# US 8,785,139 B2 · Modification-dependent activity assays

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- Number: US 8,785,139 B2
- Document ID: US8785139B2
- Status: Granted
- Kind: B2
- Application number: 13/475,634
- Filed: 2012-05-18
- Published: 2012-12-27
- Granted: 2014-07-22
- Earliest claimed: 2011-05-18
- Assignees: Baxter International Inc.; Baxter Healthcare S.A.
- Applicants: Alfred Weber; Andrea Engelmaier; Hans-Peter Schwarz
- Inventors: Alfred Weber; Andrea Engelmaier; Hans-Peter Schwarz
- Web page: https://patentgrep.com/patent/US8785139B2

## CPC

- G01N 33/6842: {Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins}
- A61P 5/00: Drugs for disorders of the endocrine system
- A61P 37/02: Immunomodulators
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- G01N 33/6857: {Antibody fragments}
- G01N 2440/00: Post-translational modifications [PTMs] in chemical analysis of biological material
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

Disclosed herein are methods, systems and kits to measure the presence and/or activity of recombinant polypeptides comprising a modification.

## Claims

### Claim 1

A method for detecting the presence of a recombinant polypeptide comprising a modification, the method comprising the steps of:
incubating a sample including the recombinant polypeptide comprising the modification with a capture agent that selectively binds the modification under conditions allowing the selective binding of the capture agent to the modification, thereby forming a polypeptide-agent complex;
purifying the polypeptide-agent complex from the sample; and
assaying for the presence of the recombinant polypeptide and/or a polypeptide activity, wherein detection of the recombinant polypeptide and/or the polypeptide activity is indicative of the presence of the recombinant polypeptide comprising the modification.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the sample includes a polypeptide without the modification and/or a polypeptide with a different pattern of degree of modification.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the recombinant polypeptide is a therapeutic polypeptide.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the recombinant polypeptide is a coagulation factor.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the coagulation factor is a Factor II, a Factor IIa, a Factor VII, a Factor VIIa, a Factor VIII, a Factor VIIIa, a Factor IX, a Factor IXa, a Factor X, or a Factor Xa.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the capture agent has an association rate constant for a polypeptide comprising the modification of more than 1×105 M−1 s−1.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the capture agent has a disassociation rate constant for a polypeptide comprising the modification of less than 1×10−3 s−1.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the capture agent has an equilibrium disassociation constant for a polypeptide comprising the modification of less than 0.500 nM.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the capture agent has an association rate constant for a polypeptide without a modification or a polypeptide with a different pattern or degree of modification of less than 1×104 M−1 s−1.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the capture agent has an association rate constant (Ka) for the recombinant polypeptide comprising a modification that is more than 1×100 M−1 s−1 relative to the association rate constant (Ka) of the capture agent for a recombinant polypeptide without such a modification and/or the association rate constant (Ka) of the capture agent for a recombinant polypeptide with a different pattern or degree of modification.

### Claim 11 (depends on 1)

The method according to claim 1, wherein the capture agent has an association rate constant (Ka) for the recombinant polypeptide comprising a modification that is at least 2-fold more then the association rate constant (Ka) of the capture agent for a recombinant polypeptide without such a modification and/or then the association rate constant (Ka) of the capture agent for a recombinant polypeptide with a different pattern or degree of modification.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the capture agent has a binding specificity ratio for a recombinant polypeptide comprising a modification relative to a recombinant polypeptide without such a modification and/or relative to a recombinant polypeptide with a different pattern or degree of modification of at least 2:1.

### Claim 13 (depends on 1)

The method according to claim 1, wherein the capture agent distinguishes the recombinant polypeptide comprising a modification from the same polypeptide but without the modification.

### Claim 14 (depends on 1)

The method according to claim 1, wherein the capture agent distinguishes the recombinant polypeptide comprising a modification from the same polypeptide but with a different pattern or degree of the same modification.

### Claim 15 (depends on 1)

The method according to claim 1, wherein the recombinant polypeptide comprising the modification is a PEGylation Factor II, a PEGylation Factor IIa, a polysialylation Factor II, a polysialylation Factor IIa, a HESylation Factor II, a HESylation Factor IIa, a Sylation Factor II, or a Sylation Factor IIa.

### Claim 16 (depends on 1)

The method according to claim 1, wherein the recombinant polypeptide comprising the modification is a PEGylation Factor VII, a PEGylation Factor VIIa, a polysialylation Factor VII, a polysialylation Factor VIIa, a HESylation Factor VII, a HESylation Factor VIIa, a Sylation Factor VII, or a Sylation Factor VIIa.

### Claim 17 (depends on 1)

The method according to claim 1, wherein the recombinant polypeptide comprising the modification is a PEGylation Factor VIII, a PEGylation Factor Villa, a polysialylation Factor VIII, a polysialylation Factor Villa, a HESylation Factor VIII, a HESylation Factor Villa, a Sylation Factor VIII, or a Sylation Factor VIIIa.

### Claim 18 (depends on 1)

The method according to claim 1, wherein the recombinant polypeptide comprising the modification is a PEGylation Factor IX, a PEGylation Factor IXa, a polysialylation Factor IX, a polysialylation Factor IXa, a HESylation Factor IX, a HESylation Factor IXa, a Sylation Factor IX, or a Sylation Factor IXa.

### Claim 19 (depends on 1)

The method according to claim 1, wherein the capture agent is an anti-PEG antibody, an anti-PSA antibody, an anti-HES antibody, or an anti-S antibody.

### Claim 20 (depends on 1)

The method according to claim 1, wherein the capture agent is attached to a solid support.

### Claim 21 (depends on 20)

The method according to claim 20, wherein the solid support is a multi-well plate, a film, a tube, a sheet, a column, or a microparticle.

### Claim 22 (depends on 1)

The method according to claim 1, wherein the assaying step is performed using a qualitative assay or a quantitative assay.

### Claim 23 (depends on 1)

The method according to claim 1, wherein the assaying step is performed using an in vitro assay, a cell-based assay, or an in vivo assay.

### Claim 24 (depends on 1)

The method according to claim 1, wherein the assaying step is performed using a non-specific polypeptide assay or a specific polypeptide assay.

### Claim 25 (depends on 24)

The method according to claim 24, wherein the non-specific polypeptide assay is a UV absorption, a biuret assay, or a Bradford assay.

### Claim 26 (depends on 24)

The method according to claim 24, wherein the specific polypeptide assay is a chromogenic assay, a colorimetirc assay, a chronometric assay, a chemiluminescense assay, an electrochemiluminescence assay, a bioluminescence assay, a fluorogenic assay, a resonance energy transfer assay, a plane polarization assay, a flow cytometry assay, an immuno-based assay or an activity assay.

### Claim 27 (depends on 24)

The method according to claim 24, wherein the activity assay is an enzymatic activity assay, an inhibitory activity assay, a coagulation activity assay, or a polymerization activity assay.

### Claim 28 (depends on 1)

The method according to claim 1, wherein selective binding of the capture agent occurs at a neutral to alkaline pH.