EP0445108A1 - A method for treating gammaglobulin - Google Patents

Abstract

Method for treating plasma by freeze-drying plasma and inactivating viruses by heating plasma.

Description

A METHOD FOR TREATING GAMMAGLOBULIN Related Applications

This is a continuation in part application of Serial No. 205,913 filed November 12, 1980, now abandoned.

Background Art Preparation, of immunoglobulin (gammaglobulin) (immunoglobulin) (serum globulin) can and do transmit non-A, non- B hepatitis (see Welch, A.G. et al Non-A, Non-B hepatitis from intravenous immunoglobulin. Lancet 1983 , pp. 1198-99; also see Lane, R.S.: Non-A, non-B hepatitis from intravenous immunoglobulin. Lancet 1983; 2:975-5} also: Lever, A.L. et al: Non-A, non-B hepatitis after intravenous immunoglobulin, Lancet 1985; 1:587; also Ochs, H.D. et al: Non-A, non-B hepatitis and intravenous immunoglobulin, Lancet 1985,

1:404-5). Also, it has been shown that the causative agent of AIDS (acquired immune deficiency syndrome) the HTLV-III virus does survive the procedure used to prepare intravenous gammaglobulin preparations (see Frine, A.M., et al: Effect of Cohn fractionation conditions on infectivity of the AIDS virus) and thus these preparations may cause AIDS in recippients. Because of these great concerns it has most recently been written, "Thus, intravenous preparations of ISG (immune serum globulin) are of particular concern, as shown, for transmission of non-A, non-B hepatitis". "Our findings suggest the desirability of applying virus-inactivation procedures to plasma subjected to Cohn ethenol fractionation, or. to the resulting plasma derivatives, in order to sterilize them against non-A, non-B hepatitis viruses and also to inactivate the agents of AIDS". (See Prince, A.M., et al: Effect of Cohn Fractination conditions on infectivity of the AIDS virus. Lancet 1986: 388-387).

It is now apparent that there is thus an important need to develop a method to inactivate any virus which may be found in immunoglobulin preparations. Disclosure of the Invention

It is thus the object of the present invention to demonstrate a method to substantially inactivate any microorganism or virus which may be present in immunologlobulin preparation. This is accomplished by using the method of lyoprrlizafcion (freeze-drying) that is the immunoglobulin preparation is lyophilized and thereafter is subjected to a heat treatment at various temperatures for various times while in the lyophilized state in order to inactivate any viruses especially non-A, non-B hepatitis and the agent causing AIDS (HTLV-III virus).

It is another object of the present invention to provide a method for heat treating immunoglobulin (gammoglobulin, or serum immuncglobulin) preparations to inactivate any hepatitis virus present therein, which method results in substantial yields of gammaglobulin. (Note the term gammaglobulin preparation, immunoglobulin preparation, serum immunoglobulin preparation, are being used denominatively.) It is still another object of the present invention to provide a method for heat-treated gammaglobulin (immunoglobulin) wherein the gammaglobulin (immunoglobulin) fraction is first prepared in lyophilized form to enhance the stability of the gammaglobulin during the heating process. It is a further object of the present invention to provide a method for heat treating lyophilized immunoglobulin (gammaglobulin) to produce a method for heat treating lyophilized immunoglobulin (gammaglobulin) to produce a hepatitis vaccine and possibly a vaccine against HTLV-III virus.

These and other objects of the present invention are achieved by lyophilizing the plasma fraction used in producing the immunoglobulin (gammaglobulin) preparation, and thereafter subjecting the lyophilized gammaglobulin to elevated temperatures for varying periods of time.

TEST PROCEDURES FOR VERIFYING RECOVERY OF GAMMAGLOBULIN A commercial lyophilized preparation (Sandoz Immune

Globulin 3 gm. lyophilized Lot No. 4.375.147.0) in bottles was obtained. The bottle held a volume of approximately 105 ml. to 130 ml. One bottle of immunoglobulin was used as a control and was not heated, while the other samples were heated in the bottle in a dry oven at several different temperatures for different times. Following heat treatments the samples were reconsituted with sterile water according to the manufacturers directions. The results of the heat treatment are contained in Table I. Another sample Lot No. 43741110 of 1 gm. Sandoz Immune Globulin was also tested.

method as described in the Beckman Laboratory Procedure Manual (see Ritchie, R.F. ed. Automated Immunoanalysis , Part I (1978), Dekker, Inc., New York; also Reimer, C.B. , et al, Clinical Chemisztry 22. No. 5, 577 (1976)).

Linear regression analysis is a statistical tool which is employed to determine how closely two analytical techniques will reproduce the same value. When the values for one technique are plotted against the values for the second technique, they will align themselves around a straight line, which is at a 45 degree angle to the x-axis. Mathematically, this straight line or regression line can be expressed in the form of the equation: y = b + mx. in the regression equation, b represents the point at which the regression line intercepts the y-axis of a graphical plotting of the comparison data. This y-intercept provides an estimate of the constant difference or bias between the two methods. The larger the derivation of the y-intercept from 0, in either a positive or negative direction, the greater is the constant difference between the two methods. This difference is expressed in terms of absolute concentration units.

In immunological reactions, antibodies produced against the same protein by different manufacturers may not react in an identical manner. The magnitude of this difference in reactivity can be observed through the amount of bias observed. In a similar manner, certain proteins, such as C3 Complement and Haptoglobin, possess multiple molecular forms. Different antibodies may recognize different forms of these proteins. This difference in selectivity may also be observeu as a bias between methods.

The slope, or m, reflects the amount of proportional bias which is evidenced by a change in the angle of the regression "line" from 45 degrees. If the two methods in question yield exactly the same values, then the m value would be 1.00x. Any value greater than 1.00x indicates that the technique plotted on the y-axis yield a proportionally higher value than the technique plotted on the x-axis. That is, if m - 1.10x, method y will yield a 10% greater value than method x. On the other hand, if m = 0.95x, this indicates that technique x will yield a 5% higher value than that produced by technique y. Generally speaking if two techniques demonstrate a value for the slope which is significantly different from 1.00, calibration or standardization differences between the two methods exist. In this situation, it is mandatory to cross check the calibrating material from one technique against the second technique. This is readily done by calibrating the first method and analyzing the second method's calibration material as an unknown. That value, when compared to the second manufacturer's stated value, will provide an assessment of the magnitude of the calibration differences between the two techniques.

When evaluating the slope (m) and y intercept (b) values from a regression analysis, discrepant values or outliers can produce a false interpretation. In these cases, all outliers must be looked at carefully as they alone could be responsible for the slope and y-intercept values obtained.

The correlation coefficient, or r, is a unitless quantity which is a measure of random error. On the graphical plot, this is observed as the relative scatter of values around the regression line. While this is the most commonly employed tool for evaluating how well two methods compare, this statistic must be interpreted with care. It is sensitive only to random error and does not reflect constant or proportional bias. Additionally, the correlation coefficient is strongly dependent upon the range of values of the sample populations being compared. Wnen setting up a correlation study, a broad range of values is generally more important than the number of values being evaluated. When interpreting the r value, the closer the value is to unity, generally, the more closely the two methods correlate. However, two methods can show excellent 4 values, e.g. 0.95 or better, yet have considerable proportional error due to differences in calibration. Additionally, the methods under consideration could possess considerable differences in antibody specificity, show a y-intercept deviating greatly from zero, yet have excellent coefficients, of: correlation.

Linearity of the Immunochemistry System is verified throughout the entire normal measuring range or dilution. It follows that linearity of the assay extends throughout the remaining dilutions. Sample concentrations that lie outside of the normal measuring range are repeated using a higher or lower dilution so that antigen concentrations within the final reaction mixture are always being analyzed in a range of values known to yield a linear response. In evaluating the results of a regression analysis, should either method be non-linear at either the high or low ends of its measuring range, a false deviation in proportional error will be observed. Since the slope of the regression line is affected, subsequent error in the y-intercept may also be observed.

The Beckman Immunochemistry System that was used combines rate nephelometry with microprocessor technology to provide a method for monitoring the immunopreipitin reactions that are used for specific protein determinations. The maximum rate of increase in forward light scatter is measured after a monospecific antisera is added to a polymeric buffer solution containing the diluted patent sample.

There are distinct advantages of utilizing a rate nephelometric metholodology compared with the more traditional diffusion and nephelometric endpoint techniques.

Immunelectropherisis of the sample heated at 65ºC (Exp-Spec.) compared to the unheated control sample (Exp-Cont) is shown in Figure I. The procedure for performing the immunoelectropheresis was performed using the following procedures.

Approximately 190 ml. buffer in Corning chambers was used. A universal agaroεe film was used. 1.0 Lambda specimen and control were added to the well and electropheresis was performed for thirty-five minutes. The film was placed in a moist incubator tray and 20 Lambda anti-serum to the trough, which was covered and incubated for seventeen to twenty-four hours at room temperature. The oven was then turned on, again was pressed for ten minutes, it was then rehydrated in the tray for ten minutes and then pressed again for ten minutes. Following this there was staining for five minutes in Amido Black, then there was a rinse in acid following, then drying in the oven for twenty minutes. Then there was cooling and specimen was put in a dirty acid for two to three minutes. It acid with distilled water, then a drying in the oven for five minutes. Specimen was labeled and photographed at a light field 30 setting. (Procedure was being used at L.A. County U.S.C.-Medical Center, L.A., California)

The results of the immunoelectropheresis is shown in Figure I.

In addition a protein electropheresis was run, comparing the unheated control sample (marked Exp-control) and the sample that was heated at 65°C (marked Exp) see Figure II. The procedure used for this was adapted from the Corning procedure. A buffer was used, approximately 190 ml. The oven was turned on, and allowed for at least thirty minutes to warm up. The agarose card was readied and then the well was filled with 0.6 Lambda specimen and then was run for thirty-five minutes, then was put in Amido Black for fifteen minutes, then was rocked gently in old acetic acid for thirty seconds, then put in the oven for twenty minutes, then cooled for one minute on towel, and was rocked gently again in old acetic acid for one minute, and was rocked gently in new acetic acid for one minute (solution should be clear) then was put in oven for ten minutes or until dry, then scanned. (This method was currently being used at the L.A. County-U.S.C. Medical Center, Los Angeles , California . ) The results of the serum protein electropheresis are shown in Figure II.

DISCUSSION OF SELECTED TEST RESULTS The results from Table I show no significant difference in recovery of the IgG following heat treatment of the lyophilized state of a commercial immunoglobulin preparation. In addition the small amounts of IgA and IgM also showed no significant difference from the unheated control. In addition (Figure 1) there was no significant difference following immunoelectropheresis of the heated sample run against normal human serum (NHS) and against IgG as compared to the unheated control. The protein electropheresis also showed no significant difference from the unheated control (Exp-cont) and the heated sample (Exp-spec) (Figure 2) . There was approximately 10% decrease in IgG following heating at 100°C for one hour. It has now been demonstrated that the plasma fraction containing IgG in large amounts which is used in preparing commercially used Immune Globulin (gammaglobulin) (immunoglobulin) can be heat treated in the lyophilized state for varying p.riods of time and for varying elevated temperatures without significant change in the recovery of immunoglobulins. currently Immune Globulin is used to treat immune deficiency such as combined immune deficiency and in primary immunoglobulin deficiency syndrome such as X-linked agammaglobulluremes. IV administered immunoglobulin has also been used for treatment of idiopathic thrombocytopemes purpera (ITP) (see Cunningham-Rundler, C. et al in: Nydegger, Al E. (Editor) Immunotherapy: A Guide to Immunoglobulin Prophylaxis and Therapy, Academic Press, London, p. 283 (1981); also see Bussel, J.B. et al Blood 62 (2), pp. 480-486 [August 1983]) and other therapeutic uses have been proposed. It should be noted that solubility was not deleteriously affected by the heat treatment and if necessary additional diluent may be added if necessary to increase the solubility.

Given the fact that hepatitis virus and other virus may be preferentially distributed in some gammaglobulin preparations suitable adjustment of the temperature and time of heating can make any hepatitis virus non-infective and immunogenicand the properly heat-treated lyophilized gamma globulin preparations may function after repeated administration to the recipients at hepatitis or HTLV-III vaccines. It should moreover be apparent that application of the heatr treatment of lyophilized gammaglobulin preparation may be instituted at any point along the process, selection of an appropriate point along the manufacturing process for applying the heat treatment may be based on pragmatic considerations such as cost or convenience. It would seem that folowing the final step of lyophilization the heat treatment process should be performed as this seems to be pragmatic and economical. The present invention is thus a cost effective and pragmatic method to substantially inactivate any viruses which may be in the gammaglobulin (immunoglobulin) preparation.

It has been shown that human AIDS-associated retro-virus (ARV) is sensitive and can inactivate infectious ARV (see Levy, J.R. et al, Lancet 1985 pp. 456-457) when heated in the dry state that is heating by a lyophilized plasma fraction (Factor VIII concentrate) in the dry state at elevated temperatures significantly inactivated infectious ARV. Thus heating gammaglobulin preparations in the lyophilized state at sufficient temperature will significantly inactivate AIDS-associated retrovirus (ARV) .

It should moreover be apparent that in addition to Hepatits-B and non-A, non-B Hepatitis being capable of being transmitted by commercial immunoglobulins, hepatitis delta virus may also be transmitted. As stated (see Ponzetto, A., et al, New England Journal of Medicine, 1986 Vol. 314, No. 8, p. 517):

"Rosina et al reported the risk of transmitting the hepatitis delta virus by transfusions of blood and blood derivatives that had been screened for HBsAg by third generation methods. The authors estimated the risk of hepatitis delta virus infection as 1 in 3000 transfusions. Since commercial immunoglobuolins are usually obtained from large pools of donors (2,000 to 5,000), transmission of hepatitis delta virus may also originate from this source."

The article further states: "In view of the high infectivity of hepatitis delta virus for carriers of HBsAg, the potential risk of transmission of hepatitis delta virus should be considered in treating such carriers with commercial preparations of immunoglobulins."

Thus, in addition to substantially decrease Hepatitis B and non-A, non-B hepatitis transmission, heat treatment according to my invention will also decrease the likelihood of transmission of hepatitis delta virus and the agent (s) causing AIDS.

Several embodiments of the present invention have been illustrated hereinabove. It is to be understood, however, that various modifications to the temperature ranges, heating periods, and purity levels, set forth in conjunction with the aforementioned embodiments can be made by those skilled in the art without departing from the scope and spirit of the present invention. It is therefore the intention of the inventor to be bounded only by the limits of the following claims .

Claims

1. A method for treating a plasma fraction containing gamma globulin in significant amounts to minimize the effect of any virus or microorganism present in the fraction, said method comprising the steps of: lyopnilizing the plasma fraction to increase the heat stability thereof; and heating the lyophilized plasma fraction for a predetermined period of time at a predetermined temperature sufficient to render any virus present in the plasma fraction non-infections.

1a. A method as in 1 wherein the plasma fraction is gamma globulin (immune globulin, immune serum globulin).

2. A method for treating a plasma fraction to minimize the effect of any hepatitis virus present in the fraction, said method comprising the steps of: lyophilizing the plasma fraction to increase the heat stability thereof; and heating the lyophilized . plasma fraction for a pre-determined period of time at a predetermined temperature sufficient to render hepatitis virus present in the plasma fraction immunogenic.

2a. A method as is 2 wherein the plasma fraction is Gamma Globulin (Immuno Globulin, Immune Serum Globulin).

3. A method as set forth in claim 1 or 2, including the further step of reconstituting the lyophilized Gamma Globulin preparation following said step of heating the lyophilized Gamma Globulin preparation for said predetermined period of time at said predetermined temperature.

4. A method as set forth in Claim 1 or 2 wherein said plasma fraction comprises Gamma Globulin (Immune Globulin; Immune Serum Globulin; Immonoglobulin) and said predetermined temperature is at least 60ºC.

5. A method as set forth in claim 4, wherein said predetermined temperature fall, within a range between 60°C. and 100°C.

6. A method for treating a plasma fraction which is gamma globulin (Immune Globulin; immunoglobuline) to minimize the effect of any virus or microorganism present in the gammaglobulin (Immune Globulin, Immunoglobulin) preparations, said method comprising the steps of: lyophilizing the gamma globulin (immune globulin, immunoglobulin) preparation to increase the heat stability thereof; heating the lyophilized gamma globulin (immune globulin, immunoglobulin) preparation for a pre-determined period of time at a predetermined temperature sufficient to inactivate hepatitis virus present in the gamma globulin (immune globulin, immunoglobuin) preparation; and adding sterile water to the lyophilized gammaglobulin (immune globuline, immunoglobulin) preparation following completion of said heating step until solullization of the lyophilized gamma globulin (immune globulin, immunoglobulin) preparation is achieved.

7. A method, as in 1, wherein the virus is any hepatitus virus, especially Hepatitis B, non-A, non-B Hepatitis viruses, and hepatitis delta virus.

8. A method, as in 1, wherein the virus is composed of any virus or microorganisms which may cause AIDS, especially HTLV-III virus.