Pure gamma globulin fraction,its preparation and pharmaceutical compositions containing it

Pure gan.ria «׳TlObulir fraction, its preparation and. pharmaceutical. compositions ;containing it_:

DR^ - Sta LOUIS . GOVAL

0? 50065 r

The invention relates to gamma globulins and particularly to a gamma globulin fraction suitable for administration by intravenous injection.

The immunoglobulin G fraction of pooled human plasma contains antibodies to many viruses and bacteria. Immunoglobulins are effective in the clinical management of a wide variety of disease states, such as

1. prophylaxis and therapy of infections, in persons with genetic and nosocomial antibody deficiency

IQ states, especially staphylococci, pneumococci,

Hemophilus streptococci and #.n.nfluenzae;

2. prophylaxis in patients with normal immunoglobulin levels, of viral infections (hepatitis, polio, measles, rubeola, rabies, herpes and parotitis), the prophylaxis of tetanus, and of Rh-incompatibility;

3. therapy of severe bacterial infections: staphylococci, coli, pseudomonas, pyocyaneaus septicemias, and also for the therapy of some viral infections such as Herpes zoster.

The full clinical potential of immunoglobulin G has not been determined because although many doses have been given intramuscularly, the intravenous preparations hitherto prepared are extremely degraded and thus low doses have /2 been used. The major antibacterial antibodies in banian gamma globulin are against microorganisms which inhabit the upper respiratory tract, the skin and the gastrointestinal tract. The quantity of gamma globulin needed to overcome an experimental. in vivo infection is proportional to the number of infectious organisms in the inoculum. This has been shwon for Pseudomonas aeruginosa, Escherichia coli, Proteus, and Staphylococcus aureus. The quantity of gamma globulin needed is also proportional to the specific antibody level in the preparation. With chloramphenicol there is a synergistic action, but with other antibiotics there is only an additive effect.

Hur.an immunoglobulins were first isolated on a large scale during the period from 1945 to 1950 at Harvard in F. J. Cohn's laboratory. It was soon observed that intravenous injection of these preparations caused shock reactions in some patients and it was subsequently established that the anticomplementary activity of IgG preparations is responsible for the shock reactions. This anticomplementary activity is cue to IgG aggregates formed during the fractionation.

In view of the shock reactions associated with the intravenous administration of the immunoglobulins, these therapeutically sr useful substances have hitherto been administered intramuscularly.

However, the intramuscular administration of immunoglobulins ' has got many limitations:

a) administration is painful;

b) the amount which can be administered is limited .

c) proteolysis at the site of injection decreases the , available IgG;

d) maximum blood levels are attained only after three or four days, which is a serious handicap in those cases requiring high blood levels of IgG immediately after injection.

Furthermore, intravenous administration of immunoglobulins has wider clinical application because the full dose of

IgG enters the blood stream immediately without being degraded at the site of injection and significantly higher blood levels can be attained. These considerations have prompted the search for methods to prepare IgG with low anticomplementary activity which is suitable for intravenous use. The methods which have been developed hitherto are based on • . - ish proteolytic or chemical treatment to abol^ii the anticomplementary properties of the aggregates.

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Examples of preparations obtained by these methods are:

1. Pepsin-treated Immunoglobulin. In this preparation the protein is extensively degraded to antibody fragments (5S ,F(ab’^) . Its usefulness for combatting bacterial infections is however limited because it has a short-life (about 30 hours compared with 20 to 30 days for negative. IgG). After combining with antigens, the 5S fragments do not fix complement. It has no application in prophylaxis.

2. Plasmin-treated Immunoglobulin. More than 60% of this preparation is degraded to fragments (Fab and Fc). The remaining 7S globulin has a normal half-life (three to four weeks), but the antibody spectrum is limited.

3. pH 4-treated Immunoglobulin. This preparation has a 15 tendency to become anticomplementary during storage.

Its compatibility is therefore restricted and high doses cannot be administered. The half-life is slightly reduced (12 to 14 days) and the antibacterial activity is reduced to an unknown degree.

4, β-propiolactone-treated Immunoglobulin. The molecules are extensively altered, probably forming new antigenic Γ determinants. The half-life is about 10 days. The bacteriolytic activity is reduced.

The four IgG subclasses have different susceptibilities to proteolysis. Thus the pepsin, plasmin and pH 4 (pepsin) preparations mentioned above differ markedly from untreated

IgG in their subclass distribution.

As noted above, the undesirable anticomplementary activity which is responsible for the shock reaction produced by the intravenous administration of IgG is due to the aggregates present therein, which are formed during the fractionation procedures used in preparation. The preparations described above are obtained by methods which use procedures to destroy these aggregates after they are formed, in most cases by either chemical or enzymatic degradation. However, such degradation procedures also result in some degradation of the IgG with consequent loss of activity. Such preparations as described above are thus not as active as desired. Little work has been done on developing methods which prevent the formation of aggregates and provide IgG preparations having substantially

2Q no anticomplementary activity.

More recently, there has been disclosed in German OffenΓ legungsschrift 2,357,800, published June, 6, 1974, a method for the preparation of a gamma globulin suitable for intravenous administration. This procedure, as well as other published procedures for the preparation of gamma globulin, requires as starting material a relatively purified gamma globulin fraction. However, of greater $ significance, the gamma globulin obtained by this method still possesses an excessively high ant!complementary activity for intravenous use.

It has also been proposed (U.S. Patent Specification Number 3,763,135) to prepare a material suitable for intravenous ₁₀ injection from Fraction III but the yield is not high and the product still exhibits i an appreciable ant!complementary activity.

Food and Drug Administration standards are available for intramuscularly administered gamma globulin, but not for intravenously administered gamma globulin. Such standards are needed to distinguish between gamma globulin which causes shock-like reactions when given by the intravenous route to sensitive individuals and gamma globulin which does not elicit such reactions.

²θ During the past fifteen years, it has been established that noclinical symptoms are observed, even in highly sensitive recipients, when the level of ant!complementary activity is sufficiently low. With the unit of the standard Mayer .

two unit assay (EXPERIMENTAL IMMUNOCHEMISTRY. By E.A. KabSt and !.'.He. Mayer 2nd Ed־, p. 133, Thomas, Springfield, Ill., 1961), the safe level is 004־ to 0.02 units or less of anticomplementary material per milligram of immunoglobulin _זי G, and may be somewhat higher than 004־, but reactions are routinely observed when the level is 0.4 units per milligram. The designation of gamma globulin preparations as suitable for intravenous use, signifying the absence of clinical reactions, is dependant on a specific low level of anticomplementary activity־ It is also necessary to preserve the physiological antibody activity and specificity, in order to provide a clinically safe and effective preparation־

DT-OS 2606118 describes the preparation of a product having less than 0.020 units per mg of anticomplementary activity as measured according to the method of KABAT and MAYER (Experimental Immunochemistry, 2nd ed., pp 224 [1961] Thomas Springfield,

Ill.) and suitable for intravenous injection. However, the anticomplementary activity is still appreciable.

U.K. Patent specification No. 1,006,258 relates to the separation, isolation and purification of plasma proteins using polyethylene glycol of molecular weight of at least 300 for the fractionation of blood plasma.

In DOS 2,364,742 a method for the purification of gamma globulin has been described in which ethanol and polyethylene glycol are used for fractional precipitation and purification.

The only single common feature between the method of the above mentioned two publications and the method of the invention is the use of ethanol and polyethylene glycol for the purification of the gamma globulin. However in the purification of biological material small changes in the process play a very important role in the purity of the desired end product. None of the two publications ♦ hints at the fact that it is possible to obtain a gamma globulin fraction of such purity as characterized in the present invention by separating the impurities from an extract of Cohn fraction II or II + III first at a pH of 5.1 by addition of 4% polyethylene glycol and then by addition of 4 - 12% ethanol and thereafter precipitating the purified gamma globulin by bringing the pH value to 8 using 12% polyethylene glycol or 25% ethanol. There are no detailed indications in the cited publications regarding the purity of the product obtained while the anticomplementary activity of the gamma globulin in accordance with the present invention exceeds by a power of ten that of the pure product obtained by the process of DE-OS 2 606 118 mentioned before.

We have now found that, using certain specific fractionation steps, a gamma globulin fraction may be isolated which not only retains the properties of native gamma globulin molecules but is also substantially free of aggregates and their anticomplementary activity and is thus suitable for

8a j administration intravenously, j - i Thus, according to the present invention, there is provided ί . ׳ ' j a process for the preparation of a gamma globulin fraction

. . ־ { i substantially free of anticomplementary activity and

I . .

I suitable for intravenous administration which comprises:

i a) extracting a paste or powder of Fraction II or II + III plasma protein or of a placental extract containing said Fraction with pyrogen-free water at a pH of from 4,9 to 6;

i . ' ! b) separating the extract thus obtained from solid material j and treating said extract with polyethylene glycol in an ] amount of about 4% weight/volume;

j c) separating the glycol-treated extract from solid material i

thus precipitated and subsequently treating with ethanol in an j amount of from 4 to 12% weight/volume at temperatures of !

ΐ from -6 to 10°C;

j d) separating solid material from the ethanol-treated extract j thus obtained to yield a further extract; and ,.

by precipitation

e) isolating the gamma globulin fraction/from said further extract at temperatures of from -6 to 20°C and at a pH of from 7 to 8.2 by means of the addition of either polyethylene glycol in an amount of up to 12% weight/volume or of ethanol in an amount of up to 30% volume/volume, said gamma globulin fraction being substantially free of anticomplementary activity.

According to a further feature of the present invention there is provided a gamma globulin fraction suitable for intravenous administration and having an anticomplementary activity of less than 0.01 units per mg

The gamma globulin fraction according to the invention is suitable for intravenous injection,shows an antibody spectrum which is substantially unaltered as compared with the start ing Fraction and shows substantially no anticomplementary activity in vitro even after lyophilization. The anticomplementary activity of the fraction is so low that it cannot be measured by the method of KABAT and MAYER (loc.cit). In a j . .

i . new assay the conditions of this method are altered so that j ‘ the number of erythrocytes is reduced by a factor of ten;

j the complement is correspondingly decreased and this assay !

! thus exhibits a ten to eleven fold increase in sensitivity.

j .

J The aht־ !complementary activity of the product produced i .

according to the invention is, measured by this method,

I

I generally from 0.0005 to 0.0025 units per mg, whereas the product of the above mentioned DT-OS 2606118 has an activity

10. of 0.010 to 0.020 units per mg.

. In addition the fraction has a pH * within the physiological

- range and a biological half-life of about 3 to 4 weeks.

Further a freeze-dried product may be produced which has i a long shelf-life and which is easy to reconstitute«, j ’ ' I ¹ ί The paste or powder of Fraction II or II + III plasma protein

I may be obtained as described by Cohn et al in J.Am□Chem.

I j . . ,,

י

Soc. 68, 459-475 (1946) and contains nearly all of the plasma immunoglobulins in addition to other proteins. Preferably the Fraction will have a protein content of from 20 to 30%. Placental extracts containing the Fraction may be obtained 5 according to conventional methods.

The pyrogen-free water is mixed with the paste or powder, preferably in an amount of from 25 to 45 litres of water per kilogram of paste or powder and optionally in the presence of a salt- e.g; sodium chloride» In addition, the pyrogen10 free water may contain about 2% polyethylene glycol and about 0.2% albumin. The suspension thus formed preferably

1 - ך 6** has a conductivity of about 300 x 10 era .ohm; Preferably the suspension is left for about 1 hour before separation of the solid material.

The treatment with polyethylene glycol is preferably effected at temperatures of from 0 to 5°C, whilst the treatment with ethanol is preferably effected at temperatures of from -6 to 0°C, more preferably -2°C₀ In addition the ethanol is preferably used in an amount of about 6% weight/volume.

Both treatments}i£. in step (b) and step (c), are preferably effected at a pH of about 5₀1₀ ' f'

The suspension of the extract in polyethylene glycol is preferably left for about one hour before separation of ,

the solid material. The suspension of the glycol-treated extract in ethanol is preferably left for from 1 to 24 hours, more preferably 2 hours.

Separation of the solid material in any of steps (b), (c) .5 and (d) may, for example, be effected by filtration or centrifugation. In the case of a placental extract particularly care is required to remove all insoluble material formed at each stage, particularly floating insoluble material.

The further extract is adjusted to a pH of from 7 to 8.2, preferably about 8 and optionally sodium chloride is added thereto in a concentration of from 0.0025 to 0.15 M e.g. 0.01 M. The gamma globulin fraction is precipitated by the addition of other polyethylene glycol in an amount of up to 12% e.g. 10 to 12% weight/volume or of ethanol in an amount of up to 30% e.g. 20 to 30% preferably about 25% volume/volume. Separation of the precipitated fraction is preferably effected by centrifugation e.g. by continuous high speed, flow thru centrifugation. Preferred temperatures for the isolation are from -6 to 0°C.

Throughout the process, the polyethylene glycol used will preferably have a molecular weight of from 4000 to 6000.

_

The isolated gamma globulin fraction may, if desired, subsequently be dissolved in a carrier or adjuvant.

The carrier or adjuvant may advantageously include human albumin, sodium acetate, potassium acetate, glycine, lactose, 5 and/or mannitol, mannitol being included if it is subsequently desired to lyophilize the solution.

The solution obtained is preferably adjusted to a pH of from 5.0 to 5.5 and may then be stored as a liquid below 10°C or״lyophilized. If the solution is to be lyophilized

1θ then preferably it will be adjusted to a pH of 6.4 to 6.6.

Lyophilization may then be effected according to conventional techniques.

Solutions of the gamma globulin fraction stored below 10°C have proved stable for at least one year whilst lyophilized powders have proved stable for at least 2 years.? As indicated above, the gamma globulin fraction c exhibits an antibody spectrum which is substantially unaltered as compared with the types and levels of gamma globulin antibodies present in the plasma Fraction whilst still showing substantially no anticomplementary activity. Advantageously the fraction is substantially free of aggregates and decomposition products F(ab)p.

F(ab)״ and F . Preferably the fraction has a sedimentation ^r׳^! G coefficient of about 7S.

' The gamma globulin fractions according to the invention are thus useful in pharmaceutical preparations suitable for intravenous administration» Thus they may be incorporated into pharmaceutical compositions in a form suitable for intravenous administration;in association with a carrier or excipient according to conventional methods. . Thus, for example they may be dissolved in an aqueous solution buffered to a pH of from 5.4 to 6.7 and containing glycine .and albumin. The concentration of the gamma globulin fraction is preferably adjusted to 5%. Suitable buffers include, for example, phosphate and sodium acetateacetic acid systems.

To prevent or reduce any denaturation at a liquid-air or liquid solid interface of the product in solution, it is advantageous to add a surfactant to the pharmaceutical composition. Suitable surfactants are non-ionic surfactants such as the block copolymers of propylene and ethylene oxides such as Pluronic 68 (poloxamer 188) and partial * a . . a esters of sorbitol and/polyoxethylene oxide of/long chain 95 fatty acid* such as the Tweens 20, 40, 60, 80 and (polysorbates 20, 40, 60, 80 and 95), water-soluble substances described in the 1973 edition of the Cosmetic, Toiletry and Fragrance Association,Inc. CTFA Cosmetic Ingredient Dictionary, and fluoro surfactants such as \ Zonyl FSA, FSB, FSC and FSN. These non-ionic surfactants stabilize proteins against surface denaturation and do not contain as part of their structure any chemical groups which may otherwise interact with or denature proteins□

The gamma globulin fraction according to the present ; ; !

invention when incorporated into pharmaceutical compositions has a longer half-life than other gamma globulin preparations now on the market. The gamma globulin fraction has proved to be useful for intravenous administration in all instances and for all conditions where intravenous administration is desired without any of the usual undesirable effects associated with the intravenous . administration of hitherto known gamma globulin fractions. The following non-limiting Examples serve to illustrate the