HK1009277A1 - Process to prepare an anti-d immunoglobulin g concentrate and pharmaceutical compositions comprising it - Google Patents

Description

The present invention relates to a method for the production of a new concentrate of anti-D (anti-resus) immunoglobulin G (anti-D IgG) and a pharmaceutical composition containing such a concentrate as an active ingredient.

Neonator haemolyticus is a disease of fetuses and newborns caused by maternal antibodies against antigens on the surface of the child's red blood cells, either of the rhesus, ABO or other blood group systems.

The most important Rhesus blood group antigens are D, the significantly less immunogenic antithetical antigens C,c and E,e, and Du; in addition, over 40 other Rhesus antigens are known. Of clinical importance in Rhesus intolerance is in particular Rhesus antigen D (RhD; Rho), a membrane protein of erythrocytes that has recently been cloned and whose primary structure has been described (Le Van Kim et al. PNAS USA, 89,10925,1992). The D antigen occurs in approximately 85% of whites in Europe; these individuals are referred to as Rhesus positive. Individuals lacking the antigen are referred to as Rhesus negative.

Since desensitization is also not possible, prophylaxis of Rhesus sensitization with immunoglobulin anti-D is crucial in Rhesus-negative women of childbearing potential.

Without treatment, up to 17% of primigravidae with Rhesus constellation (Rhesus negative mother, Rhesus positive child) would become sensitized to the Rhesus antigen during pregnancy or birth.

Anti-D preparations have been used successfully for over 30 years to prevent Rhesus sensitization of Rhesus negative and Du positive women to Rhesus factor D and thus to prevent the anti-D-related neonatal disease Rhesus erythroblastosis in all its forms.

The WHO recommended treatment dose of 200 μg Anti-D IgG as postpartum prophylaxis is sufficient to neutralize up to 10 ml of foetal erythrocytes (corresponding to approximately 20 ml of foetal blood) and thus, even with larger infusions, prevents Rhesus sensitisation in approximately 90% of cases; therefore, only 1-2% of primigravidae with Rhesus sensitisation can expect to be sensitised to Rhesus.

In addition, Anti-D is also used after mistransfusions of Rhesus-positive blood to Rhesus-negative recipients, the dosage being adjusted to the extent of Rhesus-positive erythrocyte infusion.

For some years, the use of anti-D immunoglobulin has also been discussed in the treatment of idiopathic thrombocytopenic purpura (ITP).

The pharmacokinetics of intramuscular and intravenous IgG in the body clearly indicate that intravenous administration is highly preferred. Due to the slow absorption from the muscle deposit and local proteolysis, intramuscular injection can be expected to delay maximum activity and to delay onset of action. Maximum IgG plasma concentrations are not reached until 4 days after injection in healthy subjects, 6 days after injection in bedridden subjects, and only about 30% of maximum IgG plasma concentrations in healthy subjects and 20% of the dose in bedridden subjects.

In contrast, intravenous administration has significant advantages: only in this form of administration does the entire dose of IgG, regardless of physical activity, have an immediate effect; the plasma IgG level drops to about 35-40% of the dose after 5 days, virtually independent of physical activity, and only reaches levels comparable to the maximum plasma concentration after intramuscular injection on the fifth day (Morell, A. et al. Vox Sang. 38, 272, 1980).

The production of immunoglobulin preparations at the production scale is now carried out by a variety of fractionation processes:

Cold alcohol precipitation, e.g. after Cohn, or modified methods after Cohn are particularly suitable for processing plasma volumes above 500 l per week. Special treatment, e.g. with pepsin at pH 4, can make such isolated immunoglobulins intravenously tolerant. Other precipitation methods are based on the specific precipitation of immunoglobulins by ammonium sulphate, sodium sulphate, polyethylene glycol, caprylic acid or rivanol (see Curling, J.M. in: Separation of plasma proteins, J.M. Curling ed., 1983, Pharmacia, Uppsala, Sweden).

A first method for the isolation of immunoglobulins by batch absorption on ion exchangers was described as early as 1964 (Baumstark, J.S. et al. Arch. Biochem. Biophys., 108, 514, 1964).

All known methods have achieved high yields of IgG at high purity but no specific enrichment of certain specific IgG molecules, such as anti-D.

Most antihypertensive globulin preparations are only suitable for intramuscular use. Few intravenously compatible preparations are available worldwide. One such preparation is the applicant's anti-D immunoglobulin SRK, which is manufactured using the fractionation method of Kistler and Nitschmann (Kistler, P. and Nitschmann, H. Vox Sang, 7, 414, 1962) and made intravenously tolerated by a mild pepsin treatment at pH 4. Since the yield of specific anti-D antibodies is low with such fractionation methods and at the same time anti-D hypertensive antiplasma is rare, there was a need for a new method which, in contrast to the described methods, also produces high activity anti-D antibodies with high specificity in low IgG plasma.

In Friesen, Journal of Applied Biochemistry, Vol. 3, 1981, pp. 164-175, a method for purifying anti-D (anti-D-resus) immunoglobulin G is described. Human plasma was placed on a DEAE-Sephadex column, which was first balanced with a 25 mM phosphate buffer, pH 7.5. The unbound immunoglobulins were eluted with the same buffer. It is mentioned that the immunoglobulin solution stabilized with glycine and NaCl was pure and did not contain IgA or IgM. This immunoglobulin solution is suitable for intravenous application.

One of the aims of the present invention is to provide a method for the production of a pure anti-D concentrate in which the anti-D immunoglobulins of the class IgG are specifically enriched, which has been found to be possible in a surprisingly simple manner, with simultaneous elimination of approximately 90% of the nonspecific IgG and almost all other undesirable plasma components, by further purification of blood plasma or of the immunoglobulin derived from it from specifically selected and specifically immunized blood vessels and blood donors, with a titer preferably greater than 30 μg/ml of anti-DG, which can be obtained under the conditions of an IgG exchange chromatography.

Another objective of the present invention is an anti-D preparation containing only trace amounts of immunoglobulin A, intravenously tolerated and capable of storage for several months without loss of activity with the addition of appropriate stabilizers, optionally lyophilised or preferably in solution. The pure anti-D concentrate obtained by the proposed process was found to meet these requirements, i.e. to have a specific activity not previously achieved, intravenously tolerated and extremely low IgA content.

The present invention is therefore based on the process defined in claim 1.

The method of the present invention produces immunoglobulin anti-D from human plasma. The starting material is the plasma of rhesus-negative donors sensitized to rhesus factor D. Preferably, the single donation obtained by plasmaferesis is frozen and carefully thawed and poached at 0-4°C before fractionation. The plasma pool should contain more than 10 μg of anti-DG Ig per ml, preferably more than 30 μg of anti-DG Ig per ml. The plasma fraction used for cation chromatography can be exchanged by any known method, for example by separation of cryoglobulin by fractionation or by fractionated alcohol (Cohn, E.J. et al. J. Am., 68, Soc Chem.Preferably, prior to the cation exchange chromatography of the invention, the cryoglobulin-free plasma or the plasma containing immunoglobulin, dissolved in the equilibrium buffer, is filtered and inactivated by a bio-complex solvent such as Plasma-n-butylphosphate and detonator, for example O-L-L-O1,3-tetrahydrocrylamide (Tritron) and is treated at 6°C. The result is a clear substrate called tetrahydrocrylamide (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tritron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron) (Tron (Tron) (Tron) (Tron) (Tron (Tron) (Tron) (TronIn a first main purification step, Anti-D IgG is bound from the pre-treated product to a weak acid ion exchange gel with preferably carboxymethyl (CM) as the functional group. The foreign proteins are mostly washed away; the solvents and detergents used for viral inactivation are also removed. The specific anti-D IgG obtained by this step is activated by increasing the functional capacity to preferably 10 m/cm2 by the IgE ellamine ion decoction and by a second treatment, preferably with a diethyl ion decoction (EEA), as a protective agent.The purified anti-D IgG fraction is preferably bound a second time to a CM ion exchanger gel for concentration and eluted to the maximum concentration under appropriate conditions and packaged into the final product. It is not relevant to the achievement of the conditions of the invention whether anti-D IgG is eluted by increasing the ionic strength, or by shifting the pH, or by a buffer modified in composition. To eliminate further undesirable components, such as proteins, the solution of any other stage of the process can be treated with an adsorbent solution, such as an adsorbent, in addition to an aluminium-based solution.For example, heat treatment such as pasteurisation is replaced or supplemented.

An anti-D IgG produced in accordance with the invention is liquid stable. It is further tolerated intravenously, has an IgA content of less than 5 μg/ml and has an IgG content of 1-10 mg/ml. The pH is approximately 5.2 Preferably the preparation contains up to 25 mM phosphate, up to 200 mM NaCl and up to 100 mg/ml human albumin and optionally 250-300 mM of an amino acid such as glycine or up to 100 mg/ml of a disaccharide such as sucrose or 50 mg/ml of a monosaccharide such as mannite.

An anti-D IgG produced in accordance with the invention may alternatively be lyophilised by addition of a disaccharide, such as 100 mg/ml sucrose, at a dose of 200 μg anti-D IgG at a pH of approximately 6.6.

The accompanying drawing is intended to illustrate the present invention without being intended to be a limitation and shows the typical chromatogram of the first cation exchange chromatography as shown in Example 1.

The method of the invention is explained in more detail by the following examples.

Example 1

The plasma obtained by plasmaferesis is individually frozen and carefully thawed and ice-washed at 0-4°C before fractionation. The free plasma pool is centrifuged in a centrifuge (Cepa, Virr, Germany) at 2-4°C (1 l/min; 12000 U/min). The survival is followed by a 1.2 μm (Opticon®, Haapapripore, Milford, USA) filter and then a 65,500-degree (Trinity, Hempstead, MA) filter.The clear shell is separated and filtered by a 0.2 μm filter (Sealkleen NFP 7002, Pall, Dreieich, Germany). 25 l of solvent-treated filtered plasma are filtered with a 10 mM sodium phosphate buffer on the conductivity of the sodium buffer column (50 mM sodium phosphate buffer, 5.5, 3.2 mS/cm) which is adjusted to a pH of 5.5 μm by a buffer (Opford, MA, USA) and filtered in a bed.The gel is bound to MacroPrep® 50 CM (BioRad, Hercules CA, USA) with a base area of 314 cm2 and a bed height of 16 cm, at a flow rate of 150 cm/h, the gel having been previously balanced with 50 mM sodium phosphate buffer, pH 5.5. The temperature is maintained at 20-25°C. The column is then washed with 40 column volumes of 25 mM sodium phosphate buffer, pH 7.0, and the anti-D-containing G fraction is eluted with 25 mM sodium phosphate buffer + 0.2 MG sodium chloride, pH 7.5. The typical chromatogram as obtained by this treatment is shown in the single figure. The optical diagram (IgOD) is set at 280 nm (from the time of exposure).This shows that during ordering and washing most of the unwanted products are removed, while during elution a narrow main peak appears containing the desired anti-D IgG. The eluate is diluted with water to a conductivity of 3.3 mS/cm (1 volume of eluate + approximately 5 volumes of water), the pH is set to 7.5 with 0.2 M NaOH and adsorbed in the batch process for 2.5 hours with 100 g of DEAE-Sephadex® A50 dry (Pharmacia, Uppsala, Sweden). The DEAE is first equalised with a 25 mM sodium antiphosphatespowder, pH 7.5.The DEAE filter is bound to concentrate a second time on MacroPrep® 50 CM (BioRad. Hercules CA, USA) in a column with a base area of 78.5 cm2 and a bed height of 10 cm, with a flow rate of 60 cm/h, the gel being previously balanced with 50 mM sodium phosphate buffer, pH 5.5.

Example 2

The plasma obtained by plasmaferesis is individually frozen and carefully thawed and ice-washed at 0-4°C before fractionation. The free plasma pool is centrifuged in a centrifuge (Cepa, Virr, Germany) at 2-4°C (1 l/min; 12000 U/min). The survival is followed by a 1.2 μm (Opticon®, Haapapripore, Milford, USA) filter and then a 65,500-degree (Trinity, Hempstead, MA) filter.The virin-inactivated solution is then left to stand for 10-18 hours at 37°C. The clear solution is separated and filtered through a 0.2 μm filter (Sealk NFP 7002, Pall, Dreieich, Germany). 25 l of solvent-treated filtered plasma are filtered with a 10 mM equilibrium phosphate filter to the conductivity of the sodium water vapor (50 mM phosphate filter, 5.5, 3.2 mS/cm) which is diluted by a pH filter set to 5.5 μm (Opford, MA, USA) and filtered through a bed.The column is then washed with 40 columns of 25 mM sodium phosphate buffer at pH 7.0 and the anti-DG fraction is eluted with 25 mM sodium phosphate buffer + 0.2 M sodium chloride at pH 7.5. The eluate is added at pH 6.6 to 100 mL of sucrose at a dose of 200 mg μG IgD-DG at 0.2 μM of MCGL® at pH 6.6.Millipore, Bedford MA, USA) is filtered and then lyophilized.

Example 3

5 ml of solvent-treated plasma is diluted to the conductivity of the equilibrium buffer (3.3 mS/cm) and chromatographed in a column with a base area of 2 cm2 and a bed height of 5 cm, at a flow rate of 30 cm/h via DEAE-Sephadex® A50 (Pharmacia, Uppsala, Sweden). The gel is first balanced with a 25 mM sodium phosphate buffer pH 7.5. The temperature is maintained at approximately 20-25°C. The unbound CAG fraction is re-administered for further purification with 0.2 M HCl at 5.5 and a confluent capacity of 3.2 mS/cm2, and is then placed in a base area of 0.8 and a pH of 0.50 mHg of IgM, with a pH of 6.5 and a final pH of 2.5 mHg. The gel is then mixed with a hydrophobic acid of 50 μM2 and a hydrophobic acid of at least 5.5 and a hydrophobic acid of 50 μM2 (H2O) and a hydrophobic acid of at least 0.5 and a pH of 50 μM2 (H2O). The final product is a hydrophobic acid of at least 230 μMHg of hydrophobic acid and a hydrophobic acid of at least 5.5 and a pH of at least 50 μM2 and a pH of at least 50 μM2 and a pH of at least 50 μM2 and a pH of at least 50 μM2 and a pH of at least 50 μM2 and a pH of at least 50 μM.

Example 4

The procedures in examples 1 to 3 are repeated, using as starting material, instead of plasma, a plasma fraction containing immunoglobulin produced from an appropriate amount of starting plasma by dissolving fraction I+II+III or fraction II+III according to Cohn (Cohn, E.J. et al. J. Am. Chem. Soc. 68, 459, 1949) or precipitation A or precipitation gamma globulin according to Kistler and Nitschmann (Kistler, P. and Nitschmann, H. Vox Sang, 7, 414, 1962) in the equilibrium buffer ad 30 g/l protein, with analogous results.

Example 5

Repeat the procedures in examples 1 to 4, whereby the anti-D IgG fraction is eluted alternatively with MacroPrep® 50 CM gel (BioRad, Hercules CA, USA) instead of 25 mM sodium phosphate buffer + 0.2 M sodium chloride, pH 7.5, with 0.1 M glycine + 0.5 M sodium chloride, pH 9, or generally by pH shift and/or change in ionic strength and/or changes in buffer composition, to obtain analogous results.

Example 6

The procedures in examples 1 and 2 are repeated, with the MacroPrep® 50 CM gel (BioRad, Hercules CA, USA) alternately washed with 10 mM glycine, pH 9, instead of 25 mM sodium phosphate buffer, pH 7.0, to obtain similar results.

Example 7

The concentrated anti-D eluate obtained from example 1 is adjusted as follows: Other

	flüssiges Präparat I	flüssiges Präparat II	flüssiges Präparat III	flüssiges Präparat IV
Anti-D	100 µg/ml	100 µg/ml	100 µg/ml	100 µg/ml
Glycin	20.6 mg/ml	20.6 mg/ml	20.6 mg/ml	20.6 mg/ml
Albumin	---	10 mg/ml	50 mg/ml	100 mg/ml
pH	5.20	5.20	5.20	5.20
Dosis	2 ml	2 ml	2 ml	2 ml
Dosis	2 ml	2 ml	2 ml	2 ml

	flüssiges Präparat V	flüssiges Präparat VI	flüssiges Präparat VII	flüssiges Präparat VIII
Anti-D	100 µg/ml	100 µg/ml	100 µg/ml	100 µg/ml
D-Mannit	50 mg/ml	50 mg/ml	50 mg/ml	50 mg/ml
Albumin	---	10 mg/ml	50 mg/ml	100 mg/ml
pH	5.20	5.20	5.20	5.20
Dosis	2 ml	2 ml	2 ml	2 ml

and after filtration by a 0.2 μm filter (Millipak®-20, Millipore, Bedford MA, USA) in Hypac ready-to-use glass syringes (Vetter, Ravensburg, Germany) sterile.

Example 8

The DEAE filtrate of example 1 is treated at 20-25°C for 30 minutes at a pH of about 5.5 with 0.2 g aluminium hydroxide gel per g of protein and further processed as in example 1.

Example 9

The procedures in examples 1 to 6 are repeated, using alternatively one of the following gels with the same functional group instead of MacroPrep® 50 CM: CM-Spherodex® (Sepracor IBF, Villeneuve la Garenne, France), CM-Trisacryl® (Sepracor IBF, Villeneuve la Garenne, France), CM-Sepharose® FF (Pharmacia, Uppsala, Sweden) or Fractogel® TSK CM-650 (Merck, Darmstadt, Germany) to obtain similar results.

Example 10

The procedures in examples 1 and 3 to 6 are repeated using alternately DEAE-Sephadex® A 50 MacroPrep® DEAE (BioRad, Hercules CA, USA) to obtain similar results.

The chromatographic steps in examples 1 to 6 above may be performed as batch, column or membrane chromatography, with the result that analogue results are obtained.

Claims (11)

1. Method of producing an anti-D immunoglobulin G-preparation with a specific activity of more than 0.5% anti-D IgG per gram total IgG, from human plasma, characterised in that plasma from rhesus negative blood of rhesus factor D sensitised donors or a plasma fraction containing an anti-D IgG:

   A) with a pH value in the range of pH 3.5 to 6.5 is subjected to an ion exchange chromatography, with an adsorbent which has carboxymethyl groups as functional groups, the anti-D IgG being bound to the adsorbent,

   B) the adsorbent with the bound anti-D IgG is first rinsed with a wash solution, and the anti-D IgG is subsequently eluted, and further

   C) the eluted anti-D IgG is treated with an alkaline adsorbent with ion-exchange characteristics in order to bind undesired components, and finally the anti-D IgG is concentrated.
2. Method according to claim 1, characterised in that the anti-D IgG is concentrated in stage C), in that steps A) and B) are repeated at least once.
3. Method according to claim 1 or 2, characterised in that used as the starting material is plasma which has been pre-treated with a basic adsorbent with ion-exchange characteristics in order to bind undesired components.
4. Method according to claim 1 or 2 characterised in that the protease content in the plasma or in the plasma fraction is reduced through incubation with an adsorbent such as aluminium hydroxide gel.
5. Method according to one of the claims 1 to 4, characterised in that all ion exchange chromatography steps, and if necessary further adsorption steps, are carried out alternatively as batch chromatography, column chromatography or membrane chromatography.
6. Method according to one of the claims 1 to 5, characterised in that the alkaline adsorbent in step C) has diethylaminoethyl groups as functional groups.
7. Method according to one of the claims 1 to 6, characterised in that the elution in step B) is carried out with pH shift and/or change of the buffer composition, for example by changing its ionic concentration or conductivity, respectively.
8. Method according to one of the claims 1 to 7, characterised in that it comprises at least one step of virus inactivation.
9. Method according to claim 8, characterised in that the step of virus inactivation comprises treatment of the plasma or of the fraction containing anti-D IgG with a detergent and tri(n-butyl) phosphate, the phases of the mixture containing solvent and detergent being separated and the clear lower phase being used.
10. Method according to claim 8, characterised in that the step of virus inactivation is a heat treatment.
11. Method according to one of the claims 1 to 10, characterised in that the starting plasma or the starting plasma fraction prior to the ion exchange chromatography step in stage A) is subjected to at least one of the steps mentioned in the following:

    a) freezing the plasma, the cryoprecipitate being separated by filtration and/or centrifugation after thawing and before its further use

    b) treatment with a solvent-detergent-mixture, incubation at approximately 37° C and phase separation.