Albumins: Functions and Applications

Functions:

Albumins serve important functions in the maintenance of the blood plasma system and is an essential component of regulation due to its functions of transporting physiological substances and binding toxic ones. It is the main regulator of the colloid-osmatic pressure of plasma. Albumin constitutes about 4% of the total plasma proteins. Human serum albumin (HSA) is administered to patients in various clinical situations including shock or burn patients for restoring normal blood volume and thereby alleviating certain trauma associated symptoms.

Serum albumin is the most abundant protein in mammalian sera (50 g/l, about 0.7 mM in human) and one of its functions is to bind molecules such as lipids and bilirubin. The half-life of serum albumin is directly proportional to the size of the animal where for example human serum albumin (HSA) has a half life of 19 days and rabbit serum albumin has a half-life of about 5 days.  Serum albumin is devoid of any enzymatic or immunological function and thus should not exhibit undersired side effects upon coupling to a bioactive polypeptide.. HSA is a natural carrier involved in the endogenous transport and delivery of numerous natural as well as therapeutic molecules. Several strategies have been reported to either covalently couple protiens directly to serum albumins or to a peptide or portein that will allow in vivo assocaition to serum albumins. (Ekblad (US14/427102). 

Structure:

Albumin is a single chain protein with low MW of 66.5 kDa which cotnains 585 amino acids. It is a simple protein, non-glycosylated polypeptide, hydrophobic pathces/cavities, and it lacks prosthetic groups. Human albumin gene is located on chromaosome 4 q adn mutation of this gene will end in anomalous protein. ((Rmin “overview of albumin and its purificaiton methods” Mini Review Advanced Pharmaceutical Bulletin, 2016). 

Synthesis:

Albumin syntehsis occurs in hepatocyte cells, but it is not stored by the liver. Once produced, it is secreted into the protal cirulation. The noraml concentraiton of albumin is 3.5-5 g/dl in healthy adults. (Rmin “overview of albumin and its purificaiton methods” Mini Review Advanced Pharmaceutical Bulletin, 2016)

Applications

Serum albumin is utilized udner various clinical conditions. Restoration of blood volumn emergency treatment of shock, acute management of burns, and other situations assocaited with hypovolemia are some of the clinical applicaiton sof albumin. (Rmin “overview of albumin and its purificaiton methods” Mini Review Advanced Pharmaceutical Bulletin, 2016)

Albumin Binding Proteins

Streptococca. protein G has three Ig binding motifs and three serum albumin binding domains. The structure of one of the 3 serum alumin binding domains whos a three helix bundle domain , named “ABD (albumin binidng domain) and is 46 amino acid residues in size. It has been designated as “G148-GA3”. Other bacterial albumin binding proteins other that protin G has been identified. (Ekblad (US14/427102)

Methods of Albumin Purification

Procedures using Ethanol Fractionation

–Cohn Method: The traditional method for the purification of albumin for therapeutic use has been cold ethanol fracitonation, as described by Cohn in 1946 and its later variants. Albumin has some unique properties that allow relatively simple and effective purifcation by precipitaiton methods. It has the highest solubility and the lowest isoelectric point (the pH at which it bears no net charge). Adjustments to pH, temperature, ionic strenght, ethanol concentration and protein concentration thus allow the separation of albumin from the other plasma proteins (Matejtschuk, British J. of Anaesthesia, 85(6): 887-95 (2000). The processes for obtaining albumin from human plasma normally starts with fraction V (FrV) of the alcoholic fractionation of plasma according to the Cohn method. Although less frequent, other starting materials can also be used such as supernatants (S/N) of this Cohn fracitonal, such as the S/N of Fraction IV or S/N of Fraction II+III, including an additional stage of purificaiton such as IEX (Jorquera, US 2011/0137283). 

–Kistler and Nitschmann process: The Kistler and Nitschmann process uses fewer protein precipitation steps and hence less thanol that the Cohn method. With either method, an initial low ethanol precipitaiton stage removeds fibringoen fromt he source plasma. Subsquently, by raising the ethanol concentraiton to 25% at pH 6.9 ofr the Cohn method or 19% at pH 5.85 for the Kistler and Nitchmann method, the immunogloubulins are precipitated while the albumin remains in solution. Albumin is then isolated from the majority of the other plasma contaminants (mainly alpha and beta globulins) which are precipitated by furterh addition of thanol to a final ethanol concentraiton of 40%. This is carried out in two stages in the Cohn process but as a single step in the Kistler and Nitschmann method. In a final step, the albumin is itself precipitated near its isoelectric point. The precipitate paste (fraction V) can be held frozen before futher prcoessing. 

Hao (Vox Sang, 36: 313-320 (1979) diclses a two purificaiton step method for purifying human serum alumin with a higher yield than that of the conventional ethanol prcoedure. Threefold diluted plasma is placed in a water bath at –5C and pH adjusted to 5.6 using acetate buffer. After stirring for 1 hr, precooled -5C 95% ETOH is added to a final concentraiton of 42% (v/v) with stirring. After the pH becomes 5.75, the mixture was continuously sittred for at least 1 phour prior to centifugation. The pH of the peurnatant is adjusted to 4.8 using acetate buffer. After stirring for 1 hr, the mixture was allowed to age without stirring for at least 3 h prior to centrifugation. The precipitate, similar to Cohn fraction V was albumin paste to be used for furtehr processing, whereas the supernatant, which is analogous to Cohn fraction V supernatant could be used for preparing alpha-1-acid glycoprotein as a by product. 

Procedures involving Ion Exchange

IEX is widely used for albumin production. Among them AEX has the most usage. (Rmin “overview of albumin and its purificaiton methods” Mini Review Advanced Pharmaceutical Bulletin, 2016)

–Plasma preparation -AEX:

Curling, Vox Ang, 33, 97-107 (1977) discloses algumin that is obtainable from human blood plasma by IEX in a yeild of about 95% and purity well above Pharmacopoeia requirements. Cryosupernatant, factor IX depleted plasma is preciptiated with 12-25 w/v polyethylene glycol 4000. The second precipitate is dissolved to 8% w/v protein and applied to a DEAE-Sephadex A-50 or a DEAE-Sepharose CL-6B column. Albumin is futher purified by chromatography on SP-Sephadex C-50. 

–Cohn II+III — Remove lipoprotein (e.g., SiO2) — CEX or AEX:

Fisher (US 4,228,154) discloses subjecting a cryosupernatant plasma (Cohn Rfaction II+III or equilvanet) to finely divided lipoprotein extractant, adjusting pH to 4.5-4.9 and contacting with CEX to remov albumin contaminating proteinaceous material.  Alternatively, pH is adjusted to about 5.1-5.5 and contacted with AEX to remove the proteinaceous material having and isoelectric point aove that of albumin. 

Multi-column Chromatography

–IGselect (4 columns)-HIC

Bataille (US 15/125483, published as US 2017/0073396) discloses using a an IgSelect gel from GE Healthcare (ligand si from CAC, BioAffinityCompany) which specifically binds the Fc fragments of human IgGs and  Capture Select FcXL affinity gel from Life Technologies which specifically binds the CH3 domain of the 4 human IgG subclasses on 4 columns, controlled sequentially an an automated system followed by diafiltration concentraiton of the eluate. Elution with the IgSelect was carreid out with 0.1 M glycine solution, pH 3. The IgG depleted plasma is collected (albumin not being bound by the affinity gel) and a salt tolerant mixedmode gel HEA Hypercell is used to capture albumin. 

–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. (Rmin “overview of albumin and its purificaiton methods” Mini Review Advanced Pharmaceutical Bulletin, 2016)

Expanded bed adsoprtion chromatography:

Expanded bed absorption uses high-density modified agarose/tungsten carbide beads has been used to purify alumin. (Rmin “overview of albumin and its purificaiton methods” Mini Review Advanced Pharmaceutical Bulletin, 2016)

Affinity chromatography

There are different types of affinity ligands such as prtoein, enzyme, antibody, antigen, hormone, dyes (Rmin “overview of albumin and its purificaiton methods” Mini Review Advanced Pharmaceutical Bulletin, 2016)

Removal of Albumin associated proteins and metabolites: 

As the principal transporting molcule in the blood, serum albumin has specific binding sites for lipophilic substances such as fatty acids, bilrubin, etc. The majority of albumin ligands bind to one of the two binding sties I or II. Free fatty acids, metallic ions such as copper and bilirubin bind selectively to specific binding domains. (Jorquera, US 2011/0137283). 

–Removal of Lipids: Serum albumin preparations contain variable amounts of lipid impurity. Chen describes treatment of such samples with charcoal at low pH resulted in virtually complete removal of fatty acids (Cehn, J. Biological Chemistry, 212(2), 1967, pp. 178-181). Such impurities present in commerical albumin preparations are known to influence in vitro drug binding parameters. Nakano (analytical Biochemistry 129, 64-71 (1983)) describe activated carbon beads embedded in agarose to produce column beads effective for removal of long chain fatty acids from human serum albumin. 

–Removal of Billirubin: Bilirubin which is a metabolite also has high affinity for serum albumin. The formation of this albumin-bilirubin complex is of great biological importance since a free bilirubin fraction suppresses several major enzymes and metabolic systems of the brain, causing severe toxicity. Nikolaev (International J. Artificial Organs, 14(3), 1991, pp. 179-185) discloses that high activated carbons can be promising not only for plasma adsorption but also for direct contact with whole blood (i.e., hemocarboperfusion). 

Christensen (WO/2007/063129) discloses a large scale process for the isolation of recombinant human serum albumin (rHSA) by contacting a protein solution comprising the rHSA to an adsorbent having a functionalized matrix polymer carrying a plurality of covlantly attached functional groups comprising an aromatic or heteroaromatic ring system and one or more acidic groups or the adsorbent comprises a particle with at least one high density non-porous core, surround by a porous material, optionally washing the adsorbent and obtaining the rHSA from the adsorbent.