Process for preparing immunoglobulin compositions

Abstract

A process for the preparation of pharmaceutically acceptable immunoglobulin compositions from plasma-derived immunoglobulin fractions which allows the parallel preparation of immunoglobulin compositions enriched in IgG, IgM and IgA. In this process, immunoglobulin contained in Cohn fraction I/II/III or Kistler Nitschmann fraction A+I is resolubilized at conductivities of at least 1 mS/cm, and following removal of contaminating protein the resolubilized immunoglobulin is subjected to anion exchange chromatography to obtain IgG- and IgM/IgA-enriched immunoglobulin compositions. The IgG-enriched immunoglobulin composition is further subjected to treatment with a cation exchange material to obtain an immunoglobulin composition having a reduced properdin content.

Claims

1. A process for the preparation of a pharmaceutically acceptable immunoglobulin composition[s] from a plasma-derived immunoglobulin fraction comprising or consisting of Cohn fraction I/II/III or Kistler-Nitschmann fraction A+I, said process comprising the steps of: (a) resolubilizing immunoglobulin contained in the plasma-derived immunoglobulin fraction comprising or consisting of Cohn fraction I/II/III or Kistler-Nitschmann fraction A+I by resuspending said plasma-derived immunoglobulin fraction under conditions to adjust the conductivity of the suspension to at least 1 mS/cm to obtain a suspension containing resolubilized IgG, IgM and IgA; (b) precipitating contaminating protein in the suspension obtained in step (a) and removing said contaminating protein to obtain an impurity-depleted immunoglobulin composition; (c) subjecting the impurity depleted immunoglobulin composition obtained in step (b) to ion exchange chromatography using an anion exchange resin under conditions of pH and conductivity adjusted to substantially bind IgM and IgA to the resin, and obtaining an IgG-enriched immunoglobulin composition in the flow-through fraction and/or by eluting IgG from the anion exchange resin under conditions where IgM and IgA remain bound to the anion exchange resin; and (d) subjecting contacting the IgG-enriched immunoglobulin composition obtained in step (c) to treatment with a cation exchange material under conditions of pH and conductivity where properdin is bound to said cation exchange material, comprising contacting the IgG-enriched immunoglobulin composition with said cation exchange material at a pH in the range of from 5.0 to 6.0 and at a conductivity in the range of from 16 to 30 mS/cm, and recovering IgG to obtain an IgG-enriched immunoglobulin composition having a reduced properdin content.

2. The process of claim 1 comprising the steps of: (a) resolubilizing immunoglobulin contained in the plasma-derived immunoglobulin fraction comprising or consisting of Cohn fraction I/II/III or Kistler-Nitschmann fraction A+I by resuspending said plasma-derived immunoglobulin fraction under conditions to adjust the conductivity of the suspension to at least 1 mS/cm to obtain a suspension containing resolubilized IgG, IgM and IgA; (b) precipitating contaminating protein in the suspension obtained in step (a) and removing said contaminating protein to obtain an impurity-depleted immunoglobulin composition, wherein precipitating contaminating protein comprises treating the suspension obtained in step (a) with octanoic acid; (c) subjecting the impurity depleted immunoglobulin composition obtained in step (b) to ion exchange chromatography using an anion exchange resin under conditions of pH and conductivity adjusted to bind, based on the amount of each immunoglobulin subjected to ion exchange chromatography, at least 90% by weight of each of IgM and IgA to the resin, and obtaining an IgG-enriched immunoglobulin composition[s] in the flow-through fraction and/or by eluting IgG from the anion exchange resin under conditions where IgM and IgA remain bound to the anion exchange resin; and (d) subjecting the IgG-enriched immunoglobulin composition obtained in step (c) to treatment with a cation exchange material under conditions of pH and conductivity where properdin is bound to said cation exchange material, comprising contacting the IgG-enriched immunoglobulin composition with said cation exchange material at a pH in the range of from 5.0 to 6.0 and at a conductivity in the range of from 16 to 30 mS/cm, and recovering IgG to obtain an IgG-enriched immunoglobulin composition having a reduced properdin content.

3. The process of claim 1, wherein step (c) further comprises using an anion exchange resin under conditions of pH and conductivity adjusted to also bind IgG to the resin.

4. The process of claim 2, wherein step (c) further comprises using an anion exchange resin under conditions of pH and conductivity adjusted to also bind IgG to the resin.

5. The process of claim 1, wherein the conductivity of the suspension is at least 1.5 mS/cm.

6. The process of claim 1, wherein resuspending of the plasma-derived immunoglobulin fraction is carried out using a buffer adjusted to a pH in the range of from 4.2 to 5.5.

7. The process of claim 6, wherein the buffer is an acetate buffer.

8. The process of claim 1, wherein precipitating contaminating protein in step (b) comprises treating the suspension obtained in step (a) with octanoic acid.

9. The process of claim 1, wherein removing contaminating protein in step (b) comprises filtration.

10. The process of claim 1, wherein step (b), following removal of contaminating protein, further includes subjecting the impurity-depleted immunoglobulin composition to a mild acid treatment, wherein the immunoglobulin composition is incubated at a pH in the range of from 3.8 to 4.5 before subjecting it to ion exchange chromatography with an anion exchange resin in step (c).

11. The process of claim 10, wherein step (b), following removal of contaminating protein, further includes subjecting the impurity-depleted immunoglobulin composition to a mild acid treatment, wherein the immunoglobulin composition is incubated at a temperature in the range of from 35 to 40° C. before subjecting it to ion exchange chromatography with an anion exchange resin in step (c).

12. The process of claim 1, wherein the anion exchange resin used in step (c) is a macroporous anion exchange resin.

13. The process of claim 12, wherein the anion exchange chromatography is carried out at a pH in the range of from 6.7 to 7.5.

14. The process of claim 12, wherein the anion exchange chromatography is carried out at a conductivity in the range of from 4 to 7.5 mS/cm.

15. The process of claim 12, wherein IgM and/or IgA bound to the anion exchange resin are eluted from the resin at a conductivity of at least 20 mS/cm.

16. The process of claim 15, wherein elution is carried out at a pH in the range of from 6.7 to 7.5.

17. The process of claim 1, wherein treatment of the IgG-enriched immunoglobulin composition in step (d) is carried out by contacting the IgG-enriched immunoglobulin composition with a cationic membrane adsorber under conditions of pH and conductivity where properdin is bound to the cationic membrane adsorber and IgG is recovered in the flow-through fraction.

18. The process of claim 1, wherein treatment in step (d) is carried out by contacting the IgG-enriched immunoglobulin composition with the cation exchange material at a pH in the range of from 5.2 to 5.8.

19. The process of claim 18, wherein treatment in step (d) is carried out by contacting the IgG-enriched immunoglobulin composition with the cation exchange material at a conductivity in the range of from 20 to 28 mS/cm.

20. The process of claim 1, wherein step (c) further comprises eluting from the anion exchange resin IgM and/or IgA to obtain an immunoglobulin composition enriched in IgM and/or IgA.

21. The process of claim 20, further comprising subjecting the IgG-enriched immunoglobulin composition obtained in step (d) and/or the immunoglobulin composition enriched in IgM and/or IgA obtained in step (c) to further treatment for virus inactivation to obtain a virus inactivated preparation.

22. The process of claim 20, further comprising the step of formulating the IgG-enriched immunoglobulin composition obtained in step (d) and/or the immunoglobulin composition enriched in IgM- and/or IgA obtained in step (c) into a pharmaceutical preparation.

23. A process for reducing the properdin content in a properdin-containing IgG composition, said process comprising subjecting said properdin-containing IgG composition to treatment with a cation exchange material under conditions of pH and conductivity where properdin is bound to said cation exchange material to obtain an IgG composition having a reduced properdin content, wherein treatment is carried out by contacting the IgG composition with the cation exchange material at a pH in the range of from 5.0 to 6.0 and at a conductivity in the range of from 16 to 30 m S/cm.

24. The process of claim 23, wherein treatment with the cation exchange material is carried out by subjecting the properdin-containing IgG composition to cation exchange chromatography under conditions where properdin is bound to said cation exchange material and IgG is recovered in the flow-through fraction.

25. The process of claim 23, wherein treatment of the IgG-enriched immunoglobulin composition is carried out by contacting the IgG-enriched immunoglobulin composition with a cationic membrane adsorber under conditions where properdin is bound to the cationic membrane adsorber and IgG is recovered in the flow-through fraction.

26. The process of claim 23, wherein treatment is carried out by contacting the IgG composition with the cation exchange material at a pH in the range of from 5.2 to 5.8.

27. The process of claim 23, wherein treatment is carried out by contacting the IgG composition with the cation exchange material at a conductivity in the range of from 20 to 28 mS/cm.

28. The process of claim 23, wherein the properdin-containing IgG composition is an IgG-enriched immunoglobulin composition, said IgG-enriched immunoglobulin composition having an IgG content of at least 95% by weight, based on the total weight of immunoglobulin in the properdin-containing IgG composition.

29. A process for reducing the anticomplementary activity (ACA) of a properdin-containing IgG composition by reducing the properdin content thereof, said process comprising subjecting said properdin-containing IgG composition to treatment with a cation exchange material under conditions of pH and conductivity where properdin is bound to said cation exchange material to obtain an IgG composition having a reduced ACA and properdin content, wherein treatment is carried out by contacting the IgG composition with the cation exchange material at a pH in the range of from 5.0 to 6.0 and at a conductivity in the range of from 16 to 30 mS/cm.

Description

EXAMPLES

(1) Determination of Immunoglobulin Content

(2) The immunoglobulin content was determined by capillary zone electrophoresis (CZE) according to the European Pharmacopoeia 8.0 (2.2.47—Capillary Electrophoresis). Immunoglobulin fractions were separated at pH 10 in capillaries according to their charge:mass ratio on the basis of their run time, characterized and quantified photometrically at 200 nm. A capillary electrophoresis system with UV detector (P/ACE MDQ capillary electrophoresis system, Beckman Coulter) was used for the procedure. The samples were diluted with electrophoresis buffer to a protein concentration of 2.5 g/l (borate buffer, pH 10; 14.3 g disodium tetraborate decahydrate dissolved in 1000 ml Aqua purificata and adjusted with 1 M NaOH). The mixture is used for electrophoresis without any further preparation. The electrophoresis procedure is performed according to the instrument manufacturer's instructions.

(3) Determination of Molecular Size Distribution

(4) The molecular size distribution of IgG immunoglobulins was determined by High Pressure Size Exclusion Chromatography (HPSEC) as peak area in percent of the total area of the chromatogram according to the European Pharmacopoeia 8.0 (2.2.30—Molecular size distribution of “Human normal immunoglobulin for intravenous administration”). On passing protein mixtures through hydrophilic porous gels, the molecules appear in different distribution zones depending on molecular size and pore size distribution. The largest proteins/particles migrate through the gel most rapidly while small protein molecules and low molecular weight substances migrate most slowly.

(5) A Tosoh TSK-G 3000 SW was used for separation, and a protein mass of 100 μg was injected. The separated fractions were detected and quantified at the column outlet by photometry at 280 nm. The chromatography was performed according to the equipment manufacturer's operating instructions. A Bio-Rad gel filtration standard was used as a control. An immunoglobulin preparation was used as SST-sample. The peaks are assigned to the fractions polymer, dimer, monomer and fragments, using an automated method for peak integration.

(6) Determination of Properdin Concentration

(7) A ready-to-use solid phase human properdin ELISA kit (Hycult Biotech) was used for the in vitro quantitative determination of human Properdin in IgG preparations in accordance with the manufacturer's instructions. Briefly, samples and standards are incubated in microtiter wells coated with antibodies recognizing human properdin. Biotinylated tracer antibody will bind to the captured human properdin. Streptavidin-peroxidase conjugate will bind to the biotinylated tracer antibody. Streptavidin-peroxidase conjugate will react with the substrate, tetramethylbenzidine (TMB). The enzyme reaction is stopped by the addition of oxalic acid. The absorbance at 450 nm is measured with a spectrophotometer. A standard curve is obtained by plotting the absorbance (linear) versus the corresponding concentrations of the human properdin standards (log). The human properdin concentration of samples, which are run concurrently with the standards, is determined from the standard curve.

(8) Determination of Anticomplementary Activity (ACA)

(9) Tests for ACA of immunoglobulin were performed as described in the European Pharmacopoeia 8.0 (2.6.17—Test for Anticomplementary Activity of immunoglobulin).

(10) In brief, a defined amount of test material (10 mg of immunoglobulin) is incubated with a defined amount of guinea pig complement (20 CH.sub.50). The remaining complement is titrated and incubated with red sheep blood cells that are sensitized with hemolysin. Optimally sensitized sheep red blood cells consist of sheep erythrocytes loaded with antibodies against sheep erythrocytes (hemolysin). The degree of cell lysis is determined by photometry at 541 nm. ACA is expressed as the percentage consumption of complement relative to a complement control considered as 100 percent. The hemolytic unit of complement activity (CH.sub.50) is defined as the amount of complement that, in the given reaction conditions, will produce lysis of half of the sensitized sheep red blood cells in the test. The acceptance limit for ACA in the European Pharmacopoeia is defined as such that the consumption of complement is not greater than 50 percent and 1 CH.sub.50 per milligram of immunoglobulin.

(11) Thrombogenic Activity (TGA)

(12) A fluorogenic microplate assay (Technoclone) was used to determine thrombogenic activity (TGA). Technothrombin® TGA RC High was used as reagent, Technothrombin® TGA SUB as fluorogenic substrate, and a Factor XI deficient plasma. Calibration was done with the International Standard for FXIa, 13/100 (NIBSC).

(13) Factor XI (FXI)

(14) A commercially available standard coagulation assay (Siemens Healthcare Diagnostics) was used to determine Factor XI (FXI). FXI depleted plasma, Actin FSL as activator and a CaCl.sub.2 solution were used in this assay. Calibration was done with Standard Human Plasma (Siemens Healthcare). Additional calibration points in the lower calibration range were included to improve assay sensitivity.

(15) Factor XIa (FXIa)

(16) A commercially available chromogenic assay (Hyphen Biomed) was used to determine Factor XIa employing the standard conditions of the test kit.

Example 1

Example 1a

(17) Preparation of an IgM-Enriched Immunoglobulin Composition

(18) Human blood plasma for fractionation (2000 l) from more than 500 donors was used as starting material. The plasma was transferred to the pooling area and pooled.

(19) A cryoprecipitation step was performed in order to separate coagulation factors such as Factor VIII, von Willebrand Factor, and Fibrinogen. In order to obtain the cryoprecipitate, the temperature of the plasma was adjusted under gentle stirring so that the temperature range was kept at 2±2° C. Under these conditions the cryoprecipitate remains undissolved in the thawed plasma. The cryoprecipitate was separated from the plasma by a continuously operating centrifuge such as a Westfalia separator.

(20) From the supernatant of the cryoprecipitation step the Cohn fraction I/II/III was precipitated by ethanol precipitation as follows:

(21) The temperature of the centrifugation supernatant remaining after separation of the cryoprecipitate was adjusted to 2±2° C. The pH-value of the protein solution was adjusted to pH 5.9. Subsequently, the temperature was lowered to −5° C. and ethanol was added to a final concentration of 20% by volume. Under constant slow stirring in a stainless steel vessel, Cohn Fraction I/II/III was precipitated. The Cohn Fraction I/II/III precipitate was separated from the supernatant by filtration with depth filter sheets under addition of filter aid such as Perlite or Diatomaceous Earth, using a filter press. The Cohn fraction I/II/III was recovered from the filter sheets. This Cohn fraction I/II/III precipitate comprised all immunoglobulins (IgG, IgA, IgM) in approximately the following percentages: 75% IgG, 13% IgM and 12% IgA.

(22) 90 kg of the obtained Cohn fraction I/II/III precipitate were resuspended in 450 kg of 0.1 M sodium acetate puffer pH 4.8 and mixed for 60 minutes at 22° C. The pH of the suspension was adjusted to 4.8 with acetic acid.

(23) In the following a treatment with octanoic acid was performed. The solution was treated by addition of 7.7 kg octanoic acid at room temperature. The octanoic acid was added slowly and the protein solution was further mixed for 60 minutes, using a vibrating mixer (Vibromixer®, Size 4, Graber+Pfenniger GmbH, Vibromixer adjusted to level 2-3).

(24) A calcium phosphate treatment was performed in order to complete the octanoic acid reaction as follows:

(25) Approximately 1.1 kg Ca.sub.3(PO.sub.4).sub.2 were added and the protein solution was further mixed for more than 15 minutes and filtered over depth filter sheets. The filtrate was further processed. The obtained protein solution was subjected to ultrafiltration to a protein concentration of about 50 g/l. The protein solution was diafiltered against 0.02 M sodium acetate buffer pH 4.5 and afterwards adjusted to a protein concentration of about 40 g/l.

(26) The protein solution was treated at pH 4.0 in order to inactivate viruses as follows: The pH was adjusted to pH 4.0 using 0.2 M HCl, and the resulting solution was incubated for 8 hours at 37° C. The resulting protein solution contains immunoglobulins with the following distribution: 90% IgG, 5% IgA, and 5% IgM.

(27) The obtained protein solution was further processed by anionic exchange chromatography using a macroporous anion exchange resin in order to remove accompanying proteins and to obtain an IgG- and IgM-enriched immunoglobulin compositions.

(28) Per kilogram of the intermediate protein solution 0.00121 kg of tris(hydroxymethyl)aminomethane (Tris) were added and dissolved while stirring and the conductivity was adjusted to 6 mS/cm with solid NaCl. The protein solution was adjusted to pH 7.1 by adding 1 M NaOH. A macroporous anion exchange resin (POROS® 50 HQ anion exchange resin, Life Technologies, bed height of the column: 25 cm) was equilibrated with a 10 mM Tris buffer solution (pH 7.1, 50 mM NaCl, at a linear flow rate of 800 cm/h). The protein solution was loaded on the anion exchange resin with 40 g protein per liter of resin. The column was washed with the equilibration buffer (10 mM Tris, 50 mM NaCl, pH 7.1, at 800 cm/h).

(29) An IgG-enriched immunoglobulin composition was obtained in the flow-through fraction and was further processed as described in Example 3 below.

(30) An IgM-enriched fraction was eluted by increasing the conductivity as follows: 10 mM Tris buffer solution with 300 mM NaCl at pH 7.1 is used at 800 cm/h to elute the IgM-enriched fraction. The eluted fraction contained 58% IgG, 22% IgA and 20% IgM.

(31) The protein solution was filtered through a Pall, Ultipor VF DV50 filter as a virus removal step. The filtrate was further processed by UVC light treatment at 254 nm, using a flow-through UVivatech process device (Bayer Technology Services/Sartorius) at a UVC dose of 225 J/m.sup.2 for further virus inactivation. The flow velocity through the UVC reactor was calculated using the manufacturer's instructions. The irradiated protein solution was concentrated to a protein concentration of 50 g/l by ultrafiltration (and was subjected to diafiltration (using 0.3 M glycine buffer pH 4.5). The final product was filtered through a 0.2 μm filter and was stored at 2 to 8° C.

(32) The obtained immunoglobulin composition had an IgM content of 22% by weight, an IgA content of 22% by weight and an IgG content of 56% by weight, based on the total immunoglobulin content, at an immunoglobulin concentration of 50 mg/ml. The ACA was 0.34 CH50/mg.

Example 1b

(33) Processing of Larger Amounts

(34) In order to process larger amounts of protein, multiple purification cycles on the macroporous anion exchange resin were conducted. For this purpose, cleaning steps were implemented into the chromatography cycle. Specifically, following elution of the IgM-enriched fraction from the IgG-, IgA and IgM-containing intermediate protein solution obtained as described in Example 1a, the column was stripped with 1 M NaCl solution to elute residual bound proteins. The column was further regenerated with 3 column volumes of 1 M NaOH, and a further cycle was started by the equilibration phase using equilibration buffer. In total, 12 purification cycles at a linear flow rate of 800 cm/h were conducted without loss of any purification performance.

Example 2

(35) Preparation of an IgM-Enriched Immunoglobulin Composition Using a Tentacle Resin

(36) The initial processing including the step of the pH 4 treatment was done as described in Example 1a.

(37) The obtained protein solution was further processed by anionic exchange chromatography using a tentacle anion exchange resin in order to remove accompanying proteins and to obtain a solution comprising an increased percentage of IgM relative to the other immunoglobulins, as follows:

(38) The intermediate (protein concentration: 41 g/l) was adjusted with Tris buffer (final concentration: 10 mM) to a pH of 7.1. The conductivity of the protein solution was adjusted to 6 mS/cm (at 20° C.) using NaCl.

(39) The chromatography column (Fractogel® TMAE, bed height: 39.5 cm, column volume: 80 ml) was equilibrated with 10 mM Tris buffer pH 7.1/50 mM NaCl at a linear flow rate of 150 cm/h, and the protein solution was pumped onto the chromatography column until a loading of 40 mg per ml resin was reached. The loaded column was washed with 10 mM Tris buffer pH 7.1/50 mM NaCl and the flow-through fraction was collected. The linear flow rate of 150 cm/h was kept during the experiment. The chromatography was monitored using a UV-sensor. The bound fraction was eluted by 10 mM Tris pH 7.0/300 mM NaCl. The elution fraction was collected and can be further processed as described in example 1a.

(40) The yield of the IgG-enriched flow-through fraction was 84%. In the IgG-enriched fraction, the IgA was below the limit of detection (<0.0116 g/L, Siemens BN Prospec) at a protein concentration of 9.81 g/L (determined by the Biuret assay). The IgM content was below the limit of detection (<0.00846 g/L). The IgG-4 subclass content was 2.31%.

(41) The obtained IgM-enriched immunoglobulin composition had an IgM content of 28% by weight, an IgA content of 19% by weight and an IgG content of 53% by weight based on the total immunoglobulin content, at an immunoglobulin concentration of 50 mg/ml.

Example 3

(42) Preparation of an IgG-Enriched Immunoglobulin Composition in Flow-Through Mode (Cation Exchange Chromatography)

(43) The IgG-enriched immunoglobulin composition collected as the flow through fraction of the macroporous anion exchange chromatography (POROS® 50 HQ) in Example 1a was adjusted to pH 5.5 and to a conductivity of 22-26 mS/cm with sodium acetate buffer and NaCl and then was further purified by cation exchange chromatography in a flow-through mode on a cation exchange resin (POROS® 50 HS). The binding capacity of this resin is defined as 100-3000 g/l, and chromatography was carried out at a load of 3000 g/l and a flow-rate of 800 cm/h.

(44) The cation exchange column was equilibrated with acetate buffer solution (pH 5.5, adjusted to 22, 24 and 26 mS/cm with NaCl). The protein solution was loaded to the column and washed with acetate buffer (pH 5.5, adjusted to 22-26 mS/cm with NaCl). The flow through fraction and the wash are collected and further processed. The residual protein is eluted with 1.5 M NaCl.

(45) The resulting protein solution was further processed by a nanofiltration step, in order to remove potentially present virus. A Planova BioEx 20 nm filter (Asahi Kasei) was used as a virus filter. More than 50 kg of the protein solution were filtered over a 0.1 m.sup.2 filter area at a protein concentration of 10 g/l. The maximum pressure was set according to the manufacturer's instructions. Flow rate during nanofiltration was as follows:

(46) TABLE-US-00001 Material after Mean flow-rate during POROS 50 nanofiltration HS chromatography [kg/(m.sup.2*h)] 22 mS/cm and pH 5.5 48.4 24 mS/cm and pH 5.5 53.0 26 mS/cm and pH 5.5 53.9

(47) The resulting protein solution was subjected to a concentration step to >100 g/L by ultrafiltration and diafiltered into formulation buffer (0.3 M Glycine pH 5.0). The resulting protein solution was filtered through a 0.2 μm filter in order to control sterility.

(48) The obtained immunoglobulin compositions were analysed for immunoglobulin contents, subclass distribution and ACA, and the results are shown in Table 1.

(49) TABLE-US-00002 TABLE 1 Analytical parameters for drug substances produced at lab-scale POROS ® 50 HS POROS ® 50 HS POROS ® 50 HS Parameters at 22 mS/cm at 24 mS/cm at 26 mS/cm IgG [%] 99.6 99.7 99.6 IgA [%] 0.26 0.21 0.24 IgM [%] 0.15 0.11 0.13 IgG.sub.1 [%] 62.68 63.52 63.47 IgG.sub.2 [%] 32.96 31.84 32.19 IgG.sub.3 [%] 2.58 2.40 2.53 IgG.sub.4 [%] 1.79 2.25 1.80 ACA [CH50/mg] 0.56 0.60 0.58

(50) The drug substances obtained after the POROS® 50 HS chromatography showed ACA levels in the desired range. The ratio of IgG, IgA and IgM and subclass distribution was not changed by the additional POROS® 50 HS chromatography. The subsequent nanofiltration was inconspicuous.

Example 4

(51) Investigation of Properdin Content in IgG Preparations with and without Cationic Exchange Chromatography

(52) In order to investigate the effect of cationic exchange chromatography step on the properdin levels in IgG preparations, four batches of Cohn fraction I/II/III obtained from pooled blood plasma were resuspended in 0.1 M sodium acetate buffer (pH 4.8) at manufacturing scale (100 kg of fraction I/II/III employed) and subjected to treatment with octanoic acid, tri-calcium phosphate, ultra-diafiltration, mild acid treatment and anion exchange chromatography as described in Example 1a. The flow through (IgG fractions) of batches 1 and 2 was collected and immediately subjected to ultra-/diafiltration versus 0.3 M glycine buffer, pH 4.6. The flow through of batches 3 and 4 was further subjected to cation exchange chromatography (CEX) as described in Example 3, and the flow-through was subjected to ultra-/diafiltration as above. All IgG solutions thus obtained were analysed for immunoglobulin and properdin content using a solid phase human properdin ELISA kit (Hycult Biotech) as described before. The results are shown in Table 2.

(53) TABLE-US-00003 TABLE 2 Properdin content Immuno- Properdin per mg globulin conc. Immunoglobulin Sample conc. [g/l] [μg/ml] [μg/mg] Batch 1 (without CEX) 126 229 1.82 Batch 2 (without CEX) 116 198 1.71 Batch 3 (with CEX) 113 0.23 0.0020 Batch 4 (with CEX) 126 0.14 0.0011

(54) As shown in Table 2, cation exchange chromatography results in an enormous reduction in properdin content.

Example 5

(55) Determination of Properdin Content and IgG Polymer Content in IgG-Enriched Immunoglobulin Compositions

(56) IgG preparations of the invention as described in Example 4 (Batches 3 and 4) were further tested for IgG polymer content by HPSEC as described before. Properdin and IgG polymer contents of commercially available pharmaceutical IgG compositions (CP-IgG 1 to 5) were determined by the same methods for comparison. The results are shown in Table 3.

(57) TABLE-US-00004 TABLE 3 IgG Immunoglobulin Properdin Polymer concentration Properdin [μg/mg] content Sample [g/l] [μg/ml] Immunoglobulin] [%] Batch 3 113 0.23 0.0020 0.0 Batch 4 126 0.14 0.0011 0.0 CP-IgG 1 100 1.16 0.0116 0.0 CP-IgG 2 100 5.11 0.0511 0.1 CP-IgG 3 100 13.50 0.1350 0.6 CP-IgG 4 100 4.00 0.0400 0.1 CP-IgG 5 100 0.04 0.0004 0.2 (pasteurized)

(58) As may be seen from Table 3, IgG-enriched immunoglobulin compositions obtained according to the method of the invention have a properdin content which is below that of the commercially available pharmaceutical products except for CP-IgG 5 which is a pasteurized product. Likewise, the content of IgG polymers in the IgG-enriched immunoglobulin compositions of the invention was below that of all pharmaceutical IgG compositions except for CP-IgG 1.

Example 6

(59) Determination of Thrombogenic Activity (TGA), Factor XIa and Factor XI (FXI) in IgG-Enriched Immunoglobulin Compositions

(60) IgG-enriched immunoglobulin compositions (3 batches) obtained as described in Example 4 after cation exchange chromatography were tested for TGA, FXIa and FX as described before using commercially available assays (Batches 5 to 7). The results are shown in Table 4.

(61) TABLE-US-00005 TABLE 4 Purity of Protein Ig-fraction concentration TGA FXIa FXI by CZE Sample [g/l] [mU/ml] [mU/ml] [% of norm] [%] Batch 5 100 <1.5 <2.0 <1 100 Batch 6 99 <1.5 <2.0 <1 100 Batch 7 99 <1.5 <2.0 <1 100

(62) The results show no residual TGA, FXIa and FXI (below detection limit of applied method).

Example 7

(63) Long-Term Stability of IgG-Enriched Immunoglobulin Compositions

(64) An IgG-enriched immunoglobulin composition obtained as described in Example 4 after cation exchange chromatography (118 g immunoglobulin/l) was tested for long-term stability at 5° C. and 25° C., respectively over a period of 90 weeks using HPSEC as described above. The results are shown in Table 5.

(65) TABLE-US-00006 TABLE 5 Long-term stability of IgG-enriched immunoglobulin compositions Polymer (%) after Polymer (%) after Weeks Storage at 5° C. Storage at 25° C. 0 0.0 0.0 1 0.0 0.0 4 0.0 0.0 8 0.0 0.0 24 0.0 0.0 65 0.0 0.5 90 0.0 0.6

(66) As may be seen from the results, no IgG polymers were detectable after storage at 5° C. over a period of 90 weeks. After storage at 25° C. the polymer content of IgG remains below 1.0% after a period of 90 weeks.

Example 8

(67) Preparation of an IgG-Enriched Immunoglobulin Composition in Binding Mode (Cation Exchange Chromatography)

(68) An IgG fraction obtained as described in Example 1a as a flow-through from POROS® 50 HQ anion exchange chromatography was ultra/diafiltered to 20 mM sodium acetate, pH 5.5, so as to prepare the material for cation exchange chromatography with POROS® 50 HS in a binding mode.

(69) The prepared material was successfully bound to POROS® 50 HS, and an IgG fraction was eluted with buffer (20 mM sodium acetate, 225 mM sodium chloride, pH 5.5±0.1, conductivity 24±2 mS/cm). The obtained IgG-enriched immunoglobulin composition had an ACA value of 0.58 CH50/mg protein.

Example 9

(70) ACA Break-Through Curves at Different Conductivities (Cation Exchange Chromatography)

(71) IgG fractions obtained as described in Example 1a as a flow-through from POROS® 50 HQ anion exchange chromatography were ultra/diafiltered to 10 mM Tris, 6.5 mM sodium acetate and adjusted to pH 5.5 and conductivities of 22, 24 and 26 mS/cm using NaCl. ACA break-through curves were obtained using a POROS® 50 HS column with a column volume of 0.8 ml. The protein solution was pumped over the POROS® 50 HS column at a flow-rate of 800 cm/h to remove ACA.

(72) Table 6 shows the results obtained for ACA-break-through at the intended conductivities. In all cases ACA is efficiently removed up to a load of at least 3 g protein/ml gel. The ACA levels rise again with higher loads. The higher the conductivity the faster the ACA levels rise.

(73) TABLE-US-00007 TABLE 6 ACA-break-through at 22, 24 and 26 mS/cm Load Protein ACA [g protein/l gel] concentration [g/l] [CH50/mg] 22 mS/cm Load material 69.25 1.18 3000 55.35 0.66 4000 62.64 0.74 5000 63.10 0.78 6000 59.69 0.78 7000 61.70 0.88 8000 62.03 0.90 24 mS/cm Load material 70.65 1.06 3000 54.95 0.66 4000 61.16 0.90 5000 60.11 0.88 6000 58.90 0.90 7000 59.56 1.02 8000 60.56 0.98 26 mS/cm Load material 65.50 1.20 3000 56.42 0.78 4000 62.57 0.92 5000 59.42 0.98 6000 59.03 1.02 7000 60.36 1.04 8000 57.82 1.06

Example 10

(74) Variation of Conductivity (Cation Exchange Chromatography)

(75) IgG fractions obtained as in Example 1a as a flow-through from POROS® 50 HQ anion exchange chromatography were adjusted to pH of 5.5 and conductivities ranging from 16 to 30 mS/cm using 20 mM Na acetate buffer and NaCl. The thus prepared material was applied to a POROS® 50 HS column (column load 500 g/l), and fractions were collected and the ACA level was determined. The results are shown in Table 7.

(76) TABLE-US-00008 TABLE 7 ACA values with variation of conductivity settings Conductivity settings ACA [CH50/mg [mS/cm] protein] Before POROS ® 50 HS 1.30 16 0.53 18 0.52 20 0.48 22 0.45 24 0.49 26 0.43 28 0.44 30 0.50

(77) A reduction of ACA levels could be realized over a wide range of conductivity settings. Lower conductivities have a risk of loss of IgG yield and higher conductivities have a risk of ACA break-through.

Example 11

(78) Variation of Flow Rates (Cation Exchange Chromatography)

(79) IgG fractions obtained as described in Example 1a as a flow-through from POROS® 50 HQ anion exchange chromatography were ultra/diafiltrated to 10 mM Tris, 6.5 mM sodium acetate, 225 mM sodium chloride (pH 5.5; conductivity 22 mS/cm). The thus prepared material was applied to a POROS® 50 HS column at 200, 500 and 800 cm/h (load 1.2 g/ml POROS® 50 HS). The flow-through fractions were collected and the ACA level was determined. The results are shown in Table 8.

(80) TABLE-US-00009 TABLE 8 ACA values with variation of flow rate during POROS ® 50 HS chromatography Flow-rate ACA [cm/h] [CH50/mg] Before POROS ® 50 HS 1.12 chromatography 200 0.62 500 0.66 800 0.62

(81) As may be seen from Table 8, flow-rates have no significant effect on ACA.

Example 12

(82) Membrane Adsorber as Cation Exchange Material

(83) Depletion of ACA in an IgG-enriched immunoglobulin composition obtained as described in Example 1a as a flow-through from POROS® 50 HQ anion exchange chromatography was tested using a cationic membrane adsorber (Sartorius-Sartobind S) in a non-binding mode for IgG. The IgG-enriched solution was adjusted to a pH of 5.5 using a sodium acetate buffer and a sodium chloride concentration of 225 mM (corresponding to a conductivity of 24 mS/cm). Under these conditions IgG does not bind to the cation exchange material. The membrane adsorber module was loaded with 0.5 g/ml resin. The flow-through fraction and a high-salt elution fraction (1.5 M NaCl) were collected and analyzed for ACA. The ACA value of the flow-through fraction is low (CH50/mg=0.44), whereas the bound fraction is enriched in its ACA content (CH50/mg >1.5).

Example 13

(84) Use of Cation Exchange Resin in Batch Mode

(85) An IgG-enriched immunoglobulin composition obtained as described in Example 1a as a flow-through from POROS® 50 HQ anion exchange chromatography was ultra/diafiltrated to 10 mM Tris, 6.5 mM sodium acetate, 225 mM sodium chloride (pH 5.5; conductivity 21 mS/cm) and adjusted to a protein content of 50 g/l. POROS 50 HS chromatography material was added as a powder and the suspension was gently shaken for 1 hour at room temperature. The following amounts of POROS 50 HS were thus tested in a batch mode and the results are shown in Table 9.

(86) TABLE-US-00010 TABLE 9 Use of POROS ® 50 HS in batch mode Protein load ACA [mg POROS ® 50 HS/g Protein] [CH50/mg protein] Starting material 1.12 100 0.98 250 0.70 500 0.52

(87) ACA could successfully be removed below 1 CH50/mg at load conditions of greater than 250 mg POROS® 50 HS/g protein.

Example 14

(88) Properdin Spike Experiments

(89) In order to demonstrate the correlation between properdin content and ACA, a 1 mg/ml properdin solution (obtained from Quidel) was spiked into two different 10% IgG-immunoglobulin (IVIg preparations A and B) that had been processed using a cation exchange chromatography for polishing and ACA was measured. As shown in Table 10, increasing concentrations of properdin lead to an increase in ACA in a linear dependency up to concentrations of a properdin spike of 200 μg/ml.

(90) TABLE-US-00011 TABLE 10 ACA in Properdin spiked immunoglobulin solution Properdin IgG preparation A IgG preparation B spike ACA [μg/ml] [CH50/mg] [CH50/mg] 0 0.42 0.42 25 0.52 0.60 50 0.58 0.70 100 0.82 0.92 150 0.94 1.10 200 1.08 1.16

Example 15

(91) Effect of Resolubilization Buffer on Properdin Content After Octanoic Acid Treatment.

(92) In order to demonstrate the correlation between conditions for resolubilization and properdin content after octanoic acid treatment, Cohn fraction I/II/III was resuspended in deionized water and three different resolubilization buffers. The suspensions of fraction I/II/III were subjected to treatment with octanoic acid (pH 4.8, 17.5 g/kg octanoic acid per kg suspension) and tri-calcium phosphate, as outlined in Example 1a. The precipitate was removed by depth filtration, and the resulting protein solution was subjected to ultra-/diafiltration and mild acid treatment at pH 4. The impurity depleted immunoglobulin composition thus obtained was analyzed for the properdin content. The results are shown in Table 11.

(93) TABLE-US-00012 TABLE 11 Properdin content per Conductivity Protein Properdin mg (at 22° C.) conc. concentration protein Sample Condition mS/cm [g/l] [μg/ml] [μg/mg] 1 Deiononized 0.09 46.8 5 0.107 water 2 10 mM sodium 0.77 46.8 11 0.235 acetate buffer, pH 4.8 3 50 mM sodium 2.6 42.5 36 0.847 acetate buffer, pH 4.8 4 100 mM sodium 6.9 44.6 42 0.941 acetate buffer, pH 4.8

(94) As may be seen from the results, the properdin content in the immunoglobulin compositions obtained after octanoic acid treatment increases with increasing molarity of the resolubilization buffer.

Example 16

(95) Effect of Resolubilization Buffer on Properdin Content in IgG-Enriched Immunoglobulin Compositions Obtained After Anion Exchange Chromatography

(96) In order to investigate the effect of the resolubilization buffer on the properdin content in IgG preparations obtained after anion exchange chromatography, Cohn fraction I/II/III was resuspended in either Water for Injection (WFI) or 100 mM sodium acetate buffer (pH 4.8) at laboratory scale in a fraction I/II/II to buffer ratio of 1:4. Both suspensions were treated with octanoic acid and tri-calcium phosphate, followed by ultra-diafiltration and mild acid treatment at pH 4 as described in Example 13. The resulting immunoglobulin compositions were subjected to anionic exchange chromatography on POROS 50 HQ as described in Example 1a, and the resulting flow-through fraction (IgG-enriched fraction) was subjected to ultra-/diafiltration versus 0.3 M glycine buffer, pH 4.6.

(97) The resulting IgG solutions were analysed for the protein and properdin content, and the results are shown in Table 12.

(98) TABLE-US-00013 TABLE 12 Properdin content per mg Immuno- Properdin Immuno- globulin conc. globulin ACA Sample conc. [μg/ml] [μg/mg] [CH50/mg] Cohn fraction 97 0.27 0.003 0.77 I/II/III resuspended in WFI Cohn fraction 128 214 1.67 1.18 I/II/III resuspended in 100 mM sodium acetate buffer, pH 4.8

Example 17

(99) Effect of WFI and Acetate Buffer on Suspensions of Cohn Fraction I/II/III

(100) Fraction I/II/III was resuspended at laboratory scale either in WFI (Sample A) or in 100 mM sodium acetate buffer (pH 4.8; Sample B) at a weight ratio of fraction I/II/II to buffer of 1:4 (300 g of fraction I/II/III plus 1200 g of buffer or WFI). The concentrations of IgG, IgA and IgM in the suspension as well as the distribution between the immunoglobulin classes were determined. The results are shown in Table 13.

(101) TABLE-US-00014 TABLE 13 Effect of buffer on resolubilization of immunoglobulin from Cohn fraction I/II/II Sum IgG IgA IgM IgG, IgA, IgM IgG IgA IgM Sample [g/l] [g/l] [g/l] [g/l] [%] [%] [%] A 18.0 1.9 0.69 20.6 87.3 9.3 3.4 B 19.1 2.9 2.05 24.0 79.2 12.2 8.5

(102) The concentrations of IgG, IgA and IgM in the sample resuspended in acetate buffer (sample B) increase compared to the sample resuspended in WFI (sample A). The IgM concentration raises from 0.69 g/l to 2.05 g/l; the IgA concentration is elevated from 1.9 g/l to 2.9 g/l in the suspension samples.

(103) The yields for the individual immunoglobulin classes were calculated, based on the suspension volume achieved (1500 ml) and in respect to the amount of fraction I/II/III employed. An increase in yield of 7% for IgG, 51% for IgA and 212% for IgM was observed for the suspension in acetate. The results are shown in Table 14.

(104) TABLE-US-00015 TABLE 14 Effect of buffer on immunoglobulin yields in suspension IgG per kg IgA per kg IgM per kg fraction fraction fraction IgG IgA IgM I/II/III I/II/III I/II/III Sample [g] [g] [g] [g/kg] [g/kg] [g/kg] A 27.0 2.9 1.0 90 9.7 3.3 B 28.7 4.4 3.1 96 14.7 10.3