# US 2019/0169667 A1 · Methods of decreasing trisulfide bonds during recombinant production of polypeptides

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- Number: US 2019/0169667 A1
- Document ID: US20190169667A1
- Status: Published application
- Kind: A1
- Application number: 16/185,369
- Filed: 2018-11-09
- Published: 2019-06-06
- Earliest claimed: 2016-05-10
- Assignees: Genentech, Inc.
- Applicants: Genentech, Inc.
- Inventors: Martin GAWLITZEK; Sven MARKERT; Oliver POPP; Masaru Ken SHIRATORI; Thomas TROEBS; Jessica WUU
- Web page: https://patentgrep.com/patent/US20190169667A1

## CPC

- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C12N 5/0018: {Culture media for cell or tissue culture (media for specific animal cell type C12N5/06)}
- C12N 5/0682: {Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells (oocytes C12N5/0609)}
- C12N 2500/32: Amino acids
- C12N 2500/38: Vitamins
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided herein are cell culture media and methods culturing host cells expressing polypeptides to reduce the level of trisulfide bonds in polypeptides produced by the host cells.

## Claims

### Claim 1

A method for decreasing trisulfide bond levels in a polypeptide comprising:
(a) contacting a host cell comprising a nucleic acid encoding the polypeptide with a basal medium, wherein the basal medium comprises one or more of the following components:
i) between about 2 μM to about 35 μM iron,
ii) between about 0.11 μM to about 2μM riboflavin (vitamin B2),
iii) between about 4.5μM to about 80 μM pyridoxine or pyridoxal (vitamin B6),
iv) between about 3.4 μM to about 23 μM folic acid (vitamin B9),
v) between about 0.2 μM to about 2.5 μM cyanocobalamin (vitamin B12),
vi) between about 9 mM and about 10 mM hypotaurine; and
vii) between about 0 and about 1.58 mM methionine;
(b) culturing the host cell to produce the polypeptide; and
(c) harvesting the polypeptide produced by the host cell.

### Claim 2

A method for producing a polypeptide, comprising:
(a) contacting a host cell comprising a nucleic acid encoding the polypeptide with a basal medium, wherein the basal medium comprises one or more of the following components:
i) between about 2 μM to about 35 μM iron,
ii) between about 0.11 μM to about 2μM riboflavin (vitamin B2),
iii) between about 4.5μM to about 80 μM pyridoxine or pyridoxal (vitamin B6),
iv) between about 3.4 μM to about 23 μM folic acid (vitamin B9),
v) between about 0.2 μM to about 2.5 μM cyanocobalamin (vitamin B12),
vi) between about 9 mM and about 10 mM hypotaurine; and
vii) between about 0 and about 1.58 mM methionine;
(b) culturing the host cell to produce the polypeptide; and
(c) harvesting the polypeptide produced by the host cell.

### Claim 3 (depends on 1, 2)

The method of claim 1 or claim 2, whereby the harvested polypeptide has a trisulfide bond level less than a polypeptide produced under identical conditions, except that the concentration of the one or more components differs from the concentration specified in (a).

### Claim 4 (depends on 1)

The method of claim 1, wherein the basal medium lacks cystine.

### Claim 5 (depends on 1)

The method of claim 1, wherein the basal medium comprises between about 1.4 mM to 3 mM cysteine or cystine.

### Claim 6 (depends on 1)

The method of claim 1, wherein the basal medium comprises between about 0 mM to about 1.58 mM methionine and between about 0 mM to about 3 mM cysteine.

### Claim 7 (depends on 1)

The method of claim 1, wherein the basal medium comprises about 6 mM cysteine.

### Claim 8

A method for decreasing trisulfide bond levels in a polypeptide comprising:
(a) culturing a host cell comprising a nucleic acid encoding the polypeptide in a cell culture medium, wherein the cell culture medium comprises one or more of the following components:
i) between about 2 μM to about 35 μM iron,
ii) between about 0.11 μM to about 2μM riboflavin (vitamin B2),
iii) between about 4.5μM to about 80 μM pyridoxine or pyridoxal (vitamin B6),
iv) between about 3.4 μM to about 23 μM folate/folic acid (vitamin B9),
v) between about 0.2 μM to about 2.5 μM cyanocobalamin (vitamin B12),
vi) between about 9 mM and about 10 mM hypotaurine; and
vii) between about 0 and about 4.5 mM methionine;
(b) producing the polypeptide;
(c) and harvesting the polypeptide produced by the host cell.

### Claim 9 (depends on 8)

The method of claim 8, wherein the concentration of one or more of the components in the cell culture medium is the cumulative concentration of one or more additions after inoculation.

### Claim 10 (depends on 8)

The method of claim 8, wherein the polypeptide is selected from the group consisting of: a CEA-IL2v immunocytokine, a FAP-IL2v immunocytokine, an anti-CEA/anti-CD3 bispecific antibody, an anti-VEGF/anti-angiopoietin bispecific antibody, an anti-Ang2/anti-VEGF bispecific antibody, an anti-05 antibody, and an anti-CD40 antibody.

### Claim 11 (depends on 8)

The method of claim 8, wherein the polypeptide is selected from the group consisting of: a CEA-IL2v immunocytokine, a FAP-IL2v immunocytokine, an anti-CEA/anti-CD3 bispecific antibody, an anti-VEGF/anti-angiopoietin bispecific antibody, an anti-Ang2/anti-VEGF bispecific antibody, an anti-05 antibody, and an anti-CD40 antibody.

### Claim 12 (depends on 1)

The method of claim 1, wherein the method further comprises at least one feed, and wherein the feed medium lacks one or more of the following: iron, riboflavin, pyridoxine, pyridoxal, folic acid, and cyanocobalamin.

### Claim 13 (depends on 12)

The method of claim 12, wherein the feed is a batch feed.

### Claim 14 (depends on 13)

The method of claim 13, wherein the batch feed medium lacks cystine.

### Claim 15 (depends on 13)

The method of claim 13, wherein the batch feed medium lacks cysteine.

### Claim 16 (depends on 13)

The method of claim 13, wherein the batch feed medium lacks methionine.

### Claim 17 (depends on 1)

The method of claim 1, wherein the iron is ferric iron (Fe3+) or ferrous iron (Fe2+).

### Claim 18 (depends on 1)

The method of claim 1, wherein the method further comprises:
(I) supplementing the culture of said host cell with a chelating agent and a reducing agent prior to harvest;
(II) supplementing a pre-harvest cell culture fluid (PHCCF) of said host cell with a chelating agent and a reducing agent; or
(III) supplementing a harvested cell culture fluid (HCCF) of said host cell with a chelating agent and a reducing agent following harvest.

### Claim 19

A method for decreasing level of trisulfide bonds in a polypeptide produced by a host cell comprising:
(i) supplementing a culture of said host cell with a reducing agent and a chelating agent prior to harvest;
(ii) supplementing a pre-harvest cell culture fluid (PHCCF) of said host cell with a chelating agent and a reducing agent; or
(iii) supplementing a harvested cell culture fluid (HCCF) of said host cell with a reducing agent and a chelating agent.

### Claim 20 (depends on 18)

The method of claim 18, wherein the culture, the PHCCF, or the HCCF of said host cell is supplemented with the chelating agent prior to being supplemented with the reducing agent.

### Claim 21 (depends on 20)

The method of claim 20, wherein the culture, the PHCCF, or the HCCF of said host cell is supplemented with the chelating agent between about 60 minutes to about 30 minutes prior to being supplemented with the reducing agent.

### Claim 22 (depends on 18)

The method of claim 18, wherein the chelating agent and the reducing agent are maintained in the culture, the PHCCF, or the HCCF of said host cell for about 30 minutes to about 4 days.

### Claim 23 (depends on 18)

The method of claim 18, wherein the culture, the PHCCF, or the HCCF of said host cell is maintained at a temperature between about 15° C. and about 37° C.

### Claim 24 (depends on 18)

The method of claim 18, wherein the culture, the PHCCF, or the HCCF of said host cell is maintained pH between about 6.5 to about 7.5.

### Claim 25 (depends on 18)

The method of claim 18, wherein the amount of dissolved oxygen (DO) in the culture, the PHCCF, or the HCCF of said host cell is at least about 15%.

### Claim 26 (depends on 18)

The method of claim 18, wherein the culture, the PHCCF, or the HCCF of said host cell is maintained at a temperature between about 15° C. and about 37° C. and at a pH between about 6.5 to about 7.5, and wherein the amount of dissolved oxygen (DO) in the culture or HCCF of said host cell is at least about 15%.

### Claim 27 (depends on 18)

The method of claim 18, wherein the reducing agent is selected from the group consisting of: glutathione (GSH), L-glutathione (L-GSH), cysteine, L-cysteine, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 2,3-tert-butyl-4-hydroxyanisole, 2,6-di-tert-butyl-4-methylphenol, 3-aminopropane-l-sulfonic acid, adenosylhomocysteine, anserine, B-alanine, B-carotene, butylated hydroxyanisole, butylated hydroxytoluene, carnosine, carvedilol, curcumin, cysteamine, cysteamine hydrochloride, dexamethasone, diallyldisulfide, DL-lanthionine, DL-thiorphan, ethoxyquin, gallic acid, gentisic acid sodium salt hydrate, glutathione disulfide, glutathione reduced ethyl ester, glycine, hydrocortisone, hypotaurine, isethionic acid ammonium salt, L-cysteine-glutathione Disulfide, L-cysteinesulfinic acid monohydrate, lipoic acid, reduced lipoic acid, mercaptopropionyl glycine, methionine, methylenebis(3-thiopropionic acid), oxalic acid, quercitrin hydrate, resveratrol, retinoic acid, S-carboxymethyl-L-cysteine, selenium, selenomethionine, silver diethyldithiocarbamate, taurine, thiolactic acid, tricine, vitamin C, vitamin E, vitamin B1, vitamin B2, vitamin B3, vitamin B4, vitamin B5, vitamin B6, and vitamin B11.

### Claim 28 (depends on 27)

The method of claim 27, wherein the reducing agent is selected from the group consisting of: cysteine and L-cysteine.

### Claim 29 (depends on 28)

The method of claim 28, wherein the reducing agent is L-cysteine, and wherein the L-cysteine is added to the culture or HCCF of said host cell to achieve a final concentration between about 3 mM and about 6 mM.

### Claim 30 (depends on 18)

The method of claim 18, wherein the chelating agent is selected from the group consisting of: ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), ethylenediamine-N,N′-disuccinic acid (EDDS), citrate, oxalate, tartrate, ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), 5-sulfosalicylic acid, N,N-dimethyldodecylamine N-oxide, dithiooxamide, ethylenediamine, salicylaldoxime, N-(2′-hydroxyethyl)iminodiacetic acid (HIMDA), oxine quinolinol, and sulphoxine.

### Claim 31 (depends on 30)

The method of claim 30, wherein the chelating agent is selected from the group consisting of: ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), ethylenediamine-N,N′-disuccinic acid (EDDS), and citrate.

### Claim 32 (depends on 31)

The method of claim 31, wherein the chelating agent is added to the culture or the HCCF of said host cell to achieve a final concentration of 20 mM.

### Claim 33 (depends on 1)

The method of claim 1, wherein the polypeptide is secreted into the cell culture medium.

### Claim 34 (depends on 1)

The method of claim 1, further comprising a step of purifying the harvested polypeptide.

### Claim 35 (depends on 1)

The method of claim 1, wherein the host cell is a recombinant host cell.

### Claim 36 (depends on 1)

The method of claim 1, wherein the host cell is a mammalian cell.

### Claim 37 (depends on 36)

The method of claim 36, wherein the mammalian cell is a CHO cell.

### Claim 38 (depends on 1)

The method of claim 1, wherein the method further comprises measuring the level of trisulfide bonds in the polypeptide.

### Claim 39 (depends on 1)

The method of claim 1, wherein the average % trisulfide bonds in the polypeptide is less than about 20%, less than about 10% less than about 5%, less than about 1%, less than about 0.5%, or less than about 0.1%.

### Claim 40 (depends on 1)

The method of claim 1, wherein the polypeptide is an antibody or fragment thereof.

### Claim 41 (depends on 40)

The method of claim 40, wherein the polypeptide is an antibody fragment, and wherein the antibody fragment is selected from the group consisting of: a Fab, a Fab′, an F(ab′)2, an scFv, an (scFv)2, a dAb, a complementarity determining region (CDR) fragment, a linear antibody, a single-chain antibody molecule, a minibody , a diabody, and multispecific antibody formed from antibody fragments.

### Claim 42 (depends on 40)

The method of claim 40, wherein the antibody or fragment thereof binds to an antigen selected from the group consisting of: BMPR1B, E16, STEAP1, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, HER2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, PMEL17, TMEFF1, GDNF-Ra1, Ly6E, TMEM46, Ly6G6D, LGR5, RET, LY6K, GPR19, GPR54, ASPHD1, Tyrosinase, TMEM118, GPR172A, CD33, CLL-1, C5, OX40, α4β7 and αEβ7 integrin heterodimers, IL-13, CD-20, FGFR, influenza A, influenza B, amyloid beta, HER3, complement factor D, IL-22c, PD-L1, PD-L2, PD-1, VEGF, Angiopoietin 2, CD3, FAP, CEA, and IL-6.

### Claim 43 (depends on 40)

The method of claim 40, wherein the polypeptide is an antibody, and wherein the antibody is a bispecific antibody.

### Claim 44 (depends on 43)

The method of claim 43, wherein the bispecific antibody is an anti-VEGF/anti-angiopoietin bispecific antibody, an anti-CEA/anti-CD3 bispecific antibody, or an anti-Ang2/anti-VEGF bispecific antibody.

### Claim 45 (depends on 1)

The method of claim 1, wherein the polypeptide is an immunocytokine.

### Claim 46 (depends on 45)

The method of claim 45, wherein the immunocytokine is CEA-IL2v or FAP-IL2v.

### Claim 47

Use of between about 0 and about 4.5 μM methionine in a cell culture medium for decreasing trisulfide bond levels in a polypeptide selected from the group consisting of: a CEA-IL2v immuocytokine, a FAP-IL2v immunocytokine, an anti-CEA/anti-CD3 bispecific antibody, an anti-VEGF/anti-angiopoietin bispecific antibody, an anti-Ang2/anti-VEGF bispecific antibody, an anti-C5 antibody, and an anti-CD40 antibody.

### Claim 48 (depends on 1)

A polypeptide produced according to claim 1.

### Claim 49 (depends on 48)

The polypeptide of claim 48, wherein the average % trisulfide in the polypeptide is less than about 20%, less than about 10% less than about 5%, less than about 1%, less than about 0.5%, or less than about 0.1%.