# US 10,370,407 B2 · Affinity-assisted protein modification and recycling

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- Number: US 10,370,407 B2
- Document ID: US10370407B2
- Status: Granted
- Kind: B2
- Application number: 15/545,459
- Filed: 2016-01-21
- Published: 2018-05-31
- Granted: 2019-08-06
- Earliest claimed: 2015-01-21
- Assignees: The Regents of the University of California
- Applicants: The Regents Of The University Of California
- Inventors: Richard L. Kwant; Matthew B. Francis; Christian B. Rosen; Meera Rao
- Web page: https://patentgrep.com/patent/US10370407B2

## CPC

- C07K 1/1077: {by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids}
- C07K 1/13: Labelling of peptides
- C08B 37/0012: {Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof}
- C08B 37/0015: {Inclusion compounds, i.e. host-guest compounds, e.g. polyrotaxanes}
- C08H 1/00: Macromolecular products derived from proteins (food proteins A23; glue, gelatine C09H)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C08B: POLYSACCHARIDES; DERIVATIVES THEREOF (polysaccharides containing less than six saccharide radicals attached to each other by glycosidic linkages C07H; fermentation or enzyme-using processes C12P19/00; sugar industry C13; production of cellulose D21)
- C08H: DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS (polysaccharides C08B; natural rubber C08C; natural resins or their derivatives C09F; bituminous materials C10)

## Abstract

Methods for preparing a protein conjugate having a defined number of conjugate groups are provided. The method includes: forming a mixture containing a macrocyclic matrix material and a plurality of proteins; eluting the proteins to obtain a first separated protein fraction and a second separated protein fraction, wherein substantially all of the proteins in the first separated protein fraction have the same number of handle moieties; contacting the handle moieties with a conversion reagent under conditions sufficient to convert the handle moieties in the first separated protein fraction to reactive moieties; and contacting the reactive moieties with a conjugation reagent under conditions sufficient to form a plurality of protein conjugates, wherein substantially all of the protein conjugates in the plurality have the same number of conjugate groups. Methods also include recovering enzymes and other proteins from mixtures for isolation and/or reuse of the enzymes and proteins.

## Claims

### Claim 1

A method for preparing a protein conjugate having a defined number of conjugate groups, the method comprising:
i) forming a mixture comprising a cyclodextrin matrix material and a plurality of proteins, wherein:
each protein in a first population of proteins in the plurality comprises a first number of handle moieties,
each protein in a second population of proteins in the plurality comprises a second number of handle moieties,
the first number of handle moieties and the second number of handle moieties are at least one and differ from one another, and
the mixture is formed under conditions sufficient to bind the modified proteins to the cyclodextrin matrix material;
ii) eluting the proteins from the cyclodextrin matrix material to obtain a first separated protein fraction and a second separated protein fraction, wherein:
substantially all of the proteins in the first separated protein fraction comprise the first number of handle moieties, and
substantially all of the proteins in the second separated protein fraction comprise the second number of handle moieties;
iii) contacting the handle moieties with a conversion reagent under conditions sufficient to convert the handle moieties in the first separated protein fraction to reactive moieties; and
iv) contacting the reactive moieties with a conjugation reagent under conditions sufficient to form a plurality of protein conjugates, wherein substantially all of the protein conjugates in the plurality have the same number of conjugate groups;
thereby preparing the protein conjugate
wherein the handle moieties comprise a structure according to Formula I:
or a salt thereof, wherein
m is an integer ranging from 0 to 5;
each R1 is independently selected from the group consisting of —ORa, —N(Ra)3, —SO3H, and —CO2H;
R2 is selected from the group consisting of H and —ORa;
each Ra is independently selected from the group consisting of H and C1-6 alkyl; and
L1 is a linking moiety.

### Claim 2 (depends on 1)

The method of claim 1, wherein the handle moiety comprises a structure according to Formula Ia:
or a salt thereof, wherein
m is an integer ranging from 0 to 5;
each R1 is independently selected from the group consisting of —ORa, —N(Ra)3, —SO3H, and —CO2H, wherein each Ra is independently selected from the group consisting of H and C1-6 alkyl; and
L1 is a linking moiety.

### Claim 3 (depends on 1)

The method of claim 1, wherein the linking moiety comprises a structure selected from the group consisting of:
wherein
L1a and L1b are independently selected from the group consisting of a bond, a divalent polymer moiety, and linear or branched, saturated or unsaturated C1-30 alkyl; wherein one or more carbon atoms in the C1-30 alkyl is optionally and independently replaced by O, S, NW; wherein two or more groupings of adjacent carbon atoms in the C1-30 alkyl are optionally and independently replaced by —NRa(CO)— or —(CO)NRa—; and wherein two or more groupings of adjacent carbon atoms in the C1-30 alkyl are optionally and independently replaced by a 4- to 8-membered, divalent carbocycle or a 4- to 8-membered, divalent heterocycle having one to four heteroatoms selected from the group consisting of O, S, and N; each Ra is independently selected from the group consisting of H and C1-6 alkyl; the wavy line represents the point of connection to the protein; and
the dashed line represents the point of connection to the structure of Formula I.

### Claim 4 (depends on 1)

The method of claim 1, wherein the reactive moiety comprises a structure according to Formula II:

### Claim 5 (depends on 1)

The method of claim 1, wherein the reactive moiety comprises a structure according to Formula IIa:

### Claim 6 (depends on 2)

The method of claim 2, wherein the conjugation reagent comprises a structure according to Formula III:
wherein:
R3 is selected from the group consisting of —N(Ra)2 and —ORa;
R4 is selected from the group consisting of —ORa, C1-6 alkyl, —N(Ra)2, —N3, and —NH(CO)Ra;
subscript n is an integer ranging from 0 to 3;
each Ra is independently selected from the group consisting of H and C1-6 alkyl;
L2 is a linking moiety; and
A is a prosthetic moiety.

### Claim 7 (depends on 6)

The method of claim 6, wherein the conjugation reagent comprises a structure according to Formula Ma:
wherein
L2a and L2b are independently selected from the group consisting of a bond, a divalent polymer moiety, and linear or branched, saturated or unsaturated C1-30 alkyl; wherein one or more carbon atoms in the C1-30 alkyl is optionally and independently replaced by O, S, NW; wherein two or more groupings of adjacent carbon atoms in the C1-30 alkyl are optionally and independently replaced by —NRa(CO)— or —(CO)NRa—; and wherein two or more groupings of adjacent carbon atoms in the C1-30 alkyl are optionally and independently replaced by a 4- to 8-membered, divalent carbocycle or a 4- to 8-membered, divalent heterocycle having one to four heteroatoms selected from the group consisting of O, S, and N; and
each Ra is independently selected from the group consisting of H and C1-6 alkyl.

### Claim 8 (depends on 1)

The method of claim 1, wherein the contacting is conducted in the presence of an oxidizing agent, wherein the oxidizing agent is potassium ferricyanide.

### Claim 9 (depends on 6)

The method of claim 6, wherein the protein conjugate comprises a structure according to Formula IV:

### Claim 10 (depends on 1)

The method of claim 1, wherein the cyclodextrin matrix material comprises a plurality of cyclodextrin moieties, wherein the cyclodextrin moiety comprises a structure according to Formula VI:
wherein L3 is a linking moiety, and
the wavy line represents the connection point to the matrix material.

### Claim 11 (depends on 10)

The method of claim 10, wherein the linking moiety comprises the grouping L3a-L3b-,
wherein L3a and L3b independently selected from the group consisting of a bond, a divalent polymer moiety, and linear or branched, saturated or unsaturated C1-30 alkyl; wherein one or more carbon atoms in the C1-30 alkyl is optionally and independently replaced by O, S, NW; wherein two or more groupings of adjacent carbon atoms in the C1-30 alkyl are optionally and independently replaced by —NRa(CO)— or —(CO)NRa—; and wherein two or more groupings of adjacent carbon atoms in the C1-30 alkyl are optionally and independently replaced by a 4- to 8-membered, divalent carbocycle or a 4- to 8-membered, divalent heterocycle having one to four heteroatoms selected from the group consisting of O, S, and N; and
each Ra is independently selected from the group consisting of H and C1-6 alkyl.

### Claim 12 (depends on 10)

The method of claim 10, wherein the cyclodextrin moiety comprises a structure according to Formula VIa:

### Claim 13 (depends on 1)

The method of claim 1, wherein the cyclodextrin matrix material comprises crosslinked agarose, crosslinked dextran, or a combination thereof.

### Claim 14 (depends on 1)

The method of claim 1, wherein the protein is an enzyme.