Isolation of an Endotoxin Inactivator From Human Plasma, and Methods of Use

FIG. 1--Elution profile from DEAE-Sephadex chromatography (step 1).

FIG. 2--Elution profile from Cibacron-Blue Sepharose chromatography (step 2).

FIG. 3--Elution profile from hydroxylapatite chromatography (step 3).

FIG. 4--Immunoelectropherogram of endotoxin inactivator.

FIG. 5--Polyacrylamide gel slab electropherogram of endotoxin inactivator.

FIG. 6--SDS-PAGE analysis or endotoxin inactivator.

FIG. 7--HPLC elution profile of endotoxin inactivator mixed with pyrogenic albumin.

In the method of the present invention for isolation of this endotoxin-inactivator (EI), it was carried out in a series of steps which are described hereinafter as Example 1.

The starting material can be fresh, fresh frozen, liquid, outdated plasma, cryo supernatant (after cryo precipitate is removed) or serum.

In essence, endotoxin-inactivator can be purified from plasma by three steps: namely, adsorption on DEAE-Sephadex and stepwise elution to remove the bulk of plasma proteins; adsorption on Cibacron-Blue-Sepharose and stepwise elution to remove the main contaminant, albumin; and adsorption on hydroxylapatite and stepwise elution to obtain a highly purified endotoxin-inactivator.

FIG. 1 is the elution profile of protein distribution from DEAE-Sephadex. Dialyzed plasma, 50 ml, was applied to a column, 1.8.times.20 cm. Stepwise elution using 0.07M, 0.15M and 0.25M NaCl was then carried out. Each tube collected 15 ml eluate, and every second tube was assayed for activity. Buffer (0.02M Phosphate, pH 7.35) containing 0.07M NaCl eluted the residual reddish-colored fraction (transferrin) and a yellow-colored fraction, albumin. Buffer containing 0.25M NaCl eluted one protein peak with the inactivator activity concentrated in the ascending part of peak, and a slightly bluish fraction, which is ceruloplasmin, in the descending part of the protein peak. Fractions with inactivator activity were pooled according to their endotoxin inactivating activity as assayed by the LAL test system.

While DEAE-Sephadex Chromatography is preferred, those skilled in the art will recognize that other anion exchange chromatography techinques might prove fruitful. Elution at 0.25M NaCl is preferred, but salt concentrations ranging from 0.2-0.3M may prove useful.

FIG. 2 is the elution profile of protein distribution from Cibacron-Blue Sepharose. Diafiltered DEAE eluate containing EI was applied to the CBS column (1.25.times.20 cm) previously equilibrated with 0.02M phosphate buffer, pH 7.35. Stepwise elution using 0.07M, 0.2M and 2M McCl was then carried out. Each tube collected 10 ml eluate, and every second tube was assayed for activity. At 0.07M NaCl concentration, an inactive protein peak was eluted. At 0.20M NaCl concentration, a second protein peak with inactivator activity was eluted. At 2.0M NaCl concentration, a third protein peak, mostly albumin, was eluted. It should be noted that even though each of these peaks looked more or less symmetrical, it by no means represented only one protein.

It may be possible to substitute other dye affinity chromatographic separations for the preferred Cibracron-Blue Sepharose chromatographic step described herein. While elution at 0.2M NaCl is preferred, it is believed that a range of salt concentrations such as 0.15-0.25M will be efficacious.

FIG. 3 is the elution profile of protein distribution from hydroxylapatite chromatography. Diafiltered CBS eluate was applied to a HTP column (1.times.9.5 cm) previously equilibrated in 0.02M phosphate buffer pH 6.8. In order to obtain good flow rate 2 g of Sephadex G-25 was added to 10 g of HTP. Stepwise elution using 0.14M and 0.3M phosphate buffer, pH 6.8 was carried out. Tubes of 7 ml were collected. Fractions containing EI activity was eluted at 0.14M phosphate concentration at pH 6.8. Another protein peak was eluted at 0.3M phosphate concentration at the same pH.

It will be understood that those skilled in the art may be able to effect a further purification using another adsorption medium in place of hydroxylapatite. While elution at 0.14M phosphate is preferred, those skilled in the art may wish to employ concentrations of 0.1-0.25M phosphate.

Based on the results of 5 runs each starting from 50 ml of fresh frozen plasma derived from 400 liter plasma pool an average yield of 1.4 mg of EI was obtained as assayed by Lowry (J. Biol. Chem. 193: 265, 1951). Since there was no report in the literature stating the concentration of EI in plasma, the percentage of recovery and concentration of EI are calculated as follows. Based on the activity assay, every 0.05 ml plasma inactivates 31.25 ng endotoxin standard (E. coli 055: B5). Therefore, 50 ml plasma would inactivate a total of 31,250 mg. It has also been found that every 0.05 ml EI preparation containing 0.014 mg EI would inactivate 2,560 ng which represents 8.2% recovery and the concentration of EI in plasma could therefore be calculated to contain (31,250 ng/2,560 ng).times.1.4 mg=17 mg for every 50 ml or 34 mg/100 ml.

The purified EI was subjected to immunoelectrophoresis (IEP), polyacrylamide gel slab electrophoresis (PAGE), SDS-polyacrylamide gel slab electrophoresis (SDS-PAGE) and high performance liquid chromatography (HPLC).

FIG. 4 is the IEP result of the purified EI which shows one precipitation line when reacted with anti-whole serum. The conditions of the immunoelectrophoresis of EI were as follows: Top Well, EI, 7 .mu.g; Bottom Well, Plasma (1:2), 5 .mu.g; and Trough, anti whole serum 100 ul.

FIG. 5 is the PAGE result of EI which shows one protein band (5A), midway between albumin and transferrin, when compared to that of plasma (5B). The conditions of the Polyacrylamide gel slab electrophoresis were as follows: Running gel 7.5% and stacking gel 3.5%; Endotoxin inactivator, 6 .mu.g; and Plasma (1: 17), 5 .mu.g.

FIG. 6 is the result of SDS-PAGE, which again shows one protein band (6B) having a molecular weight of approximately 61,000 when calculated from the protein standards (6A). Protein standards (6A) are (from top down): myosin (H chain), 212,000; phosphorylase B, 97,500; bovine serum albumin, 68,000; ovalbumin, 43,000; alpha-chymotrypsinogen, 25,700 and beta-lactoglobulin, 18,400. EI (6B): 24 .mu.g.

FIG. 7 is the HPLC result when EI is mixed with pyrogenic albumin. Based on these findings the molecular weight of EI is the same as albumin (HSA) which is 65,000. The test sample was made up from equal volume of human serum albumin (HSA), 0.15%, and EI, 0.12% in 0.05M phosphate, pH 7.0, containing 0.15M NaCl. The column used was TSK 3000, 7.5.times.300 mm.

Lipopolysaccharide (LPS) derived from Salmonella typhosa, Salmonella enteritides and Escherichia coli 0.55: B5 of 2-fold serial dilutions were mixed with EI and the endotoxins remained in the mixture were assayed by LAL with the sensitivity of the lysate being 0.05 ng/ml. The LAL assay is based on the discovery by Bang, John Hopkins Hosp., 98: 325 (1956) of the gelation of a lysate of amebocyte of Limulus, the horeshoe crab. The results are shown in Table 1. The last column of the table is the total nanogram of LPS inactivated per mg of EI calculated from the results in the reaction mixture (middle column).

Because of the nature of the LAL assay in which LPS solutions were prepared by 2-fold serial dilutions, the EI activity toward each LPS was expressed in a range for the given amount of EI. Although it has been known that LPS derived from one type of bacteria could be as much as 6 times more potent than the other (Greisman and Hormick, Proc. Soc. Exp. Biol. Med. 131: 1154, 1969), the purity of each LPS used in these experiments were not necessarily the same thus resulting, at least in part, in the different ranges of inactivation.

In order to ascertain that the inactivation was not a result of inhibition of the LAL test but as a result of the detoxification of the endotoxins, an aliquot of a highly pyrogenic 25% albumin solution having an endotoxin level of 12.8 ng/ml was divided into two vials, 25 ml each. To one vial, 0.125 mg of EI in 0.89 ml of 0.02M phosphate, pH 7.35, was added, and to the second vial (Control) 0.89 ml of plain buffer was added. Both vials were then incubated at 37 deg. C. for 15 min. and rabbit pyrogen tests (3 ml/kg) were carried out. The results showed that the Control, which was not treated with EI, induced an aggragate temperature rise of 2.1 deg. C. (0.2, 0.9, 1.2 deg. C.) whereas the EI-treated albumin induced an aggregate temperature rise of 0.4 deg. C. (0.0, 0.0, 0.4 deg. C.), clearly suggesting that the endotoxin had been detoxified. (Any parenteral intended for clinical use has to pass the rabbit pyrogen test with an induced temperature rise not greater than a total of 1.5 deg. C. for three rabbits, and none of the three rabbits may have a temperature rise of more than 0.5 deg. C.). The term "essentially nonpyrogenic", as used herein, means a preparation which passes the USP rabbit pyrogen test.

The term "endotoxin inactivator" encompasses substances which merely reduce the pyrogenic activity of an endotoxin sufficiently to render an endotoxin containing composition "essentially nonpyrogenic."

Preliminary results indicate that the activity of EI prepared by this procedure appears to be stable for up to 48 hours at +5 C. Whereas, samples kept at -20 C. and -80 C. remained active even after one month.

Products such as AHF and Factor IX complex are routinely isolated prior to ethanol fractionation. If there is a need of EI, it appears feasible to incorporate the present procedure into the scheme when Factor IX complex is isolated, since the procedure also involves the use of DEAE-Sephadex. (Heystek, et al., Vox Sang, 25: 113, 1973.) After DEAE-Sephadex adsorption and prior to elution of Factor IX complex at 2.0M NaCl, a wash step using 0.15M NaCl elutes most of the albumin which is to be added to the starting plasma for recovery of albumin. An additional stepwise elution of the DEAE-cake at 0.25M NaCl would yield a partially purified EI which could be further purified without interfering with the recovery of either albumin or Factor IX complex.

As described above, the first step of the purification procedure was to adsorb dialyzed plasma on DEAE-Sephadex followed by stepwise elution at different ionic strength. Partially purified EI was eluted at 0.25M NaCl in 0.02M phosphate buffer, pH 7.35. This step, with modification, could conceivably be incorporated into the procedure for isolation of Factor IX complex. One of the widely used procedures, Suomela, et al., Vox Sang, 33:37 (1977), for isolation of Factor IX complex (II, VII, IX and X) consists of adding DEAE-Sephadex (1.5 g/L plasma) to plasma or cryo supernatant, washing DEAE cake with 0.01M sodium citrate buffer containing 0.15-0.17M NaCl, pH 7.0 and eluting Factor IX complex with 0.03M sodium citrate containing 2M NaCl. The present method for isolation of EI requiring 0.25M NaCl in 0.02M phosphate buffer could be readily incorporated inbetween the washing and eluting steps. It is expected that the introduction of an additional elution step for EI would result in an insignificant reduction in the overall yield of Factor IX due to partial elution of Factor VII at this ionic strength, but the specific activity of the final products in terms of Factor IX should be higher since Factor IX remains tightly bound under such conditions.

It will be apparent that various changes may be made in the method as described herein without departing from the spirit and scope of the invention or sacrificing its material advantages, the forms hereinbefore described being merely preferred embodiments thereof.

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