METHOD FOR SEPARATING PROTEINS FROM ANIMAL OR HUMAN PLASMA, OR PLANTS, USING A PH GRADIENT METHOD

Abstract

The present subject matter is directed to a method for separating proteins of plasma using pH adjustment including the steps of reconstituting Fraction III, Fraction IV, or plasma paste, in water for injection; adjusting pH value to 1 and temperature from 1° C. to 30° C.; centrifuging the resulting suspension at 6,000 rpm at 2-8° C. for 20 min; collecting the resulting paste 1 (P1) and supernatant 1 (S1); reconstituting P1 in WFI and adjust the pH to 2; and repeating step 3) to step 5) until the pH of supernatant reaches 14. According to the method, a new formulation of immunoglobulin is prepared from plasma Fraction III and Fraction IV.

Claims

1. A method for separating proteins of plasma using pH adjustment, the method comprising the steps of: 1) reconstituting Fraction III, Fraction IV, or plasma paste, in water for injection; 2) adjusting pH value to 1 and temperature from 1° C. to 30° C.; 3) centrifuging the resulting suspension at 6,000 rpm at 2-8° C. for 20 min; 4) collecting the resulting paste 1 (P1) and supernatant 1 (S1); 5) reconstituting P1 in WFI and raising the pH by 1; and 6) repeating step 3) to step 5) until the pH of S1 reaches 14 to form a resulting solution.

2. The method of claim 1, wherein the Fraction III, Fraction IV, or plasma paste is obtained by pH adjustment in water for injection without alcohol.

3. The method of claim 1, wherein the resulting Fraction III, Fraction IV, or plasma paste is re-suspended and pH and temperature are adjusted to a proper range.

4. The method of claim 1, wherein the resulting solution has an adjusted protein concentration and pH.

5. The method of claim 1, further comprising subjecting the resulting solution to 0.22 um aseptic filtration and 20 nm nano filtration for a first virus inactivation.

6. The method of claim 1, further comprising subjecting the resulting solution to filling and low pH incubation at pH 4 for 21 days at 25° C. as a second virus inactivation.

7. A method of preventing infection of Hepatitis C virus in a patient comprising administering a Fraction III resulting solution obtained from the method of claim 1 to a patient in need thereof.

8. A method of preventing infection of HIV-1 and HIV-2 in a patient comprising administering a Fraction IV resulting solution obtained from the method of claim 1 to a patient in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Many aspects of the disclosure can be better understood with reference to the following figures.

[0020] FIG. 1 shows a sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of supernatant from Fraction III fractionation at a pH from 1-5.

[0021] FIG. 2 shows a SDS-PAGE of supernatant from Fraction III fractionation at a pH from 6-10.

[0022] FIG. 3 shows a SDS-PAGE of supernatant from Fraction III fractionation at a pH from 11-14.

[0023] FIG. 4 shows a SDS-PAGE of paste from Fraction III fractionation at a pH from 1-5.

[0024] FIG. 5 shows a SDS-PAGE of paste from Fraction III fractionation at a pH from 6-10.

[0025] FIG. 6 shows a SDS-PAGE of paste from Fraction III fractionation at a pH of 11.

[0026] FIG. 7 shows a SDS-PAGE of supernatant from Fraction IV fractionation at a pH from 1-5.

[0027] FIG. 8 shows a SDS-PAGE of supernatant from Fraction IV fractionation at a pH from 6-10.

[0028] FIG. 9 shows a SDS-PAGE of supernatant from Fraction IV fractionation at a pH from 11-14

[0029] FIG. 10 shows a SDS-PAGE of paste from Fraction IV fractionation at a pH from 1-5.

[0030] FIG. 11 shows a SDS-PAGE of supernatant from Fraction IV fractionation at a pH from 6-10.

[0031] FIG. 12 shows a SDS-PAGE of supernatant from Fraction IV fractionation at a pH of 11.

DETAILED DESCRIPTION

[0032] Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently described subject matter pertains.

[0033] Where a range of values is provided, for example, concentration ranges, percentage ranges, or ratio ranges, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the described subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also encompassed within the described subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the described subject matter.

[0034] Throughout the application, descriptions of various embodiments use “comprising” language; however, it will be understood by one of skill in the art, that in some specific instances, an embodiment can alternatively be described using the language “consisting essentially of” or “consisting of”.

[0035] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0036] According to one embodiment of the present subject matter, a method for separating proteins from fractions of plasma using a pH gradient process is provided. The method provides a process to fractionate plasma Fraction III, Fraction IV, plasma, or plants, comprising the steps of: [0037] a. reconstituting Fraction III, Fraction IV or plasma paste, in water for injection; [0038] b. adjusting pH value to 1 and temperature from 1° C. to 30° C. to form a resulting suspension; [0039] c. centrifuging the resulting suspension at 6,000 rpm at 2-8° C. for 20 min; [0040] d. collecting a resulting paste (P1) and supernatant 1 (S1); [0041] e. reconstituting P1 in water for injection and raising the pH by 1; and [0042] f. repeating steps b) to step e) until the pH of S1 reaches 14 to form a resulting solution.

[0043] In some embodiments, the method may further include the following steps: [0044] a. adjusting the protein concentration and pH of the resulting solution; [0045] b. subjecting the resulting solution to 0.22 um aseptic filtration and 20 nm nano-filtration for a first virus inactivation; and [0046] c. subjecting the resulting solution to filling and low pH incubation at a pH of 4 for 21 days at 25° C. as a second virus inactivation.

[0047] As shown in FIGS. 1-12, the method of separating proteins of plasma Fraction III and Fraction IV using a pH gradient process improves the effectiveness of the separation of proteins during plasma fractionation. In this respect, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used in order to separate proteins of plasma Fractions III and IV based on their molecular weight at different pH levels according to the pH gradient method. The SDS-PAGE results provide the average molecular weight of both the supernatant and the paste of Fractions III and IV.

[0048] In this respect, FIGS. 1-3 show the SDS-PAGE of the supernatant from Fraction III fractionation at a pH of 1-5, 6-10 and 11-14, respectively.

[0049] FIGS. 4-6 show the SDS-PAGE of the paste from Fraction III fractionation at a pH of 1-5, 6-10 and 11-14, respectively.

[0050] FIGS. 7-9 show the SDS-PAGE of supernatant from Fraction IV fractionation at a pH of 1-5, 6-10 and 11-14, respectively.

[0051] FIGS. 10-12 show the SDS-PAGE of paste from Fraction IV fractionation at a pH of 1-5, 6-10 and 11-14, respectively.

[0052] An embodiment of the present subject matter is directed to a method of stopping replication of HIV-1 and HIV-2 in a patient comprising administering Fraction IV obtained from the present method to a patient in need thereof.

[0053] Another embodiment of the present subject matter provides a method of preventing infection of Hepatitis C virus in a patient comprising administering Fraction III obtained from the present method to a patient in need thereof.

[0054] Any of these fractions or any combination of the new found proteins have the following abilities: [0055] 1) transform/repair damaged and sick cells to become good healthy cells; [0056] 2) protect cellular alterations; and [0057] 3) signal the body to produce new healthy cells immunized from intra- and extracellular damaging signals.

[0058] With the information contained herein, various departures from precise descriptions of the present subject matter will be readily apparent to those skilled in the art to which the present subject matter pertains, without departing from the spirit and the scope of the below claims. The present subject matter is not considered limited in scope to the procedures, properties, or components defined, since the preferred embodiments and other descriptions are intended only to be illustrative of particular aspects of the presently provided subject matter. Indeed, various modifications of the described modes for carrying out the present subject matter which are obvious to those skilled in chemistry, biochemistry, or related fields are intended to be within the scope of the following claims.