Removal of aggregates in 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 biomimetic, is provided. The method improves the conventional harvesting / purification process by adding a chromatography step, such as a mixed mode or an ion exchange chromatography step, near the end of the purification phase of harvesting / purification after the conventional chromatographic purification step, such as a Protein A or ion exchange chromatography step, has been completed, and the resulting eluate has been subjected to filtration, such as ultrafiltration / diafiltration. The result of return chromatography refining after filtration is surprising, i.e. Reducing all forms of high molecular weight products, thereby facilitating purification of target proteins sufficient to meet government regulations, such as quality target protein characteristics.

Description

[0001] This application claims priority to U.S. Provisional Application No. 63 / 353,776, filed on June 20, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0002] The present disclosure relates generally to the field of protein harvesting / purification, and more particularly to the field of harvesting / purification of biologic and biosimilar protein products of therapeutic value. Background Art

[0003] The continuous development of biologics and biosimilars has revealed that these molecules provide great prospects for therapeutic benefits for mankind. These molecules are large proteins typically produced using cell-based molecular expression in culture. As proteins, these molecules also often require post-translational modification and appropriate folding to maximize functionality. Although the recent progress of prokaryotic expression systems predicts the continued vitality of this protein expression pathway, such considerations often lead to the use of eukaryotic host cells to express desired products. In addition, the development of cell culture technology has led to a significant increase in the cell density of the culture producing proteins (such as biologics and biosimilars), which has shown the prospect of increasing the yield of such products, but this is at the expense of increasing the level of pollutants from cell culture operations. In particular, the use of variable eukaryotic cells is associated with a certain level of cell lysis, which produces unwanted biomolecules (such as nucleic acids, proteins, lipids, etc.) together with the presence of cell membrane fragments. In view of the potential increase in the yield from using high-density cell cultures, it is necessary to solve the harvesting or purification methods of the pollutants that increase.

[0004] Traditional purification schemes for proteins expressed in cell culture involve upstream cell culture effluent treatment, which focuses on various forms of centrifugation and / or various forms of depth filtration, with an optional initial step of flocculation or precipitation to remove some contaminants in the effluent. Downstream refining steps have involved one or more chromatographic fractionations, which involve, for example, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and other forms of chromatography. The typical affinity chromatography step involving the purification of refined target proteins (such as biologics and biosimilars) is protein A chromatography, which is well known for purifying immunoglobulin-like proteins (such as antibodies). Ion exchange chromatography generally involves anion exchange, but can also involve cation exchange. After a series of one or more chromatographic fractionations in the downstream refining steps of harvesting / purification methods, there is often a filtration step, such as an ultrafiltration step, which can involve diafiltration (a form of ultrafiltration) with solvent supplementation. After filtration, the purified protein is typically prepared for, for example, administration to a subject in need.

[0005] The use of cell culture-based expression systems to produce therapeutic biologics and biosimilars requires these multi-step purification schemes to ensure sufficient purity of products designated for administration to humans. In order to ensure sufficient purity, government regulatory agencies around the world have established strict standards that must be met, including in the United States requiring the Quality Target Protein Profiles to be met, which is a strict standard for purifying products to be administered to humans. The need to obtain government approval to administer biologics and biosimilars has created a continued demand for higher purity products expressed in culture. Therefore, there remains a need in the art for improved protein harvesting / purification methods to produce protein target products with reduced levels and types of culture-based contaminants. Summary of the invention

[0006] The present disclosure provides a method for improving the purity of a target protein (e.g., biologics and biosimilars) produced in a cell culture. The present disclosure adopts a method contrary to conventional techniques in the downstream of purifying proteins from cell cultures. More particularly, in the downstream refining stage of purifying proteins from cell cultures, the present disclosure relies on one or more chromatographic steps before the ultrafiltration / diafiltration (UF / DF) step, which is consistent with conventional purification schemes, but then adopts an unusual step of returning to the chromatographic fractionation involving at least a mixed mode chromatography or ion exchange chromatography (e.g., cation exchange chromatography) step. The purification method of the present disclosure results in a significant reduction in the undesirable high molecular weight (HMW) material of the target protein material contaminating the purification, while minimizing the reduction in the yield of the target protein. When this unusual step is added in the later stage of the refining stage of target harvesting / purification, the method improves 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 the target protein production method based on cells). The result is a target protein with higher purity, which exhibits improved features that meet the quality target protein characteristics.

[0007] In one aspect, the present disclosure provides a method for harvesting a target protein from a host cell culture fluid, the method comprising a chromatography step after an ultrafiltration / diafiltration (UF / DF) step, wherein the high molecular weight substances in the eluent from the chromatography step are reduced by at least 10% compared to the level of the high molecular weight substances in the UF / DF filtrate. In some embodiments, the method further includes at least one chromatography step before the ultrafiltration / diafiltration step. In some embodiments, at least one chromatography step includes protein A chromatography. In some embodiments, 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 an upstream batch harvesting step of centrifuging, deep filtering, or centrifuging and deep filtering the host cell culture fluid, and a downstream refining step of purifying the target protein subsequently, wherein the refining step includes 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] Still other embodiments of the method are provided, wherein the target protein subjected to the chromatography step is present in a formulation buffer (pH 5.5) comprising 10 mM glutamic acid, 250 mM threonine. In some embodiments, the discharge of the chromatography step is an eluent in which 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 eluent 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 discharge of the chromatography step is an eluent comprising 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, wherein the chromatographic medium is a mixed mode resin, a mixed mode membrane, an ion exchange resin or an ion exchange membrane. In certain embodiments, the chromatographic medium is Ca++Pure-HA, Capt0 MMC, Capto MMCImpRes, Capto SPImpRes, 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 chromatographic medium is Ca++PureHA, Capto MMC, Capto MMC ImpRes, Capto Adhere, CMM Hypercel, Eshmuno HCX, Fibro MMC, Fibro Adhere, Nuvia cPrime, ToyoPearl mX-Trp or ToyoPearlSulfate. In some embodiments, the chromatographic medium is Capto MMC ImpRes, Eshmuno HCX, Eshmuno CP-FT, Fibro MMC or Nuvia cPrime. In some embodiments, the chromatographic medium is Fibro MMC or Nuvia cPrime. In some embodiments, the chromatographic medium is a membrane. In some embodiments, a fluid containing a target protein is applied to the chromatographic medium at a loading factor of at least 400 g / L of resin, including embodiments in which the loading factor is between 400-800 g / L of resin and embodiments in which the loading factor is at least 800 g / L of resin.

[0010] The method also provides embodiments wherein the HMW% loaded is at least 0.5%. In some embodiments, the HMW% loaded is at least 0.9%, at least 1.2%, between 0 / 5%-2.6%, or at least 2.6%. In some embodiments of the method, the chromatography step produces a HMW clearance of 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%.

[0011] The method also provides embodiments in which a fluid comprising the target protein is applied to a chromatographic medium at a loading concentration of no more than 50 g / L. In some embodiments, the loading concentration is no more than 20 g / L. In some embodiments of the method, the chromatographic medium comprises a ligand having a ligand density of at least 98 mmol / mL of chromatographic medium. In some embodiments, the ligand density is between 98-157 mmol / mL, between 98-140 mmol / mL, or between 127-157 mmol / mL. In some embodiments, the eluent from the chromatographic step has a mass target protein characteristic of 0.3% or less, wherein the eluent comprises the target protein.

[0012] The present disclosure will be better understood by considering the following detailed description of the disclosure, including by considering the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 .The bar graph demonstrates the cross-molecule applicability of adding a post-UF / DF chromatography step to purification schemes for large proteins such as antibody biologics / biosimilars. The performance of the post-UF / DF chromatography step was evaluated using a worst-case loading material level of 3.4% HMW when purifying mAb4 from culture broth. Performance was evaluated by determining the HMW% as a measure of HMW clearance and by determining the step yield%. The resins tested were CaptoMMC ImpRes, Nuvia cPrime, CMM Hypercel, ToyoPearl Sulfate, and Capto Adhere. The Nuvia cPrime resin provided a 24% HMW clearance with a step yield of greater than 90%. The results demonstrate that the addition of a post-UF / DF chromatography step to a biologic / biosimilar purification scheme has broad applicability in demonstrating significant HMW reduction and desirable high step yields across a variety of purified biologics / biosimilars. HMW clearance% - salmon colored columns; step yield% - gray columns; HMW loaded% - dashed line.

[0014] Figure 2.Bar graphs showing the effect of loading factor (grams per liter of resin (g / Lr)) on HMW clearance and step yield % when a post-UF / DF chromatography step is incorporated in the purification of mAb4 from culture broth. The loading material (i.e., the target-containing fluid including any HMW material (any HMW substance)) contained 1.5% HMW. As shown, loading factors of 800 g / Lr (high) and 400 g / Lr (low) were evaluated. The resins tested were Nuvia cPrime (800 g / Lr), Nuvia cPrime (400 g / Lr), ToyoPearl Sulfate (800 g / Lr), and ToyoPearl Sulfate (400 g / Lr). Nuvia cPrime (400 g / Lr) provided 33% HMW clearance. Pool HMW % - salmon colored column; Step yield % - gray column; Loaded HMW % - dotted line. DETAILED DESCRIPTION

[0015] The data disclosed herein determine that when purifying relatively high molecular weight target molecules (such as therapeutic biologics and biosimilars), a post-ultrafiltration / diafiltration (post-UF / DF) chromatography step added relatively late in the purification scheme can significantly reduce the concentration of high molecular weight contaminants. In addition, the experimental results reveal that the post-UF / DF chromatography step can be effectively performed using a resin-based or membrane-based chromatography medium.

[0016] As used herein, "Loading Factor" is the mass of protein in the material applied to the post-UF / DF chromatography medium per liter of chromatography medium or resin. The units of loading factor are specified in grams per liter of resin. "High molecular weight" or HMW refers to material of at least 10,000 Daltons. "Loaded HMW%" is the percentage of the total mass of material applied to the post-UF / DF chromatography medium that is composed of high molecular weight material. "HMW%" is the percentage of high molecular weight material. "HMW clearance%" or "HMW reduction%" is the percentage of HW removed during post-UF / DF chromatography. HMW clearance% = (loaded HMW% - pool HMW%) / loaded HMW%. "Pool HMW% (P oo "% HMW)" is the percentage of HMW species in the eluate from post-UF / DF chromatography. "% Step Yield" is the yield of protein produced by post-UF / DF chromatography.

[0017] The following examples disclose experimental evaluation of different monoclonal antibodies (i.e., mAb3(IgG1) and mAb4(IgG1)) using various resins for post-UF / DF chromatography to reduce HMW while providing high yields of proteins (e.g., biologics and biosimilars).

[0018] Examples

[0019] Example 1

[0020] Material

[0021] Table 1

[0022] List of materials used in the experiment

[0023]

[0024]

[0025] As described in Example 2, initial screening of chromatographic media for use in post-UF / DF chromatography of mAb4 fluids yielded Figure 2 The data are shown in . The results identified Capto MMC ImpRes, Nuvia cPrime, CMM Hypercel, ToyoPearl Sulfate, and Capto Adhere as the best chromatography media for removing unwanted HMW while providing high yields of mAb4 target protein. These best chromatography media experienced a worst case HMW loading of 3.4% in the mAb4-containing fluid. The results are presented in Figure 1 and showed a reduction in HMW% while maintaining a high step yield% of at least 93.9%.

[0026] The experimental results presented in the following examples and accompanying drawings reflect a rigorous experimental analysis of multiple chromatographic modes for the post-UF / DF harvest / purification steps of target proteins (such as monoclonal antibodies). In particular, the present disclosure presents analysis of (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, CaptoAdhere, Ca++Pure HA, Capto MMC, Eshmuno HCX, Nuvia cPrime, Capto MMC ImpRes, CMM Hypercel, and Toyopearl Sulfate 650M). The results determined that multimodal or mixed-mode chromatography media were most suitable for this chromatography step in terms of % HMW removal with significant target protein yield.

[0027] Example 2

[0028] Monoclonal antibody 3 (mAb3)

[0029] Monoclonal antibody 3 (mAb3) is an IgG1 subclass antibody with a pI of 9.0. mAb3 material was produced in a large-scale run where it was buffer exchanged from a matrix of 100 mM sodium acetate, 200 mM sodium chloride, pH 5.0, into 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 Ur / DF pool was approximately 0.4%.

[0030] The mAb 3 material produced in the large-scale run was also buffer exchanged into a matrix containing 10 mM sodium phosphate, 10% (w / v) sucrose in formulation buffer, pH 7.2, and the pool was concentrated to a final concentration of approximately 19 g / L.

[0031] To generate a mAb3 UF / DF pool containing high levels of HMW (approximately 1%), a portion of the original pool (at 0.2% HMW) was kept at a low pH of 3.0 for 10 minutes at ambient temperature, neutralized to pH 6.0 using 2M Tris base, and then spiked into the original pool to increase the level of HMW%. As also mentioned in Example 3, the mAb3 UF / DF pool was stressed at 50°C for two weeks to generate a pool of 1.7% HMW for runs loaded at 1200 g / Lr.

[0032] The order of steps shown in Table 2 was followed for the chromatography workup of mAb3.

[0033] Table 2

[0034] Sequence of steps for the chromatographic procedure for mAb3

[0035]

[0036]

[0037] Example 3

[0038] Initial resin screening

[0039] Initial resin screening was performed by loading mAb3 at a loading factor of approximately 88 g / L, 800 g / Lr on the resins / membranes listed in Table 3. The chromatographic devices were pre-packed columns corresponding to a column or membrane volume of 1 mL, consistent with the volumes listed in Table 1.

[0040] HMW reduction % was calculated as ((HMW[loaded] % - HMW[pool] %) / HMW[loaded] %) to express the reduction in HMW %. The protein concentration of mAbl was measured using a CTech SoloVPE system (Repligen) (extinction coefficient of 1.54 mg / mL*cm). Table 3 summarizes the experimental findings on HMW reduction % and step yield %.

[0041] Table 3 HMW clearance % of initial resin screen for mAb3

[0042]

[0043]

[0044] Example 4

[0045] Impact of Load Factor

[0046] The initial screening experiments disclosed in Example 2 were performed at a high loading factor of 800 g / Lr. In addition, a run was performed at a lower loading factor of 400 g / Lr and two runs were performed at a higher loading factor of 1200 g / Lr. The mAb3 loading contained approximately 1.0%-1.8% HMW. As a worst case example, a 1.7% loading of HMW was achieved by stressing the mAb3 pool at 50°C for two weeks with a 1200 g / Lr run.

[0047] Table 4 summarizes the findings on the effect of loading factor on HMW clearance and step yield % for mAb3 using Nuvia cPrime, Eshmuno CP-FT, ToyoPearl MX-Trp, and FractogelCOO- (i.e., the first four resins identified from the initial screen).

[0048] Table 4

[0049] Nuvia cPrime, Eshmuno CP-FT, ToyoPearl MX-Trp and Fractogel

[0050] COO-HMW removal % and step yield % at 400, 800 and 1200 g / Lr

[0051]

[0052]

[0053] Example 5

[0054] Monoclonal antibody 4 (mAb4)

[0055] To generate a post-UF / DF pool of monoclonal antibody 4 (mAb4) containing high levels of HMW (about 3%), a portion of the original pool was kept at a low pH of 3.4 for up to 7 hours at 30°C and then neutralized to pH 7.2 using 2M Tris base. The duration of high temperature stress is described in detail for each section of the experiment. The stressed material was loaded onto the column without any spiking.

[0056] Table 5 shows the sequence of steps performed for each mAb4 experiment.

[0057] Table 5 Sequence of steps for the chromatographic procedure used for mAb4

[0058]

[0059]

[0060] Example 6

[0061] 5.1. Initial resin screening

[0062] Initial resin screening was performed by loading mAb4 at a loading factor of approximately 19 g / L, 800 g / Lr on the resin / membrane listed in Table 5. The chromatographic apparatus was a pre-packed column with the corresponding 1 mL column volumes listed in Table 1.

[0063] HMW reduction % was calculated as ((HMW [load] % - HMW [pool] %) / HMW [load] %) to express the reduction in HMW %. The protein concentration of mAb4 was measured using a CTech SoloVPE system (Repligen) (extinction coefficient of 1.40 mg / mL*cm). Table 6 summarizes the experimental findings on HMW reduction % and step yield %.

[0064] Table 6 HMW clearance % and step yield % of initial resins screened for mAb4

[0065]

[0066] Example 7

[0067] Impact of Load Factor

[0068] The initial screening experiments discussed in Example 6 were performed at a high loading factor of 800 g / Lr. Additional runs were performed at a lower loading factor of 400 g / Lr for the two resins with the highest % reduction in HMW (Nuvia cPrime and ToyoPearl Sulfate). mAb4 was loaded at pH 3.4 and stressed at 30°C for two hours and then neutralized to pH 7.2. This load contained 1.5% HMW.

[0069] Table 7 summarizes the effect of loading factor at 800 g / Lr and 400 g / Lr on HMW removal capacity and step yield % for Nuvia cPrime and ToyoPearlSulfate, the first two resins identified in the initial screen (see Example 6 and Table 6).

[0070] Table 7

[0071] HMW% and step yield% of Nuvia cPrime and ToyoPearl Sulfate loaded at 800 and 400 g / Lr

[0072]

[0073] All references cited throughout this application are hereby incorporated by reference in their entirety or in relevant part as will be apparent from the context.The present disclosure has presented embodiments that facilitate the disclosure of the present subject matter, but the only limitations imposed on the disclosure are those in the claims.

Claims

1. A method for harvesting a target protein from a host cell culture fluid, the method comprising 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.

2. The method of claim 1, further comprising at least one chromatography step before the ultrafiltration / diafiltration step.

3. The method of claim 2, wherein the at least one chromatography step comprises Protein A chromatography.

4. The method of claim 3, wherein the at least one chromatography step further comprises ion exchange chromatography, mixed mode chromatography, or both ion exchange chromatography and mixed mode chromatography.

5. The method of claim 1, further comprising an upstream bulk harvesting step of centrifuging, depth filtering, or both centrifuging and depth filtering the host cell culture fluid, and a downstream polishing step of subsequently purifying the target protein, wherein the polishing step comprises 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.

6. The method of claim 1, wherein the target protein subjected to the chromatography step is present in a formulation buffer comprising 10 mM glutamate, 250 mM threonine at pH 5.

5.

7. The method of claim 1, wherein the effluent of 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.

8. The method of claim 7, wherein high molecular weight compounds in the eluate are reduced by at least 20% compared to levels in the UF / DF filtrate.

9. The method of claim 7, wherein high molecular weight compounds in the eluate are reduced by at least 25% compared to levels in the UF / DF filtrate.

10. The method of claim 7, wherein high molecular weight compounds in the eluate are reduced by at least 30% compared to levels in the UF / DF filtrate.

11. The method of claim 7, wherein high molecular weight compounds in the eluate are reduced by 75% compared to levels in the UF / DF filtrate.

12. The method of claim 1, wherein the effluent of the chromatography step is an eluate comprising 0.3% to 1.9% high molecular weight compounds.

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

14. The method of claim 13, wherein the yield of the target protein from the chromatography step is at least 86%.

15. The method of claim 1, wherein the yield of the target protein from the chromatography step is at least 90%.

16. The method of claim 1, wherein the yield of the target protein from the chromatography step is at least 95%.

17. The method of claim 1, wherein the yield of the target protein from the chromatography step is at least 98%.

18. The method of claim 1, wherein the chromatographic medium is a mixed mode resin, a mixed mode membrane, an ion exchange resin or an ion exchange membrane.

19. The method of claim 18, wherein the chromatographic medium is Ca++Pure-HA, Capto MMC, Capto MMCImpRes, Capto SPImpRes, 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.

20. The method of claim 18, wherein the chromatographic medium is Ca++PureHA, Capto MMC, Capto MMCImpRes, Capto Adhere, CMM Hypercel, Eshmuno HCX, Fibro MMC, Fibro Adhere, NuviacPrime, ToyoPearl mX-Trp or ToyoPearl Sulfate.

21. The method of claim 20, wherein the chromatography medium is Capto MMC ImpRes, Eshmuno HCX, Eshmuno CP-FT, Fibro MMC or Nuvia cPrime.

22. The method of claim 21, wherein the chromatographic medium is Fibro MMC or Nuvia cPrime.

23. The method of claim 19, wherein the chromatographic medium is a membrane.

24. The method of claim 1, wherein the fluid comprising the target protein is applied to the chromatography medium at a loading factor of at least 400 g / L of resin.

25. The method of claim 24, wherein the loading factor is between 400-800 g / L of resin.

26. The method of claim 24, wherein the loading factor is at least 800 g / L of resin.

27. The method of claim 1, wherein the loading HMW % is at least 0.5%.

28. The method of claim 27, wherein the loading HMW % is at least 0.9%.

29. The method of claim 27, wherein the loading HMW % is at least 1.2%.

30. The method of claim 27, wherein the loading HMW % is between 0.5%-2.6%.

31. The method of claim 27, wherein the loading HMW % is at least 2.6%.

32. The method of claim 1, wherein the chromatography step produces a % HMW clearance of at least 5%.

33. The method of claim 32, wherein the HMW clearance % is at least 8%.

34. The method of claim 32, wherein the HMW clearance % is at least 24%.

35. The method of claim 32, wherein the HMW Clearance % is at least 30%.

36. The method of claim 32, wherein the HMW clearance % is at least 33%.

37. The method of claim 32, wherein the HMW Clearance % is at least 40%.

38. The method of claim 32, wherein the HMW clearance % is at least 45%.

39. The method of claim 32, wherein the HMW clearance % is at least 55%.

40. The method of claim 32, wherein the HMW Clearance % is at least 65%.

41. The method of claim 32, wherein the HMW Clearance % is at least 70%.

42. The method of claim 32, wherein the HMW clearance % is at least 75%.

43. The method of claim 1, wherein the fluid comprising the target protein is applied to the chromatography medium at a loading concentration of no more than 50 g / L.

44. The method of claim 43, wherein the loading concentration does not exceed 20 g / L.

45. The method of claim 1, wherein the chromatographic medium comprises a ligand having a ligand density of at least 98 mmol / mL of chromatographic medium.

46. ​​The method of claim 45, wherein the ligand density is between 98-157 mmol / mL.

47. The method of claim 45, wherein the ligand density is between 98-140 mmol / mL.

48. The method of claim 45, wherein the ligand density is between 127-157 mmol / mL.

49. The method of claim 1, wherein the eluate from the chromatography step has a mass target protein characteristic of 0.3% or less, wherein the eluate comprises the target protein.

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