BE1029863B1 - METHODS FOR PRODUCING IMMUNOGLOBULIN G (IgG) PREPARATIONS AND/OR SOLUTIONS - Google Patents

Abstract

The present invention relates to a method of producing immunoglobulin G (IgG) preparations or solutions comprising, inter alia, the steps of - adding caprylic acid to a first IgG suspension after which the pH of the first IgG suspension is adjusted to 4.60 to 5.00 amounts; - filtering the IgG supernatant; - preparing an IgG solution with a protein concentration of at least about 40.0 g/L and a pH of at least 6.30 to at most 6.50; - purifying the IgG solution obtained in step h) via anion exchange chromatography. Furthermore, the invention also provides an IgG preparation, preferably comprising: immunoglobulin G (IgG); - about 8.4% by weight of sucrose; - about 0.6% by weight of glycine; - about 0.3% by weight of sodium chloride; - acetate buffer; wherein the pH of the composition is about 4.40.

Description

1 BE2022/5354

METHODS OF PRODUCING IMMUNOGLOBULIN G (IgG) PREPARATIONS AND/OR

SOLUTIONS

FIELD OF THE INVENTION

Described herein are methods for producing immunoglobulin G (IgG) preparations and/or immunoglobulin G (IgG) solutions. The methods described can be used to purify IgG and/or up-concentrate IgG. Furthermore, IgG solutions and IgG preparations are also described herein.

BACKGROUND OF THE INVENTION

The use of IgG solutions and preparations in the treatment of various diseases is well known.

IgG solutions and preparations often have to be administered intravenously for this purpose. This in turn leads to a demand for extremely pure IgG solutions and preparations.

Traditionally, IgG is extracted from donor plasma by precipitating it into a paste, after which it must undergo a series of purification steps before it can be used to treat diseases and disorders. However, because the purification is so complex and involves many steps, the final yield (efficiency) is low. Thus, there is a demand for higher yield methods of IgG solutions and preparations. A demand for more efficient purification processes. Because this is a product that originates from donated plasma, even a small increase in yield is already a significant benefit.

There is also a demand for semi-finished products, i.e. IgG solutions or preparations that have to undergo further production steps or purification steps. These further production steps or purification steps are often carried out at a different location or at a different time. To prevent the IgG in the semi-finished products from destabilizing, precipitating or denaturalizing, these semi-finished products are often frozen. However, undergoing several freeze/thaw cycles does not benefit the quality and amount of IgG. And it also requires extra actions and equipment. Therefore, there is a demand for stable, preferably liquid, IgG preparations and solutions, which can either be stored and/or transported.

There is also a demand for IgG preparations and solutions that can be used in the above-mentioned treatment methods across a broad segment of the population. Preferably, the IgG preparations do not cause hemolysis.

2 BE2022/5354

SUMMARY OF THE INVENTION

The present invention provides a solution to one or more of the challenges and/or questions mentioned above. The inventors have found that the use of caprylic acid to precipitate impurities in the methods of preparing IgG solutions can provide higher efficiency. It allows the inventors to filter out impurities at a pH at which the IgG remains stable in solution, and thus less IgG precipitates and/or denatures. As a result, the pH only needs to be adjusted to a more critical value just before performing chromatography, but because many impurities have already been removed by then, more IgG can also be kept in suspension at this critical pH value.

In a first aspect, the invention provides a method of producing immunoglobulin

G (IgG) preparations or immunoglobulin G (IgG) solutions, comprising the steps of: a) providing an IgG-containing paste; b} resuspending the paste in a first buffer to form a first IgG suspension; c) adding caprylic acid to the first IgG suspension after which the pH of the first IgG suspension is at least 4.60 to at most 5.00, thereby obtaining a precipitate and an IgG supernatant; d) filtering the IgG supernatant obtained in step c) through a filter, thereby obtaining an IgG filtrate; e) optionally, concentrating the IgG filtrate obtained in step d), preferably over a first semipermeable membrane and preferably by means of a first ultrafiltration, thereby obtaining a first concentrated IgG solution; f) optionally, exchanging the buffer from the first concentrated IgG solution obtained in step d), preferably by diafiltration, against a second buffer at a pH of at least 4.60 to at most 5.00, thereby obtaining a second

IgG solution; g) optionally, concentrating the second IgG solution obtained in step f), preferably over the second semipermeable membrane and preferably by means of a second ultrafiltration, thereby obtaining a third concentrated IgG solution; h} diluting: - the IgG filtrate, if step e), step f) and step g) have not been carried out;

3 BE2022/5354 - the first concentrated IgG solution, when step e) has been performed, and steps f) and g) have not been performed; - the second IgG solution, when steps e) and f) have been performed, and step g) has not been performed; or, - the third concentrated IgG solution; when steps e), f) and g) have been performed; by adding water and/or a fourth buffer to obtain a fourth IgG solution with: - a protein concentration of at least 10.0 g/L to at most 70.0 g/L, preferably at least 15.0 g/L L to at most 65.0 g/L, preferably at least 20.0 g/L to at most 60.0 g/L, preferably at least 25.0 g/L to at most 55.0 g/L, preferably at least 30 .0 g/L to at most 50.0 g/L, preferably at least 35.0 g/L to at most 45.0 g/L, preferably at least 37.0 g/L to at most 43.0 g/L, preferably at least 38.0 g/L to at most 42.0 g/L, preferably at least 39.0 g/L to at most 41.0 g/L, preferably 40.0 g/L; and, - a pH of at least 5.80 to at most 7.00, preferably at least 5.90 at most 6.90, preferably at least 6.00 at most 6.80, preferably at least 6.10 at most 6, 70, preferably at least 6.20 to at most 6.60, preferably at least 6.30 to at most 6.50; ij) purifying the fourth IgG solution obtained in step h) via anion exchange chromatography, thereby obtaining first flow-through of IgG; j) optionally, the purification of the first IgG-containing flow-through obtained is step i) via affinity chromatography, thereby obtaining a second IgG-containing flow-through.

In one embodiment, the method further comprises: k) adjusting the pH of the first IgG-containing flow-through obtained in step i} or of the second IgG-containing flow-through when step j) has been performed, thereby obtaining a fifth IgG solution with a pH of at least 4.00 to at most 5.00, preferably at least 4.10 to at most 4.80, at least 4.20 to at most 4.70, preferably at least 4.30 to at most 4.50, preferably 4.40; and,

I) optionally, concentrating the fifth IgG solution obtained in step k), preferably over the third semipermeable membrane and preferably by means of a third ultrafiltration, thereby obtaining a sixth concentrated IgG solution.

4 BE2022/5354

In an embodiment, the method further comprises: m) stabilizing the obtained IgG solution or IgG flow-through, by adding one or more excipients and/or adjusting the pH, thereby obtaining a stable IgG preparation.

In one embodiment, the one or more excipient in step m) is selected from the list of sucrose, glycine and/or sodium chloride.

In one embodiment, the fourth IgG solution is: - a protein concentration of at least 37.0 g/L to at most 43.0 g/L, preferably at least 38.0 g/L to at most 42.0 g/L, at preferably has at least 39.0 g/L to at most 41.0 g/L, preferably 40.0 g/L; and, - has a pH of at least 6.30 to at most 6.50.

In one embodiment, the first semipermeable membrane, the second semipermeable membrane and/or the third semipermeable membrane is the same membrane. In one embodiment, the first semipermeable membrane and the second semipermeable membrane are the same semipermeable membrane. Preferably, the same semipermeable membrane is reused in the different process steps.

In one embodiment, the first semipermeable membrane, second semipermeable membrane and/or third semipermeable membrane allow molecules with a molecular weight of less than 5 kDa, preferably less than 10 kDa, preferably less than 15 kDa, preferably less than 20 kDa, to pass through. preferably less than 25 kDa, preferably less than 30 kDa, preferably less than 40 kDa, preferably less than 50 kDa by.

In one embodiment, the paste comprises fractions II+III, preferably fractions I+II+III obtained from the Cohn process.

In one embodiment, step c) comprises, after adding the caprylic acid, a maturation step of at least min, preferably at least 60 min, preferably at least 90 min, preferably at least 120 min, preferably at least 150 min, preferably at least 120 min.

In one embodiment, the anion exchange chromatography in step i} uses a strongly basic anion exchange resin.

In one embodiment, the purification via affinity chromatography uses a medium or a mixture of media, on which N-acetyl-galactosamine (GalNAc) - Galactose (Gal) - Fucose (Fuc), and Galactose-Galactose-Fucose groups are provided.

In a second aspect, the invention provides an IgG preparation, preferably an intravenous IgG preparation, obtained from a method according to at least one of the preceding claims.

In one embodiment, the IgG preparation comprises: - immunoglobulin G (IgG}; - at least 7.0 wt.% to at most 10.0 wt.%, preferably at least 8.0 wt.% to at most 9.0 wt.%, preferably at least 8.2 wt.% to at most 8.6 wt.%, preferably 8.4 wt.% sucrose - at least 0.1 wt.% to at most 1.2 wt.%, preferably at least 0.3 wt.% to at most 1.0 wt.%, preferably at least 0.5 wt.% to at most 0.8 wt.%, preferably 0.6 wt.% glycine, - at least 0.1 wt.% to at most 0.6 wt.%, preferably at least 0.3 wt.% to at most 0.4 wt.%, preferably 0.3 wt.% sodium chloride; acetate buffer, wherein the pH of the preparation is at least 4.00 to at most 5.00, at least 4.20 to at most 4.70, preferably at least 4.30 to at most 4.50, preferably 4.40.

DETAILED DESCRIPTION OF THE INVENTION

While potentially serving as a guide to understanding, any reference sign in the claims shall not be construed as limiting its scope.

As used herein, the singular forms "a," "an," and "the" include both singular and plural unless the context clearly dictates otherwise.

The terms "including", "includes" and "comprising" as used herein are synonymous with "including", "contains" or "include", "includes", and are inclusive or open-ended and exclude additional, non- - do not perform the said members, elements or method steps. The terms "comprising", "includes" and "comprising" when referring to said components, elements or method steps also include embodiments which "consist of" said recited components, elements or method steps.

6 BE2022/5354

In addition, the terms first, second, third and the like are used in the description and in the claims to distinguish between like elements and not necessarily to describe a sequential or chronological order, unless otherwise specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments described herein may operate in a different order than described or illustrated herein.

The values, as used herein to refer to a measurable value, such as a parameter, an amount, a time period, and the like, are intended to include variations of +/-10% or less, preferably +/-5% or less. less, more preferably +/-1% or less, and more preferably +/-0.1% or less of the declared value, to the extent that such variations are appropriate to one or more of the technical effects contemplated herein , to assure. It will be understood that each value as used herein is itself specifically and preferably disclosed.

Listing numerical ranges by endpoints includes all numbers and fractions that are within the respective ranges, as well as the listed endpoints.

All documents cited in the present disclosure are hereby incorporated by reference in their entirety.

Unless defined otherwise, all terms used in the concepts described herein, including technical and scientific terms, have the meanings commonly understood by one of ordinary skill in the art. For further guidance, definitions for the terms used have been included in the description to better understand the teachings of the present description. The terms and definitions used herein are intended solely to aid in the understanding of the teachings provided herein.

The term "caprylic acid" as used herein refers to a saturated carboxylic acid of molecular formula

CsH:1SO2, and can be represented by structural formula (1): a A0

OH (1) “Octanoic acid” is used synonymously with caprylic acid. Caprylic acid has 124-07-2 as CAS number.

Reference in this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or property described in connection with the embodiment is included in at least

7 BE2022/5354 one embodiment contemplated herein. Thus, the use of the terms "in one embodiment" or "in one embodiment" in different places in this description does not necessarily all refer to the same embodiment, but it may. Further, the specific features, structures or properties may be combined in any suitable manner, as would be apparent to one skilled in the art from this disclosure, in one or more embodiments. In addition, while some embodiments described herein include some but not other features included in other embodiments, combinations of features from different embodiments are also contemplated herein, and constitute different embodiments, as will be apparent to those skilled in the art. For example, in the appended claims, any of the features of the claimed embodiments may be used in any combination.

Preferred positions (features), embodiments and uses of the invention are described herein. Each statement and embodiment of the invention described herein may be combined with any other statement and/or embodiment, unless indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features or propositions indicated as being preferred or advantageous. To this end, the present invention is specifically defined by one or a combination of one or more of the theorems and embodiments numbered below, with any other aspect and/or embodiments. A method for producing immunoglobulin G (IgG) preparations or immunoglobulin

G (IgG) solutions, comprising the steps of: a) providing an IgG-containing paste; b} resuspending the paste in a first buffer to form a first IgG suspension; c) adding caprylic acid to the first IgG suspension after which the pH of the first IgG suspension is at least 4.60 to at most 5.00, thereby obtaining a precipitate and an IgG supernatant; d) filtering the IgG supernatant obtained in step c) through a filter, thereby obtaining an IgG filtrate; e) optionally, concentrating the IgG filtrate obtained in step d), preferably over a first semipermeable membrane and preferably by means of a first ultrafiltration, thereby obtaining a first concentrated IgG solution;

8 BE2022/5354 f) optionally, exchanging the buffer from the first concentrated IgG solution obtained in step d), preferably by means of diafiltration, with a second buffer at a pH of at least 4.60 to at most 5.00 , thereby obtaining a second

IgG solution; g) optionally, concentrating the second IgG solution obtained in step f), preferably over the second semipermeable membrane and preferably by means of a second ultrafiltration, thereby obtaining a third concentrated IgG solution; h} diluting: - the IgG filtrate, if step e), step f) and step g) have not been carried out; - the first concentrated IgG solution, if step e) has been carried out and steps f) and g) have not been carried out; - the second IgG solution, when steps e) and f) have been performed, and step g) has not been performed; or, - the third concentrated IgG solution; when steps e), f) and g) have been performed; by adding water and/or a fourth buffer to obtain a fourth IgG solution with: - a protein concentration of at least 10.0 g/L to at most 70.0 g/L, preferably at least 15.0 g/L L to at most 65.0 g/L, preferably at least 20.0 g/L to at most 60.0 g/L, preferably at least 25.0 g/L to at most 55.0 g/L, preferably at least 30 .0 g/L to at most 50.0 g/L, preferably at least 35.0 g/L to at most 45.0 g/L, preferably at least 37.0 g/L to at most 43.0 g/L, preferably at least 38.0 g/L to at most 42.0 g/L, preferably at least 39.0 g/L to at most 41.0 g/L, preferably about 40.0 g/L; and, - a pH of at least 5.80 to at most 7.00, preferably at least 5.90 at most 6.90, preferably at least 6.00 at most 6.80, preferably at least 6.10 at most 6, 70, preferably at least 6.20 to at most 6.60, preferably at least 6.30 to at most 6.50; ij) purifying the fourth IgG solution obtained in step h) via anion exchange chromatography, thereby obtaining first flow-through of IgG; j) optionally, the purification of the first IgG-containing flow-through obtained is step i) via affinity chromatography, thereby obtaining a second IgG-containing flow-through. 2. The method according to any one of the preceding claims, further comprising:

9 BE2022/5354 k) adjusting the pH of the first IgG-containing flow-through obtained in step i} or of the second IgG-containing flow-through when step j) has been performed, thereby obtaining a fifth IgG solution with a pH of at least 4.00 to at most 5.00, preferably at least 4.10 to at most 4.80, at least 4.20 to at most 4.70, preferably at least 4.30 to at most 4.50, preferably about 4.40; and,

I) optionally, concentrating the fifth IgG solution obtained in step k), preferably over the third semipermeable membrane and preferably by means of a third ultrafiltration, thereby obtaining a sixth concentrated IgG solution. 3. The method according to any one of the preceding claims, further comprising: - stabilizing the obtained IgG solution or IgG flow-through, by adding one or more excipients and/or adjusting the pH, thereby obtaining a stable IgG preparation. The method according to any one of the preceding claims, comprising the steps of: a) providing an IgG-containing paste; b} resuspending the paste in a first buffer to form a first IgG suspension; c) adding caprylic acid to the first IgG suspension after which the pH of the first IgG suspension is at least 4.60 to at most 5.00, thereby obtaining a precipitate and an IgG supernatant; d) filtering the IgG supernatant obtained in step c) through a filter, thereby obtaining an IgG filtrate; e) optionally, concentrating the IgG filtrate obtained in step d), preferably over a first semipermeable membrane and preferably by means of a first ultrafiltration, thereby obtaining a first concentrated IgG solution; f) optionally, exchanging the buffer from the first concentrated IgG solution obtained in step d), preferably by diafiltration, against a second buffer at a pH of at least 4.60 to at most 5.00, thereby obtaining a second

IgG solution; g) optionally, concentrating the second IgG solution obtained in step f), preferably over the second semipermeable membrane and preferably by means of a second ultrafiltration, thereby obtaining a third concentrated IgG solution; h} diluting: - the IgG filtrate, if step e), step f) and step g) have not been carried out;

BE2022/5354 - the first concentrated IgG solution, when step e) has been performed, and steps f) and g) have not been performed; - the second IgG solution, when steps e) and f) have been performed, and step g) has not been performed; or, - the third concentrated IgG solution; when steps e), f) and g) have been performed; by adding water and/or a fourth buffer to obtain a fourth IgG solution with: - a protein concentration of at least 10.0 g/L to at most 70.0 g/L, preferably at least 15.0 g/L L to at most 65.0 g/L, preferably at least 20.0 g/L to at most 60.0 g/L, preferably at least 25.0 g/L to at most 55.0 g/L, preferably at least 30 .0 g/L to at most 50.0 g/L, preferably at least 35.0 g/L to at most 45.0 g/L, preferably at least 37.0 g/L to at most 43.0 g/L, preferably at least 38.0 g/L to at most 42.0 g/L, preferably at least 39.0 g/L to at most 41.0 g/L, preferably about 40.0 g/L; and, - a pH of at least 5.80 to at most 7.00, preferably at least 5.90 at most 6.90, preferably at least 6.00 at most 6.80, preferably at least 6.10 at most 6, 70, preferably at least 6.20 to at most 6.60, preferably at least 6.30 to at most 6.50; ij) purifying the fourth IgG solution obtained in step h) via anion exchange chromatography, thereby obtaining first flow-through of IgG; j) optionally, the purification of the first IgG-containing flow-through obtained is step i) via affinity chromatography, thereby obtaining a second IgG-containing flow-through; k) adjusting the pH of the first IgG-containing flow-through obtained in step i} or of the second IgG-containing flow-through when step j) has been performed, thereby obtaining a fifth IgG solution with a pH of at least 4.00 to at most 5, 00, preferably at least 4.10 to at most 4.80, at least 4.20 to at most 4.70, preferably at least 4.30 to at most 4.50, preferably about 4.40;

I) optionally, concentrating the fifth IgG solution obtained in step k), preferably over the third semipermeable membrane and preferably by means of a third ultrafiltration, thereby obtaining a sixth concentrated IgG solution; and,

11 BE2022/5354 m) optionally, stabilizing the fifth IgG solution or the sixth concentrated IgG solution when step |) has been performed, by adding one or more excipients and/or adjusting the pH, thereby obtaining a stable IgG preparation.

The method according to any one of the preceding claims, comprising the steps of: a) providing an IgG-containing paste;

b} resuspending the paste in a first buffer to form a first IgG suspension;

c) adding caprylic acid to the first IgG suspension after which the pH of the first IgG suspension is at least 4.60 to at most 5.00, thereby obtaining a precipitate and an IgG supernatant;

d) filtering the IgG supernatant obtained in step c) through a filter, thereby obtaining an IgG filtrate;

e) concentrating the IgG filtrate obtained in step d), preferably over a first semipermeable membrane and preferably by means of a first ultrafiltration, thereby obtaining a first concentrated IgG solution;

f) exchanging the buffer from the first concentrated IgG solution obtained in step d), preferably by means of diafiltration, with a second buffer at a pH of at least 4.60 to at most 5.00, thereby obtaining a second IgG solution;

g) concentrating the second IgG solution obtained in step f), preferably over the second semipermeable membrane and preferably by means of a second ultrafiltration,

thereby obtaining a third concentrated IgG solution;

h} diluting the third concentrated IgG solution obtained in step g) by adding water and/or a fourth buffer to obtain a fourth IgG solution with:

- a protein concentration of at least 10.0 g/L to at most 70.0 g/L, preferably at least 15.0 g/L to at most 65.0 g/L, preferably at least 20.0 g/L to at most

60.0 g/L, preferably at least 25.0 g/L to at most 55.0 g/L, preferably at least 30.0 g/L to at most 50.0 g/L, preferably at least 35.0 g /L to at most 45.0 g/L, preferably at least 37.0 g/L to at most 43.0 g/L, preferably at least 38.0 g/L to at most 42.0 g/L, preferably at least 39.0 g/L to at most 41.0 g/L, preferably about 40.0 g/L; and,

- a pH of at least 5.80 to at most 7.00, preferably at least 5.90 to at most

6.90, preferably at least 6.00 to at most 6.80, preferably at least 6.10 to

12 BE2022/5354 at most 6.70, preferably at least 6.20 at most 6.60, preferably at least 6.30 at most 6.50; ij) purifying the fourth IgG solution obtained in step h) via anion exchange chromatography, thereby obtaining first flow-through of IgG; j) the purification of the first IgG-containing flow-through obtained is step i) via affinity chromatography, thereby obtaining a second IgG-containing flow-through; k) adjusting the pH of the second IgG flow-through obtained in step j}, thereby obtaining a fifth IgG solution with a pH of at least 4.00 to at most 5.00, preferably at least 4.10 to at most 4.80, preferably at least 4.20 to at most 4.70, preferably at least 4.30 to at most 4.50, preferably about 4.40;

I) concentrating the fifth IgG solution obtained in step k), preferably across the third semipermeable membrane and preferably by means of a third ultrafiltration, thereby obtaining a sixth concentrated IgG solution; and, m) stabilizing the sixth concentrated IgG solution obtained in step |), by adding one or more excipients and/or adjusting the pH, thereby obtaining a stable IgG preparation. 6. The method according to any one of the preceding statements, wherein the fourth IgG solution: - has a protein concentration of at least 37.0 g/L to at most 43.0 g/L, preferably at least 38.0 g/L to at most 42 .0 g/L, preferably at least 39.0 g/L to at most 41.0 g/L, preferably about 40.0 g/L; and, - has a pH of at least 6.30 to at most 6.50. 7. The method according to any of the preceding statements, wherein: - the fourth IgG solution: o a protein concentration of at least 37.0 g/L to at most 43.0 g/L, preferably at least 38.0 g/L to at most 42.0 g/L, preferably at least 39.0 g/L to at most 41.0 g/L, preferably about 40.0 g/L; and, o has a pH of at least 6.30 to at most 6.50; and, - the fifth IgG solution has a pH of at least 4.20 to at most 4.70. 8. The method according to any one of the preceding statements, wherein: - the fourth IgG solution:

13 BE2022/5354 o a protein concentration of at least 37.0 g/L to at most 43.0 g/L, preferably at least 38.0 g/L to at most 42.0 g/L, preferably at least 39.0 g/L up to 41.0 g/L, preferably about 40.0 g/L; and, o has a pH of at least 6.30 to at most 6.50; - the fifth IgG solution has a pH of at least 4.20 to at most 4.70; and, - the IgG preparation obtained in step m) has a pH of at least 4.20 to at most 4.70. 9. The method according to any one of the preceding claims, wherein the first semipermeable membrane, the second semipermeable membrane and/or the third semipermeable membrane is the same membrane. 10. The method according to any one of the preceding statements, wherein the one or more excipient in step m) is selected from the list of sucrose, glycine and/or sodium chloride. 11. The method according to any one of the preceding claims, wherein the IgG-containing paste was obtained from plasma; preferably human plasma. 12. The method according to at least one of the preceding claims, wherein the paste comprises fractions III, preferably fractions I+II+III obtained from the Cohn process. 13. The method according to any one of the preceding claims, wherein the IgG preparation is an intravenous

IgG preparation. 14. The method according to at least one of the preceding statements, wherein the fractions I, II and/or Ill are obtained during the Cohn process by plasma precipitation, starting at an ethanol gradient from 8% ethanol to 25% ethanol, combined with a temperature gradient over the same period of the ethanol gradient starting from -3°C to -5°C. 15. The method according to at least one of the preceding claims, wherein the IgG is human IgG. 16. The method according to at least one of the preceding claims, wherein the first buffer is an acetate buffer. 17. The method according to at least one of the preceding claims, wherein the first buffer has a pH of at least 4.0 to at most 5.6; preferably at least 4.2 to at most 5.4; preferably at least 4.4 to at most 5.2; preferably at least 4.6 to at most 5.0; preferably at least 4.7 to at most 5.9; preferably about 4.8.

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18. The method according to at least one of the preceding statements, wherein the first buffer has a pH of at least 3.50 to at most 6.00, preferably at least 3.80 to at most 5.80, preferably at least 4.00 to at most 5 .60, preferably at least 4.20 to at most 5.40, preferably at least 4.40 to at most 5.20, preferably at least 4.60 to at most 5.00, preferably at least 4.70 to at most 4.90 , preferably about 4.80.

19. The method according to at least one of the preceding statements, wherein the first buffer has a conductivity of at least 2.10 mS/cm to at most 4.30 mS/cm, preferably at least 2.40 mS/cm to at most 4.10 mS /cm, preferably at least 2.60 mS/cm to at most 3.90 mS/cm, preferably at least 2.80 mS/cm at most 3.70 mS/cm, preferably at least 3.00 mS/cm at most 3.50 mS/cm, preferably at least 3.10 mS/cm to at most 3.30 mS/cm, preferably at least 3.20 mS/cm to at most 3.40 mS/cm, preferably about 3.30 mS /cm.

20. The method according to at least one of the preceding claims, wherein the first buffer has a concentration of at least 25.0 mM to at most 150.0 mM, preferably at least 50.0 mM to at most 125.0 mM; at least 60.0 mM to at most 100.0 mM; at least 70.0 mM to at most 90.0 mM; at least 75.0 mM to at most 85.0 mM, preferably about 80.0 mM acetate buffer.

21. The method according to at least one of the preceding statements, wherein when resuspending in step b) at least 2.0 kg of first buffer/kg of paste, preferably at least 4.0 kg of buffer/kg of paste, preferably at least 6.0 kg of buffer /kg paste, preferably at least 8.0 kg buffer/kg paste is added.

22. The method according to at least one of the preceding statements, wherein the conductivity of the first IgG suspension formed in step b) is at least 2.10 mS/cm to at most 4.30 mS/cm, preferably at least 2.40 mS/cm to at most 4.10 mS/cm, preferably at least 2.60 mS/cm to at most 3.90 mS/cm, preferably at least 2.80 mS/cm to at most 3.70 mS/cm, preferably at least 3.00 mS/cm up to 3.50 mS/cm, preferably at least 3.10 mS/cm up to 3.30 mS/cm, preferably at least 3.20 mS/cm up to 3.40 mS/cm, preferably is approximately 3.30 mS/cm.

23. The method according to at least one of the preceding statements, wherein in step c) after adding the caprylic acid the pH of the first suspension is at least 4.70 to at most 4.90, preferably at least 4.75 to at most 4.85, preferably about 4.80.

15 BE2022/5354 24. The method according to at least one of the preceding statements, wherein in step c) at least 0.8 wt% (weight percent) to at most 1.6 wt%, preferably at least 1.0 wt% to at most 1.4 wt% preferably at least 1.1 wt% to at most 1.3 wt%, preferably about 1.2 wt% of caprylic acid is added relative to the weight of the first IgG suspension.

25. The method according to at least one of the preceding statements, wherein in step c) the caprylic acid is added over a period of at least 15 minutes, preferably at least 30 minutes, preferably at least 45 minutes, even more preferably at least 60 minutes.

26. The method according to at least one of the preceding statements, wherein step c) after adding the caprylic acid comprises a maturation step of at least 30 minutes, preferably at least 60 minutes, preferably at least 90 minutes, preferably at least 120 minutes, preferably at least 150 min, preferably at least 120 min. 27. The method according to at least one of the preceding statements, wherein the filter in step d) is a depth filter. 28. The method according to at least one of the preceding statements, wherein the pore size of the filter in step d} is at least 0.5 to at most 15.0 um, preferably at least 0.7 um to at most 12.0 um, preferably at least 1 .0 µm to at most 10.0 µm, preferably at least 2.0 µm to at most 9.0 µm, preferably at least 2.0 µm to at most 7.0 µm, preferably at least 2.0 µm to at most 5.0 µm, preferably at least 2.0 µm to at most 3.0 µm. 29. The method according to at least one of the preceding statements, wherein the filtration in step d) takes place at a ratio of at most 120 kg, preferably at most 100 kg, preferably at most 80 kg of suspension per square meter of filter. 30. The method according to at least one of the preceding claims, wherein the residue on the filter in step d) is washed with a third buffer. 31. The method according to at least one of the preceding claims, wherein the third buffer has a pH of at least 4.0 to at most 5.6; preferably at least 4.2 to at most 5.4; preferably at least 4.4 to at most 5.2; preferably at least 4.6 to at most 5.0; preferably at least 4.7 to at most 4.9; preferably about 4.8. 32. The method according to at least one of the preceding claims, wherein the third buffer is an acetate buffer.

16 BE2022/5354

33. The method according to at least one of the preceding claims, wherein the first buffer and the third buffer are the same.

34. The method according to any one of the preceding claims, wherein the fourth buffer is an acetate buffer.

35. The method according to any one of the preceding claims, wherein the fourth buffer contains at least 5.0 mM to at most 50.0 mM, preferably at least 7.0 mM to at most 40.0 mM; at least 10.0 mM to at most 30.0 mM; at least 12.0 mM to at most 20.0 mM; at least 12.0 mM to at most 17.0 mM, preferably about 15.0 mM acetate buffer.

36. The method according to at least one of the preceding statements, wherein at the first semipermeable membrane, second semipermeable membrane and/or third semipermeable membrane, molecules with a molecular weight of less than 5 kDa, preferably less than 10 kDa, preferably less than 15 kDa, preferably less than 20 kDa, preferably less than 25 kDa, preferably less than 30 kDa, preferably less than 40 kDa, preferably less than 50 kDa.

37. The method according to at least one of the preceding claims wherein the first semipermeable membrane, second semipermeable membrane and/or third semipermeable membrane retains molecules with a molecular weight greater than 50 kDa, preferably greater than 40 kDa, preferably greater than 30 kDa .

38. The method according to at least one of the preceding claims, wherein the first semipermeable membrane, second semipermeable membrane and/or third semipermeable membrane comprises a polyethersulfone membrane.

39. The method according to any one of the preceding claims, wherein the first semipermeable membrane, the second semipermeable membrane and/or the third semipermeable membrane is the same semipermeable membrane; preferably wherein the first semipermeable membrane, the second semipermeable membrane and the third semipermeable membrane are the same semipermeable membrane.

40. The method according to at least one of the preceding statements, wherein the first concentrated IgG solution in step e) is concentrated by at least a factor of 2, preferably at least a factor of 3, preferably at least a factor of 4, preferably at least a factor of 5, preferably at least a factor of 6 compared to the IgG filtrate obtained in step d).

17 BE2022/5354 41. The method according to at least one of the preceding statements, wherein the diafiltration in step f) takes place using at least 1 volume of second buffer, preferably at least 2 volumes of second buffer, preferably at least 3 volumes of second buffer, at least compared to 1 volume of concentrated IgG solution. 42. The method according to at least one of the preceding statements, wherein the second buffer has a pH of at least 3.50 to at most 6.00, preferably at least 3.80 to at most 5.80, preferably at least 4.00 to at most 5 .60, preferably at least 4.20 to at most 5.40, preferably at least 4.40 to at most 5.20, preferably at least 4.60 to at most 5.00, preferably at least 4.70 to at most 4.90 , preferably about 4.80. 43. The method according to at least one of the preceding statements, wherein the second buffer has a conductivity of at least 0.50 mS/cm to at most 1.90 mS/cm, preferably at least 0.60 mS/cm to at most 1.80 mS /cm, preferably at least 0.70 mS/cm to at most 1.70 mS/cm, preferably at least 0.80 mS/cm at most 1.60 mS/cm, preferably at least 0.90 mS/cm at most 1.50 mS/cm, preferably at least 1.00 mS/cm to at most 1.40 mS/cm, preferably at least 1.10 mS/cm to at most 1.30 mS/cm, preferably about 1.20 mS /cm. 44. The method according to at least one of the preceding claims, wherein the second buffer has a concentration of at least 15.0 mM to at most 50.0 mM, preferably at least 20.0 mM to at most 40.0 mM; at least 22.0 mM to at most 37.0 mM; at least 25.0 mM to at most 35.0 mM; at least 27.0 mM to at most 32.0 mM, preferably about 30.0 mM acetate buffer. 45. The method according to at least one of the preceding claims, wherein the second buffer is an acetate buffer. 46. .The method according to at least one of the preceding statements, in which the conductivity of the

IgG solution formed in step f) at least 0.5 mS/cm and at most 2.0 mS/cm, preferably at least 0.7 mS/cm and at most 1.5 mS/cm, preferably at least 1.0 mS/cm and is at most 1.4 mS/cm, preferably at least 1.1 mS/cm and at most 1.3 mS/cm, preferably about 1.2 mS/cm. 47. The method according to at least one of the preceding claims, wherein the anion exchange chromatography in step i) uses a strongly basic anion exchange resin.

18 BE2022/5354

48. The method according to at least one of the preceding claims, wherein the anion exchange chromatography in step i) uses an epoxy resin.

49. The method according to at least one of the preceding claims, wherein the anion exchange chromatography in step i) uses a resin comprising -N*(CH:)}3 functional groups.

50. The method according to at least one of the preceding claims, wherein the anion exchange chromatography in step i) uses a resin with a particle size of at least 30 µm to at most 60 µm, preferably at least 35 µm to at most 55 µm, preferably at least 38 µm to 53 µm at most.

51. The method according to at least one of the preceding claims, wherein in step i) the fourth IgG solution is loaded onto the resin after a filtration, preferably through a filter with a pore size of at least 0.5 to at most 15.0 µm, at preferably at least 0.7 µm to at most 12.0 µm, preferably at least 1.0 µm to at most 10.0 µm, preferably at least 2.0 µm to at most 9.0 µm, preferably at least 2.0 µm to at most 7.0 µm, preferably at least 2.0 µm to at most 5.0 µm, preferably at least 2.0 µm to at most 3.0 µm.

52. The method according to at least one of the preceding claims, wherein in step i) the IgG passes through the column by repellency of the resin with an anion solution, preferably an equilibration solution; preferably an acetate solution.

53. The method according to at least one of the preceding statements, wherein the anion/equilibration solution has a conductivity of at least 1.00 mS/cm and at most 3.00 mS/cm, preferably at least 1.50 mS/cm and at most 2, 50 mS/cm, preferably at least 1.80 mS/cm and at most 2.20 mS/cm, preferably at least 1.90 mS/cm and at most 2.10 mS/cm, preferably about 2.00 mS/cm .

54. The method according to at least one of the preceding claims, wherein the anion/equilibration solution has a pH of at least 5.80 to at most 7.00, preferably at least 5.90 to at most 6.90, preferably at least 6.00 to at most 6.80, preferably at least 6.10 to at most 6.70, preferably at least 6.20 to at most 6.60, preferably at least 6.30 to at most 6.50.

55. The method according to at least one of the preceding claims, wherein the anion/equilibration solution has a concentration of at least 5.0 mM to at most 50.0 mM, preferably

19 BE2022/5354 at least 7.0 mM to at most 40.0 mM; at least 10.0 mM to at most 30.0 mM; at least 12.0 mM to at most 20.0 mM; at least 12.0 mM to at most 17.0 mM, preferably about 15.0 mM acetate. 56. The method according to at least one of the preceding statements, wherein the purification via affinity chromatography uses a medium or a mixture of media, on which N-acetyl-galactosamine (GalNAc} - Galactose (Gal) - Fucose (Fuc), and Galactose Galactose

Fucose groups are provided. 57. The method according to at least one of the preceding propositions, wherein the purification via affinity chromatography uses a medium or a mixture of media, on which groups are provided corresponding to the epitopes of blood groups A and B. 58. The method according to at least one of the preceding propositions, the sixth being concentrated

IgG solution in step |) is concentrated by at least a factor 2, preferably at least a factor 3, preferably at least a factor 4, preferably at least a factor 5, preferably at least a factor 6 with respect to the fifth IgG solution obtained in step k). 59. The method according to at least one of the preceding claims, wherein the IgG preparation has a purity of at least 90%, preferably at least 95%, more preferably at least 99%. 60. An IgG preparation, preferably an intravenous IgG preparation, obtained from a method according to at least one of the preceding statements. 61. The IgG preparation of statement 60, wherein the IgG preparation has a protein level purity of at least 90.0%, preferably at least 95.0%, preferably at least 99.0%, preferably at least 99.5%, preferably at least 99.7%, preferably at least 99.9%, preferably 100.0%. 62. An IgG preparation comprising: - at least 1.0% by weight to at most 10.0% by weight, preferably at least 2.0% by weight to at most 8.0% by weight, preferably at least 3.0% by weight to at most 7.0% by weight, preferably at least 3.5% by weight to at most 6.0% by weight, preferably at least 4.0% by weight to at most 5.5% by weight, preferably at least 4.0% by weight to at most 5.0% by weight of immunoglobulin G (IgG);

20 BE2022/5354 - at least 7.0 wt.% to at most 10.0 wt.%, preferably at least 7.5 wt.% to at most 9.5 wt.%, preferably at least 8.0 wt.% to at most 9.0 wt.%, preferably at least 8, 1 wt.% up to 8.8 wt.%, preferably at least 8.2 wt.% up to 8.6 wt.%, preferably about 8.4 wt.% sucrose; - at least 0.1 wt% to at most 1.2 wt%, preferably at least 0.2 wt% at most 1.1 wt%, preferably at least 0.3 wt% at most 1.0 wt%, preferably at least 0.4 wt% at most 0.9 wt.%, preferably at least 0.5 wt.% to at most 0.8 wt.%, preferably at least 0.6 wt.% to at most 0.7 wt.%, preferably about 0.6 wt.% glycine; - at least 0.1 wt% to at most 0.6 wt%, preferably at least 0.2 wt% at most 0.5 wt%, preferably at least 0.3 wt% to at most 0.4 wt%, preferably about 0.3 wt% sodium chloride ; - acetate buffer; wherein the pH of the composition is at least 4.00 to at most 5.00, preferably at least 4.10 to at most 4.80, at least 4.20 to at most 4.70, preferably at least 4.30 to at most 4.50, preferably about 4.40. 63. An IgG preparation according to at least one of claims 60 to 62, comprising: - at least 4.0 wt% to at most 5.0 wt% immunoglobulin G (IgG); - at least 8.2 wt% to at most 8.6 wt%, preferably about 8.4 wt% sucrose; - at least 0.6 wt% to at most 0.7 wt%, preferably about 0.6 wt% glycine; - at least 0.3 wt% to at most 0.4 wt%, preferably about 0.3 wt% sodium chloride; - acetate buffer;

21 BE2022/5354 wherein the pH of the composition is at least 4.30 to at most 4.50, preferably about 4.40. 64. An IgG preparation according to at least one of claims 60 to 62, wherein the acetate buffer contains at least 5.0 mM to at most 50.0 mM, preferably at least 7.0 mM to at most 40.0 mM; at least 10.0 mM to at most 30.0 mM; at least 12.0 MM to at most 20.0 mM; at least 12.0 mM to at most 17.0 mM, preferably about 15.0 mM acetate.

In a first aspect, the invention provides a method of producing immunoglobulin G (IgG) preparations or immunoglobulin G (IgG) solutions, comprising the steps of: a) providing an IgG-containing paste; b} resuspending the paste in a first buffer to form a first IgG suspension; c) adding caprylic acid to the first IgG suspension after which the pH of the first IgG suspension is at least 4.60 to at most 5.00, thereby obtaining a precipitate and an IgG supernatant; d) filtering the IgG supernatant obtained in step c) through a filter, thereby obtaining an IgG filtrate; e) optionally, concentrating the IgG filtrate obtained in step d), preferably over a first semipermeable membrane and preferably by means of a first ultrafiltration, thereby obtaining a first concentrated IgG solution; f) optionally, exchanging the buffer from the first concentrated IgG solution obtained in step d), preferably by diafiltration, against a second buffer at a pH of at least 4.60 to at most 5.00, thereby obtaining a second

IgG solution; g) optionally, concentrating the second IgG solution obtained in step f), preferably over the second semipermeable membrane and preferably by means of a second ultrafiltration, thereby obtaining a third concentrated IgG solution; h} diluting: - the IgG filtrate, if step e), step f) and step g) have not been carried out; - the first concentrated IgG solution, if step e) has been carried out and steps f) and g) have not been carried out; - the second IgG solution, when steps e) and f) have been performed, and step g) has not been performed; or,

22 BE2022/5354 - the third concentrated IgG solution; when steps e), f) and g) have been performed; by adding water and/or a fourth buffer to obtain a fourth IgG solution with: - a protein concentration of at least 10.0 g/L to at most 70.0 g/L, preferably at least 15.0 g/L L to at most 65.0 g/L, preferably at least 20.0 g/L to at most 60.0 g/L, preferably at least 25.0 g/L to at most 55.0 g/L, preferably at least 30 .0 g/L to at most 50.0 g/L, preferably at least 35.0 g/L to at most 45.0 g/L, preferably at least 37.0 g/L to at most 43.0 g/L, preferably at least 38.0 g/L to at most 42.0 g/L, preferably at least 39.0 g/L to at most 41.0 g/L, preferably 40.0 g/L; and, - a pH of at least 5.80 to at most 7.00, preferably at least 5.90 at most 6.90, preferably at least 6.00 at most 6.80, preferably at least 6.10 at most 6, 70, preferably at least 6.20 to at most 6.60, preferably at least 6.30 to at most 6.50; ij) purifying the fourth IgG solution obtained in step h) via anion exchange chromatography, thereby obtaining first flow-through of IgG; j) optionally, the purification of the first IgG-containing flow-through obtained is step i) via affinity chromatography, thereby obtaining a second IgG-containing flow-through.

In an embodiment, the method comprises the further steps of: k) adjusting the pH of the first IgG-containing flow-through obtained in step i) or of the second IgG-containing flow-through when step j) has been performed, thereby obtaining a fifth IgG solution containing a pH of at least 4.00 to at most 5.00, preferably at least 4.10 at most 4.80, at least 4.20 at most 4.70, preferably at least 4.30 at most 4.50, preferably 4 .40; and,

I) optionally, concentrating the fifth IgG solution obtained in step k), preferably over the third semipermeable membrane and preferably by means of a third ultrafiltration, thereby obtaining a sixth concentrated IgG solution.

In one embodiment, the method comprises the further steps of: - stabilizing the obtained IgG solution or IgG flow-through, by adding one or more excipients and/or adjusting the pH, thereby obtaining a stable IgG preparation.

23 BE2022/5354

In one embodiment, the fourth IgG solution has: - a protein concentration of at least 37.0 g/L to at most 43.0 g/L, preferably at least 38.0 g/L to at most 42.0 g/L, at preferably at least 39.0 g/L to at most 41.0 g/L, preferably 40.0 g/L; and, - a pH of at least 6.30 to at most 6.50.

In one embodiment, the protein concentration of the fourth IgG solution is 40.0 g/L and the pH of the fourth IgG solution is at least 6.30 and at most 6.50.

In one embodiment, the first semipermeable membrane, the second semipermeable membrane and/or the third semipermeable membrane is the same membrane. As a result, the same membrane can be reused during the process, with or without a washing step between the different uses, preferably a washing step with a salt solution, preferably a sodium chloride salt solution.

In one embodiment, the one or more excipients is selected from the list of sucrose, glycine and/or sodium chloride. In one embodiment, sucrose, glycine and sodium chloride are added as excipients.

Immunoglobulin G (IgG) is an immunoglobulin that is produced with larger amounts or with repeated contact with the antigen. The IgG molecule is typically made up of two heavy chains and two light chains. Within the light chain are two disulfide bridges; one in the variable region and one in the constant region. Typically, there are four of these disulfide bridges in the heavy chain, which is about twice as long as the light chain. Typically, each disulfide compound forms a — peptide loop of 60 to 70 amino acid residues; when the amino acid sequences of these loops are compared, a high degree of homology is apparent. This means that each immunoglobulin peptide chain consists of a series of globular regions with a very similar secondary and tertiary structure. IgG can be divided into four subclasses: IgG1, IgG2, IgG3 and IgG4. The four subclasses of human IgG differ little from each other in amino acid sequence.

IgG can be obtained from plasma, for example from donors. A well-known process for this is the Cohn process, which combines an ethanol gradient with a temperature gradient and a pH gradient to precipitate different fractions. Typically, the different fractions are defined as shown in Table 1.

24 BE2022/5354 == B eea

Temperature -3 -5 -5 -5 -5 (°C)

Protein 5.1 3 3 3 1 fraction (%):

Table 1: Fractions from the Cohn process

In one embodiment, paste fractions II+III or fractions I+II+II are used in the process of the invention.

In one embodiment, the first buffer is an acetate buffer. In one embodiment, both the first buffer, the second buffer, the third buffer, and the fourth buffer are an acetate buffer.

In one embodiment, the first buffer has a pH of at least 3.50 to at most 6.00, preferably at least 3.80 to at most 5.80, preferably at least 4.00 to at most 5.60, preferably at least 4.20 to at most 5.40, preferably at least 4.40 to at most 5.20, preferably at least 4.60 to at most 5.00, preferably at least 4.70 to at most 4.90, preferably 4.80.

In one embodiment, the first buffer has a conductivity of at least 2.10 mS/cm to at most 4.30 mS/cm, preferably at least 2.40 mS/cm to at most 4.10 mS/cm, preferably at least 2, 60 mS/cm up to 3.90 mS/cm, preferably at least 2.80 mS/cm up to 3.70 mS/cm, preferably at least 3.00 mS/cm up to 3.50 mS/cm, at preferably at least 3.10 mS/cm to at most 3.30 mS/cm, preferably at least 3.20 mS/cm to at most 3.40 mS/cm, preferably 3.30 mS/cm.

In one embodiment, the first buffer has a concentration of at least 25.0 mM to at most 150.0 mM, preferably at least 50.0 mM to at most 125.0 mM; at least 60.0 mM to at most 100.0 mM; at least 70.0 mM to at most 90.0 mM; at least 75.0 mM to at most 85.0 mM, preferably an 80.0 mM acetate buffer.

In one embodiment, the second buffer has a pH of at least 3.50 to at most 6.00, preferably at least 3.80 to at most 5.80, preferably at least 4.00 to at most 5.60, preferably

25 BE2022/5354 at least 4.20 to at most 5.40, preferably at least 4.40 to at most 5.20, preferably at least 4.60 to at most 5.00, preferably at least 4.70 to at most 4.90, preferably 4.80.

In one embodiment, the second buffer has a conductivity of at least 0.50 mS/cm to at most 1.90 mS/cm, preferably at least 0.60 mS/cm to at most 1.80 mS/cm, preferably at least 0.70 mS/cm up to 1.70 mS/cm, preferably at least 0.80 mS/cm up to 1.60 mS/cm, preferably at least 0.90 mS/cm up to 1.50 mS/cm, preferably at least 1.00 mS/cm to at most 1.40 mS/cm, preferably at least 1.10 mS/cm to at most 1.30 mS/cm, preferably 1.20 mS/cm.

In one embodiment, the second buffer has a concentration of at least 15.0 mM to at most 50.0 mM, preferably at least 20.0 mM to at most 40.0 mM; at least 22.0 mM to at most 37.0 MM; at least 25.0 mM to at most 35.0 mM; at least 27.0 MM to at most 32.0 mM, preferably a 30.0 mM acetate buffer.

The method according to the invention allows for filtering with buffer conditions (for example the first buffer) in which IgG is stable in suspension and only just before the chromatography steps is introduced into a less stable but necessary buffer for the chromatography. This prevents loss of the IgG.

In one embodiment, the first buffer is equal to the third buffer.

In one embodiment, the conductivity of the first IgG suspension is at least 2.10 mS/cm to at most 4.30 mS/cm, preferably at least 2.40 mS/cm to at most 4.10 mS/cm, preferably at least 2, 60 mS/cm up to 3.90 mS/cm, preferably at least 2.80 mS/cm up to 3.70 mS/cm, preferably at least 3.00 mS/cm up to 3.50 mS/cm, at preferably at least 3.10 mS/cm to at most 3.30 mS/cm, preferably at least 3.20 mS/cm to at most 3.40 mS/cm, preferably 3.30 mS/cm.

In one embodiment, the conductivity of the first IgG suspension is at least 2.80 mS/cm to at most 3.40 mS/cm, preferably at least 2.90 mS/cm to at most 3.40 mS/cm, preferably at least 3, 00 mS/cm up to 3.40 mS/cm, preferably at least 3.10 mS/cm up to 3.40 mS/cm, preferably at least 3.20 mS/cm up to 3.40 mS/cm, at preferably 3.30 mS/cm.

26 BE2022/5354

In one embodiment, in step c) after adding the caprylic acid, the pH of the first suspension is at least 4.70 to at most 4.90, preferably at least 4.75 to at most 4.85, preferably 4.80.

The use of caprylic acid has the advantage that it facilitates a viral inactivation step and/or a viral removal step for both enveloped and non-enveloped viruses. Carpylic acid has prion removal capacity as evaluated for human transmissible spongiform encephalopathy (TSE) agent. Caprylic acid can further remove procoagulant activity and allows the IgG protein to remain in suspension. But at the pH range mentioned herein, these effects will not be complete and impurities will therefore remain in suspension.

In one embodiment, in step c) the caprylic acid is added over a period of at least minutes, preferably at least 30 minutes, preferably at least 45 minutes, even more preferably at least 60 minutes. Preferably, in step c) the pH is checked after this time. Adding over such a period ensures that there are no local outliers of the pH at the place where the caprylic acid is added, which could lead to a reduced yield. In one embodiment, the method in step c) comprises, after adding the caprylic acid, a maturation step of at least 30 minutes, preferably at least 60 minutes, preferably at least 90 minutes, preferably at least 120 minutes, preferably at least 150 minutes, preferably at least 120 min. Preferably, the IgG suspension is agitated during the maturation step.

In one embodiment, the conductivity of the IgG solution formed in step f) is at least 0.5 mS/cm and at most 2.0 mS/cm, preferably at least 0.7 mS/cm and at most 1.5 mS/cm, at preferably at least 1.0 mS/cm and at most 1.4 mS/cm, preferably at least 1.1 mS/cm and at most 1.3 mS/cm, preferably 1.2 mS/cm.

In one embodiment, the anion exchange chromatography in step i} uses a strongly basic anion exchanger resin or a stretched anionic anion exchanger resin.

In one embodiment, the anion exchange chromatography in step i) uses an epoxy resin.

In one embodiment, the anion exchange chromatography in step i) uses a resin comprising —N*(CH:}3 functional groups.

In one embodiment, the anion exchange chromatography in step i) uses a resin with a total ionic capacity of at least 30 jeq to at most 120 Heg, preferably at least 40 ueg at most 100 ueg, preferably at least 48 ueq at most 88 Hed.

27 BE2022/5354

In one embodiment, the anion exchange chromatography in step i) uses a resin with a particle size of at least 30 µm to at most 60 µm, preferably at least 35 µm to at most 55 µm, preferably at least 38 µm to at most 53 µm.

In one embodiment, in step i) the fourth IgG solution is loaded onto the resin after a filtration, preferably through a filter with a pore size of at least 0.5 to at most 15.0 µm, preferably at least 0.7 µm to at most 12 .0 µm, preferably at least 1.0 µm to at most 10.0 µm, preferably at least 2.0 µm to at most 9.0 µm, preferably at least 2.0 µm to at most 7.0 µm, preferably at least 2 .0 µm to at most 5.0 µm, preferably at least 2.0 µm to at most 3.0 µm. In a preferred embodiment, the same filter is used as in the first ultrafiltration.

In one embodiment, the purification via affinity chromatography uses a medium or a mixture of media, on which N-acetyl-galactosamine (GalNAc) - Galactose (Gal) - Fucose (Fuc), and Galactose-Galactose-Fucose groups are provided. This binds antibodies aimed at blood groups A and B, thus preventing hemolysis in people who are administered the IgG preparation, so that the IgG preparation can be widely used, preferably without the donors of the plasma having to be limited to donors with blood group O.

In one embodiment, the purification via affinity chromatography uses a medium or a mixture of media, on which groups are provided that correspond to the epitopes of blood groups A and B. In one embodiment, the affinity chromatography is carried out as described in EP 1 968 634 B1.

In a second aspect, the invention provides an IgG preparation, preferably an intravenous IgG preparation, obtained from a method according to an embodiment described herein.

The invention further provides an IgG preparation, comprising: - immunoglobulin G (IgG}; - at least 7.0 wt.% to at most 10.0 wt.%, preferably at least 7.5 wt.% to at most 9.5 wt.%, preferably at least 8, 0 wt% up to 9.0 wt%, preferably at least 8.1 wt% up to 8.8 wt%, preferably at least 8.2 wt% up to 8.6 wt%, preferably 8.4 wt% sucrose - at least 0.1 weight percent to at most 1.2 weight percent, preferably at least 0.2 weight percent to at most 1.1 weight percent, preferably at least 0.3 weight percent to

28 BE2022/5354 at most 1.0 weight percent, preferably at least 0.4 weight percent to at most 0.9 weight percent, preferably at least 0.5 weight percent to at most 0.8 weight percent, preferably at least 0.6 weight percent to at most 0.7 weight percent, preferably 0.6 weight percent glycine; - at least 0.1 wt.% to at most 0.6 wt.%, preferably at least 0.2 wt.% to at most 0.5 wt.%, preferably at least 0.3 wt.% to at most 0.4 wt.%, preferably 0.3 wt.% sodium chloride; - acetate buffer; wherein the pH of the composition is at least 4.00 to at most 5.00, preferably at least 4.10 to at most 4.80, at least 4.20 to at most 4.70, preferably at least 4.30 to at most 4.50, preferably 4.40.

EXAMPLES

Measurement methods

The pH of a solution, a preparation or a buffer was measured according to 07/2016:20203; Ph. EUR. 2.2.3; potentiometric determination of pH, glass electrodes.

The conductivity of a solution, a preparation or a buffer was measured according to 01/2021:20238;

Ph. EUR. 2.2.38; Equipment: resistance measuring circuit and the conductivity sensor.

The protein concentration of a solution or preparation was measured according to 01/2008:20533; Ph. EUR. 2.5.33; Total protein: 1) UV light absorption at 280nm according to Ph. EUR. 2.2.25. ABSORPTION

SPECTROPHOTOMETRY, ULTRAVIOLET AND VISIBLE.

Weight percents herein are in each case relative to the weight of the entire preparation or solution.

Example method 270 kg of a paste was provided, the paste comprising paste fractions I+II+III obtained from the Cohn process performed on human plasma. This amount of paste was resuspended in 8.0 kg of first buffer per kg of paste to form a first IgG suspension. The first buffer was an 80.0 mM acetate buffer, with a pH of 4.80 and a conductivity of 3.30 mS/cm.

To the first IgG suspension obtained above, a 1.2 wt% caprylic acid was added over a period of 60 minutes until the pH of the first IgG suspension is 4.80. After this it underwent

29 BE2022/5354 whole a maturation step of 60 minutes during which the acidified first IgG suspension was slightly agitated. During acidification with caprylic acid a precipitate was formed, however the IgG remains in solution, in other words an IgG supernatant was formed.

The precipitate was then filtered off through a depth filter with a pore size of 2 to 3 µm, for example BECOPAD® P 550 from Eaton) to obtain an IgG filtrate.

The obtained IgG filtrate was then concentrated over a first semipermeable membrane by means of a first ultrafiltration, thereby obtaining a first concentrated IgG solution with a protein concentration of 70 g/L. For example, the first semipermeable membrane was a

TangenX® FLAT SHEET MEMBRANES PES filter from Repligen with a cut of 30 kDa.

The same first semipermeable membrane was then used to exchange the buffer by diafiltration against a second buffer with a pH of 4.80, a conductivity of 1.20 mS/cm and 30 mM acetate, to obtain a second IgG solution. .

After this, the second IgG solution was further concentrated over the same semipermeable membrane by means of a second ultrafiltration, thereby obtaining a third concentrated IgG solution with a protein concentration of 90 g/L.

Subsequently, the third concentrated IgG is diluted by adding water and/or a fourth buffer to obtain a fourth IgG solution with a protein concentration of 40.0 g/L and a pH of at least 6.30 to at most 6.50. For example, the fourth buffer is a 15.0 mM acetate buffer with a pH of 6.50 and a conductivity of 2.00 mS/cm.

The fourth IgG solution is suitable for purification via anion exchange chromatography, for example with a strongly basic epoxy resin functionalized with -N*(CH3)3 groups. In this example, a Nuvia HP-Q resin from BIO-RAD was chosen. The IgG here left the resin as a first IgG-containing flow-through. The fourth buffer was used as eluent.

Subsequently, Anti-A and Anti-B antibodies were removed from the first IgG-containing flow-through via affinity chromatography using a resin functionalized with N-acetyl-galactosamine (GalNAc}, Galactose (Gal), Fucose (Fuc), and Galactose- Galactose-Fucose groups, corresponding to the epitopes of blood groups A and B. Preferably, the affinity chromatography is carried out as described in EP 1 968 634 B1 The IgG left the resin as a second flow-through comprising IgG.

30 BE2022/5354

Subsequently, the pH of the obtained second IgG flow-through was adjusted, thereby obtaining a fifth IgG solution with a pH of 4.40.

The fifth IgG solution was then concentrated over the same semipermeable membrane by means of a third ultrafiltration, thereby obtaining a sixth concentrated IgG solution with a protein concentration of 70 g/L.

Subsequently, the sixth concentrated IgG was stabilized by adding sucrose, glycine and sodium chloride, resulting in a stable IgG preparation with the following composition: - 40-55 g/L immunoglobulin G (IgG); - 8.4% by weight of sucrose; - 0.6% by weight of glycine; - 0.3 weight percent sodium chloride; in an acetate buffer and with a pH of 4.40.

The method described above achieved an efficiency between 70% and 80% each time. Getting it

IgG was 1005 pure at the protein level.

Before the semipermeable membrane was reused in the method described above, the semipermeable membrane was washed with a sodium chloride solution.

Stability testing

The above preparation was stored at room temperature for 3 months without loss of IgG.

Claims (13)

31 BE2022/5354 CONCLUSIONS (retyped) A method for producing immunoglobulin G (IgG) preparations or immunoglobulin G (IgG) solutions, comprising the steps of: a) providing an IgG-containing paste; b} resuspending the paste in a first buffer to form a first IgG suspension; c) adding caprylic acid to the first IgG suspension after which the pH of the first IgG suspension is at least 4.60 to at most 5.00, thereby obtaining a precipitate and an IgG supernatant; d) filtering the IgG supernatant obtained in step c) through a filter, thereby obtaining an IgG filtrate; e) optionally, concentrating the IgG filtrate obtained in step d), preferably over a first semipermeable membrane and preferably by means of a first ultrafiltration, thereby obtaining a first concentrated IgG solution; f) optionally, exchanging the buffer from the first concentrated IgG solution obtained in step d), preferably by diafiltration, against a second buffer at a pH of at least 4.60 to at most 5.00, thereby obtaining a second IgG solution; g) optionally, concentrating the second IgG solution obtained in step f), preferably over the second semipermeable membrane and preferably by means of a second ultrafiltration, thereby obtaining a third concentrated IgG solution; h} diluting: - the IgG filtrate, if step e), step f) and step g) have not been carried out; - the first concentrated IgG solution, if step e) has been carried out and steps f) and g) have not been carried out; - the second IgG solution, when steps e) and f) have been performed, and step g) has not been performed; or, - the third concentrated IgG solution; when steps e), f) and g) have been performed; by adding water and/or a fourth buffer to obtain a fourth IgG solution with: 32 BE2022/5354 - a protein concentration of at least 10.0 g/L to at most 70.0 g/L, preferably at least 15.0 g/L to at most 65.0 g/L, preferably at least 20.0 g/L to at most 60.0 g/L, preferably at least 25.0 g/L to at most 55.0 g/L, preferably at least 30.0 g/L to at most 50.0 g/L, preferably at least 35.0 g/L to at most 45.0 g/L, preferably at least 37.0 g/L to at most 43.0 g/L, preferably at least 38.0 g/L to at most 42.0 g/L L, preferably at least 39.0 g/L to at most 41.0 g/L, preferably 40.0 g/L; and, - a pH of at least 5.80 to at most 7.00, preferably at least 5.90 at most 6.90, preferably at least 6.00 at most 6.80, preferably at least 6.10 at most 6, 70, preferably at least 6.20 to at most 6.60, preferably at least 6.30 to at most 6.50; ij) purifying the fourth IgG solution obtained in step h) via anion exchange chromatography, thereby obtaining first flow-through of IgG; j) optionally, the purification of the first IgG-containing flow-through obtained is step i) via affinity chromatography, thereby obtaining a second IgG-containing flow-through, k) stabilizing the obtained IgG solution or IgG flow-through, by adding sucrose, glycine and sodium chloride, thereby obtaining a stable IgG preparation. The method according to claim 1, further comprising: I) adjusting the pH of the first IgG-containing flow-through obtained in step i} or of the second IgG-containing flow-through when step j) has been performed, thereby obtaining a fifth IgG solution containing a pH of at least 4.00 to at most 5.00, preferably at least 4.10 at most 4.80, at least 4.20 at most 4.70, preferably at least 4.30 at most 4.50, preferably 4 .40; and, m) optionally, concentrating the fifth IgG solution obtained in step k), preferably across the third semipermeable membrane and preferably by means of a third ultrafiltration, thereby obtaining a sixth concentrated IgG solution. The method according to any one of the preceding claims, wherein the fourth IgG solution: 33 BE2022/5354 - a protein concentration of at least 37.0 g/L to at most 43.0 g/L, preferably at least 38.0 g/L to at most 42.0 g/L, preferably at least 39.0 g/L up to 41.0 g/L, preferably 40.0 g/L; and, - has a pH of at least 6.30 to at most 6.50. The method according to any one of the preceding claims, wherein the first semipermeable membrane, the second semipermeable membrane and/or the third semipermeable membrane is the same membrane. The method according to at least one of the preceding claims, wherein at the first semipermeable membrane, second semipermeable membrane and/or third semipermeable membrane, molecules with a molecular weight of less than 5 kDa, preferably less than 10 kDa, preferably less than 15 kDa, preferably less than 20 kDa, preferably less than 25 kDa, preferably less than 30 kDa, preferably less than 40 kDa, preferably less than 50 kDa. The method according to at least one of the preceding claims, wherein the paste comprises fractions III, preferably fractions I+II+III obtained from the Cohn process. The method according to at least one of the preceding claims, wherein step c} comprises a ripening step after adding the caprylic acid, of at least 30 minutes, preferably at least 60 minutes, preferably at least 90 minutes, preferably at least 120 minutes, preferably at least 150 min, preferably at least 120 min. The method according to at least one of the preceding claims, wherein: - the first buffer and the second buffer have a pH of at least 4.60 to at most 5.00, preferably at least 4.70 to at most 4.90, preferably 4 .80; - the first buffer: o has a conductivity of at least 3.0 mS/cm to at most 3.6 mS/cm, preferably about 3.3 mS/cm; and, o has a concentration of at least 50.0 mM to at most 110.0 mM, preferably about 80.0 mM; and, 34 BE2022/5354 - the second buffer: o has a conductivity of at least 3.0 mS/cm and at most 3.6 mS/cm, preferably about 3.3 mS/cm; and, o has a concentration of at least 10.0 mM to at most 47.0 mM, preferably about 30.0 mM. The method according to at least one of the preceding claims, wherein the fourth buffer is at least 5.0 mM to at most 50.0 mM acetate buffer, preferably at least 7.0 mM to at most 40.0 mM acetate buffer; at least 10.0 mM to at most 30.0 mM acetate buffer; at least 12.0 mM to at most 20.0 mM acetate buffer; at least 12.0 mM to at most 17.0 mM acetate buffer, preferably a 15.0 mM acetate buffer. The method according to at least one of the preceding claims, wherein the anion exchange chromatography in step i) uses a strongly basic anion exchange resin. The method according to at least one of the preceding claims, wherein the purification via affinity chromatography uses a medium or a mixture of media, on which N-acetyl-galactosamine (GalNAc} - Galactose (Gal) - Fucose (Fuc), and Galactose-Galactose-Fucose groups are provided. 12. An IgG preparation, preferably an intravenous IgG preparation, obtained from a method according to at least one of the preceding claims. 13. An IgG preparation comprising: immunoglobulin G (IgG); - at least 7.0 weight percent to at most 10.0 weight percent, preferably at least 7.5 weight percent to at most 9.5 weight percent, preferably at least 8.0 weight percent to at most 9.0 weight percent, preferably at least 8.1 weight percent to at most 8.8 wt.%, preferably at least 8.2 wt.% to at most 8.6 wt.%, preferably 8.4 wt.% sucrose; 35 BE2022/5354 - at least 0.1 weight percent to at most 1.2 weight percent, preferably at least 0.2 weight percent to at most 1.1 weight percent, preferably at least 0.3 weight percent to at most 1.0 weight percent, preferably at least 0, 4 wt% up to 0.9 wt%, preferably at least 0.5 wt% up to 0.8 wt%, preferably at least 0.6 wt% up to 0.7 wt%, preferably 0.6 wt% glycine; - at least 0.1 wt.% to at most 0.6 wt.%, preferably at least 0.2 wt.% to at most 0.5 wt.%, preferably at least 0.3 wt.% to at most 0.4 wt.%, preferably 0.3 wt.% sodium chloride; - acetate buffer; wherein the pH of the composition is at least 4.00 to at most 5.00, preferably at least 4.10 to at most 4.80, at least 4.20 to at most 4.70, preferably at least 4.30 to at most 4.50, preferably 4.40.