Human serum albumin elution

The resolution of these columns for protein mixtures, however, was comparably poor. The peak capacity for human serum albumin was near 3 during 20 min gradient elution. Improvement has been reached by covalent binding of PEI (M = 400-600) onto a 330 A silica of 5 pm particle size [38], The peak capacities of ovalbumin and 2a -arid glycoprotein were 30-40 (tgradienl = 20 min). Enhanced peak capacity and resolution probably were due to the more diffuse structure of PEI coupled to silane moieties than that of strictly adsorbed on silica and cross-linked (see Sect, 2.2). Other applications of covalently adsorbed PEI are discussed in Sect. 4.1. 

Fig. 2. Gel filtration profile of I-labeled human prolactin, [ I]hPRL (VLS No. 3) io-dinated by the chloramine-T method and purified on a 1.5 x 30 cm Sephadex G-100 column, precoated with bovine serum albumin. Elution solvent was 10 mM phosphate buffer, pH 7.5, containing 0.15 M NaCl and 1 10,000 Merthiolate. The labeled hormone was purified just prior to use. The talc-resin-trichloroacetic acid (TCA) test results showed that peak III material was suitable for use in radioimmunoassay (Tower et al. ).

Human serum albumin and bovine serum albumin are closely related proteins and, consequently, the chromatographic properties of the CSPs based on these proteins are similar. The only difference between the two phases appears to be due to inherent differences in stereoselectivity between HSA and BSA. For example, on the HSA-CSP (S)-warfarin elutes before (K)-warfarin, whereas on the BSA CSP the opposite elution order is observed (85). This is consistent with the enantioselectivities of the native proteins (106). However, even though there are differences between the CSPs, the selectivity, mobile-phase effects, and chromatographic properties of the HSA CSP and BSA CSP are so similar that the two phases will be discussed together. 

Fig. 1 Examples of affinity chromatography with an epoxy-immobilized polyclonal anti-human serum albumin (HSA) antibody in a 2.1-mm-inner diameter X 30-mm POROS CO column run at 5 mL/min (8000 cm/h) using phosphate-buffered saline for loading and 12 mM HCl with 150 mM NaCl for elution. The sample was 10 jig HSA at 1 mg/mL. (a) shows the first five analyses of a relatively pure sample of HSA, where the first small peak is the unbound contaminant and the larger peak is the elution of the HSA from the affinity column, (b) shows the results of the last 5 analyses from a set of 5000 and (c) shows the calibration curve before (squares) the 5000 analyses and after (triangles).

Immobilized antibodies have also been used extensively in multidimensional liquid chromatography (MDLC) analyses. As shown in Fig. 2, an affinity column with immobilized anti-HSA is used to capture all of the human serum albumin in a sample, allowing all of the other components to flow through to waste. Then, the affinity chromatography column is eluted directly into a size-exclusion column where albumin... 

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. 

Flo. 6. EActs of changiiig n ative counterions. Proteins were cytochrome c (100/ig) human serum albumin (320/ and /Mactoglobulin B (100/ig). (A) The elution pattern of these proteins as chromatograph on a Bakerbond wide-IxMc column in 250 m3/iV-ethyl>... 

Figure 25. Chromatography of serum albumins on DEAE-cellulose using stepwise elution. The starting buffer was 0.005 M sodium phosphate buffer pH 6 2 followed by 2, 0.0175 M sodium phosphate pH 6.2 3, 0.075 M NaCl in 0.0715 M phosphate bufier pH 6.2 4, 0.2 M NaCl in 0.0175 M sodium phosphate pH 6.2. A Goat serum albumin. B Horse serum albumin. C Pig serum albumin. D Human serum albumin. From Habeeb (unpublished work).

Figure 27. Gel filtration of tryptic hydrolysates of various serum albumins. The hydrolysate (0.3 g) was applied onto a column (2.7 x 80 cm) of Sephadex G-7S and eluted with 0.01 M HN4HCO3. Fractions (7.5 ml) were analyzed continuously by an ultraviolet monitor. Tryptic hydrolysates of (A) goat serum albumin, (B) horse serum albumin, (C) pig serum albumin, and (D) human serum albumin. From Habeeb (1978a).

Figure 5 An example of a zonal elution experiment, in which small injections of L-tryptophan are made on to an immobilized human serum albumin column in the presence of increasing amounts of phenytoin in the mobile phase.

Fig. 10. Elution patterns of protein (A280) and total cholesterol (A550) for human serum. Column G5000PW+G3000SW+G3000SW+G3000SW. Sample (A), 20 pi of whole serum (B), 20 pi of the total lipoprotein fraction (d<1.210). Flow rate 1.0 ml/min for eluent 0.15 M NaCl), 0.35 ml/min for enzyme solution (Determiner TC"555"). Reaction temperature 40°C. Peaks 1, LDL 2, HDLg 3, HDL3 4, human serum albumin.

Radioiodide has also been separated from iodine-labeled proteins (human serum albumin and Bovine IgG) by thin-layer gel filtration on Sephadex G-200 and G-75 superfine layers (163). (Sephadex is a modified dextran.) The protein solutions were applied in amounts of 5-10 vJL to the Sephadex layers and the angle of slope of the layers was 15°. Elution was carried out with 0.2 M Tris buffer (Tris = tris (hydroxymethyl) aminomethane) at pH 8.0. The buffer contained Tween 80. Separations took 45-60 min on Sephadex G-75 and 3-4 h on Sephadex G-200 and were particularly good on the latter. 

Fig. 4-6. Elution diagram for process scale SEC of human serum albumin on Sephacryl S-200 HR. Sample application at an optimum interval of 40 min will make maximum use of the available column separation space. The albumin is collected in a volume of approximately 0.15 V,. (Courtesy Bergldf et al. [19].)...

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