# US 11,110,176 B2 · Composition and method for the protection of proteins, cell components and cells during temperature stress

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- Number: US 11,110,176 B2
- Document ID: US11110176B2
- Status: Granted
- Kind: B2
- Application number: 15/821,671
- Filed: 2017-11-22
- Published: 2018-05-31
- Granted: 2021-09-07
- Earliest claimed: 2016-11-25
- Assignees: The Board of Trustees of the California State University
- Applicants: Xin Wen
- Inventors: Xin Wen
- Web page: https://patentgrep.com/patent/US11110176B2

## CPC

- A61K 47/42: Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein (oligopeptides having up to five amino acids {A61K47/183}; polyamino acids A61K47/34)
- A01N 1/125: Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
- A61K 38/28: Insulins
- C12N 1/04: Preserving or maintaining viable microorganisms (immobilised microorganisms C12N11/00)
- C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Embodiments of the disclosure pertain to compositions containing (i) one or more non-antifreeze proteins or cell components or cells and (ii) one or more antifreeze proteins (AFPs), mimetics thereof, and/or analogs thereof, and a method of stabilizing a biologic (e.g., a protein, microbe, cell component or cell) against temperature stress and aggregation. The AFP may be selected from known antifreeze polypeptides and antifreeze peptides, analogs and mimetics of antifreeze proteins, active fragments of such antifreeze proteins, polypeptide and peptide mimetics, antifreeze peptoids and polymers, and combinations thereof. The non-antifreeze protein, cell component or cell comprises a known protein, cell component or cell used in the pharmaceutical, medical, agricultural, veterinary and/or food industry(ies). The AFP protects the function of the non-antifreeze protein, microbe(s), cell component(s) or cell(s) under temperature stress (e.g., a temperature of −20-60° C.).

## Claims

### Claim 1

A composition, comprising:
an antifreeze protein (AFP) selected from the group consisting of natural or engineered SEQ ID NO: 1 (DAFP-1), SEQ ID NO: 2 (DAFP-2), SEQ ID NO: 3 (DAFP-4) and SEQ ID NO: 4 (DAFP 6); and
a non-antifreeze protein selected from the group consisting of enzymes, hormones, antibodies, growth factors, therapeutic proteins, nutrient proteins and food proteins in need of protection against heat stress;
wherein the antifreeze protein and the non-antifreeze protein are present in a mass ratio of from 1:1 to about 1:1000.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the non-antifreeze protein comprises an enzyme, hormone, antibody, growth factor, or therapeutic protein.

### Claim 3 (depends on 1)

The composition of claim 1, wherein the AFP is natural or engineered SEQ ID NO:1 (DAFP-1).

### Claim 4 (depends on 1)

The composition of claim 1, wherein the AFP is natural SEQ ID NO: 1 (DAFP-1), natural SEQ ID NO: 2 (DAFP-2), natural SEQ ID NO: 3 (DAFP-4), or natural SEQ ID NO: 4 (DAFP-6).

### Claim 5 (depends on 1)

The composition of claim 1, wherein the AFP is engineered SEQ ID NO: 1 (DAFP-1), engineered SEQ ID NO: 2 (DAFP-2), engineered SEQ ID NO: 3 (DAFP-4), or engineered-SEQ ID NO: 4 (DAFP-6).

### Claim 6 (depends on 1)

The composition of claim 1, wherein the non-antifreeze protein is a nutrient or food protein selected from the group consisting of whey proteins, tofu proteins, meat proteins, milk proteins, and fermented yogurt proteins.

### Claim 7 (depends on 1)

The composition of claim 1, comprising a food selected from the group consisting of tofu, beans, nuts, lentils, seeds, meats, fish, cheese, eggs, seafood, wheat germ and quinoa, milk, whey powder, and fermented yogurt beverages, wherein the non-antifreeze protein is in the food.

### Claim 8 (depends on 2)

The composition of claim 2, wherein the non-antifreeze protein comprises alpha-1 antitrypsin, chymopapain, desmoteplase, ocriplasmin, insulin, a growth hormone, an IgG antibody, an IgE antibody, an IgA antibody, an IgM antibody or an IgD antibody.

### Claim 9 (depends on 1)

A formulation, comprising the composition of claim 1 in a pharmaceutically acceptable, nutritionally acceptable, or biologically-compatible excipient, carrier, or adjuvant or combination thereof.

### Claim 10 (depends on 9)

The formulation of claim 9, wherein the excipient comprises deionized water, distilled water, starch, a saccharide, a sugar alcohol, gelatin, polyvinylpyrrolidone (PVP), cellulose, a cross-linked polyvinylpyrrolidone, a cross-linked carboxymethyl cellulose, hydroxypropyl methylcellulose, polyethylene glycol, magnesium stearate or colloidal SiO2, and a ratio of the AFP to a volume of the formulation is 1:20 to 1:5,000,000 weight to volume.

### Claim 11

A composition, comprising:
an antifreeze protein (AFP) selected from the group consisting of natural or engineered SEQ ID NO: 1 (DAFP-1), SEQ ID NO: 2 (DAFP-2), SEQ ID NO: 3 (DAFP-4) and SEQ ID NO: 4 (DAFP-6); and
a microbe or a cell selected from the group consisting of viruses, bacteria cells, plant cells, blood cells, stem cells, egg cells, fungal cells, mold cells, and animal cells;
wherein the ratio of the mass of the AFP to the volume of the composition is in a range of 1:20 to 1:5,000,000.

### Claim 12 (depends on 11)

The composition of claim 11, wherein the microbe or the cell comprises live or dead bacteria or viruses.

### Claim 13 (depends on 11)

The composition of claim 11, wherein the AFP is engineered SEQ ID NO: 1 (DAFP-1), SEQ ID NO: 2 (DAFP-2), SEQ ID NO: 3 (DAFP-4) or SEQ ID NO: 4 (DAFP-6).

### Claim 14 (depends on 11)

The composition of claim 11, wherein the AFP is engineered SEQ ID NO: 1 (DAFP-1).

### Claim 15 (depends on 11)

The composition of claim 11, wherein the ratio of the mass of the AFP to the volume of the composition is from 1:1000 to 1:200,000.

### Claim 16 (depends on 11)

The composition of claim 11, wherein the AFP is natural SEQ ID NO: 1 (DAFP-1), SEQ ID NO: 2 (DAFP-2), SEQ ID NO: 3 (DAFP-4) or SEQ ID NO: 4 (DAFP-6).

### Claim 17 (depends on 16)

The composition of claim 16, wherein the AFP is natural SEQ ID NO: 1 (DAFP-1).

### Claim 18 (depends on 11)

The composition of claim 11, wherein the microbe or cell is a virus or bacteria cells.