Process for enriching IgA

Abstract

The invention relates to a process for enriching IgA (and IgM) from IgA-comprising material. In particular, it relates to a sequential elution process of an anion exchanger that leads to an advantageous enrichment and separation of monomeric and dimeric IgA.

Claims

1. A method for enriching IgA from an IgA-comprising composition, comprising the following steps: (a) loading the composition onto an anion exchanger under conditions that allow the IgA to bind; (b) applying an alkaline elution solution comprising a substance with at least 2 acid groups; (c) optionally applying an acidic elution solution that comprises a strong competitor for the anion exchanger, wherein protein eluted during step (b) is enriched for monomeric IgA by at least 50% and the substance with at least 2 acid groups is not phosphate.

2. The method of claim 1, wherein between steps (a) and (b), a pre-elution step (a1) is carried out by applying a low conductivity solution to the anion exchanger.

3. The method of claim 2, wherein step (a1) is performed at a weakly acidic to neutral pH.

4. The method of claim 1, wherein the substance in step (b) comprises a multivalent hydroxy-carboxylic acid with at least 2 carboxyl groups or salts thereof.

5. The method of claim 1, wherein the substance in step (b) comprises a dihydroxy-dicarboxylic acid and/or dicarboxylic acid or salt thereof, or a hydroxy-tricarboxylic acid or salt thereof.

6. The method of claim 1, wherein the substance in step (b) comprises tartaric acid/tartrate, oxalic acid/oxalate, malonic acid/malonate, or maleic acid/maleate.

7. The method of claim 1, wherein the substance in step (b) comprises tartaric acid/tartrate or oxalic acid/oxalate.

8. The method of claim 7, wherein the protein eluted in step (c) is enriched in dimeric IgA.

9. The method of claim 1, wherein the eluate collected during step (b) is essentially devoid of IgM.

10. The method of claim 1, wherein the strong competitor for the anion exchanger in step (c) comprises citrate, benzenesulfonic acid, benzoic acid or salts of hydrogen sulfates or mixtures thereof.

11. The method of claim 10, wherein the strong competitor for the anion exchanger is citrate.

12. The method of claim 1, wherein the anion exchanger is a strong anion exchanger or a weak anion exchanger.

13. The method of claim 12, wherein the anion exchanger comprises an anion exchange ligand.

14. The method of claim 13, wherein the anion exchange ligand comprises quaternary ammonium, quaternary aminoethyl, diethylaminoethyl, trimethylaminoethyl, or dimethylaminoethyl.

15. The method of claim 1, wherein the IgA-comprising composition is or is derived from blood, serum, plasma or other biological fluids.

16. The method of claim 15, wherein the IgA-comprising composition is a solution of an intermediate precipitate of plasma fractionation or a side fraction obtained during purification of IgG from plasma.

17. The method of claim 15, wherein the IgA-comprising composition is an intermediate in an IgG purification process, and wherein step (a) and (b) of the method of claim 1 are performed on an anion exchanger that is part of the IgG production process.

18. The method of claim 15, wherein the IgA-comprising composition is obtained by solubilising a precipitate.

19. The method of claim 18, wherein the precipitate is an octanoic acid precipitate obtained during IgG purification.

20. The method of claim 18, wherein the solubilisation step selectively brings IgA and IgG into solution.

21. The method of claim 19, wherein the solubilisation is carried out using a solution with a conductivity of between 1 and 15 mS/cm, and a pH of 3.5 to 6 or 7 to 9.5.

22. The method of claim 21, wherein the solubilisation is carried out with a phosphate buffer, an acetate buffer, a Tris buffer, and/or a combination of two or more of these buffers.

23. The method of claim 22, wherein the buffer is selected from 0.22 M acetate buffer or 0.15 M phosphate buffer, pH 4.8.

24. A method for enriching IgA from an IgA-comprising composition, comprising the following steps: (a) loading the IgA-comprising composition in a buffer with pH of 3 to 8 and conductivity of 5 to 50 mS/cm onto an anion exchange resin under conditions that allow the IgA to bind; (b) eluting IgA enriched by at least 75% by applying an alkaline elution solution at pH 7.4 to 7.8 comprising a dihydroxy-dicarboxylic acid and/or dicarboxylic acid or salt thereof and/or a hydroxy-tricarboxylic acid or salt thereof at a concentration of 20 to 80 mM.

25. The method of claim 24, wherein the IgA-comprising composition is a solution of an intermediate precipitate of plasma fractionation or a side fraction obtained during purification of IgG from plasma.

26. The method of claim 24, wherein the dihydroxy-dicarboxylic acid or salt thereof is tartaric acid/tartrate or the dicarboxylic acid is oxalic acid/oxalate.

Description

(1) The invention will now be illustrated by the following non-limiting examples, with reference to the following figures:

(2) FIG. 1: Flow diagram of the IgA enrichment process based on different intermediate precipitate of plasma fractionation or a side fraction obtained during purification of IgG from plasma. Two starting materials have been exemplified (Method 1 in example 1 and Method 2 in example 2).

(3) FIG. 2: HPLC analysis of the composition of the loaded material (feed stock) for the anion exchange chromatography, according to Method 1 shown in FIG. 1. The x-axis shows the elution time, the y-axis shows the absorbance at 280 nm (mAu milli-absorption unit).

(4) FIG. 3: Elution profile of the anion exchange column, showing the retention volume in milliliter on the x-axis, and the absorbance at 280 nm (mAu: milli-absorption unit) on the y-axis. The elution solutions are indicated above the profile, the vertical lines indicate the change of solution applied to the column.

(5) FIG. 4: HPLC analysis of the composition of fraction F4A, according to Method 1.

(6) FIG. 5: HPLC analysis of the composition of F4 after removal of IgG by affinity chromatography.

(7) FIG. 6: HPLC analysis of the composition of fraction F5 of IgG and IgM by affinity chromatography.

(8) FIG. 7: Elution profiles of the process of anion exchange column, using different buffers as elution buffer 1. The dashed vertical lines indicate the change of solution applied to the column. *The load for the acetate buffer elution was reduced.

(9) FIG. 8: Analysis of the content of IgA and IgM in the fractions obtained when carrying out elution 1 with different elution buffers relative to the total loaded protein.

EXAMPLES

(10) The following examples serve to illustrate the invention, but are not intended to limit the invention.

(11) In the following examples, materials that originate from the IgG purification process from plasma and/or plasma fractions are used as the starting material for the present invention. FIG. 1 shows a flow chart of the IgG purification process, and indicates examples of where in the process an IgA purification method according to the invention can be introduced.

(12) Briefly, the following steps are carried out to purify IgG from plasma: Plasma or cryo-poor plasma are subjected to cold ethanol fractionation, e.g. according to Cohn or Kistler-Nitschmann, and the immunoglobulin-containing fractions are further processed. An octanoic acid precipitation is carried out and the suspension containing most of the IgG is then subjected to filtration, virus inactivation, and anion exchange (AIEX) chromatography. Most of the IgG is collected in the AIEX flowthrough and further processed, including final formulation and packaging.

(13) As shown in FIG. 1, an IgA purification method according to the invention can be branched off from the cold ethanol fractionations, e.g. FI+II+III, the supernatant of FI, FII+III, precipitate A, or subfractions thereof. However, preferably, material solubilised from the octanoic acid precipitate (octanoic acid cake) is subjected to the methods of the invention, or the material bound to the anion exchange column is subjected to sequential elution according to the present invention to obtain plasma IgA. The latter materials are preferred as they are currently discarded. Carrying out the process for IgA purification from these materials does not interfere with the IgG process, so that the yield of IgG or other plasma proteins is not decreased or compromised in any way. Nevertheless, the invention is not limited to those starting materials.

Example 1

Sequential Elution of IgA from an Anion Exchange (AIEX) Column Using Tartrate Buffer as Elution Buffer 1

(14) The process for the purification of IgG from plasma was carried out essentially as shown in FIG. 1 and briefly described above. In the IgG purification process the AIEX column (MPHQ) is equilibrated with two buffers, using 0.78 M sodium acetate, pH 4.0±0.1, conductivity 8-10 mS/cm for the first 2 column volume (CV), followed by 8 CV 10 mM sodium acetate, pH 6.5±0.1, conductivity 0.8-1.2 mS/cm. FIG. 2 shows the AIEX feedstock composition, which is loaded on the AIEX column (about 180 g protein per L resin) and afterwards washed with 2 CV equilibration buffer 2. After this step the AIEX column was subjected to sequential elution.

(15) Therefore, a pre-elution step was carried out using 8 CV phosphate acetate buffer (10 mM phosphate+30 mM sodium acetate, pH 6.0, conductivity 2-7 mS/cm). As can be seen in FIG. 3, during the pre-elution step, fraction F3 is eluted, which contains mainly IgG.

(16) After this pre-elution step the buffer was changed to the first elution buffer (5-8 CV 55 mM tartrate, 5 mM sodium acetate, pH 7.6±0.2, conductivity 5-15 mS/cm). After switching to the tartrate buffer fraction F4 is collected, which contains IgG and IgA in a ratio of about 60:40, and is essentially devoid of IgM. IgA eluted in this fraction is mainly monomeric IgA. As can be seen in FIG. 3, several subfractions (peak fractions) can be collected in this step if desired. FIG. 4 shows the composition of the F4 subfraction F4A. However, all subfractions contain only IgG and monomeric IgA in various ratios.

(17) Thereafter, 6 CV 50 mM phosphate and 25 mM citrate (pH 5.0, conductivity 5-15 mS/cm) as second elution buffer was applied. After switching to the second elution buffer fraction F5 is eluted, which can be collected in two subfractions (peak fractions) (FIG. 3), enriched in dimeric IgA. Fraction F5 contains IgM, IgA and IgG in the ratio 20-30% IgA (predominantly di-/polymeric), 35-50% IgM and 20-35% IgG.

(18) Fractions F4 and F5 were polished by affinity chromatography (one or two steps) to selectively remove IgG and IgM. HPLC analysis showed that the obtained product from fraction F4 is highly pure monomeric IgA (FIG. 5), whereas fraction F5 is enriched in dimeric IgA (FIG. 6). Therefore, this elution method provides an excellent enrichment for monomeric IgA in fraction F4 and dimeric IgA in fraction F5.

(19) Other AIEX materials (Fractogel, HyperCel Star AX, Q HyperCel) were also tested and have given similar elution profiles.

Example 2

Testing Different Buffers for the First Elution Step

(20) Several different buffers were evaluated in the first elution step and compared to the tartrate buffer elution profile.

(21) The experiment was carried out essentially as described in Example 1, with a protein load between 100 to 180 g protein per L AIEX resin. However, for the first elution, tartrate buffer was replaced by oxalate, malonate, maleate, phosphate, acetate buffer (55 mM of each substance+5 mM sodium acetate, pH 7.6±0.2).

(22) The resulting elution profiles are shown in FIG. 7. Fraction F4 appears to be similar for all buffers as long as substances with at least 2 acid groups were used in the first elution, whereas the acetate and phosphate elution profiles look quite different. However, the first elution also affects the second elution with citrate buffer. It can be seen that oxalate buffer yields a similar F5 peak (FIG. 7). With malonate, maleate or phosphate buffers, the IgM is not eluted during the second elution. With acetate buffer as the first elution buffer, almost all IgA and the total amount of IgM is eluted in F5.

(23) The amount of IgA and IgM in these fractions relative to the total loaded protein is illustrated in FIG. 8. Tartrate and oxalate provide the preferred elution profile, where the monomeric IgA is eluted in F4, and IgM and dimeric IgA are eluted in F5. Only the elution with the hydroxydicarbon acids resulted in all 3 F4 subfractions and monomeric IgA elution. The first elution step using phosphate buffer showed only 2 subfractions and an extenuated IgA elution, whereas acetate buffer generated an F4 peak with a small amount of IgA. If the first elution is done with malonate, maleate or phosphate buffer a large peak is obtained in the strip fractions, where harsh elution conditions are used (1 M NaCl)

(24) The immunoglobulin content in the different fractions are also shown in Table 1:

(25) TABLE-US-00001 IgG* IgA** IgM** [mg] [mg] [mg] E1: Tartrate buffer Load 3250.8 139.51 47.76 pre E (F3) 137.2 3.79 0.04 E1 (F4) 117.6 90.27 0.87 E2 (F5) 37.32 26.29 49.42 STRIP 0 0.00 0.00 E1: Oxalate buffer Load 3322.7 114.78 42.60 pre E (F3) 150.2 10.55 0.09 E1 (F4) 128.8 90.38 1.72 E2 (F5) 35.77 14.94 44.14 STRIP 0 1.90 2.79 E1: Malonic acid buffer Load 3325.8 133.68 50.99 pre E (F3) 140.2 5.94 0.14 E1 (F4) 123.4 101.25 1.38 E2 (F5) 20.26 11.24 7.22 STRIP 16.13 16.30 45.70 E1: Maleic acid buffer Load 3331.5 132.98 44.98 pre E (F3) 141.2 4.95 0.06 E1 (F4) 119.7 112.80 1.23 E2 (F5) 11.43 7.17 4.75 STRIP 26.16 24.54 48.32 E1: Phosphate buffer Load 3412.2 132.19 46.98 pre E (F3) 145.15 5.69 0.05 E1 (F4) 109.1 73.61 0.34 E2 (F5B) 18.1 10.78 0.92 STRIP 34.4 42.09 45.80 E1: Acetate buffer* Load 1210 39.92 11.90 pre E (F3) 55.34 0.75 — E1 (F4) 54.68 3.03 0.02 E2 (F5) 58.55 32.81 13.64 STRIP — — — *Nephelometric analysis method, **ELISA analysis method

(26) Advantageously we found that the pH of such buffers, for example the tartrate buffer, can be stabilized by addition of Tris buffer pH 7.6±0.4, to a final concentration of 1 to 20 mM, preferably 2 to 15 mM, more preferably 5 to 10 mM.

Example 3

Solubilisation of IgA from the Octanoic Acid Precipitate

(27) As shown in FIG. 1, the octanoic acid precipitate (OA cake) is another waste fraction incurring during the IgG purification process. This precipitate was found to contain significant amounts of IgA and is therefore a good starting material for the purification of IgA.

(28) The OA cake was mixed with 0.15M phosphate buffer, pH 4.8 at a ratio of 1:6 (OA precipitate to buffer). However, other buffers can also be used for IgA extraction, e.g. Tris buffer. The solubilised material was subjected to filtration, and then loaded on an AIEX column (MPHQ). The column was then sequentially eluted essentially as described in Example 1. The elution profile looks essentially as already shown in FIG. 3.

(29) The composition of the IgA-comprising feedstock for the AIEX column varies in the ratio of IgG to IgA in comparison to the described example 1. As shown in Table 2, the ratio of IgG to IgA in fraction F4 has shifted in favor of IgA (15%:85%). Also the amount of IgA in the pre-elution fraction F3 is increased. Both fractions are devoid of IgM.

(30) The elution of dimeric/polymeric compounds is limited due to the composition of the start material.

(31) The immunoglobulin content in the different fractions is also shown in Table 2:

(32) TABLE-US-00002 IgG* IgA** IgM** [mg] [mg] [mg] Load 380.80 271.86 4.84 pre E (F3) 28.17 57.34 0.01 E1 (F4) 20.09 109.78 0.07 E2 (F5) 7.43 12.80 6.32 STRIP 0.30 1.08 0.03 *Nephelometric analysis method, **ELISA analysis method

(33) To show that this works with other IgA-comprising precipitates as well, we also tested other precipitates. IgA could also be solubilised from two different ethanol precipitates and subjected to AIEX chromatography, and very similar elution profiles were obtained with the method of the invention.

Example 4

Polishing of Monomeric IgA

(34) The sequential elution resulted in an enriched monomeric IgA fraction (F4) containing IgG as contaminant. A polishing step was carried out by a selective removal of IgG by affinity chromatography (IgSelect).

(35) The fraction F4 was collected in two subfractions F4A and F4B (FIG. 3). However, the obtained F4A fraction was subjected to ultrafiltration/diafiltration to concentrate the protein and transfer it to a common buffer system with neutral pH, preferably PBS (phosphate buffered saline). This protein solution was used as feedstock for the affinity chromatography. IgA does not bind to the affinity resin and was collected in the flow through fraction. Bound IgG was eluted with 0.1 M glycine buffer, pH 3.0. Finally the IgA product can be formulated into a stable, pharmaceutical composition. FIG. 5 shows the polished IgA fraction without IgG (and IgM).