IMPROVEMENTS TO WASH SOLUTIONS FOR ANION EXCHANGE CHROMATOGRAPHY IN A METHOD OF PURIFICATION OF RECOMBINANTLY-PRODUCED RSV PROTEINS

Abstract

The invention relates to a purification method of an RSV protein, wherein a load solution comprising the RSV protein is contacted with an anion exchange chromatography medium, whereby the RSV protein binds to the anion exchange chromatography medium, the anion exchange chromatography medium is washed with at least one wash solution and the RSV protein is eluted from the anion exchange chromatography medium.

Claims

1. Method of purification of a recombinantly-produced RSV protein, comprising an anion exchange chromatography step wherein a) a load solution obtained from a harvested cell culture fluid and comprising the RSV protein is contacted with an anion exchange chromatography medium, whereby the RSV protein binds to the anion exchange chromatography medium; b) the anion exchange chromatography medium is washed with at least one lower pH wash solution at a pH between 3.0 and 6.5, whereby the removal of host cell proteins is enhanced; and c) the RSV protein is eluted from the anion exchange chromatography medium.

2. Method according to claim 1, wherein the pH of the load solution is between 7.0 and 8.5.

3. Method according to claim 1 or 2, wherein the pH of said lower pH wash solution is between 4.0 and 6.0.

4. Method according to claim 1, wherein said lower pH wash solution comprises acetate.

5. Method according to claim 4, wherein the concentration of acetate in said lower pH wash solution is between 56 and 84 mM.

6. Method according to claim 1, wherein prior to eluting the RSV protein, the anion exchange chromatography medium is further washed with at least a first higher pH wash solution at a pH between 7.0 and 8.0.

7. Method according to claim 6, wherein said first higher pH wash solution comprises Tris at a concentration between 18 and 22 mM.

8. Method according to claim 6 or 7, wherein said first higher pH wash solution comprises NaCl at a concentration between 45 and 55 mM.

9. Method according to claim 6, wherein the wash step using said first higher pH wash solution is performed prior to the wash step using said lower pH wash solution.

10. Method according to claim 6, wherein prior to the elution of the RSV protein, the anion exchange chromatography medium is further washed with at least a second higher pH wash solution at a pH between 7.0 and 8.0.

11. Method according to claim 10, wherein said second higher pH wash solution comprises Tris at a concentration between 45 and 55 mM.

12. Method according to claim 10 or 11, wherein said second higher pH wash solution comprises NaCl at a concentration between 18 and 22 mM.

13. Method according to claim 10, wherein the wash step using said second higher pH wash solution is performed after the wash step using said lower pH wash solution.

14. Method according to claim 1, wherein the RSV protein is eluted with an elution solution having a pH between 7.0 and 8.0.

15. Method according to claim 14, wherein said elution solution comprises NaCl at a concentration between 146 and 180 mM.

16. Method according to claim 14, wherein said elution solution comprises Tris at a concentration between 18 and 22 mM.

17. Method according to claim 1, wherein the load challenge is between 7.5 and 15.0 mg per ml of the anion exchange chromatography medium.

18. Method according to claim 1, further comprising a cHA chromatography step.

19. Method according to claim 1, further comprising a HIC chromatography step.

20-28. (canceled)

29. Pharmaceutical product including an RSV protein purified by a method according to claim 1.

30-37. (canceled)

Description

[0147] FIG. 1 and FIG. 2 plot the yield values (percentage of product recovery) obtained for each wash solution, against the pH value, respectively for an RSV A and an RSV B load solution.

[0148] It will be observed on FIG. 1, for RSV A, that [0149] (i) an increased buffer strength leads to lower yields due to product losses during low pH wash; [0150] (ii) acetate and citrate show correlation of yield with wash pH; [0151] (iii) phosphate and sulfate show higher yields at pH 3.5 and are less sensitive to pH.

[0152] Data presented on FIG. 2, for RSV B, suggest that [0153] (i) a similar trend as for RSV A is observed with acetate i.e. lower pH and increased strength leading to lower yields; [0154] (ii) yield is not as sensitive to high buffer strength as for RSV A; [0155] (iii) lower recoveries are observed for RSVB as compared to RSV A.

[0156] In a further experiment, the performance of multiple wash conditions (buffer type, concentration, pH) for both RSV A and RSV B was evaluated in terms of HCP reduction and yield, and compared to the preferred wash solution: 70 mM Acetate, pH 5.0.

[0157] The data generated have been collated in Table 3 below.

TABLE-US-00005 TABLE 3 Current process condition in Expected Yields HPC removal HTS screen: based on Concentration pH observed in logs of 70 Mm Acetate historical Buffer Type Range (mM) Range screen (%) Removal (LRV) pH 5 process data RSVA [PF-06934186] Acetate  20-110 5 51-83 0.7-0.8 Yield: 51% Yield: 70% 20-50 4.5 51-59 0.8 HCP HCP Removal: Phosphate 20-50 3.5 65-80 0.6-0.7 Removal: ~0.8LRVs 0.9-1.1 LRVs  20-110 5 53-90 0.6-0.9 Sulfate 20-30 3.5-5 65-74 0.5-0.6 RSVB [PF-06937100] Citrate 20 4.5-5 42-50 0.8-1.0 Yield: 38% Yield: 65% Acetate 20 4.5 58 0.8 HCP Removal: 110-150 5 44-45 0.8-0.9 Removal: ~0.8LRVs 0.9-1.4 LRVs Phosphate 20 3.5-5 50-53 0.6 50 3.5-5 45-48 0.7 Sulfate 20-30 3.5-5 47-75 0.5

[0158] In Table 3, the data obtained for the preferred wash solution (70 mM Acetate, pH 5.0) with a high throughput screening (HTS) method—as shown in the penultimate column—have been normalized based on historical data and show: [0159] for RSV A: a yield of 70% and a log reduction value (LRV) of HCP between 0.9 and 1.1; and [0160] for RSV B: a yield of 65% and an LRV between 0.9 and 1.4.

[0161] The conducted wash screens suggest that increased buffer strengths result in yield losses during wash and decreased wash pH result in better HCP removal. RSV A and RSV B showed similar trends with yield and HCP, with RSV B showing lower yields. Different buffers showed a range of effectiveness between HCP removal and yield, in particular phosphate and sulfate which are robust options as alternatives to acetate based on normalized data in Table 3.

[0162] Finally, with a load solution having a pH between 7.0 and 8.5, and more specifically of about 7.5, and a load challenge comprising between 7.5 and 15.0 mg per ml of the anion exchange chromatography medium, the preferred low pH wash conditions for the AEX chromatography column applicable to both RSV A and RSV B is: 70 mM Acetate and pH 5.0.

[0163] Based on the aforementioned experiments, an acceptable range of pH for the low pH wash solution may be between 3.0 and 6.5, more preferably between 4.0 and 6.0, and most preferably between 4.5 and 5.5.

[0164] With these operating conditions, phosphate and sulfate are robust options as alternatives to acetate.

[0165] In the actual method, prior to loading the load solution including the target protein (RSV A or RSV B) into the AEX column, the column is equilibrated with an equilibration solution: 20 mM Tris, 50 mM NaCl pH 7.5.

[0166] After loading, the column is successively washed with three wash solutions, the second one being the lower pH wash solution, the first and third ones being the higher pH wash solutions: [0167] Wash #1: 20 mM Tris, 50 mM NaCl, pH 7.5; [0168] Wash #2: 70 mM Acetate, pH 5.0; [0169] Wash #3: 50 mM Tris, 20 mM NaCl, pH 7.5.

[0170] The aforementioned pH values and compositions for the wash solutions are those preferred, however acceptable performances in terms of HCP reduction and yield may also be obtained under the following conditions: [0171] the first higher pH wash solution (Wash #1) may have a pH between 7.0 and 8.0. Tris concentration may be between 18 and 22 mM and NaCl concentration may be between 45 and 55 mM; [0172] the concentration of acetate in the lower pH wash solution (Wash #2) may be between 56 and 84 mM, more preferably between 63 and 77 mM. The acceptable ranges of pH, as discussed above, are 3.0-6.5, preferably 4.0-6.0, and more preferably 4.5-5.5; [0173] the second higher pH wash solution (Wash #3) may have a pH between 7.0 and 8.0. Tris concentration may be between 45 and 55 mM and NaCl concentration may be between 18 and 22 mM.

[0174] After the washing step performed by washing the column successively with the three wash solutions, the RSV protein is eluted with an elution solution. The elution solution comprises NaCl at a concentration between 146 and 180 mM, preferably of about 163 mM, and Tris at a concentration between 18 and 22 mM, preferably of about 20 mM. The pH of the elution solution is between 7.0 and 8.0, and is preferably of about 7.5.

[0175] The subsequent chromatography steps of the Example are preferably operated in the following conditions.

[0176] cHA Chromatography

[0177] Prior to loading the product into the cHA chromatography column, the column is equilibrated with a first equilibration buffer 0.5 M sodium phosphate, pH 7.2 and then with a second equilibration buffer 20 mM Tris, 100 mM NaCl, 13 mM sodium phosphate, pH 7.0.

[0178] The product pool collected from the AEX chromatography column and adjusted with phosphate addition, after filtration, is loaded into the cHA chromatography column. The pH of the load is set at a value of 7.1±0.3 and the load challenge is comprised between 8.0 and 12.0 mg per ml of medium.

[0179] The column is washed with a wash solution comprising: 20 mM Tris, 100 mM NaCl, 13 mM sodium phosphate, pH 7.0.

[0180] The column is operated in a flow-through mode, meaning that, as the load fluid is loaded into the column, the target protein flows through the column while the impurities bind to the medium. The wash is intended to wash the unintentionally bound target proteins out of the column.

[0181] HIC

[0182] Prior to loading the product into the HIC column, the column is equilibrated with a first equilibration buffer comprising 20 mM potassium phosphate at pH 7.0, and then with a second equilibration buffer comprising 1.1 M potassium phosphate at pH 7.0.

[0183] The product pool collected from the cHA chromatography column and adjusted with potassium phosphate addition, after filtration, is loaded into the HIC column. The pH and the conductivity of the load are adjusted to respectively 7.0±0.3 and 104±10 mS/cm. The load challenge comprises between 8.0 and 12.0 mg per ml of medium.

[0184] The column is operated in a bind and elute mode, whereby the target proteins loaded into the column bind to the medium and then are eluted by applying an elution buffer. Before applying the elution buffer, the column is washed with a wash solution in order to wash out impurities bound to the medium.

[0185] The wash solution used in this HIC step is 1.1 M potassium phosphate, pH 7.0 and the elution buffer is 448 mM potassium phosphate, pH 7.0.

[0186] The above-described method is suitable for purifying recombinantly-produced RSV proteins with a sufficient degree of purity, such that said proteins may be used for the preparation of pharmaceutical products. In particular, such purified RSV proteins may be formulated, by addition of suitable excipients, for use as an injectable pharmaceutical product.