The method of producing immunoglobulins of the fractions formed during fractionation of human blood plasma

 

(57) Abstract:

The invention relates to a method of producing immunoglobulins. Receive immunoglobulin preparations with a high titer of the fact that carry out consistently the following stages: fractionation of blood plasma from a pool of plasma, in which the separated fraction, which can be used in industry and which contains polyclonal immunoglobulin G; preparation of protein solution protein components; carrying out affinity chromatography. The resulting antibodies can be processed into acceptable for intravenous injection, stable during storage of the product. Effect: method provides receiving immunoglobulin preparation, the use of fractions of human plasma, which is still discarded in conventional industrial methods of fractionation of plasma. 2 C. and 20 C.p. f-crystals, 14 PL.

The invention relates to a method for producing the immunoglobulins of the factions that do not use if common current method of fractionating plasma for clinical drugs or at least do not find use in preparation of immunoglobulin preparations.

who can be cleaned by fractionation and be used as therapeutic products. Known examples of the following: albumin is used to compensate oncotic deficit when gipoproteinemii or hypovolemia. The coagulation factor VIII, IX respectively, administered for the prevention and treatment of bleeding in hemophilia A, respectively B. Immunoglobulins used in the diseases associated with deficiency of antibodies for prophylaxis and therapy of infections, and in the case of idiopathic thrombocytopenic purpura. Immunoglobulins from selected donors with high contents of specific immunoglobulins are used as hyperimmunoglobulinemia drugs for the prevention and treatment of specific infections, such as hepatitis A or B.

The allocation of therapeutically applied plasma proteins can be performed, for example, known methods of fractionation in ethanol (onh. E. G. and others J. Am. Chem. Soc., 68, 459, 1946, and P. Kistler and Nitschmann H., Vox Sang, 7, 414, 1962). With both methods it is possible to allocate large amounts of functional plasma proteins as albumin or immunoglobulins, which are clinically useful in suitable formulations. However, in the processing of these methods precipitation is formed, respectively supernatant, which is not used in normal is ain A-1 (Apo A-I) during the precipitation of plasma with 19% ethanol at a pH value of 5.8 can be detected in almost equal shares in the supernatant (approximately 50% of the Apo-A-1 plasma) and in the sediment A (about 40%), the same protein after the upcoming stages of fractionation by Kistler and Nitschmann then is in those fractions, which are still not commercially used: IV sediment and sludge B (approximately 40%) (Lerch and others, in Protides of the Biological Fluids, 36, 409, 1989).

An example of this distribution is obtained for protein caeruI0-plasmin carrying copper. After fractionation find 20% of the source material in the sediment IV and 40% sludge B.

Transferrin is an example of a plasma protein, which in the case of these fractionation techniques to almost 100% accumulates in the sediment IV, while the albumin in the amount of up to 80% should be used in the present draft C.

Immunoglobulins can be obtained by fractionation by Kistler-Nitschmann or Cohn up to 50-60% in the sediment GG. The remaining 30-40% according to these methods are distributed on a sediment IV (5%), residue B (30%) and the filtrate GG (5%).

The above percentages should illustrate the gradation distribution and should not be interpreted as limiting; these data vary depending on the conditions and methods.

These examples show that a significant number of therapeutically suitable proteins molotovmcmusic fractions from fractionation of plasma by the Cohn (fraction IV-1, fraction II+III supernatant V and the supernatant II-1,2). For ethical reasons, but also because of the lack of worldwide human plasma and a known component of the plasma, it is necessary to strive for improvement is still not very high yield of immunoglobulins.

Immunoglobulins play a Central role in protection against infections. They either block virousspecificakih, neutralizing antibodies, the adsorption of virus to cellular receptors and thus prevent infection, or then opsonizing specific to bacteria, antibodies, pathogens, and thus allow their removal and destruction by neutrophils and macrophages. Pools of plasma from thousands of donors contain immunoglobulins very many different specificdate, and immunoglobulin preparations from these pools as a consequence, also contain measurable amounts of antibodies, which are directed against epitopes of viruses, bacteria, toxins, but also against autoantigens. As a result, they are effective against many infections and various other pathological conditions. Under certain circumstances, however, now it is desirable to apply the immunoglobulin is the first drug. Still getting these drugs was associated with high costs, difficulties and high cost special pools of plasma donors with elevated concentrations of specific antibodies. If, as in the case of drug against hepatitis b, there are known methods of vaccination, the need to be vaccinated donors, to make a selection and donors be treated separately. In the case of many other indications, however, immunization of donors is not carried out for ethical reasons. Only rarely can be found here by an expensive operation for the selection of donors (for example, after transferring specific disease) donors with high titres (content) and the way they are processed to obtain the drug.

Thus the present invention is a method of obtaining valuable immunoglobulin preparations of the above, still barely used fractions and precipitation, which are formed when industrial methods of fractionating plasma available and can be selected. These drugs, which correspond hyperimmunoglobulinemia drugs, then you can process in a reasonable, especially acceptable for intravenous protection from viruses, liquid or dried Penne, immunoglobulin preparations with a high content of immunoglobulins, perhaps in different ways, one of the hitherto known methods. These new methods use antibodies from shared pools of plasma, due to the use of fractions - "waste" improve the use of valuable raw materials in the blood plasma, and even have the possibility of obtaining hyperimmunoglobulinemia drugs with much higher specific activities than those hitherto known drugs. This means that using a small flow volumes and minor amounts of introduced proteins, i.e., respectively, at light loads, the recipient, in a short time you can enter high doses of specific immunoglobulins. This is possible in the proposed according to the invention the method by nicolene specific immunoglobulins due to adsorption on immobilized antigens, therefore, due to the use of processed fractions - "waste" according to the method of affinity chromatography.

The object of the present invention is specified in paragraph 1 of the claims method.

In the proposed according to the invention the method for the first time process obtained in the case of the method of the FOA is to change any affinity chromatography. Depending on the origin fraction of its processing may be different. For example, the supernatant GG can be concentrated, dialysate in an acceptable buffer and filter. Precipitation or precipitation on the filters (lists examples of such starting materials are given in table. 1 and 2), opposite the first suitable way, i.e., for example, by changing the ionic strength (less than 5 M) pH-value, temperature, by adding detergents and salts, must be suspended in such a way as to solubilisate target immunoglobulins. For example, they can be stirred overnight at pH 3.0-9.0, when the conductivity of 5-20 MS/cm, at 4oC and then clarify by centrifugation and/or filtration. Both the supernatant and the suspension at this stage can be subjected to the method of inactivation of viruses by the method of solvent/detergent Horowitz (Thrombosis and Haemostasis, 65, 1163, 1991), by the method with methylene blue (Mohr and others, Influsionsther. Influsionsmed, 20, 19 (1993)) or another method. Immunoglobulin after suspension also can be subjected to pre-treatment by pre-adsorption column matrix, or by treatment with suitable filtering means or adsorbent, such as hydroxide alumina deposition processes using conventional methods of deposition of ammonium sulfate, polyethylene glycol or ethanol, or by their combination for enrichment, concentration or removal of interfering components.

The following list indicates examples of possible ligands for the proposed according to the invention affinity chromatography:

Antigenic determinants:

Haemophilus influenzae b, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus agalactiae, Streptococcus pneumonias, Streptococcus pyogenes and other pathogenic strains of bacteria; Tetanus Toxin, Staphylococcus aureus toxic shock Toxin, and other pathogenic bacterial or other toxins, hepatitis A virus, hepatitis B virus, hepatitis C virus, varicella zoster virus and herpes zoster, cytomegalovirus, respiratory syncytial virus, parvovirus B19, herpes simplex virus 1, herpes simplex virus 2, Tollwut-virus and other pathogenic viruses,

CD2, CD3, CD4, CD5, CD28, CD40, CD72, ICAM: LFA-1, LFA-3, DNA, phospholipids, and other potential human autoantigens.

After preparation of the gel for affinity chromatography by immobilization of modified or unmodified ligands (examples of such ligands are given in the above list) using known methods "load" using the prepared supernatant liquids and slurries in concentrated or rubbable odimo, you can subsequently load the same suspension of various gels for affinity chromatography. The gels are then washed so that predominantly or in sufficient quantity to remove non-specific bound proteins. This can be accomplished, for example, by increasing the salt concentration, the addition of detergent and/or shift the pH value of the washing solution. Related proteins are now separated from the ligands, for example, by elution at low or high pH values due to the additive chaotropic salt solutions, as thiocyanate sodium or magnesium chloride, denaturing agents, as SDS or urea, solvents, such as ethylene glycol, by changing the temperature or by a combination of these measures.

In some cases, it may be desirable modification immobilizing ligands by mutagenesis or by chemical or physical means, so that specific immunoglobulins could contact their epitopes, but with less affinity, so that elution can be performed under milder conditions than provide unmodified ligands. Modification of the ligands can be done also in order to facilitate and improve their immobilization and/ilny in: P. Cuatrecasas and Anfinsen, C. B. /1971/ Ann. Rev. Biochem. 40. 259; Kull F. C. and P. Cuatrecasas /1981/, J. Immunol. 126, 1279; Ziebig and others /1994/, Vox Sang, 67, 117.

Specific, isolated immunoglobulins, if necessary, with additional filtration for virus removal process in the target product, which can be used preferably intravenously and which pyrogene free, protects against viruses and stable with addition or without stabilizers, as albumin, amino acids or carbohydrates, in liquid or lyophilized form. However, it is also possible to apply formulations that allow intramuscular or topical application.

The present invention is illustrated in more detail by the following examples which do not limit the scope of protection of the invention.

Example 1

Prepare HBS Ag-sepharose, immobilize 5 mg of recombinant surface antigen of hepatitis B (HBs Ag, Abbott Diagnostics) by binding of primary amino groups with 1 ml of activated CH-sepharose according to the method of the manufacturer (Pharmacia Biotech, Ynncana, Sweden), "Placebo"-sepharose receive the same method, associating with another aliquot of the gel, but without the addition of HBs Ag. Store the prepared gels in PBS (saline phosphate buffer) from 0.02% NaN3the t in 210 ml of solution containing 100 mm citric acid, pH of 4.0, and 0.25% Triton X-100, 10 mm N-ethylmaleimide (NEM), 1 mm phenylmethylsulfonyl (PMSF), overnight at 4oC, with continuous stirring. After separation of the insoluble components by centrifugation (5000 g, 4oC, 10 min) and filtration (pore size of 1.2 μm), at a neutral pH according to Horowitz and others (Thrombosis and Haemostasis, 65, 1163, 1991) add 1% tri-n-butyl phosphate (Merck, Darmstadt, Germany) and 1% Triton X-100 and incubated at 30oC with stirring for 4 hours. Then over night at 37oC carry out the separation of the phases, separating the clear supernatant liquid is filtered through a filter with a pore size of 0.45 μm and stored at 4oC.

130 ml of the resulting NB-suspension diluted with 280 ml of PBS (superyoung phosphate salt solution, containing 150 mm NaCl, 10 mm phosphate, pH 7,1) and 4oC is passed through the column, placebo-separate and then through a column of HBsAg-separate so that flowing through the column, the amount of liquid was again included in the collection. The flow rate is 8 ml/min for a period of 144 hours. NB-the suspension is thus pumped through both speakers as a whole 3 times. After 90 hours loading (filling) interrupt and collectionspy density of the solution at 280 nm (OD280) is less than 0.01. After the download is complete, again washed with PBS and a solution containing 200 mm NaCl, 50 mm Tris-HCl, pH 7,4. Related proteins are separated using 5 ml of 200 mm glycine-HCl, pH 2.5 and immediately neutralized and treated. The results are presented in table 3.

Example 2

From Cohn fraction II+III (instead of blood plasma as a starting material) carry out the fractionation Kistler-Nitschmann 70 g of sediment B (HB Lot 4.044.488)? from this fractionation process according to Kistler-Nitschmann overnight at 4oC in a rotating apparatus for mixing suspended in 210 ml of a solution containing 100 mm citric acid, pH of 4.0, and 0.25% Triton X-100, 10 mm NEM, 1 mm PMSF. After purification and partial removal of lipids by using ultracentrifugation (100000 g, 3 hours, 4oC: clear phase selected by introducing the side of the tube with cannula), the suspension was filtered (0.45 µm) and stored at 4oC.

125 ml NB-suspension is diluted using a 375 ml PBS and establish a pH of 7.1 with 0.1 M sodium hydroxide solution, filtered and speed of 14.5 ml/h analogously to example 1 pumped first through placebo-sepharose-gel and then through HBSAg-sepharose-gel, which is prepared analogously to example 3. Then the gels separately about the 200 mm glycine-HCl, pH=2,5. The data presented in table 4.

Example 3

30 l of the Supernatant liquid GG (Lot N X95.31.286.1) diafiltrate in PBS and concentrated to a volume of 500 ml, Respectively example 1 concentrate with a speed of 21 ml/h for 118 hours pumped through the column with placebo and HBS Ag. The column is washed and separated associated proteins like that shown in example 1 of the method, however, perform the additional step of washing with a solution containing 500 mm NaCl, 50 mm Tris-HCl, pH 7,4. The results are presented in table. 5.

Example 4

of 17.5 g of DEAE-filtercake (Lot 4.422.006.0) suspended 52,5 ml suspending buffer according to example 1 and worked up. 40 ml of the suspension was diluted with 160 ml of PBS, set pH 7.1 and then filtered. Accordingly, example 3, the suspension is pumped in over 97 hours with a speed of 21 ml/h through the column with placebo and HBS Ag. The column is washed and separated associated proteins similar to example 1 method. The results are presented in table. 6.

Example 5

Tetanus Toxoid-C-Sepharose get immobiliza of 11.5 mg of purified Tetanus Toxoid (TT) by linking carboxyl groups with 1 ml EAH-sepharose (Pharmacia Biotech. , Uppsala, Sweden) using 0.1 m N-ethyl-N' -(3-dimethylaminopropyl)-carbodiimide supernatant GG is treated similarly to the method of example 3 and used for affinity chromatography with TT-separate analogously to example 2. Download exercise with the speed of 23.5 ml/h for 159 hours at 4oC. the Results are presented in table 7.

Example 6

Tetanus Toxoid-N-Sepharose prepare because of 11.5 mg of purified Tetanus Toxoid (TT) immobilizer by binding of primary amino groups with 1 ml of activated CH-sepharose according to the manufacturer's instructions (Pharmacia Biotech., Uppsala, Sweden). "Placebo"-sepharose prepare that perform the same way binding with another aliquot of the gel, but without the addition of TT. Store the prepared gels in PBS with 0.02% of NaN3at 4oC.

Precipitate B is suspended according to example 2. After filtration (1.2 µm) 115 ml of this suspension was diluted with 200 ml PBS, set pH 7.1 and with the rate of 3.5 ml/h for 165 hours pumped first through Placebo-sepharose so that the flow of the suspension is then directly passed through a column of TT-N-separate and then got in the collection. The column is washed separately with PBS and then with a solution containing 0.5 M NaCl, 50 mm Tris-HCl, pH 7,1, up until OD280flow will not be less than 0.01. The results are presented in table. 8.

Example 7

Sediment suspended In accordance with the method of example 2. After filtration (filter pore size of 1.2 µm) 115 ml of this WM is HBSAg-sepharose so, to the flow of the suspension is then directly passed through a column of TT-separate and then got in the collection. The column is washed separately with PBS and then with a solution containing 0.5 M NaCl, 50 mm Tris-HCl, pH 7,1, up until OD280flow will not be less than 0.01. The results are presented in table. 9.

Example 8

15 mg of Sludge In the process of fractionation on Kistle-Nitschmann (Lot N 5.043.303) overnight at 4oC using fibromites suspended in 45 l of a solution containing 0.1 M citric acid, pH of 4.0, and 0.25% Triton-X-100, 10 mm NEM, 1 mm PMSF. After separation of the insoluble components by adding auxiliary filtering means and filter (pore size 1.2 µm) at a neutral pH to remove lipids and inactivation of viruses according to Horowitz add 1% tri-n-butylphosphate and 1% Triton X-100 and incubated at 30oWith stirring for 4 hours. Then over night at 37oC carry out the separation of the phases, pumped transparent supernatant liquid is filtered through a filter with a pore size of 0.45 μm and stored at 4oC.

the pH-Value of 40 ml of this HB-suspension with sodium hydroxide to bring the values of 7.1 and 4oC is pumped through the colon, which gave the collection. Columns for affinity chromatography (50 x 13 mm) is prepared by linking 250 mg recombinant HBSA, respectively Tetanus Toxoid, 25 ml Affiprep-gel (Bio-Rad Lab. Jnc. Hercules CA 94547). The velocity of the flowing fluid is 6 l/h for 62 hours. NB-the Suspension is thus pumped generally 3 times through the column. Upon termination of loading, the column is washed separately first with PBS and then with a solution containing 500 mm NaCl, 50 mm Tris-HCl, pH 7,4, up until the washing solutions will not have the optical density at 280 nm (OD280) is less than 0.01. Related proteins are separated using 200 mm glycine-HCl, pH 2.5 and immediately establish a pH-value of the fractions is equal to 5.2. The results are presented in table. 10. Processing of immunoglobulins to stable preparations carried out through pools containing IgG fractions, diafiltration and concentration of 20 mm NaCl to the solution with 100 IU/ml, respectively, 2.5 mg of anti-TT IgG/ml Add 10% sucrose and solutions lyophilizer to units with 200 IU, respectively 5 mg of anti-TT IgG.

Example 9

50 g of Sediment IV of the process fractionation of plasma by Kistler-Nitschmann suspended in 500 ml of water. Using citric acid establish a pH-value of 5.0, and establish conductivity = 13 MS with p and at 4oC reach clarification and partial removal of lipids. Carry out the determination of the concentration of the suspension dissolved proteins, Ig M, Ig A, Ig G, transferrin and Caerulo-plasmin, and data are presented in table. 11.

Example 10

50 g of Sediment B of the process fractionation of plasma by Kistler-Nitschmann (Lot 4.030.204.0) is stirred for overnight at 4oC in 100 mm citric acid solution at different pH, lighten by ultracentrifugation at 100000 g at 4oC for 3 hours and partially removing lipids. Select the transparent medium phase and determine its content of dissolved proteins, Ig M, Ig A, Ig G, transferrin and Caerulo-plasmin (PL. 12).

The IgG titer against certain viral (PL. 13) and bacterial (PL. 14) antigens determined and compared with those in the source plasma and existing immunoglobulin preparations.

Plasma 102/103 - pools of plasma; SAGL-Sando-globulin: NB, pH 4,0/5,0 - suspension of sediment B at pH of 4.0, respectively 5,0; IU = international units, PEIE - unit of the Institute of Paul Ehrlich; AU = arbitrary units; N. O. is not defined.

Plasma 102/103 - pools of plasma; SAGL-Sando-globulin, NB 4A - suspension of sediment B at pH 4,0; NB 14, a pH of 4.0 - suspension of sediment B at pH 4.0 with inactivation of the virus; kernie) units.

1. The method of obtaining the immunoglobulin preparation with a high titer, characterized in that carry out consistently the following stages: (a) fractionation of blood plasma from a pool of plasma, in which separate at least a fraction, which can be used in industry and which contains essentially polyclonal immunoglobulin C, and get at least one residual fraction, b) obtaining a solution of the protein component of the protein contained in the obtained in stage a) residual fractions or subfractions and C) the resulting protein solution at least once subjected to affinity chromatography with immobilized ligands at least one ligand type, and specific plasma protein bound with ligands, and separate the associated plasma protein, which as active components transferred to the immunoglobulin preparation with a high titer.

2. The method according to p. 1, characterized in that the fractionation of blood plasma is carried out in industrial scale, and the residual fraction is a waste.

3. The method according to p. 1 or 2, characterized in that the residual fraction according to stage a) prestolite treated with aqueous buffer solution with an ionic strength < 5M and pH 3,0 - 9,0 upon receipt of a solution of suspension.

4. The method according to p. 3, wherein the buffer solution is a phosphate buffer, Tris-HCl buffer or citrate buffer.

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

6. The method according to any of paragraphs.3 to 5, characterized in that the solution or suspension is filtered or pre-treated with an adsorbent, such as aluminum hydroxide, or adsorbent, which contains DEAE-groups.

7. The method according to any of paragraphs.3 to 5, characterized in that the solution or suspension is mixed with ammonium sulfate, polyethylene glycol or ethanol precipitate, and the supernatant or the precipitate is treated next.

8. The method according to p. 1 or 2, characterized in that the residual fraction according to stage a) is a supernatant liquid and obtaining a protein solution is performed by filtering and concentrating, for example, by diafiltration.

9. The method according to p. 8, characterized in that the supernatant liquid is filtered or pre-treated with an adsorbent, such as aluminum hydroxide, or adsorbent, khandi are natural or recombinant, viral, bacterial or cellular antigens.

11. The method according to p. 10, wherein the ligands are selected from the group antigenic determinants Haemophilus influenzae b, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus agalactiae, Streptococcus pneumonuae, Streptococcus pyogenes, Tetanus Toxin, Staphylococcus aureus toxic shock Toxin, hepatitis A virus, hepatitis B virus, hepatitis C virus, varicella zoster virus and herpes zoster, cytomegalovirus, respiratory syncytial virus, parvo virus B 19, herpes simplex virus 1 and herpes simplex virus 2, Tollwut-virus, as well as potential human antigens CD2, CD3, CD4, CD5, CD28, CD40, CD72, ICAM, LFA-1, LFA-3, DNA and phospholipids.

12. The method according to p. 10 or 11, characterized in that the ligands modified by mutagenesis or by chemical or physical methods.

13. The method according to any of paragraphs.1 - 12, characterized in that the immunoglobulin preparation with a high titer consists only of immunoglobulin G, or A, or M, or any combinations thereof.

14. The method according to any of paragraphs.1 - 13, characterized in that the immunoglobulin preparation with a high titer is subjected to viral inactivation and, if necessary, stabilize the addition of stabilizer, such as albumin, linakis is derived immunoglobulin preparation with a high titer transferred to pharmaceutically acceptable product, for example, in the drug, administered intravenously, intramuscularly or tapicerki.

16. The method of obtaining the immunoglobulin preparation with a high titer, characterized in that carry out consistently the following stages: a) get a protein solution of the protein components obtained by the fractionation of blood plasma residual fraction or her podrecca and b) the resulting protein solution at least once subjected to affinity chromatography with immobilized ligands at least one ligand type, and specific plasma protein bound with ligands, and separate the associated plasma protein, which as active components transferred to the immunoglobulin preparation with a high titer.

17. The method according to p. 16, characterized in that the immobilized ligands are natural or recombinant, viral, bacterial or cellular antigens.

18. The method according to p. 17, wherein the ligands are selected from the group antigenic determinants of group Haemophilus influenzae b, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus agalactiae, Streptococcus pneumonuae, Streptococcus pyogenes, Tetanus Toxin, Staphylococcus aureus toxic shock Toxin, hepatitis A virus, hepatitis B virus, hepatitis virus of Svilosa B 19, the herpes simplex virus 1 and herpes simplex virus 2, Tollwut-virus, as well as potential human antigens CD2, CD3, CD4, CD5, CD28, CD40, CD72, ICAM, LFA-1, LFA-3, DNA and phospholipids.

19. The method according to p. 17 or 18, characterized in that the ligands modified by mutagenesis or by chemical or physical methods.

20. The method according to any of paragraphs.16 to 19, characterized in that the immunoglobulin preparation with a high titer consists only of immunoglobulin G, or A, or M, or any combinations thereof.

21. The method according to any of paragraphs.16 to 20, characterized in that the immunoglobulin preparation with a high titer is subjected to viral inactivation and, if necessary, stabilize the addition of stabilizer, such as albumin, amino acids or carbohydrates, and/or product lyophilizer.

22. The method according to any of paragraphs.16 to 21, characterized in that the immunoglobulin preparation with a high titer transferred to pharmaceutically acceptable product, for example, in the drug, administered intravenously, intramuscularly or tapicerki.

 

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