METHOD OF PURIFICATION OF RECOMBINANTLY-PRODUCED RSV PROTEINS IN TRIMERIC FORM

Abstract

The invention relates to a purification method of a recombinantly-produced RSV F protein in trimeric form. According to the invention, the method sequentially comprises an anion exchange chromatography step, a cHA chromatography step and a HIC step. The invention is also directed to a pharmaceutical product including an RSV F protein purified by such a method.

Claims

1. Method of purification of a recombinantly-produced RSV F protein in trimeric form, sequentially comprising (a) an anion exchange chromatography step, (b) a cHA chromatography step and (c) a HIC step.

2. Method according to claim 1, wherein the anion exchange chromatography step is run in a bind and elute mode and comprises (a.1) contacting a load solution comprising the RSV F protein with an anion exchange chromatography medium, whereby the RSV F protein binds to the anion exchange chromatography medium; (a.2) washing the anion exchange chromatography medium with at least one lower pH wash solution at a pH between 3.0 and 6.5; and (a.3) eluting the RSV F protein from the anion exchange chromatography medium, thereby obtaining an anion exchange elution pool.

3. Method according to claim 2, wherein the pH of the load solution is between 7.0 and 8.5, preferably at about 7.5.

4. Method according to claim 2 or 3, wherein the pH of said lower pH wash solution is between 4.0 and 6.0, preferably between 4.5 and 5.5, preferably at about 5.0.

5. Method according to any one of claims 2 to 4, wherein said lower pH wash solution comprises acetate at a concentration between 56 and 84 mM, preferably between 63 and 77 mM, preferably at about 70 mM.

6. Method according to any one of claims 2 to 5, wherein prior to washing the anion exchange chromatography medium with the lower pH wash solution, the anion exchange chromatography medium is washed with at least a first higher pH wash solution at a pH between 7.0 and 8.0, preferably at about 7.5.

7. Method according to claim 6, wherein said first higher pH wash solution comprises Tris at a concentration between 18 and 22 mM, preferably at about 20 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, preferably at about 50 mM.

9. Method according to any one of claims 6 to 8, wherein after washing the anion exchange chromatography medium with the lower pH wash solution and prior to eluting the RSV F 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, preferably at about 7.5.

10. Method according to claim 9, wherein said second higher pH wash solution comprises Tris at a concentration between 45 and 55 mM, preferably at about 50 mM.

11. Method according to claim 9 or 10, wherein said second higher pH wash solution comprises NaCl at a concentration between 18 and 22 mM, preferably at about 20 mM.

12. Method according to any one of claims 2 to 11, wherein the RSV F protein is eluted with an elution solution having a pH between 7.0 and 8.0, preferably at about 7.5.

13. Method according to claim 12, wherein said elution solution comprises NaCl at a concentration between 146 and 180 mM, preferably at about 163 mM.

14. Method according to claim 12 or 13, wherein said elution solution comprises Tris at a concentration between 18 and 22 mM, preferably at about 20 mM.

15. Method according to any one of claims 2 to 14, wherein the cHA chromatography step is run in a flow-through mode and comprises (b.1) adding phosphate to the anion exchange elution pool, thereby obtaining a conditioned cHA load solution; (b.2) contacting said conditioned cHA load solution, comprising the RSV F protein and impurities, with a cHA medium, whereby impurities bind to the medium while the RSV F protein flows through the medium; (b.3) washing the cHA chromatography medium with a cHA wash solution at a pH between 6.0 and 8.0, preferably at about 7.0; and (b.4) collecting the RSV F protein in a flow-through pool.

16. Method according to claim 15, wherein the cHA wash solution comprises Tris at a concentration of about 20 mM, NaCl at a concentration of about 100 mM and sodium phosphate at a concentration of about 13 mM.

17. Method according to claim 15 or 16, wherein the HIC step is run in a bind and elute mode and comprises (c.1) adding phosphate to the flow-through pool from the cHA chromatography step, thereby obtaining a conditioned HIC load solution; (c.2) contacting said conditioned HIC load solution, comprising the RSV F protein, with a HIC medium, whereby the RSV F protein binds to the HIC medium; (c.3) washing the HIC medium with a HIC wash solution at a pH between 6.0 and 8.0, preferably at about 7.0; and (c.4) eluting the RSV F protein from the HIC medium with a HIC elution solution at a pH between 6.0 and 8.0, preferably at about 7.0.

18. Method according to claim 17, wherein the HIC wash solution comprises potassium phosphate at a concentration of about 1.1 M.

19. Method according to claim 17 or 18, wherein the HIC elution solution comprises potassium phosphate at a concentration of about 448 mM.

20. Method according to any one of claims 1 to 19, wherein the RSV F protein is a protein from RSV subgroup A.

21. Method according to any one of claims 1 to 19, wherein the RSV F protein is a protein from RSV subgroup B.

22. Method according to claim 20 or 21, wherein the RSV F protein is in a prefusion conformation.

23. Method according to claim 22, wherein the RSV F protein is a mutant of a wild-type F protein for any RSV subgroup that contains one or more introduced mutations.

24. Method according to claim 23, wherein the RSV F mutant is stabilized in prefusion conformation.

25. Method according to claim 23 or 24, wherein the RSV F mutant specifically binds to antibody D25 or AM14.

26. Method according to any one of claims 1 to 25, wherein the RSV F protein is formulated for use as an injectable pharmaceutical product.

27. Pharmaceutical product including an RSV F protein purified by a method according to claim 26.

28. Pharmaceutical product according to claim 27, wherein the RSV protein is a protein from RSV subgroup A.

29. Pharmaceutical product according to claim 27, wherein the RSV protein is a protein from RSV subgroup B.

30. Pharmaceutical product according to any one of claims 27 to 29, wherein the RSV protein is an RSV F protein.

31. Pharmaceutical product according to claim 30, wherein the RSV F protein is in a prefusion conformation.

32. Pharmaceutical product according to claim 31, wherein the RSV F protein is a mutant of a wild-type F protein for any RSV subgroup that contains one or more introduced mutations.

33. Pharmaceutical product according to claim 32, wherein the RSV F mutant is stabilized in prefusion conformation.

34. Pharmaceutical product according to claim 32 or 33, wherein the RSV F mutant specifically binds to antibody D25 or AM-14.

35. Pharmaceutical product according to any one of claims 27 to 34, wherein the RSV protein is formulated for use as an injectable pharmaceutical product.

Description

[0171] 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.

[0172] It will be observed on FIG. 1, for RSV A, that [0173] (i) an increased buffer strength leads to lower yields due to product losses during low pH wash; [0174] (ii) acetate and citrate show correlation of yield with wash pH; [0175] (iii) phosphate and sulfate show higher yields at pH 3.5 and are less sensitive to pH.

[0176] Data presented on FIG. 2, for RSV B, suggest that [0177] (i) a similar trend as for RSV A is observed with acetate i.e. lower pH and increased strength leading to lower yields; [0178] (ii) yield is not as sensitive to high buffer strength as for RSV A; [0179] (iii) lower recoveries are observed for RSVB as compared to RSV A.

[0180] 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.

[0181] The data generated have been collated in Table 3 below.

TABLE-US-00003 TABLE 3 Current process condition in Expected Yields HPC removal HTS screen: based on Concentration observed in logs of 70 Mm Acetate historical Buffer Type Range (mM) pH 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% HCP 20-50 4.5 51-59 0.8 HCP Removal: Removal: Phosphate 20-50 3.5 65-80 0.6-0.7 ~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: Removal: 110-150 5 44-45 0.8-0.9 ~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

[0182] In Table 3, the data obtained for the preferred wash solution (70mM Acetate, pH 5.0) with a high throughput screening (HTS) methodas shown in the penultimate columnhave been normalized based on historical data and show [0183] for RSV A: a yield of 70% and a log reduction value (LRV) of HCP between 0.9 and 1.1; and [0184] for RSV B: a yield of 65% and an LRV between 0.9 and 1.4.

[0185] 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

[0186] 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.

[0187] Finally, with a load solution having a pH between 7.0 and 8.5, and more specifically at 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 20 and pH 5.0.

[0188] 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.

[0189] With these operating conditions, phosphate and sulfate are robust options as alternatives to acetate.

[0190] 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.

[0191] 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: [0192] Wash #1: 20 mM Tris, 50 mM NaCl, pH 7.5; [0193] Wash #2: 70 mM Acetate, pH 5.0; [0194] Wash #3: 50 mM Tris, 20 mM NaCl, pH 7.5.

[0195] 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: [0196] 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; [0197] 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; [0198] 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.

[0199] 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 at about 163 mM, and Tris at a concentration between 18 and 22 mM, preferably at about 20 mM.

[0200] The pH of the elution solution is between 7.0 and 8.0, and is preferably at about 7.5.

[0201] The subsequent chromatography steps of the Example are preferably operated in the following conditions.

cHA Chromatography

[0202] 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.

[0203] The product pool collected from the AEX chromatography column (i.e. the AEx elution pool) and adjusted with phosphate addition, after filtration, is loaded into the cHA chromatography column. Conditioning the cHA load solution by adding phosphate prior to loading into the column aims to prevent the product from binding to the column. The pH of the load is set at a value of 7.10.3 and the load challenge comprises between 8.0 and 12.0 mg per ml of medium.

[0204] The column is washed with a wash solution comprising: 20 mM Tris,100 mM NaCl, 13 mM sodium phosphate at pH 7.0.

[0205] The column is operated in a flow-through mode, meaning that, as the load fluid is 15 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.

HIC

[0206] 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.

[0207] The product pool collected from the cHA chromatography column (i.e. the flow-through pool from the cHA chromatography step) and adjusted with potassium phosphate addition, after filtration, is loaded into the HIC column. Conditioning the HIC load solution by adding phosphate prior to loading into the column aims to ensure that the product binds to the column. The pH and the conductivity of the load are adjusted to 30 respectively 7.00.3 and 10410 mS/cm. The load challenge comprises between 8.0 and 12.0 mg per ml of medium.

[0208] 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.

[0209] Before applying the elution buffer, the column is washed with a wash solution in order to wash out impurities bound to the medium.

[0210] 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.

[0211] 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.