# US 6,720,165 B2 · Methods for making antibody fragments and compositions resulting therefrom

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- Number: US 6,720,165 B2
- Document ID: US6720165B2
- Status: Granted
- Kind: B2
- Application number: 09/882,814
- Filed: 2001-06-14
- Published: 2002-12-19
- Granted: 2004-04-13
- Earliest claimed: 2001-06-14
- Assignees: Zyomix, Inc.
- Inventors: Steffen Nock; David S. Wilson; Jiangchun Wu
- Web page: https://patentgrep.com/patent/US6720165B2

## CPC

- C07K 16/065: {Purification, fragmentation}
- C07K 2317/41: Glycosylation, sialylation, or fucosylation
- C07K 2317/54: F(ab')2
- Y10S 530/866: involving immunoglobulin or antibody fragment, e.g. fab', fab, fv, fc, heavy chain or light chain
- 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)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

Improved methods for making antibody fragments, preferably F(ab′)2 fragments from various classes and subclasses of antibodies is described. Pretreatment of antibodies with deglycosylases or cellular inhibition of glycosylation during expression, yields antibodies having improved susceptibility towards protease cleavage, preferably pepsinolysis, which yields F(ab′)2 antibody fragments. Compositions resulting from such methods are further disclosed.

## Claims

### Claim 1

A method for preparing a F(ab′)2 fragment from a glycosylated antibody, said method comprising the steps of:
(i) providing said glycosylated antibody, said glycosylated antibody comprising a hinge region, said hinge region comprising one or more protease cleavage sites located within said hinge region and one or more non-hinge regions adjacent to said hinge region, said non-hinge region(s) having one or more oligosaceharide groups attached thereto, said oligosaccharide group(s) causing said protease cleavage site(s) within said hinge region to be resistant to a protease treatment;
(ii) exposing said glycosylated antibody to a deglycosylation composition thereby cleaving said oligosaccharide group(s) attached to said non-hinge region(s) to form a partially or wholly deglycosylated antibody having a hinge region cleavable by said protease treatment, said deglycosylation composition comprising at least one glycosidase capable of catalyzing the hydrolysis of an N-glvcosidic or O-glycosidic linkage between a sugar unit and an amino acid; and,
(iii) exposing said partially or wholly deglycosylated antibody to said protease treatment to cause proteolytic cleavage of said hinge region cleavable by said protease treatment to form said F(ab′)2 fragment.

### Claim 2 (depends on 1)

The method of claim 1 wherein said glycosylated antibody is a plurality of glycosylated antibodies.

### Claim 3 (depends on 2)

The method of claim 2 wherein at least some of said glycosylated antibodies are polyclonal.

### Claim 4 (depends on 2)

The method of claim 2 wherein said glycosylated antibodies are monoclonal.

### Claim 5 (depends on 1)

The method of claim 1 wherein said glycosylated antibody is either an IgG1 or IgG2b glycosylated antibody.

### Claim 6 (depends on 5)

The method of claim 5 wherein said IgG1 or IgG2b antibody is from a rodent derived hybridoma cell culture or ascites.

### Claim 7 (depends on 1)

The method of claim 1 wherein said glycosylated antibody is derived from the group consisting of rat, mouse, rabbit, goat, sheep, lamb, chicken, or horse.

### Claim 8 (depends on 1)

The method of claim 1 wherein said protease treatment comprises contacting said antibody with a protease composition comprising a protease selected from the group consisting of pepsin, proteases that cleave pepsin substrates, papain, papain preactivated with cysteine, and ficin.

### Claim 9 (depends on 1)

The method of claim 1 wherein said protease is capable of producing F(ab′)2 fragments from said deglycosylated antibodies.