Method of producing intravenously injectable gamma globulin

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PATENT AUTHORITY (62) National application number (86) International filing date

86-02-24

78-06-04

77-12-02

77-12-02 (11) Publication number

443 294 (86) Filing date for European patent application

Application filed as · Swedish patent application

Q Completed international patent application with number □ European patent application converted with number (30)

76-12-03 US 747063

<td> (71)</td><td>Applicant</td><td>Myer Louis</td><td>Coval, Oakland Cal US</td>

<td> (72)</td><td>Inventor</td><td>search</td><td></td>

<td> (74)</td><td>Agent</td><td>Brann</td><td></td>

<td> (54 )</td><td>Name</td><td>Improved injectable</td><td>method for the preparation of intravenous gamma globulin</td>

(56) Published publications DE 2 606 118 DE 2 234 069 US 3 763 135 US 3 415 804 Other publications AT 329 747

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The invention relates to an improved process for the preparation of gamma globulin, suitable for intravenous administration from plasma protein paste of the readily available fractions II or II + III or from corresponding fractions derived from placental extracts. The paste fractions II or II + III are extracted with water at pH 4.9-6.0 and the impurities are fractionated by the addition of polyethylene glycol to 4% (w / v) and then 6% (w / v) ethanol. The desired gamma globulin is then precipitated at pH 8.0 by the addition of polyethylene glycol to 12% (w / v).

The present invention relates to gamma globulins. In particular, gamma globulin preparations are suitable, suitable for administration by intravenous injection and process for the preparation of said gamma globulin.

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

1st prophylaxis and therapy of infections, in people with genetic and nosocomial antibody deficiencies, especially staphylococci, pneumococci, streptococci and Η. influenzae;

2nd prophylaxis for patients with normal immunoglobulin levels, against viral infections: (hepatitis, polio, measles, rubella, rabies, herpes and parotitis), prophylaxis against tetanus and rhinocompatibility;

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

The full clinical potential of immunoglobulin G has not been determined for the following reasons: millions of doses have been given intramuscularly, the intravenous preparations are extremely impaired and low doses have been used. The major and bacterial antibodies in human gamma globulin are against microorganisms found in the upper respiratory tract, skin and gastrointestinal tract. The amount 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 shown for Pseudomonas aeruginosa, E.coli, proteus and Staphylococcus aureus. The amount of gamma globulin needed is also proportional to the specific antibody level in the preparation. With chloramphenicol, a synergistic effect is achieved, but with other antibodies only an additive effect is obtained.

Human immunoglobulins were first isolated on a large scale during a period from 1945 to 1950 at Harvard in FJ Cohn's laboratory. It was soon observed that intravenous injection of these preparations caused shock reactions in some patients and it was subsequently determined that the anti-complement activity of the IgG preparations is responsible for the shock reactions. This anti-complement activity is due to IgG aggregates formed during fractionation.

In view of these shock reactions associated with the intravenous administration of the immunoglobulins, these therapeutically valuable substances are instead administered intramuscularly.

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

a) they are painful;

b) the amount that can be administered is limited;

c) proteolysis at the site of injection reduces available IgG;

d) maximum blood levels are only reached after 3 or days, which is a serious handicap in cases where high blood IgG levels are required immediately after injection.

In addition, intravenous administration of the immunoglobulins has further clinical application, since the entire dose of IgG enters the blood stream immediately without deterioration at the injection site and significantly higher blood levels can be achieved. These views have provided that methods for the preparation of IgG with low anticomplementary activity suitable for intravenous use have been sought. The methods that have been developed are based on proteolytic or chemical treatment to eliminate the anti-complement properties of the aggregates.

Examples of preparations prepared using these methods are:

1st Pepsin-treated immunoglobulin. In this preparation, the protein is extensively degraded to antibody fragments (5S, F (ab ')₂). Its usefulness to attack bacterial infections is limited, as it is short-lived (about 30 hours, compared with 20-30 days for negative IgG). After being combined with antigens, the 5S fragments do not fix complement. It has no use in prophylaxis.

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

3rd pH 4-treated immunoglobulin. This preparation tends to become anti-complementary during storage. Its tolerability is therefore limited 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.

4th 3-propiolactone-treated immunoglobulin. The molecules are extensively aged, and are likely to form new antigen determinations. The half-life is approximately · 10 days. The bacteriolytic activity is reduced.

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The four IgG subclasses are different sensitive to proteolysis. Accordingly, pepsin, plasmin and pH 4 (pepsin) preparations differ markedly from untreated IgG with respect to subclass distribution.

As stated above, the undesirable anticomplementary activity responsible for the shock reaction induced by intravenous administration of IgG depends on the aggregates present therein, which are formed during the friction procedures used in the preparation. The above-described preparations are obtained by methods which use methods to destroy these aggregates as they are formed, in most cases either by chemical or enzymatic degradation. However, such degradation methods also provide some degradation of IgG with subsequent loss of activity, so that the preparations described above do not become as active as desired. So far, little work has been done on developing methods that prevent the formation of aggregates and provide IgG preparations without any significant anti-complement activity.

Very recently, in German Publication No. 2,357,800, a method for the preparation of gamma globulin has been described, suitable for intravenous administration. This process, like other published methods, for the preparation of gamma globulin, requires as a starting material a relatively purified gamma globulin fraction. However, it is of great importance that the gamma globulin obtained by this method still has an extremely high anti-complement activity for intravenous use.

It has also been proposed (US Patent 3,763,135) to produce a material suitable for intravenous injection from Fraction III, but the method of the present invention provides much greater yield and the product has a much smaller amount of anti-complementary material.

The Food and Drug Administration standard is available for intramuscular gamma globulin but not for intravenous gamma globulin. Such standards are needed to distinguish gamma globulin which results in shock-like reactions when administered intravenously to sensitive individuals and gamma globulin that does not elicit such reactions.

Over the past 15 years, no clinical symptoms have been observed, even in very sensitive recipients, when the level of anti-complementary activity is low enough. With the unit of the standard Mayer two-unit assessment (Experimental Immunochemistry by EA Kabat and Μ M Mayer 2nd edition, p. 133, Thomas, Springfield, Ill., 1961), the safe level is 0.04 to 0.02 units or less of conjugate material per mg of immunoglobulin G and may be slightly higher

7713719-8 than 0.04, but reactions are routinely observed when the level is 0.4 units per mg. The design of gamma globulin preparations suitable for intravenous use and suggesting the absence of clinical responses depends on a specific low level of anti-complement activity. It is also necessary to preserve the physiological antibody activity and species (specificity) to provide clinically safe and effective preparations.

The methods of the present invention have been shown to provide a product which retains the properties of the native gamma globulin molecules and is substantially free of aggregates and their anti-complement activity, thereby providing product safety and efficacy for intravenous use.

Thus, it is an object of the present invention to provide a gamma globulin preparation suitable for intravenous injection.

Another object of the present invention is to provide gamma globulin preparations suitable for intravenous injection, essentially without in vitro complementary activity.

It is a further object of the present invention to provide a gamma globulin preparation suitable for intravenous injection which has a biological half-life of about 3-4 weeks.

It is a further object of the present invention to provide a gamma globulin preparation suitable for intravenous injection which is capable of fixing complement when combined with the corresponding antigen and having a substantially un-aged antibody spectrum, as compared to the types and levels of gamma globulin antibodies present. in the starting plasma pool and in standard gamma globulin obtained by Cohn's classic ethanol fractionation of plasma.

It is a further object of the present invention to provide a method of preparing a gamma globulin preparation suitable for intravenous administration.

It is a further object of the present invention to provide a method for preparing a gamma globulin preparation suitable for intravenous injection from readily available blood protein fractions.

It is a further object of the present invention to provide a method for preparing a gamma globulin preparation suitable for intravenous injection, according to which substantially no formation of aggregates occurs.

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Another method is known from DT-OS 2606118, which provides a product, suitable for intravenous injection, containing less than 0.020 units of anti-complementary material, evaluated by the method of Kabat and Mayer (Experimental Immunochemistry, 2nd edition, p. 224 (1961, Thcmas Springfield, Ill.)). According to the process of the present invention, by changing fractionation conditions, a product whose pH is within the physiological range and whose anti-complementary activity is not even increased by lyophilization is obtained, which was a disadvantage in all known products.

The anti-complement activity of the product of the present process is so low that it cannot be measured by the above method. According to a new measurement method, the conditions of this method are changed so that the number of erythrocytes is reduced by a factor of 10; the complement is reduced correspondingly, and this measurement method indicates a 10 to 11 fold increase in sensitivity. The anti-complement activity of this new product, as measured by this method, is 0.0005 to 0.0025 units per mg, while the product of the above-mentioned German disclosure has an anti-complement activity of 0.010 to 0.020 units per mg. Therefore, according to the present invention, a freeze-dried product can be prepared which has a long shelf life and is easy to reshape.

According to one aspect of the process of the present invention, the gamma globulin is obtained from the readily available fraction protein II + III of Cohn et al., As described in J. Am.Chem. Soc. 68, (1946), p. 459-475. This fraction, which contains almost all immunoglobulins except for other proteins, is subjected to new fractionation technique which prevents the formation of aggregates which are formed during the prior art fractionation procedures and which provide an active gamma globulin, essentially without anticomplementary activity and suitable for intravenous administration.

Another useful source of raw material is the fraction II material, which is readily available as immune serum globulin. This material is economical, stable as freeze-dried powder or frozen paste and free from hepatitis virus. It can be processed in the same way as the fraction II + III material.

Yet another useful starting material is a placenta extract containing corresponding fractions.

In the process of the invention, a paste of plasma proteins of fractions II or II + III is extracted with water at a pH of about 4.9-6.0, preferably about 5.0. Pyrogen-free water in an amount of about 25-45 liters, preferably 30 liters per kg of paste, with

7713719-8 a protein content of about 25-30% was used. All non-toxic pharmaceutically acceptable organic or inorganic acids such as acetic acid, lactic acid, citric acid, hydrochloric acid, sulfuric acid and the like can be used to adjust the ρΗ value. The water-insoluble material is separated and the filtrate is then subjected to fractional precipitation, first with polyethylene glycol at a concentration of 4, then with ethanol at concentrations of 4-12%, preferably at about 6%, and finally with PEG at 12%, the last at a pH of about 8.0. The first two fractionation precipitates remove impurities, and the final precipitate provides the desired gamma globulin of the present invention. The preferred polyethylene glycol has a molecular weight of about 4000-12000. All non-toxic, pharmaceutically acceptable inorganic bases can be used to adjust the pH to about 8.0. The process can be carried out at a temperature of about -10 to + 20 ° C.

The invention is further illustrated by the following examples, wherein the temperatures relate to degrees Celsius.

Example 1

Pastes of Cohn Fraction II + III were suspended in pyrogen-free distilled water at 0-5 ° at a concentration of 30 l / kg. An amount of 20-50 l can be used. The pH of the suspension was then adjusted to pH 5.1 (range 4.9-6.0) with dilute acetic acid (other acids such as the above may also be used). The suspension was then filtered or centrifuged at 0-5 ° and the precipitate discarded. The filtrate was transferred to 4% polyethylene glycol 4000 (PEG 4000) (PEG 6000 and 12000 can be used at other concentrations). The precipitate formed in one hour is removed as in the previous step. The filtrate is then transferred to 6% in ethanol (range 4-12%) by gentle addition at -2 ° (range 0 to -6 °). The precipitate is removed again after 1 to 24 hours, preferably 2 hours.

The solution is then made 0.01 M in NaCl and the pH is adjusted to 8.0 (range Ί to 8.2) with sodium hydroxide (1%). The precipitate formed by the addition of ethanol to 25% or by polyethylene glycol 4000 to 10-12%, preferably 12%, is removed by continuous high-speed throughput centrifugation. The resulting paste is dissolved in the following solution: heated human albumin, 5-25 mg / 1, preferably 5-10 mg / ml, sodium acetate 0.025 M, glycine 0.15 M, mannitol 1-2%, preferably 2%, all adjusted with acetic acid to pH 5.1. Lactose can replace mannitol.

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The resulting solution containing 5-6% IgG can be lyophilized or stored as liquid below 10 °. If stored as a liquid, the mannitol is excluded from the dissolving solution. The solution can be lyophilized, lyophilized as follows: pH adjusted to 6.4-6.6 with 1% sodium hydroxide; after dispensing in small bottles in the desired amounts of shell-frozen (solution frozen) · the solution quickly and lyophilization is carried out according to known methods, where care should be taken to avoid overheating when the water is removed. If the product is lyophilized, it is re-formed to form a 5-6% solution of gamma globulin. The solution is stable, frozen or at 10 ° for at least one year, and the freeze-dried powder for at least two years. The purity obtained is at least 97% with the only detectable impurity albumin. The anti-complement activity is below 0.01 units per mg of gamma globulin, range 0.00025-0.0015 units. The units are measured by the two-unit assessment of Mayer, compare above.

Example 2

In pyrogen-free distilled water containing 1-4%, preferably 2% polyethylene glycol 4000 and 0.1-1%, preferably 0.2%, human albumin, Cohn Fraction II paste or powder is dissolved at 0-5 ° to a protein solution of 1- 5%, preferably 2%. The pH is adjusted to

5.1 (range 4.9-6.0, preferably 5.0-5.8) and the resulting precipitate formed after one hour is removed by filtration or by centrifugation.

The PEG concentration in the filtrate is increased to 4% with a 50% PEG-4000 solution or with dry PEG-4000 powder. The precipitate, formed in one hour, is removed. Ethanol was then added to 6% by slow addition so that the temperature did not exceed 2 °. The formed precipitate is removed after 1-12 hours, preferably 12 hours. The pH is then raised to 8.0 (range 6.8-8.1). The precipitate obtained is collected by centrifugation after increasing the ethanol to 25% and dissolving it under the same conditions as in the first example.

The product contains over 99% gamma globulin when measured prior to the addition of albumin in the dissolving solution. There is no detectable inhibition of hemolysis when 50 mg of the gamma globulin per ml (a 5% solution) is evaluated in the standard Mayer assay of less than 0.01 units per mg of IgG. The final solution contains only the added albumin previously heated by conventional methods to remove any hepatitis B virus. No other proteins can be detected by conventional techniques such as cellulose acetate electrophoresis, immunoelectrophoresis or immunodiffusion.

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Example 3

Placental gamma globulin isolated by conventional procedures can be treated as in Example 2. Since placental gamma globulin isolated in the conventional manner contains a few percent impurities which are plasma proteins, additional care should be taken to remove all insoluble material at each step, especially liquid insoluble material before The pH is adjusted in the first step and all other steps. The 6% ethanol filtrate is made 0.01 M in NaCl. as in the first example. All other steps are as in Example 2. The purity of the product is at least 98% gamma globulin when measured (prior to the addition of the dissolving solution containing albumin, 5-10 mg / ml) by the same technique as in Example 2.

The same dissolving solutions are used as in the first example, with mannitol excluded if the product is desired as a solution and should not be lyophilized.

Example 4

Immunoglobulin G paste or fraction II or dried fraction II powder of placental origin can be greatly purified and also used for the preparation of a product suitable for intravenous use by:

1st Suspension of the paste or powder at 1% concentration (range 0.3-5%) in water containing 2% polyethylene glycol 4000 (PEG 2000, 6000, 8000 and 12000 as average molecular weight may also be used) and 0.2% heated human albumin at 1 ° (range 0-5 °). The insoluble liquid material is removed by foaming.

2nd The pH is then adjusted to 5.1 (range 4.9-6.0) with acetic or hydrochloric or citric or other acids. The precipitate is removed at 1 ° after one hour by filtration or centrifugation.

3rd The polyethylene glycol 4000 concentration is then increased

%. The liquid material is removed again. Then, the precipitate is removed by filtration or centrifugation.

4th The ethanol is added to 6% (range 2-12%) by slow addition at -6 ° (range -2 to -15 °).

5th The precipitate and liquid material are removed as in the previous step.

6th Sodium chloride is added to 0.01 N (range 0.0025-0.15).

7th The pH is then raised to 8.0 (range 7-8).

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Eighth Alcohol is added slowly to 25% final concentration slowly at -6 °.

9th The precipitate is collected by centrifugation.

10th The precipitate is dissolved to a final concentration of 5-6% based on determinations with known weights of the paste and known volumes of the following solution: 0.5% human heated albumin (range 0.3-5%), sodium acetate (0.0125 or 0.025 N), glycine 0.15 N, pH 5.1 with acetic acid. The pH of the dissolved paste is adjusted to 6.6 with alkali such as 1% sodium hydroxide or potassium hydroxide or tris (hydroxymethyl) aminomethane.

11th If the solution is to be lyophilized, 2% mannitol (range 1-3%) is added.

12th The resulting solution or freeze-dried powder contains no known impurities and has less than 0.01 units of anti-complementary activity in the 2-unit complement determination of Mayer.

13th If the precipitate from step 9 is analyzed, it contains more than 98% gamma globulin.

The gamma globulin of the present invention can be readily incorporated into pharmaceutical preparations suitable for intravenous administration. In the preparation of such preparations, the gamma globulin is dissolved in a / aqueous solution buffered to above 5.4-6.7 and containing glycine, albumin and a non-ionic surfactant. The pH of the preparation is then adjusted as desired to a pH between 5.4-6.7 and the concentration of the gamma globulin in the preparation is adjusted to 5%. Suitable buffer agents include phosphate and sodium acetate-acetic acid system.

In order to prevent or reduce any denaturation at a liquid-air or liquid-solid surface of the product in solution, it is advantageous to add a surfactant to the pharmaceutical composition. Suitable surfactants are nonionic surfactants such as block copolymers of propylene and ethylene oxide such as Pluronic 68 (poloxamer 188) and partial esters of sorbitol and polyoxyethylene oxide of long chain fatty acids such as Tweens 20, 40, 60, 80 and 85 (polysorbate 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 fluoride surfactants such as Zonyl FSA, FSB, FSC and FSN. These nonionic surfactants stabilize proteins against surface denaturation and do not, as part of their structure, contain any chemical groups that otherwise interact with or denature the proteins.

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The gamma globulin of the present invention, when stored in the pharmaceutical compositions of the present invention, has a longer half-life than other gamma globulin preparations currently on the market. The gamma globulin of the present invention has been proven to be useful for intravenous administration at all times and under all conditions where intravenous administration is desirable without any of the usual undesirable effects associated with intravenous administration of gamma globulins.

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