A method for equilibrating a chromatography medium

Abstract

The present invention relates to a chromatography equilibration method that accelerates equilibration dynamics and reduces the consumption of equilibration solution through a bolus addition of a buffering / titrating agent for on-column titration. The method is particularly useful as a component in a chromatography medium cleaning process.

Description

[0001]10151‐WO01‐SEC    Electronically Filed July 26, 2024    TITLE A METHOD FOR EQUILIBRATING A CHROMATOGRAPHY MEDIUM This application claims the benefit of U.S. Provisional Application No.63 / 529,398, filed July 28, 2023, which is hereby incorporated by reference in its entirety and for all purposes as if fully set forth herein. FIELD OF DISCLOSURE The present invention relates to the field of biopharmaceutical manufacturing. In particular, the invention relates to a chromatography equilibration method that accelerates equilibration dynamics and reduces the consumption of equilibration solution through a bolus addition of a buffering / titrating agent for on-column titration, useful particularly during cleaning of chromatography media. BACKGROUND Protein A affinity chromatography has become the preferred capture chromatography operation for recombinant immunoglobulins and other Fc-containing proteins. Protein A is a surface protein of the Staphylococcus aureus cell wall that has affinity for and binds within the fragment crystallizable (Fc) region of the immunoglobulin heavy chain without regard to antigen specificity. Protein A chromatography media makes use of the native protein as well as variant and engineered Protein A ligands. It has been estimated that up to 60% of the cost of a protein purification process comes from the chromatography operations, with Protein A resin being a significant cost contributor. Resin cost and the number of cycles before resin replacement is required are the two most important factors for a Protein A affinity chromatography unit operation (Danielsson, Chapter 17 Affinity Chromatography, page 375; in Biopharmaceutical Processing: Development, Design, and Implementation of Manufacturing Processes, Jagschies, Lindskog, Lacki and Galliher eds., Elsevier, 2018). A typical Protein A affinity resin can be cycled 100-200 times during its lifetime. Cleaning recycled Protein A chromatography resin is an integral part of clinical and commercial manufacturing processes and validating the efficacy of cleaning methods is of importance for product safety. Typically, Protein A chromatography resin is cleaned following the elution of the bound protein products of interest. The purpose of the cleaning step is to minimize carryover of product, impurities and / or contaminants from one cycle to another, reduce fouling and bioburden, minimize deterioration of the packed column, allow for recycling of the chromatography resin, and to prolong the lifetime of the resin (i.e., of the number of times that the resin may be recycled without significant degradation of performance that affects the yield, purity, or other critical attributes of the purified protein). Cleaning Protein A chromatography resin also contributes to the safety and quality of the purified protein by removing impurities and contaminants that could be released into the product stream. Therefore, there exists a need to develop cleaning procedures for Protein A chromatography medium that not only reduce contaminants and impurities between cycles or lots but also contribute to the 1      extension of the chromatography medium lifespan, while not negatively impacting cost, resources, and time. The invention described herein provides a method using on-column titration to accelerate chromatography medium equilibrium dynamics that meets this need. BRIEF SUMMARY OF THE INVENTION The invention provides a method for on-column titration to accelerate chromatography medium equilibrium dynamics comprising the steps of (a) passing a first equilibration flush solution through the chromatography medium; (b)passing a bolus of a titrating / buffering agent through the chromatography medium; and (c) passing a second equilibrium flush solution through the chromatography medium. In one embodiment the first equilibrium flush solution comprises Tris, Phosphate, Bis-Tris, Mes, Carbonate, or Acetate. In another embodiment the pH of the first equilibration flush solution is at least 5.0 to 8.0. In another embodiment the conductivity of the first equilibrium flush solution is at least 5 to 20 mS / cm. In another embodiment the first equilibration flush solution is passed through the chromatography medium until one or more desired process parameters, selected from effluent pH, effluent conductivity, contact time, and / or number of column volumes of the first equilibration flush solution passed through the chromatography medium, is achieved. In a related embodiment at least one column volume of the first equilibrium flush solution is passed through the chromatography medium. In another related embodiment the contact time for the first equilibration flush solution with the chromatography medium is at least 5 minutes. In another related embodiment the first equilibration flush solution is passed through the chromatography medium until the conductivity of the effluent is at least 5 to 20 mS / cm. In another related embodiment the first equilibration flush solution is passed through the chromatography medium until the pH of the effluent is at least 2.5 to 5.0. In one embodiment the titrating / buffering agent is a strong titrating species. In a related embodiment the strong titrating species is a strong base. In another related embodiment the strong titrating species is Sodium Hydroxide, Potassium Hydroxide, Tris Base, or Sodium Carbonate. In one embodiment the bolus of titrating / buffering agent is passed through the chromatography medium until one or more desired process parameters, selected from one or more of effluent pH, effluent conductivity, contact time, and / or column volumes of the bolus of denaturing agent passed through the chromatography medium, is achieved. In a related embodiment the titrating / buffering agent is passed through the chromatography medium until the pH of the effluent is at least 2.5 to 5.0. In another related embodiment the titrating / buffering agent is passed through the chromatography medium until the conductivity of the effluent is at least 1.0 m to 1.0 mS / cm to 20 mS / cm. In another related embodiment the contact time for the bolus of titrating / buffering agent and the chromatography medium is at least 5 seconds. In another related embodiment at least 0.01 column volume of titrating / buffering agent is passed through the chromatography medium. In one embodiment a sufficient bolus of titrating / buffering agent is passed through the chromatography medium to not overshoot a desired effluent pH and / or conductivity. 2        In one embodiment the second equilibrium flush solution comprises Tris, Phosphate, Bis-Tris, Mes, Carbonate, or Acetate. In another embodiment the pH of the second equilibration flush solution is at least 5.0 to 8.0. In another embodiment the conductivity of the second equilibrium flush solution is at least 5 to 20 mS / cm. In another embodiment the first equilibrium flush solution and the second equilibrium flush solution are the same. In another embodiment the first equilibrium flush solution and the second equilibrium flush solution are different. In another embodiment the second equilibration flush solution is passed through the chromatography medium until one or more desired process parameters selected from selected from one or more of pH, conductivity, contact time, and / or column volumes of second equilibrium buffer passed through the chromatography medium is achieved. In a related embodiment at least one column volume of the second equilibrium flush solution is passed through the chromatography medium. In a related embodiment the contact time for the second equilibrium flush solution with the chromatography medium is at least 5 minutes. In another related embodiment the second equilibration flush is passed through the chromatography medium until the pH of the second equilibrium flush solution is at least 6.5 to 8.0. In another related embodiment the second equilibration flush is passed through the chromatography medium until the conductivity of the second equilibrium flush solution is at least 5.0 mS / cm to 20.0 mS / cm. In one embodiment the invention further comprises passing a water flush through the chromatography medium prior to step (a). In a related embodiment the water flush is passed through the chromatography medium until one or more desired process parameters selected from selected from one or more of pH, conductivity, contact time, and / or column volumes of second equilibrium buffer passed through the chromatography medium is achieved. In a related embodiment the water flush is passed through the chromatography medium until the pH of the effluent is about 2.5 to 3.0. In a related embodiment the water flush is passed through the chromatography medium until the conductivity of the effluent is about 0 to 1.0 mS / cm. In a related embodiment at least one column volume of water is passed through the chromatography medium. In another related embodiment the contact time for the water flush with the chromatography medium is less than or equal to 1 hour. In another related embodiment the number of column volumes of water flush is the same as the first equilibration solution flush. In another related embodiment the number of column volumes of water flush is different from the number of column volumes for the first equilibration solution flush. In a related embodiment the contact time for the water flush is the same as the contact time for first equilibration solution flush. In a related embodiment the contact time for the water flush is different from the contact time for the first equilibration solution flush. In one embodiment the pH, conductivity, number of column volumes, or contact time for two or more of the water flush, first equilibrium flush, bolus, and / or second equilibrium flush steps are the same. In one embodiment the water flush is purified water or water for injection. In one embodiment the chromatography medium is an affinity chromatography medium. In one embodiment the chromatography medium is a high capacity chromatography medium. In a related embodiment the chromatography medium is a Protein A or Protein G chromatography medium. 3        The invention also provides method for cleaning a chromatography medium for reuse comprising the steps of (a) passing an acidic agent through the chromatography medium; (b) passing a first equilibration solution through the chromatography medium; (c) passing a bolus of a titrating / buffering agent through the chromatography medium; and (d) passing a second equilibrium solution through the chromatography medium. In one embodiment the acidic agent is non-denaturing. In one embodiment the pH of the acidic agent is at least 2.0 to 3.5. In one embodiment the acidic agent is phosphoric acid. In one embodiment the acidic agent is passed through the chromatography medium until one or more desired process parameters, selected from effluent pH, effluent conductivity, contact time, and / or number or column volumes of the acidic agent passed through the chromatography medium, is achieved. In a related embodiment the contact time for the acidic agent with the chromatography medium is at least 10 to 60 minutes. In a related embodiment the acidic agent is passed through the chromatography medium until the conductivity of the effluent is at least 5 to 20 mS / cm. In a related embodiment the acidic agent is passed through the chromatography medium until the pH of the effluent is at least 3 to 5. In a related embodiment the chromatography medium is subjected to at least one column volume of the acidic agent. In one embodiment the invention further comprises a regeneration step following step (d). In one embodiment the regeneration step comprising passing at least one denaturing agent through the chromatography medium. In a related embodiment the denaturing agent is an alkali agent. In a related embodiment the alkali agent is a strong base. In one embodiment the denaturing agent is a chaotropic agent. In one embodiment the denaturing agent is passed through the chromatography medium until one or more desired process parameters, selected from selected from one or more of pH, conductivity, contact time, and / or number of column volumes of denaturing agent passed through the chromatography medium, is achieved. In a related embodiment the contact time for the denaturing agent with the chromatography medium is at least 10 minutes. In a related embodiment at least one column volume of the denaturing agent is passed through the chromatography medium. In a related embodiment the denaturing agent is passed through the chromatography medium until the pH from the effluent of the regeneration step is at least 9.0 to 11.0. In a related embodiment the denaturing agent is passed through the chromatography medium until conductivity of the effluent of the regeneration step is at least 10.0 mS / cm to 200.0 mS / cm. In one embodiment the titrating / buffering agent and the denaturing agent are the same. In one embodiment the titrating / buffering agent and the denaturing agent are different. In one embodiment the invention further comprises storing the cleaned chromatography medium in a 2% Benzyl Alcohol, 50 mM Sodium Citrate solution. In one embodiment the invention further comprises passing a water flush through the chromatography medium prior to step (b). The invention also provides a method for reducing the consumption of equilibrium solution during the cleaning of a chromatography medium comprising the steps of (a) a strip step; (b) a water flush step; 4        (c) a first equilibrium flush step; (d) a bolus of a titrating / buffering agent; and (e) a second equilibrium flush step; wherein the amount of equilibrium solution consumed is less than the amount of equilibrium solution consumed during a cleaning method consisting only of a strip step and a single equilibrium flush step. The invention also provides a method for purifying a recombinant protein comprising the steps of (a) initiating a cell culture in a bioreactor; (b) culturing cells engineered to recombinantly express a protein; (c) harvesting the cell culture containing the recombinant protein; (d) binding the recombinant protein onto an affinity chromatography medium; (e) eluting and collecting the bound recombinant protein; (f) striping the chromatography medium with an acidic agent; (g) optionally flushing the chromatography medium with water; (h) flushing the chromatography medium with a first equilibration solution; (i) passing a bolus of a titrating / buffering agent through the chromatography medium; (j) flushing the chromatography medium with a second equilibrium solution; and (k) repeating steps d-j until all the harvested cell culture has been loaded and the recombinant protein has been collected. In one embodiment the invention further comprises a regeneration step comprising a denaturing agent. In one embodiment the regeneration step occurs before or after step (k). In one embodiment the invention further comprises subjecting the collected recombinant protein to a low pH or detergent viral inactivation. In one embodiment the invention further comprises neutralizing the viral inactivated recombinant protein. In one embodiment the invention further comprises at least one additional chromatography step. In one embodiment the chromatography step is selected from ion exchange chromatography, hydrophobic interaction chromatography, or multimodal chromatography. In one embodiment the invention further comprises at least one viral filtration step. In one embodiment the invention further comprises at least one UFDF step. In one embodiment is provided the recombinant protein made by the method of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the process flow for a typical Protein A chromatography cleaning and regeneration operation (left column) and a cleaning / regeneration method that makes use of accelerated equilibration dynamics that reduce the consumption of equilibration solution through a bolus addition of a buffering / titrating agent for on-column titration (right column). Briefly, for either process an equilibrated chromatography medium is loaded with a product feed stream. Once the loading is complete, the chromatography medium is washed with one or more solutions to re-equilibrate the chromatography medium, remove non-binding impurities and / or contaminants, and / or prepare the chromatography medium for elution. The product is eluted, and the purified protein collected in the eluate. The chromatography medium is then cleaned with a low pH strip solution and re-equilibrated prior to reloading. A regeneration step can be used after one or more cleaning cycles and / or prior to storage. For the typical cleaning process, following the low pH strip step, the chromatography medium is neutralized with equilibration solution until the desired pH and / or conductivity in the column effluent is achieved. 5        For the inventive method, a low pH strip step is followed by an optional water flush then a flush with a first equilibration solution. The chromatography medium is then subjected to a single discrete (bolus) addition of a titrating / buffering agent, (e.g., a strong titrating species), followed by continued equilibration with a second equilibration flush solution until a desired process parameter is achieved. For both processes, after a desired number of cycles (“X cycles”) or at the end of each lot, the cleaned chromatography medium is regenerated with a denaturing solution. The column may continue in use or be stored until used again. Figure 2 shows pH trends with and without the use of a titrating / buffering agent during the equilibration step. DETAILED DESCRIPTION OF THE INVENTION Proteins that contain an Fc region, such as monoclonal and multispecific antibodies as well as Fc fusion proteins, remain a preferred biologic therapeutic platform. Downstream purification operations for such Fc-containing proteins typically include at least one affinity chromatography capture operation that makes use of a Fc-substrate binding ligand from Staphylococcus aureus, such as Protein A. Protein A affinity chromatography is highly selective for proteins having an Fc region and is regularly used as an initial capture step following the harvest of recombinantly expressed Fc-containing proteins of interest. Protein A affinity chromatography is used to isolate, concentrate, and stabilize the protein of interest, removing as many critical impurities as possible. An initial Protein A affinity chromatography capture step can result in purity levels greater than 90%. This simplifies downstream processing and can result in a more robust manufacturing process. Protein A affinity chromatography media are widely used from bench scale to large scale commercial manufacturing. Due to its critical role in downstream purification operations, as well as its high cost, much effort is taken to make Protein A affinity chromatography operations as economical and efficient as possible, and cleaning is critical to extending the life of the chromatography medium. In a typical Protein A affinity chromatography operation a load feed containing a protein of interest may be derived from harvest or storage pools, eluants, effluents, and the like from cell culture harvest, capture and / or polish chromatography, virus inactivation, neutralization, depth filtration, virus filtration, and / or ultrafiltration / diafiltration (UFDF) operations. The chromatography medium is subjected to one or more washes to re-equilibrate the chromatography medium, remove any unbound contaminants and / or impurities, and / or prepare the chromatography medium for elution. The bound protein is then eluted from the chromatography medium. The chromatography medium is then subjected to a cleaning step. Cleaning consists of a strip and equilibration step. A regeneration step typically follows the cleaning step, see Figure 1, right column. The strip step removes impurities and / or contaminants from the chromatography medium, typically with an acidic formulation. The strip is conducted following elution of the protein of interest from the chromatography medium after one or more chromatography cycles. A “cycle” comprises the steps from loading the chromatography medium with a feed comprising the protein of interest and at least one impurity, 6        washing the chromatography medium and eluting the protein of interest and collecting the purified protein in the chromatography eluate. One or more cycles may be required to process a batch. A “batch”, or “lot”, comprises all the chromatography cycles necessary to process a fixed quantity of material containing the protein of interest. Frequent cleaning helps control the accumulation of product, impurities and / or contaminants that could be carried over from one cycle or batch to the next and released into the product stream. Cleaning the chromatography medium following a cycle reduces chromatography medium fouling, minimize deterioration of the packed column, allows for recycling of the chromatography medium, and / or prolongs the lifespan of the chromatography medium. One or more cleaning steps used in combination with one or more regeneration steps may be performed together or separately after one or more cycles, after one or more batches, and / or prior to storage. Having a cleaning method that gets the most out of these chromatography media with as little impact as possible on the cost, resources, environment, and / or production time is beneficial and necessary for a robust manufacturing process. Multiple chromatography cycles increase the risk of contamination and product carryover. Load material used in Protein A chromatography, such as harvest pools, may contain components that can have a deteriorating effect on the chromatography medium, resulting in a decrease in chromatography medium performance. Cleaning the chromatography medium is an integral part of a downstream platform for protein purification. Cleaning minimizes carryover of impurities and / or contaminants from one cycle to another, reduces fouling and bioburden, minimizes deterioration of the packed column, and prolongs the lifetime of the medium i.e., the reusability or recycling. Cleaning increases the duration the chromatography medium, increasing its usability without significant degradation of performance that affects the yield, purity, or other product quality attributes of the purified protein. Cleaning the chromatography medium also contributes to the safety and quality of the purified protein by removing impurities and contaminants that could be released into the product stream. To meet the demands of ever increasing titers from upstream operations, advancements in Protein A resin technology has included improvements to both the affinity ligands and the supporting matrix, resulting in chromatography media suitable for process intensification to achieve increased productivity and efficiency. Protein A ligand-containing resins having enhanced or improved qualities including binding affinity, binding capacity, equilibrium capacity, and the like are known in the art and are commercially available. These high capacity chromatography media enable cycling the load material through smaller columns, reducing cost by reducing the amount of chromatography medium needed, and as such are a desirable component of an affinity chromatography operation and are increasingly relied upon in clinical and commercial manufacturing platforms. However, this high capacity Protein A chromatography medium was found to have an unexpected drawback when it comes to cleaning. As described herein, high capacity Protein A chromatography medium required at least twice as much equilibration solution to reach pH equilibrium following a traditional cleaning strategy that made use of a low pH strip and a single equilibration step. The increased 7        volume of equilibration solution was due to the slow dynamics of the pH equilibration of the chromatography medium which was attributed to the higher capacity of the Protein A ligand that increases the effective buffer capacity of the chromatography medium in addition to its intended effect of increasing product binding capacity. Increased equilibration solution consumption impacts run rate and media preparation resourcing, increases the time required for cleaning the chromatography medium between cycles, and results in increasing wastewater emissions. Described herein is a chromatography equilibration method that accelerates equilibration dynamics and reduces the consumption of equilibration solution through a bolus addition of a buffering / titrating agent for on-column titration. The addition of the bolus of the titrating / buffering agent during the equilibration process reduced both the consumption of equilibration solution and the time needed to reach process targets (e.g., pH, conductivity, contact time, and / or column volumes passed through the chromatography medium) by rapidly titrating the chromatography medium through its buffering capacity. The method makes use of an optional water flush, followed by an initial brief first equilibration flush to begin neutralizing the acidic environment of the chromatography medium following a low pH strip typical of a chromatography medium cleaning process. The chromatography medium is then subjected to a small bolus feed of a buffering / titrating agent, such as a strong titrating species, in particular, a strong base. The initial neutralization is necessary as adding a strong base to an acidic environment is not desirable. Adding the bolus a strong titrating species during the equilibration step was found to accelerate the equilibration dynamics of the chromatography medium compared to traditional cleaning practice that make use of only an equilibration solution. Following the bolus addition of the buffering / titrating agent, the equilibration continues until one or more desired process parameter(s), e.g., pH, conductivity, contact time, and / or column volumes of equilibrium buffer is achieved, see Figure 1, left column. In addition to accelerating the equilibrated state, the method described herein also reduced the consumption of equilibration solution, in some cases by at least 4 or more column volumes, compared to equilibrium without the bolus addition of the buffering / titrating agent. For example, for a 500L chromatography column operated for 5 cycles per lot, reducing the amount of equilibrium solution needed by 1 CV would save ~2,500L of solution / lot. Saving 4CVs would reduce the volume of equilibrium solution that must be prepared, stored, and disposed of, by ~10,000L / lot. This represents a great savings in terms of time, resources, storage, and raw materials, as well as decreasing environmental impact. Protein A chromatography media are typically used as a first capture step in a downstream process and as such are subjected to feed material that may contain high levels of impurities and contaminants. As the chromatography medium is cycled during a batch, it becomes fouled from remaining impurities and / or contaminants that are carried over from cycle to cycle. The chromatography medium fouling decreases binding capacity which reduces yield, impacts the equilibration of the chromatography medium, and decreases the chromatography medium’s functional lifetime. Cleaning the chromatography medium removes strongly bound product which could precipitate and further foul the chromatography medium, reduces contaminant accumulation, and / or removes impurities including product-related impurities such as 8        product fragments, modified product, product aggregates, etc. Process-related impurities such as HCP, DNA, lipids, endotoxins, etc., that could be carried over to the next cycle or batch or could be released into the product stream and impact product safety and / or efficacy are also removed during cleaning. Cleaning also reduces the bioburden risk due to microbial and / or viral contamination. Bioburden contamination could require that the production lot and / or the chromatography medium be discarded, which is costly and would negatively impact production timelines. Strip Step The cleaning method described herein includes at least one strip step, performed at a pH lower than that of the elution solution. Strong denaturing agents are sometimes used for cleaning chromatography medium. However, while such agents can remove strongly bound impurities, the denatured proteins can foul a chromatography medium, decrease binding capacity, reduce flow rate, carryover to subsequent runs. The caustic exposure can also diminish the functional life span of the chromatography medium. As described herein, the strip step is performed with a non-denaturing agent. In one embodiment the strip step is performed using an acidic agent. In one embodiment the acidic agent is phosphoric acid. In one embodiment the acidic agent is 100mM phosphoric acid. In one embodiment the pH of the acidic agent is at least 2.0. In one embodiment the pH of the acidic agent is at least 2.0 to about 3.5. In one embodiment the pH of the acidic agent is at least 2.0 to about 3.0. In one embodiment the pH of the acidic agent is at least 2.0 to about 2.5. In one embodiment, the pH of the acidic agent is at least 2.0. In one embodiment, the pH of the acidic agent is at least 2.1. In one embodiment, the pH of the acidic agent is at least 2.2. In one embodiment, the pH of the acidic agent is at least 2.3. In one embodiment, the pH of the acidic agent is at least 2.4. In one embodiment, the pH of the acidic agent is at least 2.5. In one embodiment, the pH of the acidic agent is at least 2.6. In one embodiment, the pH of the acidic agent is at least 2.7. In one embodiment, the pH of the acidic agent is at least 2.8. In one embodiment, the pH of the acidic agent is at least 2.9. In one embodiment, the pH of the acidic agent is at least 3.0. In one embodiment, the pH of the acidic agent is at least 3.1. In one embodiment, the pH of the acidic agent is at least 3.2. In one embodiment, the pH of the acidic agent is at least 3.3. In one embodiment, the pH of the acidic agent is at least 3.4. In one embodiment, the pH of the acidic agent is at least 3.5. The acidic agent is passed through the chromatography medium until one or more desired process parameters are achieved. The process parameters are selected from one or more of effluent pH, effluent conductivity, contact time, and / or column volumes of the acidic agent passed through the chromatography material. In one embodiment the acidic agent is passed through the chromatography medium until the pH of the effluent is at least 3 to 5. In one embodiment the pH is at least 4 to 5. In one embodiment the pH at least 3. In one embodiment the pH is at least 4. In one embodiment the pH is 5. In one embodiment acidic agent is passed through the chromatography medium until the conductivity of the effluent is at least 5 to 20 mS / cm. In one embodiment the conductivity of the effluent 9        is 5 to 15 mS / c. In one embodiment the conductivity of the effluent is 5 to 10 mS / c. In one embodiment the conductivity of the effluent is at least 5 mS / cm. In one embodiment the conductivity of the effluent is at least 6 mS / cm. In one embodiment the conductivity of the effluent is at least 7 mS / cm. In one embodiment the conductivity of the effluent is at least 8 mS / cm. In one embodiment the conductivity of the effluent is at least 9 mS / cm. In one embodiment the conductivity of the effluent is at least 10 mS / cm. In one embodiment the conductivity of the effluent is at least 11 mS / cm. In one embodiment the conductivity of the effluent is at least 12 mS / cm. In one embodiment the conductivity of the effluent is at least 13 mS / cm. In one embodiment the conductivity of the effluent is at least 14 mS / cm. In one embodiment the conductivity of the effluent is at least 15 mS / cm. In one embodiment the conductivity of the effluent is at least 16 mS / cm. In one embodiment the conductivity of the effluent is at least 17 mS / cm. In one embodiment the conductivity of the effluent is at least 18 mS / cm. In one embodiment the conductivity of the effluent is at least 19 mS / cm. In one embodiment the conductivity of the effluent is at least 20 mS / cm. The chromatography medium is subjected to at least one column volume (CV) of the acidic agent following the elution of the bound protein of interest from the chromatography medium. In one embodiment, at least 2 CVs of the acidic agent is passed through the chromatography medium. In one embodiment, at least 2 to 10 CVs of the acidic agent is used. In one embodiment, at least 3 to 5 CVs of the acidic agent is used. In one embodiment, at least 3 CVs of the acidic agent is used. In one embodiment, at least 3 CVs of the acidic agent is used. In one embodiment, at least 4 CVs of the acidic agent is used. In one embodiment, at least 5 CVs of the acidic agent is used. In one embodiment, at least 6 CVs of the acidic agent is used. In one embodiment, at least 7 CVs of the acidic agent is used. In one embodiment, at least 8 CVs of the acidic agent is used. In one embodiment, at least 9 CVs of the acidic agent is used. In one embodiment, at least 10 CVs of the acidic agent is used. The contact time of the acidic agent with the chromatography medium is less than or equal to 1 hour. Contact time is defined as (bed height / linear flow rate) * Column Volumes. In one embodiment the contact time is at least 10 minutes to 60 minutes. In one embodiment the contact time is at least 10 minutes to 30 minutes. In one embodiment the contact time is at least 10 minutes to 15 minutes. In one embodiment the contact time is at least 10 minutes to 20 minutes. In one embodiment the contact time is about 10 minutes. In one embodiment the contact time is about 11 minutes. In one embodiment the contact time is about 12 minutes. In one embodiment the contact time is about 13 minutes. In one embodiment the contact time is about 14 minutes. In one embodiment the contact time is about 15 minutes. In one embodiment the contact time is about 16 minutes. In one embodiment the contact time is about 17 minutes. In one embodiment the contact time is about 18 minutes. In one embodiment the contact time is about 19 minutes. In one embodiment the contact time is about 20 minutes. In one embodiment the contact time is about 25 minutes. In one embodiment the contact time is about 30 minutes. In one embodiment the contact time is about 35 minutes. In one embodiment the contact time is about 40 minutes. In one embodiment the contact time is about 45 minutes. In one embodiment the contact time is about 50 minutes. In one embodiment the contact time is about 55 minutes. In one embodiment the contact time is about 60 minutes. 10        Water Flush The low pH strip step may be followed by an optional water flush. The water flush contributes to the neutralization of the pH of the chromatography medium in preparation for the addition of a bolus of a titrating / buffering agent. The water flush may be performed using purified water, water for injection, or any other appropriate water source. The water flush is passed through the chromatography medium until one or more desired process parameters are achieved. The process parameters are selected from one or more of effluent pH, effluent conductivity, contact time, and / or column volumes of the water passed through the chromatography material. In one embodiment the water flush is passed through the chromatography medium until the pH of the effluent is at least 3. In one embodiment the effluent pH is about 2.5 to 3.0. In one embodiment the effluent pH is at least 2.5. In one embodiment the effluent pH is at least 2.6. In one embodiment the effluent pH is at least 2.7. In one embodiment the effluent pH is at least 2.8. In one embodiment the effluent pH is at least 2.9. In one embodiment the effluent pH is at least 3.0. In one embodiment water flush is passed through the chromatography medium until the conductivity of the effluent is at most 1 mS / cm. In one embodiment the conductivity of the effluent is 0 to 1 mS / cm. In one embodiment the conductivity of the effluent is 0.1 mS / cm. In one embodiment the conductivity of the effluent is 0.2 mS / cm. In one embodiment the conductivity of the effluent is 0.3 mS / cm. In one embodiment the conductivity of the effluent is 0.4 mS / cm. In one embodiment the conductivity of the effluent is 0.5 mS / cm. In one embodiment the conductivity of the effluent is 0.6 mS / cm. In one embodiment the conductivity of the effluent is 0.7 mS / cm. In one embodiment the conductivity of the effluent is 0.8 mS / cm. In one embodiment the conductivity of the effluent is 0.9 mS / cm. In one embodiment the conductivity of the effluent is 1.0 mS / cm. In one embodiment at least 1 to 5 CVs of water flush is passed through the chromatography medium. In one embodiment at least 1 CV of water flush is passed through the chromatography medium. In one embodiment at least 2 CVs of water flush is passed through the chromatography medium. In one embodiment at least 3 CVs of water flush is passed through the chromatography medium. In one embodiment at least 4 CVs of water flush is passed through the chromatography medium. In one embodiment at least 5 CVs of water flush is passed through the chromatography medium. The contact time of the water flush with the chromatography medium is less than or equal to 1 hour. In one embodiment the contact time is at least 5 minutes to 1 hour. In one embodiment the contact time is at least 5 minutes to 30 minutes. In one embodiment the contact time is at least 5 minutes to 15 minutes. In one embodiment the contact time is at least 5 minutes to 20 minutes. In one embodiment the contact time is about 5 minutes. In one embodiment the contact time is about 11 minutes. In one embodiment the contact time is about 12 minutes. In one embodiment the contact time is about 13 minutes. In one embodiment the contact time is about 14 minutes. In one embodiment the contact time is about 15 minutes. 11        In one embodiment the contact time is about 16 minutes. In one embodiment the contact time is about 17 minutes. In one embodiment the contact time is about 18 minutes. In one embodiment the contact time is about 19 minutes. In one embodiment the contact time is about 20 minutes. In one embodiment the contact time is about 25 minutes. In one embodiment the contact time is about 30 minutes. In one embodiment the contact time is about 35 minutes. In one embodiment the contact time is about 40 minutes. In one embodiment the contact time is about 45 minutes. In one embodiment the contact time is about 50 minutes. In one embodiment the contact time is about 55 minutes. In one embodiment the contact time is about 60 minutes. First Equilibrium Flush Step The low pH strip step or the water flush may be followed by a first equilibration flush step. The first equilibration flush solution contributes to the neutralization of the chromatography medium pH in preparation for the addition of the bolus of a titrating / buffering agent. Suitable buffers include, but are not limited to, Tris, Phosphate, Bis-Tris, Mes, Carbonate, and Acetate. Suitable salts include, but are not limited to, sodium chloride, sodium phosphate, and ammonium sulfate. In one embodiment the pH of the first equilibrium flush solution is at least 5.0. In one embodiment the pH of the first equilibration flush solution is at least 5.0 to 8.0. In one embodiment the pH of the first equilibration solution is at least 6.0 to 7.0. In one embodiment the pH of the first equilibration flush solution is at least 5.0. In one embodiment the pH of the first equilibration flush solution is at least 5.5. In one embodiment the pH of the first equilibration flush solution is at least 6.0. In one embodiment the pH of the first equilibration solution is at least 6.5. In one embodiment the pH of the first equilibration flush solution is at least 6.6. In one embodiment the pH of the first equilibration flush solution is at least 6.7. In one embodiment the pH of the first equilibration flush solution is at least 6.8. In one embodiment the pH of the first equilibration flush solution is at least 6.9. In one embodiment the pH of the first equilibration flush solution is at least 7.0. In one embodiment the pH of the first equilibration flush solution is at least 7.1. In one embodiment the pH of the first equilibration flush solution is at least 7.2. In one embodiment the pH of the first equilibration flush solution is at least 7.3. In one embodiment the pH of the first equilibration flush solution is at least 7.4. In one embodiment the pH of the first equilibration flush solution is at least 7.5. In one embodiment the pH of the first equilibration flush solution is at least 7.6. In one embodiment the pH of the first equilibration flush solution is at least 7.7. In one embodiment the pH of the first equilibration flush solution is at least 7.8. In one embodiment the pH of the first equilibration flush solution is at least 7.9. In one embodiment the pH of the first equilibration flush solution is at least 8.0. In one embodiment the conductivity of the first equilibrium flush solution is at least 5.0 mS / cm. In one embodiment the pH of the first equilibrium flush solution is at least 5 to 20 mS / cm. In one embodiment the pH of the first equilibrium flush solution is at least 5 to 10 mS / cm. In one embodiment the pH of the first equilibrium flush solution is at least 10 to 20 mS / cm. In one embodiment the conductivity of the first equilibrium flush solution is at least 6.0 mS / cm. In one embodiment the conductivity of the first 12        equilibrium flush solution is at least 7.0 mS / cm. In one embodiment the conductivity of the first equilibrium flush solution is at least 8.0 mS / cm. In one embodiment the conductivity of the first equilibrium flush solution is at least 9.0 mS / cm. In one embodiment the conductivity of the first equilibrium flush solution is at least 10.0 mS / cm. In one embodiment the conductivity of the first equilibrium flush solution is at least 11.0 mS / cm. In one embodiment the conductivity of the first equilibrium flush solution is at least 12.0 mS / cm. In one embodiment the conductivity of the first equilibrium flush solution is at least 13.0 mS / cm. In one embodiment the conductivity of the first equilibrium flush solution is at least 14.0 mS / cm. In one embodiment the conductivity of the first equilibrium flush solution is at least 15.0 mS / cm. In one embodiment the conductivity of the first equilibrium flush solution is at least 16.0 mS / cm. In one embodiment the conductivity of the first equilibrium flush solution is at least 17.0 mS / cm. In one embodiment the conductivity of the first equilibrium flush solution is at least 18.0 mS / cm. In one embodiment the conductivity of the first equilibrium flush solution is at least 19.0 mS / cm. In one embodiment the conductivity of the first equilibrium flush solution is at least 20.0 mS / cm. In one embodiment the first equilibration solution is 25mM Tris, 100mM NaCl, pH 7.4. The first equilibrium flush solution is passed through the chromatography medium until one or more desired process parameters are achieved. The process parameters are selected from one or more of pH, conductivity, contact time, and / or column volumes of the first equilibrium flush buffer passed through the chromatography material. In one embodiment the first equilibrium solution is passed through the chromatography medium until the pH of the effluent is at least 2.5. In one embodiment the first equilibrium solution is passed through the chromatography medium until the pH of the effluent is at least 3. In one embodiment the first equilibrium solution is passed through the chromatography medium until the pH of the effluent is at least 3.5. In one embodiment the first equilibrium solution is passed through the chromatography medium until the pH of the effluent is at least 4. In one embodiment the first equilibrium solution is passed through the chromatography medium until the pH of the effluent is at least 4.5. In one embodiment the first equilibrium solution is passed through the chromatography medium until the pH of the effluent is at least 5. In one embodiment the first equilibrium solution is passed through the chromatography medium until the conductivity of the effluent is at least 1 mS / cm. In one embodiment the first equilibrium solution is passed through the chromatography medium until the conductivity of the effluent is at least 2 mS / cm. In one embodiment the first equilibrium solution is passed through the chromatography medium until the conductivity of the effluent is at least 3 mS / cm. In one embodiment the first equilibrium solution is passed through the chromatography medium until the conductivity of the effluent is at least 5 mS / cm. In one embodiment the first equilibrium solution is passed through the chromatography medium until the conductivity of the effluent is at least 10 mS / cm. In one embodiment the first equilibrium solution is passed through the chromatography medium until the conductivity of the effluent is at least 15 mS / cm. In one embodiment the first equilibrium solution is passed through the chromatography medium until the conductivity of the effluent is at least 20 mS / cm. 13        In one embodiment at least 1 to 3 CVs of the first equilibrium solution is passed through the chromatography medium. In one embodiment at least 1 CV of first equilibrium solution is passed through the chromatography medium. In one embodiment at least 2 CVs of first equilibrium solution is passed through the chromatography medium. In one embodiment at least 3 CVs of first equilibrium solution is passed through the chromatography medium. In one embodiment the contact time of the first equilibration flush with the chromatography medium is at least 5 minutes to 1 hour. In one embodiment the contact time is at least 5 minutes to 30 minutes. In one embodiment the contact time is at least 5 minutes to 15 minutes. In one embodiment the contact time is at least 5 minutes to 20 minutes. In one embodiment the contact time is about 5 minutes. In one embodiment the contact time is about 6 minutes. In one embodiment the contact time is about 7 minutes. In one embodiment the contact time is about 8 minutes. In one embodiment the contact time is about 9 minutes. In one embodiment the contact time is about 10 minutes. In one embodiment the contact time is about 11 minutes. In one embodiment the contact time is about 12 minutes. In one embodiment the contact time is about 13 minutes. In one embodiment the contact time is about 14 minutes. In one embodiment the contact time is about 15 minutes. In one embodiment the contact time is about 16 minutes. In one embodiment the contact time is about 17 minutes. In one embodiment the contact time is about 18 minutes. In one embodiment the contact time is about 19 minutes. In one embodiment the contact time is about 20 minutes. In one embodiment the contact time is about 25 minutes. In one embodiment the contact time is about 30 minutes. In one embodiment the contact time is about 35 minutes. In one embodiment the contact time is about 40 minutes. In one embodiment the contact time is about 45 minutes. In one embodiment the contact time is about 50 minutes. In one embodiment the contact time is about 55 minutes. In one embodiment the contact time is about 1 hour. In one embodiment the number of CVs of water from the water flush and the number of CVs of first equilibration flush solution are the same. In one embodiment the number of CVs of water from the water flush and the number of CVs of first equilibration flush solution are different. In one embodiment the contact time for the water flush is the same as for the contact time for the first equilibration flush. In one embodiment the contact time for the water flush is different from than the contact time for the first equilibration flush. Bolus of Titrating / Buffering Agent It was found that the equilibrium dynamics of a high capacity Protein A chromatography medium was accelerated by the addition of a bolus addition of a titrating / buffering agent during the equilibrium step. Adding the bolus of titrating / buffering agent to the cleaning method reduced the amount of equilibrium solution consumed during the cleaning step compared to a cleaning method consisting only of a low pH strip step and a single equilibrium flush step. This on-column titration decreased equilibration solution consumption and reduced the time needed for equilibration by rapidly titrating through the chromatography medium buffering capacity. Many Protein A chromatography media are now more tolerant of alkali agents. 14        The bolus addition of a titrating / buffering agent used during equilibration did not have a negative impact on the chromatography media or its performance. In one embodiment, the titrating / buffering agent is a strong titrating species. In one embodiment the strong titrating species is a strong base species. Strong titrating species include but are not limited to Sodium Hydroxide, Potassium Hydroxide, Tris Base, and Sodium Carbonate. Higher volumes of a weaker titrating species can also be used. In one embodiment the titrating / buffering agent is a chaotropic agent. In one embodiment the chaotropic agent is guanidine or urea. In one embodiment, the strong titrating species is Sodium Hydroxide. Sodium Hydroxide is commonly used for regenerating chromatography media and cleaning and sanitizing hardware. However, the concentration and contact time of the bolus load are not sufficient to regenerate the column. Sodium Hydroxide is cost effective, easy to detect and remove and is not subject to disposal restrictions at large scale. The bolus of titrating / buffering agent is passed through the chromatography medium until one or more desired process parameters are achieved. The process parameters are selected from one or more of pH, conductivity, contact time, and / or column volumes of the bolus of a titrating / buffering agent are passed through the chromatography material. Since there is a delay between the titrating / buffering agent addition and measurement of the eluate, measurement of effluent pH and / or conductivity may not be conducted before moving to the second equilibration flush. In one embodiment a sufficient volume of titrating / buffering agent is passed through the chromatography medium to not overshoot the effluent pH and / or conductivity. In one embodiment the bolus of titrating / buffering agent is passed through the chromatography medium until the pH of the effluent is at least 2.5 to 5.0. In one embodiment the effluent pH is at least 2.5. In one embodiment the effluent pH is at least 3.0. In one embodiment the bolus of titrating / buffering agent is passed through the chromatography medium until the pH of the effluent is at least 3.5. In one embodiment the bolus of titrating / buffering agent is passed through the chromatography medium until the pH of the effluent is at least 4.0. In one embodiment the bolus of titrating / buffering agent is passed through the chromatography medium until the pH of the effluent is at least 4.5. In one embodiment the bolus of titrating / buffering agent is passed through the chromatography medium until the pH of the effluent is at least 5.0. In one embodiment the bolus of titrating / buffering agent is passed through the chromatography medium until the conductivity of the effluent is at least 1.0 mS / cm to 20.0 mS / cm. In one embodiment the bolus of titrating / buffering agent is passed through the chromatography medium until the conductivity of the effluent is at least 1.0 mS / cm. In one embodiment the bolus of titrating / buffering agent is passed through 15        the chromatography medium until the conductivity of the effluent is at least 2.0 mS / cm. In one embodiment the bolus of titrating / buffering agent is passed through the chromatography medium until the conductivity of the effluent is at least 3.0 mS / cm. In one embodiment the bolus of titrating / buffering agent is passed through the chromatography medium until the conductivity of the effluent is at least 5.0 mS / cm. In one embodiment the bolus of titrating / buffering agent is passed through the chromatography medium until the conductivity of the effluent is at least 10.0 mS / cm. In one embodiment the bolus of titrating / buffering agent is passed through the chromatography medium until the conductivity of the effluent is at least 15.0 mS / cm. In one embodiment the bolus of titrating / buffering agent is passed through the chromatography medium until the conductivity of the effluent is at least 20.0 mS / cm. In one embodiment at least 0.01 to 1.0 CVs of titrating / buffering agent is used. In one embodiment at least 0.01 CVs of titrating / buffering agent is used. In one embodiment at least 0.05 CVs of titrating / buffering agent is used. In one embodiment at least 0.03 CVs of titrating / buffering agent is used. In one embodiment at least 0.1 CVs of titrating / buffering agent is used. In one embodiment at least 0.5 CVs of titrating / buffering agent is used. In one embodiment at least 0.09 CVs of titrating / buffering agent is used. In one embodiment at least 1.0 CVs of titrating / buffering agent is used. In one embodiment at least 0.01 to 1.0 CVs of a strong titrating agent is used. In one embodiment at least 0.01 to 1.0 CVs of strong base is used. In one embodiment the strong base is at least 10 mM to 1.0 M Sodium Hydroxide. In one embodiment the strong base is at least 0.1 M to 0.5 M Sodium Hydroxide. In one embodiment the strong base is at least 0.5 M to 1.0 M Sodium Hydroxide. In one embodiment the strong base is 10mM Sodium Hydroxide. In one embodiment the strong base is 50 mM Sodium Hydroxide. In one embodiment the strong base is 0.1 M Sodium Hydroxide. In one embodiment the strong base is 0.2 M Sodium Hydroxide. In one embodiment the strong base is 0.3 M Sodium Hydroxide. In one embodiment the strong base is 0.4 M Sodium Hydroxide. In one embodiment the strong base is 0.5 M Sodium Hydroxide. In one embodiment the strong base is 0.6 M Sodium Hydroxide. In one embodiment the strong base is 0.7 M Sodium Hydroxide. In one embodiment the strong base is 0.8 M Sodium Hydroxide. In one embodiment the strong base is 0.9 M Sodium Hydroxide. In one embodiment the strong base is 1.0 M Sodium Hydroxide. In one embodiment at least 0.01 to 1.0 CVs of Sodium Hydroxide is used. In one embodiment at least 0.01 CVs of Sodium Hydroxide is used. In one embodiment at least 0.02 CVs of Sodium Hydroxide is used. In one embodiment at least 0.03 CVs of Sodium Hydroxide is used. In one embodiment at least 0.04 CVs of Sodium Hydroxide is used. In one embodiment at least 0.05 CVs of Sodium Hydroxide is used. In one embodiment at least 0.06 CVs of Sodium Hydroxide is used. In one embodiment at least 0.07 CVs of Sodium Hydroxide is used. In one embodiment at least 0.08 CVs of Sodium Hydroxide is used. In one embodiment at least 0.09 CVs of Sodium Hydroxide is used. In one embodiment at least 0.1 CVs of Sodium Hydroxide is used. In one embodiment at least 0.5 CVs of Sodium Hydroxide is used. In one embodiment at least 1 CVs of Sodium Hydroxide is used. In one embodiment the contact time of the bolus of titrating / buffering agent with the chromatography medium is at least 5 seconds to 30 minutes. In one embodiment the contact time is at least 16        5 seconds to 10 minutes. In one embodiment the contact time is at least 5 seconds to 5 minutes. In one embodiment the contact time is at least 5 seconds to 4 minutes. In one embodiment the contact time is about 5 seconds. In one embodiment the contact time is about 10 seconds. In one embodiment the contact time is about 15 seconds. In one embodiment the contact time is about 20 seconds. In one embodiment the contact time is about 25 seconds. In one embodiment the contact time is about 30 seconds. In one embodiment the contact time is about 1 minute. In one embodiment the contact time is about 2 minutes. In one embodiment the contact time is about 3 minutes. In one embodiment the contact time is about 4 minutes. In one embodiment the contact time is about 5 minutes. In one embodiment the contact time is about 10 minutes. In one embodiment the contact time is about 15 minutes. In one embodiment the contact time is about 20 minutes. In one embodiment the contact time is about 25 minutes. In one embodiment the contact time is about 30 minutes. Second Equilibrium Flush Following the addition of the bolus of a titrating / buffering agent, a second equilibrium flush solution is added to continue the neutralization of the chromatography medium. A second equilibrium flush solution is passed through the chromatography medium until one or more desired process parameters are achieved. The process parameters are selected from one or more of pH, conductivity, contact time, and / or column volumes of the second equilibrium solution passed through the chromatography material. Suitable buffers include, but are not limited to, Tris, Phosphate, Bis-Tris, Mes, Carbonate, and Acetate. Suitable salts include, but are not limited to, sodium chloride, sodium phosphate, and ammonium sulfate. In one embodiment the equilibration solution is 25mM Tris, 100mM NaCl, pH 7.4. In one embodiment the first equilibrium solution and the second equilibrium solution are the same. In one embodiment the first equilibrium solution and the second equilibrium solution are the different. The second equilibrium flush solution is passed through the chromatography medium until one or more desired process parameters are achieved. The process parameters are selected from one or more of pH, conductivity, contact time, and / or column volumes of the first equilibrium flush buffer passed through the chromatography material. In one embodiment second equilibrium flush solution is passed through the chromatography medium until the pH of the effluent is at least 6.5 to 8.0. In one embodiment the pH of the effluent is at least 6.5. In one embodiment the pH of the effluent is at least 6.6. In one embodiment the pH of the effluent is at least 6.7. In one embodiment the pH of the effluent is at least 6.8. In one embodiment the pH of the effluent is at least 6.9. In one embodiment the pH of the effluent is at least 7.0. In one embodiment the pH of the effluent is at least 7.1. In one embodiment the pH of the effluent is at least 7.2. In one embodiment the pH of the effluent is at least 7.3. In one embodiment the pH of the effluent is at least 7.4. In one embodiment the pH of the effluent is at least 7.5. In one embodiment the pH of the effluent is at least 7.6. In one embodiment the pH of the effluent is at least 7.7. In one embodiment the pH of the effluent is at 17        least 7.8. In one embodiment the pH of the effluent is at least 7.9. In one embodiment the pH of the effluent is at least 8.0. In one embodiment the second equilibrium flush solution is passed through the chromatography medium until the conductivity of the effluent matches the load conductivity. In one embodiment the second equilibrium flush solution is passed through the chromatography medium until the conductivity of the effluent is at least 5.0 mS / cm to 20.0ms / cm. In one embodiment the conductivity of the eluate is 5.0 mS / cm. In one embodiment the second equilibrium solution is passed through the chromatography medium until the conductivity of the effluent is at least 6.0 mS / cm. In one embodiment the second equilibrium solution is passed through the chromatography medium until the conductivity of the effluent is at least 7.0 mS / cm. In one embodiment the second equilibrium solution is passed through the chromatography medium until the conductivity of the effluent is at least 8.0 mS / cm. In one embodiment the second equilibrium solution is passed through the chromatography medium until the conductivity of the effluent is at least 9.0 mS / cm. In one embodiment the second equilibrium solution is passed through the chromatography medium until the conductivity of the effluent is at least 10.0 mS / cm. In one embodiment the second equilibrium solution is passed through the chromatography medium until the conductivity of the effluent is at least 11.0 mS / cm. In one embodiment the second equilibrium solution is passed through the chromatography medium until the conductivity of the effluent is at least 12.0 mS / cm. In one embodiment the second equilibrium flush solution is passed through the chromatography medium until the conductivity of the effluent is at least 13.0 mS / cm. In one embodiment the second equilibrium solution is passed through the chromatography medium until the conductivity of the effluent is at least 14.0 mS / cm. In one embodiment the second equilibrium solution is passed through the chromatography medium until the conductivity of the effluent is at least 15.0 mS / cm. In one embodiment the second equilibrium solution is passed through the chromatography medium until the conductivity of the effluent is at least 20.0 mS / cm. In a preferred embodiment, the conductivity of the effluent is at least 12.0 mS / cm. In one embodiment at least 1 to 5 CVs of the second equilibrium flush solution is passed through the chromatography medium. In one embodiment at least 1 CV of second equilibrium flush solution is passed through the chromatography medium. In one embodiment at least 2 CVs of second equilibrium flush solution is passed through the chromatography medium. In one embodiment at least 3 CVs of second equilibrium flush solution is passed through the chromatography medium. In one embodiment at least 4 CVs of second equilibrium flush solution is passed through the chromatography medium. In one embodiment 5 CVs of second equilibrium flush solution is passed through the chromatography medium. In one embodiment 5 CVs of 25mM Tris, 100mM NaCl, pH 7.4 is passed through the chromatography medium. The contact time of the second equilibration flush solution with the chromatography medium is less than or equal to 8 hours. In one embodiment the contact time is at least 5 minutes. In one embodiment the contact time is at least 10 minutes. On one embodiment the contact time is at least 15 minutes. On one embodiment the contact time is 20 minutes. The contact time of the second equilibration flush with the chromatography medium is less than or equal to 30 minutes. In one embodiment the contact time is at least 18        30 minutes. In one embodiment the contact time is at least 60 minutes. In one embodiment the contact time is at least 90 minutes. In one embodiment the contact time is at least 2 hours. In one embodiment the contact time is at least 3 hours. In one embodiment the contact time is at least 4 hours. In one embodiment the contact time is at least 5 hours. In one embodiment the contact time is at least 6 hours. In one embodiment the contact time is at least 7 hours. In one embodiment the contact time is at least 8 hours. In one embodiment a sufficient volume of the second equilibrium agent is passed through the chromatography medium to not overshoot the effluent pH and / or conductivity. Overshooting the effluent pH and / or conductivity expose the chromatography medium results in caustic exposure that could result in the chromatography medium losing capacity. To mitigate this risk, the denaturing agent can be set to a pH that with the operating parameters of the chromatography material. In one embodiment, Sodium carbonate / Tris which has a higher pH than the target but lower than the pH of Sodium Hydroxide can be used. Regeneration Step In addition to the strip, optional water flush, first and second equilibration flushes, and bolus addition of a denaturing agent, the cleaning method may also include one or more regeneration steps to reduce and / or remove any remaining impurities and contaminants from the chromatography medium. Regeneration contributes to the reduction of fouling, minimizes deterioration of the packed column, allows for recycling of the chromatography medium, and / or prolonging the lifetime of the medium. The regeneration step also reduces and / or removes the contaminants that contribute to the bioburden load, such as microbial and / or viral contaminants. Bioburden management is required to minimize contamination of the product as well the manufacturing environment and for complying with regulatory requirements. The regeneration step is performed under harsher conditions than the strip step. These harsh conditions may cause degradation of the chromatography medium, so care is needed to select conditions that provide sufficient cleaning with minimal degradation of the chromatography medium. The regeneration step may be performed as an isocratic gradient or as a linear gradient. The regeneration conditions include the use of a denaturing agent. In one embodiment, the denaturing agent includes alkali agents. In one embodiment the alkali agents are strong titrating species. In one embodiment, the strong titrating species is a strong base. In one embodiment a strong base includes Sodium Hydroxide. The denaturing agent also includes chaotropic agents. In one embodiment chaotropic agents include guanidine hydrochloride, urea, and organic solvents such as isopropanol. In one embodiment chaotropic agents are used in combination with strong titrating species. In one embodiment the titrating / buffering agent used for the bolus and the denaturing agent used for the regeneration step are the same. In one embodiment the titrating / buffering agent used for the bolus and the denaturing agent used for the regeneration step are the different. 19        In one embodiment the denaturing agent is Sodium Hydroxide. In one embodiment the agent is least 0.5M to 1.5M M Sodium Hydroxide. In one embodiment the denaturing agent is at least 0.5 M Sodium Hydroxide. In one embodiment the agent is at least 0.6 M Sodium Hydroxide. In one embodiment the denaturing agent is at least 0.7 M Sodium Hydroxide. In one embodiment the denaturing agent is at least 0.8 M Sodium Hydroxide. In one embodiment the denaturing agent is at least 0.9 M Sodium Hydroxide. In one embodiment the denaturing agent is at least 1.0 M Sodium Hydroxide. In one embodiment the denaturing agent is at least 1.1 M Sodium Hydroxide. In one embodiment the denaturing agent is at least 1.2 M Sodium Hydroxide. In one embodiment the denaturing agent is at least 1.3 M Sodium Hydroxide. In one embodiment the denaturing agent is at least 1.4 M Sodium Hydroxide. In one embodiment the denaturing agent is 1.5 M Sodium Hydroxide. The denaturing agent is passed through the chromatography medium until one or more desired process parameters selected from one or more of effluent pH, effluent conductivity, contact time, and / or column volumes of denaturing agent passed through the chromatography material are achieved. . In one embodiment the denaturing agent is passed through the chromatography medium until the pH of the effluent is at least 9.0. In one embodiment the denaturing agent is passed through the chromatography medium until the pH of the effluent is at least 9.5. In one embodiment the denaturing agent is passed through the chromatography medium until the pH of the effluent is at least 10.0. In one embodiment the denaturing agent is passed through the chromatography medium until the pH of the effluent is at least 10.5. In one embodiment the denaturing agent is passed through the chromatography medium until the pH of the effluent is at least 11. In one embodiment the denaturing agent is passed through the chromatography medium until the conductivity of the effluent is at least 10 mS / cm. In one embodiment the denaturing agent is passed through the chromatography medium until the conductivity of the effluent is at least 15 mS / cm. In one embodiment the denaturing agent is passed through the chromatography medium until the conductivity of the effluent is at least 20 mS / cm. In one embodiment the denaturing agent is passed through the chromatography medium until the conductivity of the effluent is at least 50 mS / cm. In one embodiment the denaturing agent is passed through the chromatography medium until the conductivity of the effluent is at least 75 mS / cm. In one embodiment the denaturing agent is passed through the chromatography medium until the conductivity of the effluent is at least 100 mS / cm. In one embodiment the denaturing agent is passed through the chromatography medium until the conductivity of the effluent is at least 125 mS / cm. In one embodiment the denaturing agent is passed through the chromatography medium until the conductivity of the effluent is at least 150 mS / cm. In one embodiment the denaturing agent is passed through the chromatography medium until the conductivity of the is at least 175 mS / cm. In one embodiment the denaturing agent is passed through the chromatography medium until the conductivity of the effluent is at least 200 mS / cm. 20        In one embodiment at least 1 CV to 5 CV of denaturing agent is used. In one embodiment at least 1 CV is used. In one embodiment at least 2 CV is used. In one embodiment at least 3 CV is used. In one embodiment at least 4 CV is used. In one embodiment 5 CV is used. The contact time of the denaturing agent with the chromatography medium is less than or equal to 8 hours. In one embodiment the contact time of the denaturing agent with the chromatography medium is at least 10 minutes to 4 hours. In one embodiment the contact time is at least 10 minutes. In one embodiment the contact time is at least 15 minutes. In one embodiment the contact time is at least 20 minutes. In one embodiment the contact time is at least 30 minutes. In one embodiment the contact time is at least 1 hour. On one embodiment the contact time is at least 2 hours. On one embodiment the contact time is at least 3 hours. On one embodiment the contact time is at least 4 hours. One or more regeneration steps may be performed following a chromatography batch. Preferably, one or more cleaning steps are performed following one or more cycles of the chromatography medium. One or more strip and equilibration steps and one or more regeneration steps may be performed together or separately after one or more cycles, after one or more batches, and / or prior to column storage. In one embodiment, at least one regeneration step is performed in combination with the cleaning steps. In one embodiment, at least one regeneration step is performed following two or more cleaning steps. In one embodiment a regeneration step is performed in combination with a cleaning step following one or more cycles of the chromatography medium. In one embodiment, a regeneration step is performed in combination with a cleaning step prior to column storage. Following a desired number of cycles or batches, the chromatography medium may be prepared for short- or long-term column storage by using the cleaning method described herein. In one embodiment, the chromatography medium may be prepared for column storage after one or more cycles. In one embodiment the chromatography medium is prepared for column storage following completion of one or more batches. In one embodiment the chromatography medium is stored in ethanol. In one embodiment the chromatography medium is stored in 2% Benzyl Alcohol, 50mM Sodium Citrate. In one embodiment anti-microbial, anti-viral, and / or other suitable solutions may be used in addition. Storage temperature is up to 20°C. Depending on the chromatography medium, storage may be up to 5 years. Following cleaning, the chromatography medium is assessed for residual impurities and contaminants. It is essential to detect any carryover between batches, especially when the chromatography medium is use for purification of two or more different protein products. Blank runs with no sample added can be used to assess product carryover throughout the cleaning process. In one embodiment a blank run is performed after 1 or more cycles. In one embodiment a blank run is performed after 2 or more cycles. In one embodiment a blank run is performed after 3 or more cycles. In one embodiment a blank run is performed after 4 or more cycles. In one embodiment a blank run is performed after 5 or more cycles. In one embodiment a blank run is performed after 6 or more cycles. In one embodiment a blank run is performed after 7 or more cycles. In one embodiment a blank run is performed after 8 or more cycles. 21        Monitoring and validation of the cleaning are routine production processes. Such monitoring and validation can include maintenance of cleaning agent contact time and measuring product purity and impurity profiles. Process analytical techniques (PAT) can also be for monitoring and validation. The method described herein can be used for chromatography medium where the slow dynamics of chromatography medium pH equilibration increases the effective buffer capacity of the chromatography medium in addition to its intended effect of increasing product binding capacity. The method can be used in single column systems, multicolumn column systems, such as periodic counter current culture, expanded bed chromatography systems, and the like. Also, membrane and monolith systems as well. In one embodiment the chromatography medium is an affinity chromatography medium. In particular, the method may be used with affinity chromatography medium such as Protein A chromatography medium. Examples include Protein A chromatography resins such as MABSELECTTM, Sepharose FAST FLOW™, PrismA, (Cytiva, Marlborough, MA), PROSEP-A™(ProSep, Houston, TX), TOYOPEARL™AF-rProtein A-650F, AP Plus (Tosoh Bioscience GmbH Darmstadt-Griesheim, Germany). “Cell” or “Cells” include any prokaryotic or eukaryotic cell. Cells can be either ex vivo, in vitro, or in vivo, either separate or as part of a higher structure such as a tissue or organ. In one embodiment, the cell is a host cell. Host cells, when cultured under appropriate conditions, express a product 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). Selection of appropriate host cells is well within the ability of one ordinarily skilled in the art. Host cells may be genetically engineered to express a protein of commercial and / or scientific interest. Host cells are typically derived from a lineage arising from a primary culture that can be maintained under cell culture conditions for an unlimited time. 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. A host cell can be any prokaryotic cell (for example, Escherichia coli) or eukaryotic cell (for example, yeast, insect, or animal cells, in particular mammalian cells (e.g., Chinese Hamster Ovary cells (CHO cells)). In some embodiments, the cells are eukaryotic cells, preferably mammalian cells. The mammalian cells can be, for example, human, primate, avian, rodent, or bovine cell lines or cell strains. As non-limiting examples, a wide variety of mammalian cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and other vendors. Cells may be adherent, attached to surfaces such as beads or adapted to growth in suspension culture. In some embodiments the cells, cell lines, or cell strains include, but are not limited to, mouse myeloma (NSO)-cell lines, IT 1080, Η9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, VERO, SP2 / 0, ΥΒ2 / 0, Υ0, C127, L cell, COS, e.g., COS1, QC1-3, HEK-293, VERO, PER.C6, HeLa, ΕΒ1, ΕΒ2, ΕΒ3, ΕΒ14, ΕΒ24, ΕΒ26, ΕΒ66, or ΕΒν13, CLIO cells from CFIO-S, oncolytic, or hybridoma-cell lines. Other embodiments include monkey kidney CVl line 22        transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, (Graham et al, 1977, J. Gen Virol.36:59); baby hamster kidney cells (BHK, ATCC CCL 10), BF1K; mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod.23:243-251); monkey kidney cells (CVl ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatoma cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., 1982, Annals N.Y Acad. Sci.383:44-68); MRC 5 cells or FS4 cells and mammalian myeloma cells. In some embodiments, the mammalian cells are Chinese hamster cells (CHO) and CHO-cell lines. In some embodiments, the mammalian cells are chosen from CHO-K1 cells, CHO-K1 SV cells, DG44 CHO cells, DUXB11 CHO cells, CHOS cells, CHO GS knock-out cells (GSKO), CHO-K1 SV GS knockout cell, CHO FUT8 GS knock-out cells, POTELLIGENT®CHOK1 SV (Lonza, Inc.), CHOZN cells. CHO cells, including CHOK1 cells (ATCC CCL61), are widely used to produce complex recombinant proteins. In some embodiments, the dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al., 1980, Proc Natl Acad Sci USA 77: 4216-4220), DXB11 and DG-44, are desirable CHO host cell lines because the efficient DHFR selectable and amplifiable gene expression system allows high level recombinant protein expression in these cell lines (Kaufman R. J., 1990, Meth Enzymol 185:537-566). Also included are the glutamine synthase (GS)-knockout CHOK1SV cell lines, making use of glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection. Other suitable CHO host cells for use in a bioreactor of a flexible facility of the present disclosure include, but are not limited to, the following (ECACC accession numbers in parenthesis): CHO (85050302); CHO (PROTEIN FREE) (00102307); CHO-K1 (85051005); CHO-K1 / SF (93061607); CHO / dhFr-(94060607); CHO / dhFr-AC-free (05011002); and RR-CHOKI (92052129). Expression systems and constructs in the form of plasmids, expression vectors, transcription, or expression cassettes that comprise at least one nucleic acid encoding a product of interest are provided herein, as well host cells comprising such expression systems or constructs. As used herein, “vector” refers to 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 desired 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. A person of ordinary skill in the art may select elements appropriate for their use. Vector DNA can be introduced into the cells via conventional techniques. Expression systems and constructs in the form of expression vectors 23        such as plasmids or transcription or expression cassettes that comprise one or more polynucleotides encoding a desired protein, such as those identified herein, are provided, as well as host cells comprising such expression systems or constructs. Methods and vectors for genetically engineering cells and / or cell lines to express a desired proteins are well known to those of skill in the art. Genetically engineering the host cell involves transfecting, transforming or transducing the cells with a recombinant polynucleotide molecule, and / or otherwise 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 protein. 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 (including components effective in controlling the timing of recombinant protein production), 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 5A 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 cultures can also be supplemented with independent concentrated feeds of particular nutrients which may be difficult to formulate or are quickly depleted in cell cultures. Such nutrients may be amino acids such as tyrosine, cysteine and / or cystine (see e.g., WIPO Publication No.2012 / 145682). Cell culture media can be serum- free, protein-free, growth factor-free, and / or peptone-free media. Cell culture media may also be enriched by the addition of nutrients at greater than their usual, recommended concentrations. In preferred embodiments, cell culture media is tailored to the growth of host cells to cell densities greater than 106cells / ml. Various media formulations can be used during the life of the culture, for example, to initiate the culture, 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 feed media provided during perfusion culture). A basal media formulation containing components essential for cell survival and growth, is typically used to initiate the cell culture. A growth media formulation is typically used to promote cell growth and minimize protein expression. A production media formulation is typically used to promote production of the desired protein and maintenance of the cells, with minimal new cell growth). A feed media, typically a concentrated media formulation containing components such as 24        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 perfusion mode. Such a concentrated feed media may contain some, if not all, of the components of the cell culture media at, for example, about 5×, 6×, 7×, 8×, 9×, 10×, 12×, 14×, 16×, 20×, 30×, 50×, 100×, 200×, 400×, 600×, 800×, or even about 1000× of their normal amount or concentration. Cells are cultured in distinct stages. Each stage can be conducted in its own bioreactor vessel or other vessel suitable for cell culture. Alternatively, more than one stage can be conducted in a common vessel. For commercial production of recombinant proteins by mammalian cells, there are commonly multiple, for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 growth stages that occur in different culture vessels preceding the final production bioreactor(s). For example, cells may be cultured in various bioreactors for one or more growth stages prior to the N-1 seed stage, and then cultured in one or more N- 1 bioreactors. The duration of the N-1 stage can range from, e.g., 3 to 14 days, or be continuous, and can be designed to maintain the cells in exponential growth prior to inoculation of a production bioreactor (N). The cells from the N-1 bioreactor are transferred to a production bioreactor and grown under conditions that maximize protein production. Various methods that may be used in commercial processes for the production of recombinant proteins by mammalian cell culture include batch culture, fed-batch cultures, and perfusion cultures. Batch cell culture is executed in a closed bioreactor system that contains a fixed volume of a culture medium which serves as the source of nutrients to support the culture over its duration. Since no additional media will be added, a typical batch culture begins with a culture medium containing all essential nutrient components added at or near 100% of the bioreactor working volume. Large-scale bioreactors operating with volumes up to tens of thousands of liters are favored. The culture is inoculated at a low cell density with cells in the growth phase to maintain waste product levels below a threshold that would impact cell culture viability and productivity for as long as possible. "Cell density" refers to the number of living cells in a given volume of culture medium. Harvest typically begins when the product quality, cell viability, and / or productivity declines. Cells may be cultured in suspension or in an adherent form, attached to a solid substrate. Cells can be established with or without microcarriers. Cells may be cultured in bioreactor, a vessel suitable to conduct a cell culture. The bioreactor may comprise a disposable container, e.g. made of plastic material, or a reusable container, e.g. made of glass or stainless steel. In some embodiments, the method of the disclosure is conducted in stainless-steel bioreactors, particularly built-in-place large-scale stainless-steel bioreactors. The bioreactor can have a volume between 100 mL and 50,000 L. Unless otherwise indicated, a bioreactor can be of any size so long as it is useful for the culturing of cells; typically, a bioreactor is sized appropriate to the volume of cell culture being grown inside of it. In non-limiting embodiments and unless 25        otherwise indicated by context, a bioreactor may be at least 500, 1,000, 1500, 2000, 2,500, 5,000, 8,000, 10,000, 12,000, 18,000, 20,000 liters or more, or any volume in between. The internal conditions of the bioreactor, including, but not limited to, pH and temperature, can be controlled during the culturing period. Those of ordinary skill in the art will be aware of, and will be able to select, suitable bioreactors for use in the flexible facilities disclosed herein based on the relevant considerations. In some embodiments, an example bioreactor can perform one or more (e.g., one, two, three, all) of the following steps: feeding of nutrients and / or carbon sources, injection of suitable gas (such as, e.g., oxygen), inlet and outlet flow of cell culture medium, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and CO2levels, maintenance of pH level, agitation (e.g., stirring), and / or cleaning / sterilizing. Any suitable bioreactor diameter can be used. The bioreactor system maintains conditions within the bioreactor to support cell culture. Suitable culture conditions for mammalian cells are known in the art. See e.g. Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). By “running” a bioreactor system is meant maintaining conditions in the bioreactor system to support cell culture. A bioreactor “run” typically comprises the steps of inoculating a prepared bioreactor with a seed culture, subjecting the cells to one or more growth phase and / or production phases until one or more predetermined parameters are met (time, viable cell density, packed cell volume) and then harvesting the contents of the bioreactor. To increase production of a desired protein by the mammalian cells, cell growth can be limited or arrested. Such methods include temperature shifts, pH shifts, use of chemical inducers of protein production and cell cycle inhibitors, nutrient limitation, or starvation, either alone or in combination. For example, lower culture temperature(s) may be used to decrease cell growth and to promote protein production and culture longevity. Use of temperature shifts from optimal growth phase temperatures to optimal production phase temperatures are often employed in cell culture strategies. To optimize mammalian cell growth, the culture temperature is typically maintained at physiological temperature, 35°C to 37°C, during the growth phase. The temperature during the production phase is typically from about 28°C to about 35°C. Temperature modulation can be used throughout the duration of the culture to achieve desired growth and production objectives. Combinations of temperature shifts may also be used to go from a first growth phase to a first production phase, to second growth phase, followed by a second production phase, and so on. One embodiment the temperature during the growth phase is from about 35°C to about 38°C. In one embodiment the temperature is from about 34°C to about 36°C. In one embodiment the temperature is at least 35°C, 36°C or 37°C ± 0.5°C. In one embodiment the temperature is 38°C, 37°C, 36.5°C, 36°C 35.5°C, 35°C, or 34.5°C. One embodiment of the invention the temperature during the production phase is from about 32°C to about 35°C. In one embodiment the temperature is from about 30°C to about 35°C. In one embodiment the temperature is from about 32°C to about 34°C. In one embodiment the temperature 26        is at least 30°C, 31°C, 32°C, 33°C or 34°C ± 0.5°C. In one embodiment the temperature is 32°C, 32.5°C, 33°C, or 33.5°C. In one embodiment, the production phase begins when the culture temperature is decreased. In one embodiment, a pH shift may be used alone or in combination with a temperature shift. Another method to maintain cells at a desired physiological state is to induce cell growth-arrest by exposure of the cell culture to low L-asparagine conditions and / or asparagine starvation (see e.g., WIPO Publication No. WO 2013 / 006479). Cell growth-arrest may be achieved and maintained through a culture media that contains a limiting concentration of L-asparagine and maintaining a low concentration of L- asparagine in the cell culture. Maintaining the concentration of L-asparagine at 5 mM or less can be used to induce and maintain cells in a growth-arrested state whereby productivity is increased. In addition, chemical inducers of protein production, such as caffeine, butyrate, and hexamethylene bisacetamide (HMBA), may be added before, at the same time as, 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. Cell cycle inhibitors, compound known or suspected to regulate cell cycle progression and the associated processes of transcription, DNA repair, differentiation, senescence and apoptosis related to this are also useful to induce cell growth-arrest. Cell cycle inhibitors that interact with the cycle machinery, such as cyclin-dependent kinases (CDKs) are useful as are those molecules that interact with proteins from other pathways, such as AKT, mTOR, and other pathways that affect, directly or indirectly, the cell cycle. Chemical inducers of protein production, for example, caffeine, butyrate, and hexamethylene bisacetamide (HMBA), may be added at the same time as, before, and / or after a temperature shift, or in place of 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. Shifts in pH may also be used alone or in combination with temperature and / or chemical inducers. The production phase continues until one or more desired target criteria including a culture volume, final culture volume, bioreactor working volume, final bioreactor working volume, time point, titer, cell density, packed cell volume, product attribute, process, production and / or a plant schedule, is met. The cell culture is sampled as needed to monitor the culture and operation. One or more conditions, attributes, characterizations, etc., of the culture may be monitored, such as cell count, viability, viable cell density, packed cell volume, bioreactor volume, pH, pCO2, DO, glucose, lactate, ammonia, osmolality, titer, amino acids, and product quality. Various methods that may be used in commercial processes for the production of recombinant proteins by mammalian cell culture include batch culture, fed-batch cultures, and perfusion cultures. Batch cell culture is executed in a closed bioreactor system that contains a fixed volume of a culture medium which serves as the source of nutrients to support the culture over its duration. Since no additional media will be added, a typical batch culture begins with a culture medium containing all essential nutrient 27        components added at or near 100% of the bioreactor working volume. Large-scale bioreactors operating with volumes up to tens of thousands of liters are favored. The culture is inoculated at a low cell density with cells in the growth phase to maintain waste product levels below a threshold that would impact cell culture viability and productivity for as long as possible. "Cell density" refers to the number of living cells in a given volume of culture medium. Harvest typically begins when the product quality, cell viability, and / or productivity declines. Fed batch culture offers an improvement over batch culture methods by supplementing the culture with fresh nutrient media to replenish depleted nutrients during the course of the culture, but like batch culture, the spent media containing waste products and the like is not removed from the bioreactor and accumulates over time. Like batch cultures, large-scale bioreactors operating at volumes up to tens of thousands of liters are favored for fed batch operations due to low titers and to meet product demands. Harvest typically begins when the product quality, cell viability, and / or productivity of the cells declines. is flexible and accommodates a wide range of bioreactor types and sizes and can be scaled up or down to meet supply demands. The fed batch culture is flexible and accommodates a wide range of bioreactor types and sizes and can be scaled up or down to meet supply demands. Perfusion culture makes use of media exchange (i.e., the addition of fresh culture media (feed media) to the bioreactor and removal of a similar volume of spent culture media, as permeate, through the separation system(s)) These separation systems comprise pumps used to direct the contents of the bioreactor through separation systems that typically comprise one or more membrane filters (often operated in tangential flow mode) that selectively retain or return cell culture components back to the bioreactor. Spent culture media, waste products and impurities, and other components including recombinant proteins, can be selectively removed in the permeate depending on the selection criteria of the separation system. Like batch and fed batch cultures, in perfusion cultures the cells are typically retained in the bioreactor for the duration of the culture. Unlike batch and fed batch cultures, perfusion cultures typically have a higher cell mass which in turn produces a larger amount of waste and byproducts over the course of the culture compared to a batch or fed batch process, while in turn requiring large volumes of fresh media replenishment. Perfusion cultures can be continuous and / or semi-continuous. Cell densities achieved during a perfusion culture are typically higher than those for batch or fed-batch cultures. Another type of perfusion culture, lean perfusion, operates at a high cell density during the growth phase(s) and a low cell density during the production phase(s). A low medium exchange rate sufficient to ensure removal of waste byproducts to avoid culture toxicity while delivering sufficient nutrients via the fresh media to maintain essential cellular functions is maintained throughout the culture, see US Provisional Application No.63 / 403,896. The harvesting operation fully or partially clarifies and / or purifies the target protein away from at least one impurity with which it is found in the cell culture fluid, such as remaining cell culture media, cells, cell debris, undesired cell, or media components, and / or product- and / or process-related impurities. 28        Methods for harvesting recombinant proteins from suspension cell cultures 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, ultrafilters, microfilters, tangential flow, alternative tangential flow, depth filters, and alluvial filtration filters. Centrifugation is used to clarify cell culture pool. The centrifuge is one that is suitable for the volume and type of material to be separated. Intermittent solids discharge centrifuge and continuous solids discharge centrifuge are typically used for cell culture harvest operations. In one embodiment the centrifuge is a continuous solid discharge, disc stack centrifuge. Such centrifuges are known in the art and available commercially from a variety of vendors, such as Alfa Laval (Uppsala, Sweden) and Westfalia (Oelde, Germany). The bulk cell culture fluid is continuously fed (feed flow) into the centrifuge which separates solids and liquids simultaneously. The denser solids (heavy phase) move outwards towards the rotating bowl wall and are removed continuously (heavy phase flow) and discarded. The less dense liquid phase (light phase) for the centrate that will be collected from the centrifuge further processed by one or more downstream purification operations. The centrate may be collected into a vessel, a surge tank or holding vessel or transferred directly to the next unit operation. Batch and fed batch cultures are typically bulk harvested, the contents of the bioreactor is drained and subjected to a variety of harvest methods. Perfusion cultures are typically harvested by equipping the separation system with a filter having a pore size or MWCO that allows a desired protein to pass through into the permeate where it is collected for harvest. The high cell density, high packed cell volume, and lower harvest day viability associated with perfusion cell culture make it a challenge for harvest by centrifugation, especially intermittent or continuous solid discharge centrifugation. The lower cell densities and / or lower packed cell volumes make the cultures from the lean perfusion method described herein more suitable for the capabilities and capacities of a centrifuge-based harvest operation. Making use of a centrifugation-based harvest method frees the lean perfusion method from the issues related harvesting through the separation system and the low recovery titers common with that method. The cell culture fluid can be subjected one or more additional clarification methods known in the art, including but not limited to, acid precipitation, accelerated sedimentation such as flocculation, acoustic wave separation and capture chromatography. The collected centrate may be subjected to one or more depth filtration trains to further reduce the centrate particulate content and remove impurities. To facilitate a continuous flow, a surge tank or hold vessel may be located between the centrifuge and the depth filtration train or the centrate can directly transferred to the filtration train(s) in-line. 29        The depth filter train may include one or more depth filters of the same or different type. Depth filters suitable for use in the methods are known in the art and are commercially available. Such filters include, but are not limited to, cellulose, pre-treated filtration matrix, synthetic fiber meshes or a combination all. In some embodiments, the filtration step is depth filtration or tangential flow filtration. Exemplary depth filters include, but are not limited to, a VIRESOLVE®Pro Shield, a VIRESOLVE®Pro Shield H, a MILLISTAK+®D0HC filter, a MILLISTAK+®X0HC filter, a MILLISTAK+®C0HC filter, a MILLISTAK+®C0SP filter, a MILLISTAK+®X0SP filter, Clarisolve 20MS filter, a Clarisolve 40MS filter, a Clarisolve 60HX filter, (Millipore, Burlington, MA), a SARTOCLEAR®DL60 filter, a SARTOCLEAR®DL75 filter (Sartorius, Göttingen, Germany), a 3M™ Zeta Plus™ Filter, diatomaceous earth, or a 3M™ Emphaze™ AEX Hybrid Purifier (EM, Meriden, CT). In addition, the depth filtration may be followed by one or more sterile or bioreduction control filters. Such filters are known in the art and commercially available and include, but are not limited to, Millipore EXPRESS®SHC hydrophilic polyetheresulfone filter (Millipore) and SARTOPORE®2 polyethersulfone (PES) liquid filters (Sartorius). Pre-filters can be used in combination with the filters to help eliminate certain contaminants in centrate and to extend the life of the filters. The harvested cell culture fluid (HCCF) from the filtration train(s) can be stored in surge tanks, holding tanks, bags, or other containers that are adapted to provide feed to a chromatography column skid. The harvested cell culture fluid may be subjected to one or more downstream operations to purify and / or polish the target protein. The downstream operations make use of capture chromatography that includes resins and / or membranes containing agents that will bind and / or interact in some manner with at least one desired protein, impurity, or contaminant. Examples of capture chromatography include affinity chromatography, size exclusion chromatography, ion exchange chromatography (IEX) such as cation exchange (CEX) and anion exchange (AEX) chromatography, hydrophobic interaction chromatography (HIC), multimodal or mixed-modal (MMC), hydroxyapatite chromatography (HA), reverse phase chromatography and gel filtration, among others and the like. Such materials are known in the art and are commercially available. Affinity chromatography is commonly used in biomanufacturing processes, typically as an initial capture step to isolate and concentrate desired proteins from harvested cell culture fluid. Examples of such affinity chromatography materials include those that make use of Staphylococcus proteins such as Protein A, Protein G, Protein A / G, and Protein L; substrate-binding capture mechanisms; antibody- or antibody fragment-binding capture mechanisms; aptamer-binding capture mechanisms; cofactor-binding capture mechanisms; and the like. Immobilized metal affinity chromatography (IMAC) can be used to capture proteins that have or have been engineered to have affinity for metal ions. Protein A affinity chromatography is typically used as in first line, bulk purification operations. Protein A ligands are highly 30        selective for a wide range of proteins containing an antibody Fc region and provides a robust removal of process-related impurities and high target protein yields. Protein A material is available commercially from a number of vendors. For example, MaBSelectTMSURE Protein A, Protein A Sepharose Fast Flow™, MaBSelect™PrismA, PRAESTO®AP+, APc, AC, Jetted A50 (Cytiva, Marborough, MA), ProSep-A™(Merck Millipore, U.K), Toyopearl™AF-rProtein A-650F Protein A resin (TosoHass Co., Philadelphia, PA), and AP Plus, Purolite, King of Prussia, PA). Intermediate and / or polishing unit operations make use of various chromatography methods for the continued purification of the desired protein and clearance of contaminants and impurities such as DNA, host cell proteins, removal of process- and product-related impurities, virus, and the like. These chromatography unit operations makes use of chromatography media and / or membranes containing agents that can be operated in a variety of configurations, such as flow-through mode where the desired protein is contained in the eluent and the contaminants and impurities are bound to the chromatography medium; frontal or overloaded chromatography mode where a solution containing the desired protein is loaded onto a column until adsorption sites on are occupied and the species with the least affinity for the stationary phase (the desired protein) starts to elute; and bind and elute mode, where the desired protein is bound to the chromatography medium and eluted after the contaminants and impurities have flowed through or been washed off the chromatography medium, or any other method. Examples of chromatography media used in such processes include, ion exchange chromatography (IEX), such as anion exchange chromatography (AEX) and cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed modal or multimodal chromatography (MM), hydroxyapatite chromatography (HA). Multiple chromatography unit operations, usually one, two, or three, each typically performing a different function or operating in different modes, are combined depending on the requirements of the manufacturing process. Ion exchange chromatography, based on electrostatic interactions between charged surfaces, separates desired proteins from impurities based on differential absorption and desorption. 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). Cation exchange chromatography is typically operated in bind and elute mode, the high pI of many desired proteins enable binding to the chromatography material. Cation exchange chromatography may also be run in flow through mode. CEX chromatography is typically used to remove high molecular weight (HMW) contaminants, process related impurity, and / or viral clearance. Commercially available cation exchange media are available and include but are not limited to sulphopropyl (SP) immobilized on agarose (e.g. SP-SEPHAROSE FAST FLOW™, SP-SEPHAROSE FAST FLOW XL™ or SP-SEPHAROSE HIGH PERFORMANCE™, CAPTO S™, CAPTO SP ImpRes™, CAPTO S ImpAct™ (Cytiva), FRACTOGEL-SO3™, FRACTOGEL-SE HICAP™, and FRACTOPREP™ (EMD Merck, Darmstadt, Germany), TOYOPEARL®XS, 31        TOYOPEARL®HS (Tosh Bioscience, King of Prussia, PA), UNOsphere™ (BioRad, Hercules, CA), S Ceramic Hyper™DF (Pall, Port Washington, NY), POROS™ (ThermoFisher, Waltham, MA), and ESHMUNO®CSP (Millipore Sigma, Darmstadt, Germany). Anion exchange chromatography refers to chromatography performed on a solid phase medium that is positively charged and has free anions for exchange with anions in an aqueous solution passed over or through the solid phase. In biomanufacturing, anion exchange chromatography is typically operated in flow through mode due to a high pI of many therapeutic proteins. The therapeutic proteins do not to bind to the AEX chromatography material. AEX chromatography is used, for example, for viral clearance and impurity removal. Commercially available anion exchange media are available and include, but are not limited to, sulphopropyl (SP) immobilized on agarose (e.g. Source 15 Q, Capto™ Q, Q-SEPHAROSE FAST FLOW™ (Cytiva), FRACTOGEL EDM TMAE™, FRACTOGEL EDM DEAE™ (EMD Merck), TOYOPEARL Super Q®(Tosh Bioscience), POROS HQ™, POROS XQ™, (ThermoFisher). Mixed-mode or multi-mode chromatography (MMC) refers to chromatography that makes use of more than one form of interaction between the stationary phase and analyte to achieve their separation. MMC differs from single mode chromatography in that two or more types of interactions, such as electrostatic, hydrogen bonding and hydrophobic interactions, contribute significantly to the retention of solutes. Commercially available multi-modal chromatography media are available and include, but are not limited to, CaptoTMAdhere, Capto™ MMC Impress, Capto MMC, (Cytiva), PPA Hypercel, MEP Hypercell, HEA Hypercell (Pall Corporation, Port Washington, NY). Eshmuno HCX, (Merk Millipore), Toyopearl MX-Trp-650M (Tosoh Bioscience). Hydrophobic interaction chromatography refers to chromatography performed on a solid phase medium that makes use of the interaction between hydrophobic ligands and hydrophobic residues on the surface of a desired protein. Commercially available hydrophobic interaction chromatography media includes but are not limited to Phenyl SephroseTM(Cytiva), Tosoh hexyl (Tosoh Bioscience), and CaptoTMphenyl (Cytiva). Hydroxyapatite chromatography refers to chromatography performed on a solid phase medium that makes use of positively charged calcium and negatively charged phosphate and depending on the pI of the protein and the pH of the buffer, can act as a cation or anion. Commercially available hydroxyapatite media are available and include, but are not limited to, CA++Pure-HA, Tosho Bioscience, HA ULTROGEL®, Sartorius). Unit operations directed towards inactivating, reducing and / or eliminating viral contaminants may include processes that mitigate viral risk by manipulating the environment and / or through use of filtration. Viral mitigation measures are critical to ensure the safety of protein therapeutics and may be performed one or more times throughout the downstream purification. Viral contaminants can arise from a variety of sources including use of reagents of animal origin, adventitious viral contaminants in host cell lines, or 32        system failures at GMP manufacturing sites. Viruses are classified as enveloped and non-enveloped viruses. With enveloped viruses, the envelope allows the virus to identify, bind, enter, and infect target host cells. As such, enveloped viruses are susceptible to inactivation methods. Various methods can be employed for virus inactivation and include heat inactivation / pasteurization, UV and gamma ray irradiation, use of high intensity broad spectrum white light, addition of chemical inactivating agents, surfactants, and solvent / detergent treatments. Surfactants, such as detergents, solubilize membranes and therefore can be very effective in specifically inactivating enveloped viruses. One method for achieving virus inactivation is incubation at low pH (e.g., pH<4). Low pH virus inactivation can be followed with by neutralization that readjusts the viral inactivated solution to a pH more compatible with the requirements of the following downstream unit operations. Low pH viral inactivation is typically performed following purification of the harvest cell culture fluid with affinity chromatography, in particular affinity chromatography that makes use of a substrate binding ligand from Staphylococcus aureus, such as Protein A chromatography, since elution is usually performed at a low pH. Exemplary low pH viral inactivation methods are described in US Application 63 / 168,608 and US Application 63 / 159,217. An exemplary detergent inactivation is described in World International Patent Organization Publication No: WO 2020 / 190985. Low pH viral inactivation may also be followed by filtration, such as depth filtration, for clarification of the neutralized fluid. Non-enveloped viruses are more difficult to inactivate without risk to the protein being manufactured and are removed by filtration methods. An exemplary process is described in World International Patent Organization Publication No: WO2020 / 159838. Viral filtration can be performed using micro- or nano-filters, such as those available from PLAVONA® (Asahi Kasei, Chicago, IL), VIROSART® (Sartorius, Goettingen, Germany), VIRESOLVE® Pro (MilliporeSigma, Burlington, MA), PegasusTM Prime (Pall Biotech, Port Washington, NY), CUNO Zeta Plus VR, (3M, St. Paul, Mn). Viral filtration may occur at one or more steps in the downstream operations of a biomanufacturing process. Typically, viral filtration precedes the ultrafiltration / diafiltration (UFDF) operation but may also take place following UFDF. Unit operations may also comprise product concentration and solution exchange of the desired protein into a desired formulation solution for bulk storage of the drug substance can be accomplished using known methods for ultrafiltration and diafiltration (UFDF). Instead of storage, unit operations related to drug product fill / finish can also immediately follow a UFDF operation. The method further optionally comprises concentrating the protein product using ultrafiltration and diafiltration (UFDF). The purified protein is subjected to an ultrafiltration and diafiltration operation comprising concentrating or diluting the purified protein by ultrafiltration; solution exchanging the purified concentrated / diluted protein into a desired formulation by diafiltration; further diluting or concentrating the formulated purified protein by a second round of ultrafiltration until a target protein concentration is achieved. One or more stability- enhancing excipients may optionally be added directly to the UFDF retentate feed tank containing the formulated purified protein resulting in formulated drug substance or added to the UFDF eluate pool. An 33        exemplary UFDF process is described in WIPO Published Application NO. WO 2020 / 159838. Filters for use in a UFDF operation are well known and common in the art and are commercially available from many sources. There are many types of materials available, regenerated cellulose Pellicon (MilliporeSigma, Danvers, MA), stabilized cellulose, Sartocon®Slice, Sartocon®ECO Hydrosart®(Sartorius, Goettingen, Germany), polyethersulfone (PES) membrane, Omega (Pall Corporation, Port Washington, NY). In all chromatography processes, multiple filters can be used to the capacity that holders, skids, or the physical set up of the UFDF system will allow or are needed to achieve the desired objectives of a production process. Critical attributes and performance parameters of the purified desired protein can be measured to better inform decisions regarding performance of each step during manufacture. These critical attributes and parameters can be monitored real-time, near real-time, and / or after the fact. Key critical parameters such as cell culture 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, packed cell volume, pH, osmolality, appearance, color, aggregation, percent yield and titer may be monitored during appropriated stages in the manufacturing process. Monitoring and measurements can be done using known techniques and commercially available equipment. The terms “polypeptide”, “protein” or “product” are used interchangeably throughout and refer to a molecule comprising two or more amino acid residues joined to each other by peptide bonds. The polypepties, proteins or products also include macromolecules having one or more deletions from, insertions to, and / or substitutions of the amino acid residues of the native sequence, that is, a polypeptide, protein, or product produced by a naturally-occurring and non-recombinant cell; or is produced by a genetically-engineered or recombinant cell, and comprise molecules having one or more deletions from, insertions to, and / or substitutions of the amino acid residues of the amino acid sequence of the native protein. The polypeptides, proteins or products also include amino acid polymers in which one or more amino acids are chemical analogs of a corresponding naturally-occurring amino acid and polymers. The polypeptides, proteins, or products are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. The polypeptides, proteins, or products can be of scientific and / or commercial interest, including protein-based therapeutics. Products of interest include, but are not limited to, secreted proteins, non-secreted proteins, intracellular proteins, or membrane-bound proteins. Polypeptides, proteins, and products of interest can be produced by cell lines (e.g., host cells) 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 34        term “isolated protein” or “isolated recombinant protein” refers to a polypeptide or product of interest that is purified away from polypeptides, proteins, or other impurities that would interfere with its therapeutic, diagnostic, prophylactic, research, and / or other use. Products 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 herein, including targets derived therefrom, targets related thereto, and modifications thereof. Products 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 to which it binds (e.g., the 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 et al., 2010, Protein Eng Des Sel 23:221-228)), hetero-IgG (see, e.g., Liu et al., 2015, J Biol Chem 290:7535-7562), muteins, and XMAB®(Xencor, Inc., Monrovia, CA). Also included are bispecific T cell engager molecules (BITE®molecules), bispecific T cell engagers having extensions, and others, chimeric antigen receptors (CARs, CAR Ts), and T cell receptors (TCRs). In some embodiments, products of interest may include colony stimulating factors, such as 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, EPOGEN®(epoetin alfa), ARANESP®(darbepoetin alfa), DYNEPO®(epoetin delta), MIRCERA®(methyoxy polyethylene glycol- 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. In some embodiments, products 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, p150,95, VLA-4, ICAM-1, VCAM, 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, growth hormone releasing factor, parathyroid hormone, mullerian-inhibiting substance, human macrophage inflammatory protein (MIP-1-alpha), erythropoietin (EPO), nerve 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-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5, insulin-like growth factors-I and -II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I), and osteoinductive 35        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 hormone receptors, 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, regulatory proteins, 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 / hCGβ, hepatitis-C virus, mesothelin dsFv[PE38] conjugate, Legionella pneumophila (Ily), 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, α4β7, platelet specific (platelet glycoprotein IIb / IIIb (PAC-1), transforming growth factor beta (TFGβ), Zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet derived growth factor receptor alpha (PDGFRα), sclerostin, and biologically active fragments or variants of any of the foregoing. In some embodiments, products of interest include abciximab, acapatamab, 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, efaveleukin alfa, epratuzumab, erenumab, etanercept, evolocumab, galiximab, ganitumab, gemtuzumab, golimumab, ibritumomab infliximab, ipilimumab, lerdelimumab, letikafusp, lumiliximab, lxdkizumab, mapatumumab, motesanib diphosphate, muromonab- 36        CD3, natalizumab, nesiritide, nimotuzumab, nivolumab, ocrelizumab, ofatumumab, omalizumab, oprelvekin, ordesekimab, palivizumab, panitumumab, pembrolizumab, pertuzumab, pexelizumab, ranibizumab, rilotumumab, rituximab, rocatinlimab, romiplostim, romosozumab, rozibafusp alfa, sargamostim, tarlatamag, tezepelumab, tocilizumab, tositumomab, tiuxetan, trastuzumab, ustekinumab, vedolizumab, visilizumab, volociximab, zanolimumab, and zalutumumab, as well as biosimilars of any of the foregoing. As used herein, the terms “a” and “an” mean “one or more” unless specifically indicated otherwise. Additionally, “one or more” and “at least one” are used interchangeably herein. Furthermore, unless otherwise required by context, singular terms include pluralities and plural terms include the singular. 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. What is described in an embodiment of the disclosure can be combined with one or more other embodiments of the disclosure unless context clearly indicates otherwise. The disclosed subject matter is not intended to be limited in scope by the specific embodiments described herein, which are instead intended as non-limiting illustrations of individual aspects of the disclosure. Functionally equivalent methods and components are within the scope of the disclosure. Indeed, various modifications of the disclosed subject matter, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description and accompanying drawing(s). Such modifications are intended to fall within the scope of the disclosed subject matter. The descriptions of the various embodiments and / or examples of the disclosed subject matter have been presented for purposes of illustration but are not intended to be exhaustive or limiting in any way. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein was chosen to best explain the principles of the embodiments, the practical application or technical improvement over technologies found in the marketplace, and / or to enable others of ordinary skill in the art to understand the disclosed subject matter. 37        EXAMPLES Example 1 The equilibration dynamics of the chromatography medium was evaluated when a bolus of a titrating / buffering solution was used during equilibration, such as would be performed during the cleaning of the medium. The experiment compared the amount of solution required to equilibrate the chromatography medium in the absence and presence of the titrating / buffering solution. The conditions are outlined in Table 1. Three Protein A chromatography resins (MabSelect™ PrismA, Cytiva, Marlborough, MA) (1 cm diameter x 25 cm bed height) were used. The experiment consisted of a strip step and an equilibration step, such as would be performed during a cleaning. For the strip step each of the resins was subjected to a low pH phosphoric acid followed by: Column 1: an equilibration flush until the effluent pH was 7.4 (Control) Column 2: a first equilibration solution flush followed by a bolus of the titrating / buffering solution (0.5 M NaOH), followed by a second equilibrium flush until the effluent pH was 7.4 (Condition 1) Column 3: a water flush, then a first equilibration solution flush, followed by a bolus of the titrating / buffering solution (0.5 M NaOH), and followed by a second equilibrium flush until the effluent pH was 7.4 (Condition 2), see Table 1. Table 1. Conditions for Protein A resin cleaning with and without a bolus of a titrating / buffering species. Condition Strip Water 1stEquilibrium Bolus of 2ndEquilibrium (CV) Flush Solution Flush Titrating / Solution .4 Introduction of the bolus of the titrating / buffering solution reduced the consumption of equilibration solution by approximately 4 to 5 column volumes (CVs) relative to the control, as shown in Table 2 and Figure 2. The equilibration efficiency was further improved by adding a brief water flush and a brief equilibration flush following the low pH strip phase. Use of a water flush followed by an equilibration solution flush prior to the base bolus addition (condition 2, Figure 2 (C)) resulted in a faster 38        pH equilibration than flushing with equilibration solution alone prior to the base bolus (condition 1, Figure 2 (B)) or flushing with equilibration solution only (Control, Figure 2 (A)). Table 2. Effect of a bolus of a strong titrating species on efficiency of Protein A chromatography equilibration ConditionWaterReduction in Figure 2 EQ NaOH Required flush flush equilibration l m flush CVs of EQ l ti n Condition 2 led to a slight overshoot in the resin effluent pH (<2 pH units) which was minimal relative to a sodium hydroxide treatment during aperiodic regeneration phase and correlated with significantly shorter equilibration periods. Note that equilibration times for conductivity at the column effluent are generally much shorter than pH and are therefore not shown for the purposes of this analysis. 39

Claims

What is claimed is:

1. A method for on-column titration to accelerate chromatography medium equilibrium dynamics comprising the steps of a. passing a first equilibration flush solution through the chromatography medium; b. passing a bolus of a titrating / buffering agent through the chromatography medium; and c. passing a second equilibrium flush solution through the chromatography medium.

2. The method according to claim 1, wherein the first equilibrium flush solution comprises Tris, Phosphate, Bis-Tris, Mes, Carbonate, or Acetate.

3. The method according to claim 1, wherein the pH of the first equilibration flush solution is at least 5.0 to 8.

0.

4. The method according to claim 1, wherein the conductivity of the first equilibrium flush solution is at least 5 to 20 mS / cm.

5. The method according to claim 1, wherein the first equilibration flush solution is passed through the chromatography medium until one or more desired process parameters, selected from effluent pH, effluent conductivity, contact time, and / or number of column volumes of the first equilibration flush solution passed through the chromatography medium, is achieved.

6. The method according to claim 5, wherein at least one column volume of the first equilibrium flush solution is passed through the chromatography medium.

7. The method according to claim 5, wherein the contact time for the first equilibration flush solution with the chromatography medium is at least 5 minutes.

8. The method according to claim 5, wherein the first equilibration flush solution is passed through the chromatography medium until the conductivity of the effluent is at least 5 to 20 mS / cm.

9. The method according to claim 5, wherein the first equilibration flush solution is passed through the chromatography medium until the pH of the effluent is at least 2.5 to 5.

0.

10. The method according to claim 1, wherein the titrating / buffering agent is a strong titrating species.

11. The method according to claim 10, wherein the strong titrating species is a strong base.

12. The method according to claim 10, wherein the strong titrating species is Sodium Hydroxide, Potassium Hydroxide, Tris Base, or Sodium Carbonate.

13. The method according to claim 1 wherein the bolus of titrating / buffering agent is passed through the chromatography medium until one or more desired process parameters, selected from one or more of effluent pH, effluent conductivity, contact time, and / or column volumes of the bolus of denaturing agent passed through the chromatography medium, is achieved.

14. The method according to claim 13, wherein the titrating / buffering agent is passed through the chromatography medium until the pH of the effluent is at least 2.5 to 5.

0.

15. The method according to claim 13, wherein the titrating / buffering agent is passed through the chromatography medium until the conductivity of the effluent is at least 1.0 m to 1.0 mS / cm to 20 mS / cm. 40     16. The method according to claim 13, wherein the contact time for the bolus of titrating / buffering agent and the chromatography medium is at least 5 seconds.

17. The method according to claim 13, wherein at least 0.01 column volume of titrating / buffering agent is passed through the chromatography medium.

18. The method according to claim 1, wherein a sufficient bolus of titrating / buffering agent is passed through the chromatography medium to not overshoot a desired effluent pH and / or conductivity.

19. The method according to claim 1, wherein the second equilibrium flush solution comprises Tris, Phosphate, Bis-Tris, Mes, Carbonate, or Acetate.

20. The method according to claim 1, wherein the pH of the second equilibration flush solution is at least 5.0 to 8.

0.

21. The method according to claim 1, wherein the conductivity of the second equilibrium flush solution is at least 5 to 20 mS / cm.

22. The method according to claim 1, wherein the first equilibrium flush solution and the second equilibrium flush solution are the same.

23. The method according to claim 1, wherein the first equilibrium flush solution and the second equilibrium flush solution are different.

24. The method according to claim 1, wherein the second equilibration flush solution is passed through the chromatography medium until one or more desired process parameters selected from selected from one or more of pH, conductivity, contact time, and / or column volumes of second equilibrium buffer passed through the chromatography medium is achieved.

25. The method according to claim 24, wherein at least one column volume of the second equilibrium flush solution is passed through the chromatography medium.

26. The method according to claim 24, wherein the contact time for the second equilibrium flush solution with the chromatography medium is at least 5 minutes.

27. The method according to claim 24, wherein the second equilibration flush is passed through the chromatography medium until the pH of the second equilibrium flush solution is at least 6.5 to 8.

0.

28. The method according to claim 24, wherein the second equilibration flush is passed through the chromatography medium until the conductivity of the second equilibrium flush solution is at least 5.0 mS / cm to 20.0 mS / cm.

29. The method according to claim 1, further comprising passing a water flush through the chromatography medium prior to step (a).

30. The method according to claim 29, wherein the water flush is passed through the chromatography medium until one or more desired process parameters selected from selected from one or more of pH, conductivity, contact time, and / or column volumes of second equilibrium buffer passed through the chromatography medium is achieved.

31. The method according to claim 30, wherein the water flush is passed through the chromatography medium until the pH of the effluent is about 2.5 to 3.

0. 41   32. The method according to claim 30, wherein the water flush is passed through the chromatography medium until the conductivity of the effluent is about 0 to 1.0 mS / cm.

33. The method according to claim 30, wherein at least one column volume of water is passed through the chromatography medium.

34. The method according to claim 30, wherein the contact time for the water flush with the chromatography medium is less than or equal to 1 hour.

35. The method according to claim 30, wherein the number of column volumes of water flush is the same as the first equilibration solution flush.

36. The method according to claim 30, wherein the number of column volumes of water flush is different from the number of column volumes for the first equilibration solution flush.

37. The method according to claim 30, wherein the contact time for the water flush is the same as the contact time for first equilibration solution flush.

38. The method according to claim 30, wherein the contact time for the water flush is different from the contact time for the first equilibration solution flush.

39. The method according to claim 30 wherein the pH, conductivity, number of column volumes, or contact time for two or more of the water flush, first equilibrium flush, bolus, and / or second equilibrium flush steps are the same.

40. The method according to claim 38, wherein the water flush is purified water or water for injection.

41. The method according to claim 1, wherein the chromatography medium is an affinity chromatography medium.

42. The method according to claim 1, wherein the chromatography medium is a high capacity chromatography medium.

43. The method according to claim 41, wherein the chromatography medium is a Protein A or Protein G chromatography medium.

44. A method for cleaning a chromatography medium for reuse comprising the steps of a. passing an acidic agent through the chromatography medium; b. passing a first equilibration solution through the chromatography medium; c. passing a bolus of a titrating / buffering agent through the chromatography medium; and d. passing a second equilibrium solution through the chromatography medium.

45. The method according to claim 44, wherein the acidic agent is non-denaturing.

46. The method according to claim 44, wherein the pH of the acidic agent is at least 2.0 to 3.

5.

47. The method according to claim 44, wherein the acidic agent is phosphoric acid.

48. The method according to claim 44, wherein the acidic agent is passed through the chromatography medium until one or more desired process parameters, selected from effluent pH, effluent conductivity, contact time, and / or number or column volumes of the acidic agent passed through the chromatography medium, is achieved.

49. The method according to claim 48, wherein the contact time for the acidic agent with the chromatography medium is at least 10 to 60 minutes. 42   50. The method according to claim 48, wherein the acidic agent is passed through the chromatography medium until the conductivity of the effluent is at least 5 to 20 mS / cm.

51. The method according to claim 48, wherein the acidic agent is passed through the chromatography medium until the pH of the effluent is at least 3 to 5.

52. The method according to claim 48, wherein the chromatography medium is subjected to at least one column volume of the acidic agent.

53. The method according to claim 44, further comprises a regeneration step following step (d).

54. The method according to claim 53, wherein the regeneration step comprising passing at least one denaturing agent through the chromatography medium.

55. The method according to claim 53, wherein the denaturing agent is an alkali agent.

56. The method according to claim 55, wherein the alkali agent is a strong base.

57. The method according to claim 53, wherein the denaturing agent is a chaotropic agent.

58. The method according to claim 53, wherein the denaturing agent is passed through the chromatography medium until one or more desired process parameters, selected from selected from one or more of pH, conductivity, contact time, and / or number of column volumes of denaturing agent passed through the chromatography medium, is achieved.

59. The method according to claim 58, wherein the contact time for the denaturing agent with the chromatography medium is at least 10 minutes.

60. The method according to claim 58, wherein at least one column volume of the denaturing agent is passed through the chromatography medium.

61. The method according to claim 58, wherein the denaturing agent is passed through the chromatography medium until the pH from the effluent of the regeneration step is at least 9.0 to 11.

0.

62. The method according to claim 58, wherein the denaturing agent is passed through the chromatography medium until conductivity of the effluent of the regeneration step is at least 10.0 mS / cm to 200.0 mS / cm.

63. The method according to claim 53, wherein the titrating / buffering agent and the denaturing agent are the same.

64. The method according to claim 53, wherein the titrating / buffering agent and the denaturing agent are different.

65. The method according to claim 53, further comprising storing the cleaned chromatography medium in a 2% Benzyl Alcohol, 50 mM Sodium Citrate solution.

66. The method of claim 53, further comprising passing a water flush through the chromatography medium prior to step (b).

67. A method for reducing the consumption of equilibrium solution during the cleaning of a chromatography medium comprising the steps of a. a strip step; b. a water flush step; 43   c. a first equilibrium flush step; d. a bolus of a titrating / buffering agent; and e. a second equilibrium flush step; wherein the amount of equilibrium solution consumed is less than the amount of equilibrium solution consumed during a cleaning method consisting only of a strip step and a single equilibrium flush step.

68. A method for purifying a recombinant protein comprising the steps of a. initiating a cell culture in a bioreactor; b. culturing cells engineered to recombinantly express a protein; c. harvesting the cell culture containing the recombinant protein; d. binding the recombinant protein onto an affinity chromatography medium; e. eluting and collecting the bound recombinant protein; f. striping the chromatography medium with an acidic agent; g. optionally flushing the chromatography medium with water; h. flushing the chromatography medium with a first equilibration solution; i. passing a bolus of a titrating / buffering agent through the chromatography medium; j. flushing the chromatography medium with a second equilibrium solution; and k. repeating steps d-j until all the harvested cell culture has been loaded and the recombinant protein has been collected.

69. The method according to claim 68 further comprising a regeneration step comprising a denaturing agent.

70. The method according to claim 69, wherein the regeneration step occurs before or after step k.

71. The method according to claim 68, further comprising subjecting the collected recombinant protein to a low pH or detergent viral inactivation.

72. The method according to claim 68, further comprising neutralizing the viral inactivated recombinant protein.

73. The method according to claim 68, further comprising at least one additional chromatography step.

74. The method according to claim 68, wherein the chromatography step is selected from ion exchange chromatography, hydrophobic interaction chromatography, or multimodal chromatography.

75. The method according to claim 68, further comprising at least one viral filtration step.

76. The method according to claim 68, further comprising at least one UFDF step.

77. The recombinant protein made by the method of claim 67. 44

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