique for industrial purposes is an important drawback which causes the new technology 7][78] Moreover, membrane capacity to bind antibodies is relatively low in comparison with advanced resins. 78

Simulated moving bed chromatography (SMB)
This is a multi-column method based on reversed-phase chromatography in which the sorbent and solvent consumption is decreased.Among the advantages of this method, increased efficiency (due to the countercurrent between solid and liquid phases), higher purity and yielding amount are the most outstanding.Nevertheless it has two disadvantages; on one hand the number of fractions is not more than two and each product stream contains more than one compound.On the other hand, the limitation of isocratic elution mode is a major problem.To overcome this problem, the pressure, pH and other factors can be modified.This four sectioned method is a promoted form of the "triangle method" by Storti et al. 79

Steric exclusion chromatography (SXC)
The new method for purification of large proteins is the steric exclusion chromatography (SXC) method which is based on PEG-induced precipitation and it is made using crogel monolith column.The polyethylene glycol (PEG) in the mobile phase helps proteins to restrain on the hydrophilic stationary phase.In 2014, Wang et al. reported that the crogels are very useful for protein separation.The advantage of cryogel monolith column compared to polymethacrylate monoliths is having the large pores of aproximtely 10-100 µm which leads to a high flow rate along with low back pressure.Moreover, the retention capacity is supposed to be higher than adsorption-based chromatography.They could purify albumin and also globulin with high purity from bovine serum sample. 80

Expanded bed adsorption chromatography
The traditional fractionation process of plasma (the Cohn`s cold ethanol method) is an isolating method for crucial plasma components.In 2009, Lihme et al. developed a new fractionation process containing five expanded bed adsorption steps using high-density modified agarose/tungsten carbide beads.Through this method, albumin was purified up to 99% purity following a recovery step using 6% agarose-10% tungsten carbide beads coupled with an acidic mixedmode ligand under the acidic condition (pH 4.5).The EBA chromatography method might be an applicable alternative for core plasma fractionation method. 81

Affinity Chromatography
Affinity chromatography is a type of liquid chromatography in which, a biological agent named affinity ligand is used as stationary phase in column.3][84][85] Affinity chromatography was first introduced for the purpose of purification by Cuatecasas, et al. in 1968. 86There are different types of affinity ligands such as protein, enzyme, enzyme substrate or inhibitor, antibody, antigen, hormone, biomimetic dye and etc. Depending on the purpose, selected type of ligand is immobilized within the column and further purification process is done following selective binding. 82Here we describe some: Dye ligand affinity chromatography Another important protein purification method is dyeligand affinity chromatography.The biomimetic monocholoro or dicholorotriazinyl dyes are able to bind most type of proteins, especially enzymes. 87,88It has so many advantages over other methods. 89This method is economically and widely available and its immobilization and scale up is easy.Beside its medium specificity, the capacity is very high.Moreover, the matrix can be stored without losing its activity. 90eneral dyes used in dye-ligand affinity chromatography can be noted to Reactive Red 120, CB F3GA, Congo Red, R. Green HE4BD, Reactive Blue 4, and Mimetic Blue 1 A6XL. 57, Immobzed Metal-ion Affinity Chromatography (IMAC) In 2001, Denizli, et al. developed a novel and new method of purification to increase the protein adsorption capacity.They used the PHEMA beads containing 2-methacryloamidohistidine in metal chelate affinity chromatography.This survey indicated a noteworthy growth in the HSA adsorption capacities from human plasma (up to 94.6 mg/g).Moreover, High desorption ratios (up to 98% of the adsorbed HSA) were noted.It was possible to reuse Cu (II) chelated (HEMA-co-MAH) beads without significant decreases in the adsorption capabilities. 112ronate Affinity Chromatography Boronate as an affinity ligand is used for affinity chromatography for separation.Targets, like carbohydrate-containing compounds, which have cisdiol groups, can bind to the boronate at a basic pH.82 Synthetic dyes are also used as ligands and the method is known as dye-ligand affinity chromatography.For this purpose, triazine dyes are used in columns to purify albumin and various proteins.113,114 Immunoaffinity chromatography (IAC) We applied IAC through Cyanogen bromide (CNBr)activated Sepharose to facilitate albumin purification and improving its purity.Initially, the pure HSA was injected to the three white New Zealand rabbits to produce of polyclonal antibody.Antibody purification was done by IEC and protein G affinity chromatography.Then the purified antibody was attached to the CNBr-activated Sepharose and eventually it used for purification of albumin protein from human serum.Western blotting analysis and heatinduced insolubility were applied for functional and stability assessment of immunoaffinity purified HSA, respectively. 11AC is mentioned as a robust method for albumin purification while joining with appropriate matrix.CNBr may be a most proper matrices for protein purification due to working as an activation reagent.We produced HSA with a purity of 98% through CNBractivated sepharose.The purity of prepared albumin was high and no more differences was shown in heatinduced insolubility at 60-90 °C between purified and commercial albumin.Also IAC is a cost-effective and monophase technique which could be a preferred method for plasma proteins purification.11 Albumin purification methods was summarized in Table 3.

Conclusion
Regarding to albumin protein priority, different methods were applied for albumin purification; Cohn method which combined with chromatographic techniques is the most well-known among them.In this method other worthwhile proteins of plasma such as coagulation factors, immunoglobulins and etc. would be separated in upstream, and albumin will be seen in Advanced Pharmaceutical Bulletin, 2016, 6(4), 495-507 the last fraction.Due to the robustness and high purity of plasma in large volumes, this technique, is costeffective for plasma therapeutic protein production.Hence, this method currently seems to be a major technique in albumin production.The aforementioned methods such as purification from placenta, ammonium sulfate precipitation combined with LC, Heat shock, TCA/Acetone precipitation, different dye ligand affinity chromatographies, Simulated moving bed chromatography, Steric exclusion chromatography, Expanded bed adsorption chromatography and etc for albumin purification are suitable for lab-scale and don't have enough efficacy.As respects, the raw material of these techniques is human serum with related safety limitation.Therefore efforts have been done to produce albumin via recombination technology.

Figure 1 .
Figure 1.Albumin structure.Albumin protein is composed of three domains (elucidated by X-ray crystallography).

Figure 4 .
Figure 4. Purification and polishing methods for human serum albumin (HSA).The purification process consists of two anion and cation exchange chromatography steps.The polishing process also consists of three steps containing two ultrafiltration and one gel filtration steps.

Table 1 .
Types of albumin.

Table 2 .
Side effect of intravenous injection of human serum albumin.

Table 3 .
Comparison of various methods for albumin production.