Clearance of aggregates from UF / DF pools in downstream antibody purification

Abstract

A method for improving the harvesting or purification of a target protein such as a biologic or biosimilar is provided. The method improves conventional harvesting / purification methodologies by adding a chromatography step, such as a mixed mode or ion exchange chromatography step, toward the end of the polishing phase of harvesting / purification after subjecting the resulting eluate from the completion of a conventional chromatography polishing step, such as a Protein A or ion exchange chromatography step, to filtration, such as ultrafiltration / diafiltration. The surprising result of returning to chromatography polishing after filtration is that all forms of high molecular weight product are pulled down, facilitating purification of the target protein sufficient to meet government regulations such as Quality Target Protein Profiles.

Description

Technical Field

[0001] This application claims the priority of U.S. Provisional Patent Application No. 63 / 353,776, filed on June 20, 2022, which is hereby incorporated by reference in its entirety.

[0002] The present disclosure generally relates to the field of protein harvesting / purification, and more particularly to the field of harvesting / purifying therapeutic valuable biological protein products and biosimilar protein products.

Background Art

[0003] The continued development of biologics and biosimilars has revealed great promise for these molecules to provide therapeutic benefits to humans. These molecules are typically large proteins produced using cell-based expression of the molecule in culture. Also, as proteins, these molecules often require post-translational modifications and proper folding to maximize functionality. Such considerations often lead to the use of eukaryotic host cells for expressing the desired product, although recent advances in prokaryotic expression systems hint at the continued viability of this avenue of protein expression. In addition, the development of cell culture technology has led to a significant increase in the cell density of the cultures producing proteins such as biologics and biosimilars, which shows promise for increasing the yield of such products, but at the expense of an increase in the level of contaminants from cell culture operations. In particular, the use of unstable eukaryotic cells is associated with a certain level of cell lysis that results in the presence of unwanted biomolecules such as nucleic acids, proteins, lipids, etc. along with cell membrane fragments. Considering the potential increase in yield from the use of high-density cell culture, there is a need for harvesting or purification methods to address the increased presence of contaminants.

[0004] Conventional purification protocols for proteins expressed in cell culture require upstream cell culture harvest processing that focuses on various forms of centrifugation and / or various forms of depth filtration, along with an optional initial step of flocculation or precipitation to remove some of the contaminants in the harvest. The downstream polishing steps have typically included one or more chromatographic fractions, including, for example, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and other forms of chromatography. A typical affinity chromatography step included in polishing the purification of a target protein such as a biologic and biosimilar is protein A chromatography, which is well known for use in the purification of immunoglobulin-like proteins such as antibodies. Ion exchange chromatography often includes anion exchange, but can also include cation exchange. After a series of one or more chromatographic fractions in the downstream polishing step of the harvest / purification methodology, there is often a filtration step such as ultrafiltration, which may include diafiltration, which is a form of ultrafiltration with solvent replenishment. After filtration, the purified protein is typically formulated for use, such as administration to the desired subject.

[0005] The use of cell culture-based expression systems to produce therapeutic biologics and biosimilars has required these multi-step purification protocols to ensure sufficient purity of the product intended for human administration. To ensure sufficient purity, government regulatory agencies around the world have developed strict specifications that must be met, including the requirement to meet the stringent Quality Target Protein Profiles for the purification of products to be administered to humans in the United States. The need to obtain government approval to administer biologics and biosimilars has resulted in a continuing requirement for greater purity of the products expressed in culture. Therefore, improvements to protein harvest / purification methodologies to yield protein target products with reduced levels and types of culture-based contaminants are continuously needed in the art.

Summary of the Invention

Means for Solving the Problems

[0006] The present disclosure provides a methodology for improving the purity of target proteins such as biopharmaceuticals and biosimilars produced in cell culture. The present disclosure takes an approach that competes with the prior art in the downstream aspects of protein purification from cell culture. More specifically, in the downstream polishing phase of protein purification from cell culture bodies, the present disclosure relies on one or more chromatographic steps prior to the ultrafiltration / diafiltration (UF / DF) step that is consistent with conventional purification protocols, but subsequently takes an unusual step of returning to a chromatographic fractionation that includes at least a mixed-mode chromatography or ion exchange chromatography (e.g., cation exchange chromatography) step. The purification methodology of the present disclosure results in a significant reduction in the undesired high molecular weight (HMW) materials contaminating the purified target protein material, while minimizing the reduction in the yield of the target protein. By adding this unusual step late in the polishing phase of target harvest / purification, the method achieves an improvement in the removal of all forms of high molecular weight compounds, including nucleic acids, proteins, lipids, and other forms of high molecular weight compounds found in cell-based target protein production methods. The result is a target protein of greater purity that exhibits an improved profile compliant with the Quality Target Protein Profile.

[0007] In one aspect, the present disclosure provides a method for harvesting a target protein from a host cell culture supernatant that includes a chromatography step after an ultrafiltration / diafiltration (UF / DF) step, wherein the high molecular weight species in the eluate from the chromatography step are reduced by at least 10% compared to the level of high molecular weight species in the UF / DF filtrate. In some embodiments, the method further includes at least one chromatography step prior to the ultrafiltration / diafiltration step. In some embodiments, the at least one chromatography step includes protein A chromatography. In some embodiments, the at least one chromatography step further includes ion exchange chromatography, mixed mode chromatography, or both ion exchange chromatography and mixed mode chromatography. In some embodiments, the method further includes a downstream polishing step for purifying the target protein following an upstream bulk harvesting step of centrifuging, depth filtering, or both centrifuging and depth filtering the host cell culture supernatant, the polishing step including a protein A chromatography step, a low pH virus inactivation step, a cation exchange step, a mixed mode anion exchange chromatography step, a virus filtration step, an ultrafiltration / diafiltration step, a chromatography step after the ultrafiltration / diafiltration step, a polysorbate 80 addition step, and a final filtration step.

[0008] Further embodiments of the method are provided where the target protein subjected to the chromatography step is present in a formulation buffer containing 10 mM glutamic acid, 250 mM threonine, pH 5.5. In some embodiments, the product from the chromatography step is an eluate in which the high molecular weight compounds are reduced by at least 9% compared to the levels in the UF / DF filtrate. In some embodiments, the high molecular weight compounds in the eluate are reduced by at least 20%, at least 25%, at least 30%, or at least 75% compared to the levels in the UF / DF filtrate. In some embodiments, the product from the chromatography step is an eluate containing 0.3 - 1.9% high molecular weight compounds. In some embodiments, the yield of the target protein from the chromatography step is at least 58%, 60%, 65%, 70%, 75%, 80%, 86%, at least 90%, at least 95%, or at least 98%.

[0009] Some embodiments of the method are disclosed where the chromatography medium is a mixed-mode resin, a mixed-mode membrane, an ion-exchange resin, or an ion-exchange membrane. In some embodiments, the chromatography medium is Ca++Pure-HA, Capto MMC, Capto MMC ImpRes, Capto SP ImpRes, Capto Adhere, CIMultus PrimaS, CIMultus Hbond, CMM Hypercel, Eshmuno CP-FT, Eshmuno HCX, Fibro MMC, Fibro Adhere, Fractogel COO-(M), Fractogel SO3-(M), Mustang XT S, Nuvia S, Nuvia HR-S, Nuvia cPrime, Sartobind Phenyl, ToyoPearl MX-Trp, ToyoPearl Sulfate 650M, or UNOsphere S. In some embodiments, the chromatography medium is Ca++Pure HA, Capto MMC, Capto MMC ImpRes, Capto Adhere, CMM Hypercel, Eshmuno HCX, Fibro MMC, Fibro Adhere, Nuvia cPrime, ToyoPearl mX-Trp, or ToyoPearl Sulfate. In some embodiments, the chromatography medium is Capto MMC ImpRes, Eshmuno HCX, Eshmuno CP-FT, Fibro MMC, or Nuvia cPrime. In some embodiments, the chromatography medium is Fibro MMC or Nuvia cPrime. In some embodiments, the chromatography medium is a membrane. In some embodiments, the fluid containing the target protein is applied to the chromatography medium at a loading rate of at least 400 grams / resin liter, and embodiments where the loading rate is from 400 to 800 grams / resin liter and embodiments where the loading rate is at least 800 grams / resin liter are included.

[0010] The method also provides embodiments where the load HMW% is at least 0.5%. In some embodiments, the load HMW% is at least 0.9%, at least 1.2%, from 0.5 to 2.6%, or at least 2.6%. In some embodiments of the method, the chromatography step results in at least 5%, at least 8%, at least 24%, at least 30%, at least 33%, at least 40%, at least 45%, at least 55%, at least 65%, at least 70%, or at least 75% HMW clearance%.

[0011] The method also provides embodiments where a fluid containing a target protein is applied to a chromatography medium at a load concentration of 50 g / L or less. In some embodiments, the load concentration is 20 g / L or less. In some embodiments of the method, the chromatography medium contains a ligand at a ligand density of at least 98 mmol / mL of chromatography medium. In some embodiments, the ligand density is from 98 to 157 mmol / mL, from 98 to 140 mmol / mL, or from 127 to 157 mmol / mL. In some embodiments, the eluate from the chromatography step has a Quality Target Protein Profile of 0.3% or less and the eluate contains the target protein.

[0012] The present disclosure will be better understood in view of the following detailed description of the present disclosure, including consideration of the figures.

Brief Description of the Drawings

[0013]

Figure 1

Figure 2

Mode for Carrying Out the Invention

[0014] The data disclosed herein establish that post ultrafiltration / diafiltration (post UF / DF) chromatography steps, which are added relatively late in the purification regimen, can significantly reduce the concentration of high molecular weight contaminants when purifying target molecules such as relatively high molecular weight therapeutic biologics and biosimilars. Further, the results of the experiments have shown that the post UF / DF chromatography steps can be effectively carried out using resin-based or membrane-based chromatography media.

[0015] As used herein, "load rate" is the amount of protein in the material applied to the post UF / DF chromatography media per liter of chromatography media or resin. The load rate is specified in units of grams / resin liter. "High molecular weight" or "HMW" refers to species that are at least 10,000 daltons. "Load HMW%" is the percentage of the total mass of the material applied to the post UF / DF chromatography media that is composed of high molecular weight species. "HMW%" is the percentage of high molecular weight species. "HMW clearance%" or "HMW reduction percentage" is the percentage of HMW removed during post UF / DF chromatography. HMW clearance% = (Load HMW% - Pool HMW%) / Load HMW%. "Pool HMW%" is the percentage of HMW species in the eluate from post UF / DF chromatography. "Process yield%" is the yield of protein derived from post UF / DF chromatography.

[0016] The following examples disclose an experimental evaluation of subjecting various monoclonal antibodies, namely mAb3 (IgG1) and mAb4 (IgG1), to post UF / DF chromatography using various resins to reduce HMW while achieving a high yield of proteins such as biologics and biosimilars.

Example

[0017] Example 1 Materials

[0018]

Table 1

[0019] The initial screen of the chromatography media used in the post UF / DF chromatography of mAb4 fluid gave the data shown in Figure 2, described in Example 2. The results established that Capto MMC ImpRes, Nuvia cPrime, CMM Hypercel, ToyoPearl Sulfate, and Capto Adhere were the top chromatography media for removing undesired HMW while achieving a high yield of the mAb4 target protein. These top chromatography media were subjected to the worst-case load HMW% of 3.4% in the mAb4-containing fluid. The results are shown in Figure 1, which shows a reduction in HMW% and maintenance of a high process yield% of at least 93.9%.

[0020] The results of the experiments shown in the following examples and figures reflect a rigorous experimental analysis of a number of chromatography modes used in the post UF / DF harvest / purification process for target proteins such as monoclonal antibodies. In particular, the present disclosure relates to (1) cation exchange chromatography (Eshmuno CP-ft, Fractogel COO-(M), Mustang XT S, CIMultus PrimaS, Nuvia S, Fractogel SO3-(M), Nuvia HR-S, Fibro Adhere, Fibro MMC, and UNOsphere S), (2) hydrophobic interaction chromatography (Sartobind Phenyl), and (3) mixed-mode exchange chromatography (ToyoPearl MX-Trp, Capto Adhere, Ca ++Analysis of Pure HA, Capto MMC, Eshmuno HCX, Nuvia cPrime, Capto MMC ImpRes, CMM Hypercel, and Toyopearl Sulfate 650M is shown. The results identify multimode or mixed-mode chromatography media as being optimal for use in this chromatography step with respect to high HMW removal % and high target protein yield.

[0021] Example 2 Monoclonal antibody 3 (mAb3) Monoclonal antibody 3 (mAb3) is an IgG1 subclass antibody with a pI of 9.0. The mAb3 material was generated in a large-scale run. Here, it was buffer-exchanged from a matrix of 100 mM sodium acetate, 200 mM sodium chloride, pH 5.0 to a formulation buffer containing 6.4 mM L-histidine, 7.6% sucrose, pH 6.0 (see Table 2), and the pool was concentrated to a final concentration of approximately 88 g / L. The HMW% in the mAb3 UF / DF pool was approximately 0.4%.

[0022] Also, the monoclonal antibody 3 material generated in a large-scale run was buffer-exchanged into a matrix of a formulation buffer containing 10 mM sodium phosphate, 10 (w / v)% sucrose, pH 7.2, and the pool was concentrated to a final concentration of approximately 19 g / L.

[0023] To generate an mAb3 UF / DF pool containing a high level of HMW (approximately 1%), a portion of the original pool (0.2 HMW%) was held at ambient temperature for 10 minutes at a low pH of 3.0, neutralized to pH 6.0 using 2 M tris base, and then spiked back into the original pool to raise the level of HMW%. Also, as mentioned in Example 3, the mAb3 UF / DF pool was stressed at 50 °C for 2 weeks to generate a pool with 1.7 HMW% for a run loaded at 1200 g / Lr.

[0024] The sequence of steps shown in Table 2 was continued for the chromatography operations for mAb3.

[0025] [Table 2]

[0026] Example 3 Initial resin screening The initial resin screen was performed by loading mAb3 at approximately 88 g / L, and the loading rate was 800 g / Lr on the resin / membrane listed in Table 3. The chromatography apparatus was a packed column, and the corresponding column or membrane volume was 1 mL, which matched the volume listed in Table 1.

[0027] The % HMW reduction was calculated as ((% HMW [load] - % HMW [pool]) / % HMW [load]), and the reduction % was expressed as % HMW. The protein concentration of mAb1 was measured using a CTech SoloVPE System (Repligen) (extinction coefficient 1.54 mg / mL*cm). Table 3 summarizes the experimental investigation results regarding the % HMW reduction and the % process yield.

[0028] [Table 3]

[0029] Example 4 Effect of loading rate The initial screening experiment disclosed in Example 2 was performed at a high loading rate of 800 g / Lr. In addition, runs were performed at a lower loading rate of 400 g / Lr, and two runs were performed at a higher loading rate of 1200 g / Lr. The mAb3 load contained approximately 1.0 - 1.8% HMW. For the 1200 g / Lr run as the worst-case example, 1.7 load % HMW was achieved by stressing the mAb3 pool at 50 °C for 2 weeks.

[0030] Table 4 summarizes the results of an investigation into the effect of the loading rate on the HMW clearance capacity and the % process yield of mAb3 using Nuvia cPrime, Eshmuno CP-FT, ToyoPearl MX-Trp, and Fractogel COO- (i.e., the top 4 resins identified from the initial screen).

[0031]

Table 4

[0032] Example 5 Monoclonal Antibody 4 (mAb4) To generate a monoclonal antibody 4 (mAb4) post UF / DF pool containing a high level of HMW (about 3%), a portion of the original pool was held at 30 °C for up to 7 hours at a low pH of 3.4 and then neutralized to pH 7.2 using 2M Tris base. Each section of the experiment details the period of high temperature stress. The stressed material was loaded onto the column without any spiking.

[0033] Table 5 shows the sequence of steps performed for each mAb4 experiment.

[0034]

Table 5

[0035] Example 6 5.1 First Resin Screen The first resin screen was performed by loading mAb4 at approximately 19 g / L, and the loading rate was 800 g / Lr on the resins / membranes listed in Table 5. The chromatography apparatus was a packed column. The corresponding 1 mL column volume is listed in Table 1.

[0036] The HMW reduction % was calculated as ((HMW [load]% - HMW [pool]%) / HMW [load]%), and the reduction % was expressed in HMW%. The protein concentration of mAb4 was measured using a CTech SoloVPE System (Repligen) (extinction coefficient 1.40 mg / mL*cm). Table 6 summarizes the experimental investigation results regarding the HMW reduction % and the process yield %.

[0037]

Table 6

[0038] Example 7 Effect of Loading Rate The first screening experiment disclosed in Example 6 was carried out at a high loading rate of 800 g / Lr. In addition, runs were carried out at a lower loading rate of 400 g / Lr for the two resins (Nuvia cPrime and ToyoPearl Sulfate) with the highest HMW reduction %. Stress was applied to the mAb4 load at 30 °C and pH 3.4 for 2 hours and then neutralized to pH 7.2. This load contained 1.5 HMW%.

[0039] Table 7 summarizes the effect of the loading rate on the HMW clearance ability and the process yield % for Nuvia cPrime and ToyoPearl Sulfate (i.e., the top 2 resins identified in the first screen) (see Example 6 and Table 6) at both 800 g / Lr and 400 g / Lr.

[0040]

Table 7

[0041] All references cited throughout the application are hereby incorporated by reference in their entirety or, where relevant, in part, as if fully set forth herein, as if clear from the context. The present disclosure has shown embodiments to facilitate the disclosure of the subject matter, but the only limitations that apply to the present disclosure are those found in the claims.

Claims

1. A method for harvesting a target protein from a host cell culture medium, comprising an ultrafiltration / diafiltration (UF / DF) step followed by a chromatography step, wherein the high molecular weight species in the eluate derived from the chromatography step are reduced by at least 10% compared to the level of high molecular weight species in the UF / DF filtrate.

2. The method according to claim 1, further comprising at least one chromatography step prior to the ultrafiltration / dialysis filtration step.

3. The method according to claim 2, wherein the at least one chromatography step prior to the ultrafiltration / diafiltration step includes protein A chromatography.

4. The method according to claim 3, wherein the at least one chromatography step prior to the ultrafiltration / diafiltration step further includes ion exchange chromatography, mixed-mode chromatography, or both ion exchange chromatography and mixed-mode chromatography.

5. The method according to claim 1, further comprising an upstream bulk harvesting step of centrifuging, depth filtration, or centrifuging and depth filtration of the host cell culture medium, followed by a downstream polishing step for purifying the target protein, wherein the polishing step includes a protein A chromatography step, a low pH virus inactivation step, a cation exchange step, a mixed-mode anion exchange chromatography step, a virus filtration step, an ultrafiltration / dialysis filtration step, a chromatography step after the ultrafiltration / dialysis filtration step, a polysorbate 80 addition step, and a final filtration step.

6. The method according to claim 1, wherein the chromatography step after the ultrafiltration / diafiltration (UF / DF) step includes mixed-mode chromatography.

7. The method according to claim 1, wherein the high molecular weight compound in the eluate is reduced by at least 20% compared to the level in the UF / DF filtrate.

8. The method according to claim 7, wherein the high molecular weight compound in the eluate is reduced by at least 30% compared to the level in the UF / DF filtrate.

9. The method according to claim 7, wherein the high molecular weight compound in the eluate is reduced by 75% compared to the level in the UF / DF filtrate.

10. The method according to claim 1, wherein the product derived from the chromatography step is an eluate containing 0.3 to 1.9% of high molecular weight compounds.

11. The method according to claim 1, wherein the yield of the target protein from the chromatography step is at least 58%.

12. The method according to claim 11, wherein the yield of the target protein from the chromatography step is at least 86%.

13. The method according to claim 11, wherein the yield of the target protein from the chromatography step is at least 90%.

14. The method according to claim 1, wherein the chromatography step after the ultrafiltration / diafiltration (UF / DF) step includes using a chromatography medium, the chromatography medium being a mixed-mode resin, a mixed-mode membrane, an ion exchange resin, or an ion exchange membrane.

15. The chromatography media include Ca++Pure-HA, Capto MMC, Capto MMC ImpRes, Capto SP ImpRes, Capto Adhere, CIMultus PrimaS, CIMultus Hbond, CMM Hypercel, Eshmuno CP-FT, Eshmuno HCX, Fibro MMC, Fibro Adhere, Fractogel COO-(M), Fractogel SO3-(M), Mustang XT S, Nuvia S, Nuvia HR-S, Nuvia cPrime, Sartobind The method according to claim 14, wherein the material is Phenyl, ToyoPearl MX-Trp, ToyoPearl Sulfate 650M, or UNOsphere S.

16. The method according to claim 15, wherein the chromatographic medium is Ca++Pure HA, Capto MMC, Capto MMC ImpRes, Capto Adhere, CMM Hypercel, Eshmuno HCX, Fibro MMC, Fibro Adhere, Nuvia cPrime, ToyoPearl mX-Trp, or ToyoPearl Sulfate.

17. The method according to claim 16, wherein the chromatographic medium is Capto MMC ImpRes, Eshmuno HCX, Eshmuno CP-FT, Fibro MMC, or Nuvia cPrime.

18. The method according to claim 17, wherein the chromatography medium is Fibro MMC or Nuvia cPrime.

19. The method according to claim 14, wherein the chromatography medium is a film.

20. The method according to claim 1, wherein the load rate is 400 to 800 grams / liter of resin.

21. The method according to claim 1, wherein the load rate is at least 800 grams / liter of resin.

22. The method according to claim 1, wherein the load HMW% is at least 0.9%.

23. The method according to claim 1, wherein the load HMW% is at least 1.2%.

24. The method according to claim 1, wherein the load HMW% is 0.5 to 2.6%.

25. The method according to claim 1, wherein the load HMW% is at least 2.6%.

26. The method according to claim 1, wherein the fluid containing the target protein is applied to a chromatography medium at a load concentration of 50 g / L or less.

27. The method according to claim 26, wherein the load concentration is 20 g / L or less.

28. The method according to claim 1, wherein the chromatography medium contains the ligand at a ligand density of at least 98 mmol / mL of chromatography medium.

29. The method according to claim 1, wherein the eluate derived from the chromatography step has a Quality Target Protein Profile of 0.3% or less, and the eluate contains the target protein.

Images