ANIONIC EXCHANGE-HYDROPHOBIC MIXED MODE CHROMATOGRAPHY RESIN

Abstract

Chromatography resins having anionic exchange-hydrophobic mixed mode ligands, that are useful for purifying target biomolecules using anionic exchange (i.e., where the ligand is positively charged) and hydrophobic mixed mode chromatography. The chromatography resins allow for efficient purification of target biomolecules (e.g., recombinant proteins, antibodies, antibody-drug conjugates, or antibody derivatives including, but not limited to, antibody fragments and antibody fusions) from a sample, and have been found to be useful in purifying monomeric target biomolecules from aggregate target biomolecules. In an embodiment, the chromatography resins are useful for separating antibodies from one or more components (e.g., contaminants) in the sample.

Description

BACKGROUND

The extraction of immunoglobulins from source liquids, which are primarily mammalian bodily fluids or cell culture harvest, is of value in obtaining the immunoglobulins in a sufficiently concentrated or purified form for diagnostic and therapeutic uses as well as laboratory studies in general. Similarly, purification of other types of proteins and other molecules from biological samples can be of value.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the dynamic binding capacity (DBC) for various mixed mode chromatography resins for monoclonal antibody S (mAb S).

FIG. 2 is the chromatographic elution profile of mAb S from Prototype 13.

FIG. 3 shows a flow-through mode purification chromatogram of monoclonal antibody T (mAb T) on Prototype 13.

FIG. 4 is a bind-elute mode purification chromatogram of mAb S on Prototype 12.

FIG. 5 is a bind-elute mode purification chromatogram of mAb S on Prototype 13.

SUMMARY

Chromatography resins comprising chromatography matrices linked to an anionic exchange-hydrophobic mixed mode ligand are provided. In some embodiments, the chromatography resin has the formula:

Chromatography matrix-(X)—N(R¹)(R²)—(R³-L)_n-Ar

• • or an anionic salt thereof,
  • wherein:
    • X is a spacer;
    • R¹ and R² are each independently C₁ to C₆ alkyl optionally substituted with —OH;
    • R³ is C₂ to C₆ alkyl or C₄ to C₆ cycloalkyl;
    • L is NR⁴, O, or S, wherein R⁴ is hydrogen or C₁ to C₄ alkyl;
    • n=1 or 2; and
    • Ar is a 6-10 membered ring and:
      • if Ar is aryl, the aryl is optionally substituted with up to five C₁ to C₃ unsubstituted alkyl, C₃ to C₆ branched alkyl, unsubstituted aryl, or fluorine groups; or
      • if Ar is heteroaryl, the heteroaryl is optionally substituted with up to four alkyl groups.

In some embodiments of the chromatography resin:

• • X is selected from the group consisting of —O—CH₂—, —O—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—CH₂—, —O—CH₂—CH(CH₂—OH)—(O—CH₂—CH(OH)—CH₂)₂—, —O—CH₂—CH₂—CH(CH₂—OH)—(O—CH₂—CH₂—CH(OH)—CH₂)₂—, —O—CH₂—CH(OH)—CH₂—, —O—CH₂—CH₂—CH(OH)—CH₂—CH₂—, —O—CH₂—CH(OH)—CH₂—O—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH(OH)—CH₂—, and —CO—NH—C(CH₃)₂—CO—;
  • R¹ and R² are each independently C₁ to C₃ alkyl;
  • R³ is C₂ to C₄ alkyl;
  • L is O;
  • n=1; and
    • Ar is a 6 membered ring and:
      • if Ar is aryl, the aryl is optionally substituted with up to four C₁ to C₂ unsubstituted alkyl, C₃ to C₄ branched alkyl, or fluorine groups or
      • if Ar is heteroaryl, the heteroaryl is optionally substituted with up to three alkyl groups.

DETAILED DESCRIPTION

Provided are chromatography resins that are useful for purifying target biomolecules using anionic exchange (i.e., where the ligand is positively charged) and hydrophobic mixed mode chromatography. The chromatography resins allow for efficient purification of target biomolecules (e.g., recombinant proteins, antibodies, antibody-drug conjugates, or antibody derivatives including, but not limited to, antibody fragments and antibody fusions) from a sample, and have been found to be useful in purifying monomeric target biomolecules from aggregate target biomolecules. In an embodiment, the chromatography resins are useful for separating antibodies from one or more components (e.g., contaminants) in the sample.

Definitions

Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions given below. As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Definition of standard chemistry terms can be found in reference works, including Carey and Sundberg (2007) “Advanced Organic Chemistry 5th Ed.” Vols. A and B, Springer Science+Business Media LLC, New York. The practice of the present invention will employ, unless otherwise indicated, conventional methods of synthetic organic chemistry, mass spectroscopy, preparative and analytical methods of chromatography, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology.

“Antibody” refers to an immunoglobulin, composite (e.g., fusion), or fragmentary form thereof. The term includes but is not limited to polyclonal or monoclonal antibodies of the classes IgA, IgD, IgE, IgG, and IgM, derived from human or other mammalian cell lines, including natural or genetically modified forms such as humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies. “Antibody” also includes composite forms including but not limited to fusion proteins containing an immunoglobulin moiety. “Antibody” also includes antibody fragments such as Fab, F(ab′)2, Fv, scFv, Fd, dAb, Fc, whether or not they retain antigen-binding function.

As used herein, the term “alkyl” refers to a straight or branched, saturated, aliphatic radical having between 1-10 carbon atoms. For example, C₁-C₆ alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, and/or hexyl. Alkyl can include any number of carbons, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6 and 5-6. The alkyl group is typically monovalent, but can be divalent, such as when the alkyl group links two chemical groups together.

As used herein, the term “cycloalkyl” refers to monocyclic alkyl having the number of carbon atoms indicated. Monocyclic rings include, for example, cyclobutyl, cyclopentyl, and cyclohexyl.

As used herein, the term “aryl” refers to a monocyclic or fused bicyclic aromatic ring assembly. For example, aryl can be phenyl, naphthyl, or pyridyl. Aryl groups can optionally be substituted by one, two, three, four, or five unsubstituted alkyl groups, unsubstituted aryl groups, or fluorine groups.

The term “heteroatom” refers to N, O and S.

As used herein, the term “heteroaryl group” refers to aromatic groups that include one heteroatom as a ring member. Examples include, but are not limited to, pyrrole, furan, thiophene, and pyridine. Heteroaryl groups can optionally be substituted by one, two, three, or four alkyl groups.

An “anionic salt” is formed at a basic (e.g., alkylamino) group in the ligands. Anionic salts include, but are not limited to, halides, sulfonates, sulfates, carboxylates, phosphates, acetates, citrates and nitrates. Examples of acid-addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, sulfate, acetate, citrate, and nitrate.

As used herein, the term “spacer” refers to a molecule having 1-30 atoms selected from H, C, N, O and S. The spacer has a neutral charge and can include cyclic groups. The spacer links the chromatographic ligand to the chromatography matrix. The types of bonds used to link the spacer to the chromatography matrix include, but are not limited to, amides, amines, ethers, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonate and thioureas. In some embodiments, the bonds used to link the spacer to the chromatography matrix are amines, ethers or amides.

“Biological sample” refers to any composition containing a target molecule of biological origin (a “biomolecule) that is desired to be purified. In some embodiments, the target molecule to be purified is an antibody or a non-antibody protein (e.g., hormones or enzymes).

“Bind-elute mode” refers to an operational approach to chromatography in which the buffer conditions are established so that target molecules and, optionally undesired contaminants, bind to the ligand when the sample is applied to the ligand. Fractionation of the target can be achieved subsequently by changing the conditions such that the target is eluted from the support. In some embodiments, contaminants remain bound following target elution. In some embodiments, contaminants either flow-through or are bound and eluted before elution of the target.

“Flow-through mode” refers to an operational approach to chromatography in which the buffer conditions are established so that the target molecule to be purified flows through the chromatography support comprising the ligand, while at least some sample contaminants are selectively retained, thus achieving their removal from the sample.

Chromatography Resins

In a first embodiment, a chromatography resin has the formula:

Chromatography matrix-(X)—N(R¹)(R²)—(R³-L)_n-Ar

or an anionic salt thereof,wherein:

• • X is a spacer;
  • R¹ and R² are each independently C₁ to C₆ alkyl optionally substituted with —OH;
  • R³ is C₂ to C₆ alkyl or C₄ to C₆ cycloalkyl;
  • L is NR⁴, O, or S, wherein R⁴ is hydrogen or C₁ to C₄ alkyl;
  • n is 1 or 2; and
  • Ar is a 6-10 membered ring and:
    • if Ar is aryl, the aryl is optionally substituted with up to five C₁ to C₃ unsubstituted alkyl, C₃ to C₆ branched alkyl, unsubstituted aryl, or fluorine groups; or
    • if Ar is heteroaryl, the heteroaryl is optionally substituted with up to four alkyl groups.

The nitrogen adjacent to the spacer carries a positive charge as a consequence of its structure and thus the charge is independent of pH. Therefore these resins provide strong ion exchange.

In a first aspect of the first embodiment, R¹ and R² are each independently C₁ to C₃ alkyl. Alternatively, R¹ and R² are each independently C₁ or C₂ alkyl.

In a second aspect of the first embodiment, R³ is C₂ to C₄ alkyl. Alternatively, R³ is C₂ or C₃ alkyl.

In a third aspect of the first embodiment, L is NR⁴ or O; or NR⁴ or S. Alternatively, L is O.

In a fourth aspect of the first embodiment, n is 1.

In a fifth aspect of the first embodiment, Ar is a 6 membered ring and if Ar is aryl, the aryl is optionally substituted with up to four C₁ to C₂ unsubstituted alkyl, C₃ to C₄ branched alkyl, or fluorine groups or if Ar is heteroaryl, the heteroaryl is optionally substituted with up to three alkyl groups. Alternatively, Ar is phenyl, napthyl, or pyridyl optionally substituted with up to three C₁ to C₂ unsubstituted alkyl or fluorine groups. Alternatively, Ar is phenyl optionally substituted with one or two C₁ to C₂ unsubstituted alkyl. Alternatively, Ar is unsubstituted phenyl. Alternatively, Ar is heteroaryl and a heteroatom in the heteroaryl is N. Alternatively, Ar is unsubstituted heteroaryl. In yet another alternative, Ar is pyridyl.

In a sixth aspect of the first embodiment, X is attached to chromatography matrix via a bond selected from an amide, amine, ether, ester, carbamate, urea, thioether, thiocarbamate, thiocarbonate and thiourea. Alternatively the bond is an amine, ether or amide.

In an seventh aspect of the first embodiment, X is selected from the group consisting of

—O—CH₂—, —O—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—CH₂—, —O—CH₂—CH(CH₂—OH)—(O—CH₂—CH(OH)—CH₂)₂—, —O—CH₂—CH₂—CH(CH₂—OH)—(O—CH₂—CH₂—CH(OH)—CH₂)₂—, —O—CH₂—CH(OH)—CH₂—, —O—CH₂—CH₂—CH(OH)—CH₂—CH₂—, —O—CH₂—CH(OH)—CH₂—O—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH(OH)—CH₂—, and —CO—NH—C(CH₃)₂—CO—. Alternatively, X is selected from the group consisting of —O—CH₂—, —O—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—CH₂—, and —O—CH₂—CH(CH₂—OH)—(O—CH₂—CH(OH)—CH₂)₂—.

In a second embodiment, the chromatography resin has the formula:

Chromatography matrix-(X)—N(R¹)(R²)—(R³-L)_n-Ar

or an anionic salt thereof,wherein:

• • X is selected from the group consisting of —O—CH₂—, —O—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—CH₂—, —O—CH₂—CH(CH₂—OH)—(O—CH₂—CH(OH)—CH₂)₂—, —O—CH₂—CH₂—CH(CH₂—OH)—(O—CH₂—CH₂—CH(OH)—CH₂)₂—, —O—CH₂—CH(OH)—CH₂—, —O—CH₂—CH₂—CH(OH)—CH₂—CH₂—, —O—CH₂—CH(OH)—CH₂—O—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH(OH)—CH₂—, and —CO—NH—C(CH₃)₂—CO—;
  • R¹ and R² are each independently C₁ to C₃ alkyl;
  • R³ is C₂ to C₄ alkyl;
  • L is O;
  • n is 1; and
  • Ar is a 6 membered ring and:
    • if Ar is aryl, the aryl is optionally substituted with up to four C₁ to C₂ unsubstituted alkyl, C₃ to C₄ branched alkyl, or fluorine groups or
    • if Ar is heteroaryl, the heteroaryl is optionally substituted with up to three alkyl groups.

In a first aspect of the second embodiment, R¹ and R² are each independently C₁ or C₂ alkyl.

In a second aspect of the second embodiment, R³ is C₂ or C₃ alkyl.

In a third aspect of the second embodiment, Ar is phenyl, napthyl, or pyridyl optionally substituted with up to three C₁ to C₂ unsubstituted alkyl. Alternatively, Ar is phenyl optionally substituted with one or two C₁ to C₂ unsubstituted alkyl. Alternatively, Ar is unsubstituted phenyl. Alternatively, Ar is heteroaryl and a heteroatom in the heteroaryl is N. Alternatively, Ar is unsubstituted heteroaryl.

In a third embodiment, the chromatography resin has the formula:

Chromatography matrix-(X)—N(R¹)(R²)—(R³-L)_n-Ar

or an anionic salt thereof,wherein:

• • X is selected from the group consisting of —O—CH₂—, —O—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—CH₂—, and —O—CH₂—CH(CH₂—OH)—(O—CH₂—CH(OH)—CH₂)₂—;
  • R¹ and R² are each independently C₁ or C₂ alkyl;
  • R³ is C₂ or C₃ alkyl;
  • L is O;
  • n is 1; and
  • Ar is phenyl, napthyl, or pyridyl optionally substituted with up to three C₁ to C₂ unsubstituted alkyl.

In a first aspect of the third embodiment, Ar is phenyl optionally substituted with one or two C₁ to C₂ unsubstituted alkyl. Alternatively, Ar is unsubstituted phenyl.

In a fourth embodiment, —(X)—N(R¹)(R²)—(R³-L)_n-Ar is any one of the ligands of Table 1.

TABLE 1
		Structure of Ligand
		Attached to Matrix
		(Spheres represent	Resin
Ligand	Structure of Ligand	matrix and Spacer X)	Number
N,N-dimethyl-2- phenoxyethan-1-amine			13
N,N-dimethyl-3- phenoxypropan-1- amine			94
N,N-dimethyl-2-(2- phenoxyethoxy)ethan- 1-amine			34-1
2-(methyl(2- phenoxyethyl)amino) ethan-1-ol			34-2
2-(3,5- dimethylphenoxy)- N,N-dimethylethan-1- amine			86-1
2-([1,1′-biphenyl]-4- yloxy)-N,N- dimethylethan-1-amine			86-2
N,N-dimethyl-2-(p- tolyloxy)ethan-1-amine			97-1
2-(4-ethylphenoxy)- N,N-dimethylethan-1- amine			97-2
2-(4- isopropylphenoxy)- N,N-dimethylethan-1- amine			98-1
2-(4-fluorophenoxy)- N,N-dimethylethan-1- amine			32-1
2-(2,5- difluorophenoxy)-N,N- dimethylethan-1-amine			91-1
3-(3,5- difluorophenoxy)-N,N- dimethylpropan-1- amine			91-2
2-(3-fluorophenoxy)- N,N-dimethylethan-1- amine			91-3
2-(3,5- difluorophenoxy)-N,N- dimethylethan-1-amine			91-4
2-(3,4- difluorophenoxy)-N,N- dimethylethan-1-amine			91-5
N,N-dimethyl-2-(3,4,5- trifluorophenoxy)ethan- 1-amine			91-6
2-(4-(tert- butyl)phenoxy)-N,N- dimethylethan-1-amine			32-2
N,N-dimethyl-2- (naphthalen-1- yloxy)ethan-1-amine			CB216
N,N-dimethyl-2- (perfluorophenoxy)ethan- 1-amine			50-1
N,N-dimethyl-2- (pyridin-4-yloxy)ethan- 1-amine			71-2
N,N-dimethyl-2- (pyridin-3-yloxy)ethan- 1-amine			71-4
2-((2,6- dimethylpyridin-4- yl)oxy)-N,N- dimethylethan-1-amine			71-3
N,N-dimethyl-3- (pyridin-4- yloxy)propan-1-amine			71-1
N,N-dimethyl-3- phenoxycyclobutan-1- amine
N,N-dimethyl-3- phenoxycyclopentan-1- amine
N,N-dimethyl-3- phenoxycyclohexan-1- amine

In a fifth embodiment, the chromatography resin has the formula:

Chromatography matrix-(X)—N(R¹)—[(R³-L)_n-Ar]₂

or an anionic salt thereof,wherein:

• • X is a spacer;
  • R¹ is C₁ to C₆ alkyl optionally substituted with —OH;
  • R³ is C₂ to C₆ alkyl or C₄ to C₆ cycloalkyl;
  • L is NR⁴, O, or S, wherein R⁴ is hydrogen or C₁ to C₄ alkyl;
  • n is 1 or 2; and
  • Ar is a 6-10 membered ring and:
    • if Ar is aryl, the aryl is optionally substituted with up to five C₁ to C₃ unsubstituted alkyl, C₃ to C₆ branched alkyl, unsubstituted aryl, or fluorine groups; or
    • if Ar is heteroaryl, the heteroaryl is optionally substituted with up to four alkyl groups.

In a first aspect of the fifth embodiment, R¹ is C₁ to C₃ alkyl. Alternatively, R¹ is C₁ or C₂ alkyl.

In a second aspect of the fifth embodiment, R³ is C₂ to C₄ alkyl. Alternatively, R³ is C₂ or C₃ alkyl.

In a third aspect of the fifth embodiment, L is NR⁴ or O or NR⁴ or S. Alternatively, L is O.

In a fourth aspect of the fifth embodiment, n is 1.

In a fifth aspect of the fifth embodiment, Ar is a 6 membered ring and if Ar is aryl, the aryl is optionally substituted with up to four C₁ to C₂ unsubstituted alkyl, C₃ to C₄ branched alkyl, or fluorine groups or if Ar is heteroaryl, the heteroaryl is optionally substituted with up to three alkyl groups. Alternatively, Ar is phenyl, napthyl, or pyridyl optionally substituted with up to three C₁ to C₂ unsubstituted alkyl or fluorine groups. Alternatively, Ar is phenyl optionally substituted with one or two C₁ to C₂ unsubstituted alkyl. Alternatively, Ar is unsubstituted phenyl. Alternatively, Ar is heteroaryl and a heteroatom in the heteroaryl is N. Alternatively, Ar is unsubstituted heteroaryl.

In a sixth embodiment, the chromatography resin has the formula:

Chromatography matrix-(X)—N(R¹)—[(R³-L)_n-Ar]₂

or an anionic salt thereof,wherein:

• • X is selected from the group consisting of —O—CH₂—, —O—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—CH₂—, —O—CH₂—CH(CH₂—OH)—(O—CH₂—CH(OH)—CH₂)₂—, —O—CH₂—CH₂—CH(CH₂—OH)—(O—CH₂—CH₂—CH(OH)—CH₂)₂—, —O—CH₂—CH(OH)—CH₂—, —O—CH₂—CH₂—CH(OH)—CH₂—CH₂—, —O—CH₂—CH(OH)—CH₂—O—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH(OH)—CH₂—, and —CO—NH—C(CH₃)₂—CO—;
  • R¹ is C₁ to C₃ alkyl;
  • R³ is C₂ to C₄ alkyl;
  • L is O;
  • n=1; and
  • Ar is a 6 membered ring and:
    • if Ar is aryl, the aryl is optionally substituted with up to four C₁ to C₂ unsubstituted alkyl, C₃ to C₄ branched alkyl, or fluorine groups or
    • if Ar is heteroaryl, the heteroaryl is optionally substituted with up to three alkyl groups.

In a first aspect of the sixth embodiment, R¹ is C₁ or C₂ alkyl.

In a second aspect of the sixth embodiment, R³ is C₂ or C₃ alkyl.

In a third aspect of the sixth embodiment, Ar is phenyl, napthyl, or pyridyl optionally substituted with up to three C₁ to C₂ unsubstituted alkyl. Alternately, Ar is phenyl optionally substituted with one or two C₁ to C₂ unsubstituted alkyl. Alternately, Ar is unsubstituted. Alternatively, Ar is heteroaryl and a heteroatom in the heteroaryl is N. Alternatively, Ar is unsubstituted heteroaryl.

In a seventh embodiment, the chromatography resin has the formula:

Chromatography matrix-(X)—N(R¹)—[(R³-L)_n-Ar]₂

or an anionic salt thereof,wherein:

• • X is selected from the group consisting of —O—CH₂—, —O—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—CH₂—, and —O—CH₂—CH(CH₂—OH)—(O—CH₂—CH(OH)—CH₂)₂—;
  • R¹ is C₁ or C₂ alkyl;
  • R³ is C₂ or C₃ alkyl; and
  • L is O;
  • n is 1; and
  • Ar is phenyl, napthyl, or pyridyl optionally substituted with up to three C₁ to C₂ unsubstituted alkyl.

In a first aspect of the seventh embodiment, Ar is phenyl optionally substituted with one or two C₁ to C₂ unsubstituted alkyl. Alternately, Ar is unsubstituted.

In an eighth embodiment, —(X)—N(R¹)—[(R³-L)_n-Ar]₂ is any one of the ligands of Table 2.

TABLE 2
		Structure of Ligand Attached to
		Matrix
		(Spheres represent matrix and
Ligand	Structure of Ligand	Spacer X)
N-methyl-2- phenoxy-N-(2- phenoxyethyl)ethan- 1-amine
N-methyl-2-(2- phenoxyethoxy)-N- (2- phenoxyethoxy) ethyl)ethan-1-amine
2-(bis(2- phenoxyethyl)amino) ethan-1-ol
2-(3,5- dimethylphenoxy)- N-(2-(3,5- dimethylphenoxy) ethyl)-N- methylethan-1- amine
2-([1,1′-biphenyl]- 4-yloxy)-N-(2- ([1,1′-biphenyl]-4- yloxy)ethyl)-N- methylethan-1- amine
N-methyl-2-(p- tolyloxy)-N-(2-(p- tolyloxy)ethyl)ethan- 1-amine
2-(4-ethylphenoxy)- N-(2-(4- ethylphenoxy)ethyl)- N-methylethan-1- amine
2-(4- isopropylphenoxy)-N- (2-(4- isopropylphenoxy)ethyl)- N-methylethan-1- amine
2-(4-(tert- butyl)phenoxy)-N- (2-(4-(tert- butyl)phenoxy)ethyl)- N-methylethan-1- amine
N-methyl-2- (naphthalen-1- yloxy)-N-(2- (naphthalen-1- yloxy)ethyl)ethan- 1-amine
2-(4- fluorophenoxy)-N- (2-(4- fluorophenoxy)ethyl)- N-methylethan-1- amine
2-(2,5- difluorophenoxy)- N-(2-(2,5- difluorophenoxy) ethyl)-N- methylethan-1- amine
2-(3- fluorophenoxy)-N- (2-(3- fluorophenoxy)ethyl)- N-methylethan-1- amine
2-(3,5- difluorophenoxy)- N-(2-(3,5- difluorophenoxy) ethyl)-N- methylethan-1- amine
2-(3,4- difluorophenoxy)- N-(2-(3,4- difluorophenoxy) ethyl)-N- methylethan-1- amine
N-methyl-2-(3,4,5- trifluorophenoxy)- N-(2-(3,4,5- trifluorophenoxy) ethyl)ethan-1-amine
N-methyl-2- (perfluorophenoxy)- N-(2- (perfluorophenoxy) ethyl)ethan-1-amine
2-((2,6- dimethylpyridin-4- yl)oxy)-N-(2-((2,6- dimethylpyridin-4- yl)oxy)ethyl)-N- methylethan-1- amine
N-methyl-2- (pyridin-4-yloxy)- N-(2-(pyridin-4- yloxy)ethyl)ethan- 1-amine
N-methyl-2- (pyridin-3-yloxy)- N-(2-(pyridin-3- yloxy)ethyl)ethan- 1-amine
N-methyl-3- phenoxy-N-(3- phenoxycyclobutyl) cyclobutan-1-amine
N-methyl-3- phenoxy-N-(3- phenoxycyclopentyl) cyclopentan-1- amine
N-methyl-4- phenoxy-N-(4- phenoxycyclohexyl) cyclohexan-1-amine

In some embodiments, the anionic salt is hydrochloride or sulfate.

The chromatography matrix is a polymer that is functionalized so that a bond can be formed to the spacer, X. Preferably, the polymer is a hydrophilic polymer. The polymer is insoluble in water. Suitable polymers are polyhydroxy polymers, e.g. based on polysaccharides, such as agarose, dextran, cellulose, starch, pullulan, and completely synthetic polymers, such as polyacrylic amide, polymethacrylic amide, poly(hydroxyalkylvinyl ethers), poly(hydroxyalkylacrylates) and polymethacrylates (e.g. polyglycidylmethacrylate), polyvinyl alcohols and polymers based on styrenes and divinylbenzenes, and copolymers in which two or more of the monomers corresponding to the above-mentioned polymers are included. Suitable synthetic polymers include, but are not limited to, Fractogel from Toso-Haas, POROS media from ThermoFisher Scientific, Bio-Gel P and Macro Prep from Bio-Rad, HEMA and Separon from TESSEK, and Hyper D and Trisacryl media from Pall. Polymers, which are soluble in water, may be derivatized to become insoluble, e.g. by cross-linking and by coupling to an insoluble body via adsorption or covalent binding. Hydrophilic groups can be introduced on hydrophobic polymers (e.g. on copolymers of monovinyl and divinylbenzenes) by polymerisation of monomers exhibiting groups which can be converted to a hydroxyl group, or by hydrophilization of the final polymer, e.g. by adsorption of suitable compounds, such as hydrophilic polymers. Examples of monomers that can be polymerized to achieve useful matrices are vinyl acetate, vinyl propylamine, acrylic acid, methacrylate, butyl acrylate, acrylamide, methacrylamide, vinyl pyrrolidone (vinyl pyrrolidinone), with functional groups in some cases. Cross-linking agents are also of use in many embodiments, and when present can in some embodiments constitute a mole ratio of from about 0.1 to about 0.7 relative to total monomer. Examples of crosslinking agents are dihydroxyethylenebisacrylamide, diallyltartardiamide, triallyl citric triamide, ethylene diacrylate, bisacrylylcystamine, N,N′-methylenebisacrylamide, and piperazine diacrylamide. In some embodiments, the matrix is an UNOsphere™ Support, a polymer produced from water-soluble hydrophilic monomers (Bio-Rad, Hercules, Calif.).

The chromatography matrix can be in the form of a particle, chips, a membrane, or a monolith, i.e., a single block, pellet, or slab of material. Preferably, the chromatography matrix is porous. Particles when used as matrices can be spheres or beads and are either smooth-surfaced or with a rough or textured surface. In some cases, some of the pores are through-pores, extending through the particles to serve as channels large enough to permit hydrodynamic flow or fast diffusion through the pores. When in the form of spheres or beads, the median particle diameter, where the term “diameter” refers to the longest exterior dimension of the particle, is about 25 microns to about 150 microns. Disclosures of exemplary matrices and the processes by which they are made are found in Lihme et al., U.S. Pat. No. 6,498,236, Hjerten et al., U.S. Pat. No. 5,645,717, Liao et al., U.S. Pat. No. 5,647,979, Liao et al., U.S. Pat. No. 5,935,429, and Liao et al., U.S. Pat. No. 6,423,666.

The ligands are linked to the chromatography matrix via the spacer X. Linkage to the chromatography matrix will depend on the specific chromatography matrix used and the chemical group to be linked to the chromatography matrix. Ligands can be linked to the chromatography matrix by performing a reaction between the ligand and a functional group on the chromatography matrix. For chromatography matrices that do not have a suitable functional group, the chromatography matrix is reacted with a suitable activating reagent to create a suitable functional group to which the ligand can be attached. Reductive amination, epoxide chemistry or azalactone chemistry are examples of chemistries acting on aldehyde, epoxide, or azalactone functional groups, respectively.

In some embodiments, the chromatography matrix comprises an epoxide group and a tertiary amine in the ligand is linked to the epoxide group via epoxide chemistry by the scheme below. In this scheme, the spacer X is —O—CH₂—CH(OH)—CH₂—. In this and other synthetic schemes in this disclosure, the square represents the matrix and all coupling chemistry is shown separately.

In some embodiments, the chromatography matrix comprises an azlactone ring and a primary amine in the ligand is linked to the azlactone ring by the scheme below. In this scheme, the spacer X is —CO—NH—C(CH₃)₂—CO—.

In some embodiments, the chromatography matrix comprises a diol and a tertiary amine is linked to an —OH group by activating the resin with two activating reagents, allylglydicylether (AGE) and bromine, by the scheme below. In this scheme, the spacer X is —O—CH₂—CH(CH₂—OH)—(O—CH₂—CH(OH)—CH₂)₂—.

In certain embodiments, the chromatography matrix comprises an —OH group and a tertiary amine is linked to the —OH group by activating the resin with epichlorohydrin by the scheme below. In this scheme, the spacer X is —O—CH₂—CH(OH)—CH₂—.

In some embodiments, the chromatography matrix comprises an —OH group and a tertiary amine is linked to the —OH group by activating the resin with 1,4 butanedioldiglycidyl ether by the scheme below. In this scheme, the spacer X is —O—CH₂—CH(OH)—CH₂—O—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH(OH)—CH₂—.

Other activating reagents include, but are not limited to, epibromohydrin (reacts with an —OH functional group on the chromatography matrix to create an epoxide group), poly(ethylene glycol) diglycidyl ether (reacts with an —OH functional group on the chromatography matrix to create an epoxide group), and sulfonyl chlorides such as tosyl chlorides and tresyl chlorides (react with an —OH functional group on the chromatography matrix to create a sulfonate ester).

Other spacers can include, but are not limited to, —O—CH₂—, —O—CH₂—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—CH₂—, —O—CH₂—CH₂—CH(CH₂—OH)—(O—CH₂—CH₂—CH(OH)—CH₂)₂—, and —O—CH₂—CH₂—CH(OH)—CH₂—CH₂—.

The chromatography matrix can be utilized in any conventional configuration, including packed columns and fluidized or expanded-bed columns, monoliths or porous membranes, and by any conventional method, including batchwise modes for loading, washes, and elution, as well as continuous or flow-through modes. In some embodiments, a column can range in diameter from 1 cm to 1 m, and in height from 1 cm to 30 cm or more.

Methods

Also provided are methods of purifying a target biomolecule. In an embodiment, the method comprises contacting a sample comprising the biomolecule to a chromatography resin, thereby separating the biomolecule from a contaminant. The resulting purified biomolecule is subsequently collected. In some embodiments, the target biomolecule is a monomeric antibody and the method comprises purifying the monomeric antibody from aggregated antibodies in the sample.

The chromatography resins are useful for purifying target biomolecules using anionic exchange (i.e., where the ligand is positively charged) and hydrophobic mixed mode chromatography. The conditions can be adjusted so as to run the chromatography in bind-elute mode or flow-through mode.

Protein preparations to which the methods can be applied can include unpurified or partially purified antibodies (e.g. IgG) from natural, synthetic, or recombinant sources. Unpurified antibody preparations, for example, can come from various sources including, but not limited to, plasma, serum, ascites fluid, milk, plant extracts, bacterial lysates, yeast lysates, or conditioned cell culture media. Partially purified protein preparations can come from unpurified preparations that have been processed by at least one chromatography, precipitation, other fractionation step, or any combination of the foregoing. In some embodiments, the chromatography step or steps employ any method, including but not limited to size exclusion, affinity, anion exchange, cation exchange, protein A affinity, hydrophobic interaction, immobilized metal affinity chromatography, or hydroxyapatite chromatography. The precipitation step or steps can include salt or polyethylene glycol (PEG) precipitation, or precipitation with organic acids, organic bases, or other agents. Other fractionation steps can include but are not limited to crystallization, liquid:liquid partitioning, or membrane filtration.

As will be appreciated in the art, load, wash and elution conditions for use in the mixed mode chromatography will depend on the specific chromatography media/ligands used.

In some bind-elute mode embodiments, loading (i.e., binding the antibodies to the chromatography resin), and optionally washing, is performed at a pH above 7, e.g., between 7-8, 7-9, etc. Some exemplary bind-elute conditions are:

binding condition: 0-1000 mM NaCl or 100-300 mM NaCl, pH 6.5-8.5 in an appropriate buffer (e.g., Tris, Bis-Tris or phosphate);elution condition: 1-1000 mM NaCl or 0-150 mM NaCl, pH 3-8.5 or 4.0-6.0, using an appropriate buffer having sodium acetate, citrate, arginine, or glycine.

Optionally, the chromatography resin can be washed under conditions such that some components of the sample are removed from the chromatography resin but the target biomolecules remain immobilized on the chromatography resin. In some embodiments, the target biomolecule is subsequently eluted by changing (e.g., decreasing or increasing) the salt concentration and/or reducing the pH of the solution in contact with the matrix.

Alternatively, the sample can be applied in flow through mode in which some components of the sample are immobilized to the chromatography resin but the target biomolecules flow through (i.e., flow passed) the chromatography resin, and are collected. Some exemplary flow through conditions are 0-150 mM NaCl, pH 4.0-8.0; appropriate buffers can include, e.g., 2-(N-morpholino)ethanesulfonic acid (MES), Bis-Tris, sodium acetate or citrate-phosphate.

EXAMPLES

The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results.

Example 1—Preparation of Chromatography Matrices Having the Ligands of Table 1

Reaction with AGE (allylglydicylether): For all the ligands in Table 2, UNOspherem™ Diol (100 mL), a copolymer of 3-allyloxy-1,2-propanediol and vinyl pyrrolidinone, crosslinked with N,N′-methylenebisacrylamide and with a diol density of 200-300 μmol/mL, was used in the form of spherical beads. The beads were suspended in 30 mL of water, 30 mL ION NaOH and 16 g Na₂SO₄ at 50° C. in a 250 RPM shaker for 10 min. 100 mL AGE was added and the mixture was kept at 50° C. in the same shaker overnight. The resulting resin was washed with 3×2 column volumes (CV) of isopropyl alcohol (IPA) and 30 CV water.

Bromination: The above AGE modified resin was mixed with 100 mL water and 3.4 g NaOAC. Bromine liquid was added drop-wise to the slurry until an orange color remained (indicating the completion of reaction between double bond and bromine). Na₂SO₃ was then added until the orange color disappeared (reduction of excess bromine to bromide). The resin was washed with 30 CV water and was ready for ligand coupling.

Coupling of prototype ligands to UNO sphere Diol bromide: For each prototype ligand (i.e., 16 ligands), 100 mL UNOsphere Diol bromide was mixed with 50 mL water and 50 mL IPA. Then 12.5 g prototype ligand was added. Each mixture was incubated at 50° C. in a 250 RPM shaker overnight. At the end of the reaction, the resin was washed with 2 CV IPA, 2 CV water, 2 CV 1N HCl, 2 CV water, 2 CV 1N NaOH, and then 30 CV water to obtain each prototype resin.

The number, structure, and the density of each ligand attached to UNOsphere Diol bromide resin is listed in Table 3.

TABLE 3
	Structure of Ligand
	Attached to Matrix
Resin Number	(Spheres represent matrix	Ligand Density
from Table 1	and Spacer X)	(μmol/mL)
13		100
94		113
34-1		92
34-2		73
86-1		86
86-2		64
97-1		108
97-2		102
98-1		69
32-1		102
91-1		73
91-2		103
91-3		109
91-4		102
91-5		112
91-6		112
32-2		83
50-1		53
71-2		46
71-4		108
71-3		87
71-1		86

Example 2—Preparation of Chromatography Resin with 2-Phenoxyethylamine as a Comparative Ligand (Prototype 12)

A chromatography resin with Prototype 12 ligand was prepared for comparison purposes. The ligand, 2-phenoxyethylamine, has the following structure:

The nitrogen of Prototype 12 can be protonated and thus positively charged based on pH and is thus a weak ion exchanger in contrast to the strong ion exchange ligands of the disclosure.

To attach 2-phenoxyethylamine to UNOsphere™ Diol (20 mL), 82 mol/mL of the chromatography matrix was used in the form of spherical beads. UNOsphere™ Diol is a copolymer of 3-allyloxy-1, 2-propanediol and vinyl pyrrolidinone, crosslinked with N,N′-methylenebisacrylamide and with a diol density of 200-300 μmol/mL.

The beads were suspended in 20 mL of either 0.1 M sodium acetate or water. Sodium periodate was added to a concentration within the range of 50 to 100 mM, and the resulting mixture was incubated at room temperature (approximately 70° F. (21° C.)) for 3-24 hours. The reaction resulted in conversion of the diol groups to aldehyde groups in the range of 150-250 μmol/mL. The resulting aldehyde-functionalized resin was transferred to a 20 mL column where it was washed with 100 mL of water.

Twenty milliliters of UNOsphere aldehyde resin was then suspended in 20 ml of 0.20 M sodium phosphate containing 0.6 g of 2-phenoxyethylamine at pH 7.0. After this mixture was incubated (shaking, 200 rpm) at room temperature for 15 minutes, 200 mg NaBH₃CN was then added and the reaction was allowed to continue for 3-20 hours. The 2-phenoxyethylamine concentration in the reaction was determined to be in the range of 25-200 mM. At the end of the reaction, resin prototype 12 was transferred to a 20 ml column, washed with 3 CV of water followed by 1-2 CV of 0.1N HCl, and then washed with 5 CV water. The 2-phenoxyethylamine ligand density was in the range of 25-100 μmol/ml.

Example 3—Resin Evaluation in Bind-Elute Mode

Dynamic binding capacity (DBC) and recovery of an acidic mAb, mAb S (pI˜7.2) was determined for three mixed mode resins including two commercial resins and prototype resin 13.

Materials

• • 1. Chromatography media: Ceramic Hydroxyapatite (CHT) Type I (Bio-Rad, 40 μm bead size), CHT Type II (Bio-Rad, 40 μm bead size), and Prototype 13 (Bio-Rad, 50 μm bead size)
  • 2. Test protein monoclonal antibody S (mAb S): Purified from tissue culture fluid with UNOsphere SUPrA™ affinity chromatography media (Bio-Rad), about 25% of mAb S molecules existed as aggregates.
  • 3. Packed columns: Bio-Scale Mini cartridge (Bio-Rad), 0.56×4 cm, about 1 ml bed volume with 0.63 g CHT I and II without compression, or 1 mL of Prototype 13 with a compression factor of 1.2.
  • 4. Binding buffer for CHT I and II: 5 mM NaPO4, 25 mM NaCl, pH 6.5
  • 5. Binding buffer for Prototype 13: 20 mM NaPO4, pH 7.8
  • 6. Elution buffer for CHT I and II: 5 mM NaPO4, 550 mM NaCl, pH 6.5
  • 7. Elution buffer for Prototype 13: 20 mM NaOAc, pH6.0
  • 8. Stripping buffer: 20 mM NaOAc, pH 4.0
  • 9. BioLogic Duoflow 10 system (Bio-Rad)

Methods:

DBC determination on a BioLogic Duoflow 10 (Bio-Rad): A solution containing about 1.0 mg/mL mAb S in binding buffer was applied onto each column at a flow rate of 0.62 mL/min. For each column, the time at which the column effluent optical density at 280 nm (OD280) reached a value equal to 5 or 10% of the OD280 of the original mAb solution (i.e., the 5 or 10% “breakthrough”) was determined. When 5 or 10% breakthrough was reached, sample loading was stopped. The column was then washed with binding buffer. DBC was determined by multiplying the retention time at 5 or 10% breakthrough by the flow rate and mAb concentration.Target mAb recovery: A solution containing about 1.0 mg/mL of mAb S in binding buffer was injected onto a 1-mL column of resin. The mAb S was eluted with elution buffer at pH 6.0. The collected antibody elution fractions of each column were analyzed by size exclusion high performance liquid chromatography (HPLC-SEC) to determine the percent aggregate content of antibody in the elution fractions. The percent monomer, dimer, and tetramer content for the samples was determined by integrating the respective monomer, dimer, and tetramer peak areas and is listed in Table 4.

Results: The results for dynamic binding capacity (DBC) are shown in FIG. 1, the chromatographic elution profile of mAb S from the resin is shown in FIG. 2, and the monomer content of load, eluate, and strip fractions is in Table 4.

TABLE 4
Monomer Content of mAB S from Prototype 13 Resin
		Monomer	Dimer	Tetramer
	Sample	(%)	(%)	(%)
	Load	93.1	6.5	0.4
	Eluate	100	0	0
	Strip	76.8	23.2	0

Referring to FIG. 1, the DBC of Prototype 13 for mAb S is higher than the DBC of CHT I and CHT II.

Referring to Table 4 and FIG. 2, mAb S monomer was eluted from Prototype 13 at about pH 6 (FIG. 2). Thus, Prototype 13 can be used to remove aggregates from mAb S in bind-elute mode.

Example 4—Evaluation of Prototype 13 Resin in Flow Through Mode

Materials

• • 1. Chromatography media: Prototype 13 (Bio-Rad, 50 μm bead size)
  • 2. Sample containing monoclonal antibody T (mAb T): pI of mAb T is about 8.45 (DrugBank DB00072). The mAb T starting material contained 98.3 98.9% monomer when purified from CHO cell culture harvest with UNOsphere SuprA. The mAb T concentration of 24-26 mg/mL was determined by Bradford using bovine IgG as a standard. The starting material had 1-2 ng dsDNA and 150-200 ng/mg host cell protein (HCP) as determined by CHO-CM HCP ELISA kit (Cygnus Technologies).
  • 3. Packed columns: Bio-Scale Mini cartridge (Bio-Rad), 0.56×4 cm, 1 mL of Prototype 13 with a compression factor of 1.2.
  • 4. Binding buffer: 20 mM NaPO4, 100 mM NaCl, pH 6.5
  • 5. Stripping buffer: 20 mM NaOAc, pH 4.0

Methods: The resin was packed into a column and was equilibrated with binding buffer. A solution containing 24-26 mg/mL mAb T was applied to the column at a flow rate of 0.62 mL/minute. The mAb T flowed through the column. Stripping buffer was then applied to the column to remove bound substances.

Results: A chromatogram for the resin is shown in FIG. 3. Prior to applying stripping buffer to the column, an increase in OD280 was observed in the chromatogram, indicating that a portion of the mAb T bound to the resin. The results show that Prototype 13 resin can be used to purify mAb T in flow-through mode.

Example 5—Testing of Prototype 13 for mAb Aggregate Removal in Flow Through Mode

Materials: The same materials as in Example 4 were used in this experiment.

Methods: The resin was packed into a column and was equilibrated with binding buffer. A solution containing 24-26 mg/mL mAb T was applied to the column at a flow rate of 0.62 mL/minute. The mAb T flowed through the column. Stripping buffer was then applied to the column to remove bound substances. Monomeric mAb T recovered in eluate was quantified by OD280, an absorption efficiency of 1.4 and the following equation:

Monomer Recovery (%)=(Total monomer in eluate/Total loaded monomer)×100.

Results: The results are shown in Table 5 below. Recovery of the mAb T was the highest with prototype 13 resin at pH 6.5. The results show that prototype 13 resin can be used to purify mAb T in flow-through mode.

TABLE 5
mAb T Recovery
	Monomer	Flow Through
	Content	Monomer Recovery	HCP	dsDNA
Media	(%)	(%)	(ng/mg)	(ng/mg)
Prototype 13	99.4	85.9	5.2	0
Flow-through
at pH 7.5
Prototype 13	99.3	94.1	4.0	0
Flow-through
at pH 6.5

Example 6—Testing of Prototype Resins for mAb Aggregate Removal in Bind-Elute Mode

Materials

• • 1. Chromatography media: each of the prototype resins listed in Table 3.
  • 2. Test protein mAb S: Purified from tissue culture fluid with UNOsphere SUPrA™ affinity chromatography media (Bio-Rad), about 25% of mAb S molecules existed as aggregates.
  • 3. Packed columns: Bio-Scale Mini cartridge (Bio-Rad), 0.56×4 cm, ˜1 ml bed volume with 0.63 g CHT, or 1 mL of Prototype 13 with a compression factor of 1.2.
  • 4. Binding buffer: 20 mM sodium phosphate, pH 7.8
  • 5. Elution buffer: 20 mM sodium acetate (pH4.0)

Methods:

Target mAb recovery: A solution containing about 1.0 mg/mL of mAb S in binding buffer was injected onto a 1-mL column of each of the prototype resins listed in Table 3. The flow rate was 150 cm per hour. The mAb S was eluted with a gradient of 0-100% elution buffer in 10 column volumes. The collected antibody elution fractions of each column were analyzed by size exclusion high performance liquid chromatography (HPLC-SEC) to determine the percent aggregate content of antibody in the elution fractions. The percent monomer content for the samples was determined by subtracting the percent aggregate content from 10000 and is listed in Table 6. Monomeric mAb S recovered in eluates was quantified by OD280, an absorption efficiency of 1.4 and the following equation:

Monomer Recovery (%)=(Total protein in eluate/Total loaded protein)×100.

Results: The eluate monomer content and monomer recovery results for mAb Sare listed in Table 6 for each prototype resin. The data show that each of the resins can be used to recover (or purify) monomeric mAb S.

TABLE 6
Prototype Resin mAb S Monomer Recovery
	Eluate Monomer Content	Monomer Recovery
Prototype Resin	(%)	(%)
13	83.4	96.5
94	94.0	80.0
34-2	84.2	89.8
34-1	79.5	80.7
97-1	92.4	100
97-2	99.0	94.3
98-1	100	75.6
86-1	100	61.9
86-2	100	17.7
32-2	100	68.5
32-1	84.8	89.5
91-1	87.2	90.0
91-2	97.2	53.7
91-3	97.1	67.1
91-4	97.6	63.2
91-5	97.8	75.3
91-6	97.5	77.6
50-1	78.2	100
71-1	0 (100% in flow through)	0
71-2	0 (100% in flow through)	0
71-3	50	68.9
71-4	68	85.7

Example 7—Comparison of Prototype 12 and Prototype 13 Resins in Bind-Elute Mode

Materials and Methods

Target mAb recovery: A solution containing 1.18 mg/mL of mAb S in binding buffer (20 mM sodium phosphate, pH7.8) was injected onto a 1-mL column of each of the prototype resins 12 and 13. The flow rate was 150 cm per hour. The mAb S was eluted with a gradient of 0-100% elution buffer (20 mM sodium acetate, pH4.0) in 10 column volumes. The collected antibody elution fractions of each column were analyzed by size exclusion high performance liquid chromatography (HPLC-SEC) to determine the percent monomer and aggregate content of antibody in the elution fractions. The percent monomer, dimer, and tetramer content for the samples with each resin was determined by integrating the respective monomer, dimer, and tetramer peak areas and is listed in Tables 7 and 8.

Results: The monomer content results for monomeric mAb S with the two resins are listed in Tables 7 and 8.

TABLE 7
Monomer Recovery of mAb S with Prototype 12
		Monomer
	Time range	Content	Dimer	Tetramer
Sample	(min)	(%)	(%)	(%)
Starting Material	—	74.1	20.3	5.6
Flow Through	45-64	96.2	3.8	0
Eluate 1	74-77	77.5	19.3	3.2
Eluate 2	77-80	70.3	23.2	3.5
Eluate 3	80-82	69.9	22.9	7.2
Combined	74-82	72.8	22.8	4.4
Eluates 1 to 3

TABLE 8
Monomer Recovery of mAb S with Prototype 13
		Monomer
	Time range	Content	Dimer	Tetramer
Sample	(min)	(%)	(%)	(%)
Starting Material	—	74.1	20.3	5.6
Flow Through	82-100	100	0	0
Eluate 0	100-104	100	0	0
Eluate 1	104-107	97.8	2.2	0
Eluate 2	107-110	72.3	22.9	4.8
Eluate 3	110-113	60.3	29.1	10.6
Combined	100-110	83.6	13.6	2.8
Eluates 0 to 2

Referring to Tables 7 and 8, prototype 13 resin gave better aggregate clearance efficiency as evidenced by early elution fractions containing highmonomer content (i.e., there was monomer enrichment in the early elution fractions Eluate 0 and Eluate 1). The recovery results of mAb S with prototype 12 showed no significant difference in monomer content among the elution fractions; thus, no monomer enrichment was observed in the early elution fractions (Eluate 1 and Eluate 2).

FIGS. 4 and 5 are bind-elute mode purification chromatograms of mAb S on prototype 12 resin and prototype 13 resin, respectively. During purification, Absorbance Units (AU on the left y-axis) and pH (pH units are on the right y-axis) was monitored as a function of time. Referring to FIG. 4, as the pH decreased from 7.8 to 4, mAb S eluted from prototype 12 resin, but no enrichment in monomeric mAb S was observed in the elution fractions (as evidenced by the monomer content results in Table 7). Thus, for prototype 12 resin, a pH gradient from 7.8 to 4 did not result in purified mAb S. Referring to FIG. 5, as the pH decreased from 7.8 to 4, mAb S monomer was eluted from prototype 13 resin and elution of mAb S monomer was complete at about pH 6 (or during elution fraction Eluate 3). Given that elution of mAb S monomer was complete by elution fraction Eluate 3 and that monomer enrichment occurred in elution fractions Eluate 0 and Eluate 1 (as evidenced by the monomer content results in Table 8), pH 6 instead of pH 4 can be used to elute monomeric mAb S from prototype 13 resin. This is supported by the bind-elute results for mAb S with prototype 13 resin at pH 6 shown in Example 3.

All patents, patent applications, and other published reference materials cited in this specification are hereby incorporated herein by reference in their entirety.

Additional Disclosure and Claimable Subject Matter

Item 1. A chromatography resin having the following formula:

Chromatography matrix-(X)—N(R¹)(R²)—(R³-L)_n-Ar

• or an anionic salt thereof,
• wherein:
• X is a spacer;
• R¹ and R² are each independently C₁ to C₆ alkyl optionally substituted with —OH;
• R³ is C₂ to C₆ alkyl or C₄ to C₆ cycloalkyl;
• L is NR⁴, O, or S, wherein R⁴ is hydrogen or C₁ to C₄ alkyl;
• n is 1 or 2; and
• Ar is a 6-10 membered ring and:
  • if Ar is aryl, the aryl is optionally substituted with up to five C₁ to C₃ unsubstituted alkyl, C₃ to C₆ branched alkyl, unsubstituted aryl, or fluorine groups; or
  • if Ar is heteroaryl, the heteroaryl is optionally substituted with up to four alkyl groups.

Item 2. The chromatography matrix of item 1, wherein:

• X is selected from the group consisting of —O—CH₂—, —O—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—CH₂—, —O—CH₂—CH(CH₂—OH)—(O—CH₂—CH(OH)—CH₂)₂—, —O—CH₂—CH₂—CH(CH₂—OH)—(O—CH₂—CH₂—CH(OH)—CH₂)₂—, —O—CH₂—CH(OH)—CH₂—, —O—CH₂—CH₂—CH(OH)—CH₂—CH₂—, —O—CH₂—CH(OH)—CH₂—O—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH(OH)—CH₂—, and —CO—NH—C(CH₃)₂—CO—;
• R¹ and R² are each independently C₁ to C₃ alkyl;
• R³ is C₂ to C₄ alkyl;
• L is O;
• n is 1; and
• Ar is a 6 membered ring and:
  • if Ar is aryl, the aryl is optionally substituted with up to four C₁ to C₂ unsubstituted alkyl, C₃ to C₄ branched alkyl, or fluorine groups or
  • if Ar is heteroaryl, the heteroaryl is optionally substituted with up to three alkyl groups.

Item 3. The chromatography resin of item 2, wherein:

• X is selected from the group consisting of —O—CH₂—, —O—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—CH₂—, and —O—CH₂—CH(CH₂—OH)—(O—CH₂—CH(OH)—CH₂)₂—;
• R¹ and R² are each independently C₁ or C₂ alkyl;
• R³ is C₂ or C₃ alkyl;
• L is O;
• n is 1; and
• Ar is phenyl, napthyl, or pyridyl optionally substituted with up to three C₁ to C₂ unsubstituted alkyl.

Item 4. The chromatography resin of any one of items 1-3, wherein Ar is phenyl optionally substituted with one or two C₁ to C₂ unsubstituted alkyl.

Item 5. The chromatography resin of any one of items 1-4, wherein —(X)—N(R¹)(R²)—(R³-L)_n-Ar is any one of the ligands of Table 1.

Item 6. A chromatography resin having the following formula:

Chromatography matrix-(X)—N(R¹)—[(R³-L)_n-Ar]₂

• or an anionic salt thereof,
• wherein:
• X is a spacer;
• R¹ is C₁ to C₆ alkyl optionally substituted with —OH;
• R³ is C₂ to C₆ alkyl or C₄ to C₆ cycloalkyl;
• L is NR⁴, O, or S, wherein R⁴ is hydrogen or C₁ to C₄ alkyl;
• n is 1 or 2; and
• Ar is a 6-10 membered ring and:
  • if Ar is aryl, the aryl is optionally substituted with up to four C₁ to C₃ unsubstituted alkyl, C₃ to C₆ branched alkyl, unsubstituted aryl, or fluorine groups; or
  • if Ar is heteroaryl, the heteroaryl is optionally substituted with up to four alkyl groups.

Item 7. The chromatography resin of item 6, wherein:

• X is selected from the group consisting of —O—CH₂—, —O—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—CH₂—, —O—CH₂—CH(CH₂—OH)—(O—CH₂—CH(OH)—CH₂)₂—, —O—CH₂—CH₂—CH(CH₂—OH)—(O—CH₂—CH₂—CH(OH)—CH₂)₂—, —O—CH₂—CH(OH)—CH₂—, —O—CH₂—CH₂—CH(OH)—CH₂—CH₂—, —O—CH₂—CH(OH)—CH₂—O—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH(OH)—CH₂—, and —CO—NH—C(CH₃)₂—CO—;
• R¹ is C₁ to C₃ alkyl;
• R³ is C₂ to C₄ alkyl;
• L is O;
• n is 1; and
• Ar is a 6 membered ring and:
  • if Ar is aryl, the aryl is optionally substituted with up to four C₁ to C₂ unsubstituted alkyl, C₃ to C₄ branched alkyl, or fluorine groups or
  • if Ar is heteroaryl, the heteroaryl is optionally substituted with up to three alkyl groups.

Item 8. The chromatography resin of item 7, wherein:

• X is selected from the group consisting of —O—CH₂—, —O—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—, —O—CH₂—CH₂—CH₂—CH₂—, and —O—CH₂—CH(CH₂—OH)—(O—CH₂—CH(OH)—CH₂)₂—;
• R¹ is C₁ or C₂ alkyl;
• R³ is C₂ or C₃ alkyl; and
• L is O;
• n is 1; and
• Ar is phenyl, napthyl, or pyridyl optionally substituted with up to three C₁ to C₂ unsubstituted alkyl.

Item 9. The chromatography resin of any one of items 6-8, wherein Ar is phenyl optionally substituted with one or two C₁ to C₂ unsubstituted alkyl.

Item 10. The chromatography resin of any one of items 6-9, wherein —(X)—N(R¹)—[(R³-L)_n-Ar] is any one of the ligands of Table 2.

Item 11. The chromatography resin of any one of items 1-10, wherein Ar is unsubstituted.

Item 12. The chromatography resin of item 1 or 6, wherein Ar is heteroaryl and a heteroatom in the heteroaryl is N.

Item 13. The chromatography resin of any one of items 1-12, wherein the anionic salt is a hydrochloride salt or a sulfate salt.

Item 14. The chromatography resin of any one of items 1-13, wherein X is attached to chromatography matrix via an amine, ether or amide bond.

Item 15. A chromatography resin prepared by reacting any one of the ligands of Table 1 with a chromatography matrix by any one of reductive amination, epoxide chemistry, or azalactone chemistry.

Item 16. The chromatography resin of item 15, wherein the chromatography matrix comprises an aldehyde group and any one of the ligands of Table 1 is reacted with the chromatography matrix by reductive amination.

Item 17. The chromatography resin of item 15, wherein the chromatography matrix comprises an epoxide group and any one of the ligands of Table 1 is reacted with the chromatography matrix by epoxide chemistry.

Item 18. The chromatography resin of any one of items 15-17 wherein prior to reacting the chromatography matrix with the ligand, the chromatography matrix is reacted with allylglydicylether and bromine; 1,4-butanedioldiglycidyl; or epichlorohydrin.

Item 19. The chromatography resin of item 18, wherein the chromatography matrix comprises a —OH group and is reacted with allylglydicylether and bromine.

Item 20. A chromatography resin prepared by reacting any one of the ligands of Table 2 with a chromatography matrix by epoxide chemistry.

Item 21. A method of purifying a biomolecule, the method comprising:

• contacting a sample comprising the biomolecule to a chromatography resin of any one of claims 1-20, thereby separating the biomolecule from a contaminant; and
• collecting a purified biomolecule.

Item 22. The method of item 21, wherein the purified biomolecule is a protein.

Item 23. The method of item 22, wherein the protein is an antibody.

Item 24. The method of any one of items 21-23, wherein the sample comprises a monomeric antibody and antibody aggregates, the method comprises separating the monomeric antibody from the antibody aggregates, and the purified biomolecule comprises the monomeric antibody.

Item 25. The method of any one of items 24, wherein the purified biomolecule is a monomeric antibody.

Item 26. The method of item 25, wherein the contacting step comprises immobilizing the monomeric antibody to the chromatography matrix and the collecting step comprises eluting the monomeric antibody from the chromatography matrix.

Item 27. The method of item 26, wherein the monomeric antibody is eluted by a step comprising reducing a pH of a solution in contact with the ligand from about 7-9 to about 4-6.

Item 28. The method of item 25, wherein the contacting step comprises flowing the monomeric antibody through the chromatography matrix and the collecting step comprises collecting the monomeric antibody in the flow through.

Claims

1-28. (canceled)

29. A chromatography resin having the following formula: Chromatography matrix-(X)—N(R1)(R2)—(R3-L)n-Aror an anionic salt thereof,wherein: X is a spacer;R1 and R2 are each independently C1 to C6 alkyl optionally substituted with —OH;R3 is C2 to C6 alkyl or C4 to C6 cycloalkyl;L is NR4, O, or S, wherein R4 is hydrogen or C1 to C4 alkyl;n is 1 or 2; andAr is a 6-10 membered ring and: if Ar is aryl, the aryl is optionally substituted with up to five C1 to C3 unsubstituted alkyl, C3 to C6 branched alkyl, unsubstituted aryl, or fluorine groups; orif Ar is heteroaryl, the heteroaryl is optionally substituted with up to four alkyl groups.

30. The chromatography matrix of claim 29, wherein: X is selected from the group consisting of —O—CH2—, —O—CH2—CH2—, —O—CH2—CH2—CH2—, —O—CH2—CH2—CH2—CH2—, —O—CH2—CH(CH2—OH)—(O—CH2—CH(OH)—CH2)2—, —O—CH2—CH2—CH(CH2—OH)—(O—CH2—CH2—CH(OH)—CH2)2—, —O—CH2—CH(OH)—CH2—, —O—CH2—CH2—CH(OH)—CH2—CH2—, —O—CH2—CH(OH)—CH2—O—CH2—CH2—CH2—CH2—O—CH2—CH(OH)—CH2—, and —CO—NH—C(CH3)2—CO—;R1 and R2 are each independently C1 to C3 alkyl;R3 is C2 to C4 alkyl;L is O;n is 1; andAr is a 6 membered ring and: if Ar is aryl, the aryl is optionally substituted with up to four C1 to C2 unsubstituted alkyl, C3 to C4 branched alkyl, or fluorine groups orif Ar is heteroaryl, the heteroaryl is optionally substituted with up to three alkyl groups.

31. The chromatography resin of claim 30, wherein: X is selected from the group consisting of —O—CH2—, —O—CH2—CH2—, —O—CH2—CH2—CH2—, —O—CH2—CH2—CH2—CH2—, and —O—CH2—CH(CH2—OH)—(O—CH2—CH(OH)—CH2)2—;R1 and R2 are each independently C1 or C2 alkyl;R3 is C2 or C3 alkyl;L is O;n is 1; andAr is phenyl, napthyl, or pyridyl optionally substituted with up to three C1 to C2 unsubstituted alkyl.

32. The chromatography resin of claim 29, wherein Ar is phenyl optionally substituted with one or two C1 to C2 unsubstituted alkyl.

33. The chromatography resin of claim 29, wherein —(X)—N(R1)(R2)—(R3-L)n-Ar is any one of the ligands of Table 1.

34. The chromatography resin of claim 29, wherein Ar is unsubstituted.

35. The chromatography resin of claim 29, wherein Ar is heteroaryl and a heteroatom in the heteroaryl is N.

36. The chromatography resin of claim 29, wherein the anionic salt is a hydrochloride salt or a sulfate salt.

37. The chromatography resin of claim 29, wherein X is attached to chromatography matrix via an amine, ether or amide bond.

38. A chromatography resin having the following formula: Chromatography matrix-(X)—N(R1)—[(R3-L)n-Ar]2 or an anionic salt thereof,wherein: X is a spacer;R1 is C1 to C6 alkyl optionally substituted with —OH;R3 is C2 to C6 alkyl or C4 to C6 cycloalkyl;L is NR4, O, or S, wherein R4 is hydrogen or C1 to C4 alkyl;n is 1 or 2; andAr is a 6-10 membered ring and: if Ar is aryl, the aryl is optionally substituted with up to four C1 to C3 unsubstituted alkyl, C3 to C6 branched alkyl, unsubstituted aryl, or fluorine groups; orif Ar is heteroaryl, the heteroaryl is optionally substituted with up to four alkyl groups.

39. The chromatography resin of claim 38, wherein: X is selected from the group consisting of —O—CH2—, —O—CH2—CH2—, —O—CH2—CH2—CH2—, —O—CH2—CH2—CH2—CH2—, —O—CH2—CH(CH2—OH)—(O—CH2—CH(OH)—CH2)2—, —O—CH2—CH2—CH(CH2—OH)—(O—CH2—CH2—CH(OH)—CH2)2—, —O—CH2—CH(OH)—CH2—, —O—CH2—CH2—CH(OH)—CH2—CH2—, —CH2—CH(OH)—CH2—O—CH2—CH2—CH2—CH2—O—CH2—CH(OH)—CH2—, and —CO—NH—C(CH3)2—CO—;R1 is C1 to C3 alkyl;R3 is C2 to C4 alkyl;L is O;n is 1; andAr is a 6 membered ring and: if Ar is aryl, the aryl is optionally substituted with up to four C1 to C2 unsubstituted alkyl, C3 to C4 branched alkyl, or fluorine groups orif Ar is heteroaryl, the heteroaryl is optionally substituted with up to three alkyl groups.

40. The chromatography resin of claim 39, wherein: X is selected from the group consisting of —O—CH2—, —O—CH2—CH2—, —O—CH2—CH2—CH2—, —O—CH2—CH2—CH2—CH2—, and —O—CH2—CH(CH2—OH)—(O—CH2—CH(OH)—CH2)2—;R1 is C1 or C2 alkyl;R3 is C2 or C3 alkyl; andL is O;n is 1; andAr is phenyl, napthyl, or pyridyl optionally substituted with up to three C1 to C2 unsubstituted alkyl.

41. The chromatography resin of claim 38, wherein Ar is phenyl optionally substituted with one or two C1 to C2 unsubstituted alkyl.

42. The chromatography resin of claim 38, wherein —(X)—N(R1)—[(R3-L)n-Ar] is any one of the ligands of Table 2.

43. A chromatography resin prepared by reacting any one of the ligands of Table 1 with a chromatography matrix by any one of reductive amination, epoxide chemistry, or azalactone chemistry.

44. The chromatography resin of claim 43, wherein the chromatography matrix comprises an aldehyde group and any one of the ligands of Table 1 is reacted with the chromatography matrix by reductive amination.

45. The chromatography resin of claim 43, wherein the chromatography matrix comprises an epoxide group and any one of the ligands of Table 1 is reacted with the chromatography matrix by epoxide chemistry.

46. The chromatography resin of claim 43, wherein prior to reacting the chromatography matrix with the ligand, the chromatography matrix is reacted with allylglydicylether and bromine; 1,4-butanedioldiglycidyl; or epichlorohydrin.

47. The chromatography resin of claim 46, wherein the chromatography matrix comprises a —OH group and is reacted with allylglydicylether and bromine.

48. A chromatography resin prepared by reacting any one of the ligands of Table 2 with a chromatography matrix by epoxide chemistry.

49. A method of purifying a biomolecule, the method comprising: contacting a sample comprising the biomolecule to a chromatography resin of claim 29, thereby separating the biomolecule from a contaminant; andcollecting a purified biomolecule.

50. The method of claim 49, wherein the purified biomolecule is a protein.

51. The method of claim 50, wherein the protein is an antibody.

52. The method of claim 49, wherein the sample comprises a monomeric antibody and antibody aggregates, the method comprises separating the monomeric antibody from the antibody aggregates, and the purified biomolecule comprises the monomeric antibody.

53. The method of claim 52, wherein the purified biomolecule is a monomeric antibody.

54. The method of claim 53, wherein the contacting step comprises immobilizing the monomeric antibody to the chromatography matrix and the collecting step comprises eluting the monomeric antibody from the chromatography matrix.

55. The method of claim 54, wherein the monomeric antibody is eluted by a step comprising reducing a pH of a solution in contact with the ligand from about 7-9 to about 4-6.

56. The method of claim 53, wherein the contacting step comprises flowing the monomeric antibody through the chromatography matrix and the collecting step comprises collecting the monomeric antibody in the flow through.