Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/18—Ion-exchange chromatography
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/06—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies from serum
  • C07K16/065—Purification, fragmentation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/46—Hybrid immunoglobulins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components

Definitions

• the present invention relates to the field of biopharmaceutical manufacturing.
• the invention relates to methods for removal of low isoelectric point product-related impurities during cation exchange purification operations.
• Antibody products are the largest sector of the biopharmaceuticals market and could easily reach hundreds of billions in sales over the next decade.
• the commercial development of therapeutic monoclonal antibodies began in the 1980s with the approval of the first therapeutic monoclonal antibody and has continued to evolve and expand ever since. While monoclonal antibodies bind a target with high affinity and specificity and have been very successful therapeutic treatments for some indications, they also have limitations. Monoclonal antibodies bind a single target; however many diseases are multifactorial. In cancer immunotherapy, a single target treatment may not be sufficient to destroy or immobilize cancer cells. In addition, some patients receiving monoclonal antibody therapies may fail to respond to treatment or develop drug resistance.
• New antibody-like structures such as antibody Fab fragments, Fc-fusion proteins, antibody- drug conjugates, glycol-engineered antibodies, and most especially, bispecific and other multispecific antibody-like structures have been developed to meet these challenges.
• Bispecific antibodies are the most diverse group of these antibody -like structures with an ever- increasing variety of frameworks to meet the needs of therapeutic indications. These structures combine the binding properties of antibodies with additional molecular properties engineered to suit desired disease indications. Bispecific antibodies are being developed for a variety of indication and uses, such as redirecting immune effector cells to tumor cells for immune response against cancer, blocking signaling pathways, targeting tumor angiogenesis, blocking cytokines, crossing the blood-brain barrier, diagnostic assays, treatment of pathogens, and as delivery agents.
• Product-related impurities having similar charge (isoelectric point) to a multispecific protein of interest may co-elute with the multispecific protein during cation exchange chromatography unit operations, complicating purification and lowering yield. It would be beneficial to separate the low pi product-related impurities prior to elution.
• the invention described herein meets this need by providing high salt load conditioning for removal of these low pi impurities during cation exchange chromatography.
• the invention provides a method of purifying a multispecific protein from a composition comprising the multispecific protein and at least one product-related impurity, the method comprising equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 94-105 mM Sodium chloride; loading the composition on to the cation exchange medium in a load buffer comprises 94-105 mM Sodium chloride; washing the column with at least one wash buffer comprising 94-105 mM Sodium chloride; and eluting the multispecific protein from the cation exchange chromatography medium.
• the load buffer comprises 94-96 mM Sodium chloride.
• the load buffer comprises 96-105 mM Sodium chloride.
• the load buffer comprises 94 mM sodium chloride. In a related embodiment the load buffer comprises 96 mM sodium chloride. In a related embodiment the load buffer comprises 105 mM sodium chloride. In one embodiment the load buffer comprises acetate. In a related embodiment the load buffer comprises acetate, pH 4.9-5.1. In a related embodiment the load buffer comprises acetate, pH 5.0 ⁇ 0.05 to 5.0 ⁇ 0.1. In a related embodiment the load buffer comprises 100 mM acetate. In one embodiment the load buffer comprises acetate and 94 mM-105 mM sodium chloride. In one embodiment at least one wash buffer comprises 94-96 mM sodium chloride.
• At least one wash buffer comprises 96-105 mM sodium chloride. In a related embodiment at least one wash buffer comprises 94 mM sodium chloride. In a related embodiment at least one wash buffer comprises 96 mM sodium chloride. In a related embodiment at least one wash buffer comprises 105 mM sodium chloride. In one embodiment at least one wash buffer comprises acetate. In one embodiment at least one wash buffer comprises acetate, pH 4.9-5.1. In a related embodiment, at least one wash buffer comprises acetate, pH 5.0 ⁇ 0.05 to 5.0 ⁇ 0.1. In a related embodiment at least one wash buffer comprises 100 mM acetate. In one embodiment at least one wash buffer comprises acetate and 94 mM-105 mM sodium chloride.
• At least one additional wash buffer comprises 0-26 mM sodium chloride. In one embodiment at least one wash buffer comprises acetate and 94-96 mM sodium chloride, followed by at least one additional wash buffer comprising acetate and 25 mM sodium chloride. In one embodiment at least one wash buffer comprises acetate and 105 mM sodium chloride, followed by at least one additional wash buffer comprising acetate. In one embodiment at least one equilibration buffer comprises 94-96 mM sodium chloride. In a related embodiment at least one equilibration buffer comprises 96-105 mM sodium chloride. In a related embodiment at least one equilibration buffer comprises 94 mM sodium chloride.
• At least one equilibration buffer comprises 96 mM sodium chloride. In a related embodiment at least one equilibration buffer comprises 105 mM sodium chloride. In one embodiment the equilibration buffer comprises acetate. In a related embodiment the equilibration buffer comprises acetate, pH 4.9-5.1. In a related embodiment the equilibration buffer comprises acetate, pH 5.0 ⁇ 0.05 to 5.0 ⁇ 0.1. In a related embodiment the equilibration buffer comprises 100 mM acetate. In one embodiment the equilibration buffer comprises acetate and 94 mM-105 mM sodium chloride. In one embodiment the composition is loaded at 10-27 g/L.
• the composition is loaded at 15-27 g/L.
• the multispecific protein is eluted from the cation exchange resin by a gradient. In a related embodiment the gradient is linear. In one embodiment the gradient is a salt gradient.
• the multispecific protein is a bispecific protein. In one embodiment the multispecific protein is a bispecific antibody. In one embodiment is provided a purified, multispecific protein prepared by the method described above. In one embodiment the cation exchange chromatography medium is a resin.
• the invention provides a method of reducing low pi impurities in the eluate from cation exchange chromatography, the method comprising equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 94-105 mM Sodium chloride; loading the composition on to the cation exchange medium in a load buffer comprises 94-105 mM Sodium chloride; washing the column with at least one wash buffer comprising 94-105 mM Sodium chloride; and eluting the multispecific protein from the cation exchange chromatography medium; wherein the cation exchange chromatography eluate has reduced low pi impurities compared to the cation exchange chromatography eluate recovered in a corresponding method in which no sodium chloride is used in the equilibration, load, and wash steps.
• the low pi impurity is a product-related impurity.
• at least one product-related impurity is a half antibody or 2X, 3X, or 4X light chain-mis- assembly.
• the invention provides a method of performing cation exchange chromatography under high salt loading conditions to reduce product-related impurities, the method comprising equilibrating a cation exchange chromatography medium with an equilibration buffer; loading the composition on to the cation exchange medium in a load buffer; washing the column with a first and a second wash buffer; and eluting the multispecific protein from the cation exchange chromatography medium; wherein the equilibration, loading and first wash buffers comprise 94-105 mM Sodium chloride.
• the second wash buffer comprises 0-26 mM.
• the invention provides a method of producing an isolated, purified, recombinant multispecific protein, the method comprising establishing a cell culture in a bioreactor with a host cell expressing the multispecific protein; culturing the host cells to express the multispecific protein; harvesting the recombinant multispecific protein; affinity purifying the harvested recombinant multispecific protein; inactivating virus at low pH in the eluate pool from the affinity purification and neutralizing the pool; equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 94- 105 mM Sodium chloride; loading the neutralized affinity purified recombinant multispecific protein on to the equilibrated cation exchange medium in a load buffer comprises 94-105 mM Sodium chloride; washing the cation exchange medium with a wash buffer comprising 94-105 mM Sodium chloride, followed by a second wash buffer comprising 0-26 mM Sodium chloride; eluting the multispecific protein
• the second chromatography resin is selected from an anion exchange chromatography resin, cation exchange chromatography resin, multi-modal chromatography resin, hydrophobic interaction chromatography resin, and hydroxyapatite chromatography resin.
• an isolated, purified, recombinant multispecific protein prepared by the method described above.
• a pharmaceutical composition comprising the isolated, purified, recombinant multispecific protein prepared by the method described above.
• Figure 1 Shows that impurities (half antibodies and 2X LC) are eluting with the main product, Bi- specific #1.
• Figure 2 Shows that following high salt load conditioning, the low pi impurities flowed through the column between the load and first wash steps.
• the second wash returned the UV baseline to zero before the elution.
• the elution peak was reduced from four peaks to one peak.
• Figure 3 Shows one elution peak resulting from the high load density, no salt load conditioning, at a steep elution gradient for Bi-specific #2.
• the low pi product-related impurities did not resolve from the main product under the high load density and are mostly in fractions 1-3.
• Figure 4 Shows that a lower loading density (10 vs 25 g/L) and shallower gradient (8 vs 16 mM/CV) allowed for separation of the main low pi product-impurities into a distinct peak formed by fractions 1- 4 fir Bi-specific #2.
• multispecific proteins may make them susceptible to formation of product-related impurities and cell culture conditions may impact the amount of such impurities. These impurities complicate purification and can lower the yield and activity of the desired multispecific protein. It was found that a high salt loading strategy improved the yield of the main product in the CEX eluate pool by removing the lower pi impurities prior to the elution step. By targeting a final sodium chloride concentration of 94-105 mM for the equilibration buffer, the final conditioned load buffer, and the first wash buffer, the lower pi impurities flowed through the column, reducing the number peaks in the elution profile and reducing the amount of product-related impurities in the CEX eluate.
• a second wash step was also added to ensure complete binding conditions for the desired multispecific protein and to reestablish the UV baseline to zero before the start of the elution, thereby tightening the elution profile, resulting in a much more efficient collection and better quality of the main product.
• the high salt load conditioning unexpectedly reduced length of the elution for a bi-specific protein from 44 column volumes (CVs) to 20.3 CVs, a savings in time and resources as well as a reduction in CEX eluate pool volume which is highly desirable for process efficiency and robustness in intermediate downstream unit operations.
• the high salt loading conditions allowed for efficient removal of impurities prior to elution, reducing the amount of product-related impurities in the CEX eluate, as well as simplifying the elution collection criteria, thus providing a more robust manufacturing process.
• the invention provides a method of purifying a multispecific protein from a composition comprising the multispecific protein and at least one product-related impurity, the method comprising equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 94- 105 mM sodium chloride; loading the composition on to the cation exchange medium in a load buffer comprises 94-105 mM sodium chloride; washing the column with at least one wash buffer comprising 94-105 mM sodium chloride; and eluting the multispecific protein from the cation exchange chromatography medium.
• the invention also provides a method of reducing low pi impurities in the eluate from cation exchange chromatography, the method comprising equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 94-105 mM sodium chloride; loading the composition on to the cation exchange medium in a load buffer comprises 94-105 mM sodium chloride; washing the column with at least one wash buffer comprising 94-105 mM sodium chloride; and eluting the multispecific protein from the cation exchange chromatography medium; wherein the cation exchange chromatography eluate has reduced low pi impurities compared to the cation exchange chromatography eluate recovered in a corresponding method in which no sodium chloride is used in the equilibration, load, and wash steps.
• the invention also provides a method of performing cation exchange chromatography under high salt loading conditions to reduce product-related impurities, the method comprising equilibrating a cation exchange chromatography medium with an equilibration buffer; loading the composition on to the cation exchange medium in a load buffer; washing the column with a first and second wash buffer; and eluting the multispecific protein from the cation exchange chromatography medium; wherein the equilibration, loading and first wash buffers comprise 94-105 mM sodium chloride.
• the invention also provides a method of producing an isolated, purified, recombinant multispecific protein of interest, the method comprising establishing a cell culture in a bioreactor with a host cell expressing the multispecific protein of interest; culturing the host cells to express the multispecific protein; harvesting the recombinant multispecific protein; affinity purifying the harvested recombinant multispecific protein; inactivating virus at low pH in the eluate pool from the affinity purification and neutralizing the pool; equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 94-105 mM sodium chloride; loading the neutralized affinity purified recombinant multispecific protein on to the equilibrated cation exchange medium in a load buffer comprises 94-105 mM sodium chloride; washing the cation exchange medium with a wash buffer comprising 94-105 mM sodium chloride, followed by a second wash buffer comprising 0-26 mM Sodium chloride; eluting the multispec
• the load buffer comprises 94-105 mM sodium chloride. In one embodiment the load buffer comprises 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105 mM sodium chloride. In one embodiment the load buffer comprises 94 mM sodium chloride. In one embodiment the load buffer comprises 96 mM sodium chloride. In one embodiment the load buffer comprises 98 mM sodium chloride. In one embodiment the load buffer comprises 105 mM sodium chloride.
• the load buffer comprises acetate. In one embodiment the load buffer comprises acetate, pH 4.9-5.1. In one embodiment the load buffer comprises acetate at pH, 4.9, 5.0, or 5.1. In one embodiment the load buffer comprises acetate at pH, 4.9, 4.95, 5.0, 5.05, or 5.1. In one embodiment the load buffer comprises acetate, pH of 5.0 ⁇ 0.05 to 5.0 ⁇ 0.1. In one embodiment the load buffer comprises acetate at pH, 5.0. In one embodiment the load buffer comprises 100 mM acetate. In one embodiment the load buffer comprises 100 mM acetate, pH 4.9-5.1. In one embodiment the load buffer comprises 100 mM acetate at pH, 4.9, 5.0, or 5.1.
• the load buffer comprises 100 mM acetate at pH, 4.9, 4.95, 5.0, 5.05, or 5.1. In one embodiment the load buffer comprises 100 mM acetate, pH 5.0 ⁇ 0.05% to pH 5.0 ⁇ 0.1%.
• the load buffer comprises acetate and 94 mM-105 mM sodium chloride. In one embodiment the load buffer comprises acetate and 94 mM-96 mM sodium chloride. In one embodiment the load buffer comprises acetate and 96 mM-105 mM sodium chloride. In a related embodiment the load buffer comprises acetate and 94 mM sodium chloride. In a related embodiment the load buffer comprises acetate and 96 mM sodium chloride. In a related embodiment the load buffer comprises acetate and 98 mM sodium chloride. In a related embodiment the load buffer comprises acetate and 105 mM sodium chloride. In a related embodiment, the acetate concentration is 100 mM.
• the load buffer comprises acetate, 94 mM-105 mM sodium chloride, pH 4.9-5.1. In a related embodiment the load buffer comprises acetate, 94 mM-105 mM sodium chloride, pH of 4.9, 5.0 or 5.1. In a related embodiment the load buffer comprises acetate, 94-105 mM sodium chloride, pH of 4.9, 4.95, 5.0, 5.05, or 5.1. In a related embodiment the load buffer comprises acetate, 94 mM-105 mM sodium chloride, pH of 5.0. In arelated embodiment, the acetate concentration is 100 mM.
• the load buffer comprises 100 mM acetate, 94 mM-105 mM sodium chloride, pH 5.0 ⁇ 0.05 to 5.0 ⁇ 0.1. In a related embodiment the load buffer comprises 100 mM acetate, 94 mM-105 mM sodium chloride, pH 4.9-5.1. In a related embodiment the load buffer comprises 100 mM acetate, 94 mM-105 mM sodium chloride, pH 4.9, 4.95, 5.0, 5.05, or 5.1. In a related embodiment the load buffer comprises 100 mM acetate, 94 mM-105 mM sodium chloride, pH 4.9, 5.0, or 5.1. In a related embodiment the load buffer comprises 100 mM acetate, 94 mM-105 mM sodium chloride, pH 5.0.
• At least one wash buffer comprises 94-96 mM sodium chloride. In one embodiment at least one wash buffer comprises 96-105 mM sodium chloride. In one embodiment at least one wash buffer comprises 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105 mM sodium chloride. In one embodiment at least one wash buffer comprises 94 mM sodium chloride. In one embodiment at least one wash buffer comprises 96 mM sodium chloride. In one embodiment at least one wash buffer comprises 98 mM sodium chloride. In one embodiment at least one wash buffer comprises 105 mM sodium chloride.
• At least one wash buffer comprises acetate. In one embodiment at least one wash buffer comprises acetate, pH 4.9-5.1. In one embodiment at least one wash buffer comprises acetate at pH, 4.9, 5.0, or 5.1. In one embodiment the wash buffer comprises acetate at pH, 4.9, 4.95, 5.0, 5.05, or 5.1. In one embodiment at least one wash buffer comprises acetate, pH of 5.0 ⁇ 0.05 to 5.0 ⁇ 0.1. In one embodiment at least one wash buffer comprises 100 mM acetate. In one embodiment at least one wash buffer comprises 100 mM acetate, pH 4.9-5.1. In one embodiment at least one wash buffer comprises 100 mM acetate at pH, 4.9, 5.0, or 5.1.
• the wash buffer comprises 100 mM acetate at pH, 4.9, 4.95, 5.0, 5.05, or 5.1. In one embodiment at least one wash buffer comprises 100 mM acetate, pH 5.0 ⁇ 0.05% to pH 5.0 ⁇ 0.1%.
• At least one wash buffer comprises acetate, 94 mM-105 mM sodium chloride. In one embodiment at least one wash buffer comprises acetate, 94 mM-96 mM sodium chloride. In one embodiment at least one wash buffer comprises acetate, 96 mM-105 mM sodium chloride. In a related embodiment at least one wash buffer comprises acetate, 94 mM sodium chloride. In a related embodiment at least one wash buffer comprises acetate, 96 mM sodium chloride. In a related embodiment at least one wash buffer comprises acetate, 98 mM sodium chloride. In a related embodiment at least one wash buffer comprises acetate, 105 mM sodium chloride. In a related embodiment, the acetate concentration is 100 mM.
• At least one wash buffer comprises acetate, 94 mM-105 mM sodium chloride, pH 5.0 ⁇ 0.05 to 5.0 ⁇ 0.1. In a related embodiment at least one wash buffer comprises acetate, 94 mM-105 mM sodium chloride, pH 4.9-5.1. In a related embodiment at least one wash buffer comprises acetate, 94 mM-105 mM sodium chloride, pH of 4.9, 5.0 or 5.1. In a related embodiment at least one wash buffer comprises acetate, 94 mM-105 mM sodium chloride, pH 4.9, 4.95, 5.0, 5.05, or 5.1. In a related embodiment at least one wash buffer comprises acetate, 94 mM-105 mM sodium chloride, pH of 5.0. In a related embodiment, the acetate concentration is 100 mM.
• At least one wash buffer comprises 100 mM acetate, 94 mM-105 mM sodium chloride, pH 5.0 ⁇ 0.05 to 5.0 ⁇ 0.1. In one embodiment at least one wash buffer comprises 100 mM acetate, 94 mM-105 mM sodium chloride, pH 4.9-5.1. In a related embodiment at least one wash buffer comprises 100 mM acetate, 94 mM-105 mM sodium chloride, pH 4.9, 5.0, or 5.1. In a related embodiment at least one wash buffer comprises 100 mM acetate, 94 mM-105 mM sodium chloride, pH 4.9, 4.95, 5.0, 5.05, or 5.1. In a related embodiment at least one wash buffer comprises 100 mM acetate, 94 mM-105 mM sodium chloride, pH 5.0.
• at least one additional wash is a second wash.
• at least one additional wash buffer comprises 0-26 mM sodium chloride.
• at least one additional wash buffer comprises 0, 23, 24, 25, or 26 mM sodium chloride.
• at least one additional wash buffer comprises 0 mM sodium chloride.
• at least one additional wash buffer comprises 23 mM sodium chloride.
• at least one additional wash buffer comprises 24 mM sodium chloride.
• at least one additional wash buffer comprises 25 mM sodium chloride.
• at least one additional wash buffer comprises 26 mM sodium chloride.
• At least one additional wash buffer comprises acetate. In one embodiment at least one additional wash buffer comprises acetate, pH 4.9-5.1. In one embodiment at least one additional wash buffer comprises acetate at pH, 4.9, 5.0, or 5.1. In one embodiment at least one additional wash buffer comprises acetate at pH, 4.9, 4.95, 5.0, 5.05, or 5.1. In one embodiment at least one additional wash buffer comprises acetate, pH of 5.0 ⁇ 0.05 to 5.0 ⁇ 0.1. In one embodiment at least one additional wash buffer comprises 100 mM acetate. In one embodiment at least one additional wash buffer comprises 100 mM acetate, pH 4.9-5.1.
• At least one additional wash buffer comprises 100 mM acetate at pH, 4.9, 5.0, or 5.1. In one embodiment at least one additional wash buffer comprises 100 mM acetate at pH, 4.9, 4.95, 5.0, 5.05, or 5.1. In one embodiment at least one additional wash buffer comprises 100 mM acetate, pH 5.0 ⁇ 0.05% to pH 5.0 ⁇ 0.1%.
• At least one wash buffer comprises acetate and sodium chloride followed by at least one additional wash.
• at least one wash buffer comprises acetate, 94-105 mM sodium chloride, followed by at least one additional wash.
• at least one wash buffer comprises acetate, 94-105 mM sodium chloride, followed by at least one additional wash buffer comprising 0-26 mM sodium chloride.
• at least one additional wash buffer comprises 23-26 mM sodium chloride.
• at least one additional wash buffer comprises 0 mM sodium chloride.
• at least one additional wash buffer comprises 23 mM sodium chloride.
• at least one additional wash buffer comprises 24 mM sodium chloride.
• at least one additional wash buffer comprises 25 mM sodium chloride.
• at least one additional wash buffer comprises 26 mM sodium chloride.
• At least one wash buffer comprises acetate and sodium chloride followed by at least one additional wash.
• at least one wash buffer comprises acetate, 94-105 mM sodium chloride, followed by at least one additional wash comprising acetate.
• at least one wash buffer comprises acetate, 94-105 mM sodium chloride, followed by at least one additional wash buffer comprising acetate, 0-26 mM sodium chloride.
• at least one additional wash buffer comprises acetate, 23-26 mM sodium chloride.
• at least one additional wash buffer comprises acetate, 0 mM sodium chloride.
• at least one additional wash buffer comprises acetate, 23 mM sodium chloride.
• At least one additional wash buffer comprises acetate, 24 mM sodium chloride. In a related embodiment at least one additional wash buffer comprises acetate, 25 mM sodium chloride. In a related embodiment at least one additional wash buffer comprises acetate, 26 mM sodium chloride. In one embodiment at least one wash buffer comprises acetate, 94 mM sodium chloride, followed by an additional wash buffer comprising acetate, 23-24 mM sodium chloride. In one embodiment at least one wash buffer comprises acetate, 96 mM sodium chloride, followed by an additional wash buffer comprising acetate, 25 mM sodium chloride.
• At least one wash buffer comprises acetate, 98 mM sodium chloride, followed by an additional wash buffer comprising acetate, 26 mM sodium chloride. In one embodiment at least one wash buffer comprises acetate, 105 mM sodium chloride, followed by an additional wash buffer comprising acetate, 0 mM sodium chloride. In one embodiment, the acetate concentration is 100 mM.
• the additional wash is a second wash.
• the second wash buffer comprises 0-26 mM sodium chloride.
• least one wash buffer comprises acetate, 94-105 mM sodium chloride, followed by a second wash buffer comprising acetate, 0-26 mM sodium chloride.
• the acetate concentration is 100 mM.
• At least one wash buffer comprises acetate, 94-105 mM sodium chloride, followed by at least one additional wash buffer comprising acetate, 0-26 mM sodium chloride where the pH of the buffers is the same or different.
• the pH of one or more of the wash buffers is pH 4.9-5.1.
• the pH of one or more of the wash buffers is pH 4.9, 4.95, 5.0, 5.05, or 5.1.
• the pH of one or more of the wash buffers is pH 4.9, 5.0, or 5.1.
• the pH of one or more of the wash buffers is pH 5.0.
• the pH of one or more of the wash buffers is pH 5.0 ⁇ 0.05% to pH 5.0 ⁇ 0.1%.
• the acetate concentration of one or more of the wash buffers is 100 mM.
• the equilibration buffer comprises 94-96 mM sodium chloride. In one embodiment the equilibration buffer comprises 96-105 mM sodium chloride. In one embodiment the equilibration buffer comprises 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105 mM sodium chloride. In one embodiment the equilibration buffer comprises 94 mM sodium chloride. In one embodiment the equilibration buffer comprises 96 mM sodium chloride. In one embodiment the equilibration buffer comprises 105 mM sodium chloride.
• the equilibration buffer comprises acetate. In one embodiment the equilibration buffer comprises acetate, pH 4.9-5.1. In one embodiment the equilibration buffer comprises acetate at pH 4.9, 5.0, or 5.1. In one embodiment the equilibration buffer comprises acetate at pH 5.0. In one embodiment the equilibration buffer comprises acetate, pH of 5.0 ⁇ 0.05 to 5.0 ⁇ 0.1. In one embodiment the equilibration buffer comprises 100 mM acetate. In one embodiment the equilibration buffer comprises 100 mM acetate, pH 4.9-5.1.
• the equilibration buffer comprises 100 mM acetate at pH, 4.9, 4.95, 5.0, 5.05, or 5.1. In one embodiment the equilibration buffer comprises 100 mM acetate at pH, 4.9, 5.0, or 5.1. In one embodiment the equilibration buffer comprises 100 mM acetate, pH 5.0 ⁇ 0.05% to pH 5.0 ⁇ 0.1%.
• the equilibration buffer comprises acetate, 94 mM-105 mM sodium chloride. In one embodiment the equilibration buffer comprises acetate, 94 mM-96 mM sodium chloride. In one embodiment the equilibration buffer comprises acetate, 96 mM-105 mM sodium chloride. In a related embodiment the equilibration buffer comprises acetate, 94 mM sodium chloride. In a related embodiment the equilibration buffer comprises acetate, 96 mM sodium chloride. In a related embodiment the equilibration buffer comprises acetate, 105 mM sodium chloride. In one embodiment, the acetate concentration is 100 mM.
• the equilibration buffer comprises acetate, 94 mM-105 mM sodium chloride, pH 5.0 ⁇ 0.05 to 5.0 ⁇ 0.1. In a related embodiment the equilibration buffer comprises acetate, 94 mM-105 mM sodium chloride, pH 4.9-5.1. In a related embodiment the equilibration buffer comprises acetate, 94 mM-105 mM sodium chloride, pH of 4.9, 4.95, 5.0, 5.05, or 5.1. In a related embodiment the equilibration buffer comprises acetate, 94 mM-105 mM sodium chloride, pH of 4.9, 5.0 or 5.1. In a related embodiment the equilibration buffer comprises acetate, 94 mM-105 mM sodium chloride, pH of 5.0. In one embodiment, the acetate concentration is 100 mM.
• the equilibration buffer comprises 100 mM acetate, 94 mM-105 mM sodium chloride, pH 5.0 ⁇ 0.05 to 5.0 ⁇ 0.1. In a related embodiment the equilibration buffer comprises 100 mM acetate, 94 mM-105 mM sodium chloride, pH 4.9-5.1. In a related embodiment the equilibration buffer comprises 100 mM acetate, 94 mM-105 mM sodium chloride, pH of 4.9, 4.95, 5.0, 5.01, or 5.1. In a related embodiment the equilibration buffer comprises 100 mM acetate, 94 mM-105 mM sodium chloride, pH of 4.9, 5.0 or 5.1. In a related embodiment the equilibration buffer comprises 100 mM acetate, 94 mM-105 mM sodium chloride, pH of 5.0.
• the composition is loaded at 10-27 g/L. In a related embodiment the composition is loaded at 10-25 g/L. In a related embodiment the composition is loaded at 10-23 g/L. In a related embodiment the composition is loaded at 10-15 g/L. In a related embodiment the composition is loaded at 15-27 g/L. In a related embodiment the composition is loaded at 15-25 g/L. In a related embodiment the composition is loaded at 15-23 g/L. In a related embodiment the composition is loaded at 23-27 g/L. In a related embodiment the composition is loaded at 23-25 g/L. In a related embodiment the composition is loaded at 25-27 g/L. In one embodiment the composition is loaded at 10, 15, 23, 25 or 27 g/L.
• the composition is loaded at 10 g/L. In one embodiment the composition is loaded at 15 g/L. In one embodiment the composition is loaded at 23 g/L. In one embodiment the composition is loaded at 25 g/L. In one embodiment the composition is loaded at 27 g/L.
• the multispecific protein is eluted from the cation exchange resin by a gradient.
• the gradient is linear.
• the gradient is a salt gradient.
• the low pi impurity is a product-related impurity.
• at least one product-related impurity is a half antibody or 2X, 3X, or 4X light chain-mis- assembly.
• the multispecific protein is a bispecific protein. In one embodiment the multispecific protein is a bispecific antibody.
• the cation exchange chromatography medium is a resin.
• the second chromatography medium is a resin.
• the second chromatography resin is selected from an anion exchange chromatography resin, cation exchange chromatography resin, multi-modal chromatography resin, hydrophobic interaction chromatography resin, and hydroxyapatite chromatography resin.
• the invention provides a purified, multispecific protein produced according to the methods described herein.
• the invention provides an isolated, purified, recombinant multispecific proteins made according to the methods described herein.
• the invention provides a pharmaceutical composition comprising the isolated, purified, recombinant multispecific protein of interest according to the methods described herein.
• “Multispecific”, “multispecific protein”, and “multispecific antibody” are used interchangeably herein to refer to proteins that are recombinantly engineered to simultaneously bind and neutralize at least two different antigens or at least two different epitopes on the same antigen.
• multispecific proteins can be engineered to target immune effectors and cytotoxic agents to tumors or infectious agents.
• multispecific proteins have been found useful for a variety of applications such as in cancer immunotherapy by redirecting immune effector cells to tumor cells, modifying cell signaling by blocking signaling pathways, targeting tumor angiogenesis, blocking cytokines, and as pre-targeted delivery vehicles for drugs, such as delivery of chemotherapeutic agents, radiolabels (to improve detection sensitivity) and nanoparticles (directed to specific cells/tissues, such as cancer cells).
• bispecific proteins can be grouped in two broad categories: immunoglobulin G (IgG)-iike molecules and non-IgG-like molecules.
• IgG-like molecules retain Fc-mediated effector functions, such as antibody-dependent cell mediated cytotoxicity (ADCC). complement-dependent cytotoxicity (CDC), and antibody -dependent cellular phagocytosis (AD CP), the Fc region helps improve solubility and stability and facilitate some purification operations.
• Non-IgG-like molecules are smaller, enhancing tissue penetration.
• Bispecific proteins are sometimes used as a framework for additional components having binding specificities to different antigens or numbers of epitopes, increasing the binding specificity of the molecule.
• bispecific proteins which include bispecific antibodies, are constantly evolving and include, but are not limited to, quadromas, knobs-in-holes, cross-Mabs, dual variable domains IgG (DVD-IgG), IgG-single chain Fv (scFv), scFv-CFG KIH, dual action Fab (DAF), half-molecule exchange, kl-bodies, tandem scFv, scFv-Fc, diabodies, single chain diabodies (scDiabodies), scDiabodies-CH3, triple body, miniantibody, minibody, TriBi minibody, tandem diabodies, scDiabody- HAS, Tandem scFv-toxin, dual-affinity retargeting molecules (DARTs), nanobody, nanobody-HSA, dock and lock (DNL), strand exchange engineered domain SEEDbody, Triomab, leucine zipper (LUZ- Y), XmAb ® ;
• bispecific proteins may include blinatumomab, catumaxomab, ertumaxomab, solitomab, targomiRs, lutikizumab (ABT981), vanucizumab (RG7221), remtolumab (ABT122), ozoralixumab (ATN103), floteuzmab (MGD006), pasotuxizumab (AMG112, MT112), lymphomun (FBTA05), (ATN-103), AMG211 (MT111, Medi-1565), AMG330, AMG420 (B 1836909), AMG-110 (MT110), MDX-447, TF2, rM28, HER2Bi-aATC, GD2Bi-aATC, MGD006, MGD007, MGD009, MGD010, MGD011 (JNJ64052781), IMCgplOO, indium-labele
• Multispecific proteins also include trispecific antibodies, tetravalent bispecific antibodies, multispecific proteins without antibody components such as dia-, tria- or tetrabodies, minibodies, and single chain proteins capable of binding multiple targets. Coloma, M.J., et. al., Nature Biotech. 15 (1997) 159-163.
• multispecific proteins of interest bind, neutralize and/or interact specifically to one or more CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factors, fibroblast growth factors, transforming growth factors (TGF), insulin-like growth factors, osteoinductive factors, insulin and insulin-related proteins, coagulation and coagulation- related proteins, colony stimulating factors (CSFs), other blood and serum proteins blood group antigens; receptors, receptor-associated proteins, growth hormones, growth hormone receptors, T-cell receptors; neurotrophic factors, neurotrophins, relaxins, interferons, interleukins, viral antigens, lipoproteins, integrins, rheumatoid factors, immunotoxins, surface membrane proteins, transport proteins, homing receptors, addressins, regulatory proteins, and immunoadhesins.
• CD proteins CD proteins
• HER receptor family proteins cell adhesion molecules
• growth factors nerve growth factors, fibroblast growth factors, transforming growth factors (TGF), insulin-like growth factors,
• multispecific proteins of interest bind, neutralize and/or interact with one or 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, pl50,95, 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
• multispecific proteins of interest may include bispecific antibodies that specifically bind to combinations including CD3 and CD19, EpCAM, CEA, PSA, CD33, BCMA, Her2, CD20, P-cadherin, CD123, gpA33, or B7H3.
• bispecific antibodies of interest may include bispecific antibodies that specifically bind to combinations including ILla + I LI b .
• the multispecific proteins can be of scientific or commercial interest, particularly bispecific- based therapeutics. Multispecific proteins can be produced in various ways, most commonly by recombinant animal cell lines using cell culture methods. The multispecific proteins may be produced intracellularly or secreted into the culture medium from which it can be recovered and/or collected and may be referred to interchangeably as “recombinant multispecific protein”, “recombinant multispecific antibody.
• isolated multispecific protein “isolated recombinant multispecific antibody” refer to a multispecific protein that that have been purified away from proteins, polypeptides, DNA, and/or other contaminants or impurities that would interfere with its therapeutic, diagnostic, prophylactic, research, or other use.
• Multispecific proteins of interest include multispecific antibodies that exert a therapeutic effect by binding two or more targets, particularly targets among those listed below, including targets derived therefrom, targets related thereto, and modifications thereof.
• the invention provides a method of purifying a multispecific protein from a composition comprising the multispecific protein and at least one product-related impurity, the method comprising equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 94- 105 mM Sodium chloride; loading the composition on to the cation exchange medium in a load buffer comprises 94-105 mM Sodium chloride; washing the column with at least one wash buffer comprising 94-105 mM Sodium chloride; and eluting the multispecific protein from the cation exchange chromatography medium.
• purifying is meant increasing the degree of purity of the multispecific protein in the composition by removing (partially or completely) at least one product-related impurity from the composition.
• Recovery and purification of multispecific proteins is accomplished by the downstream unit operations, in particular, those operations involving ion exchange chromatography, resulting in a more “homogeneous” multispecific protein composition that meets yield and product quality targets (such as reduced product-related impurities, increased product quality and the like).
• Product-related impurity refers to product-related variants of the multispecific protein of interest. In some instances, these impurities have a pi that is lower than the main product in an elution peak.
• Product-related impurities include, for example, homodimers, half antibodies, aggregates, antibody fragments and various combinations of antibody fragments, and light chain mis-assemblies, such as 2XLC, 3XLC, or 4XLC, high molecular weight (HMW) species, low molecular weight (LMW) species.
• HMW high molecular weight
• LMW low molecular weight
• Half antibodies refer to a product-related impurity that can form, for example, due to incomplete assembly or disruption of the interaction between the two heavy chain polypeptides.
• Half antibodies comprise a single light chain polypeptide and a single heavy chain polypeptide.
• “Homodimers” refer to a product-related impurity that can, for example, form when heavy and light chains having specificity for the same target recombine with each other instead of pairing with heavy and light chains that have specificity to a different target to form a desired bispecific heterodimer. This typically occurs during expression in the host cell.
• LCs light chains
• engineered residues such as charged paired mutations, knob-hole, etc
• the multispecific protein is bivalent, having two sites for binding to each antigen of interest, it is possible to have 3X LC1, 4X LC1, and other combinations of mispaired species.
• the invention provides a method of reducing low pi impurities in the eluate from cation exchange chromatography, the method comprises equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 94-105 mM Sodium chloride; loading the composition on to the cation exchange medium in a load buffer comprises 94-105 mM Sodium chloride; washing the column with at least one wash buffer comprising 94-105 mM Sodium chloride; and eluting the multispecific protein from the cation exchange chromatography medium; wherein the cation exchange chromatography eluate has reduced low pi impurities compared to the cation exchange chromatography eluate recovered in a corresponding method in which no sodium chloride is used in the equilibration, load, and wash steps.
• the pI of product-related impurities may be similar to the pi of the desired multispecific protein. These product-related impurities are found in the eluate peak with the main product. They have a slightly lower pi so they elute just prior to the main product, as pre-peaks.
• isoelectric point or “pI” of a protein, refers to the pH at which the positive charge balances the negative charge of the protein.
• the pI can be calculated/determined using known methods such as from the net charge of the amino acid residues of the protein or by isoelectric focusing.
• Product-related impurities having it lower pi than the main product are more acidic than the main product.
• the invention provides method of producing an isolated, purified, recombinant multispecific protein of interest, the method comprising establishing a cell culture in a bioreactor with a host cell expressing the multispecific protein; culturing the host cells to express the multispecific protein; harvesting the recombinant multispecific protein; affinity purifying the harvested recombinant multispecific protein; inactivating virus at low pH in the eluate pool from the affinity purification and neutralizing the pool; equilibrating a cation exchange chromatography medium with an equilibration buffer comprising 94-105 mM Sodium chloride; loading the neutralized affinity purified recombinant multispecific protein on to the equilibrated cation exchange medium in a load buffer comprises 94-105 mM Sodium chloride; washing the cation exchange medium with a wash buffer comprising 94-105 mM Sodium chloride, followed by a second wash buffer comprising 0-26 mM Sodium chloride; eluting the multispecific protein from
• vector means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, transposon, cosmid, chromosome, virus, virus capsid, virion, naked DNA, complexed DNA and the like) suitable for use to transfer and/or transport multispecific protein encoding information into a host cell and/or to a specific location and/or compartment within a host cell.
• vector means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, transposon, cosmid, chromosome, virus, virus capsid, virion, naked DNA, complexed DNA and the like) suitable for use to transfer and/or transport multispecific protein encoding information into a host cell and/or to a specific location and/or compartment within a host cell.
• Vectors can include viral and non-viral vectors, non- episomal mammalian vectors.
• Vectors are often referred to as expression vectors, for example, recombinant expression vectors and cloning vectors.
• the vector may be introduced into a host cell to allow replication of the vector itself and thereby amplify the copies of the polynucleotide contained therein.
• the cloning vectors may contain sequence components that generally include, without limitation, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements may be selected as appropriate by a person of ordinary skill in the art.
• 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.
• Cells include “host cells”, also referred to as “cell lines”, which are genetically engineered to express a multispecific protein of commercial or scientific interest. Host cells are typically derived from a lineage arising from a primary culture that can be maintained in culture for an unlimited time.
• Genetically engineering the host cell involves transfecting, transforming or transducing the cells with a recombinant polynucleotide molecule, and/or 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 recombinant multispecific protein.
• Methods and vectors for genetically engineering cells and/or cell lines to express multispecific proteins of interest are well known to those of skill in the art.
• a host cell can be any prokaryotic cell (for example, Escherichia coli (E. coli)) or eukaryotic cell (for example, yeast, insect, or animal cells, in particular mammalian cells (e.g., CHO cells)).
• Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
• the cell is a host cell.
• Host cells when cultured under appropriate conditions, express the multispecific protein of interest that can be subsequently collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted).
• the selection of an appropriate host cell will depend upon various factors, such as desired expression levels, protein modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule.
• 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 used interchangeably to refer to media suitable for growth of a host cell during in vitro cell culture.
• cell culture media contains a buffer, salts, energy source, amino acids, vitamins and trace essential elements. Any media capable of supporting growth of the appropriate host cell in culture can be used.
• Cell culture media which may be further supplemented with other components to maximize cell growth, cell viability, and/or recombinant protein production in a particular cultured host cell, are commercially available and include RPMI-1640 Medium, RPMI-1641 Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K Medium, Ham's F12 Medium, Iscove's Modified Dulbecco's Medium, McCoy's 5A Medium, Leibovitz's L-15 Medium, and serum-free media such as EX-CELLTM 300 Series, among others, which can be obtained from the American Type Culture Collection or SAFC Biosciences, as well as other vendors.
• Cell culture media can be serum-free, protein-free, growth factor-free, and/or peptone -free media. Cell culture may also be enriched by the addition of nutrients and used at greater than its usual, recommended concentrations.
• Various media formulations can be used during the life of the culture, for example, to facilitate the transition from one stage (e.g., the growth stage or phase) to another (e.g., the production stage or phase) and/or to optimize conditions during cell culture (e.g. concentrated media provided during perfusion culture).
• a growth medium formulation can be used to promote cell growth and minimize protein expression.
• a production medium formulation can be used to promote production of the protein of interest and maintenance of the cells, with a minimal of new cell growth).
• a feed media typically a media containing more concentrated components such as nutrients and amino acids, which are consumed during the course of the production phase of the cell culture may be used to supplement and maintain an active culture, particularly a culture operated in fed batch, semi-perfusion, or perfusion mode.
• Such a concentrated feed medium can contain most of the components of the cell culture medium at, for example, about 5 c , 6 c , 7 c , 8 c , 9 c , 10 c , 12 c , 14 c , 16 c , 20 c , 30 c , 50 c , 100 c , 200 c , 400 c , 600 c , 800 x, or even about 1000/ of their normal amount.
• a growth phase may occur at a higher temperature than a production phase.
• a growth phase may occur at a first temperature from about 35° C. to about 38° C.
• a production phase may occur at a second temperature from about 29° C. to about 37° C., optionally from about 30° C. to about 36° C. or from about 30° C. to about 34° C.
• chemical inducers of protein production such as, for example, caffeine, butyrate, and hexamethylene bisacetamide (HMBA) may be added at the same time as, before, and/or after a temperature shift. If inducers are added after a temperature shift, they can be added from one hour to five days after the temperature shift, optionally from one to two days after the temperature shift. pH may also be shifted during culture, either independently or in combination with other methods.
• Host cells may be cultured in suspension or in an adherent form, attached to a solid substrate.
• Cell cultures can be established in fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors, with or without microcarriers
• Cell cultures can be operated in a batch, fed batch, continuous, semi-continuous, or perfusion mode.
• Mammalian cells such as CHO cells, may be cultured in bioreactors at a smaller scale of less than 100 ml to less than 1000 mis. Alternatively, larger scale bioreactors that contain 1000 mis to over 20,000 liters of media can be used. Large scale cell cultures, such as for clinical and/or commercial scale biomanufacturing of protein therapeutics, may be maintained for weeks and even months, while the cells produce the desired protein(s).
• product-related impurities such as homodimers, half antibodies, 2X LC-mis alignments and the like can resemble the desired multispecific protein
• strategies and techniques such as knob and hole, CrossMab, DVD IgG, and others have been developed to increase the selectivity for the desired multispecific protein during cell culture.
• knob and hole such as knob and hole, CrossMab, DVD IgG, and others have been developed to increase the selectivity for the desired multispecific protein during cell culture.
• knob and hole such as knob and hole, CrossMab, DVD IgG, and others
• the resulting expressed recombinant multispecific protein can then be harvested from the cell culture media.
• Methods for harvesting proteins from suspension cells include, but are not limited to, acid precipitation, accelerated sedimentation such as flocculation, separation using gravity, centrifugation, acoustic wave separation, filtration, including membrane filtration, using ultrafilters, microfilters, tangential flow filters, alternating tangential flow, depth, and alluvial filtration filters.
• Recombinant proteins expressed by prokaryotes are retrieved from inclusion bodies in the cytoplasm by redox folding processes known in the art.
• the harvested multispecific protein can then be purified, or partially purified, away from any impurities, such as remaining cell culture media, cell extracts, undesired components, host cell proteins, improperly expressed proteins, product-related impurities, and the like, through one or more downstream purification operations.
• impurities such as remaining cell culture media, cell extracts, undesired components, host cell proteins, improperly expressed proteins, product-related impurities, and the like.
• Purification of the multispecific protein from the harvested cell culture fluid can begin with capture chromatography that makes use of resins and/or membranes containing agents that will bind to the recombinant multispecific protein of interest, for example affinity chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography (HIC), immobilized metal affinity chromatography (IMAC), and the like.
• capture chromatography makes use of resins and/or membranes containing agents that will bind to the recombinant multispecific protein of interest, for example affinity chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography (HIC), immobilized metal affinity chromatography (IMAC), and the like.
• Affinity chromatography options may comprise a substrate-binding capture mechanism, an aptamer-binding capture mechanism, and a cofactor-binding capture mechanism, for example.
• an antibody- or antibody fragment-binding capture mechanism such as Protein A, Protein G, Protein A/
• virus inactivation and/or virus filtration can be performed to remove viral matter from the purified multispecific protein solution.
• One method for achieving virus inactivation is incubation at low pH or other solution conditions for achieving the inactivation of viruses.
• Low pH virus inactivation can be followed with a neutralization unit operation that readjusts the viral inactivated solution to a pH more compatible with the requirements of the following unit operations.
• neutralization is at pH 5-7.
• Viral inactivated or neutralized viral inactivated pools may also be followed by filtration, such as depth filtration, to remove any resulting turbidity or precipitation.
• Sterile filtration is typically performed along with depth filtration.
• Viral filtration can be performed using micro- or nano-filters, such as those available from Asahi Kasei (Plavona ® ) and EDM Millipore (VPro ® ).
• polishing is used herein to refer to one or more chromatographic steps performed to remove remaining contaminants and impurities such as DNA, host cell proteins, product-specific impurities, variant products and aggregates, and virus adsorption from a fluid including a recombinant multispecific protein that is close to a final desired purity.
• Polish chromatography makes use of resins and/or membranes containing agents that can be used in a flow-through mode (where the protein of interest flows through the resin/membrane and the contaminants and impurities are bound to the chromatography medium and the protein of interest is contained in the eluent), frontal or overloaded chromatography mode (where a solution containing the protein of interest is loaded onto a column until adsorption sites on are occupied and the species with the least affinity for the stationary phase (the protein of interest) starts to elute), or bind and elute mode (where the protein of interest is bound to the chromatography medium and eluted after the contaminants and impurities have flowed through or been washed off the chromatography medium).
• a flow-through mode where the protein of interest flows through the resin/membrane and the contaminants and impurities are bound to the chromatography medium and the protein of interest is contained in the eluent
• frontal or overloaded chromatography mode where a solution containing the protein of
• chromatography methods include ion exchange chromatography (IEX), including anion exchange chromatography (AEX) and/or cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed modal or multimodal chromatography (MM), hydroxyapatite chromatography (HA); reverse phase chromatography, and gel filtration.
• IEX ion exchange chromatography
• AEX anion exchange chromatography
• CEX cation exchange chromatography
• HIC hydrophobic interaction chromatography
• MM mixed modal or multimodal chromatography
• HA hydroxyapatite chromatography
• reverse phase chromatography reverse phase chromatography
• gel filtration gel filtration.
• the chromatographic method is cation exchange chromatography.
• the cation exchange medium is a resin.
• the invention provides a method of performing cation exchange chromatography under high salt loading conditions to reduce product-related impurities, the method comprising equilibrating a cation exchange chromatography medium with an equilibration buffer, loading the composition on to the cation exchange medium in a load buffer, washing the column with a first and second wash buffer, and eluting the multispecific protein from the cation exchange chromatography medium, wherein the equilibration, loading, and first wash buffer comprise 94-105 mM Sodium chloride.
• “Cation exchange chromatography” refers to chromatography performed on a solid phase material that is negatively charged and has free cations for exchange with cations in an aqueous solution passed over or through the solid phase.
• the charge may be provided by attaching one or more charged ligands to the solid phase, e.g. by covalent linking. Alternatively, or in addition, the charge may be an inherent property of the solid phase (e.g. as is the case for silica, which has an overall negative charge).
• Commercially available cation exchange materials are available and include, but are not limited to, resin and membrane absorber medium, weak cation exchangers, strong cation exchangers, sulphopropyl (SP) immobilized on agarose (e.g.
• SP-SEPHAROSE FAST FLOWTM, SP-SEPHAROSE FAST FLOW XLTM or SP-SEPHAROSE HIGH PERFORMANCETM from GE Healthcare
• CAPTO STM, CAPTO SP ImpResTM, CAPTO S ImpActTM GE Healthcare
• FRACTOGEL-S03TM, FRACTOGEL-SE HICAPTM FRACTOPREPTM (EMD Merck)
• Fractogel ® EMD SE Hicap M
• Eshmuno ® CPX Eshmuno ® S resins
• Mustang S Acrodisc with Mustang S AcroPrep with Mustang S CM Ceramic HyperD ® F AcroSep with CM Ceramic HyperD ® F, among others.
• cation exchange chromatography is performed in bind and elute mode.
• An eluate or storage pool containing the multispecific protein of interest is loaded onto an equilibrated cation exchange medium such that the multispecific protein of interest is bound to the cation exchange medium.
• binding the multispecific protein to the cation exchange material is meant exposing the multispecific protein to the cation exchange material under appropriate conditions (pH/conductivity) such that the multispecific protein is reversibly immobilized in or on the cation exchange material by virtue of ionic interactions between the multispecific protein of interest and a charged group or charged groups of the cation exchange material.
• the multispecific protein may be in an eluate or pool originating from a previous unit operation, such as affinity chromatography, neutralized low pH viral inactivation, depth filtration, or a polish chromatography operation.
• the performance of cation exchange chromatography in bind and elute mode for the inventive method consists of several steps.
• the medium In preparation for loading of the multispecific protein on to the cation exchange medium, the medium is equilibrated prior to loading with the same buffer composition as the multispecific protein composition.
• An “equilibration buffer” is the buffer used to equilibrate the chromatography material prior to loading the composition comprising a multispecific protein of interest.
• an eluate or storage pool containing a multispecific protein of interest from a previous unit operation is titrated with a high salt buffer formulation such at the final conditioned load buffer of the composition contains sodium chloride at a desired concentration.
• a high salt buffer formulation such at the final conditioned load buffer of the composition contains sodium chloride at a desired concentration.
• Load buffer and “final conditioned load buffer” are used interchangeably herein.
• the load buffer has a suitable formulation such that the multispecific protein of interest binds to the cation exchange material.
• the loaded and bound cation exchange chromatography material is then subjected to a plurality of washes. Washing the cation exchange material means passing an appropriate wash buffer through or over the cation exchange material.
• the wash buffer removes one or more contaminants, including product-related impurities, from the cation exchange material, without substantial elution of the multispecific protein of interest.
• the wash buffer is used to wash or re-equilibrate she cation exchange material prior to eluting the multispecific protein of interest.
• One or more of the wash buffer formulations may be the same as the equilibration and/or final conditioned load buffer formulations.
• first wash and second wash should not be interpreted as excluding the use of one or more additional washes or other buffers between the load and the first and/or second wash steps.
• the U V baseline has returned to or is very near to zero, prior to the start of foe elution.
• the invention provides that the equilibration buffer, final conditioned load buffer, and at least one wash buffer has a high salt buffer formulation, in one embodiment, they all have the same high salt formulation buffer.
• the equilibration buffer, final conditioned load buffer, and at least one wash buffer comprises 94-105 mM Sodium chloride.
• a “buffer” is a solution that resists changes in pH by the action of its acid-base conjugate components.
• the buffer is an acetate buffer.
• the buffer is a 100 mM acetate buffer.
• the pH of the buffer is in the range of 5 ⁇ 0.05 to 5.0 ⁇ 0.1.
• the range is pH 4.9 to 5.1.
• the range is pH 4.95 to 5.05.
• the equilibration buffer, final conditioned load buffer, and at least one wash buffer each also comprise a salt.
• the salt is sodium chloride.
• the salt is sodium chloride in an amount from about 94 mM to about 105 mM.
• the load buffer comprises 96-105 mM Sodium chloride. In one embodiment the load buffer comprises 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104 or 105 mM Sodium chloride. In one embodiment the load buffer comprises 94 mM Sodium chloride. In one embodiment the load buffer comprises 96 mM Sodium chloride. In one embodiment the load buffer comprises 105 mM Sodium chloride.
• the load buffer comprises acetate. In one embodiment the load buffer comprises acetate, pH 4.9-5.1. In one embodiment the load buffer comprises acetate at pH, 4.9, 5.0, or 5.1. In one embodiment the load buffer comprises acetate, pH of 5.0 ⁇ 0.05 to 5.0 ⁇ 0.1. In one embodiment the load buffer comprises 100 mM acetate. In one embodiment the load buffer comprises 100 mM acetate, pH 4.9-5.1. In one embodiment the load buffer comprises 100 mM acetate at pH, 4.9, 5.0, or 5.1. In one embodiment the load buffer comprises 100 mM acetate, pH 5.0 ⁇ 0.05% to pH 5.0 ⁇ 0.1%.
• the load buffer comprises acetate and 94 mM-105 mM Sodium chloride. In one embodiment the load buffer comprises acetate and 94 mM-96 mM Sodium chloride. In one embodiment the load buffer comprises acetate and 96 mM-105 mM Sodium chloride. In a related embodiment the load buffer comprises acetate and 94 mM Sodium chloride. In a related embodiment the load buffer comprises acetate and 96 mM Sodium chloride. In a related embodiment the load buffer comprises acetate and 105 mM Sodium chloride. In one embodiment the load buffer comprises acetate, 94 mM-105 mM Sodium chloride, pH 5.0 ⁇ 0.05 to 5.0 ⁇ 0.1.
• the load buffer comprises acetate, 94-96 mM Sodium chloride, pH 5.0 ⁇ 0.05. In a related embodiment the load buffer comprises acetate, 96-105 mM Sodium chloride, pH 5.0 ⁇ 0.05. In a related embodiment the load buffer comprises acetate, 96 mM Sodium chloride, pH 5.0 ⁇ 0.05. In a related embodiment the load buffer comprises acetate, 105 mM Sodium chloride, pH 5.0 ⁇ 0.1. In a related embodiment the load buffer comprises acetate, 94 mM-105 mM Sodium chloride, pH 4.9-5.1.
• the load buffer comprises acetate, 94 mM-105 mM Sodium chloride, pH of 4.9, 5.0 or 5.1. In a related embodiment the load buffer comprises acetate, 94 mM-105 mM Sodium chloride, pH of 5.0. In a related embodiment the load buffer comprises acetate, 94-96 mM Sodium chloride, pH of 4.9-5.1. In a related embodiment the load buffer comprises acetate, 96 mM Sodium chloride, pH of 5.0 ⁇ 0.05. In a related embodiment the load buffer comprises acetate, 105 mM Sodium chloride, pH of 5.0 ⁇ 0.1.
• the load buffer comprises 100 mM acetate, 94 mM-105 mM Sodium chloride, pH 5.0 ⁇ 0.05 to 5.0 ⁇ 0.1. In a related embodiment the load buffer comprises 100 mM acetate, 94-96 mM Sodium chloride, pH 5.0 ⁇ 0.05. In a related embodiment the load buffer comprises 100 mM acetate, 96-105 mM Sodium chloride, pH 5.0 ⁇ 0.05. In a related embodiment the load buffer comprises 100 mM acetate, 96 mM Sodium chloride, pH 5.0 ⁇ 0.05. In a related embodiment the load buffer comprises 100 mM acetate, 105 mM Sodium chloride, pH 5.0 ⁇ 0.1.
• the load buffer comprises 100 mM acetate, 94 mM-105 mM Sodium chloride, pH 4.9-5.1. In a related embodiment the load buffer comprises 100 mM acetate, 94 mM-105 mM Sodium chloride, pH of 4.9, 5.0 or 5.1. In a related embodiment the load buffer comprises 100 mM acetate, 94 mM-105 mM Sodium chloride, pH of 5.0. In a related embodiment the load buffer comprises 100 mM acetate, 94-96 mM Sodium chloride, pH of 4.9-5.1. In a related embodiment the load buffer comprises 100 mM acetate, 96 mM Sodium chloride, pH of 5.0 ⁇ 0.05. In a related embodiment the load buffer comprises 100 mM acetate, 105 mM Sodium chloride, pH of 5.0 ⁇ 0.1.
• At least one wash buffer comprises 94-96 mM Sodium chloride. In one embodiment at least one wash buffer comprises 96-105 mM Sodium chloride. In one embodiment at least one wash buffer comprises 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104 or 105 mM Sodium chloride. In one embodiment at least one wash buffer comprises 94 mM Sodium chloride. In one embodiment at least one wash buffer comprises 96 mM Sodium chloride. In one embodiment at least one wash buffer comprises 105 mM Sodium chloride.
• At least one wash buffer comprises acetate. In one embodiment at least one wash buffer comprises acetate, pH 4.9-5.1. In one embodiment at least one wash buffer comprises acetate at pH, 4.9, 5.0, or 5.1. In one embodiment at least one wash buffer comprises acetate, pH of 5.0 ⁇ 0.05 to 5.0 ⁇ 0.1. In one embodiment at least one wash buffer comprises 100 mM acetate. In one embodiment at least one wash buffer comprises 100 mM acetate, pH 4.9-5.1. In one embodiment at least one wash buffer comprises 100 mM acetate at pH, 4.9, 5.0, or 5.1. In one embodiment at least one wash buffer comprises 100 mM acetate, pH 5.0 ⁇ 0.05% to pH 5.0 ⁇ 0.1%.
• At least one wash buffer comprises acetate, 94 mM-105 mM Sodium chloride. In one embodiment at least one wash buffer comprises acetate, 94 mM-96 mM Sodium chloride. In one embodiment at least one wash buffer comprises acetate, 96 mM-105 mM Sodium chloride. In a related embodiment at least one wash buffer comprises acetate, 94 mM Sodium chloride. In a related embodiment at least one wash buffer comprises acetate, 96 mM Sodium chloride. In a related embodiment at least one wash buffer comprises acetate, 105 mM Sodium chloride.
• At least one wash buffer comprises acetate, 94 mM-105 mM Sodium chloride, pH 5.0 ⁇ 0.05 to 5.0 ⁇ 0.1. In a related embodiment at least one wash buffer comprises acetate, 94-96 mM Sodium chloride, pH 5.0 ⁇ 0.05. In a related embodiment at least one wash buffer comprises acetate, 96-105 mM Sodium chloride, pH 5.0 ⁇ 0.05. In a related embodiment at least one wash buffer comprises acetate, 96 mM Sodium chloride, pH 5.0 ⁇ 0.05. In a related embodiment at least one wash buffer comprises acetate, 105 mM Sodium chloride, pH 5.0 ⁇ 0.1.
• At least one wash buffer comprises acetate, 94 mM-105 mM Sodium chloride, pH 4.9-5.1. In a related embodiment at least one wash buffer comprises acetate, 94 mM-105 mM Sodium chloride, pH of 4.9, 5.0 or 5.1. In a related embodiment at least one wash buffer comprises acetate, 94 mM-105 mM Sodium chloride, pH of 5.0. In a related embodiment at least one wash buffer comprises acetate, 94-96 mM Sodium chloride, pH of 4.9-5.1. In a related embodiment at least one wash buffer comprises acetate, 96 mM Sodium chloride, pH of 5.0 ⁇ 0.05. In a related embodiment at least one wash buffer comprises acetate, 105 mM Sodium chloride, pH of 5.0 ⁇ 0.1.
• At least one wash buffer comprises 100 mM acetate, 94 mM-105 mM Sodium chloride, pH 5.0 ⁇ 0.05 to 5.0 ⁇ 0.1. In a related embodiment at least one wash buffer comprises 100 mM acetate, 94-96 mM Sodium chloride, pH 5.0 ⁇ 0.05. In a related embodiment at least one wash buffer comprises 100 mM acetate, 96-105 mM Sodium chloride, pH 5.0 ⁇ 0.05. In a related embodiment at least one wash buffer comprises 100 mM acetate, 96 mM Sodium chloride, pH 5.0 ⁇ 0.05.
• At least one wash buffer comprises 100 mM acetate, 105 mM Sodium chloride, pH 5.0 ⁇ 0.1. In a related embodiment at least one wash buffer comprises 100 mM acetate, 94 mM-105 mM Sodium chloride, pH 4.9-5.1. In a related embodiment at least one wash buffer comprises 100 mM acetate, 94 mM-105 mM Sodium chloride, pH of 4.9, 5.0 or 5.1. In a related embodiment at least one wash buffer comprises 100 mM acetate, 94 mM-105 mM Sodium chloride, pH of 5.0.
• At least one wash buffer comprises 100 mM acetate, 94-96 mM Sodium chloride, pH of 4.9-5.1. In a related embodiment at least one wash buffer comprises 100 mM acetate, 96 mM Sodium chloride, pH of 5.0 ⁇ 0.05. In a related embodiment at least one wash buffer comprises 100 mM acetate, 105 mM Sodium chloride, pH of 5.0 ⁇ 0.1.
• at least one additional wash is a second wash.
• at least one additional wash buffer comprises 0-26 mM Sodium chloride.
• at least one additional wash buffer comprises 23-26 mM Sodium chloride.
• at least one additional wash buffer comprises 23, 24, 25, or 26 mM Sodium chloride.
• at least one additional wash buffer comprises 23 mM Sodium chloride.
• at least one additional wash buffer comprises 24 mM Sodium chloride.
• at least one additional wash buffer comprises 25 mM Sodium chloride.
• at least one additional wash buffer comprises 26 mM Sodium chloride.
• At least one additional wash buffer comprising 25 mM sodium chloride, pH of 5.0 ⁇ 0.05.
• at least one wash buffer comprises acetate and sodium chloride followed by at least one additional wash.
• least one wash buffer comprises acetate, 94-105 mM sodium chloride, followed by at least one additional wash.
• least one wash buffer comprises acetate, 94-105 mM sodium chloride, followed by at least one additional wash buffer comprising 0-26 mM Sodium chloride.
• the additional wash is a second wash. In a related embodiment the second wash buffer comprises 0 - 26 mM Sodium chloride.
• least one wash buffer comprises acetate, 94-96 mM sodium chloride, followed by at least one additional wash buffer comprising acetate, 25 mM sodium chloride. In one embodiment at least one wash buffer comprises acetate, 105 mM sodium chloride, followed by at least one additional wash buffer comprising acetate.
• least one wash buffer comprises 100 mM acetate, 94-105 mM sodium chloride, pH of 5.0 ⁇ 0.05, followed by at least one additional wash.
• the additional wash is a second wash.
• the second wash buffer comprises 0-26 mM Sodium chloride.
• least one wash buffer comprises 100 mM acetate, 94-96 mM sodium chloride, pH of 5.0 ⁇ 0.05, followed by at least one additional wash buffer comprising 100 mM acetate, 25 mM sodium chloride, pH of 5.0 ⁇ 0.05.
• at least one wash buffer comprises 100 mM acetate, 105 mM sodium chloride, pH of 5.0 ⁇ 0.1, followed by at least one additional wash buffer comprising 100 mM acetate, pH of 5.0 ⁇ 0.1.
• the bound multispecific protein is then eluted from the cation exchange chromatography material.
• the multispecific protein may be eiuted by a gradient.
• the multispecific protein may be eluted from the cation exchange material by a linear or step gradient.
• the gradient is a salt gradient.
• the gradient is created using at least two elution buffers, wherein the combination of the buffers has a substantially increased conductivity such that the multispecific protein of interest is eluted from the cation exchange material.
• the conductivity of the gradient is greater than that of the equilibrium and of each of the preceding buffers.
• the cation exchange chromatography eluate can be subjected to further polish chromatography purification operations. Preferably at least one additional polish chromatography operation. Preferably the multispecific protein of interest is applied to the chromatography material in flow-through mode.
• Concentration of the purified multispecific protein and buffer exchange into a desired formulation buffer for bulk storage of the drug substance can be accomplished by an ultrafiltration and diafiltration operation. Viral filtration can also be performed at any time dining the downstream process.
• Critical attributes and performance parameters of the purified multispecific 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 media components that are consumed (such as glucose), levels of metabolic by-products (such as lactate and ammonia) that accumulate, as well as those related to cell maintenance and survival, such as dissolved oxygen content can be measured during cell culture. Critical attributes such as specific productivity, viable cell density, pH, osmolality, appearance, color, aggregation, percent yield, titer, concentration, viability, activity, and the like may be monitored during appropriated stages in the manufacturing process. Monitoring and measurements can be done using known techniques and commercially available equipment.
• compositions may include one or more of the following: buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives; sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylened
• Neutralized Protein A pool containing a fully human bi-specific, engineered immunoglobulin (Bi-specific #1) in an acetate buffer was loaded onto an Eshmuno CP-FT ® cation exchange chromatography resin (GE Healthcare Bio-Science, Marlborough, MA) under the conditions outlined in Table 1.
• Figure 1 shows that multiple impurities remained on the column and were eluted with the main product. These impurities were similar in charge (isoelectric point) to the main product and included half antibodies, 2X light chain-mis-assemblies and high molecular weight (HMW).
• HMW high molecular weight
• a neutralized virus inactivated pool (lOOmM Acetate, pH 5.0) containing Bi-specific #1 was combined with a load buffer (100 mM Acetate 350 mM sodium chloride pH 5.00) at a ratio of 1:0.378, giving a final conditioned load of 100 mM Acetate 96 mM Sodium Chloride pH 5.00.
• the conditioned load was loaded onto a Capto-SP ImpRes ® cation exchange chromatography resin under the conditions described in Table 2.
• Figure 2 shows that high salt loading conditions resulted in a reduction in the number of impurity peaks in the elution profde, from four peaks to a single peak (shows CenterPoint results).
• the majority of the impurities were removed between the load and second wash steps.
• the half mAbs flowed through the resin during the load or were minimally bound to the column.
• the 2X LCs were removed from the column or minimally bound to the column following the first wash step.
• the second wash step provided complete binding conditions for the remaining protein species and reestablished the UV baseline to zero before the start of the elution, tightening the elution profile, resulting in a much more efficient collection and better quality of the main product.
• the equilibration buffer, the final conditioned load, and the first wash buffer were tested at 94 - 98 mM sodium chloride, with similar results.
• the pH of the equilibration buffer, the final conditioned load, and the two wash buffer formulations were tested at various concentrations from 4.95 to 5.05, with similar results.
• the load concentration was tested at 5-27 mg/ml, with similar results. Overall the high salt loads had a lower yield (—60%) compared to the no salt load conditions.
• the elution could be fractionated, allowing higher accuracy and precision in controlling the final product quality.
• the no salt load had a steep gradient of 8 mM[Sodium chloride]/CV, which is not optimal for a robust manufacturing process
• the high salt loads had a salt gradient of 16 mM [Sodium chloride]/CV.
• the high salt load conditioning allowed for a reduction in the length of the elution from 44 column volumes to 20 column volumes. This reduction in column volume saves time and resources since the elution gradient is shorter, which also results in a more robust manufacturing process.
• Figure 3 shows one elution peak resulting from the high load density, no salt load conditioning, at a steep elution gradient.
• the low pi product-related impurities did not resolve from the main product under the high load density and are mostly in fractions 1, 2, and 3 as shown by a reduced CE-SDS LC1 to LC2 ratios of 4 to 7 (mispaired LC1 species) and LMW species of 2 to 4%.
• Example 4 Lower load density, no salt load conditioning, Bi-specific #2
• the neutralized Protein A eluate pool containing the fully human, engineered IgG/Fab fusion protein (Bi-specific #2) was loaded onto a Capto-SP ImpREs ® cation exchange chromatography resin (GE Healthcare Bio-Science, Marlborough, MA) under the conditions outlined in Table 4.
• FIG. 4 shows that a lower loading density (10 vs 25 g/L) and shallower gradient (8 vs 16 mM/CV) allowed for separation of the main low pi product-impurities into a distinct peak formed by fractions 1 to 4. This fraction showed a LC 1 to LC2 ratio of 3 to 10, indicating mispaired LC1 species. In contrast, the main peak showed a cumulative LC1 to LC2 ratio of 1.2.
• Neutralized virus inactivated pool containing Bi-specific # 2 was combined with a high salt load buffer (100 mM Acetate 500 mM Sodium Chloride pH 5.00) giving a final conditioned load buffer concentration of 100 mM Acetate 105 mM Sodium Chloride pH 5.00.
• the conditioned load was loaded onto a Capto-SP ImpREs ® cation exchange chromatography resin (GE Healthcare Bio-Science, Marlborough, MA) under the conditions outlined in Table 4. Table 5 Conditions for cation exchange chromatography under high salt load.
• the second wash step also reestablished the UV baseline to zero before the start of the elution tightening the elution profile, resulting in a much more efficient collection and better quality of the main product.
• the optimized procedure with lower load level and shallower gradient, combined with a high salt conditioned load allowed an increased CEX purification yield from 44% to 58%, while enabling an elution profile for collecting a purified pool with low levels of mispaired LCl species (as evidence by the LC1 to LC2 ratio close to 1), HMW and LMW, and an absorbance- based pooling criteria.

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