Method for preparing a concentrate of polyvalent immunoglobulin

Abstract

The present invention relates to a method for preparing a concentrate of polyvalent immunoglobulins with view to therapeutic use, from an initial solution of blood plasma or a plasma fraction enriched with immunoglobulins, comprising the steps for removing the protein contaminants by precipitation with caprylic acid in order to obtain a solution free of proteases, and for separating by chromatography on a fluidized bed the solution free of proteases, said method allowing a concentrate of human polyvalent immunoglobulins with a yield of more than 4.5 g of immunoglobulins per liter of blood plasma applied to be obtained.

Claims

1. A method for preparing a concentrate of human polyvalent immunoglobulins from an initial solution of blood plasma or a fraction of plasma enriched with immunoglobulins, comprising: (a) removing protein contaminants by contacting the initial solution of blood plasma or a fraction of plasma enriched with immunoglobulins with caprylic acid in order to obtain a solution free of proteases, wherein the concentration of caprylic acid ranges from 0.5 to 1.5% by volume of caprylic acid per volume of initial solution of blood plasma or a fraction of plasma enriched with immunoglobulins to be treated, and (b) fluidized bed chromatography of the mixed-mode type of the solution free of proteases, to obtain a concentrate of human polyvalent immunoglobulins with a yield of more than 4.5 g of immunoglobulins per liter of blood plasma.

2. The method according to claim 1, wherein the step for removing protein contaminants with caprylic acid (a) is carried out at a pH between 4.3 and 4.9.

3. The method according to claim 2, wherein the step for removing protein contaminants with caprylic acid (a) is carried out at a pH between 4.6 and 4.8.

4. The method according to claim 1, further comprising between the step for removing protein contaminants with caprylic acid (a) and the fluidized bed chromatography step (b), a clarification step at an acid pH.

5. The method according to claim 4, wherein the clarification step is by depth filtration.

6. The method according to claim 1, wherein the fluidized bed chromatography step of the mixed-mode type comprises: loading on a chromatography column equilibrated beforehand with a buffer at a pH between 4.5 and 8, the solution having undergone the clarification step by depth filtration, adjusted beforehand to the same pH, washing the loaded column with a buffer solution until all non-adsorbed proteins on the column are removed, eluting polyvalent immunoglobulins adsorbed on the column with an elution buffer adjusted to a pH between 8 and 10, and recovering the solution enriched with human polyvalent immunoglobulins.

7. The method according to claim 1 further comprising, after step (b), one or more of the following steps: (i) a viral inactivation step, (ii) a step for anion exchange chromatography of the solution obtained at the end of step (i), (iii) a step for removing anti-A and anti-B antibodies from the solution obtained at the end of step (ii), (iv) a filtration step through nanometric filters with decreasing porosity from 100 to 15 nm, (v) a concentration step by ultrafiltration of the solution from step (iv) associated with a formulation step, (vi) and then a conventional sterilizing filtration step.

8. The method according to claim 1, wherein the initial solution is a plasma fraction enriched with immunoglobulins by fractionation with ethanol or by separation with chromatography.

9. The method according to claim 8, wherein the initial solution is a I+II+III precipitate or a II+III precipitate obtained from blood plasma fractionated with ethanol, and put back into solution.

10. The method according to claim 9, wherein the I+II+III precipitate or the II+III precipitate is put back into solution in purified water for injection or in a solution containing ions.

11. The method according to claim 10, wherein the solution containing ions is a solution comprising NaCl at a concentration of less than or equal to 20 mM.

12. The method according to claim 11, wherein the solution containing ions is a solution comprising NaCl at a concentration between 5 and 15 mM.

13. The method according to claim 12, wherein the solution containing ions is a solution comprising NaCl at a concentration equal to 10 mM.

14. The method according to claim 9, wherein the I+II+III precipitate or the II+III precipitate is treated with CaCl.sub.2 solution, the concentration of which is less than or equal to 20 mM.

15. The method according to claim 14, wherein the I+II+III precipitate or the II+III precipitate is treated with CaCl.sub.2 solution, the concentration of which is between 5 and 15 mM.

16. The method according to claim 15, wherein the I+II+III precipitate or the II+III precipitate is treated with a 10 mM CaCl.sub.2 solution.

17. The method according to claim 1, comprising: (i) a step for removing protein contaminants with caprylic acid, (ii) a clarification step by depth filtration, (iii) a fluidized bed chromatography step of the “mixed-mode” type, (iv) a viral inactivation step with solvent/detergent treatment, (v) an anion exchange chromatography step, (vi) a step for removing anti-A and anti-B antibodies, (vii) filtration through nanometric filters with decreasing porosity from 100 to 15 nm, (viii) a step for concentrating by ultrafiltration the solution from the preceding step associated with a formulation step, and then a conventional sterilizing filtration step.

18. The method according to claim 1, further comprising the following steps: (a) adding to the concentrate of human polyvalent immunoglobulins obtained according to claim 1, one or several pharmaceutically acceptable stabilizers, and (b) optionally, freezing or freeze-drying the pharmaceutical preparation obtained in the preceding step; so that the pharmaceutical preparation is in a liquid, frozen or freeze-dried form.

19. The method according to claim 1, wherein the concentration of caprylic acid ranges from 0.8 to 1.2%.

20. The method according to claim 19, wherein the concentration of caprylic acid ranges from 0.9 to 1.1%.

21. A method for preparing a concentrate of human polyvalent immunoglobulins from an initial solution of blood plasma or a fraction of plasma enriched with immunoglobulins, comprising: (a) removing protein contaminants by contacting the initial solution of blood plasma or the fraction of plasma enriched with immunoglobulins with caprylic acid in order to obtain a solution free of proteases, wherein the concentration of caprylic acid ranges from 0.9 to 1.1% by volume of caprylic acid per volume of initial solution of blood plasma or the fraction of plasma enriched with immunoglobulins to be treated, and (b) fluidized bed chromatography of the mixed-mode type of the solution free of proteases, to obtain a concentrate of human polyvalent immunoglobulins with a yield of more than 4.5 g of immunoglobulins per liter of blood plasma.

Description

EXAMPLES

Examples 1

(1) 1.1 Putting the I+II+III Precipitate Back into Solution

(2) As a starting material the I+II+III precipitate is used, the one obtained from blood plasma fractionated with ethanol according to the Cohn or Kistler and Nitschmann method (1962, Vox Sang. 7, 414). This precipitate is put back into solution in an amount of 56.7 g for 228 mL of demineralized water or equivalent proportions. The mixture is stirred for 20 minutes at 15° C.±5° C. The temperature is then raised to 22° C.±2° C. and caprylic acid (1% weight/volume) is slowly added into the I+II+III precipitate put back into solution. The pH of the obtained solution after adding caprylic acid is adjusted to 4.8 and the solution remains with stirring for 60 minutes.

(3) 1.2 Depth Filtration

(4) The resulting solution is clarified by depth filtration on a SEITZ® T 2600 filter (Pall Corporation). This filtration retains both the filtration adjuvants present in the I+II+III precipitate and the protein precipitate generated by the addition of caprylic acid.

(5) The pH of the sample is also advantageously studied in order to optimize the filtration yield.

(6) The pH of the protein solution is adjusted to 6.0 only after filtration. Indeed, the adjustment of the pH from 4.8 to 6.0 generates a slight precipitate (insolubility of polyvalent immunoglobulins) retained by the filter and therefore a reduction in the filtration yield of polyvalent immunoglobulins.

(7) The rinsing volume of the filter (apyrogenous purified water) is equivalent to the volume of the initial sample in order to optimize the final yield.

(8) 1.3 Chromatography of the Mixed-mode Type on a Fluidized Bed

(9) The operating conditions are the following:

(10) Equilibration and washing buffer: 20 mM Na/Na.sub.2PO.sub.4 at pH 6. Elution buffer: 20 mM glycine/20 mM NaCl at pH 9.8.
By selecting elution by a pH effect in a Gly/NaCl buffer, it is possible by simple dilution (adjustment of the conductivity and pH) to obtain a sample ready to be injected onto the ion exchange column (TMAE Fractogel).

(11) 1.4 Treatment with Solvent/Detergent

(12) The eluate from the chromatography of the mixed-mode type on a fluidized bed according to example 1.3 is subject to a viral inactivation treatment with solvent/detergent as described by Neurath and Horowitz (U.S. Pat. No. 4,764,369).

(13) The 10 times concentrated solvent/detergent mixture contains 3% of TnBP (tri(n-butyl)phosphate) and 10% of Octoxinol. The final concentration in the eluate is 0.3% of TnBP and 1% of Octoxinol.

(14) After 1 hour of inactivation, the eluate is adjusted to pH 9.0 and diluted in water in order to obtain a conductivity of less than 1,100 μS/cm.

(15) 1.5 Ion Exchange Chromatography

(16) The ion exchange gel applied is TMAE-Fractogel®, Merck. Pre-equilibration buffer: glycine 80 mM/NaCl 80 mM at pH 9. Equilibration buffer: glycine 9 mM/NaCl 9 mM at pH 9.

(17) After loading the sample, the column is washed with the equilibration buffer and it is then eluted in a 20 mM Na/Na.sub.2PO.sub.4 buffer at pH 6.2.

(18) 1.6 Affinity Chromatography

(19) The applied affinity gel is Iso A/Iso B Hypercel, Pall Corporation.

(20) The eluate of TMAE chromatography is subject to this affinity chromatography, in order to remove the anti-A and anti-B antibodies. The polyvalent immunoglobulins of interest cross the column without being retained. Equilibration of the gel with demineralized water Recovery of the non-adsorbed fraction without washing the column.

(21) 1.7 Filtration

(22) The protein solution from the affinity chromatography is filtered on a depth filter 90LA CUNO, 3M and then on a 0.2 μm filter. The filters are rinsed with water and the rinsing solution is incorporated to the filtrate.

(23) 1.8 Nanofiltration

(24) The preceding filtrate, adjusted to pH 4.5 is pre-filtered on line on a Fluorodyne II filter 0.1 μm, Pall Corporation and nanofiltered on a DV50 filter, Pall Corporation, and then on a Planova 20N filter from Asahi.

(25) 1.9 Formulation

(26) The product is pre-concentrated to about 80 g/L by ultrafiltration on a cassette with a cut-off threshold of 30 kDa, and then diafiltered at constant volume until the conductivity is <600 μS/cm. The product is then formulated and adjusted to 50 g/L of proteins.

Example 2

Obtaining a Concentrate of Polyvalent Immunoglobulins by Applying the Method of the Invention on a Pilot Scale

(27) 2.1 Putting Back the I+II+III Precipitate Back into Solution

(28) The precipitate I+II+III is used as a starting material, obtained from blood plasma fractionated with ethanol according to the Kistler and Nitschmann method (1962, Vox Sang. 7, 414). This precipitate is put back into solution in an amount of 3 kg for 12 L of demineralized water. The mixture is stirred for 20 minutes at least at 10° C.±3° C. Next, the temperature is raised to 22° C.±2° C. and caprylic acid (1% weight/volume) is slowly added into the I+II+III precipitate put back into solution. The pH of the obtained solution after addition of caprylic acid is adjusted to 4.8 and the solution remains with stirring for 60 minutes.

(29) 2.2 Depth Filtration

(30) The resulting solution is clarified by depth filtration on a SEITZ® T 2600 (Pall Corporation) filter. This filtration retains both the filtration adjuvants present in the I+II+III precipitate and the protein precipitate generated by addition of caprylic acid. For this test, the surface area used is 8.7 kg of precipitate/m.sup.2 of filtering medium.

(31) The rinsing volume of the filter (apyrogenous purified water) is equivalent to the volume of the initial sample i.e. 15 L.

(32) 2.3 Chromatography of the Mixed-mode Type on a Fluidized Bed

(33) The chromatography gel applied is Rhobust IGIV gel, Upfront.

(34) 4.3 L of gel in a column with a diameter of 10 cm.

(35) The operating conditions are the following:

(36) Equilibration and washing buffer: 20 mM Na/Na.sub.2PO.sub.4 at pH 6. Elution buffer: 20 mM glycine/20 mM NaCl at pH 9.8.

(37) 2.4 Treatment with Solvent/Detergent

(38) The eluate from chromatography of the mixed-mode type on a fluidized bed undergoes a viral inactivation treatment with solvent/detergent as described by Neurath and Horowitz (U.S. Pat. No. 4,764,369).

(39) The 10 times concentrated solvent/detergent mixture contains 3% of TnBP (tri-n-butylphosphate) and 10% of Octoxinol. The final concentration in the eluate is 0.3% of TnBP and 1% of Octoxinol.

(40) After at least 1 hour of inactivation, the eluate is adjusted to pH 9.0 and diluted with water in order to obtain a conductivity of less than 1,100 μS/cm.

(41) 2.5 Ion Exchange Chromatography

(42) The ion exchange gel applied is EMD-TMAE Fractogel®, Merck.

(43) 4 L of gel in a column with a diameter of 12.7 cm.

(44) Pre-equilibration buffer: glycine 80 mM/NaCl 80 mM at pH 9. Equilibration buffer: glycine 9 mM/NaCl 9 mM at pH 9. After loading the sample, the column is washed in the equilibration buffer.
Next, it is eluated in a 20 mM Na/Na.sub.2PO.sub.4 buffer, at pH 6.2.

(45) 2.6 Affinity Chromatography

(46) The affinity gel applied is Iso A/Iso B Hypercel, Pall Corporation.

(47) 154 mL of gel in a column with a diameter of 7 cm.

(48) The eluate from TMAE chromatography is subject to this affinity chromatography, in order to remove the anti-A and anti-B antibodies. The polyvalent immunoglobulins of interest cross the column without being retained. Equilibration of the gel in demineralized water Recovery of the non-adsorbed fraction without washing the column.

(49) 2.7 Filtration

(50) The protein solution from Iso A/Iso B Hypercel affinity chromatography is filtered on a depth filter 90LA CUNO, 3M, and then on a 0.2 μm filter. The rinsing of the filters in water is incorporated to the filtrate.

(51) 2.8 Nanofiltration

(52) The preceding filtrate adjusted to pH 4.5 is pre-filtered on line on a Fluorodyne II 0.1 μm filter, Pall Corporation and nanofiltered on a DV50 filter, Pall Corporation, and then on a Planova 20N filter from Asahi.

(53) 2.9 Formulation

(54) The product is pre-concentrated to about 80 g/L by ultrafiltration on a cassette with a cut-off threshold of 30 kDa, and then diafiltered at constant volume until the conductivity is less than <600 μS/cm. The product is then formulated and adjusted to 50 g/L of proteins.

Example 3

Yield of the Method

(55) Three batches were made with three different I+II+III precipitates according to the method in accordance with the invention described in Example 1 and specified in Example 3. Removal of the proteases is obtained with 1% of caprylic acid. Filtration is carried out on a SEITZ T2600 plate filter-press.

(56) A control batch is treated according to the reference method, as described in patent application EP 1 385 886.

(57) The yield results of the novel method are shown in the table below:

(58) TABLE-US-00001 Accumulated yields in g/L of proteins or of plasma Ig at the formulated concentrated stage Batch Control batch according treated according to the to the method invention as described in 10AXTO1064 EP 1 385 886 015 (Batch 1) 10AXTO1064 038 Yield in g of proteins per liter of 5.22 4.67 treated plasma. Yield in g of polyvalent Igs per 4.75 4.49 liter of treated plasma

(59) The method according to the invention allows an increase in the yield in g of polyvalent immunoglobulins per liter of treated plasma, as compared with the reference method (EP 1 385 886).

(60) The gain in the example above is 0.26 g of IgG/L of plasma, between the batch according to the invention and the control batch made from the same precipitate.

Example 4

Anti-complementary Activity

(61) Three exploratory batches were made according to the method of the invention at a pilot scale from three different I+II+III precipitates. These batches were compared with a batch made on the same scale (pilot size), but according to the industrial method for producing immunoglobulins by intravenous injection as described in patent EP 1 385 886, and three industrial batches prepared according to the method described in application EP 1 385 886, made from the same precipitates.

(62) A batch made with the pilot size corresponds to a batch for which the size represents at least 10% of the industrial batch.

(63) The batches according to the invention have an anti-complementary activity (ACA) measured according to the test of European Pharmacopeia 7.5, edition 01/2012:0918, paragraph 2.6.17, always less than those of the batches made according to the method described in application EP 1 385 886. Moreover, the progression observed within 18 months on this criterion is less for the batches made according to the invention.

(64) The anti-complementary activity (ACA) results are shown in the table below:

(65) TABLE-US-00002 T = +18 Progression T = 0 months (%) Exploratory batches made to the pilot ACA % ACA % size prepared according to the method of the invention: Batch 1* 28 31 11% Batch 2** 27 31 15% Batch 3*** 27 31 15% Batch made to the pilot size* prepared 33 40 21% according to the method described in application EP 1 385 886: Industrial batches made according to the method described in application EP 1 385 886: Industrial batch 1* 36 38 6% Industrial batch 2** 32 39 22% Industrial batch 3*** 32 37 16% *, **, ***batches made with the same precipitates

(66) The method of the invention therefore gives the possibility of obtaining a concentrate of polyvalent immunoglobulins for which the anti-complementary activity is less than 30%.

Example 5

Purity of Immunoglobulins Produced According to the Method of the Invention

(67) The analytic purity data for the pilot batches described in Example 2 are summarized in the table below.

(68) TABLE-US-00003 Average of the industrial batches obtained according to the method described Measurement Pilot in application EP Analysis method Pilot batch No. 1 batch No. 2 Pilot batch No. 3 1 385 886 pH Ph. Eur (2.2.35) 5.0 4.8 4.8 4.9 Osmolality Ph. Eur (2.2.29) 297 294 299 297 mosmol/kg DTM polymers Ph. Eur (2.2.29) <0.4 <0.4 <0.4 0.5 (%) Dimers (%) Ph. Eur (2.2.29) 2.7 3.7 2.4 ND Monomers (%) Ph. Eur (2.2.29) 96.3 95.3 96.4 98.7* Fragments (%) Ph. Eur (2.2.29) 1 1.1 1.1 1 Total proteins Ph. Eur (2.5.33) 51 49 49 50 (g/l) Protein level Ph. Eur (2.5.33) 98 98 98 (%) % % % % proteins proteins proteins proteins IgG g/L Nephelometry** 46.4 91 46 95 47.6 96 45.7 92 IgG1 g/L Nephelometry** 29.5 58 27.1 56 26.7 54 28 56 IgG2 g/L Nephelometry** 18 35 17.8 37 16.7 34 16.1 32 IgG3 g/L Nephelometry** 1.4 3 1.2 2 1.3 3 1.1 2 IgG4 g/L Nephelometry** 1.1 2 1 2 1 2 0.9 2 IgGA g/L ELISA*** 7.4 0.01 7.1 0.01 6 0.01 8.6 0.02 IgGE g/L Nephelometry** <0.8 ND <0.8 ND <0.8 ND <0.8 ND IgGM g/L Nephelometry** 0.23 0.2 0.18 0.16 Antibody anti- ELISA*** 5.1 3.5 3.3 4.6 Hbs Ag (IU/mL) Albumin mg/L Nephelometry** <2.2 <2.2 <2.2 <2.2 Transferrin Nephelometry** <2.1 <2.1 <2.1 <2.1 mg/L Activator of Ph. Eur (2.6.15) <1 <1 <1 <2 prekallikrein Anti-A Ph. Eur (2.6.20) 4 16 8 8 hemagglutinins Anti-B Ph. Eur (2.6.20) 2 4 8 4 hemagglutinins Anti- Ph. Eur (2.6.17) 28 27 27 33 complementary activity (%) Ph. Eur: European Pharmacopeia 6.sup.th edition *Monomers + dimers. **Pressac M, Later R. “Dosages sériques d'IgG, IgA, IgM, transferrine et haptoglobine. II Précision analytique et comparaison des résultats fournis par différents analyseurs”. Ann Biol Clin 1995; 53: 273-81. ***R. A. Goldsby, T. J. Kindt, B. A. Osborne et J. Kuby, “Enzyme-Linked Immunosorbent Assay” in Immunology, 5e édition, pages 148-150, W. H. Freeman, New York, 2003

(69) The sum “monomers+dimers”, the distribution of the sub-classes, the levels of contaminants and the anti-complementary activity are equivalent for the immunoglobulins from the method of the invention or from the reference method according to patent EP 1 385 886. These characteristics meet the criteria of the European Pharmacopeia.

Example 6

Stability of the Immunoglobulins Produced According to the Method of the Invention

(70) The formulated products from pilot batches described in Example 2 were kept at 4° C. and aliquots of the industrial batches were made with the same initial precipitates and kept at the same stage under the same conditions. Samplings carried out every six months gave the possibility of observing on three criteria the differences between the batches obtained according to the invention (pilot batches 1, 2 and 3) and the batches obtained according to the reference industrial method EP 1 385 886 (industrial batches 1, 2 and 3). The three measured criteria were the following: measurement of the concentration of immunoglobulins G (IgG) and that of the concentration of anti-Hbs immunoglobulins according to the method described in European Pharmacopeia 6.7 (Pharmeuropa, volume 21, number 2, April 2009), paragraph 2.7.1, and determination of the molecular size (polymers, dimers, monomers and fragments) according to the method described in European Pharmacopeia 6.7 (Pharmeuropa, Volume 21, Number 2, April 2009), paragraph 2.2.29.

(71) The table below accounts for these differences:

(72) TABLE-US-00004 Ig DTM % (aggregates/ Ig DTM % (aggregates Batches prepared according to αHbs dimers/monomers/ IgG αHbs dimers/monomers/ the method of the invention: IgG g/L IU/mL fragments) g/L IU/mL fragments) T = 0 T = +6 months Pilot batch 1* 46.4 5.1 <0.4/2.7/96.3/1.0 43.7 5.2 <0.4/5.7/93.4/<1.0 Pilot batch 2** 46.0 3.5 <0.4/3.7/95.3/1.1 42.7 3.5 <0.4/5.2/93.7/1.1 Pilot batch 3*** 47.6 3.3 <0.4/2.4/96.4/1.1 43.5 3.0 <0.4/4.3/94.6/1.0 Batch made to the pilot size* 49.6 6.1 <0.4/3.6/95.7/<1 45.3 5.4 <0.4/5.1/93.9/<1.0 prepared according to the method described in EP1385886: Industrial batches prepared according to the method described in EP1385886: Industrial batch 1* 45.9 5.1 0.4/98.8/1.1 46.6 5.5 <0.4/4.8/94.1/1.0 Industrial batch 2** 46.0 4.7 0.7/98.6/1.0 44.9 4.4 0.9/5.1/93.2/<1.0 Industrial batch 3*** 45.2 3.9 0.4/98.6/1.0 44.4 3.5 <0.4/4.6/94.3/<1.0 T = +12 months T = +18 months Pilot batch 1* 51.7 5.2 <0.4/5.6/93.3/ 48.5 5.0 <0.4/4.8/94.1/1.0 1.0 Pilot batch 2** 49.7 3.7 <0.4/5.3/93.7/<1.0 45.7 3.3 <0.4/4.4/94.5/1.1 Pilot batch 3*** 49.5 3.2 <0.4/4.7/94.3/ 47.6 3.1 <0.4/4.4/94.5/1.1 1.0 Batch made to the pilot size* 51.8 5.7 <0.4/5.4/93.6/<1.0 49.1 5.4 <0.1/5.2/93.8/1.0 prepared according to the method described in EP1385886 Industrial batches prepared according to the method described in EP1385886: Industrial batch 1* 51.4 5.9 <0.4/4.8/93.6/ 46.5 5.3 <0.4/4.1/94.8/1.0 1.0 Industrial batch 2** 48.2 4.5 0.7/4.8/93.6/1.0 46.2 4.3 0.7/4.9/93.5/1.0 Industrial batch 3*** 49.5 3.7 <0.4/4.4/94.4/ 47.1 3.4 <0.4/4.4/94.4/1.0 1.0 *, **, ***batches made with the same precipitates.
Analysis of the data shows: stability of the IgG concentration over 18 months and homogeneity of the 7 batches, stability of the concentration of the anti-Hbs Igs over 18 months: each batch being specific to this criterion, a constant monomers+dimers sum greater than the 85% required by the pharmacopeia and this for each batch over 18 months.

Example 7

A Method Comprising a Step for Caprylic Precipitation, a Clarification Step and a Chromatography Step of the Mixed Mode Type on a Fluidized Bed

(73) The treatment with caprylic acid has the purpose of removing by precipitation (the immunoglobulins remaining in solution) part of the contaminating proteins from the plasma and most particularly proteases. Depending on the percentage of caprylic acid used and on the method for separating the supernatant and the precipitate, a more or less significant portion of the immunoglobulins may be lost. Experimental conditions were tested so as to have the most coarse clarification possible compatible with fluidized bed chromatography and generating the smallest loss of immunoglobulins. For this, filters with increasing porosity were compared without caprylic precipitation in a first phase, and then confirmed subsequently with treatment with caprylic acid at different concentrations.

(74) Test on a filter with a diameter of 90 i.e. 50 cm.sup.2 (with rinsing),

(75) No precipitation with caprylic acid

(76) TABLE-US-00005 Test number 440 077 440 078 440 079 Filter type Pall Pall/Seitz Pall/Seitz Supradur50P T 5500 T 2600 Porosity 4-8 μm 25-70 μm 15-40 μm pH 4.8 4.8 4.8 Flow volume mL 150 150 150 Flow rinsing 0 75 75 Recovered volume 9 (water) 208 216 mL Initial IgG g/L 14.35 15.05 15.05 Filtrate IgG g/L ND* 10.5 10.2 Yield % ND* 97 98 Volume/surface l/m.sup.2 ND* 30 30 *clogging of the filter, no metering carried out.

(77) These results show that a minimum porosity of the order of 10-20 μm has to be applied in order to limit the IgG loss because of clogging. The filter T2600 was selected relatively to the filter T5500 for its lower porosity at an equivalent IgG yield.

(78) The percentage of added caprylic acid having an impact on the precipitation of the proteases and on the filterability of the solution, a range of caprylic acid from 0.5 to 1% without adding any filtration adjuvant was tested. The obtained results are compared with those resulting from control clarification by simple centrifugation.

(79) TABLE-US-00006 Test - Re-suspending (RES) 1% caprylic acid - filter with diameter 90 i.e. 50 cm.sup.2, load 210 mL RES pH Clarified 4.8 (1) RES (2) Test A   1% SEITZ IgG g/L 13.7 9.7 2600 filtration Yield (YLD) % 100 76 (3) Proteases mOD/mL 570 13 Test B 0.5% SEITZ IgG g/L 15 9.4 2600 filtration YLD % 100 51 (3) Proteases mOD/mL NR NR Test C 0.5% centrifugation IgG g/L 14.1 12.4 YLD % 100 72 Proteases mOD/mL 967 250 (1) Precipitate put back into solution, adjusted to pH 4.8 before clarification. (2) Precipitate put back into solution pH-adjusted and clarified. (3) Rinsing of the filters with a volume corresponding to half of the product to be clarified.

(80) The control by centrifugation shows that part of the immunoglobulins is lost in the precipitate, if the latter is not rinsed, this technique was not retained. The most interesting tested condition is the 1% concentration of caprylic acid: protease contamination is low and the immunoglobulin loss is less than 30%. A concentration of 1% of caprylic acid therefore appears to be the best compromise between filterability, yield and removal of the proteases.

(81) In order to increase the yield, the test below was conducted with a smaller load (180 mL instead of 210 mL) and by increasing the washing volume of the precipitate with the rinsing buffer as described in the table below.

(82) TABLE-US-00007 Test 09AXTO440 126 - RES 1% caprylic acid - filter de diameter 90 i.e. 50 cm.sup.2, load 180 mL. IgG Steps Volume (mL) (g/L) Amount (mg) Yield (YLD) % RES pH 4.8 210 13.65 2866.5 100 RES filter 180 11.2 2016.0 70.3 Rinsing 1 30 3.71 111.3 3.9 Rinsing 2 30 2.26 67.8 2.4 Rinsing 3 30 2.28 68.4 2.4 Rinsing 4 30 1.71 51.3 1.8 Rinsing 5 30 1.16 34.8 1.2 Rinsing 6 30 0.91 27.3 1 Rinsing 7 30 0.91 27.3 1 Filtered pool 385 6.95 2675.8 93.3

(83) By reducing the load volume on the filter and by rinsing the precipitate with a washing volume equivalent to the volume of product to be clarified (180 mL), no clogging is observed and the totality of the immunoglobulins was recovered. The totality of the immunoglobulins is available for the affinity chromatography step which follows in the purification method.

Example 8

Improvement in Putting the IgG Back into Solution

(84) Upon making pilot size batches according to the novel method as described in Example 2, comparison with the batch prepared according to the method described in patent application EP1385886 at the same scale (10AXTO1064015) shows that it is possible to improve the putting of the I+II+III precipitate back into solution. Indeed, a difference of at least 1 g/L of IgG is observed in this step (the volumes being equivalent) as demonstrated by the table below.

(85) TABLE-US-00008 Putting back into a non-filtered solution Pilot size batch prepared according to the Pilot size batches prepared according to method described in patent the method of the invention (g/L) application EP1385886 (g/L)* Batch 1* Batch 2 Batch 3 12.8 11.6 10.3 11.7 *Batches made with the same precipitate

(86) Complementary tests have shown that, advantageously, it is possible to put the immunoglobulins present in the precipitate back into solution with a solution containing ions rather than with purified water. In particular, by using a 10 mM NaCl solution.

(87) Moreover, the precipitation of protein contaminants with caprylic acid may also be improved at pHs of less than 4.8. In particular, a pH of 4.6 allows this improvement.

(88) A pilot size batch (novel batch) was made by taking into account these improvements and compared with the Robust Chromatography Eluate stage with the three batches (batches 1, 2 and 3) already made according to the invention.

(89) The table below accounts for these differences:

(90) TABLE-US-00009 Yield of the novel method at the   Fluidized bed chromatography eluate   stage Novel batch Batch 1 Batch 2 Batch 3 pH of RES 4.6 4.8 4.8 4.8 g IgG/L plasma 6.00 5.96 5.62 5.77 IgG/proteins 0.910 0.820 0.812 0.893

(91) The novel batch made with putting back into solution with a solution having a 10 mM NaCl concentration, treated by caprylic acid and adjusted to a pH equal to 4.6, has: better yield of immunoglobulins per liter of plasma applied, better purity as regards the immunoglobulins-total proteins ratio

Example 9

Application of the Invention to the II+III Precipitate

(92) The “II+III” precipitate is an alternative to the “I+II+III” precipitate as a source of raw material for purification of polyclonal IgGs.

(93) On this basis, two batches according to the invention were made from different II+III precipitates. The results of the first evaluations obtained from both of these raw materials were compared to the “fluidized bed chromatography eluate” stage.

(94) The table below accounts for these results:

(95) TABLE-US-00010 Batch Batch Batch  I + II + III   II + III   II + III  pH of RES 4.6 4.6 4.6 g IgG/L of plasma 6.00 5.17 5.1 Purification yield % 98 88 95 IgG/proteins 0.910 0.837 0.859

(96) These results show that first steps of the method of the invention are performed in an equivalent way with both types of precipitate: as regards extraction yield: the cumulated yields of the clarification and robust affinity chromatography steps are comparable even if one of the batches is a little set back. However, the II+III precipitates which have undergone an additional fractionation step with ethanol as compared with the I+II+III precipitate have a yield in grams of IgG/L of plasma of 15% less than the one obtained with the I+II+III precipitate. This deficit is related to a loss of a portion of the IgGs during the additional fractionation step with ethanol. as regards purity: the IgG/total proteins ratios obtained with the II+III precipitates put back into solution, treated by caprylic acid and adjusted to a pH of 4.6 are comparable with those obtained with the I+II+III precipitates put back into solution, treated by caprylic acid and adjusted to a pH of 4.8.