Purification method

Abstract

Disclosed herein are methods for mitigating viscous fingering during cation exchange chromatography and reducing the number of peaks in a cation exchange chromatography isocratic elution profile.

Description

PURIFICATION METHOD CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 654,298, filed May 31, 2024.FIELD OF DISCLOSURE

[0002] The present invention relates to the field of biopharmaceutical manufacturing. In particular, the invention relates to methods for mitigating viscous fingering and reducing the number of peaks in a cation exchange chromatography isocratic step gradient elution profile.BACKGROUND

[0003] In biologies drug substance manufacturing, the dow stream operations are critical to purify the recombinant protein from product- and process-related impurities and contaminants to meet product qualify and regulator}' requirements for the final bulk drug substance. Downstream operations from capture to polishing make use of a variety of preparative chromatography methods which have become the predominant tool for purification of recombinant proteins. It is critical that these chromatography methods operate in a consistent and predictable manner. Drug substance manufacturing operations must consistently provide high-quality products that meet or exceed attribute requirements for safety, potency, purity, and efficacy, as well as compliant with regulatory requirements.

[0004] There is an increased demand for preparative chromatography methods that are consistent and reliable. Such methods are described herein.BRIEF SUMMARY

[0005] In one aspect, a method comprises a) loading a recombinant protein to an equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; b) washing the cation exchange chromatography medium with at least one wash buffer comprising 10% to 50% w / v sucrose; and c) releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising 10% to 50% w / v sucrose. In some embodiments, at least one wash buffer comprises 10% to 20% w / v sucrose and the elution buffer comprises 10 to 20% w / v sucrose. In some embodiments, at least one wash buffer comprises sodium citrate and sodium chloride. In some embodiments, at least one w ash buffer comprises 10% to 20% w / v sucrose, pH 4.8-5.2. In some embodiments, at least one wash buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8-5.2. In some embodiments, at least one wash buffer comprises 10-40mM sodium citrate. 0-50mM sodium chloride. 10% w / v sucrose, pH 4.8-5.2.In some embodiments, the conductivity of the wash buffer is 6.0-7.0 mS / cm. In some embodiments, at least one wash buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, 20% w / v sucrose, pH 4.8-5.2. In some embodiments, the conductivity of the wash buffer is 5.0-6.0 mS / cm. In some embodiments, the measured conductivity of the wash buffer is 2.0-6.0 mS / cm. In some embodiments, the elution buffer comprises sodium citrate and sodium chloride. In some embodiments, the elution buffer comprises 10 to 20% w / v sucrose, pH 4.8-5.2. In some embodiments, the elution buffer further comprises 10-40mM sodium citrate, 270-500mM sodium chloride, pH 4.8-5.2. In some embodiments, the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, 10% w / v sucrose, pH 4.8- 5.2. In some embodiments, the conductivity of the elution buffer is 25.5-27.5 mS / cm. In some embodiments, the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, 20% w / v sucrose, pH 4.8-5.2. In some embodiments, the conductivity’ of the elution buffer is 20-22 mS / cm. In some embodiments, the measured conductivity’ of elution buffer is 8.0-20.5 mS / cm. In some embodiments, the cation exchange chromatography medium is equilibrated with an equilibration buffer having pH 4.8-5.2. In some embodiments, the cation exchange chromatography medium is equilibrated with an equilibration buffer comprising 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8-5.2. In some embodiments, the number of peaks in the cation exchange chromatography elution profile are reduced compared to a method wherein only the wash buffer comprises 10% to 20% w / v sucrose or only the elution buffer comprises 10% to 20% w / v sucrose. In some embodiments, the method results in reduced viscous fingering compared to washing the cation exchange chromatography medium with wash buffer without sucrose and releasing the recombinant protein with an elution buffer without sucrose. In some embodiments, the method results in a single peak in the cation exchange chromatography isocratic step gradient elution profile. In some embodiments, the cation exchange chromatography medium comprises a polymer porous resin, an agarose porous resin, or a tentacular resin. In some embodiments, the equilibration buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8 to 5.2; the load buffer has a pH from 4.8 to 5.2; at least one wash buffer comprises 10-40mM sodium citrate and 0-50mM sodium chloride, pH 4.8 to 5.2. and 10% to 50% w / v sucrose; and the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, pH 4.8 to 5.2, and 10% to 50% w / v sucrose. In some embodiments, the equilibration buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8 to 5.2; the load buffer has a pH from 4.8 to 5.2; at least one wash buffer comprises 10-40mM sodium citrate and 0- 50mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% w / v sucrose; and the elution buffercomprises 10-40mM sodium citrate, 270-500mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% w / v sucrose.

[0006] In one aspect, a method comprises a) equilibrating a cation exchange chromatography medium with a buffer comprising sodium citrate and sodium chloride, pH 4.8-5.2; b) loading a recombinant protein on to the equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; c) washing the cation exchange chromatography medium with at least one wash buffer comprising sodium citrate, sodium chloride, 10-50% w / v sucrose, pH 4.8-5.2; and d) releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising sodium citrate, sodium chloride, 10% to 50% w / v sucrose, pH 4.8- 5.2. In some embodiments, at least one wash buffer comprises 10 to 20% w / v sucrose and the elution buffer comprises 10-20% w / v sucrose. In some embodiments, the method results in reduced viscous fingering compared to washing the cation exchange chromatography medium with wash buffer comprising sodium citrate and sodium citrate, pH 4.8-5.2, without sucrose and releasing the recombinant protein with an elution buffer comprising sodium citrate, sodium chloride. pH 4.8-5.2. without sucrose. In some embodiments, the equilibration buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8 to 5.2; the load buffer has a pH from 4.8 to 5.2; at least one wash buffer comprises 10-40mM sodium citrate and 0-50mM sodium chloride, pH 4.8 to 5.2, and 10% to 50% w / v sucrose; and the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, pH 4.8 to 5.2, and 1 % to 50% w / v sucrose. In some embodiments, the equilibration buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8 to 5.2; the load buffer has a pH from 4.8 to 5.2; at least one wash buffer comprises 10-40mM sodium citrate and 0-50mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% w / v sucrose; and the elution buffer comprises 10- 40mM sodium citrate, 270-500mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% w / v sucrose.

[0007] In one aspect, a method comprises a) equilibrating a cation exchange chromatography medium with a buffer comprising 10-40mM sodium citrate, 0-50mM sodium chloride. pH 4.8-5.2; b) loading a recombinant protein on to the equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; c) washing the cation exchange chromatography medium with at least one wash buffer comprising 10-40mM sodium citrate, 0-50mM sodium chloride, 10% - 50% w / v sucrose, pH 4.4-5.3; and d) releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising 10-40mM sodium citrate, 270-500mMsodium chloride, 10% - 50% w / v sucrose, pH 4.8-5.2. In some embodiments, at least one wash buffer comprises 10 to 20% w / v sucrose and the elution buffer comprises 10-20% w / v sucrose. In some embodiments, the method results in fewer peaks in the cation exchange chromatography isocratic step gradient elution profile compared to washing the cation exchange chromatography medium with wash buffer comprising sodium citrate and sodium citrate, pH 4.8-5.2, without sucrose and releasing the recombinant protein with an elution buffer comprising sodium citrate, sodium chloride, pH 4.8-5.2, without sucrose. In some embodiments, the method results in a single peak in the cation exchange chromatography isocratic step gradient elution profile.

[0008] In one aspect, a method comprises a) establishing a cell culture in a bioreactor with a host cell expressing a protein; b) culturing the host cells to express the protein; c) harvesting the protein from the cell culture; d) affinity purifying the protein; e) equilibrating a cation exchange chromatography medium with a buffer comprising sodium citrate, sodium chloride, pH 4.8-5.2; 1) loading the purified protein onto an equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; g) washing the cation exchange chromatography medium with at least one wash buffer comprising sodium citrate, sodium chloride, 10%-20% w / v sucrose, pH 4.8-5.2; and h) releasing recombinant protein by isocratic step gradient elution with an elution buffer comprising sodium citrate, sodium chloride, 10% to 20% w / v sucrose, pH 4.8- 5.2 into the effluent stream or collecting in an eluant pool. In some embodiments, at least one wash buffer comprises 10 to 20% w / v sucrose and the elution buffer comprises 10-20% w / v sucrose. In some embodiments, the method further comprises one or more unit operations selected from viral inactivation, viral filtration, neutralization, anion exchange chromatography, cation exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, ultrafiltration, diafiltration, depth filtration, sterile filtration, and fill / finish. In some embodiments, a, isolated, purified, recombinant protein is produced by the method. In some embodiments, a pharmaceutical composition comprising the isolated, purified, recombinant protein produced by the method. In some embodiments, the equilibration buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride. pH 4.8 to 5.2; the load buffer has a pH from 4.8 to 5.2; at least one wash buffer comprises 10- 40mM sodium citrate and 0-50mM sodium chloride, pH 4.8 to 5.2, and 10% to 50% w / v sucrose; and the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, pH 4.8 to 5.2, and 10% to 50% w / v sucrose. In some embodiments, the equilibration buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8 to 5.2; the loadbuffer has a pH from 4.8 to 5.2; at least one wash buffer comprises 10-40mM sodium citrate and 0-50mM sodium chloride. pH 4.8 to 5.2, and 10% to 20% w / v sucrose; and the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% w / v sucrose.

[0009] In one aspect, a method for purifying a recombinant protein, the method comprises loading and binding the recombinant protein to the equilibrated cation exchange chromatography medium, washing the cation exchange chromatography medium with at least one wash buffer comprising 10 to 20% v / v sucrose, and releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising 10 to 20% v / v sucrose. In one embodiment at least one wash buffer further comprises sodium citrate and sodium chloride. In a related embodiment at least one wash buffer comprises lOmM sodium citrate. 50mM sodium chloride, pH 4.8-5.2. In one embodiment at least one wash buffer comprises 20mM sodium citrate, 50mM sodium chloride, 10% v / v sucrose pH 4.8-5.2. In a related embodiment the conductivity of the buffer is 6.0-7.0 mS / cm. In one embodiment at least one wash buffer comprises 20mM sodium citrate, 50mM sodium chloride, 20% v / v sucrose pH 4.8-5.2. In a related embodiment the conductivity of the wash buffer is 5.0-6.0 mS / cm. In another embodiment the elution buffer further comprises 20mM sodium citrate, 300mM sodium chloride, pH 4.8-5.2. In one embodiment the elution buffer comprises 20mM sodium citrate, 300mM sodium chloride, 10% v / v sucrose pH 4.8-5.2. In a related embodiment the conductivity of the elution buffer is 25.5-27.5 mS / cm. In one embodiment the elution buffer comprises 20mM sodium citrate, 300mM sodium chloride, 20% v / v sucrose pH 4.8-5.2. In one embodiment the conductivity of the elution buffer is 20-22 mS / cm. In another embodiment the equilibration buffer comprises 20mM sodium citrate, 50mM sodium chloride, pH 4.8-5.2. In another embodiment the number of peaks in the cation exchange chromatography elution profile are reduced compared to a method that makes use of only a wash buffer comprising 20% v / v sucrose or only an elution buffer comprising 20% v / v sucrose.

[0010] In one aspect, a method for mitigating viscous fingering while purifying an recombinant protein, the method comprising equilibrating a cation exchange chromatography medium with a buffer comprising sodium citrate, sodium chloride, and 10%-20% v / v sucrose, loading and binding the recombinant protein on to the equilibrated cation exchange chromatography medium, washing the cation exchange chromatography medium with at least one wash buffer comprising sodium citrate, sodium chloride, and 10-20% v / v sucrose, andreleasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising sodium citrate, sodium chloride, and 10% to 20% v / v sucrose.

[0011] In one aspect, a method for reducing the number of peaks in the cation exchange chromatography isocratic step gradient elution profile, the method comprising equilibrating a cation exchange chromatography medium with a buffer comprising 20mM sodium citrate, 50mM sodium chloride, 10% - 20% v / v sucrose, pH 4.8-5.2, loading and binding the recombinant protein on to the equilibrated cation exchange chromatography medium, washing the cation exchange chromatography medium with at least one wash buffer comprising 20mM sodium citrate, 50mM sodium chloride, 10% - 20% v / v sucrose, pH 4.8- 5.2, and releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising 20mM sodium citrate, 300mM sodium chloride, 10% - 20% v / v sucrose, pH 4.8-5.2.

[0012] In one aspect, a method for producing an isolated, purified, recombinant protein, the method comprising establishing a cell culture in a bioreactor with a host cell expressing the protein, culturing the host cells to express the protein, harvesting the recombinant protein from the cell culture, affinity purifying the recombinant protein, equilibrating a cation exchange chromatography medium with a buffer comprising sodium citrate, sodium chloride, loading and binding the purified recombinant protein onto an equilibrated cation exchange chromatography medium, washing the cation exchange chromatography medium with at least one wash buffer comprising sodium citrate, sodium chloride, pH 4.8-5.2. and 10%-20% v / v sucrose, and releasing recombinant protein by isocratic step gradient elution with an elution buffer comprising sodium citrate, sodium chloride, pH 4.8-5.2, and 10% to 20% v / v sucrose into the effluent stream or collecting in an eluant pool. In one embodiment the method further comprising one or more unit operations selected from viral inactivation, viral filtration, neutralization, anion exchange chromatography, cation exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, ultrafiltration, diafiltration, depth filtration, sterile filtration, and fill / finish. In one embodiment is provided the isolated, purified, recombinant protein produced by the method. In one embodiment is provided a pharmaceutical composition comprising the isolated, purified, recombinant protein produced by the method.

[0013] Further aspects and advantages will be apparent to those of ordinary' skill in the art from a review of the following detailed description. The description hereafter includes specific cases, embodiments, and examples with the understanding that the disclosure isillustrative and is not intended to limit the embodiments of the present disclosure to the specific cases, embodiments, and examples described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 shows the elution profiles overlayed for Resin 1 (Fractogel® SE Hicap, light gray) and Resin 2 (Fractogel* SOa’ dark gray) (UV 300nM).

[0015] Figure 2 shows the monoclonal antibody elution profile on Resin 2 at different wavelengths (UV 280 light gray, UV 300 dark gray). Fractions #1, #2, and #3 are highlighted.

[0016] Figure 3 shows the elution profiles of the monoclonal antibody from Resin 2 (UV 300) as a function of column load factor (lOg / L-r to 70 g / L-r) confirming viscous fingering.

[0017] Figure 4 shows a comparison of the functional groups on Resin 1 (Fractogel® SE Hicap) and Resin 2 (Fractogel® SCU )

[0018] Figures 5A-5K provide an overview of the process parameters tested to mitigate the viscous fingering phenomenon during cation exchange chromatography elution.

[0019] Figures 6A-6G provide monoclonal antibody elution profiles for wash and elution buffers different concentrations of sucrose.DETAILED DESCRIPTION

[0020] Surprising multi-peak elution behavior was observed during cation exchange chromatography. No impact was observed on pool product quality characteristics. Direct correlation of column loading to multi-peak elution behavior confirmed viscous fingering phenomenon was responsible for the multi-peak behavior under isocratic step gradient elution conditions.

[0021] It was found that, when the concentration of the protein elution band is very' high, there can be a difference in viscosity7between the mobile phase (<?.g., an elution buffer) and the protein elution band, and viscous fingering can occur. Once the high-viscosity protein band displaces the low-viscosity mobile phase, the mobile phase at the rear end can push the protein band, creating fingers. Cation exchange resins with high binding capacities can create high concentration zones (high viscosity) within the column during elution. Viscous fingering in turn can lead to multi-peak elution. Multiple peaks can interfere with collection of the protein of interest during manufacturing. For example, collection of eluted protein may be stopped after a peak maximum. However, when there are multiple peaks, collection may be stopped too soon or too late. Stopping collection too soon can result in incompletecollection of the protein (e.g., low yield). Stopping collection too late can result in collection of impurities (e.g, low purity). The claimed methods can be particularly effective in removing product-related impurities such as high molecular weight (HMW) species.

[0022] Features of the resin and protein can contribute to higher binding capacities and / or high viscosity within the column during elution. The hydrophobicity and overall charge of the resin can contribute to higher binding capacities and / or high viscosity within the column during elution. The complimentary charge and similar hydrophobicity of the protein can also contribute to higher binding capacities and / or high viscosity within the column during elution. For example, a highly charged protein can bind more strongly to a cation exchange resin, leading to increased viscosity of the protein elution band. For example, as shown in Example 1. a protein with hydrophobic regions can bind more strongly to a cation exchange resin that is more hydrophobic, leading to increased viscosity of the protein elution band. Additionally, as shown in Example 1, a tentacular resin structure can also contribute to higher binding capacities and / or high viscosity within the column during elution, particularly when a resin is both tentacular and hydrophobic. A high concentration of protein can also contribute to high viscosity of the protein elution band. High concentration of the protein is often desirable in biomanufacturing, which decreases the overall pool volume, thus helping with facility fit.

[0023] It was found that the viscosity of the mobile phase w as modulated when sucrose (e.g.. 10% to 50% v / v or 10% to 50% w / v or 10% to 20% v / v or 10 to 20% w / v) was used in both the wash and elution buffers, resulting in a single elution peak. It was found in Example 1 that adding sucrose to the w ash and elution buffers increased the viscosity of the mobile phase (e.g.. wash and elution buffers) and reduced the viscosity difference between the mobile phase and the protein elution band. By increasing the viscosity of the mobile phase (e.g., wash and elution buffers) to more closely match the high viscosity of the protein elution band, the effects of viscous fingering (e.g., multipeak elution) could be reduced. It was found that adding sucrose did not affect the pH of the wash or elution buffers. It was also found that adding sucrose to the elution buffer did not affect the salt concentration needed for elution. It was found that sucrose could be added at concentrations (e.g.. 10% to 50% v / v or 10% to 50% w / v) that increase the viscosity of the mobile phase sufficiently to mitigate viscous fingering while also maintaining a sufficiently low back pressure to be compatible with manufacturing (e.g., differential pressure on the column less than 0.3 MPa or less than 45 psi). For example, as shown in Example 1, low back pressures were associated with 10 to 20% w / v conditions. It was found that sucrose could be added at concentrations (e.g, 10% to50% v / v or 10% to 50% w / v or 10% to 20% v / v or 10 to 20% w / v) that resulted in single peak elution.

[0024] Concentrations of sucrose expressed as v / v % can be related approximately to concentrations expressed as w / v % based on the density of sucrose (1.58 g / mL) and the density of water (1 g / mL). For example, 10 % v / v sucrose in water is approximately 15.8 % w / v sucrose. In another example, 20% v / v sucrose in water is approximately 31.6% w / v sucrose. In another example, 50% v / v sucrose in water is approximately 79% w / v sucrose.

[0025] This disclosure provides a method comprising a) loading a recombinant protein to an equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; b) washing the cation exchange chromatography medium with at least one wash buffer comprising 10% to 50% w / v sucrose: and c) releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising 10% to 50% w / v sucrose. In some embodiments, at least one wash buffer comprises 10% to 20% w / v sucrose and the elution buffer comprises 10 to 20% w / v sucrose. In some embodiments, at least one wash buffer comprises sodium citrate and sodium chloride. In some embodiments, at least one wash buffer comprises 10% to 20% w / v sucrose, pH 4.8-5.2. In some embodiments, at least one wash buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8-5.2. In some embodiments, at least one wash buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, 10% w / v sucrose, pH 4.8-5.2. In some embodiments, the conductivity of the wash buffer is 6.0-7.0 mS / cm. In some embodiments, at least one wash buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, 20% w / v sucrose, pH 4.8-5.2. In some embodiments, the conductivity7of the wash buffer is 5.0-6.0 mS / cm. In some embodiments, the measured conductivity of the wash buffer is 2.0-6.0 mS / cm. In some embodiments, the elution buffer comprises sodium citrate and sodium chloride. In some embodiments, the elution buffer comprises 10 to 20% w / v sucrose, pH 4.8-5.2. In some embodiments, the elution buffer further comprises 10-40mM sodium citrate, 270-500mM sodium chloride, pH 4.8-5.2. In some embodiments, the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, 10% w / v sucrose, pH 4.8- 5.2. In some embodiments, the conductivity of the elution buffer is 25.5-27.5 mS / cm. In some embodiments, the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, 20% w / v sucrose, pH 4.8-5.2. In some embodiments, the conductivity7of the elution buffer is 20-22 mS / cm. In some embodiments, the measured conductivity of elution buffer is 8.0-20.5 mS / cm. In some embodiments, the cation exchange chromatography medium is equilibrated with an equilibration buffer having pH 4.8-5.2. In some embodiments, the cationexchange chromatography medium is equilibrated with an equilibration buffer comprising 10-40mM sodium citrate, 0-5 OmM sodium chloride, pH 4.8-5.2. In some embodiments, the number of peaks in the cation exchange chromatography elution profile are reduced compared to a method wherein only the wash buffer comprises 10% to 20% w / v sucrose or only the elution buffer comprises 10% to 20% w / v sucrose. In some embodiments, the method results in reduced viscous fingering compared to washing the cation exchange chromatography medium with wash buffer without sucrose and releasing the recombinant protein with an elution buffer without sucrose. In some embodiments, the method results in a single peak in the cation exchange chromatography isocratic step gradient elution profile. In some embodiments, the cation exchange chromatography medium comprises a polymer porous resin, an agarose porous resin, or a tentacular resin. In some embodiments, the equilibration buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8 to 5.2; the load buffer has a pH from 4.8 to 5.2; at least one wash buffer comprises 10-40mM sodium citrate and 0-50mM sodium chloride, pH 4.8 to 5.2, and 10% to 50% w / v sucrose; and the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, pH 4.8 to 5.2, and 10% to 50% w / v sucrose. In some embodiments, the equilibration buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8 to 5.2; the load buffer has a pH from 4.8 to 5.2; at least one wash buffer comprises 10-40mM sodium citrate and 0- 50mM sodium chloride, pH 4.8 to 5.2. and 10% to 20% w / v sucrose; and the elution buffer comprises 10-40mM sodium citrate. 270-500mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% w / v sucrose.

[0026] This disclosure also provides a method comprising a) equilibrating a cation exchange chromatography medium with a buffer comprising sodium citrate and sodium chloride, pH 4.8-5.2; b) loading a recombinant protein on to the equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; c) washing the cation exchange chromatography medium with at least one wash buffer comprising sodium citrate, sodium chloride, 10-50% w / v sucrose, pH 4.8-5.2; and d) releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising sodium citrate, sodium chloride, 10% to 50% w / v sucrose. pH 4.8- 5.2. In some embodiments, at least one wash buffer comprises 10 to 20% w / v sucrose and the elution buffer comprises 10-20% w / v sucrose. In some embodiments, the method results in reduced viscous fingering compared to washing the cation exchange chromatography medium with wash buffer comprising sodium citrate and sodium citrate, pH 4.8-5.2, withoutsucrose and releasing the recombinant protein with an elution buffer comprising sodium citrate, sodium chloride, pH 4.8-5.2. without sucrose.

[0027] This disclosure also provides a method comprising a) equilibrating a cation exchange chromatography medium with a buffer comprising 10-40mM sodium citrate, 0- 50mM sodium chloride, pH 4.8-5.2; b) loading a recombinant protein on to the equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; c) washing the cation exchange chromatography medium with at least one wash buffer comprising 10-40mM sodium citrate, 0-50mM sodium chloride, 10% - 50% w / v sucrose, pH 4.4-5.3; and d) releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising 10-40mM sodium citrate, 270-500mM sodium chloride, 10% - 50% w / v sucrose, pH 4.8-5.2. In some embodiments, at least one wash buffer comprises 10 to 20% w / v sucrose and the elution buffer comprises 10- 20% w / v sucrose. In some embodiments, the method results in fewer peaks in the cation exchange chromatography isocratic step gradient elution profde compared to washing the cation exchange chromatography medium with wash buffer comprising sodium citrate and sodium citrate. pH 4.8-5.2. without sucrose and releasing the recombinant protein with an elution buffer comprising sodium citrate, sodium chloride, pH 4.8-5.2, without sucrose. In some embodiments, the method results in a single peak in the cation exchange chromatography isocratic step gradient elution profile.

[0028] This disclosure also provides a method comprising a) establishing a cell culture in a bioreactor with a host cell expressing a protein; b) culturing the host cells to express the protein; c) harvesting the protein from the cell culture; d) affinity purifying the protein; e) equilibrating a cation exchange chromatography medium with a buffer comprising sodium citrate, sodium chloride, pH 4.8-5.2; f) loading the purified protein onto an equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; g) washing the cation exchange chromatography medium with at least one wash buffer comprising sodium citrate, sodium chloride, 10%-20% w / v sucrose, pH 4.8-5.2; and h) releasing recombinant protein by isocratic step gradient elution with an elution buffer comprising sodium citrate, sodium chloride, 10% to 20% w / v sucrose, pH 4.8-5.2 into the effluent stream or collecting in an eluant pool. In some embodiments, at least one wash buffer comprises 10 to 20% w / v sucrose and the elution buffer comprises 10-20% w / v sucrose. In some embodiments, the method further comprises one or more unit operations selected from viral inactivation, viral filtration, neutralization, anion exchange chromatography, cation exchange chromatography, multimodalchromatography, hydrophobic interaction chromatography, ultrafiltration, diafiltration. depth filtration, sterile filtration, and fill / finish. In some embodiments, provided herein is an isolated, purified, recombinant protein produced by the method. In some embodiments, provided herein is a pharmaceutical composition comprising the isolated, purified, recombinant protein produced by the method. In some embodiments, the equilibration buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8 to 5.2; the load buffer has a pH from 4.8 to 5.2; at least one wash buffer compnses 10-40mM sodium citrate and 0- 50mM sodium chloride, pH 4.8 to 5.2, and 10% to 50% w / v sucrose; and the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, pH 4.8 to 5.2, and 10% to 50% w / v sucrose. In some embodiments, the equilibration buffer comprises 10-40mM sodium citrate. 0-50mM sodium chloride, pH 4.8 to 5.2; the load buffer has a pH from 4.8 to 5.2; at least one wash buffer comprises 10-40mM sodium citrate and 0-50mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% w / v sucrose; and the elution buffer comprises 10- 40mM sodium citrate, 270-500mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% w / v sucrose.

[0029] This disclosure also provides a method for purifying a recombinant protein, the method comprises loading and binding the recombinant protein to the equilibrated cation exchange chromatography medium, washing the cation exchange chromatography medium with at least one wash buffer comprising 10 to 20% v / v sucrose, and releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising 10 to 20% v / v sucrose. In some embodiments at least one wash buffer further comprises sodium citrate and sodium chloride. In a related embodiment at least one wash buffer comprises lOmM sodium citrate, 50mM sodium chloride, pH 4.8-5.2. In some embodiments at least one wash buffer comprises 20mM sodium citrate, 50mM sodium chloride, 10% v / v sucrose pH 4.8-5.2. In a related embodiment the conductivity of the buffer is 6.0-7.0 mS / cm. In some embodiments at least one wash buffer comprises 20mM sodium citrate, 50mM sodium chloride, 20% v / v sucrose pH 4.8-5.2. In a related embodiment the conductivity of the w ash buffer is 5.0-6.0 mS / cm. In another embodiment the elution buffer further comprises 20mM sodium citrate. 300mM sodium chloride, pH 4.8-5.2. In some embodiments the elution buffer comprises 20mM sodium citrate, 300mM sodium chloride, 10% v / v sucrose pH 4.8- 5.2. In a related embodiment the conductivity of the elution buffer is 25.5-27.5 mS / cm. In some embodiments the elution buffer comprises 20mM sodium citrate, 300mM sodium chloride, 20% v / v sucrose pH 4.8-5.2. In some embodiments the conductivity of the elution buffer is 20-22 mS / cm. In another embodiment the equilibration buffer comprises 20mMsodium citrate, 50mM sodium chloride, pH 4.8-5.2. In another embodiment the number of peaks in the cation exchange chromatography elution profile are reduced compared to a method that makes use of only a wash buffer comprising 20% v / v sucrose or only an elution buffer comprising 20% v / v sucrose.

[0030] This disclosure also provides a method for mitigating viscous fingering while purifying a recombinant protein, the method comprising equilibrating a cation exchange chromatography medium with a buffer comprising sodium citrate, sodium chloride, and 10% to 20% v / v sucrose, loading and binding the recombinant protein on to the equilibrated cation exchange chromatography medium, washing the cation exchange chromatography medium with at least one wash buffer comprising sodium citrate, sodium chloride, and 10 to 20% v / v sucrose, and releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising sodium citrate, sodium chloride, and 10% to 20% v / v sucrose.

[0031] This disclosure also provides a method for reducing the number of peaks in the cation exchange chromatography isocratic elution profile, the method comprising equilibrating a cation exchange chromatography medium with a buffer comprising 20mM sodium citrate. 50mM sodium chloride, 10% to 20% v / v sucrose. pH 4.8-5.2. loading and binding the recombinant protein on to the equilibrated cation exchange chromatography medium, washing the cation exchange chromatography medium with at least one wash buffer comprising 20mM sodium citrate, 50mM sodium chloride, 10% to 20% v / v sucrose, pH 4.8- 5.2, and releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising 20mM sodium citrate, 300mM sodium chloride, 10% to 20% v / v sucrose, pH 4.8-5.2.

[0032] This disclosure further provides a method for producing an isolated, purified, recombinant protein, the method comprising establishing a cell culture in a bioreactor with a host cell expressing the protein, culturing the host cells to express the protein, harvesting the recombinant protein from the cell culture, affinity purifying the recombinant protein, equilibrating a cation exchange chromatography medium with a buffer comprising sodium citrate, sodium chloride, loading and binding the purified recombinant protein onto an equilibrated cation exchange chromatography medium, washing the cation exchange chromatography medium with at least one wash buffer comprising sodium citrate, sodium chloride, pH 4.8-5.2, and 10% to 20% v / v sucrose, and releasing recombinant protein by isocratic step gradient elution with an elution buffer comprising sodium citrate, sodium chloride, pH 4.8-5.2. and 10% to 20% v / v sucrose into the effluent stream or collecting in an eluant pool. In some embodiments the method further comprising one or more unitoperations selected from viral inactivation, viral filtration, neutralization, anion exchange chromatography, cation exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, ultrafiltration, diafiltration, depth filtration, sterile filtration, and fill / finish. In some embodiments is provided the isolated, purified, recombinant protein produced by the method. In some embodiments is provided a pharmaceutical composition comprising the isolated, purified, recombinant protein produced by the method.

[0033] This disclosure provides a purification process that includes a unit operation comprising cation exchange chromatography preformed according to the methods disclosed herein.

[0034] “Unit operation” refers to a functional step that is performed as part of the process of purifying a recombinant protein of interest. For example, a unit operation can include steps in operations such as, but not limited to, harvest, capture, purification, polish, viral inactivation, virus filtration, concentration and / or formulation the recombinant protein of interest. Unit operations can be designed to achieve a single objective or multiple objectives, such as a combination of capture and virus inactivation steps. Unit operations can also include holding or storing steps between processing steps.

[0035] The method described herein can be used to purify polypeptides and proteins of interest. The polypeptides and proteins can be of scientific or commercial interest. including protein-based therapeutics. Proteins of interest include, but are not limited to, secreted proteins, non-secreted proteins, intracellular proteins, or membrane-bound proteins. Polypeptides and proteins of interest can be produced by recombinant animal cell lines using cell culture methods described herein and may be referred to as “recombinant proteins.” The expressed protein(s) may be produced intracellularly or secreted into the culture medium from which it can be recovered and / or collected. The term “isolated protein” or “isolated recombinant protein” refers to a polypeptide or protein of interest, that is purified away from proteins or polypeptides or other contaminants that would interfere with its therapeutic, diagnostic, prophylactic, research, or other use. Proteins of interest include, but are not limited to, proteins that exert a therapeutic effect by binding a target, such as, e.g., a target among those listed below, including targets derived therefrom, targets related thereto, and modifications thereof.

[0036] Proteins of interest may include, but are not limited to, “antigen-binding proteins.” An “antigen-binding protein” refers to a protein or polypeptide that comprises an antigen-binding region or antigen-binding portion that has affinity for another molecule towhich it binds (antigen). Antigen-binding proteins include, but are not limited to. antibodies, peptibodies, antibody fragments, antibody derivatives, antibody analogs, fusion proteins (including, e.g., single-chain variable fragments (scFvs), double-chain (divalent) scFvs, and IgGscFv (see, e.g., Orcutt etal., 2010, Protein Eng Des Sei 23:221-228)), hetero-IgG (see, e.g., Liu et al., 2015, J Biol Chem 290:7535-7562), muteins, and proteins made using XmAb® technology (Xencor, Inc., Monrovia, CA). Also included are BiTE® molecules, bispecific T cell engagers, bispecific T cell engagers having extensions, and others, chimeric antigen receptors (CARs, CAR Ts), and T cell receptors (TCRs).

[0037] As used herein, the term “antibody” generally refers to a tetrameric immunoglobulin protein comprising two light chain polypeptides (about 25 kDa each) and two heavy chain polypeptides (about 50-70 kDa each). The term “light chain” or “immunoglobulin light chain” refers to a polypeptide comprising, from amino terminus to carboxyl terminus, a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL). The immunoglobulin light chain constant domain (CL) can be a human kappa (K) or human lambda (A,) constant domain. The term “heavy chain” or “immunoglobulin heavy chain” refers to a polypeptide comprising, from amino terminus to carboxyl terminus, a single immunoglobulin heavy chain variable region (VH), an immunoglobulin heavy chain constant domain 1 (CHI), an immunoglobulin hinge region, an immunoglobulin heavy chain constant domain 2 (CH2), an immunoglobulin heavychain constant domain 3 (CH3), and optionally an immunoglobulin heavy chain constant domain 4 (CH4). Heavy chains are classified as mu (p), delta (A), gamma (y), alpha (a), and epsilon (c), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG-class and IgA-class antibodies are further divided into subclasses, namely, IgGl, IgG2, IgG3, and IgG4. and IgAl and IgA2, respectively. The heavy chains in IgG, IgA, and IgD antibodies have three constant domains (CHI, CH2, and CH3), whereas the heavy chains in IgM and IgE antibodies have four constant domains (CHI, CH2, CH3, and CH4). The immunoglobulin heavy chain constant domains can be from any immunoglobulin isoty pe, including subtypes. The antibody chains are linked together via inter-polypeptide disulfide bonds between the CL domain and the CHI domain (i.e., between the light and heavy chain) and betw een the hinge regions of the two antibody heavy chains.

[0038] Variable regions of immunoglobulin chains generally exhibit the same overall structure, comprising relatively conserved framework regions (FR) joined by three hypervariable regions, more often called “complementarity determining regions” or CDRs. The CDRs from the tw o chains of each heavy chain and light chain pair typically are alignedby the framework regions to form a structure that binds specifically to a specific epitope on the target protein. From N-terminus to C-terminus, naturally-occurring light and heavy chain variable regions both typically conform with the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering system has been devised for assigning numbers to amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD). or Chothia & Lesk. 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. The CDRs and FRs of a given antibody may be identified using this system. Other numbering systems for the amino acids in immunoglobulin chains include IMGT® (the international ImMunoGeneTics information system; Lefranc et al., Dev. Comp. Immunol. 29: 185-203; 2005) and AHo (Honegger and Pluckthun. J. Mol. Biol. 309(3):657-670; 2001).

[0039] As used in the context of this disclosure, an “antigen-binding fragment,” used interchangeably herein with “binding fragment” or “antibody fragment,” is a portion of an antibody that lacks at least some of the amino acids present in a full-length heavy’ chain and / or light chain, but which is still capable of specifically binding to an antigen. An antigenbinding fragment includes, but is not limited to, a single-chain variable fragment (scFv), a nanobody (e.g., VH domain of heavy chain only antibodies (e.g., camelid heavy chain antibodies); VHH fragment, see C ortez-Re tamozo et al., Cancer Research, Vol. 64:2853-57, 2004), a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a Fd fragment, and a CDR fragment, and can be derived from any mammalian source, such as human, mouse, rat, rabbit, or camelid. Antigen-binding fragments may compete for binding of a target antigen with an intact antibody, and the fragments may be produced by the modification of intact antibodies (e.g., enzymatic or chemical cleavage) or synthesized de novo using recombinant DNA technologies or peptide synthesis. In some embodiments, the antigenbinding fragment comprises at least one CDR from an antibody that binds to the antigen, for example, the heavy chain CDR3 from an antibody that binds to the antigen. In other embodiments, the antigen-binding fragment comprises all three CDRs from the heavy chain of an antibody that binds to the antigen or all three CDRs from the light chain of an antibody that binds to the antigen. In still other embodiments, the antigen-binding fragment comprises all six CDRs from an antibody that binds to the antigen (three from the heavy chain and three from the light chain).

[0040] Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual“Fc” fragment which contains all but the first domain of the immunoglobulin heavy chain constant region. The Fab fragment contains the variable domains from the light and heavy chains, as well as the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Thus, a “Fab fragment” is comprised of one immunoglobulin light chain (light chain variable region (VL) and constant region (CL)) and the CHI domain and variable region (VH) of one immunoglobulin heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. The “Fd fragment” comprises the VH and CHI domains from an immunoglobulin heavy chain. The Fd fragment represents the heavy chain component of the Fab fragment.

[0041] The “Fc fragment” or “Fc domain” of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. The Fc domain may be an Fc domain from an IgGl, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc domain comprises CH2 and CH3 domains from a human IgGl or human IgG2 immunoglobulin. The Fc domain may retain effector function, such as Clq binding, complement dependent cytotoxicity (CDC). Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC). and phagocytosis. In other embodiments, the Fc domain may be modified to reduce or eliminate effector function.

[0042] A “Fab1fragment” is a Fab fragment having at the C-terminus of the CHI domain one or more cysteine residues from the antibody hinge region.

[0043] A “F(ab')2 fragment” is a bivalent fragment including two Fab' fragments linked by a disulfide bridge between the heavy chains at the hinge region.

[0044] The “Fv” fragment is the minimum fragment that contains a complete antigen recognition and binding site from an antibody. This fragment consists of a dimer of one immunoglobulin heavy chain variable region (VH) and one immunoglobulin light chain variable region (VL) in tight, non-covalent association. It is in this configuration that the three CDRs of each variable region interact to define an antigen binding site on the surface of the VH-VL dimer. A single light chain or heavy chain variable region (or half of an Fv fragment comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site comprising both VH and VL.

[0045] A “single-chain variable fragment” or “scFv fragment” comprises the VH and VL regions of an antibody, wherein these regions are present in a single polypeptide chain, and optionally comprising a peptide linker between the VH and VL regions that enables theY1Fv to form the desired structure for antigen binding (see e.g., Bird et al., Science, Vol. 242:423-426, 1988; and Huston etal., Proc. Natl. Acad. Sci. USA, Vol. 85:5879-5883. 1988).

[0046] A “nanobody’’ is the heavy chain variable region of a heavy-chain antibody. Such variable domains are the smallest fully functional antigen-binding fragment of such heavy-chain antibodies, with a molecular mass of only 15 kDa. See Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004. Functional heavy-chain antibodies devoid of light chains are naturally occurring in certain species of animals, such as nurse sharks, wobbegong sharks, and Camelidae, such as camels, dromedaries, alpacas and llamas. The antigen-binding site is reduced to a single domain, the VHH domain, in these animals. These antibodies form antigen-binding regions using only heavy chain variable region, i.e., these functional antibodies are homodimers of heavy chains only (referred to as “heavy -chain antibodies” or “HCAbs”). Camelized VHH reportedly recombines with IgG2 and IgG3 constant regions that contain hinge, CH2, and CH3 domains and lack a CHI domain. Camelized VHH domains have been found to bind to antigen with high affinity (Desmyter et al., J. Biol. Chem, Vol. 276:26285-90, 2001) and possess high stability in solution (Ewert et al., Biochemistry. Vol. 41 :3628-36, 2002). Methods for generating antibodies having camelized heavy chains are described in, for example, U.S. Patent Publication Nos. 2005 / 0136049 and 2005 / 0037421. Alternative scaffolds can be made from human variable-like domains that more closely match the shark V-NAR scaffold and may provide a framework for a long penetrating loop structure. Human heavy-chain antibodies can be produced by transgenic animals expressing human immunoglobulin genes, such as Uni Ab™ antibodies produced by UniRat™ transgenic rats.

[0047] In some embodiments, proteins of interest may include colony stimulating factors, such as, e.g.. granulocyte colony-stimulating factor (G-CSF). Such G-CSF agents include, but are not limited to, Neupogen® (filgrastim) and Neulasta® (pegfilgrastim). Also included are erythropoiesis stimulating agents (ESA), such as, e.g., Epogen® (epoetin alfa), Aranesp® (darbepoetin alfa), Dynepo® (epoetin delta), Mircera® (methyoxy polyethylene gly col-epoetin beta), Hematide®, MRK-2578, INS-22, Retacrit® (epoetin zeta).Neorecormon® (epoetin beta), Silapo® (epoetin zeta), Binocrit® (epoetin alfa), epoetin alfa Hexal, Abseamed® (epoetin alfa), Ratioepo® (epoetin theta), Eporatio* (epoetin theta), Biopoin® (epoetin theta), epoetin alfa, epoetin beta, epoetin zeta, epoetin theta, and epoetin delta, epoetin omega, epoetin iota, tissue plasminogen activator, and GLP-1 receptor agonists, as well as variants or analogs thereof and biosimilars of any of the foregoing.

[0048] In some embodiments, proteins of interest bind, neutralize and / or interact specifically to one or more CD proteins. HER receptor family proteins, cell adhesion molecules, growth factors, nerve grow th factors, fibroblast growth factors, transforming growth factors (TGF), insulin-like growth factors, osteoinductive factors, insulin and insulin- related proteins, coagulation and coagulation-related proteins, colony stimulating factors (CSFs), other blood and serum proteins blood group antigens; receptors, receptor-associated proteins, growth hormones, growth hormone receptors, T-cell receptors; neurotrophic factors, neurotrophins, relaxins, interferons, interleukins, viral antigens, lipoproteins, integrins, rheumatoid factors, immunotoxins, surface membrane proteins, transport proteins, homing receptors, addressins, regulatory proteins, and immunoadhesins.

[0049] In some embodiments, proteins of interest bind to one of more of the following, alone or in any combination: CD proteins including, but not limited to, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, and CD174, HER receptor family proteins, including, for instance, HER2, HER3, HER4, and the EGF receptor. EGFRvIII. cell adhesion molecules, for example. LFA-1. Mol. pl 50.95, VLA-4. ICAM-1. VC AM, and alpha v / beta 3 integrin. growth factors, including but not limited to, for example, vascular endothelial growth factor (“VEGF”); VEGFR2, growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, grow th hormone releasing factor, parathyroid hormone, mullerian-inhibiting substance, human macrophage inflammatory protein (MIP-1 -alpha), erythropoietin (EPO), nen e growth factor, such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factors, including, for instance, aFGF and bFGF, epidermal growth factor (EGF), Cripto. transforming growth factors (TGF), including, among others, TGF-a and TGF-|3. including TGF-01. TGF- 2. TGF-|33, TGF-|34, or TGF-|35, insulin-like growth factors-I and -II (IGF-I and IGF-II), des(l-3)-IGF-I (brain IGF-I), and osteoinductive factors, insulins and insulin-related proteins, including, but not limited to, insulin, insulin A-chain, insulin B-chain, proinsulin, and insulin-like growth factor binding proteins;(coagulation and coagulation-related proteins, such as, among others, factor VIII, tissue factor, von Willebrand factor, protein C. alpha- 1 -antitrypsin, plasminogen activators, such as urokinase and tissue plasminogen activator (“t-PA”), bombazine, thrombin, thrombopoietin, and thrombopoietin receptor, colony stimulating factors (CSFs), including the following, among others, M-CSF, GM-CSF, and G-CSF, other blood and serum proteins, including but not limited to albumin. IgE, and blood group antigens, receptors and receptor-associated proteins, including, for example, flk2 / flt3 receptor, obesity (OB) receptor, growth hormonereceptors, and T-cell receptors; neurotrophic factors, including but not limited to. bone- derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5. or -6 (NT-3, NT-4. NT-5, or NT-6); relaxin A-chain, relaxin B-chain, and prorelaxin, interferons, including for example, interferon-alpha, -beta, and -gamma, interleukins (ILs), e.g., IL-1 to IL-10, IL-12, IL-15, IL- 17, IL-23, IL-12 / IL-23, IL-2Ra, IL1-R1, IL-6 receptor, IL-4 receptor and / or IL-13 to the receptor, IL-13RA2, or IL-17 receptor, IL-1RAP; viral antigens, including but not limited to, an AIDS envelope viral antigen, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, lung surfactant, tumor necrosis factor-alpha and -beta, enkephalinase, BCMA, IgKappa, ROR-1, ERBB2, mesothelin, RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-associated peptide, DNase, FR-alpha. inhibin, and activin, integrin, protein A or D, rheumatoid factors, immunotoxins, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane proteins, decay accelerating factor (DAF), AIDS envelope, transport proteins, homing receptors, MIC (MIC -a, MIC-B), ULBP 1-6, EPCAM, addressins, regulatory7proteins, immunoadhesins, antigen-binding proteins, somatropin, CTGF, CTLA4. eotaxin-1, MUC1, CEA, c-MET, Claudin-18, GPC-3. EPHA2, FPA. LMP1. MG7, NY-ESO-1, PSCA, ganglioside GD2. ganglioside GM2, BAFF, OPGL (RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, c-Kit, B7RP-1, PSMA, NKG2D-1, programmed cell death protein 1 and ligand, PD1 and PDL1, mannose receptor / hCGp, hepatitis-C virus, mesothelin dsFv[PE38] conjugate. Legionella pneumophila (lly). IFN gamma, interferon gamma induced protein 10 (IP10), IFNAR, TALL-1 , thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin / Kexin Type 9 (PCSK9), stem cell factors, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, a4p7, platelet specific (platelet glycoprotein Ilb / IIIb (PAC-1), transforming growth factor beta (TFGP), Zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet derived growth factor receptor alpha (PDGFRa), sclerostin, and biologically active fragments or variants of any of the foregoing.

[0050] In some embodiments, proteins of interest include abciximab, adalimumab, adecatumumab, aflibercept, alemtuzumab, alirocumab, anakinra, atacicept, basiliximab, belimumab, bemarituzumab. bevacizumab, biosozumab, blinatumomab, brentuximab vedotin, brodalumab, cantuzumab mertansine, canakinumab, cetuximab, certolizumab pegol, conatumumab, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, epratuzumab, erenumab, etanercept, evolocumab, galiximab, ganitumab, gemtuzumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, lerdelimumab, lumiliximab. Ixdkizumab, mapatumumab, motesanib diphosphate, muromonab-CD3, natalizumab, nesiritide,nimotuzumab, nivolumab, ocrelizumab, ofatumumab, omalizumab, oprelvekin, ordeskimab, palivizumab. panitumumab, pembrolizumab, pertuzumab, pexelizumab, ranibizumab, rilotumumab, rituximab, romiplostim, romosozumab, sargamostim, tocilizumab, tositumomab, tarlatamab, trastuzumab, ustekinumab, vedolizumab, visilizumab, volociximab, zanolimumab, and zalutumumab, as well as biosimilars of any of the foregoing.

[0051] In some embodiments, a recombinant protein produced by a method described herein is selected from bemarituzumab, denosumab, erenumab. evolocumab. ordeskimab, panitumumab, romosozumab, and tarlatamab.

[0052] In some embodiments, proteins of interest can also include genetically engineered receptors, such as, e.g., chimeric antigen receptors (CARs) and T-cell receptors (TCRs), as well as other proteins comprising an antigen binding molecule that interacts with that targeted antigen. CARs can be engineered to bind to an antigen (such as, e.g., a cell-surface antigen) by incorporating an antigen-binding molecule that interacts with that targeted antigen. CARs ty pically incorporate an antigen binding domain (such as scFv) in tandem with one or more costimulatory ( "signaling") domains and one or more activating domains.

[0053] In some embodiments, the protein of interest has a positive charge at pH 4.5-5.5 or pH 4.7-5.3 or pH 4.8-5.2. A highly positive charge on the protein of interest can contribute to higher binding capacities and / or high viscosity within the column during elution. In some embodiments, the surface of the protein of interest includes amino acids with positively charged side chains (e.g., arginine, histidine, lysine). In some embodiments, the protein of interest has a pl of 7.9-9. 1. In some embodiments, the protein of interests has a highly positive charge at pH 4.7-5.3. In some embodiments, the surface of the protein has at least about 100 amino acids with positively charged side chains. In some embodiments, the protein as a net positive charge of about 44 to about 67 at a pH between 4.7 and 5.3. In some embodiments, the protein as a net positive charge of about 53.5 ± 0.5. The proportion of positively charged side chains on the protein or the charge of the protein can be estimated using software such as Molecular Operating Environment (MOE).

[0054] In some embodiments, the protein of interest is hydrophobic. In some embodiments, the surface of the protein of interest includes amino acids that are hydrophobic (e.g., glycine, alanine, cysteine, proline, leucine, isoleucine, methionine, tryptophan, phenylalanine). Hydrophobic of amino acids at the surface of the protein can contribute to higher binding capacities and / or high viscosity within the column during elution. For example, hydrophobic amino acids on the surface can contribute to stronger hydrophobicinteractions with the column or to stronger hydrophobic interactions among the protein after elution.

[0055] By ‘'purify’; ‘'purified’ or '‘purifying” is meant increasing the degree of purity of the recombinant protein in the composition by removing (partially or completely) at least one product-related impurity from the composition. Recovery and purification of recombinant proteins is accomplished by the downstream unit operations, in particular, those operations involving ion exchange chromatography, resulting in a more “homogeneous” recombinant protein compositions that meets yield and product qualify targets.

[0056] Product-related impurity refers to product-related variants of the protein of interest and include, for example, homodimers, high molecular weight (HMW) species, half antibodies, aggregates, low molecular weight (LMW) species, antibody fragments and various combinations of antibody fragments, and light chain mis-assemblies, such as 2XLC, 3XLC, or 4XLC. Process-related impurities refers to cell substrates, host cell proteins, cellular DNA, leached Protein A, cell culture or chromatography components such as media components, antibiotics, inducers, solvents, buffer components, and the like.

[0057] Expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes that comprise one or more polynucleotides encoding an antigen binding protein of interest as provided herein, as well host cells comprising such expression systems or constructs. As used herein, “vector” means any molecule or entity (e.g.. nucleic acid, plasmid, bacteriophage, transposon, cosmid. chromosome, virus, virus capsid, virion, naked DNA, complexed DNA and the like) suitable for use to transfer and / or transport protein encoding information into a host cell and / or to a specific location and / or compartment within a host cell. Vectors can include viral and non-viral vectors, non-episomal mammalian vectors. Vectors are often referred to as expression vectors, for example, recombinant expression vectors and cloning vectors. The vector may be introduced into a host cell to allow replication of the vector itself and thereby amplify the copies of the polynucleotide contained therein. The cloning vectors may contain sequence components that generally include, without limitation, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements may be selected as appropriate by a person of ordinary skill in the art.

[0058] “Cell” or “Cells” include any prokaryotic or eukary otic cell. Cells can be either ex vivo, in vitro, or in vivo, either separate from or as a part of a higher structure such as a tissue or organ. Cells include “host cells”, also referred to as “cell lines”, which are genetically engineered to express an protein of commercial or scientific interest. Host cellsare typically derived from a lineage arising from a primary culture that can be maintained in culture for an unlimited time. Genetically engineering the host cell involves transfecting, transforming or transducing the cells with a recombinant polynucleotide molecule, and / or otherw ise altering (e.g., by homologous recombination and gene activation or fusion of a recombinant cell with a non-recombinant cell) to cause the host cell to express a desired recombinant protein. Methods and vectors for genetically engineering cells and / or cell lines to express proteins of interest are well known to those of skill in the art.

[0059] A host cell can be any prokaryotic cell (for example, E. coll) or eukaryotic cell (for example, yeast, insect, or animal cells (e.g., CHO cells)). Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.

[0060] Host cells, when cultured under appropriate conditions, express the protein of interest that can be subsequently collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). The selection of an appropriate host cell will depend upon various factors, such as desired expression levels, protein modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule.

[0061] By “culture” or “culturing” is meant the growth and propagation of cells outside of a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. Cell culture media and tissue culture media are interchangeably used to refer to media suitable for growth of a host cell during in vitro cell culture. Typically, cell culture media contains a buffer, salts, energy source, amino acids, vitamins and trace essential elements. Any media capable of supporting growth of the appropriate host cell in culture can be used. Cell culture media, which may be further supplemented with other components to maximize cell growth, cell viability, and / or recombinant protein production in a particular cultured host cell, are commercially available and include RPMI-1640 Medium, RPMI-1641 Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K Medium, Ham's F12 Medium, Iscove's Modified Dulbecco's Medium, McCoy's 5 A Medium, Leibovitz's L-15 Medium, and serum-free media such as EX-CELL™ 300 Series, among others, which can be obtained from the American Type Culture Collection or SAFC Biosciences, as well as other vendors. Cell culture media can be serum-free, protein-free, growth factor-free, and / or peptone-free media. Cell culture may also be enriched by the addition of nutrients and used at greater than its usual, recommended concentrations.

[0062] Various media formulations can be used during the life of the culture, for example, to facilitate the transition from one stage (e.g., the growth stage or phase) to another (e.g., the production stage or phase) and / or to optimize conditions during cell culture (e.g. concentrated media provided during perfusion culture). A growth medium formulation can be used to promote cell growth and minimize protein expression. A production medium formulation can be used to promote production of the protein of interest and maintenance of the cells, with a minimal of new cell growth). A feed media, typically a media containing more concentrated components such as nutrients and amino acids, which are consumed during the course of the production phase of the cell culture may be used to supplement and maintain an active culture, particularly a culture operated in batch, fed batch, semi- continuous, continuous, or perfusion mode. Such a concentrated feed medium can contain most of the components of the cell culture medium at, for example, about 5x, 6x, 7x, 8x, 9x, 10x, 12x, 14x, 16x, 20x, 30 , 50*, 100x, 200 , 400x, 600*, 800 , or even about 1000x of their normal amount.

[0063] A growth phase may occur at a higher temperature than a production phase. For example, a growth phase may occur at a first temperature from about 35° C. to about 38° C.. and a production phase may occur at a second temperature from about 29° C. to about 37° C., optionally from about 30° C. to about 36° C. or from about 30° C. to about 34° C. In addition, chemical inducers of protein production, such as, for example, caffeine, butyrate, and hexamethylene bisacetamide (HMBA), may be added at the same time as, before, and / or after a temperature shift. If inducers are added after a temperature shift, they can be added from one hour to five days after the temperature shift, optionally from one to two days after the temperature shift.

[0064] Host cells may be cultured in suspension or in an adherent form, attached to a solid substrate. Cell cultures can be established in fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors, with or without microcarriers

[0065] Cell cultures can be operated in a batch, fed batch, continuous, semi-continuous, perfusion mode, or combination of modes. Mammalian cells, such as CHO cells, may be cultured in bioreactors at a smaller scale of less than 100 mL to less than 1000 mLs. Alternatively, larger scale bioreactors that contain 1000 mLs to over 20,000 liters of media can be used. Large scale cell cultures, such as for clinical and / or commercial scale biomanufacturing of protein therapeutics, may be maintained for weeks and even months, while the cells produce the desired protein(s).

[0066] The resulting expressed recombinant proteins can then be harvested from the cell culture media. Methods for harvesting proteins from suspension cells are known in the art and include, but are not limited to, acid precipitation, accelerated sedimentation such as flocculation, separation using gravity, centrifugation, acoustic wave separation, filtration, including membrane filtration, using ultrafilters, microfilters, tangential flow filters, alternative tangential flow, depth, and alluvial filtration filters. Recombinant proteins expressed by prokaryotes are retrieved from inclusion bodies in the cytoplasm by redox folding processes known in the art.

[0067] The harv ested recombinant proteins can then be purified, or partially purified, away from any impurities, such as process-related impurities, cell culture media, cell extracts, undesired components, host cell proteins, improperly expressed proteins, product-related impurities, and the like, through one or more downstream unit operations.

[0068] Purification of the recombinant proteins from the harvested cell culture fluid typically begins with capture chromatography. Capture chromatography makes use of mediums, such as resins, membranes, gels and the like, that will bind to the recombinant protein, for example affinity chromatography, immobilized metal affinity chromatography (IMAC), and the like. Such materials are known in the art and are commercially available. Affinity' chromatography options may comprise a substrate-binding capture mechanism, an aptamer-binding capture mechanism, or a cofactor-binding capture mechanism, for example. For proteins having a Fc component, an antibody- or antibody fragment-binding capture mechanism such as Protein L, Protein A, Protein A / L, Protein G, or Protein A / G, for example, can be used. The recombinant protein of interest can also be tagged with a polyhistidine tag or an epitope, such a FLAG® and subsequently purified by using a specific antibody directed to such epitope.

[0069] At any point in the downstream process one or more virus inactivation and / or virus filtration unit operations can be performed to remove viral matter from the composition comprising the recombinant protein. One method for achieving virus inactivation is by incubation at low pH. Low pH virus inactivation can be followed with a neutralization operation that readjusts the viral inactivated solution to a pH more compatible with the requirements of the follow ing unit operations. Other methods for viral inactivation include use of solvents and / or detergents. Viral inactivated or neutralized viral inactivated pools may also be followed by filtration, such as depth filtration, to remove any resulting turbidity or precipitation. Viral filtration can be performed using suitably sized micro- or nano-filters, such as those available from Asahi Kasei (Plavona®) and EDM Millipore (VPro®).

[0070] The term "polishing" is used herein to refer to one or more chromatographic unit operations performed to remove remaining contaminants and impurities such as DNA, host cell proteins, product-related impurities, variant products and aggregates, process-related impurities, and biological contaminants, from a fluid composition comprising the recombinant protein that is close to a final desired urity. Examples of such chromatography operations include ion exchange (IEX) chromatography, including anion exchange (AEX) chromatography and / or cation exchange (CEX) chromatography; hydrophobic interaction chromatography (HIC); mixed modal or multimodal (MM) chromatography, hydroxyapatite (HA) chromatography; reverse phase chromatography (RPC), size exclusion chromatography (SEC), and gel filtration. Chromatography media include resins and membrane absorbers.

[0071] Cation exchange chromatography refers to chromatography performed on a solid phase medium that is negatively charged and has free cations for exchange with cations in an aqueous solution passed over or through the solid phase. The charge may be provided by attaching one or more charged ligands to the solid phase, e.g. by covalent linking. Alternatively, or in addition, the charge may be an inherent property of the solid phase (e.g. as is the case for silica, which has an overall negative charge). Commercially available cation exchange media are available and include but are not limited to FRACTOGEL-SO3™, FRACTOGEL-SE HICAP™, and FRACTOPREP™ (EMD Merck), SP-SEPHAROSE FAST FLOW™, SP-SEPHAROSE FAST FLOW XL™ or SP-SEPHAROSE HIGH PERFORMANCE™ (GE Healthcare), CAPTO S™, CAPTO SP ImpRes™, CAPTO S ImpAct™ (GE Healthcare), Eshmuno®-CPTF. In some embodiments, the cation exchange chromatography medium is a resin. In some embodiments, the cation ion exchange chromatography medium comprises a polymer porous resin. In some embodiments, the cation ion exchange chromatography medium comprises an agarose porous resin. In some embodiments, the cation ion exchange chromatography medium comprises a tentacular resin.

[0072] As described herein, the cation exchange chromatography unit operation is performed in bind and elute mode. The protein (e.g. , a recombinant protein) is loaded onto the medium in a loading buffer such that it binds to the medium. By “binding" the protein (e.g.. a recombinant protein) to the cation exchange medium is meant exposing the protein to the cation exchange medium under appropriate conditions (e g. pH, ionic strength, and / or conductivity) such that the protein (e.g, a recombinant protein) can be reversibly immobilized in or on the cation exchange medium by virtue of ionic interactions between the protein of interest and a charged group or charged groups of the cation exchange medium. For example, binding includes exposing the protein to the cation exchange medium a first setof conditions (e.g, pH, ionic strength, and / or conductivity) such that the protein can be reversibly immobilized in or on the cation exchange groups by virtue of ionic interactions between positive charge on the protein of interest and a negatively charged group or groups of the cation exchange medium. The protein can then be eluted or released from the cation exchange medium under a second set conditions (e.g, pH, ionic strength, and / or conductivity).

[0073] The protein can bind to or release from the cation exchange medium by exposure of the protein and / or cation exchange medium to different buffer solutions (e.g, equilibration buffers, loading buffers, wash buffers, elution buffers). In some embodiments, one or more of the buffers can include one or more salts that can be used to modify pH and / or conductivity. In some embodiments, one or more of the buffers can include a salt of a weak acid (e.g, sodium citrate) that can maintain the pH of the buffer (e.g. within ±0.2). In some embodiments, the concentration of salt of a weak acid (e.g., sodium citrate) can be selected to maintain the pH of the buffer solution. In some embodiments, one or more of the buffers can include one or more salts (e.g, sodium chloride). In some embodiments, the concentration of salt (e.g. sodium chloride) can be selected to obtain a particular ionic strength. In some embodiments, adjusting the concentration of salt (e.g, sodium chloride), thereby adjusting the ionic strength, can alter the ionic interaction between the protein and the cation exchange medium (e.g., increasing concentration of salt can decrease ionic interactions between the protein and the cation exchange medium, thereby releasing the protein).

[0074] The protein (e g , a recombinant protein) may have originated from a previous unit operation, such as affinity chromatography, neutralized low pH viral inactivation, depth filtration, or a harvest and / or a polish chromatography operation. Typically, the loading buffer is the same or similar buffer formulation in which the protein (e.g, a recombinant protein) was obtained from the previous unit operation, for example, a chromatography elution buffer. The loading buffer may be adjusted for a desired pH and / or conductivity prior to loading. In some embodiments the pH of the loading buffer is from 4.0 to 6.0. In some embodiments the pH of the loading buffer is from 4.5 to 5.5. In some embodiments the pH of the loading buffer is from 4.7 to 5.3. In some embodiments the pH of the loading buffer is from 4.8 to 5.2. In some embodiments the pH of the loading buffer is 5.0. In some embodiments, the pH of the loading buffer is such that the protein of interest has a positive charge in the loading buffer. Additional buffer(s) may be added such that the final load of the recombinant protein is at a desired concentration.

[0075] For loading, the protein (e.g. , a recombinant protein) may be in the form of an effluent stream from a previous unit operation that may be loaded in a connected and semi- continuous or continuous manner. The load may be in the form of an eluant pool collected from a previous unit operation that may be loaded in a batch, multi-batch, semi-continuous, or continuous mode.

[0076] Prior to loading of the protein (e.g., a recombinant protein) on to the cation exchange chromatography medium, the chromatography medium can be equilibrated with an equilibration buffer. The equilibration buffer may have the same or similar composition as the loading buffer. In some embodiments the equilibration buffer includes a citrate buffer and sodium chloride. In some embodiments the equilibration buffer formulation is the same as at least one wash buffer formulation, but without sucrose. The equilibration buffer may have the same or similar pH as the loading buffer. In some embodiments the pH of the equilibration buffer is from 4.5 to 5.5. In some embodiments the pH of the equilibration buffer is from 4.7 to 5.3. In some embodiments the pH range of the equilibration buffer is 4.8 to 5.2. In some embodiments the pH of the equilibration buffer is 5.0. In some embodiments the equilibration buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride. pH 4.7 to 5.3. In some embodiments the equilibration buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8 to 5.2. In some embodiments the equilibration buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, pH 5.0. In some embodiments the equilibration buffer comprises 20mM sodium citrate, 50mM sodium chloride, pH 4.7 to 5.3. In some embodiments the equilibration buffer comprises 20mM sodium citrate, 50mM sodium chloride, pH 4.8 to 5.2. In some embodiments the equilibration buffer comprises 20mM sodium citrate, 50mM sodium chloride, pH 5.0. In some embodiments the equilibration buffer comprises 40mM sodium citrate, 50mM sodium chloride, pH 4.7 to 5.3. In some embodiments the equilibration buffer comprises 40mM sodium citrate, 50mM sodium chloride, pH 4.8 to 5.2. In some embodiments the equilibration buffer comprises 40mM sodium citrate, 50mM sodium chloride, pH 5.0.

[0077] The loaded cation exchange chromatography medium is subjected to at least one wash step. The loaded cation exchange chromatography medium is subjected to at least one wash step that includes a wash buffer that includes sucrose. The wash step(s) typically take place following loading and prior to elution of the protein of interest. The w ash step(s) may be used for equilibrating the loaded chromatography medium, pre-equilibrating prior to elution, removing or at least reducing those weakly bound or free impurities and / or contaminants that have less or no affinity for the chromatography medium, prior to elution.In some embodiments at least one wash buffer comprises sodium citrate and sodium chloride. In some embodiments at least one wash buffer comprises 10-40mM sodium citrate and 0- 50mM sodium chloride. At least one wash buffer may have the same or similar pH as the loading buffer. In some embodiments the pH of at least one wash buffer is from 4.5 to 5.5. In some embodiments the pH of at least one wash buffer is from 4.7 to 5.3. In some embodiments the pH of at least one wash buffer is from 4.8 to 5.2. In some embodiments the pH of the wash buffer is 5.0. In some embodiments at least one wash buffer includes 10% to 50% v / v sucrose. In some embodiments at least one wash buffer includes 10% to 50% w / v sucrose. In some embodiments at least one wash buffer includes 10% to 20% v / v sucrose. In some embodiments at least one wash buffer includes 10% to 20% w / v sucrose. In some embodiments at least one wash buffer includes 10% w / v sucrose. In some embodiments at least one wash buffer includes 20% w / v sucrose. In some embodiments, at least one wash buffer includes 10%, 20%, 25%, 30%, 35%, 50% w / v sucrose or any sucrose concentration within a range bounded by any two values disclosed herein.

[0078] In some embodiments, adding sucrose to a wash buffer can alter the measured conductivity of the wash buffer. For example, adding sucrose can decrease the measured conductivity of the wash buffer. However, addition of sucrose (and associated change in measured conductivity ) does not alter the concentrations of sodium chloride at which the protein of interest binds. Therefore, when adding sucrose to a wash buffer, it is generally not necessary’ to change the concentration of sodium chloride in the wash buffer. Instead, the acceptance criteria for the conductivity of the wash buffer may be modified. A modified acceptance criteria can be determined by measuring the conductivity’ of the w ash buffer at different concentrations of sucrose to determine how' sucrose alters the measured conductivity.

[0079] In some embodiments at least one wash buffer comprises 20mM sodium citrate, 50mM sodium chloride, pH 4.7 to 5.3, and 10% to 20% v / v sucrose. In some embodiments at least one wash buffer comprises 20mM sodium citrate, 50mM sodium chloride, pH 4.7 to 5.3, and 10% to 20% w / v sucrose. In some embodiments at least one wash buffer comprises 20mM sodium citrate, 50mM sodium chloride pH 4.7 to 5.3. and 10% to 50% w / v sucrose. In some embodiments at least one wash buffer comprises 20mM sodium citrate, 50mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% v / v sucrose. In some embodiments at least one w ash buffer comprises 20mM sodium citrate, 50mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% w / v sucrose. In some embodiments at least one wash buffer comprises 20mM sodium citrate, 50mM sodium chloride, pH 4.8 to 5.2, and 10% to 50% w / v sucrose. In someembodiments at least one wash buffer comprises 20mM sodium citrate, 50mM sodium chloride, pH 5.0 and 10% v / v sucrose. In some embodiments at least one wash buffer comprises 20mM sodium citrate, 50mM sodium chloride, pH 5.0 and 10% w / v sucrose. In a related embodiment, the measured conductivity of the wash buffer without sucrose is 6.0 to 7.0 mS / cm. In a related embodiment, the measured conductivity7of the wash buffer with sucrose is 2.0 to 6.0 mS / cm. In some embodiments at least one wash buffer comprises 20mM sodium citrate. 50mM sodium chloride, pH 5.0. and 20% v / v sucrose. In some embodiments at least one wash buffer comprises 20mM sodium citrate, 50mM sodium chloride, pH 5.0, and 20% w / v sucrose. In a related embodiment, the measured conductivity7of the wash buffer without sucrose is 5.0 to 6.0 mS / cm. In a related embodiment, the measured conductivity of the wash buffer with sucrose is 2.0 to 6.0 mS / cm.

[0080] In some embodiments at least one wash buffer comprises 40mM sodium citrate, 50mM sodium chloride, pH 4.7 to 5.3, and 10% to 20% v / v sucrose. In some embodiments at least one wash buffer comprises 40mM sodium citrate, 50mM sodium chloride, pH 4.7 to 5.3, and 10% to 20% w / v sucrose. In some embodiments at least one wash buffer comprises 40mM sodium citrate, 50mM sodium chloride, pH 4.7 to 5.3. and 10% to 50% w / v sucrose. In some embodiments at least one wash buffer comprises 40mM sodium citrate, 50mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% v / v sucrose. In some embodiments at least one wash buffer comprises 40mM sodium citrate, 50mM sodium chloride, pH 4.8 to 5.2. and 10% to 20% w / v sucrose. In some embodiments at least one wash buffer comprises 40mM sodium citrate, 50mM sodium chloride, pH 4.8 to 5.2, and 10% to 50% w / v sucrose. In some embodiments at least one wash buffer comprises 40mM sodium citrate, 50mM sodium chloride, pH 5.0 and 10% v / v sucrose. In some embodiments at least one wash buffer comprises 40mM sodium citrate, 50mM sodium chloride. pH 5.0 and 10% w / v sucrose. In a related embodiment, the measured conductivity of the wash buffer without sucrose is 6.0 to 7.0 mS / cm. In a related embodiment, the measured conductivity of the w ash buffer with sucrose is 2.0 to 6.0 mS / cm. In some embodiments at least one wash buffer comprises 40mM sodium citrate, 50mM sodium chloride, pH 5.0, and 20% v / v sucrose. In some embodiments at least one wash buffer comprises 40mM sodium citrate. 50mM sodium chloride, pH 5.0. and 20% w / v sucrose. In a related embodiment, the measured conductivity7of the wash buffer without sucrose is 5.0 to 6.0 mS / cm. In a related embodiment, the measured conductivity of the w ash buffer with sucrose is 2.0 to 6.0 mS / cm.

[0081] In some embodiments at least one wash buffer comprises 10-40mM sodium citrate and 0-50mM sodium chloride, pH 4.7 to 5.3, and 10% to 20% v / v sucrose. In someembodiments at least one wash buffer comprises 10-40mM sodium citrate and 0-50mM sodium chloride. pH 4.7 to 5.3, and 10% to 20% w / v sucrose. In some embodiments at least one wash buffer comprises 10-40mM sodium citrate and 0-50mM sodium chloride, pH 4.7 to 5.3, and 10% to 50% w / v sucrose. In some embodiments at least one wash buffer comprises 10-40mM sodium citrate and 0-50mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% v / v sucrose. In some embodiments at least one wash buffer comprises 10-40mM sodium citrate and 0-50mM sodium chloride. pH 4.8 to 5.2, and 10% to 20% w / v sucrose. In some embodiments at least one wash buffer comprises 10-40mM sodium citrate and 0-50mM sodium chloride, pH 4.8 to 5.2, and 10% to 50% w / v sucrose. In some embodiments at least one wash buffer comprises 10-40mM sodium citrate and 0-5 OmM sodium chloride, pH 5.0 and 10% v / v sucrose. In some embodiments at least one wash buffer comprises 10-40mM sodium citrate and 0-50mM sodium chloride, pH 5.0 and 10% w / v sucrose. In a related embodiment, the measured conductivity of the wash buffer without sucrose is 6.0 to 7.0 mS / cm. In a related embodiment, the measured conductivity of the wash buffer with sucrose is 2.0 to 6.0 mS / cm. In some embodiments at least one wash buffer comprises 10-40mM sodium citrate and 0-50mM sodium chloride, pH 5.0. and 20% v / v sucrose. In some embodiments at least one wash buffer comprises 20mM sodium citrate, 50mM sodium chloride, pH 5.0, and 20% w / v sucrose. In a related embodiment, the measured conductivity of the wash buffer without sucrose is 5.0 to 6.0 mS / cm. In a related embodiment, the measured conductivity of the wash buffer with sucrose is 2.0 to 6.0 mS / cm.

[0082] In some embodiments, a wash buffer containing sucrose does not contain a nonionic polymer, in particular, the elution buffer containing sucrose does not contain poly (ethylene glycol) (PEG), poly (propylene glycol) (PPG), PEG-PPG copolymers, or triblock copolymers composed of poly (oxypropylene) (poly (propylene oxide)) flanked by poly (oxy ethylene) (poly (ethylene oxide).

[0083] Following one or more washes, the bound recombinant protein is released from the solid phase of the cation exchange chromatography medium by isocratic step gradient elution. The recombinant buffer is released using an elution buffer that includes sucrose. In some embodiments the elution buffer comprises sodium citrate and sodium chloride. The elution buffer may have the same or similar pH as the loading buffer. In some embodiments the pH of the elution buffer is from 4.5 to 5.5. In some embodiments the pH of the elution buffer is from 4.7 to 5.3. In some embodiments the pH of the elution buffer is from 4.8 to 5.2. In some embodiments the pH of the elution buffer is 5.0. In some embodiments the elution buffer includes 10% to 50% v / v sucrose. In some embodiments the elution bufferincludes 10% to 50% w / v sucrose. In some embodiments the elution buffer includes 10% to 20% v / v sucrose. In some embodiments the elution buffer includes 10% to 50% w / v sucrose. In some embodiments the elution buffer includes 10% to 20% w / v sucrose. In some embodiments the elution buffer includes 10% v / v sucrose. In some embodiments the elution buffer includes 10% w / v sucrose. In some embodiments the elution buffer includes 20% v / v sucrose. In some embodiments the elution buffer includes 20% w / v sucrose. In some embodiments, at least one elution buffer includes 10%, 20%, 25%, 30%, 35%, 50% w / v sucrose or any sucrose concentration within a range bounded by any two values disclosed herein.

[0084] In some embodiments, adding sucrose to an elution buffer can alter the measured conductivity of the elution buffer. For example, adding sucrose can decrease the measured conductivity of the elution buffer. However, addition of sucrose (and associated change in measured conductivity) does not alter the concentrations of sodium chloride at which the protein of interest is eluted. Therefore, when adding sucrose to an elution buffer, it is generally not necessary to change the concentration of sodium chloride in the elution buffer. Instead, the acceptance criteria for the conductivity of the elution buffer may be modified. A modified acceptance criteria can be determined by measuring the conductivity of the elution buffer at different concentrations of sucrose to determine how sucrose alters the measured conductivity.

[0085] In some embodiments the elution buffer comprises 20mM sodium citrate. 300mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% v / v sucrose. In some embodiments the elution buffer comprises 20mM sodium citrate, 300mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% w / v sucrose. In some embodiments the elution buffer comprises 20mM sodium citrate, 300mM sodium chloride, pH 4.8 to 5.2, and 10% to 50% w / v sucrose. In some embodiments the elution buffer comprises 20mM sodium citrate, 300mM sodium chloride, pH 4.7 to 5.3, and 10% to 20% v / v sucrose. In some embodiments the elution buffer comprises 20mM sodium citrate, 300mM sodium chloride, pH 4.7 to 5.3, and 10% to 20% w / v sucrose. In some embodiments the elution buffer comprises 20mM sodium citrate, 300mM sodium chloride, pH 4.7 to 5.3, and 10% to 50% w / v sucrose. In some embodiments the elution buffer comprises 20mM sodium citrate, 300mM sodium chloride, pH 5.0 and 10% v / v sucrose. In some embodiments the elution buffer comprises 20mM sodium citrate, 300mM sodium chloride, pH 5.0 and 10% w / v sucrose. In a related embodiment, the measured conductivity of the elution buffer without sucrose is 20 to 30 mS / cm In a related embodiment, the measured conductivity of the elution buffer without sucrose is 25.5 to 27.5mS / cm. In a related embodiment, the measured conductivity of the elution buffer with sucrose is 8.0 to 20.5 mS / cm. In some embodiments the elution buffer comprises 20mM sodium citrate, 300mM sodium chloride, pH 5.0 and 20% v / v sucrose. In some embodiments the elution buffer comprises 20mM sodium citrate, 300mM sodium chloride, pH 5.0 and 20% w / v sucrose. In a related embodiment, the measured conductivity of the elution buffer without sucrose is 20 to 22 mS / cm. In a related embodiment, the measured conductivity of the elution buffer with sucrose is 8.0 to 20.5 mS / cm.

[0086] In some embodiments the elution buffer comprises 10-40mM sodium citrate, 270- 500mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% v / v sucrose. In some embodiments the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% w / v sucrose. In some embodiments the elution buffer comprises 10- 40mM sodium citrate, 270-500mM sodium chloride, pH 4.8 to 5.2, and 10% to 50% w / v sucrose. In some embodiments the elution buffer comprises 10-40mM sodium citrate, 270- 500mM sodium chloride, pH 4.7 to 5.3, and 10% to 20% v / v sucrose. In some embodiments the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride. pH 4.7 to 5.3, and 10% to 20% w / v sucrose. In some embodiments the elution buffer comprises 10- 40mM sodium citrate, 270-500mM sodium chloride, pH 4.7 to 5.3, and 10% to 50% w / v sucrose. In some embodiments the elution buffer comprises 10-40mM sodium citrate, 270- 500mM sodium chloride, pH 5.0 and 10% v / v sucrose. In some embodiments the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, pH 5.0 and 10% w / v sucrose. In a related embodiment, the measured conductivity7of the elution buffer without sucrose is 20 to 30 mS / cm. In a related embodiment, the measured conductivity7of the elution buffer without sucrose is 25.5 to 27.5 mS / cm. In a related embodiment, the measured conductivity of the elution buffer with sucrose is 8.0 to 20.5 mS / cm. In some embodiments the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, pH 5.0 and 20% v / v sucrose. In some embodiments the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, pH 5.0 and 20% w / v sucrose. In a related embodiment, the measured conductivity of the elution buffer without sucrose is 20 to 22 mS / cm. In a related embodiment, the measured conductivity of the elution buffer with sucrose is 8.0 to 20.5 mS / cm.

[0087] In some embodiments the elution buffer comprises 10-40mM sodium citrate, 270- 330mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% v / v sucrose. In some embodiments the elution buffer comprises 10-40mM sodium citrate, 270-330mM sodium chloride. pH 4.8 to 5.2, and 10% to 20% w / v sucrose. In some embodiments the elution buffer comprises 10-40mM sodium citrate, 270-330mM sodium chloride, pH 4.8 to 5.2, and 10% to 50% w / v sucrose. In some embodiments the elution buffer comprises 10-40mM sodium citrate, 270- 330mM sodium chloride, pH 4.7 to 5.3, and 10% to 20% v / v sucrose. In some embodiments the elution buffer comprises 10-40mM sodium citrate, 270-330mM sodium chloride, pH 4.7 to 5.3, and 10% to 20% w / v sucrose. In some embodiments the elution buffer comprises 10- 40mM sodium citrate, 270-330mM sodium chloride, pH 4.7 to 5.3, and 10% to 50% w / v sucrose. In some embodiments the elution buffer comprises 10-40mM sodium citrate. 270- 330mM sodium chloride, pH 5.0 and 10% v / v sucrose. In some embodiments the elution buffer comprises 10-40mM sodium citrate, 270-330mM sodium chloride, pH 5.0 and 10% w / v sucrose. In a related embodiment, the measured conductivity of the elution buffer without sucrose is 20 to 30 mS / cm. In a related embodiment, the measured conductivity of the elution buffer without sucrose is 25.5 to 27.5 mS / cm. In a related embodiment, the measured conductivity of the elution buffer with sucrose is 8.0 to 20.5 mS / cm. In some embodiments the elution buffer comprises 10-40mM sodium citrate, 270-330mM sodium chloride, pH 5.0 and 20% v / v sucrose. In some embodiments the elution buffer comprises 10-40mM sodium citrate, 270-330mM sodium chloride, pH 5.0 and 20% w / v sucrose. In a related embodiment, the measured conductivity of the elution buffer without sucrose is 20 to 22 mS / cm. In a related embodiment, the measured conductivity of the elution buffer w ith sucrose is 8.0 to 20.5 mS / cm.

[0088] In some embodiments, the elution buffer does not contain a non-ionic polymer, in particular, the elution buffer does not contain poly (ethylene glycol) (PEG), poly (propylene glycol) (PPG), PEG-PPG copolymers, or triblock copolymers composed of poly (oxypropylene) (poly (propylene oxide)) flanked by poly (oxyethylene) (poly (ethylene oxide).

[0089] In some embodiments, the equilibration buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8 to 5.2, the load buffer has a pH from 4.8 to 5.2, at least one wash buffer comprises 10-40mM sodium citrate and 0-5 OmM sodium chloride, pH 4.8 to 5.2, and 10% to 50% w / v sucrose, and the elution buffer comprises 10-40mM sodium citrate. 270-500mM sodium chloride, pH 4.8 to 5.2. and 10% to 50% w / v sucrose.

[0090] In some embodiments, the equilibration buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8 to 5.2; the load buffer has a pH from 4.8 to 5.2; at least one wash buffer comprises 10-40mM sodium citrate and 0-50mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% w / v sucrose; and the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% w / v sucrose.

[0091] In some embodiments, the equilibration buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8 to 5.2; the load buffer has a pH from 4.8 to 5.2; at least one wash buffer comprises 10-40mM sodium citrate and 0-5 OmM sodium chloride, pH4.7 to 5.2, and 10% to 50% w / v sucrose; and the elution buffer comprises 10-40mM sodium citrate, 270-330mM sodium chloride, pH 4.8 to 5.2, and 10% to 50% w / v sucrose.

[0092] In some embodiments, the equilibration buffer comprises 10-40mM sodium citrate. 0-50mM sodium chloride, pH 4.8 to 5.2; the load buffer has a pH from 4.8 to 5.2; at least one wash buffer comprises 10-40mM sodium citrate and 0-50mM sodium chloride, pH4.8 to 5.2, and 10% to 20% w / v sucrose; and the elution buffer comprises 10-40mM sodium citrate, 270-330mM sodium chloride, pH 4.8 to 5.2, and 10% to 20% w / v sucrose.

[0093] One or more polish chromatography unit operations can be performed before or following the cation exchange chromatography step. Such chromatography operations include anion exchange chromatography, cation exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography.

[0094] The cation exchange chromatography eluate can be subjected to further downstream polish chromatography purification unit operations. In some embodiments, following cation exchange chromatography in bind and elute mode, the protein (e.g., a recombinant protein) is applied to at least one polish chromatography medium in flow- through mode.

[0095] Following the polish chromatography operations, the concentration of the purified protein (e.g., a recombinant protein) and buffer exchange into a desired formulation buffer for bulk storage of the drug substance can be accomplished by an ultrafiltration and diafiltration operation. The bulk drug substance may then be subjected to a fill / finish unit operation to prepare the drug product for distribution and use.

[0096] Critical attributes and performance parameters of the purified protein (e.g. , a recombinant protein) can be measured to better inform decisions regarding performance of each step during manufacture. These critical attributes and parameters can be monitored realtime, near real-time, and / or after the fact. Key critical parameters such as media components that are consumed (such as glucose), levels of metabolic by-products (such as lactate and ammonia) that accumulate, as well as those related to cell maintenance and survival, such as dissolved oxygen content can be measured during cell culture. Critical attributes such as specific productivity, viable cell density, pH, osmolality, appearance, color, aggregation, percent yield and titer may be monitored during appropriated stages in the manufacturingprocess. Monitoring and measurements can be done using known techniques and commercially available equipment.

[0097] The pharmaceutical compositions (solutions, suspensions or the like), may include one or more of the following: buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g.. aluminum hydroxide); and preservatives; sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben: antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0098] While the terminology used in this application is standard within the art, definitions of certain terms are provided herein to assure clarity7and definiteness to the meaning of the claims. Units, prefixes, and symbols may be denoted in their SI accepted form. Numeric ranges recited herein are inclusive of the numbers defining the range and include and are supportive of each integer within the defined range. The methods and techniques described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 3rd ed.. Cold Spring Harbor Laboratory7Press, Cold Spring Harbor, N.Y. (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory7Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990). All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference.

[0099] The present invention is not to be limited in scope by the specific embodiments described herein that are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention.What is described in an embodiment of the invention can be combined with other embodiments of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0100] Provided herein as Embodiment 1 is a method comprising a) loading a recombinant protein to an equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; b) washing the cation exchange chromatography medium wi th at least one wash buffer comprising 10% to 50% w / v sucrose; and c) releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising 10% to 50% w / v sucrose.

[0101] Provided herein as Embodiment 2 is the method of Embodiment 1, wherein at least one wash buffer comprises 10% to 20% w / v sucrose and the elution buffer comprises 10 to 20% w / v sucrose.

[0102] Provided herein as Embodiment 3 is the method of any one of Embodiments 1-2, wherein at least one wash buffer comprises sodium citrate and sodium chloride.

[0103] Provided herein as Embodiment 4 is the method of any one of Embodiments 1-3, wherein at least one wash buffer comprises 10% to 20% w / v sucrose, pH 4.8-5.2.

[0104] Provided herein as Embodiment 5 is the method of any one of Embodiments 1-4, wherein at least one wash buffer comprises 10-40mM sodium citrate. 0-50mM sodium chloride, pH 4.8-5.2.

[0105] Provided herein as Embodiment 6 is the method of any one of Embodiments 1-5, wherein at least one wash buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, 10% w / v sucrose, pH 4.8-5.2.

[0106] Provided herein as Embodiment 7 is the method of any one of Embodiments 1-6, wherein the conductivity of the wash buffer is 6.0-7.0 mS / cm.

[0107] Provided herein as Embodiment 8 the method of any one of Embodiments 1-7, wherein at least one wash buffer comprises 10-40mM sodium citrate. 0-50mM sodium chloride. 20% w / v sucrose, pH 4.8-5.2.

[0108] Provided herein as Embodiment 9 is the method of any one of Embodiments 1-8, wherein the conductivity of the wash buffer is 5.0-6.0 mS / cm.

[0109] Provided herein as Embodiment 10 is the method of any one of Embodiments 1-9, wherein the measured conductivity of the wash buffer is 2.0-6.0 mS / cm.

[0110] Provided herein as Embodiment 11 is the method of any one of Embodiments 1-10, wherein the elution buffer comprises sodium citrate and sodium chloride.

[0111] Provided herein as Embodiment 12 is the method of any one of Embodiments 1-11, wherein the elution buffer comprises 10 to 20% w / v sucrose, pH 4.8-5.2.

[0112] Provided herein as Embodiment 13 is the method of any one of Embodiments 1-12, wherein the elution buffer further comprises 10-40mM sodium citrate, 270-500mM sodium chloride. pH 4.8-5.2.

[0113] Provided herein as Embodiment 14 is the method of any one of Embodiments 1-13, wherein the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, 10% w / v sucrose, pH 4.8-5.2.

[0114] Provided herein as Embodiment 15 is the method of any one of Embodiments 1-14, wherein the conductivity of the elution buffer is 25.5-27.5 mS / cm.

[0115] Provided herein as Embodiment 16 is the method of any one of Embodiments 1-15, wherein the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, 20% w / v sucrose, pH 4.8-5.2.

[0116] Provided herein as Embodiment 17 is the method of any one of Embodiments 1-16, wherein the conductivity of the elution buffer is 20-22 mS / cm.

[0117] Provided herein as Embodiment 18 is the method of any one of Embodiments 1-17, wherein the measured conductivity of elution buffer is 8.0-20.5 mS / cm.

[0118] Provided herein as Embodiment 19 is the method of any one of Embodiments 1-18, wherein the cation exchange chromatography medium is equilibrated with an equilibration buffer having pH 4.8-5.2

[0119] Provided herein as Embodiment 20 is the method of any one of Embodiments 1 -19, wherein the cation exchange chromatography medium is equilibrated with an equilibration buffer comprising 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8- 5.2.

[0120] Provided herein as Embodiment 21 is the method according to any one of Embodiments 1-20, wherein the number of peaks in the cation exchange chromatography elution profile are reduced compared to a method wherein only the wash buffer comprises 10% to 20% w / v sucrose or only the elution buffer comprises 10% to 20% w / v sucrose.

[0121] Provided herein as Embodiment 22 is the method of any one of Embodiments 1- 21, wherein the method results in reduced viscous fingering compared to washing the cation exchange chromatography medium with wash buffer without sucrose and releasing the recombinant protein with an elution buffer without sucrose.

[0122] Provided herein as Embodiment 23 is the method of any one of Embodiments 1-22, wherein the method results in a single peak in the cation exchange chromatography isocratic step gradient elution profile.

[0123] Provided herein as Embodiment 24 is the method of any one of Embodiments 1-23, wherein the cation exchange chromatography medium comprises a polymer porous resin, an agarose porous resin, or a tentacular resin.

[0124] Provided herein as Embodiment 25 is a method comprising a) equilibrating a cation exchange chromatography medium with a buffer comprising sodium citrate and sodium chloride, pH 4.8-5.2; b) loading a recombinant protein on to the equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; c) washing the cation exchange chromatography medium wi th at least one wash buffer comprising sodium citrate, sodium chloride, 10-50% w / v sucrose, pH 4.8-5.2; and d) releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising sodium citrate, sodium chloride, 10% to 50% w / v sucrose, pH 4.8-5.2.

[0125] Provided herein as Embodiment 26 is the method of Embodiment 25. wherein at least one wash buffer comprises 10 to 20% w / v sucrose and the elution buffer comprises 10- 20% w / v sucrose.

[0126] Provided herein as Embodiment 27 is the method of any one of Embodiments 25- 26. wherein the method results in reduced viscous fingering compared to washing the cation exchange chromatography medium with wash buffer comprising sodium citrate and sodium citrate, pH 4.8-5.2, without sucrose and releasing the recombinant protein with an elution buffer comprising sodium citrate, sodium chloride, pH 4.8-5.2, without sucrose.

[0127] Provided herein as Embodiment 28 is a method comprising a) equilibrating a cation exchange chromatography medium with a buffer comprising 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8-5.2; b) loading a recombinant protein on to the equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; c) washing the cation exchange chromatography medium with at least one wash buffer comprising 10-40mM sodium citrate, 0-5 OmM sodium chloride, 10% - 50% w / v sucrose, pH 4.4-5.3; and d) releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising 10-40mM sodium citrate, 270-500mM sodium chloride, 10% - 50% w / v sucrose, pH 4.8-5.2.

[0128] Provided herein as Embodiment 29 is the method of Embodiment 28, wherein at least one wash buffer comprises 10 to 20% w / v sucrose and the elution buffer comprises 10- 20% w / v sucrose.

[0129] Provided herein as Embodiment 30 is the method of any one of Embodiments 28-29, wherein the method results in fewer peaks in the cation exchange chromatography isocratic step gradient elution profile compared to washing the cation exchange chromatography medium with wash buffer comprising sodium citrate and sodium citrate, pH 4 8-5.2, without sucrose and releasing the recombinant protein with an elution buffer comprising sodium citrate, sodium chloride, pH 4.8-5.2, without sucrose.

[0130] Provided herein as Embodiment 31 is the method of any one of Embodiments 28-30, wherein the method results in a single peak in the cation exchange chromatography isocratic step gradient elution profile.

[0131] Provided herein as Embodiment 32 is a method comprising a) establishing a cell culture in a bioreactor with a host cell expressing a protein; b) culturing the host cells to express the protein; c) harvesting the protein from the cell culture; d) affinity purifying the protein; e) equilibrating a cation exchange chromatography medium with a buffer comprising sodium citrate, sodium chloride, pH 4.8-5.2; f) loading the purified protein onto an equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; g) washing the cation exchange chromatography medium with at least one wash buffer comprising sodium citrate, sodium chloride, 10%-20% w / v sucrose, pH 4.8-5.2; and h) releasing recombinant protein by isocratic step gradient elution with an elution buffer comprising sodium citrate, sodium chloride, 10% to 20% w / v sucrose, pH 4.8-5.2 into the effluent stream or collecting in an eluant pool.

[0132] Provided herein as Embodiment 33 is the method of Embodiment 32, wherein at least one wash buffer comprises 10 to 20% w / v sucrose and the elution buffer comprises 10- 20% w / v sucrose.

[0133] Provided herein as Embodiment 34 is the method according to any one of Embodiments 32-33. further comprising one or more unit operations selected from viral inactivation, viral filtration, neutralization, anion exchange chromatography, cation exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, ultrafiltration, diafiltration, depth filtration, sterile filtration, and fill / finish.

[0134] Provided herein as Embodiment 35 is the isolated, purified, recombinant protein produced by any one of Embodiments 23-34.

[0135] Provided herein as Embodiment 36 is a pharmaceutical composition comprising the isolated, purified, recombinant protein produced by the method of Embodiment 35.

[0136] Provided herein as Embodiment 37 is a method for purifying a recombinant protein, comprising a) loading the recombinant protein to an equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; b) washing the cation exchange chromatography medium with at least one wash buffer comprising 10% to 20% v / v sucrose; and c) releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising 10% to 20% v / v sucrose.

[0137] Provided herein as Embodiment 38 is the method of Embodiment 37, wherein at least one wash buffer comprises sodium citrate and sodium chloride.

[0138] Provided herein as Embodiment 39 is the method of any one of Embodiments 37-38, wherein at least one wash buffer comprises 10% to 20% v / v sucrose, pH 4.8-5.2.

[0139] Provided herein as Embodiment 40 is the method of any one of Embodiments 37-39, wherein at least one wash buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride. 10% v / v sucrose. pH 4.8-5.2.

[0140] Provided herein as Embodiment 41 is the method of any one of Embodiments 37-40, wherein at least one wash buffer comprises lOmM sodium citrate, 50mM sodium chloride, pH 4.8-5.2.

[0141] Provided herein as Embodiment 42 is the method of any one of Embodiments 35-41 , wherein at least one wash buffer comprises 20mM sodium citrate, 50mM sodium chloride, 10% v / v sucrose, pH 4.8-5.2.

[0142] Provided herein as Embodiment 43 is the method of any one of Embodiments 37-42, wherein the conductivity of the wash buffer is 6.0-7.0 mS / cm.

[0143] Provided herein as Embodiment 44 is the method of any one of Embodiments 37-43, wherein at least one wash buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, 20% v / v sucrose, pH 4.8-5.2.

[0144] Provided herein as Embodiment 45 is the method of any one of Embodiments 37-44, wherein at least one wash buffer comprises 20mM sodium citrate, 50mM sodium chloride, 20% v / v sucrose, pH 4.8-5.2.

[0145] Provided herein as Embodiment 46 is the method of any one of Embodiments 37-45, wherein the conductivity of the wash buffer is 5.0-6.0 mS / cm.

[0146] Provided herein as Embodiment 47 is the method of any one of Embodiments 37-46, wherein the elution buffer comprises sodium citrate and sodium chloride.

[0147] Provided herein as Embodiment 48 is the method of any one of Embodiments 37-47, wherein the elution buffer comprises 10 to 20% w / v sucrose, pH 4.8-5.2.

[0148] Provided herein as Embodiment 49 is the method of any one of Embodiments 37-48, wherein the elution buffer further comprises 10-40mM sodium citrate, 270-500mM sodium chloride, pH 4.8-5.2.

[0149] Provided herein as Embodiment 50 is the method of any one of Embodiments 37-49, wherein the elution buffer further comprises 20mM sodium citrate, 300mM sodium chloride, pH 4.8-5.2.

[0150] Provided herein as Embodiment 51 is the method of any one of Embodiments 37-50, wherein the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, 10% v / v sucrose. pH 4.8-5.2.

[0151] Provided herein as Embodiment 52 is the method of any one of Embodiments 37-51, wherein the elution buffer comprises 20mM sodium citrate, 300mM sodium chloride, 10% v / v sucrose, pH 4.8-5.2.

[0152] Provided herein as Embodiment 53 is the method of any one of Embodiments 37-52, wherein the conductivity of the elution buffer is 25.5-27.5 mS / cm.

[0153] Provided herein as Embodiment 54 is the method of any one of Embodiments 37-53, wherein the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, 20% v / v sucrose, pH 4.8-5.2.

[0154] Provided herein as Embodiment 55 is the method of any one of Embodiments 37-54, wherein the elution buffer comprises 20mM sodium citrate, 300mM sodium chloride, 20% v / v sucrose, pH 4.8-5.2.

[0155] Provided herein as Embodiment 56 is the method of any one of Embodiments 37-55, wherein the conductivity of the elution buffer is 20-22 mS / cm.

[0156] Provided herein as Embodiment 57 is the method of any one of Embodiments 37-56, wherein the cation exchange chromatography medium is equilibrated with an equilibration buffer having pH 4.8-5.2

[0157] Provided herein as Embodiment 58 is the method of any one of Embodiments 37-57, wherein the cation exchange chromatography medium is equilibrated with an equilibration buffer comprising 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8- 5.2

[0158] Provided herein as Embodiment 59 is the method of any one of Embodiments 37-58, wherein the cation exchange chromatography medium is equilibrated with an equilibration buffer comprising 20mM sodium citrate, 50mM sodium chloride, pH 4.8-5.2.

[0159] Provided herein as Embodiment 60 is the method according to any one of Embodiments 37-59. wherein the number of peaks in the cation exchange chromatography elution profile are reduced compared to a method wherein only the wash buffer comprises 20% v / v sucrose or only the elution buffer comprises 20% v / v sucrose.

[0160] Provided herein as Embodiment 61 is a method for mitigating viscous fingering while purifying a recombinant protein, the method comprising a) equilibrating a cation exchange chromatography medium with a buffer comprising sodium citrate and sodium chloride, pH 4.8-5.2; b) loading the recombinant protein on to the equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; c) washing the cation exchange chromatography medium with at least one wash buffer comprising sodium citrate, sodium chloride, 10% to 20% v / v sucrose, pH 4.8-5.2; and d) releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising sodium citrate, sodium chloride, 10% to 20% v / v sucrose, pH 4.8-5.2.

[0161] Provided herein as Embodiment 62 is the method of any one of Embodiments 61, wherein the method results in reduced viscous fingering compared to washing the cation exchange chromatography medium with wash buffer comprising sodium citrate and sodium citrate, pH 4.8-5.2, without sucrose and releasing the recombinant protein with an elution buffer comprising sodium citrate, sodium chloride, pH 4.8-5.2, without sucrose.

[0162] Provided herein as Embodiment 63 is a method for reducing the number of peaks in the cation exchange chromatography isocratic step gradient elution profile, the method comprising a) equilibrating a cation exchange chromatography medium with a buffer comprising 20mM sodium citrate, 50mM sodium chloride, pH 4.8-5.2; b) loading the recombinant protein on to the equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; c) washing the cation exchange chromatography medium with at least one wash buffer comprising 20mM sodium citrate, 50mM sodium chloride, 10% to 20% v / v sucrose, pH 4.8- 5.2; and d) releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising 20mM sodium citrate, 300mM sodium chloride, 10% - 20% v / v sucrose, pH 4.8-5.2.

[0163] Provided herein as Embodiment 64 is the method of Embodiment 63, wherein the method results in few er peaks in the cation exchange chromatography isocratic step gradient elution profile compared to washing the cation exchange chromatography medium with wash buffer comprising 20mM sodium citrate, 50mM sodium chloride, pH 4.8-5.2, without sucroseand releasing the recombinant protein with an elution buffer comprising 20mM sodium citrate, 300mM sodium chloride, pH 4.8-5.2, without sucrose.

[0164] Provided herein as Embodiment 65 is the method of any one of Embodiments 63- 64, wherein the method results in a single peak in the cation exchange chromatography isocratic step gradient elution profile.

[0165] Provided herein as Embodiment 66 is a method for producing an isolated, purified, recombinant protein, the method comprising a) establishing a cell culture in a bioreactor with a host cell expressing the protein; b) culturing the host cells to express the protein; c) harvesting the recombinant protein from the cell culture; d) affinity purifying the recombinant protein; e) equilibrating a cation exchange chromatography medium with a buffer comprising sodium citrate, sodium chloride, pH 4.8-5.2; f) loading the purified recombinant protein onto an equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; g) washing the cation exchange chromatography medium with at least one wash buffer comprising sodium citrate, sodium chloride. 10% to 20% v / v sucrose, pH 4.8-5.2; and h) releasing recombinant protein by isocratic step gradient elution with an elution buffer comprising sodium citrate, sodium chloride, 10% to 20% v / v sucrose, pH 4.8-5.2 into the effluent stream or collecting in an eluant pool.

[0166] Provided herein as Embodiment 67 is the method according to Embodiment 66, further comprising one or more unit operations selected from viral inactivation, viral filtration, neutralization, anion exchange chromatography, cation exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, ultrafiltration, diafiltrarion, depth filtration, sterile filtration, and fill / finish.

[0167] Provided herein as Embodiment 68 is the isolated, purified, recombinant protein produced by any one of Embodiments 66-67.

[0168] Provided herein as Embodiment 69 is a pharmaceutical composition comprising the isolated, purified, recombinant protein produced by the method of any one of Embodiments 66-68.

[0169] The following examples, including the experiments conducted and the results achieved, are provided for illustrative purposes only and are not to be construed as limiting the scope of the appended claims.

[0170] Any of the embodiments disclosed herein may be properly combined with any other embodiment disclosed herein. The combination of any one the embodiments disclosed herein with any other embodiments is expressly contemplated.

[0171] Although the systems, methods, devices, and components thereof, have been described in terms of exemplary embodiments, they are not limited thereto. The detailed description is to be construed as exemplary only and does not describe every possible embodiment of the invention because describing every possible embodiment would be impractical, if not impossible. Numerous alternative embodiments could be implemented, using either current technology or technology developed after the filing date of this patent that would still fall within the scope of the claims defining the invention.

[0172] Those skilled in the art will recognize that a wide variety of modifications, alterations, and combinations can be made with respect to the above described embodiments without departing from the scope of the invention, and that such modifications, alterations, and combinations are to be viewed as being within the ambit of the inventive concept.EXAMPLESExample 1

[0173] When replacing the chromatography medium used in a purification process for a fully human monoclonal antibody from one strong cation exchanger chromatography resin (Resin 1) (Fractogel® SE Hicap, EMD Millipore, Burlington, MA) to a second strong cation exchanger chromatography resin (Resin 2) (Fractogel® SO EMD Millipore) operating under the same isocratic step elution conditions, it was surprisingly discovered that the elution profile from the second chromatography resin showed multi -peak elution behavior (Figure 1). The monoclonal antibody had a pl of 9. 1. The monoclonal antibody had a net positive charge of + 66.64 at pH 4.7, + 53.7 at pH 5.0. and + 44.89 at pH 5.3. At pH 5.0, the monoclonal antibody had 146 positively charged amino acids that contributed to the net positive charge of the antibody (22 histidine, 90 lysine, and 34 arginine).

[0174] The resulting multi peak elution behavior was further evaluated to study the impact on product quality. Figure 2 shows the monoclonal antibody profile from Resin 2 at different wavelengths, highlighting Fractions #1, #2, and #3. The elution behavior was hypothesized to be due to on-column aggregation based on previous literature (Gau et al., J Chrom a, 1356 (2014) pages 117-128). However, contrary to that theory, no impact was observed on pool product quality characteristics. Later eluting fractions did not show' enrichment of high molecular weight species, as shown in Table 1.Table 1 Analytical results from Fractions #1, #2, and #3

[0175] A correlation between column loading and multi-peak elution behavior was then tested and confirmed viscous fingering phenomenon for the monoclonal antibody under isocratic step gradient elution from Resin 2, see Figure 3. As protein loading is increased (10 g / L-r, 20 g / L-r, 35 g / L-4, 50 g / L-r, 70 g / L-r), a higher amount of protein is loaded on the same column. As a result, the protein concentration in the elution band keeps increasing with loading and thus causing viscous fingering and multipeak elution at higher loads. During elution, the protein band exhibits higher viscosity than the mobile phase surrounding the band. Once the high-viscosity solute plug displaces the low-viscosity mobile phase, the mobile phase at rear end pushes the solute plug creating fingers. Strong cation exchange resins with high binding capacities tend to create high concentration zones (high viscosity) within the column during elution. While both Resin 1 and Resin 2 are strong cation exchangers, the sulfoisobutyl moiety on Resin 2 provides higher hydrophobicity, possibly resulting in stronger monoclonal antibody binding to the resin. The tentacular nature of the Resin 2 matrix coupled with strong binding of the monoclonal antibody on the SOs" moiety possibly resulted in higher concentration zones during protein elution, thus causing viscous fingering during elution, Figure 4.

[0176] Relevant process conditions were evaluated to understand and mitigate the unusual multi-peak elution behavior. A multimodal chromatography eluate pool containing a fully human monoclonal antibody in a citrate buffer was loaded onto a Fractogel® SOs" cation exchange chromatography resin (EMD Millipore) under a variety of conditions as outlined in Table 2. The elution profiles for these parameter variations are shown in Figs. 5A-5K and Figs. 6A-6G. In Figs. 5A-5K and Figs. 6A-6G, the x-axis shows column volumes, while the y-axis shows absorbance units (AU). These parameter variations included four different wash treatments: increasing column volumes (Fig. 5E), increasing salt (Fig. 5F), doubling buffer salt (Fig. 5G). and including 20% w / v sucrose (Fig. 5H). The sequence of the chromatography steps tested was equilibration of the chromatography resin with 20 mMsodium citrate, 50 mM sodium chloride, pH 5.0 and loading the antibody containing pool onto the equilibrated chromatography column. A single wash step was performed, followed by isocratic step gradient elution of the monoclonal antibody from the chromatography column. Unless otherwise indicated in Table 2, the wash buffer was 20mM sodium citrate, 50 mM sodium chloride, pH 5, and the elution buffer was 20 mM sodium citrate, 300 mM sodium chloride, pH 5

[0177] The chromatography resin was regenerated and stored using 0.2 M NaOH.Table 2 Parameters tested

[0178] Of the first 10 parameters tested (shown in Figs. 5A-5I). only the elution pH and the elution salt molarity improved the chromatographic elution profiles. However, both cases still exhibited a two peak elution behavior with significant tailing. It was found that the viscosity of the mobile phase was modulated when sucrose (10%-20% or 10%-50% w / v) was used in both the wash and elution buffer. Sucrose concentrations of 10%, 20%, 25%, 30%, 35%, and 50% w / v for both the wash and elution buffers were tested. Figs. 6A-6F show the chromatograms for sucrose concentrations of 10% (Fig. 6A), 20% (Fig. 6B). 25% (Fig. 6C). 30% (Fig. 6D), 35% (Fig. 6E), 40% (6F), and 50% (Fig. 6G) w / v. In Figs. 6A-6G, the x-axis shows column volumes, while the y-axis shows absorbance units (AU). These concentrations of sucrose resulted in a manufacturing-friendly single elution peak, Figs. 5J-5K, Figs. 6A-6G. As shown in Table 3 below, the back pressure increased as the concentration of sucrose increased. It was found that 10% w / v of sucrose decreased column back pressure while also maintaining a single peak elution profile. As shown in Table 3, addition of sucrose also decreased the measured conductivity of the elution buffer (for an elution buffer of 20mM sodium citrate, 300mM sodium chloride, pH 5.0). Without sucrose, the measured conductivity of the elution buffer was 30 mS / cm. Addition of sucrose, however, did not affect the concentration of sodium chloride needed for binding and elution. Therefore, this change in measured conductivity indicates that, rather than modifying the salt concentration as sucrose is added, the acceptance criteria for conductivity of the elution / wash buffer can be modified.Table 3: Effect of sucrose concentration on column back pressure

Claims

What is claimed is1. A method comprising a) loading a recombinant protein to an equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; b) washing the cation exchange chromatography medium with at least one wash buffer comprising 10% to 50% w / v sucrose; and c) releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising 10% to 50% w / v sucrose.

2. The method of claim 1, wherein at least one wash buffer comprises 10% to 20% w / v sucrose and the elution buffer comprises 10 to 20% w / v sucrose.

3. The method of any one of claims 1-2, wherein at least one wash buffer comprises sodium citrate and sodium chloride.

4. The method of any one of claims 1-3, wherein at least one wash buffer comprises 10% to 20% w / v sucrose, pH 4.8-5.2.

5. The method of any one of claims 1-4, wherein at least one wash buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8-5.2.

6. The method of any one of claims 1-5, wherein at least one wash buffer comprises 10-40mM sodium citrate, 0-5 OmM sodium chloride, 10% w / v sucrose, pH 4.8-5.2.

7. The method of any one of claims 1-6. wherein the conductivity of the wash buffer is 6.0-7.0 mS / cm.

8. The method of any one of claims 1-7, wherein at least one wash buffer comprises 10-40mM sodium citrate, 0-50mM sodium chloride, 20% w / v sucrose, pH 4.8-5.2.

9. The method of any one of claims 1-8, wherein the conductivity of the wash buffer is 5.0-6.0 mS / cm.

10. The method of any one of claims 1-9, wherein the measured conductivity of the wash buffer is 2.0-6.0 mS / cm.

11. The method of any one of claims 1-10, w herein the elution buffer comprises sodium citrate and sodium chloride.

12. The method of any one of claims 1-11. wherein the elution buffer comprises 10 to 20% w / v sucrose, pH 4.8-5.2.

13. The method of any one of claims 1-12, wherein the elution buffer further comprises 10- 40mM sodium citrate, 270-500mM sodium chloride, pH 4.8-5.2.

14. The method of any one of claims 1-13, wherein the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, 10% w / v sucrose, pH 4.8-5.2.

15. The method of any one of claims 1-14. wherein the conductivity of the elution buffer is 25.5-27.5 mS / cm.

16. The method of any one of claims 1-15, wherein the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, 20% w / v sucrose, pH 4.8-5.2.

17. The method of any one of claims 1-16, wherein the conductivity of the elution buffer is 20- 22 mS / cm.

18. The method of any one of claims 1-17, wherein the measured conductivity of elution buffer is 8.0-20.5 mS / cm.

19. The method of any one of claims 1-18, wherein the cation exchange chromatography medium is equilibrated with an equilibration buffer having pH 4.8-5.2.

20. The method of any one of claims 1-19, wherein the cation exchange chromatography medium is equilibrated with an equilibration buffer comprising 10-40mM sodium citrate, 0- 50mM sodium chloride, pH 4.8-5.2.

21. The method according to any one of claims 1-20, wherein the number of peaks in the cation exchange chromatography elution profile are reduced compared to a method wherein only the wash buffer comprises 10% to 20% w / v sucrose or only the elution buffer comprises 10% to 20% w / v sucrose.

22. The method of any one of claims 1-21, wherein the method results in reduced viscous fingering compared to washing the cation exchange chromatography medium with wash buffer without sucrose and releasing the recombinant protein with an elution buffer without sucrose.

23. The method of any one of claims 1-22, wherein the method results in a single peak in the cation exchange chromatography isocratic step gradient elution profile.

24. The method of any one of claims 1-23. wherein the cation exchange chromatography medium comprises a polymer porous resin, an agarose porous resin, or a tentacular resin.

25. A method comprising a) equilibrating a cation exchange chromatography medium with a buffer comprising sodium citrate and sodium chloride, pH 4.8-5.2; b) loading a recombinant protein on to the equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; c) washing the cation exchange chromatography medium with at least one wash buffer comprising sodium citrate, sodium chloride, 10-50% w / v sucrose, pH 4.8-5.2; and d) releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising sodium citrate, sodium chloride, 10% to 50% w / v sucrose, pH 4.8-5.2.

26. The method of claim 25, wherein at least one wash buffer comprises 10 to 20% w / v sucrose and the elution buffer comprises 10-20% w / v sucrose.

27. The method of any one of claims 25-26, wherein the method results in reduced viscous fingering compared to washing the cation exchange chromatography medium with wash buffer comprising sodium citrate and sodium citrate, pH 4.8-5.2, without sucrose and releasing the recombinant protein with an elution buffer comprising sodium citrate, sodium chloride, pH 4.8- 5.2, without sucrose.

28. A method comprising a) equilibrating a cation exchange chromatography medium with a buffer comprising 10-40mM sodium citrate, 0-50mM sodium chloride, pH 4.8-5.2; b) loading a recombinant protein on to the equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium;c) washing the cation exchange chromatography medium with at least one wash buffer comprising 10-40mM sodium citrate, 0-50mM sodium chloride, 10% - 50% w / v sucrose, pH 4.4-5.3; and d) releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising 10-40mM sodium citrate, 270-500mM sodium chloride, 10% - 50% w / v sucrose, pH 4.8-5.2.

29. The method of claim 28, wherein at least one wash buffer comprises 10 to 20% w / v sucrose and the elution buffer comprises 10-20% w / v sucrose.

30. The method of any one of claims 28-29, wherein the method results in fewer peaks in the cation exchange chromatography isocratic step gradient elution profde compared to washing the cation exchange chromatography medium with wash buffer comprising sodium citrate and sodium citrate. pH 4.8-5.2, without sucrose and releasing the recombinant protein with an elution buffer comprising sodium citrate, sodium chloride, pH 4.8-5.2, without sucrose.

31. The method of any one of claims 28-30. wherein the method results in a single peak in the cation exchange chromatography isocratic step gradient elution profile.

32. A method comprising a) establishing a cell culture in a bioreactor with a host cell expressing a protein; b) culturing the host cells to express the protein; c) harvesting the protein from the cell culture; d) affinity purifying the protein; e) equilibrating a cation exchange chromatography medium with a buffer comprising sodium citrate, sodium chloride. pH 4.8-5.2; f) loading the purified protein onto an equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; g) washing the cation exchange chromatography medium with at least one wash buffer comprising sodium citrate, sodium chloride. 10%-20% w / v sucrose, pH 4.8-5.2; andh) releasing recombinant protein by isocratic step gradient elution with an elution buffer comprising sodium citrate, sodium chloride, 10% to 20% w / v sucrose, pH 4.8-5.2 into the effluent stream or collecting in an eluant pool.

33. The method of claim 32, wherein at least one wash buffer comprises 10 to 20% w / v sucrose and the elution buffer comprises 10-20% w / v sucrose.

34. The method according to any one of claims 32-33, further comprising one or more unit operations selected from viral inactivation, viral filtration, neutralization, anion exchange chromatography, cation exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, ultrafiltration, diafiltration, depth filtration, sterile filtration, and fill / finish.

35. The isolated, purified, recombinant protein produced by any one of claims 32-34.

36. A pharmaceutical composition comprising the isolated, purified, recombinant protein produced by the method of claim 35.

37. A method for purifying a recombinant protein, comprising a) loading the recombinant protein to an equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; b) washing the cation exchange chromatography medium with at least one wash buffer comprising 10% to 20% v / v sucrose; and c) releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising 10% to 20% v / v sucrose.

38. The method of claim 37, wherein at least one wash buffer comprises sodium citrate and sodium chloride.

39. The method of any one of claims 37-38, wherein at least one wash buffer comprises 10% to 20% v / v sucrose, pH 4.8-5.2.

40. The method of any one of claims 37-39, wherein at least one wash buffer comprises 10- 40mM sodium citrate, 0-50mM sodium chloride, 10% v / v sucrose, pH 4.8-5.2.

41. The method of any one of claims 37-40, wherein at least one wash buffer comprises lOmM sodium citrate, 50mM sodium chloride, pH 4.8-5.2.

42. The method of any one of claims 37-41, wherein at least one wash buffer comprises 20mM sodium citrate. 50mM sodium chloride, 10% v / v sucrose, pH 4.8-5.2.

43. The method of any one of claims 37-42, wherein the conductivity of the wash buffer is 6.0- 7.0 mS / cm.

44. The method of any one of claim 37-43, wherein at least one wash buffer comprises 10- 40mM sodium citrate, 0-50mM sodium chloride, 20% v / v sucrose, pH 4.8-5.2.

45. The method of any one of claim 37-44, wherein at least one wash buffer comprises 20mM sodium citrate, 50mM sodium chloride, 20% v / v sucrose, pH 4.8-5.2.

46. The method of any one of claims 37-45, wherein the conductivity of the wash buffer is 5.0- 6.0 mS / cm.

47. The method of any one of claims 37-46, wherein the elution buffer comprises sodium citrate and sodium chloride.

48. The method of any one of claims 37-47, wherein the elution buffer comprises 10 to 20% w / v sucrose, pH 4.8-5.2.

49. The method of any one of claims 37-48, wherein the elution buffer further comprises 10- 40mM sodium citrate, 270-500mM sodium chloride, pH 4.8-5.2.

50. The method of any one of claims 37-49, wherein the elution buffer further comprises 20mM sodium citrate, 300mM sodium chloride, pH 4.8-5.2.

51. The method of any one of claims 37-50, wherein the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, 10% v / v sucrose, pH 4.8-5.2.

52. The method of any one of claims 37-51, wherein the elution buffer comprises 20mM sodium citrate. 300mM sodium chlonde, 10% v / v sucrose, pH 4.8-5.2.

53. The method of any one of claims 37-52, wherein the conductivity of the elution buffer is 25.5-27.5 mS / cm.

54. The method of any one of claims 37-53, wherein the elution buffer comprises 10-40mM sodium citrate, 270-500mM sodium chloride, 20% v / v sucrose, pH 4.8-5.2.

55. The method of any one of claims 37-54, wherein the elution buffer comprises 20mM sodium citrate. 300mM sodium chloride, 20% v / v sucrose, pH 4.8-5.2.

56. The method of any one of claims 37-55, wherein the conductivity of the elution buffer is 20-22 mS / cm.

57. The method of any one of claims 37-56, wherein the cation exchange chromatography medium is equilibrated with an equilibration buffer having pH 4.8-5.

258. The method of any one of claims 37-57, wherein the cation exchange chromatography medium is equilibrated with an equilibration buffer comprising 10-40mM sodium citrate, 0- 50mM sodium chloride, pH 4.8-5.

259. The method of any one of claims 37-58, wherein the cation exchange chromatography medium is equilibrated with an equilibration buffer comprising 20mM sodium citrate. 50mM sodium chloride, pH 4.8-5.2.

60. The method according to any one of claims 37-59, wherein the number of peaks in the cation exchange chromatography elution profile are reduced compared to a method wherein only the wash buffer comprises 20% v / v sucrose or only the elution buffer comprises 20% v / v sucrose.

61. A method for mitigating viscous fingering while purifying a recombinant protein, the method comprising a) equilibrating a cation exchange chromatography medium with a buffer comprising sodium citrate and sodium chloride, pH 4.8-5.2; b) loading the recombinant protein on to the equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; c) washing the cation exchange chromatography medium with at least one wash buffer comprising sodium citrate, sodium chloride, 10% to 20% v / v sucrose, pH 4.8-5.2; and d) releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising sodium citrate, sodium chloride, 10% to 20% v / v sucrose, pH 4.8-5.2.

62. The method of any one of claims 61. wherein the method results in reduced viscous fingering compared to washing the cation exchange chromatography medium with wash buffer comprising sodium citrate and sodium citrate, pH 4.8-5.2, without sucrose and releasing the recombinant protein with an elution buffer comprising sodium citrate, sodium chloride, pH 4.8- 5.2, without sucrose.

63. A method for reducing the number of peaks in the cation exchange chromatography isocratic step gradient elution profile, the method comprising a) equilibrating a cation exchange chromatography medium with a buffer comprising 20mM sodium citrate, 50mM sodium chloride, pH 4.8-5.2; b) loading the recombinant protein on to the equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; c) washing the cation exchange chromatography medium with at least one wash buffer comprising 20mM sodium citrate. 50mM sodium chloride. 10% to 20% v / v sucrose, pH 4.8- 5.2; and d) releasing the recombinant protein by isocratic step gradient elution with an elution buffer comprising 20mM sodium citrate, 300mM sodium chloride, 10% - 20% v / v sucrose, pH 4.8-5.2.

64. The method of claim 63, wherein the method results in fewer peaks in the cation exchange chromatography isocratic step gradient elution profile compared to washing the cation exchange chromatography medium with wash buffer comprising 20mM sodium citrate, 50mM sodium chloride, pH 4.8-5.2, without sucrose and releasing the recombinant protein with an elution buffer comprising 20mM sodium citrate. 300mM sodium chloride, pH 4.8-5.2, without sucrose.

65. The method of any one of claims 63-64, wherein the method results in a single peak in the cation exchange chromatography isocratic step gradient elution profile.

66. A method for producing an isolated, purified, recombinant protein, the method comprising a) establishing a cell culture in a bioreactor with a host cell expressing the protein; b) culturing the host cells to express the protein; c) harvesting the recombinant protein from the cell culture; d) affinity purifying the recombinant protein; e) equilibrating a cation exchange chromatography medium with a buffer comprising sodium citrate, sodium chloride, pH 4.8-5.2;f) loading the purified recombinant protein onto an equilibrated cation exchange chromatography medium under conditions such that the recombinant protein binds to the chromatography medium; g) washing the cation exchange chromatography medium with at least one wash buffer comprising sodium citrate, sodium chloride, 10% to 20% v / v sucrose, pH 4.8-5.2; and h) releasing recombinant protein by isocratic step gradient elution with an elution buffer comprising sodium citrate, sodium chloride, 10% to 20% v / v sucrose, pH 4.8-5.2 into the effluent stream or collecting in an eluant pool.

67. The method according to claim 66. further comprising one or more unit operations selected from viral inactivation, viral filtration, neutralization, anion exchange chromatography, cation exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, ultrafiltration, diafiltration, depth filtration, sterile filtration, and fill / finish.

68. The isolated, purified, recombinant protein produced by any one of claims 66-67.

69. A pharmaceutical composition comprising the isolated, purified, recombinant protein produced by the method of any one of claims 66-68.

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