High salt washes during cation exchange chromatography to remove product-realated impurities

Abstract

The invention relates to methods for removal of low isoelectric point product-related impurities during cation exchange purification operations.

Description

It was found that when a high salt wash step was added, (Wash 2), the impurities were removed from the CEX resin before elution. These impurities likely corresponded to mispaired LC1 species given that the LC1 to LC2 ratio on the collected wash 2 and wash 3 was 8.0, as compared to the expected ratio of 1 when LC1 and LC2 correctly assemble and pair. Figure 7 shows that the high salt wash resulted in a reduction in the number of impurity peaks in the elution profile, from two peaks to a single peak with a small shoulder that still contained mispaired species (LC1 / LC2 = 2 to 4). The majority of the impurities were removed during the second and third wash steps. The third wash step also reestablished the UV baseline to zero before the start of the elution, tightening the elution profile, resulting in a much more efficient collection and better quality of the main product. This optimized procedure with a higher salt wash, combined with a lower load level and shallower gradient, is suitable for use on the manufacturing floor. CEX purification yield increased from 44% to 66%, with an elution profile for collecting a purified pool with low levels of mispaired LC1 species (as evidence by the LC1 to LC2 ratio close to 1), HMW and LMW, and an absorbance-based pooling criteria. Example 8 High Salt Wash Bi-specific #4 Neutralized low pH viral inactivated Protein A eluate pool containing a fully human engineered immunoglobulin bi-specific antibody (Bi-specific #4), was loaded onto a Capto-SP ImpREs® cation exchange chromatography resin under the conditions described in Table 7. Table 7 Conditions for cation exchange chromatography under high salt wash conditions Bi-specific #4 Column rate 20cm, 180 cm / h linear velocity Concentration g / L 35 Pre Equilibration 200 mM Acetate 1.0 M Sodium Chloride pH 5.0 Equilibration 100 mM Acetate pH 5.0 Wash 1 100 mM Acetate pH 5.0 Wash 2 100 mM Acetate 125 mM Sodium Chloride pH 5.0 Elution Buffer A 100 mM Acetate 125 mM Sodium Chloride pH 5.0 Elution Buffer B 100 mM Acetate 500 mM Sodium Chloride pH 5.0 Elution Gradient Start, %B 0 Elution Gradient End, %B 100 Elution Gradient Length, CV 10 Elution Salt Gradient, mM / CV 37.5 Average Pool Column Volumes (CV) 2.5 2-3 (second run) Yield 66% 77.7% (second run) Impurities in the recovered eluate SE-HPLC HMW = 1.3% (second run 1.2) SE HPLC LMW = 0.6 (second run 0.5) % Main Peak = 98.1% (second run 98.3) % Post Peak = 0.0 for both runs % Pre Peaks = 0.0 for both runs It was found that when a wash step at a high salt concentration was added (Wash 2), the low pl product-related impurities (homodimers and aggregated species) were removed from the cation exchange medium prior to elution. Figure 8 shows that the high salt wash resulted in a reduction in the number of 5 impurity peaks in the elution profile to a single peak. A first wash without sodium chloride brought the conductivity to base line. Low pl product-related impurities were removed during the following high salt wash step and returned the conductivity baseline to zero prior to elution. The conductivity was maintained with Elution Buffer A, which had the same high salt formulation as the high salt wash buffer. This allowed for maintaining stable conductivity before elution started and tightening the elution profile, resulting in a 10 much more robust and efficient collection and better quality of the main product, since the separation was based on pl. In addition, 0.1 pH units above and below pH 5.0 of the wash buffers (pH 4.9 - 5.1) were tested and found to be equally effective for the removal of low pl product-related impurities. In addition, load concentration from 25 to 40 g / L-r of Bi-specific #4 were tested and found to have 15 similar impurity clearance as the 35 g / L-r condition described above.

Claims

1. A method for purifying a bispecific antibody comprisingloading a sample comprising a bispecific antibody onto a cation exchange chromatography medium;washing the cation exchange chromatography medium with at least one wash buffer of pH 4.9 to 5.1 comprising acetate and 100 - 147 mM sodium chloride; andeluting the bispecific antibody from the cation exchange chromatography medium.

2. The method according to claim 1, wherein at least one wash buffer comprises 100 - 125 mM, 100 - 105 mM, 105 - 147 mM, 105 - 125 mM or 125 - 147 mM sodium chloride.

3. The method according to claim 1, wherein at least one wash buffer comprises 100 mM acetate.

4. The method according to claim 1, wherein the cation exchange chromatography medium is washed with at least two wash buffers.

5. The method according to claim 1, wherein the cation exchangechromatography medium is washed with at least three wash buffers.

6. The method according to claim 4, wherein the cation exchangechromatography medium is washed with at least one of the wash buffers comprising 0-70 mM sodium chloride.

7. The method according to claim 1, wherein the cation exchange chromatography medium is washed with at least two wash buffers, at least one wash buffer comprising acetate, 0 mM sodium chloride, followed by a wash buffer comprising acetate, 100-147 mM sodium chloride.

8. The method of claim 7, wherein the cation exchange chromatography medium is washed with a wash buffer comprising acetate, 0 mM sodium chloride, followed by a wash buffer selected from the group consisting of a wash buffer comprising acetate, 100 mM sodium chloride, a wash buffer comprising acetate, 105 mM sodium chloride, or a wash buffer comprising acetate, 125 mM sodium chloride;2020377919   12 Aug 20269. The method of claim 7, wherein the cation exchange chromatography medium is washed with a wash buffer comprising acetate, 100-147 mM sodium chloride, followed by a wash buffer comprising acetate, 0-70 mM sodium chloride.

10. The method of claim 9, wherein the cation exchange chromatography medium is washed with a wash buffer comprising acetate, 100-147 mM sodium chloride, followed by a wash buffer comprising acetate, 0 mM or 70 mM sodium chloride.

11. The method according to claim 5, wherein the cation exchange chromatography medium is washed witha first wash buffer comprising acetate, 0 mM sodium chloride, followed bya second wash buffer comprising acetate, 100-147 mM sodium chloride, followed bya third wash buffer comprising acetate, 0 mM sodium chloride or a wash buffer comprising acetate, 70 mM sodium chloride.

12. The method of claim 11, wherein the cation exchange chromatography medium is washed witha first wash buffer comprising acetate, 0 mM sodium chloride, followed bya second wash buffer selected from the group consisting of a wash buffer comprising acetate, 100 mM sodium chloride, a wash buffer comprising acetate, 105 mM sodium chloride, or a wash buffer comprising acetate, 125 mM sodium chloride, followed bya third a wash buffer comprising acetate, 0 mM sodium chloride.

13. The method of claim 11, wherein the cation exchange chromatography medium is washed witha first wash buffer comprising acetate, 0 mM sodium chloride, followed bya second wash buffer comprising acetate, 147 mM sodium chloride, followed bya third wash buffer comprising acetate, 70 mM sodium chloride.

14. The method according to claim 1, wherein the cation exchange chromatography medium is washed with 2.5 mM / CV of a wash buffer comprising 147 mM sodium chloride.

15. The method according to claim 1, wherein the bispecific antibody is eluted from the cation exchange chromatography medium by a salt gradient.2020377919   12 Aug 202616. The method according to claim 15, wherein(i) the gradient is a linear or step gradient; or(ii) at least one of the buffers used to form the elution gradient comprises 0-1M sodium chloride, optionally 70-500 mM sodium chloride, optionally 125 mM sodium chloride.

17. The method according to claim 1, wherein at least one wash buffer and one elution buffer comprise 125 mM sodium chloride.

18. The method according to claim 1, wherein the cation exchange chromatography medium is loaded with at least 10 g / L, 10 g / L to 40 g / L,15 g / L to 30 g / L, or 25 g / L - 40 g / L of the bispecific antibody.

19. The method according to claim 1, wherein the cation exchange chromatography medium is loaded with:(i) 10 g / L of the bispecific antibody, washed with a wash buffer comprising 105 mM sodium chloride and eluted in a salt gradient at 8 mM / CV;(ii) 15g / L to 30 g / L of the bispecific antibody, washed with a wash buffer comprising 147 mM sodium chloride; or(iii) 25g / L to 40 g / L of the bispecific antibody, wherein at least one wash buffer and one elution buffer comprise 125 mM sodium chloride.

20. The method according to any one of claims 1 to 11, wherein at least one product-related impurity is a homodimer, high molecular weight species, half antibody, aggregate, low molecular weight species, antibody fragment, or a light chain mis-assembly.

21. The method according to any one of claims 1 to 16, further comprising, before and / or after the cation exchange chromatography step, one or more unit operations for purifying the bispecific antibody, comprising affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography column, and / or mixed-mode chromatography column.

22. The method according to any one of claims 1 to 17, wherein the cation exchange chromatography medium is a resin.

23. The method according to claim 1, wherein the cation exchange chromatography eluate has reduced low pI product-related impurities compared to the2020377919   12 Aug 2026cation exchange chromatography eluate recovered in a corresponding method comprising loading a composition comprising the bispecific antibody onto the cation exchange chromatography medium, washing the cation exchange chromatography medium, and eluting the bispecific antibody from the cation exchange chromatography medium in which no sodium chloride is included in a wash buffer formulation.

24. The method of claim 1, wherein the method comprisesestablishing a cell culture in a bioreactor with a host cell expressing the bispecific antibody;culturing the host cells to express the bispecific antibody;harvesting the bispecific antibody from the cell culture;affinity purifying the bispecific antibody;wherein the composition loaded onto the cation exchange chromatography medium in claim 1 is the affinity purified bispecific antibody, andwherein the method further comprisesloading the cation exchange chromatography eluate eluted in claim 1 onto an additional chromatography medium in flow through mode.Amgen Inc.Patent Attorneys for the Applicant / Nominated Person SPRUSON & FERGUSON

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