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EP-4739416-A1 - METHOD OF PURIFICATION

EP4739416A1EP 4739416 A1EP4739416 A1EP 4739416A1EP-4739416-A1

Abstract

The present disclosure relates to methods of isolating a protein-of-interest enriched solution, such as an immunoglobulin G (IgG), and a plurality of protein-of-interest depleted fractions from a protein mixture, such as plasma or a fraction thereof, using continuous chromatography. The present disclosure also relates to a continuous chromatography system for separating a protein-of-interest from a protein mixture, formulations and uses of the purified products thereof.

Inventors

  • DOLLINGER, PETER
  • HEERING, Dorota
  • PAL CHOWDHURY, Nilanjan
  • PILZ, Tessa

Assignees

  • CSL Behring AG

Dates

Publication Date
20260513
Application Date
20240704

Claims (15)

  1. 1. A method of isolating a protein-of-interest enriched solution and a plurality of protein - of-interest depleted fractions from a protein mixture comprising a protein-of-interest using a continuous chromatography system comprising at least four separation units, each separation unit comprising a respective chromatography medium capable of separating and retaining a portion of a protein-of-interest from a protein mixture, wherein the method comprises: performing at least one chromatography cycle comprising a series of cycle segments, wherein a cycle segment comprises: a. loading a feed comprising the protein mixture comprising the protein-of-interest onto a feed separation unit, wherein a portion of a protein-of-interest content of the feed is retained in the feed separation unit, thereby producing a feed flow through; b. directing the feed flow through from the feed separation unit onto a feed loopback separation unit via a feed flow through feedback loop, wherein at least a portion of a protein-of-interest content of the feed flow through is retained in the feed loopback separation unit, thereby producing a protein-of-interest depleted feed flow through fraction; c. loading a wash buffer onto a wash separation unit containing retained protein-of- interest from a previous cycle segment, thereby producing a wash flow through; d. directing the wash flow through from the wash separation unit onto a wash loopback separation unit via a wash flow through feedback loop, wherein a portion of a protein-of-interest content of the wash flow through is retained in the wash loopback separation unit, thereby producing a protein-of-interest depleted wash flow through fraction; and e. loading an elution buffer onto the wash separation unit and/or onto an elution separation unit to elute retained protein-of-interest from the respective chromatography medium, thereby producing a protein-of-interest enriched solution, wherein the feed flow through and the wash flow through are maintained separate from each other.
  2. 2. The method of claim 1, wherein a function of one or more of the at least four separation units varies between successive cycle segments such that each separation unit performs phases of the chromatography cycle, the phases including: loading, washing, eluting, equilibrating, feed loopback, wash loopback, and/or combinations thereof, optionally wherein the functions of each separation unit vary such that each separation unit performs each of the phases in a predetermined sequence.
  3. 3. The method of claim 1 or claim 2, wherein performing the chromatography cycle comprises loading the feed comprising the protein mixture onto each of the at least four separation units successively, wherein the loading of the feed onto each successive separation unit demarks a respective cycle segment, optionally wherein loading the feed is performed substantially continuously across successive cycle segments such that a flow rate of the feed during the chromatography cycle is substantially constant.
  4. 4. The method of any one of claims 1 to 3, comprising determining a protein concentration of one or more of the feed flow-through, the wash flow through, the protein-of-interest depleted feed flow through fraction, the protein-of-interest depleted wash flow through fraction and the protein-of-interest enriched solution, optionally wherein determining the protein concentration comprises performing a spectroscopic analysis, optionally wherein determining the protein concentration comprises one or more of: (a) performing absorbance spectroscopy; (b) performing reflection spectroscopy; (c) detecting an optical density (OD), optionally wherein determining the protein concentration comprises detecting a UV absorption, preferably comprising measuring UV absorption at one or more of 300 nm, 280 nm, 245 nm, 224 nm, and 214 nm; (d) performing Raman spectroscopy; and/or (e) performing near-infrared (NIR) spectroscopy.
  5. 5. The method of any one of claims 1 to 4, wherein the protein-of-interest depleted wash flow through fraction is further fractionated into a low protein concentration fraction and a high protein concentration fraction, optionally wherein the further fractionation is based on a determined protein concentration of the protein-of-interest depleted wash flow through fraction and/or a volume of wash buffer passed over the chromatography media.
  6. 6. The method of claim 5, wherein the method further comprises pooling the protein-of- interest depleted feed flow through fraction and the high protein concentration fraction from the further fractionated protein-of-interest depleted wash flow through fraction to produce a protein-of-interest depleted preparation.
  7. 7. The method of any one of claim 6, wherein the protein-of-interest depleted preparation comprises one or more plasma protein products, for example immunoglobulin G (IgG), an apolipoprotein Al, an albumin, a serine protease, a plasmin, plasminogen, a FXa, an alpha- 1- antitrypsin, an IgA, an IgM, a factor VIII, a fibrinogen, a von Willebrand factor, an activated clotting factor, factor XIII, a contact system factor, a PKA, a factor IX, a prothrombin complex, a Cl esterase inhibitor, a protein C, an anti-thrombin III, a RhD immunoglobulin protein product, alpha acid glycoprotein, haptoglobin, hemopexin, transferrin, Factor H, coagulation factors such as Factor VII, Factor VIII and Factor IX and combinations thereof, optionally wherein the method further comprises purifying one or more of the plasma protein products from the protein-of-interest depleted preparation, for example, wherein the one or more plasma protein products is further purified from the protein-of-interest depleted preparation by one or more purification steps selected from the group consisting of precipitation, such as ethanol, ammonium sulphate and octanoic acid fractionation; chromatography; viral inactivation; viral filtration; ultrafiltration/diafiltration and combinations thereof, for example, wherein the one or more plasma protein products is further purified from the protein-of-interest depleted preparation using a continuous chromatography system.
  8. 8. The method of any one of claims 1 to 7, wherein the continuous chromatography system is selected from the group consisting of simulated moving bed (SMB) chromatography and periodic counter-current chromatography (PCC) and/or wherein one or more of the chromatography media is selected from the group consisting of an ion exchange chromatography medium, an affinity chromatography medium, a hydrophobic interaction chromatography medium, a mixed mode chromatography medium, an adsorption chromatography medium and a partition chromatography medium, wherein, for example: (a) the ion exchange chromatography medium is an anion exchange chromatography medium or a cation exchange chromatography medium; (b) one or more of the chromatography media is selected from the group consisting of a membrane, a monolith and a resin; (c) one or more of the chromatography media comprise a ligand capable of specifically binding the protein-of-interest.
  9. 9. The method of any one of claims 1 to 8, wherein one or more of the chromatography media comprise: (A) a ligand capable of specifically binding to human IgG, optionally wherein the ligand is capable of specifically binding to a constant domain of human IgG, optionally wherein the ligand is capable of specifically binding to a CHI domain, a CH2 domain, a CH3 domain, a CH4 domain, or combinations thereof; for example, a camelid-derived single domain [VHH] antibody fragment, optionally wherein the ligand comprises: (a) an amino acid sequence set forth in SEQ ID NO: 1 or a sequence comprising at least 50% amino acid identity to a sequence set forth in SEQ ID NO: 1; (b) a framework region comprising an amino acid sequence set forth in SEQ ID NO: 1 or a sequence comprising at least 50% amino acid identity to a sequence set forth in SEQ ID NO: l; or (c) an amino acid sequence that comprises framework regions (FR)1, 2, 3 and 4, and complementarity determining regions (CDRs)l, 2 and 3, that are operably linked in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein: i. the CDR1 has an amino acid sequence selected from the group consisting of SEQ ID No: 2 or an amino acid sequence that differs from SEQ ID NO: 2 in one or two of the amino acid residues; ii. the CDR2 has an amino acid sequence having at least 80% sequence identity with an amino acid sequence of SEQ ID NO: 3; and, iii. the CDR3 has an amino acid sequence having at least 80% sequence identity with an amino acid sequence of SEQ ID NO: 4; and, wherein each of the framework regions has at least 50% amino acid identity with the framework amino acid sequence of any one of SEQ ID NO: 1, and iv. wherein each of the framework regions has at least 50% amino acid identity with the framework amino acid sequence of SEQ ID NO: 1, optionally wherein the antigen binding protein specifically binds to the Fc domain of a human IgG molecule and does not bind to an IgG molecule of murine origin or bovine origin; and/or (B) a matrix selected from the group consisting of a cross-linked poly(styrene- divinylbenzene) matrix and an agarose-based matrix.
  10. 10. The method of claim 8 or claim 9, wherein one or more of the separation units comprises an affinity chromatography medium, for example a ligand capable of specifically binding to human IgG, optionally wherein the ligand is capable of specifically binding to a constant domain of human IgG, optionally wherein the ligand is capable of specifically binding to a CHI domain, a CH2 domain, a CH3 domain, a CH4 domain, or combinations thereof, and one or more of the separation units comprises an ion exchange chromatography medium.
  11. 11. The method of any one of claims 1 to 10, wherein: (a) one or more of the separation units is loaded with the protein mixture at a concentration above a dynamic binding capacity (DBC) of the chromatography medium; (b) one ore more of the separation units are loaded with the protein-of-interest at a concentration up to the DBC of the chromatography medium; (c) the chromatography media have a total bed height of between 2 cm and 30 cm; (d) one or more of the separation units are selected from the group consisting of a column, a cassette, a capsule and a filter holder; for example, a column, such as a column with a diameter of between 5 cm and 200 cm; (e) the wash buffer has a pH of between 5 and 10 and a dissociation constant (pKa) between 6.8 and 8.5 at 25°C, preferably wherein the wash buffer comprises a buffering agent selected from a group consisting of sodium dihydrogen phosphate, imidazole, Tris, glycylglycine, 3-morpholinopropane-l-sulfonic acid (MOPS), piperazine -N,N'-bis(2- ethanesulfonic acid) (PIPES), 2-[(2-Hydroxy-l,l- bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES), bis[(2- hydroxyethyl)amino] acetic acid (Bicine), 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES), sulfurous acid, 4-(2-Hydroxyethyl)-l- piperazinepropanesulfonic acid (EPPS), N-(Hydroxyethyl)piperazine-N'-2- hydroxypropanesulfonic acid (HEPPSO), 4-(N-Morpholino)butanesulfonic acid (MOBS), Piperazine -N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), N- [Tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid (TAPSO), Tricine, triethanolamine (TEA) and combinations thereof; and optionally wherein the buffering agent is at a concentration of between 5 mM to 200 mM; (f) the wash buffer further comprises sodium chloride and/or a divalent salt at a concentration of up to 1000 mM, for example, 20 mM sodium dihydrogen phosphate, 500 mM sodium chloride and is at a pH of 7.4; (g) the elution buffer has a pH of between 3 and 5, optionally wherein the elution buffer is or comprises a phosphate buffer, an acetate buffer or an acetic acid buffer; (h) the elution buffer contacts the chromatography medium for up to 5 minutes; (i) the method further comprises equilibrating at least one chromatography medium with an equilibration buffer before loading the protein mixture, optionally wherein the equilibration buffer has a pH of between 7 and 8, such as pH of 7.4, optionally wherein the equilibration buffer comprises 20 mM sodium dihydrogen phosphate, 1000 mM sodium chloride and is at a pH of 7.4; and/or (j) the method further comprises regenerating one or more of the chromatography media.
  12. 12. The method of any one of claims 1 to 11, wherein: (a) the protein-of-interest is a plasma protein, for example a plasma protein selected from the group consisting of immunoglobulin G (IgG), an apolipoprotein Al, an albumin, a serine protease, a plasmin, plasminogen, a FXa, an alpha- 1- antitrypsin, an IgA, an IgM, a factor VIII, a fibrinogen, a von Willebrand factor, an activated clotting factor, factor XIII, a contact system factor, a PKA, a factor IX, a prothrombin complex, a Cl esterase inhibitor, a protein C, an anti-thrombin III, a RhD immunoglobulin protein product, alpha acid glycoprotein, haptoglobin, hemopexin, transferrin, Factor H, a coagulation factor, a serine protease inhibitor and combinations thereof; (b) the protein mixture is plasma or a plasma fraction, for example , the plasma fraction is selected from a group consisting cryo-rich plasma, cryo-poor plasma, Supernatant I (SN I), Cohn Fraction II (Fr II), Cohn Fraction II+III (Fr II+III), Cohn Fraction I+II+III (FrI+II+III), Kistler/Nitschmann Precipitate A (KN A), Kistler/Nitschmann Precipitate B (KN B), Kistler/Nitschmann Precipitate of Supernatant B (KN B+l), and combinations thereof, optionally wherein the plasma or plasma fraction is thawed at a temperature of at least 32°C, for example, the plasma or plasma fraction is at a temperature in the range of from 2°C to 28°C before loading, such as a temperature of 21°C; (c) at least 75% of the protein-of-interest is recovered from the protein mixture; (d) the protein-of-interest is IgG, and at least 75% of IgG is recovered from the plasma or fraction thereof; (e) the protein-of-interest is IgG, and the protein-of-interest enriched solution comprises a purity of at least 95% IgG; (f) the protein-of-interest is IgG, and the protein-of-interest enriched solution comprises polyvalent IgG; and/or (g) the protein-of-interest is IgG, and the IgG is further purified from the protein-of-interest enriched solution, for example, the IgG is further purified from the protein-of-interest enriched solution by one or more purification steps selected from the group consisting of precipitation, such as ethanol, ammonium sulphate and octanoic acid fractionation; chromatography; viral inactivation; viral filtration; ultrafiltration/diafiltration and combinations thereof.
  13. 13. A pharmaceutical composition comprising: (a) a protein-of-interest purified or produced by a method of any one of claims 1 to 12; (b) IgG purified or produced as a protein of interest by a method of any one of claims 1 to 12; or (c) one or more plasma protein products from the protein-of-interest depleted preparation purified or produced by a method of claims 6 or 7, for example wherein the plasma protein product is selected from a group consisting of an apolipoprotein Al, an albumin, a serine protease, a plasmin, plasminogen, a FXa, an alpha- 1- antitrypsin, an IgA, an IgM, a factor VIII, a fibrinogen, a von Willebrand factor, an activated clotting factor, factor XIII, a contact system factor, a PKA, a factor IX, a prothrombin complex, a Cl esterase inhibitor, a protein C, an anti-thrombin III, a RhD immunoglobulin protein product, alpha acid glycoprotein, haptoglobin, hemopexin, transferrin, Factor H, coagulation factors such as Factor VII, Factor VIII and Factor IX and combinations thereof.
  14. 14. The pharmaceutical composition of claim 13, for use in treating, preventing and/or delaying progression of a condition in a subject, such as a condition selected from a group consisting of immune conditions, particular autoimmune diseases and certain neurological diseases. These conditions include Rheumatoid arthritis, Systemic Lupus Erythematosus (SLE), Antiphospholipid syndrome, immune thrombocytopenia (ITP), Kawasaki disease, Guillain Barre syndrome (GBS), multiple sclerosis (MS), chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy (MMN), myasthenia gravis (MG), skin blistering diseases, scleroderma, Dermatomyositis, Polymyositis, Alzheimer's Disease, Parkinson's Disease, Alzheimer's Disease related to Downs Syndrome, cerebral amyloid angiopathy, Dementia with Lewy bodies, Fronto- temporal lobar degeneration, vascular dementia, cell and organ transplant and combinations thereof.
  15. 15. A continuous chromatography system for separating a protein-of-interest from a protein mixture, wherein the system comprises: at least four separation units, each separation unit comprising: an inlet; an outlet; a fluid flow path extending through the separation unit from the inlet to the outlet; and a chromatography medium in contact with the fluid flow path and capable of separating at least a portion of the protein-of-interest from the protein mixture; a switching valve arrangement configured to: selectively direct flow of one or more of a feed comprising the protein mixture, a wash buffer and an eluting agent to the inlet of any one of the separation units; selectively direct flow from the outlet of each separation unit to: at least one feedback loop in fluid communication with the inlet of another one of the separation units; at least one collection flow path; and a waste flow path; and a valve control system operable to control the switching valve arrangement, wherein the valve control system is configured to operate the switching valve arrangement such that: the feed is directed to the inlet of a selected first separation unit; the wash buffer is directed to the inlet of a selected second separation unit; a feed flow through from the outlet of the first separation unit and a wash flow through from the outlet of the second separation unit are directed to respective feedback loops, wherein the feed flow through and the wash flow through are maintained separate from each other.

Description

METHOD OF PURIFICATION RELATED APPLICATION DATA This application claims priority from European Patent Application No 23183376.5 filed on 4 July 2023 and entitled “Method of Purification”, the entire contents of which are hereby incorporated by reference. SEQUENCE LISTING The present application is filed together with a Sequence Listing in electronic form. The entire contents of the Sequence Listing are hereby incorporated by reference. FIELD The present disclosure relates to methods of isolating a protein-of-interest enriched solution, such as an immunoglobulin G (IgG), and a plurality of protein-of- interest depleted fractions from a protein mixture, such as plasma or a fraction thereof, using continuous chromatography. The present disclosure also relates to a continuous chromatography system for separating a protein-of-interest from a protein mixture, formulations and uses of the purified products thereof. BACKGROUND Protein purification is one of the most costly aspects of therapeutic protein production. Existing methods of protein purification include chromatography (e.g. affinity chromatography, anion exchange chromatography, hydrophobic interaction chromatography, SE-HPLC) and non-chromatography (e.g. precipitation and liquid extraction) purification methods. Major obstacles of existing methods are the high cost and time involved in purification and the need to ensure that the product is of a suitable quality (e.g. purity and stability) for therapeutic use. For example, affinity resins used in affinity chromatography can have relatively low binding capacity and chromatography purification from an average size batch can reach volumes of several hundred litres, being a huge capital investment in the amount of resin used, the infrastructure to handle and pack the chromatography columns, along with the running costs. In some instances, co-purification of one or more proteins from the same protein mixture is required. For example, during plasma fractionation, blood plasma fractions containing one or more plasma proteins (e.g., immunoglobulin G (IgG), albumin and coagulation factors) are obtained from the same blood plasma sample. Each of these plasma fractions can then be processed for therapeutic use. To date, several methods of plasma fractionation have been developed. Current methods require extremely high volumes of raw material and can often result in poor yield of some plasma protein fractions due to additional processing steps required to remove protein aggregates. At present, up to 70-75% of the IgG present in plasma may be recovered from plasma using existing technologies. It will therefore be apparent to the skilled person that there is a need in the art for improved methods of fractionating proteins from protein mixtures, e.g., purifying IgG from plasma or fractions thereof. SUMMARY The present disclosure is based on the inventors’ identification of a method of purifying immunoglobulin G (IgG) from plasma or a fraction thereof with efficient isolation of undiluted IgG depleted fractions. The method allows IgG to be recovered from plasma or a fraction thereof at high yields (e.g., > 75%) and high purity (e.g., > 95%), whilst also permitting a high recovery rate (e.g., >90%) of IgG depleted fractions with low dilution (e.g., < 10%) for further downstream purification. In particular, the inventors have found that use of continuous affinity chromatography (e.g., simulated moving bed (SMB) chromatography) comprising at least four columns and two feedback loops permits separation of two outlet streams (i.e., feed/load flow-through and wash flow-through) in parallel, enabling the efficient fractionation of IgG depleted undiluted plasma material from the diluted wash buffer fraction. The inventors found that directing the load flow-through onto another column over the first loopback enables recovery of the IgG product at high yield and high purity and also permits collection of the flow through undiluted for the isolation of further proteins of interest. Additionally, the inventors found that directing the wash flow-through over the second loopback permits fractionation of a low diluted IgG-depleted flow-through fraction. The low-diluted IgG- depleted flow-through fraction can then be pooled with the main IgG-depleted flowthrough fraction, increasing the recovery rate. The method advantageously enables the protein-of-interest to be isolated as well as the isolated protein-of-interest depleted plasma (i.e., the undiluted IgG depleted fractions) to be further processed for isolation and processing of further plasma products with high efficiency and yields. Accordingly, the findings by the inventors provide the basis for a method of isolating a protein-of-interest enriched solution and a plurality of protein-of-interest depleted fractions from a protein mixture using continuous chromatography. It will be apparent to the skilled person from the disclosure herein that the methods of the disclosure permit