Human serum albumin separation from

B. Subfile and N. Thuaud, Determination of tryptophan-human serum albumin binding from retention data and separation of tryptophan enantiomers by high-performance liquid chromatography, J. Litj. Chromatogr., 3 299 (1980). 

Figure 5. Separation of human serum albumin (HSA) from bovine serum albumin (BSA) on anti-HSA-silic HPAC column, anti-HSA-silica (4 mg/g), 10 cm X 0.5 cm sample applied, a mixture of HSA and BSa< (each -50 ug) flow-rate, 1.5 ml/min starting solvent, 0.1 M sodium phosphate buffer (pH 7.5) eluant (A). glycine-HCl buffer, 0.2 M, pH 2.2. The protein content was monitored at 280 nm. Reproduced fronjj (17) by permission.

Proteins have, to date, only rarely been purified by SMB. The first attempt was made by Huang et al. in 1986 [42]. They isolated trypsin from porcine pancreas extracts using an SMB made of only six columns. In addition, this example also demonstrates that SMB systems with a very limited number of columns can be efficient. Another example for a successful protein-separation by SMB is the purification of human serum albumin (HSA) using two SMB-systems connected in series [43]. The first SMB was used for removing the less strongly retained components and the second one for removing the more strongly retained components of the sample matrix. 

Proteins, amino acids bonded through peptide linkages to form macromolecular biopolymers, used as chiral stationary phases for hplc include bovine and human serum albumin, OL-acid glycoprotein, ovomucoid, avidin, and cellobiohydrolase. The bovine serum albumin column is marketed under the name Resolvosil and can be obtained from Phenomenex. The human serum albumin column can be obtained from Alltech Associates, Advanced Separation Technologies, Inc., and J. T. Baker. The a1-acid glycoprotein and cellobiohydrolase can be obtained from Advanced Separation Technologies, Inc. or J. T. Baker, Inc. 

Protein samples used to conduct a protein transfer were bovine serum albumin (BSA, 67,000 daltons), chicken egg ovalbumin (Ovalb, 45,000 daltons), and human serum cryo-precipitate. These agents were obtained from plasma containing a mixture of proteins including Fibrinogen (340,000 daltons), human serum albumin (HSA, 67,000 daltons), and immunoglobulin G (IgG, 47,000-56,000 daltons). The cryo-precipitate was diluted with 20 ml of buffer solution prior to separation. 

The specific interaction of Cibacron Blue and its derivatives to dinucleotides, mainly to NAD, NADP and ATP offers the possibility of purifing all enzymes which are dependent on these coenzymes. According to Mosbach < - > there are 163 enzymes requiring NAD, 141 enzymes requiring NADP, about 40 enzymes requiring NADP or NAD and 225 enzymes dependent on ATP. Besides these specific interactions non-specific interactions of Cibacron Blue and its derivatives with. proteins can also be applied for separation purposes. The non-specific interaction of Cibacron Blue with human serum albumin, for instance, enables albumin to be removed from transferrin, ceruloplasmin or other plasma proteins in order to purify human... 

Fig. 2. pH Activity curves of two anodal-moving components separated from concentrated human gastric juice in the Tiselius apparatus. , Major, faster component, mobility 2.99 X 10— cm /volt/sec o, minor, slower component. Electrophoresis was carried out in 33 mM acetate buffer, pH 2.5. Protein concentration, 0.51 g/100 ml. Human serum albumin as substrate temp. 37° time 3 hr. From Taylor (T15). 

There are several reports about the application of the disk monolithic columns for the separation and purification of plasma proteins. " Purification and monitoring of clotting Factor IX (FIX) from human plasma were performed using the ion-exchange monolithic disks. In addition, separation of vitronectin from FIX was possible. A similar system was used for the separation of a complex between clotting Factor VIII and von Willebrand factor (FVIII-vWF). Another application was the monitoring of oti-antitrypsin production. Separation of impurities such as human serum albumin and transferrin was achieved in a few minutes. 

Ohlson et al. (05) were the first to describe the combination of a bioaffinity support and HPLC. They used a silica support coated with amino-hexyl AMP groups to separate horse liver alcohol dehydrogenase and porcine lactate dehydrogenase. Antibodies to human serum albumin bonded to silica separated bovine from human serum albumin within 5 minutes. The recovery of human serum albumin added to the column was quantitative (>95%). 

Sample preparation Separate the buffer containing the drug from human serum albumin by centrifuging at 37° at 700 g for 3 min using a Micropartition System MPS-1 (Amicon) unit, iiyect a 10-20 p-L ahquot of the ultrafiltrate. 

It was shown that the chromatography method was suitable to separate recombinant EPO from amounts of human serum albumin commonly present as a stabilizer in various pharmaceutical preparations. In addition, it was possible to obtain different elution profiles for EPO products with variations in the glycoforms. Fluorescence detection was applied for quantification and showed linear signals over the range of 10-200-pg/mL EPO. 

FIGURE 22.5 CZE electropherograms of rhEPO alpha formulated with human serum albumin (HSA), (a) without immunodepletion of HSA, (b) after HSA immunodepletion. Separation conditions Uncoated fused-silica capillary 87 cm x 50 p,m i.d. Separation buffer 0.01 M Tricine, 0.01 M NaCl, 0.01 M sodium acetate, 7 M urea, and 3.9 mM putrescine, pH 5.5 pressure injection 30 s at 0.5 psi 25 kV temperature 35 C detection at 214 nm. (Adapted from Lara-Quintanar, P. et al., J. Chromatogr. A, 1153, 227, 2007. With permission.)... 

The first industrial application of SEC for protein solutions were for desalting dairy products (66). Large columns (2500 liters) were used to separate proteins in whey or skim milk from low-molecular-weight sugars and salts. SEC is also used in the deethanolization of human serum albumin (HSA) (67) produced by the Cohn cold ethanol procedure. The purification of insulin was the first successful industrial application of SEC for protein fractionation (68), followed by the fractionation of HSA proteins (69). 

---