# US 12,600,748 B2 · Affinity chromatography of immunoglobulins by using pre-capture flocculation

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- Number: US 12,600,748 B2
- Document ID: US12600748B2
- Status: Granted
- Kind: B2
- Application number: 17/601,197
- Filed: 2020-03-25
- Published: 2022-06-23
- Granted: 2026-04-14
- Earliest claimed: 2019-04-04
- Assignees: Richter Gedeon Nyrt.
- Applicants: Richter Gedeon Nyrt.
- Inventors: Zsuzsanna Keszey; Zoltán Sütö
- Web page: https://patentgrep.com/patent/US12600748B2

## CPC

- C07K 1/22: Affinity chromatography or related techniques based upon selective absorption processes
- B01D 15/12: relating to the preparation of the feed
- B01D 15/3809: {of the antigen-antibody type, e.g. protein A, G or L chromatography}
- C07K 1/18: Ion-exchange chromatography
- C07K 1/34: by filtration, ultrafiltration or reverse osmosis
- C07K 1/36: by a combination of two or more processes of different types
- C07K 16/065: {Purification, fragmentation}
- C07K 16/22: against growth factors {; against growth regulators}
- C07K 16/32: against translation products of oncogenes
- 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)
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)

## Abstract

The present invention relates to new methods for the purification of immunoglobulins from cell culture harvest. These methods are characterized by cleaning steps preceding an initial capture chromatography step. Preferably, the initial capture chromatography is an affinity chromatography and the pre-capture cleaning effect is obtained by flocculation and filtration. By using such pre-capture cleaning steps, an improved quality of the eluted immunoglobulin from the capture matrix is achieved. Furthermore, the enhanced clarification of the cell culture liquid results in reduced precipitations during affinity chromatography and thus increases the lifetimes of the expensive affinity resin. This is an important improvement for immunoglobulin purification in large scale production. The present invention further relates to sequential polishing chromatographies and filtration steps downstream to the capture chromatography, yielding immunoglobulins of high purity.

## Claims

### Claim 1

A method for prevention of precipitations in the course of a capture affinity chromatography of an immunoglobulin from a cell culture fluid, the method comprising the following steps in the following order:
(a) adding a flocculation-inducing compound to the cell culture fluid;
(b) depth filtration of the mixture of step (a);
(c) exposing the filtrate obtained in step (b) to affinity chromatography, wherein the immunoglobulin is bound to the affinity chromatography medium;
(d) washing the affinity chromatography medium by adding a washing buffer with a pH value of 5 to 9 and a ionic strength of 0.1 to 5.0 mol/L to the affinity chromatography medium of step (c); and
(e) eluting the immunoglobulin from the affinity chromatography resin by adding an elution buffer with a pH value of 2.5 to 4.5 to the affinity chromatography medium, wherein the flocculation-inducing compound is poly (diallyldimethylammonium chloride) and wherein except the poly (diallyldimethylammonium chloride) no further substances are added for performing the flocculation, wherein the method is carried out at large scale, in which the immunoglobulin eluted from the column of the capture step amounts to more than 50 g.

### Claim 2 (depends on 1)

The method of claim 1 wherein step (a) comprises the following steps:
(a1) centrifugation and/or filtration of the cell culture fluid; and
(a2) adding a flocculation-inducing compound to the supernatant or filtrate obtained in step (a1).

### Claim 3 (depends on 1)

The method of claim 1, wherein the poly (diallyldimethylammonium chloride) has a molecular mass of 10 kDa to 10 000 kDa.

### Claim 4 (depends on 1)

The method of claim 1, wherein the poly (diallyldimethylammonium chloride) is added in a final concentration of 0.01 to 1.0% (w/v).

### Claim 5 (depends on 1)

The method of claim 1, wherein the flocculation with poly (diallyldimethylammonium chloride) is performed without further adjustment of pH or conductivity.

### Claim 6 (depends on 1)

The method of claim 1, wherein the affinity chromatography is a Protein A chromatography.

### Claim 7 (depends on 1)

The method of claim 1, wherein between step (b) and (c) microfiltration is carried out.

### Claim 8 (depends on 1)

The method of claim 1, wherein step (c) is carried out at most 8 h after step (b).

### Claim 9 (depends on 1)

The method of claim 1, wherein the immunoglobulin is IgG1 or IgG2.

### Claim 10 (depends on 1)

A method for purifying an immunoglobulin, comprising the steps as defined in claim 1 and comprising one or more further steps following the affinity chromatography wherein the one or more further steps are selected from the group consisting of virus inactivation, anion exchange chromatography, cation exchange chromatography, mixed-mode chromatography, nanofiltration, and ultrafiltration/diafiltration.

### Claim 11 (depends on 10)

The method according to claim 10, wherein the one or more further steps comprise: (f) incubating the eluate of step (e) for virus inactivation at a low pH value of 2.5 to 4.5, for at least 10 minutes.

### Claim 12 (depends on 10)

The method according to claim 10, wherein the one or more further steps comprise:
(f) incubating the eluate of step (e) for virus inactivation at a low pH value of 2.5 to 4.5, for at least 10 minutes;
(g) performing a cation exchange chromatography;
(h) performing a mixed-mode chromatography or an anion exchange chromatography;
(i) exposing the eluate of step (h) to nanofiltration, optionally wherein the eluate of step (h) is exposed to one or more further processing steps prior to nanofiltration; and
(j) exposing the filtrate of step (i) to ultrafiltration/diafiltration, optionally wherein the filtrate of step (i) is exposed to one or more further processing steps prior to the ultrafiltration/diafiltration.

### Claim 13 (depends on 12)

The method according to claim 12, wherein the cation exchange chromatography (g) is performed in a bind-elute mode, optionally including one or more washing steps.

### Claim 14 (depends on 12)

The method according to claim 12, wherein the mixed mode chromatography in step (h) is performed in a bind-elute mode, optionally including one or more washing steps.

### Claim 15 (depends on 12)

The method according to claim 12, wherein the chromatography (h) is performed in flow through mode.

### Claim 16 (depends on 12)

The method according to claim 12, wherein the cation exchange chromatography of step (g) is performed with sulfopropyl (—CH2CH2CH2SO3—) as a ligand.

### Claim 17 (depends on 12)

The method according claim 12, wherein the mixed-mode chromatography medium of step (h) is performed with a positively charged mixed-mode medium.

### Claim 18 (depends on 2)

The method of claim 2, wherein the flocculation-inducing compound is a cationic compound selected from the group consisting of a divalent metal ion salt, a water-soluble organic polymer, and a water-insoluble organic polymer.

### Claim 19 (depends on 18)

The method of claim 18, wherein the water-soluble organic polymer is poly (diallyldimethylammonium chloride).

### Claim 20 (depends on 19)

The method of claim 19, wherein the poly (diallyldimethylammonium chloride) has a molecular mass of 10 kDa to 10 000 kDa.

### Claim 21 (depends on 19)

The method of claim 19, wherein the poly (diallyldimethylammonium chloride) is added in a final concentration of 0.01 to 1.0% (w/v).

### Claim 22 (depends on 19)

The method of claim 19, wherein except the poly (diallyldimethylammonium chloride) no further substances are added for performing the flocculation.

### Claim 23 (depends on 19)

The method of claim 19, wherein the flocculation with poly (diallyldimethylammonium chloride) is performed without further adjustment of pH or conductivity.

### Claim 24 (depends on 8)

The method of claim 8, wherein step (c) is carried out at most 3 h after step (b).

### Claim 25 (depends on 12)

The method according to claim 12, wherein the cation exchange chromatography of step (g) is performed with a medium comprising sulfopropyl (—CH2CH2CH2SO3—) as a ligand.

### Claim 26 (depends on 12)

The method according to claim 12, wherein the mixed-mode chromatography medium of step (h) is performed with a positively charged mixed-mode medium, comprising N-benzyl-N-methyl ethanolamine as a ligand, bound to a cross-linked agarose matrix.