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EP-3674310-B1 - IMMUNOGLOBULIN PURIFICATION USING PRE-CLEANING STEPS

EP3674310B1EP 3674310 B1EP3674310 B1EP 3674310B1EP-3674310-B1

Inventors

  • FELFÖLDI, Ferenc
  • BENKÖ, Zsuzsa
  • GÁSPÁR, Melinda

Dates

Publication Date
20260506
Application Date
20150309

Claims (15)

  1. Method for purifying an immunoglobulin from a sample comprising the immunoglobulin and at least one impurity, the method comprising the following steps in the following order: (a) exposing the sample to anion exchange chromatography and obtaining the immunoglobulin, which is not bound to the anion exchange chromatography resin, in the flow-through; (b) exposing the flow-through obtained in step (a) either to Protein A affinity chromatography, wherein the immunoglobulin is bound to the Protein A affinity chromatography resin, and obtaining the immunoglobulin in the eluate by eluting the protein from the Protein A affinity chromatography resin, or to Mixed Mode chromatography, wherein the immunoglobulin is bound to the Mixed Mode chromatography resin, and obtaining the immunoglobulin in the eluate by eluting the protein from the Mixed Mode chromatography resin; (c) exposing the eluate obtained in step (b), or a composition derived therefrom and obtained after one or more further processing steps performed after step (b), to cation exchange chromatography, wherein the immunoglobulin is bound to the cation exchange chromatography resin, and obtaining the immunoglobulin in the eluate by eluting the protein from the cation exchange chromatography resin, wherein the sample is harvested cell culture fluid, cell culture supernatant or pretreated cell culture supernatant.
  2. Method of claim 1, wherein the anion exchange chromatography of step (a) is strong anion exchange chromatography, wherein the ligand of the strong anion exchange chromatography resin preferably is selected from the group consisting of quaternary aminoethyl (QAE) moieties, quaternary ammonium moieties and trimethylammonium moieties, wherein more preferably the ligand of the strong anion exchange chromatography resin is trimethylammonium (-N(CH3)3+).
  3. Method of any of the preceding claims, wherein the Protein A affinity chromatography resin comprises an alkali-stabilized Protein A derivative bound to highly cross-linked agarose.
  4. Method of any of the preceding claims, wherein the Mixed Mode chromatography resin of step (b) comprises 4-mercapto-ethyl-pyridine as a ligand.
  5. Method of any of the preceding claims, wherein the flow through of step (a) is not temporarily stored in a collecting vessel but is immediately passed to the chromatography resin of step (b).
  6. Method of any of the preceding claims, wherein step (c) is followed by the following step: (d) exposing the eluate obtained in step (c) to Mixed Mode chromatography, wherein the immunoglobulin is bound to the Mixed Mode chromatography resin, and obtaining the immunoglobulin in the eluate by eluting the protein from the Mixed Mode chromatography resin.
  7. Method of claim 6, wherein the Mixed Mode chromatography resin of step (d) comprises positively charged ligands, preferably wherein the positively charged ligands are N-Benzyl-N-methyl ethanol amine moieties with the following formula:
  8. Method of any of the preceding claims, wherein eluting in step (b) is performed by applying a pH shift or pH gradient which elutes the immunoglobulin.
  9. Method of any of the preceding claims, wherein eluting of step (c) is performed by applying a pH shift or pH gradient which elutes the immunoglobulin.
  10. Method of any of the preceding claims, wherein step (a) is preceded by the following steps: (i) centrifuging the sample, wherein the immunoglobulin is obtained in the supernatant; (ii) filtrating the supernatant obtained in step (i), wherein the immunoglobulin is obtained in the filtrate.
  11. Method of claim 10, wherein filtrating in step (ii) is depth filtrating and/or sterile filtrating.
  12. Method of any of the preceding claims, wherein the method for purifying an immunoglobulin is carried out on a large scale.
  13. Method of any of the preceding claims, wherein the immunoglobulin is IgG1 and wherein the Fc part of the IgG1 is human.
  14. Method of any of the preceding claims, wherein the cell culture fluid is obtained from recombinant CHO cell culture.
  15. Method of any of the preceding claims, wherein the method comprises a further step of incubating the eluate of step (b) at low pH of 2.5 to 4.5 for a defined time, wherein step (a), the step of incubation of the eluate of step (b) at low pH and step (c) result in a cumulative log10 reduction factor of at least 15 with respect to enveloped viruses.

Description

FIELD OF THE INVENTION The present invention relates to methods of purifying antibodies from a cell culture-derived composition using pre-cleaning steps prior to a capture step and/or further polishing steps subsequent to the capture step. BACKGROUND OF THE INVENTION The selection of efficient and economic downstream sequences for purification of polypeptides produced by recombinant DNA technology is a crucial step in the development of every new biopharmaceutical intended for therapeutic use. In the recent past the need for large scale purification processes for monoclonal antibodies (mabs), due to their exceptionally high therapeutic dosages in medical use, has been further intensified with the use of improved cell culture methods resulting in higher cell densities and higher expression rates. The increasing concentrations in the culture fluids of product and contaminants set higher demands on the capture chromatography, on its preceding sample clarification steps and on the subsequent polishing chromatographies. The entire downstream process has to: (i) manage an increased mass of product, (ii) efficiently remove increased process- and product-related impurities to below defined acceptance criteria, and (iii) maintain economic yields and sufficient quality of the mab. Usually, the downstream process accounts for a major part of the total manufacturing costs of therapeutic antibodies. The mabs in crude fractions are typically associated with impurities such as host cell proteins (HCP), host cell DNA, viruses, aggregates, other undesired product variants, and various leachates from process materials. The presence of these impurities is a potential health risk for patients, and hence their absence from the final product is a regulatory requirement. Only very low residual amounts will be tolerated. The classical procedure for purifying cell-culture derived polypeptides follows the sequence of capture-intermediate-polishing chromatographies, accompanied by filtration, concentration or dialysis steps at various positions of the downstream sequence. In recent years platform approaches have been successfully established in the field of mab purification. Since mabs are a well-defined class of glycoproteins possessing common physicochemical properties, the use of a generic platform process is reasonable (Kelly B 2009). Such a universal process, with more or less product-specific adaptions, can be applied to many mabs, especially for those immunoglobulins of the same class or subclass, e.g. IgG1. One of the most frequent capture steps used for mab purification is affinity chromatography with Protein A. This capture offers exceptional selectivity for Fc-bearing molecules, thereby removing more than 99.5% of contaminants in a single step. However, besides its advantages, two disadvantages should also be mentioned. One drawback is the undesired leaching of Protein A or fragments of Protein A which are known to be toxic (Gagnon P 1996). The other disadvantage is the high cost of this type of resin, particularly at the industrial scale necessary to purify therapeutic antibodies. A Protein A resin is approximately 30 times more expensive than an ion exchange resin. It was calculated that for the downstream processing of a 10 m3 cell culture the cost for the Protein A affinity chromatography is about 4-5 million USD (Farid SS 2009). Many solutions have been published to overcome the problem of leached Protein A (Gagnon P 1996; Fahrner RL 2001). Several approaches related to post-Protein A chromatographic steps which remove leached Protein A, such as anion exchange chromatography used in binding mode (EP0345549) or flow-through mode (WO2004076485), cation exchange chromatography (WO2009058812), hydrophobic interaction chromatography (WO9522389), or combinations of chromatographies, for instances ion exchange chromatography followed by hydrophobic interaction chromatography (WO2010141039), anion exchange chromatography followed by cation exchange chromatography (WO2011090720), or cation exchange chromatography and Mixed Mode chromatography in any order (WO2011150110). Since the required overall degree of purity for a therapeutic antibody is extremely high, a typical platform purification scheme consists of at least two post-Protein A chromatographies which are usually selected from cation exchange chromatography, anion exchange chromatography in flow-through, and hydrophobic interaction chromatography (Fahrner RL 2001, Kelly B 2009, WO9522389, WO2009138484, WO2010141039, WO2011017514, WO2011090720). Other approaches reduce the leachates already during the Protein A affinity chromatography by using special wash steps removing leached Protein A prior to eluting the immunoglobulin. Many intermediate wash buffers were developed containing salts or additional components, for example hydrophobic electrolytes such as tetramethylammonium chloride (Fahrner RL 2001). Some methods take effect closer to the source of the Protein A leac