AU715427B2 - Method of recovering immunoglobulin from fractions produced during fractionation of human blood plasma - Google Patents

Abstract

Prodn. of a high-titre immunoglobulin (Ig) prepn. comprises: (a) fractionating blood plasma from a plasma pool to separate ≥ 1 industrially utilisable polyclonal IgG fraction from a residual fraction; (b) preparing a protein soln. from the protein components of the residual fraction or its subfractions; (c) subjecting the protein soln. to ≥ 1 affinity chromatography step in which specific plasma proteins are bound to immobilised ligands of ≥ 1 type and the bound proteins are then released, and (d) converting the released proteins into a high-titre Ig prepn.

Description

a 1 1- P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE

SPECIFICATION

STANDARD PATENT Invention Title: METHOD OF RECOVERING

IMMUNOGLOBULIN

FROM FRACTIONS PRODUCED

DURING

FRACTIONATION OF HUMAN BLOOD PLASMA 4

S

S

The following statement is a full description of this invention, including the best method of performing it known to us: GH REF: P23806-C:DAA:RK METHOD OF RECOVERING IMMUNOGLOBULIN FROM FRACTIONS PRODUCED DURING FRACTIONATION OF HUMAN BLOOD PLASMA This invention relates to the preparation of immunoglobulin, and more particularly to a method of producing a high-titer immunoglobulin preparation. This method may be utilized for recovering immunoglobulins from fractions which, in the methods of plasma fractionation customary until now, are not made use of, or at least are not used in the production of immunoglobulin preparations.

There are over one hundred different proteins in human blood plasma. Some of them can be purified by fractionation from pools of donor plasma and used as therapeutic products as in the following examples: albumin is used for compensating an oncotic deficit in hypoproteinemia or hypovolemia; blood coagulation factors VIII and IX are administered as hemorrhage prophylaxis and therapy in hemophilia A and B, respectively; 15 immunoglobulins are made use of as infection prophylaxis and therapy in antibody-deficiency diseases, as well as in idiopathic thrombocytopenic purpura; immunoglobulins from selected donors having high titers of specific immunoglobulins are used as hyperimmunoglobulin preparations for the prophylaxis and treatment of specific infections such as hepatitis A or B.

20 Therapeutically usable plasma proteins can be isolated, for example, according to known methods of ethanol fractionation (Cohn, E. et al., J. Am.

Chem. Soc., 68, 459, 1946; Kistler, and Nitschmann, Vox Sang., 7, 414, 1962). With both methods, it is possible to isolate large amounts of functional plasma proteins such as albumin or immunoglobulins which, in suitable 25 formulations, can be profitably utilized clinically. However, when working according to these methods, precipitates and/or supernatants occur which cannot be used in conventional processing. The composition of these fractions varies greatly.

Whereas, for example, in the precipitation of blood plasma with 19% ethanol at a pH of 5.8, human apolipoprotein A-I (apoA-I) is to be found in approximately equal parts in the supernatant a (about 50% of the plasma 2 apoA-l) and in precipitate A (about the same protein is then found after the next fractionation steps according to Kistler and Nitschmann in those fractions which have not been used commercially until now: precipitate IV and precipitate B (about 40% each) (Lerch et al., Protides of the Biological Fluids, 36, 409, 1989).

A similar distribution pattern results for the copper-binding protein ceruloplasmin. After fractionation, 20% of the starting material is found in precipitate IV and 40% in precipitate B.

Transferrin is an example of a plasma protein which, in the same fractionation method, is substantially 100% concentrated in precipitate IV, whereas 80% albumin is to be found in the precipitate C currently used.

From 50-60% of immunoglobulins may be recovered in precipitate GG by means of Kistler-Nitschmann or Cohn fractionation. The remaining 40% is distributed among precipitate IV precipitate B and filtrate GG in these methods.

The aforementioned percentages are intended to illustrate the order of magnitude of the distribution and are not to be taken as restrictive; they are variable depending upon the conditions and methods used.

These examples show that in some instances considerable amounts S: 20 of therapeutically usable proteins are to be found in the fractions precipitate IV, precipitate B, supernatant c, and supernatant GG, or in the corresponding fractions of the plasma fractionation according to Cohn (fraction IV-1, fraction I1+111, supernatant V, and supernatant 11-1,2). For ethical reasons, but also because of the worldwide shortage of human blood plasma and certain plasma components, an improvement should be sought in the yield of immunoglobulins, which has not been very high until now.

Immunoglobulins play a pivotal r6le in warding off infections. Either virus-specific, neutralizing antibodies block the adsorption of viruses on the cellular receptors and thus prevent infection, or bacteria-specific antibodies opsonize the pathogen and thus allow it to be eliminated and killed by neutrophils and macrophages. Plasma pools from several thousand donors contain immunoglobulins of very many different specificities, and immunoglobulin preparations from such pools consequently also contain measurable titers of immunoglobulins directed against epitopes on viruses, bacteria, and toxins, but also against autoantigens. Hence they are effective against many infections and in the most varied other pathological conditions.

Now, under certain circumstances, however, it is desirable to make use of an immunoglobulin preparation having high titers of specific antibodies, a so-called hyperimmunoglobulin preparation. Until now, such preparations have been prepared at great expenditure of time and money from special plasma pools of donors having increased titers of specific antibodies. For various reasons, this involves difficulties. If, as in the case of an anti-hepatitis B preparation, there are recognized vaccination procedures, then the donors must be inoculated and selected, and the donated blood must be separately processed. In the case of many other indications, however, immunization of the donor cannot take place for ethical reasons. Here it is only rarely possible to locate high-titer blood through an involved selection of the donors after their having recovered from a specific disease) and to obtain a preparation through processing of the donated blood.

It is therefore an aim of one or more aspects of this invention to provide a method of recovering immunoglobulins by means of which valuable immunoglobulin preparations can be made accessible and isolated from the aforementioned fractions and precipitates, hardly made use of until now, which become available during industrial plasma-fractionation methods.

Subsequently, it should be possible to process these preparations, corresponding to hyperimmunoglobulin preparations, into a well-tolerated, especially intravenously virus-proof, liquid or freeze-dried preparation.

It has now been found that it is possible to produce hyperimmunoglobulin preparations or high-titer immunoglobulin preparations by a method differing from the prior art methods. This novel method uses immunoglobulins from the general plasma pools, improves the exploitation of the valuable raw material blood plasma through the use of "waste" fractions, S:23806C and even permits the production of hyperimmunoglobulin preparations having far greater specific activities than previous preparations. This means that with small quantities administered IV, and with low amounts of IV administered proteins, a correspondingly low burden on the recipient, high doses of specific immunoglobulins can be given within a short time. This is made possible in the inventive method through the concentration of the specific immunoglobulins through adsorption on immobilized antigens, thus through the use of processed "waste" fractions in affinity chromatographic techniques.

According to the present invention there is provided a method of producing a hyperimmunoglobulin preparation, comprising the successive steps of: preparing a protein solution from the protein components of a waste fraction obtained during the fractionation of blood plasma, or a subfraction thereof; subjecting the resulting protein solution at least once to affinity chromatography with immobilized S' 20 ligands of at least one ligand type to obtain specific i. plasma proteins; and preparing said hyperimmunoglobulin preparation from said plasma proteins.

In the inventive method, a waste fraction obtained in 25 the Cohn plasma fractionation process or the Nitschmann plasma fractionation process, is first processed in such a way that it can be used in affinity chromatography. As used throughout the specification and claims references to the "Cohn plasma fractionation process" or the "Cohn 30 process" or words to that effect shall be taken as a reference to the abovementioned standard methods of Cohn et al and variants thereof, and references to the "Nitschmann plasma fractionation process" or the "Nitschmann process" or words to that effect shall be taken as a reference to the abovementioned methods of Nitschmann et al and variants thereof. Depending upon the provenance of the fraction, this processing may vary greatly. Thus, for example, the H:\MaraR\Keep\Speci\P29477.doc 5/11/1999 supernatant GG may be concentrated down, dialyzed against a suitable buffer, and filtered. On the other hand, precipitates or filter cakes (lists of examples of such starting materials are given in Tables 1 and 2 below) must first be suitably suspended, through variation of the ionic strength, the pH, and/or the temperature, or through the addition of detergents and salts, in such a way that immunoglobulins are specifically solubilized. They may, for example, be stirred overnight within a pH range of from 3-9, at a conductance of from 5-20 mS/cm, at 4 0 C and then be clarified by centrifugation and/or filtration. Both supernatants and suspensions may at this stage be subjected to a virus-inactivation process according to the solventdetergent method of Horowitz ff ftf ft f t ft ft **fotf ft ftf H:\MaraR\Keep\Speci\P29477.doc 5/11/1999 (Thrombosis and Haemostasis, 65, 1163, 1991), the methylene blue method (Mohr et al., Infusionsther. Infusionsmed. 20, 19 [1993]), or some other method.

The immunoglobulins may also be subjected after suspension to prepurification by preadsorption on a matrix in a column or through treatment with a suitable filter aid or adsorbent such as aluminum hydroxide, chromatography on protein A or G for concentration, or one or more precipitations by the common methods of ammonium sulfate, polyethylene glycol, or ethanol precipitation or combinations thereof for enrichment, concentration, or depletion of disturbing components.

O

Supernatants Precipitates and Residues Supernatant c Precipitate B Supernatant GG Precipitate IV Residue after DEAE treatment Residue after aluminum hydroxide treatment Residues after clarifying filtrations I, II and III Table 1: Examples of possible starting materials coming from fractionation according to Kistler and Nitschmann or from other processing steps resulting from the preparation of IV administrable, stable plasma products.

Supernatants Precipitates and Residues Supernatant V Precipitate II+111 Supernatant 11-1,2 Precipitate IV-1 Residue after DEAE treatment Residue after aluminum hydroxide treatment Residues after clarifying filtrations I, II, and III Table 2: Examples of possible starting materials coming from the fractionation according to Cohn or from other processing steps resulting from the preparation of IV administrable, stable plasma products.

The following list gives examples of possible ligands for the inventive affinity chromatography: Antigenic determinants of Haemophilus influenza b Staphylococcus aureus Staphylococcus epidermidis Staphylococcus agalactiae Streptococcus pneumoniae Streptococcus pyogenes and other pathogenic strains of bacteria tetanus toxin Staphylococcus aureus toxic shock toxin and further pathogenic bacterial or other toxins hepatitis A virus hepatitis B virus hepatitis C virus varizella zoster virus cytomegalo virus respiratory syncytial virus 20 parvovirus B19 herpes simplex virus 1 herpes simplex virus 2 rabies virus and other pathogenic viruses 25 CD2, CD3, CD4, CD28, CD40, CD72 ICAM, LFA-1, LFA-3,

DNA

and other potential human autoantigens 0*l Since affinity gels have been prepared by immobilizing modified or unmodified ligands (examples of such ligands are given in the above list) by means of methods known per se, they are loaded with the processed supernatants and suspensions in concentrated or diluted form, if necessary also by repeated application of the flow. If desired, various affinity gels may be activated in succession with the same suspension. The gels are thereafter washed in such a way that unspecifically binding proteins are removed preponderantly or to a sufficient extent. This can be done, for example, by increasing the saline concentration, by addition of a detergent, and/or by shifting the pH in the washing solution. The bound proteins are now separated from the ligands, by elution at a low or high pH, by addition of chaotropic saline solutions such as sodium thiocyanate or magnesium chloride, denaturing agents such as SDS or urea, solvents such as ethylene glycol, by modifying the temperature, or by combinations of the foregoing.

In some cases, it may be desirable to modify the ligands to be immobilized by mutagenesis or by chemical or physical methods in such a way that the specific immunoglobulins can still bind to their epitopes, but with reduced affinity, so that elution can take place under milder conditions than with the unmodified ligands. Modification of the ligands may also take place in order to facilitate and improve their immobilization and/or their epitope presentation. Technical details and basic principles of the affinity chromatography process in general are described in Cuatrecasas, and Anfinsen, C. B. (1971), Ann. Rev. Biochem. 40, 259; Kull, F. and Cuatrecasas, P. (1981), J. Immunol. 126, 1279; Liebing et al. (1994), Vox Sang. 67, 117.

The specific, separated immunoglobulins, also with additional filtration for eliminating viruses, if need be, are processed into an end product which can preferably be administered IV and which is free of pyrogens, virusproof, and stable with or without the addition of stabilizers such as albumin, amino acids, or carbohydrates in liquid or freeze-dried form. However, formulations may also be used which make possible intramuscular or topical administration.

25 Preferred embodiments of the present invention are described and illustrated below by means of the following examples, which are not intended to be limitative Example 1 HBsAg-Sepharose was prepared by immobilizing 5 mg of recombinant hepatitis B virus surface antigen (HBsAg, Abbott Diagnostics) through coupling of the primary amino groups to 1 ml of activated CH- 8 Sepharose as directed by the manufacturer (Pharmacia Biotech, Uppsala, Sweden). "Placebo"-Sepharose was prepared by carrying out the same coupling process with another gel aliquot, but without adding HBsAg. The finished gels were stored in PBS with 0.02% NaN 3 at 4 0

C.

Seventy grams of precipitate B from the Kistler-Nitschmann plasma fractionation (NB Lot 4.030.216) were suspended overnight in 210 ml of 100 mM citric acid, pH 4.0, 0.25% Triton X-100, 10 mM N-ethyl maleimide (NEM), 1 mM phenylmethylsulfonyl fluoride (PMSF) at 4 0 C on a RotaryMix appliance.

After separation of the insoluble components by centrifugation (5000g, 4 0 C, min.) and filtration (pore size: 1.2 pm), 1% tri-n-butylphosphate (Merck, Darmstadt, Germany) and 1% Triton X-100 were added at a neutral pH according to Horowitz et al. (Thrombosis and Haemostasis, 65, 1163, 1991) and incubated at 30 0 C for 4 hrs. with mixing. A phase separation was then carried out overnight at 37 0 C, the clear lower phase was removed, filtered 15 through a 0.45 pm filter, and stored at 4 0

C.

One hundred thirty ml of such an NB suspension were diluted with 280 ml of PBS (phosphate-buffered saline solution: 150 mM NaCI, 10 mM sodium phosphate, pH 7.1) and pumped over the placebo-Sepharose column, then over the HBsAg-Sepharose column at 4°C in such a way that the column 20 flow passed back into the storage vessel. The rate of flow was 8 ml/hr for 144 hrs. Thus the NB suspension was pumped over the columns a total of three times. After 90 hrs., the loading was interrupted, and the columns were washed individually, first with PBS and then with 200 mM NaCI, 50 mM tris-HCI, pH 7.4, until the wash solution had an optical density of less than 0.01 at 280 nm (OD 2 80 After conclusion of the activation, washing was again carried out with PBS and 200 mM NaCI, 50 mM tris-HCI, pH 7.4. Bound proteins were separated with 5 ml of 200 mM glycine-HC, pH 2.5, immediately neutralized, and processed.

Gel HBsAg Placebo Total loading: Protein 1775 mg 1775 mg IgG 450 mg 450 mg Anti-HBs IgG 4560 mlU 4560 mlU Flow: Anti-HBs IgG 1140 mlU 1140 mlU Eluate: IgG 0.13 mg 0.08 mg Anti-HBs IgG 5603 mlU 114 mlU Table 3: Anti-HBsAg affinity chromatography with NB suspension Example 2 Starting from the Cohn fraction 11+111 (instead of blood plasma as the starting material), a Kistler-Nitschmann fractionation was carried out. Seventy grams of precipitate B (NB Lot 4.044.488) from this Kistler-Nitschmann fractionation were suspended overnight in 210 ml of 100 mM citric acid, pH 0.25% Triton X-100, 10 mM NEM, 1 mM PMSF, at 4 0 C on a RotaryMix 10 appliance. After clarification and partial delipidation by ultracentrifugation (100,000g, 3 hrs., 4 0 C: the clear phase was withdrawn by piercing the side of the tube with a syringe), the suspension was filtered (0.45 [lm) and stored at One hundred twenty-five ml of this NB suspension were diluted with in Example 1, first over placebo-Sepharose gel and then over HBsAg- Sepharose gel prepared analogously to Example 3. The gels were then washed separately with PBS and 200 mM NaCl, 50 mM tris-HCI, pH 7.4. The bound proteins were removed with 5 ml of 200 mM glycin-HCI, pH 2.5. Table 4 gives a summary of the data.

S0* HBsAg Placebo Protein____ 1400 mg 1400 mg 619 mg 619 mg Anti-HBs IgG 10OIU Anti-H~s IgG 151IU 15 IU Eluate: I IgG 0.18 mg 0.35 mg Anti-HBs lgG j3.3 IU 0.08 IU Table 4: Anti-HBsAg affinity chromatography with NB suspension (Cohn 11+111) Example 3 Thirty liters of supernatant GG (Lot No. X95.31.286.1) were diafiltered in PBS and concentrated down to 500 ml. As in Example 1, the concentrate was pumped over placebo and HBsAg columns at 21 mI/hr for 118 hrs. Washing of the columns and separation of the bound proteins likewise took place analogously to Example 1, but an additional washing step with 500 10 mM NaCI, 50 mM tris-HCI, pH 7.4, was carried out. The results are shown in Table Gel IHBsAg IPlacebo Total loading: Protein j8800 mg 8800 mg IgG j320 mg 320 mg Anti-HBs IgG 15000 mlU 5000 mlU Flow: I Anti-HBs lgG <DL [<DL Eluate: IgG J0.05 mg 0.14 mg r Anti-HBs IgO 3035 mlU 7 mlU Table 5: Anti-HBsAg affinity chromatography with supernatant GG concentrate DL: detection limit 11 Example 4 DEAE filter cake in an amount of 17.5 g (Lot 4.422.006.0) was suspended in 52.5 ml of suspension buffer according to Example 1 and processed. Forty ml of suspension were diluted with 160 ml of PBS, the pH adjusted to 7.1, and then filtered. As in Example 3, the suspension was pumped over placebo and HBsAg columns at 21 ml/hr for 97 hrs. Washing of the columns and separation of the bound proteins also took place analogously to Example 1. The results are shown in Table 6.

Gel HBsAg Placebo Total loading: Protein 548 mg 548 mg lgG 280 mg 280 mg Anti-HBs IgG 400 mlU 400 mlU Flow: Anti-HBs IgG <DL

<DL

Eluate: IgG 0.07 mg 0.11 mg Anti-HBs IgG 331 mlU 14 mlU Table 6: Anti-HBsAg affinity chromatography with DEAE filter-cake suspension DL: detection limit Example Tetanus toxoid C-Sepharose was prepared by immobilizing 11.5 mg of purified tetanus toxoid (TT) through coupling of the carboxy groups to 1 ml of EAH-Sepharose (Pharmacia Biotech, Uppsala, Sweden) with the aid of 0.1 M N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide-HCI, as directed by the manufacturer. The finished gels were stored in PBS with 0.02% NaN 3 at 4 0

C.

A concentrate of supernatant GG was prepared as in Example 3 and used for affinity chromatography with TT-Sepharose analogously to Example 2.

Activation took place at 23.5 ml/hr for 159 hrs. at 4 0 C. The results are shown in Table 7.

B

Gel Tetanus Toxoid C Total loading: Protein 15000 mg IgG 320 mg Anti-TT IgG 36 pg Flow: Anti-TT IgG 16 pg Eluate: IgG 0.16 mg Anti-TT IgG 76 jg Table 7: Anti-tetanus toxoid affinity chromatography with supernatant GG concentrate Example 6 Tetanus toxoid N-Sepharose was prepared by immobilizing 11.5 mg of purified tetanus toxoid (TT) through coupling of the primary amino groups to 1 ml of activated CH-Sepharose as directed by the manufacturer (Pharmacia Biotech, Uppsala, Sweden). "Placebo"-Sepharose was prepared by carrying out the same coupling process with another gel aliquot, but without adding TT.

The finished gels were stored in PBS with 0.02% NaN 3 at 4 0

C.

:Precipitate B was suspended according to Example 2.

After filtration (1.2 Jm), 115 ml of this suspension were diluted with 200 ml of PBS, the pH adjusted to 7.1, and pumped first over the placebo- Sepharose at 3.5 ml/hr for 165 hrs. in such a way that its flow passed 15 immediately thereafter over the TT N-Sepharose column, then back into the storage vessel. The columns were washed separately with PBS and then with M NaCI, 50 mM tris-HCI, pH 7.1, until the OD 2 80 of the flow was less than 0.01. The results are shown in Table 8.

Gel ITetanus Toxoid N Placebo Total loading: Protein 1334 mg 11334 mg IgG 333 mg j333 mg Anti-UT lgG 0.509 mg 0.509 mg Flow: I_ Anti-TT IgG 10.213 mg 10.213 mg Eluate: IgG 0.35 mg 0.004 mg Anti-fl IgG 0.091 mg j0.003 mg Table 8: Anti-tetanus toxoid affinity chromatography with NB suspension Example 7 Precipitate B was suspended according to Example 2.

After filtration (1.2 pm), 115 ml of this suspension were diluted with 200 ml of PBS, the pH adjusted to 7. 1, and pumped first over the HBsAG- Sepharose column at 5 mI/hr for 165 hrs. in such a way that its flow passed immediately thereafter over the TT-Sepharose column and then back into the storage vessel. The columns were washed separately with PBS and then with 10 0.5 M NaCI, 50 mM tris-HCI, pH 7.1, until the 0D 28 0 of the flow was less than 0.01. The results are shown in Table 9.

Gel JHBsAg ITetanus Toxoid Total Protein 1334 mg 1334 mg 19G 333 mg 333 mg Anti-HBsAg 945 mlU 945 mlU Anti-UT IgG 0.509 mg 0.509 mg F low: Anti-HBsAg j536 mlU 536 mlU Anti-fl IgG J0.265 mg i 0.265 mg Eluate: IgG .0.13 mg 0.25 mg Anti-H BsAg 1152 Anti-fl lgG j0.18 mg

S

S

S. S S. S

*SS*

S.

S. S 555555 .5 5 14 Table 9: Anti-HBsAb and anti-tetanus toxoid affinity chromatography with NB suspension Example 8 Fifteen kg of precipitate B from the Kistler-Nitschmann fractionation (Lot No. 5.043.303) were suspended in 45 liters of 0.1 M citric acid, pH 0.25% Triton X-100, 10 mM NEM, 1 mM PMSF overnight at 4*C with a vibromixer. After separation of the insoluble components by adding filter aids and filtering (pore size: 1.2 lm), 1% tri-n-butyl phosphate and 1% Triton X-100 were added at neutral pH for delipidation and virus inactivation according to Horowitz and incubated for 4 hours at 30 0 C with mixing. Thereafter, a phase separation was carried out overnight at 37°C, the clear lower phase pumped off, filtered through a 0.45 im filter, and stored at 4 0

C.

The pH of 40 liters of this NB suspension was adjusted with NaOH to a value of 7.1, and it was pumped at 4 0 C over a HBsAg column and a tetanus toxoid Affiprep column in such a way that the column flow passed back into the storage vessel. The Affiprep columns (50x13 mm) were prepared by coupling 250 mg of recombinant HBsAg and tetanus toxoid, respectively, with 25 ml of Affiprep gel (BioRad Lab. Inc., Hercules, CA 94547. The rate of flow was 6 It/hr for 62 hrs. Thus the NB suspension was pumped over the columns 20 a total of three times. After conclusion of the activation, the columns were washed separately, first with PBS and then with 500 mM NaCI, 50 mM tris-HCI, pH 7.4, until the washing solutions had an optical density of less than 0.01 at 280 nm (OD 280 Bound proteins were removed with 200 mM glycin-HCI, pH and the pH of the fractions immediately adjusted to 5.2. The results are 25 compiled in Table 10. The immunoglobulins were processed into stable preparations by pooling the fractions containing IgG, diafiltration, and concentration with 20 mM NaCI into solutions of 100 IU/ml and 2.5 mg anti-TT- IgG/ml, respectively. Ten percent saccharose was added, and the solutions were lyophilized in units of 200 IU, and 5 mg of anti-TT-IgG, respectively.

Example 9 Fifty grams of precipitate IV from the Kistler-Nitschmann plasma fractionation were suspended in 500 ml of water. The pH was adjusted to with citric acid, and the conductance to 13 mS with NaCI. After stirring overnight at 4 0 C, clarification and partial delipidation were achieved by centrifugation for 30 min. at 30,000g and 4°C. The separated protein, IgM, IgA, IgG, transferrin, and ceruloplasmin content of the suspension was determined and is indicated in Table 11.

Gel HBsAg Tetanus Toxoid Total loading: Protein 448 g 448 g IgG 204 g 204 g Anti-HBs IgG 1120 IU 1120 IU Anti-TT IgG 317 mg 317 mg Flow: Anti-HBs IgG 221 IU 221 IU Anti-TT IgG 172 mg 172 mg Eluate: IgG 39.8 mg 171 mg Anti-HBs IgG 1368 IU Anti-TT IgG 89 mg a a a Table 10: Anti-HBsAg and anti-tetanus toxoid 10 suspension affinity chromatography with NB Total Protein IgM IgA I gG Transferrin Caeruloplasmin 5.5 0.063 0.628 0.488 2.582 0.09 Table 11: Suspension from precipitate IV (all values in mg/ml) Example Fifty grams of precipitate B from the Kistler-Nitschmann plasma 15 fractionation (Lot No. 4030.204.0) in 100 mM citric acid at various pH values and at 4 0 C were stirred overnight, clarified by ultracentrifugation at 100,000g and 4 0 C for 3 hrs. and partially delipidated. The clear middle phase was removed, and its separated protein, IgM, IgA, IgG, transferrin, and ceruloplasmin content was determined (Table 12).

Total Protein IgM IgA IgG Transferrin Caeruloplasmin 4.9 1.2 1.3 1.9 <DL 0.06 pH 5.0 6.1 1.2 1.0 2.1 <DL <DL Table 12: Suspension from precipitate B (all values in mg/ml); <DL: below detection limit The titer of igG against certain viral (Table 13) and bacterial (Table 14) antigens was determined and compared with thatin starting plasma and existing immunoglobulin preparations.

Plasma Plasma SAGL NB pH 4.0 NB pH 102 103 Anti-HBsAg 0.02 0.05 0.01 0.05 0.05 IU/mg IgG Anti-CMV 0.28 0.16 0.3 0.52 0.34 PEIE/mg IgG Anti-VZV 0.08 0.09 n.d. 0.21 0.1 IU/mg IgG Anti-measles 0.9 0.2 0.02 2.1 0.81 IU/mg IgG Anti-HSV1 1037 n.d. n.d. 2317 749 AU/mg 1gG Table 13: Antiviral IgG in plasma pools, SAGL, and NB suspensions Plasma 102/103: plasma pools; SAGL: Sandoglobulin®; NB pH 4.0/5.0: suspension from precipitate B at pH 4.0 and 5.0, respectively; IU: International Units; PEIE: Paul Ehrlich Institute units; AU: arbitrary units; not determined.

Plasma 102 Plasma 103 SAGL NB 4A NB 14 pH anti- 162 286 436 100 283 tig/g IgG HiBOAg anti-TT 1855 4159 2740 2928 2923 pg/g IgG anti-SEB 412 689 783 918 670 AU/g IgG Table 14: Antibacterial IgG in plasma pools, SAGL, and NB suspensions Plasma 102/103: plasma pools; SAGL: Sandoglobulin®; NB 4A: suspension from precipitate B at pH 4.0; NB 14 pH 15 suspension from precipitate B at pH 4.0 with virus inactivation; HiBOAg: haemophilius influenza B-oligosaccharide antigen; TT: tetanus toxoid; SEB: Staphylococcus enterotoxin B; AU: arbitrary units.

9

Claims (21)

1. A method of producing a hyperimmunoglobulin preparation, comprising the successive steps of: preparing a protein solution from the protein components of a waste fraction obtained during the Cohn process or the Nitschmann process for the fractionation of blood plasma, or a subfraction thereof; subjecting the resulting protein solution at least once to affinity chromatography with immobilized ligands of at least one ligand type to obtain specific plasma proteins; and preparing said hyperimmunoglobulin preparation from said plasma proteins.

2. A method according to claim 1, wherein the fractionation of blood plasma is carried out on an industrial scale.

3. A method according to claim 1 or 2, wherein the waste fraction according to step is a precipitate or a filter 20 cake, and the protein components are put into solution by treating the precipitate or the filter cake with an aqueous S. buffer solution having an ionic strength of <5M and a pH of from 3.0 to 9.0 so as to form a solution or suspension. 25

4. A method according to claim 3, wherein the buffer solution is a phosphate buffer, a tris-HCl buffer, or a citrate buffer.

5. A method according to claim 3 or 4, wherein the buffer solution contains a detergent, one or more protease inhibitors, and/or a salt.

6. A method according to one of the claims 3 to wherein the solution or suspension is filtered or is pretreated with an adsorbent such as aluminum hydroxide or an adsorbent containing DEAE groups. H:\MaraR\Keep\Spei\P29477,doc 5/11/1999

7. A method according to one of the claims 3 to wherein the solution or suspension is reacted with ammonium sulfate, polyethylene glycol, or ethanol so as to form a precipitate, and the supernatant or the precipitate is processed.

8. A method according to claim 1 or 2, wherein the waste fraction according to step is a supernatant, and the protein solution is prepared by filtration and concentration.

9. A method according to claim 8, wherein the protein solution is prepared by diafiltration.

10. A method according to claim 8 or claim 9, wherein the supernatant is filtered or is pretreated with an adsorbent such as aluminum hydroxide or an adsorbent containing DEAE o° groups. sooo 20

11. A method according to one of the claims I to wherein the immobilized ligands are natural or recombinant viral, bacterial, or cellular antigens. i

12. A method according to claim 11, wherein the ligands are selected from the group of antigenic determinants of Haemophilus influenza b, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, tetanus :toxin, Staphylococcus aureus toxic shock toxin, hepatitis A virus, hepatitis B virus, hepatitis C virus, varizella zoster virus, cytomegalo virus, respiratory syncytial virus, parvovirus B19, herpes simplex virus 1 and 2, rabies virus, and the potential human autoantigens CD2, CD3, CD4, CD28, CD40, CD72 ICAM, LFA-I, LFA-3, DNA, and phospholipids. H: \MaraR\Keep\Speci\P29477 .doc 5/11111999

13. A method according to claim 11 or 12, wherein the ligands have been modified by mutagenesis or chemical or physical methods.

14. A method according to one of the claims 1 to 13, wherein said hyperimmunoglobulin preparation consists of immunoglobulins of the classes G or A or M or any combination thereof.

15. A method according to one of the claims 1 to 14, wherein said hyperimmunoglobulin preparation obtained is subjected to virus inactivation and, if necessary, stabilized through the addition of a stabilizer such as albumin, amino acids, or carbohydrates and/or the product 15 is freeze-dried. S

16. A method according to one of the claims 1 to wherein said hyperimmunoglobulin preparation is converted 0e into a pharmaceutically acceptable product.

17. A method according to claim 16, wherein said hyperimmunoglobulin preparation is converted into an intravenously, intramuscularly or topically administrable preparation.

18. A hyperimmunoglobulin preparation produced by a method as defined in any one of claims 1 to

19. A pharmaceutically acceptable product produced by a method as defined in claim 16 or claim 17. A method of producing a hyperimmunoglobulin preparation substantially as herein described with reference to any one of the Examples, excluding Comparative or Control Methods.

H:\MaraR\Keep\Speci\P29477.doc 17/09/99

21. A hyperinimunoglobulin preparation produced by a method substantially as herein described with reference to any one of the Examples, excluding Comparative or Control Methods. Dated this 5th day of November 1999 ROTKREUZSTIFTUNG ZENTRALLABORATORIUM BLUTSPENDEDIENST SRK By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia 4. 464 *s S H: \MaraR\Keep\Speci\P29477 .doc 5/11/1999