Methods of purifying immunoglobulins comprising n-terminal glutamine

Abstract

The present invention relates to a method for the purification of immunoglobulins comprising N- terminal glutamine. In particular, the method comprises an incubation step embedded in a sequence of chromatographic methods comprising a capture step, an intermediate chromatography step, and a polishing step. The implementation of an incubation step embedded in the sequence of chromatographic purification methods promotes the conversion of N-terminal glutamine to pyro glutamate, thereby reducing the amount of N-terminal glutamine to be depleted by cation exchange chromatography while overall product yield is increased. In addition, the incubation may be conducted prior or following the intermediate chromatography step.

Description

[0001] Methods of purifying immunoglobulins comprising N-terminal glutamine

[0002] Field of the invention

[0003] The present invention pertains to downstream purification methods of immunoglobulins comprising N-terminal glutamine.

[0004] Background of the invention

[0005] An efficient and economic downstream sequence for the purification of monoclonal antibodies is a crucial step in every biopharmaceutical development. The need for large scale purification processes for monoclonal antibodies increased in the recent years because of the high therapeutic dosage in medical use. The classical downstream purification procedure for cellculture derived monoclonal antibodies follows the sequence of capture chromatography, intermediate chromatography, and polishing chromatography, accompanied by filtration, concentration, and dialysis at various steps of the downstream sequence. The downstream process has to (i) manage the increased amount of product, (ii) efficiently remove the process- and product-related impurities, (iii) maintain economic yields, and (iv) ensure the quality of monoclonal antibodies.

[0006] Therapeutic monoclonal antibodies may contain a large number of post-translational modifications, some of which may impact efficacy, safety, and immunogenicity. Pyroglutamate (PyroGlu) is a cyclic amino acid formed at the N-terminus of some proteins and biological peptides. PyroGlu formation occurs through the rearrangement of the originally synthesized glutamine (Gin) or glutamate (Glu) residues at this position, however, PyroGlu formation is more prevalent at N-terminal Gin residues (Liu et al. 2011). Although the reaction can occur spontaneously, the enzyme glutaminyl cyclase which can catalyse the reaction is found in plants and animals (Schilling et al. 2008). PyroGlu is a common post-translational modification at the N-terminus of monoclonal antibodies. An incomplete conversion of Gin to PyroGlu results in heterogeneity in monoclonal antibodies which can be detected as multiple peaks in ion exchange chromatography or isoelectric focusing. Differences in heterogeneity may indicate a lack of process control. Moreover, charge variants such as PyroGlu are critical quality attributes as defined in ICHQ8-11 just like size-related variants, oxidation-related variants, glycosylation, structural variants, or process-related impurities, which must be within an appropriate limit, range, or distribution to ensure product quality.

[0007] Based on differences in the isoelectric point of N-terminal Gin and PyroGlu, the two charge variants can be separated by ion exchange chromatography, e.g., cation exchange chromatography. Typically, the ratio of N-terminal Gin can be reduced by discarding the cation exchange chromatography fractions which are rich in N-terminal Gin. However, cutting of the N- terminal Gin containing fractions drastically reduces the overall production yield.

[0008] Various studies address the formation of PyroGlu from N-terminal Gin, with a focus on pharmaceutical stability studies. Chelius et al. (2006) identified the formation of PyroGlu from N-terminal Glu in the heavy chains and light chains of several antibodies that occurs after a few weeks of incubation at increased temperatures of 37 °C and 45 °C, respectively. A buffer analysis showed a pH dependency of the PyroGlu formation with less PyroGlu formation at a pH of 6 but increased PyroGlu formation at a pH of 4 and a pH of 8. Yu et al. (2006) showed PyroGlu formation at the N-terminus of the light and heavy chain of antibodies over a time of three months and considered the PyroGlu formation as part of a degradation pathway during antibody production and storage depending on pH, time, and temperature. Dick et al. (2007) demonstrated that the formation of PyroGlu from N-terminal Gin occurred spontaneously. Most of the conversion occurred because of bioreactor conditions, with less contribution of the purification process. An alternative approach was demonstrated by Xu et al. (2013) in which glutaminyl-peptide cyclase, an enzyme found in plants, animals, and humans, was used to convert the N-terminal Gin to PyroGlu to identify charge variants of monoclonal antibodies of other chemical nature.

[0009] The amino acid sequence of tocilizumab reveals that the molecule contains a Gin amino acid at the N-terminus which may be spontaneously converted to PyroGlu. Tocilizumab is a recombinant, humanized, anti-human monoclonal antibody of the immunoglobulin G1 subclass directed against membrane-bound and soluble interleukin-6 receptors (IL-6R) (Sheppard et al. 2017). The molecule was generated by grafting the complementarity determining regions of mouse anti-human IL-6 receptor onto human lgG1 (Tanaka et al. 2011). Tocilizumab inhibits the binding of IL-6 to its receptor, by doing so it inhibits the pro-inflammatory activity of cytokine by competing with both, the soluble and the membrane-bound forms of the human IL-6 receptor (Scheinecker et al. 2009, Sheppard et al. 2017). The human IL-6 is referred to as a pleiotropic cytokine. IL-6 contributes to host defence against pathogens and is involved in a broad spectrum of biological events, such as immune responses, haematopoiesis, and acute-phase reactions, whereas dysregulation of IL-6 production has been implicated in the pathogenesis of various autoimmune and chronic inflammatory diseases (Tanaka et al. 2011). IL-6 exerts its signalling by a receptor system which consists of the ligand-binding membrane protein IL-6R and the signal-transducing transmembrane protein gp130. On target cells, IL-6 binds to IL-6R. Signalling occurs when the IL-6 / IL-6R complex associates with gp130 and gp130 activation leads to the expression of acute phase protein genes. A soluble form of IL-6R which lacks the cytoplasmic domain can also bind to gp130 following IL-6 binding and thus trigger downstream signalling events (Sheppard et al. 2017, Tanaka et al. 2011). Tocilizumab is approved in Europe and the US for the treatment of rheumatoid arthritis, systemic juvenile idiopathic arthritis, polyarticular juvenile idiopathic arthritis, giant cell arteritis, and cytokine release syndrome. A process for downstream purification of an anti-IL-6 receptor antibody was disclosed in W02019043096A1 . Via the sequence of Protein A affinity chromatography followed by a first mixed-mode chromatography and a second mixed-mode chromatography, both in flow-through mode, product-related and process-related impurities were reduced. However, N-terminal PyroGlu formation was not addressed by the purification process.

[0010] It is an object of the present invention to obtain an immunoglobulin with N-terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine.

[0011] It is an object of the present invention to obtain an immunoglobulin with N-terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine and thus, preventing heterogeneity in the final immunoglobulin formulation.

[0012] It is also an object of the present invention to provide a method of obtaining an immunoglobulin with N-terminal pyro glutamate from a composition comprising the immunoglobulin with N- terminal glutamine while maintaining acceptable levels of yield.

[0013] Summary of the invention

[0014] The present invention relates to a method for the purification of immunoglobulins with N-terminal glutamine. The method comprises an incubation step embedded in a sequence of chromatographic methods comprising a capture step, an intermediate chromatography step, and a polishing step.

[0015] The key problem of purifying N-terminal glutamine containing immunoglobulins lies in the fact that the incomplete conversion of N-terminal glutamine to pyro glutamate produces heterogeneity in the antibody which may impact the product quality and quantity.

[0016] The implementation of an incubation step embedded in the sequence of chromatographic purification methods promotes the conversion of N-terminal glutamine to pyro glutamate, thereby reducing the amount of N-terminal glutamine to be depleted by cation exchange chromatography while overall product yield is increased.

[0017] The methods of the invention comprise specific conditions to reduce the amount of N-terminal glutamine variants.

[0018] The incubation may be conducted prior or following the intermediate chromatography step. Preferably, the capture step is a protein A chromatography step. Preferably, the intermediate chromatography step is a mixed-mode anion exchange chromatography or an anion exchange chromatography in flow-through mode. Preferably, the incubation may be performed with the load or the flow-through of the mixed-mode anion exchange chromatography. Preferably, the incubation is performed with the flow-through of the mixed-mode anion exchange chromatography. Preferably, the polishing step is a mixed-mode cation exchange chromatography or cation exchange chromatography. The incubation may be conducted prior to the polishing step. Preferably, the polishing chromatography step is a cation exchange chromatography in bind-elute mode. By using a strong cation exchange chromatography, the desired charge variant profile of the immunoglobulin within a composition can be achieved. Performing the incubation step before the cation exchange chromatography results in a large amount of glutamine being converted to pyro glutamate, therefore less N-terminal glutamine needs to be removed in the subsequent cation exchange chromatography step, thereby increasing the overall product yield.

[0019] In one aspect, the present invention provides a method for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0020] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0021] (ii) a pH adjustment step to obtain a pH adjusted composition,

[0022] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or

[0023] (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0024] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixedmode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0025] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), said adjusted pH is maintained throughout steps (ii) to (v) within 2.0 units, preferably 1 .8 units such as 1 .6 units of said adjusted pH.

[0026] In another aspect, the present invention provides a method for obtaining an immunoglobulin with N-terminal pyro glutamate from a composition comprising the immunoglobulin with N- terminal glutamine, the method comprising the following sequential steps:

[0027] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0028] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition, (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or

[0029] (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0030] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0031] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v).

[0032] In another aspect, the present invention provides a method for obtaining an immunoglobulin with N-terminal pyro glutamate from a composition comprising the immunoglobulin with N- terminal glutamine, the method comprising the following sequential steps:

[0033] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0034] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0035] (iii) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0036] (iv) subjecting the incubated flow-through obtained in step (iii) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0037] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v). In preferred embodiments, the immunoglobulin is an lgG1 , preferably an anti-IL-6 receptor antibody.

[0038] In preferred embodiments, the immunoglobulin is tocilizumab. Tocilizumab is further described in WO9219759A1.

[0039] In preferred embodiments, the light chain of tocilizumab is characterized by SEQ ID NO: 1 and the heavy chain of tocilizumab is characterized by SEQ ID NO: 2.

[0040] SEQ ID NOs: 1 and 2 are as follows:

[0041] SEQ ID NO: 1 >Tocilizumab light chain:

[0042] DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPS RFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0043] SEQ ID NO: 2 >Tocilizumab heavy chain:

[0044] QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTY NPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG

[0045] In preferred embodiments, the immunoglobulin comprises an amino acid sequence having at least 90% identity to SEQ ID NO:1 and an amino acid sequence having at least 90% identity to SEQ ID NO:2.

[0046] In preferred embodiments, the immunoglobulin comprises an amino acid sequence having at least 99% identity to SEQ ID NO:1 and an amino acid sequence having at least 99% identity to SEQ ID NO:2.

[0047] In preferred embodiments, the incubation step reduces the N-terminal glutamine by at least 70 %, at least 80 %, at least 90%, at least 95% or at least 99%.

[0048] In a preferred embodiment, the yield of the immunoglobulin is higher as compared to the same method without an incubation step of (iii-a) or (iii-b), wherein the yield of the immunoglobulin is given by the main peak in % as measured by ion exchange-high-performance liquid chromatography (HPLC-IEX).

[0049] In another preferred embodiment, the yield of the immunoglobulin is higher as compared to the same method without an incubation step of (iii-b), wherein the yield of the immunoglobulin is given by the main peak in % as measured by ion exchange-high-performance liquid chromatography (HPLC-IEX).

[0050] In preferred embodiments, the pH adjustment step (ii) above, comprises adjusting the pH to 5.0 to 7.0. In preferred embodiments, the yield of the immunoglobulin is higher as compared to the same method in which the pH is not maintained within 5.0 to 7.0, wherein the yield of the immunoglobulin is given by the main peak in % as measured by ion exchange-high-performance liquid chromatography (HPLC-IEX).

[0051] Brief Description of Drawings

[0052] Figure 1 - Depiction of one embodiment of the invention in which the pH is maintained within 5.0 to 7.0.

[0053] Figure 2 - Depiction of one embodiment of the invention in which the pH is maintained within 5.0 to 7.0. The incubation step is integrated into the process prior to the intermediate chromatography.

[0054] Figure 3 - Depiction of one embodiment of the invention in which the pH is maintained within 5.0 to 7.0. The incubation step is integrated into the process prior to the polishing chromatography.

[0055] Definitions

[0056] The term “cell culture harvest” refers to a harvested cell culture fluid, cell culture supernatant or pre-treated cell culture supernatant. The cell culture fluid can be obtained directly from a host cell or organism producing the immunoglobulin. The cell culture fluid may have been partially clarified or purified by centrifugation and / or filtration, for example microfiltration, diafiltration, ultrafiltration, and depth filtration. In a preferred embodiment, the sample is derived from a cell culture fluid, which is obtained from recombinant CHO cell culture. Preferably, the cell culture fluid is obtained from a recombinant cell culture during the growth phase.

[0057] The term “chromatography medium” or “chromatography material” refers to a chromatography material or medium in form of beads, plates, crystals, monoliths, membranes, fibers, meshwork of fibers or any other solid phase. The chromatography media bears functional groups referred to as ligands bound to a backbone, directly or via spacer, referred to as matrix. An exception are gel chromatography resins for size exclusion chromatography which are typically without any attached ligand. The term “chromatography media” does not limit the methods of the invention to column chromatography employing chromatography resins but also includes other types of chromatography, e.g., membrane chromatography employing membrane adsorbers. In particular, the term “ion exchange chromatography” used in the present invention comprises both an ion chromatography exchange resin and an ion exchange chromatography membrane adsorber.

[0058] The term “resin” refers to any chromatographic media or material in form of beads comprising a matrix with a bound functional group referred to as ligand which may interact with the protein or at least one contaminant. An exception are gel chromatography resins for size exclusion chromatography which are typically commercially available without any attached ligand. Resins may be supplied as beads of different sizes and are packed in columns. Alternatively, prepacked columns may be utilized.

[0059] The term “matrix” or “solid phase” represents a non-aqueous, non-soluble matrix to which a bound functional group referred to as ligand can adhere. The matrix of interest herein is generally one which comprises glass, ceramic, silica, cellulose, agarose, methacrylate polymer or polystyrene.

[0060] The term “ligand” as used herein refers to any functional group, which interacts with the Fc- peptide fusion protein or with at least one contaminant and which is covalently bound to the matrix.

[0061] “Protein A” encompasses Protein A recovered from a native source thereof, Protein A produced synthetically or biosynthetically (e.g., by peptide synthesis or by recombinant techniques), and variants thereof which retain the ability to bind proteins which have CH2 / CH3 and / or Fc regions. The term "loading" as used in the present invention refers to a sample containing the immunoglobulin of interest and applied to the chromatography. The term "loading" is independent of chromatography conditions in which a sample containing the immunoglobulin of interest is purified in bind-elute or flow-through mode.

[0062] The term “binding mode” or “bind -elute mode” as used herein refers to chromatography conditions in which a sample containing the immunoglobulin to be purified is applied to a chromatography medium, and wherein the immunoglobulin binds to the chromatography medium. Thus, the immunoglobulin is retained on the chromatography medium, whereas the impurities of the sample may be present in the non-binding fraction, also called the flow-through fraction. When a chromatography step is carried out in the binding mode, one or more washing steps may be performed after the binding of the immunoglobulin to the chromatography medium and prior to eluting the immunoglobulin from the medium. To obtain the immunoglobulin, the immunoglobulin is then eluted and obtained in the eluate, which may be further purified in a further chromatographic step if desired. “Elution” of the immunoglobulin may be performed using selective conditions permitting contaminants to remain bound to the medium while the immunoglobulin is eluted. Performing a chromatography step in the binding mode does not necessarily mean that 100 % of the Fc-peptide fusion protein is bound to the chromatography medium. In the context of the present invention ’’bound to the chromatography resin” or ’’bound to the chromatography medium” means that at least 50 % of the immunoglobulin is bound, preferably at least 75 % of the immunoglobulin is bound, more preferably at least 85 % of the immunoglobulin is bound, and most preferably more than 95 % of the immunoglobulin is bound to the resin or chromatography medium.

[0063] The term “flow-through mode” as used herein refers to chromatography conditions in which a sample containing the immunoglobulin of interest is applied to the chromatography resin or medium, and wherein the immunoglobulin does not bind to the chromatography resin but is mainly present in the fraction that is not bound to the resin or medium and thus contained in the flow-through. The intermediate chromatography of the present invention makes use of the flow- through mode. Impurities may bind to the resin or medium in this mode.

[0064] In the context of the present invention, it is understood, that the capture affinity chromatography step is performed in the binding mode, and wherein the capture step is considered the first chromatography step which is performed in the binding mode. The intermediate mixed-mode chromatography step of the present invention is performed in flow-through mode. The final polishing cation exchange chromatography step of the present invention is performed in binding mode.

[0065] The term “wash step” or “washing step” as used herein refers to a step performed in a chromatography in binding mode, after the immunoglobulin is loaded onto the chromatography column, but before the immunoglobulin is eluted from the column. The wash step additionally removes contaminants less tightly or non-specifically bound to the matrix, to the immunoglobulin, and / or to the ligand, without significantly eluting the immunoglobulin from the resin. During the “wash step” or “washing step” as used herein, the resin is washed with the desired wash buffer (e.g., the wash buffer is passed through the chromatography column until the UV absorption measured in the outlet of the column returns to baseline). During the wash steps the immunoglobulin of interest remains bound to the chromatography medium.

[0066] The term “elution” refers to as a process, which desorbs an immunoglobulin of interest from a chromatography medium by altering the solution conditions such that buffer components compete with the immunoglobulin of interest for the ligand site on the chromatography resin. Another mode of elution occurs in affinity chromatography, for example using Protein A. In this case, the elution buffer may alter the conformation of the ligand or the immunoglobulin, thereby loosening their binding capabilities. An immunoglobulin may be eluted from ion exchange resins by altering the ionic strength of the buffer surrounding the ion exchange material such that the buffer ions in the mobile phase compete with the molecule for the charged ionic sites of the ion exchange resin. Alternatively, a change in the pH influences the amphoteric protein and a pH increase above the isoelectric point (pl) of the immunoglobulin henceforth prevent its binding to a cation exchange resin and the immunoglobulin elutes. The same effect occurs on an anion exchange chromatography resin when the pH is decreased below the pl of the immunoglobulin. As understood herein the term “elution” comprises isocratic elution, single step elution, and gradient elution, with or without preceding wash steps. The elution of the immunoglobulin may be conducted by increasing the ionic strength or conductivity in the mobile phase, which is affected by increasing the salt concentration in the buffer solution. Alternatively, an increase or decrease in the pH value may be suitable. Discontinuous step gradients, linear gradients, nonlinear gradients, or a suitable combination of such gradients may be employed.

[0067] As used herein the term “buffer” refers to a solution that resists changes in the pH by the action of acid-base conjugate components. Buffers suitable for washing and for elution can be selected from acetate, citrate, succinate, maleate, malonate, Tris-HCI, Tris-phosphoric acid, Tris-acetate, Tris-glycine, phosphate, succinate, MES, MOPS, PIPES, HEPES, ethanolamine, BisTris, glycine, histidine, and other suitable buffers with the addition of salts such as phosphates, sulfates, or chlorides, such as NaCI or KCI. The ionic strength and the salt concentration, by means of which the elution is achieved, are dependent on the pH value of the buffer solution and the pl of the immunoglobulin. The wash buffer may further comprise a detergent (e.g., polysorbate), a solvent (e.g., hexylene glycol, isopropanol, or ethanol), or a polymer (e.g., polyethylene glycol). Furthermore, the wash buffer may include chaotropic reagents (e.g., urea or arginine) and / or protease inhibitors (e.g., EDTA).

[0068] The terms “immunoglobulin” and “antibody” are used interchangeably herein.

[0069] The term “N-terminal” or „N-terminus” as used herein refers to the free amino group (-NH2) of an amino acid located at the end of a polypeptide and represents the start of the polypeptide. Within a peptide, the amine group is bonded to the carboxylic group (-COOH) of another amino acid creating a chain. The free carboxylic group at one end of the peptide is called the C- terminus. By convention, the peptide sequence is written from N-terminus to C-terminus correlating to the translation direction of the messenger RNA. The N-terminus can be modified co- or post-translationally, for example by the attachment of small chemical groups such as acetyl and methyl or the addition of membrane anchors, such as palmitoyl and myristoyl groups. The term “glutamine” or “Gin” or“Q” as used herein refers to one of the 20 amino acids encoded by the genetic code. Glutamine is synthesized from glutamic acid by glutamine-synthetase. Instead of the carboxy group of glutamic acid, glutamine has an acid amide group, which is an uncharged polar side chain, i.e . , it has two amino groups.

[0070] The term “glutamic acid”, “Glu” or“E” as used herein refers to one of the 20 amino acids encoded by the genetic code. The salt and ester of glutamic acid are called “glutamate”. Glutamic acid possesses carboxyl group as side chain which is negatively charged at physiological pH conditions.

[0071] The term “pyroglutamate”, “pyroglutamic acid”, “PyroGlu” or“PyroE” as used herein refers to a derivate of glutamic acid. Pyroglutamate does not belong to proteinogenic amino acids because there is no basic amino group in the molecule in addition to the acidic carboxy group, since the free amino group is cyclized to a lactam. The formation of pyroglutamate may occur through a cyclization of N-terminal pyroglutamate under dehydration as well as through a cyclization of N- terminal glutamine under subsequent loss of the amino group. Pyroglutamate has been identified at the N-terminus of a variety of proteins, including the light and heavy chains of antibodies, fibrinogen, collagen, and kinines.

[0072] The method of the invention may be suitable for immunoglobulin purification on a small and large scale. Preferably, the method is carried out on a large scale.

[0073] “Small scale”, also called “laboratory scale” as used herein refers to the purification of samples containing less than 50 g immunoglobulin, less than 10 g immunoglobulin, or less than 1 g immunoglobulin. Small scale also refers to the purification processes in which the protein eluted from the column of the capture step amounts to less than 50 g immunoglobulin, less than 10 g immunoglobulin, or less than 1 g immunoglobulin.

[0074] “Large scale”, also called “production scale” or “manufacturing scale” or “commercial scale”, as used herein refers to the purification of samples containing more than 50 g immunoglobulin, more than 100 g immunoglobulin, more than 200 g immunoglobulin or more than 300 g immunoglobulin. Large scale also refers to the purification processes in which the protein eluted from the column of the capture step amounts to more than 50 g immunoglobulin, more than 100 g immunoglobulin, more than 200 g immunoglobulin or more than 300 g immunoglobulin.

[0075] Detailed description of the invention

[0076] Immunoglobulin

[0077] The immunoglobulin may be a monoclonal antibody, polyclonal antibody, multispecific antibody (e.g., bispecific antibody) and fragments thereof exhibiting the desired antigen binding activity. Naturally occurring antibodies are molecules with varying structures. For example, native IgG antibodies are hetero tetrameric glycoproteins of about 150,000 Daltons, composed of two identical light chains and two identical heavy chains that are linked by disulfide bonds. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable domain followed by three or four constant domains (CH1 , CH2, CH3 and optionally CH4). Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable domain followed by a constant light chain (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda ( ), based on the amino acid sequence of its constant domain.

[0078] Preferably the immunoglobulin is a monoclonal antibody. The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. In contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0079] The term “monoclonal antibodies” as used herein include “chimeric” antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0080] The monoclonal antibodies described herein also include “humanized” antibodies. Such antibodies are obtained by “humanization” of non-human (for example murine) antibodies and contain only minimal sequences derived from the animal immunoglobulin. Most of the molecule is derived from a human sequence. Residues from a hypervariable region of the human recipient antibody are replaced by residues from a hypervariable region of a non-human donor antibody having the desired binding properties.

[0081] The monoclonal antibodies as referred to herein also include fully “human” antibodies, which may be obtained by screening of a human antibody gene library.

[0082] The immunoglobulin may be of the murine class lgG1 , lgG2a, lgG2b, IgM, IgA, IgD or IgE, the human classes lgG1 , lgG2, lgG3, lgG4, IgM, lgA1 , lgA2, IgD or IgE, or combinations or fragments thereof.

[0083] The immunoglobulin may recognize any one or a combination of proteins including, but not limited to the following antigens: CD2, CD3, CD4, CD8, CD11 a, CD14, CD18, CD19, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80 (B7.1), CD79b, CD86 (B7.2), CD147, CD152, IL-la, IL-1 B, IL-1 , IL-2, IL-3, IL-7, IL-4, IL-5, IL-8, IL-10, IL-12, IL-13, IL-17a, IL-17F, IL- 23, IL23a, IL-2 receptor, IL-4 receptor, IL-6 receptor, IL-12 receptor, IL-13 receptor, IL-18 receptor subunits, PDGF-P, and analogues thereof, PLGF, VEGF, TGF, TGF-P2, TGF-p1 , EGF receptor, PLGF receptor, VEGF receptor, platelet receptor gpllb / llla, thrombopoietin receptor, apoptosis receptor PD-1 , hepatocyte growth factor, osteoprotegerin ligand, interferon alpha, interferon beta, interferon gamma, B lymphocyte stimulator BLyS, T-cell activation regulator CTLA-4, C5 complement, IgE, tumor antigen CA125, tumor antigen MUC1 , PEM antigen, ErbB2 / HER-2, tumor-associated epitopes that are present in elevated levels in the sera of patients, cancer-associated epitopes or proteins expressed on breast, colon, squamous cell, prostate, pancreatic, lung, and / or kidney cancer cells and / or on melanoma, glioma, or neuroblastoma cells, the necrotic core of a tumor, integrin alpha 4 beta 7, the integrin VLA-4, B2 integrins, a4pi and a4p7 integrin, TRAIL receptors 1 ,2,3, and 4, RANK, a RANK ligand (RANKL), TNF-a, the adhesion molecule VAP-1 , epithelial cell adhesion molecule (EpCAM), intercellular adhesion molecule-3 (ICAM-3), leukointegrin adhesin, the platelet glycoprotein gp llb / llla, cardiac myosin heavy chain, parathyroid hormone, sclerostin, MHC I, carcinoembryonic antigen (CEA), calcitonin gene-related peptide (CGRP), alpha-fetoprotein (AFP), tumor necrosis factor (TNF), Fc-y-1 receptor, HLA-DR 10 beta, HLA-DR antigen, L-selectin, Kallikrein, Kirin, and IFN-y.

[0084] In a preferred embodiment, the immunoglobulin contains one or more N-terminal glutamine or glutamate residues. The immunoglobulin may be for example, but without limitation abciximab, adalimumab, alemtuzumab, basiliximab, bevacizumab, cetuximab, daclizumab, eculizumab, efalizumab, gemtuzumab, ibritumomab, infliximab, natalizumab, ocrelizumab, ofatumumab, omalizumab, palivizumab, panitumumab, ranibizumab, rituximab, Tocilizumab, and trastuzumab.

[0085] The immunoglobulin according to the present invention is preferably an IgG molecule, such as lgG1 , lgG2, lgG3, or lgG4 molecule. More preferably, the immunoglobulin is lgG1. The immunoglobulin may be a humanized lgG1 form a murine progenitor. Most preferably, the humanized antibody is tocilizumab. Tocilizumab is a humanized anti-IL-6 receptor antibody, which is described in detail in, e.g., EP0628639B1.

[0086] In a preferred embodiment, the immunoglobulin is derived from a cell culture fluid, which is obtained from recombinant CHO cell culture.

[0087] Preferably, the immunoglobulin contains N-terminal Gin residues. More preferably, the immunoglobulin is an lgG1. Preferably, the lgG1 is an antibody, more preferably, the lgG1 is an anti-IL-6 receptor antibody. In a preferred embodiment, the anti-IL-6 receptor antibody is tocilizumab.

[0088] In preferred embodiments, the light chain of tocilizumab is characterized by SEQ ID NO: 1 and the heavy chain of tocilizumab is characterized by SEQ ID NO: 2.

[0089] In preferred embodiments, the immunoglobulin comprises an amino acid sequence having at least 90% identity to SEQ ID NO:1 and an amino acid sequence having at least 90% identity to SEQ ID NO:2.

[0090] In preferred embodiments, the immunoglobulin comprises an amino acid sequence having at least 99% identity to SEQ ID NO:1 and an amino acid sequence having at least 99% identity to SEQ ID NO:2.

[0091] Affinity chromatography step

[0092] The term affinity chromatography step is used interchangeably with capture chromatography step and refers to the first chromatography step in the purification train. The capture step for purification of an immunoglobulin is usually carried out as an affinity chromatography step. It refers to a method of selectively binding the immunoglobulin of the culture fluid based on a highly specific interaction between a ligand and the immunoglobulin. In the present invention, affinity chromatography was successfully used to capture immunoglobulins. In one embodiment, one or more filtration steps precede the affinity chromatography step. In a more preferred embodiment, the affinity chromatography step is preceded by a depth filtration and a microfiltration step for cell culture clarification.

[0093] Protein A or derivatives or analogues thereof are mostly used as affinity capture. Protein A affinity chromatography is a widely used affinity chromatography step which employs as ligands natural or recombinant proteins of microbial origin (e.g., Staphylococcus aureus, Streptococcus, Peptostreptococcus magnus) or variants derived thereof, or synthetic peptides that may be of microbial origin with the ability to bind to Fc-moieties of immunoglobulins. Exemplary immunoglobulin binding proteins may be Protein A, Protein G, Protein L, or Protein A / G. Preferably, the immunoglobulin-Fc binding protein or peptide is Protein A. The ligands can comprise one or more of the E, D, A, B, and C domains of Protein A. More preferably, the ligands comprise domain B of protein A or the engineered protein Z. An exemplary resin employing as ligand a 14 kDa peptide recombinantly produced with Saccharomyces cerevisiae is IgSelect (GE Healthcare). This ligand was specifically designed for high affinity to all subclasses of human IgG-Fc.

[0094] In preferred embodiments, the affinity chromatography step of the present invention is a Protein A affinity chromatography step. In preferred embodiments, the Protein A chromatography resin used for the capture step comprises an alkali-tolerant Protein A derivative as a ligand, bound to highly cross-linked agarose. In a preferred embodiment, the alkali-tolerant Protein A derivative is an alkali-stabilized tetramer variant of domain B of Protein A.

[0095] To make the Protein A affinity chromatography resin more resistant to harsh cleaning conditions and to provide protection against inter-run cross-contamination effects, it is common to use improved Protein A affinity resins, bearing ligands specially engineered to ensure alkali tolerance, high binding capacity, and low ligand leakage. One major drawback of these improved resins is, however, that they are significantly costlier than conventional Protein A resins. It is an important advantage of the method of the present invention that both conventional Protein A resins as well as the more recent new generation Protein A resin products can be used.

[0096] In embodiments of the invention, examples of common Protein A resins that can be used may include, without limitation, Unosphere SUPrA (Bio-Rad), Protein A Ceramic HyperD F (Pall Corporation), Poros MabCapture A (Applied Biosystems), ProSep HC, ProSep Ultra, and ProSep Ultra Plus (EMD Millipore), Protein A Sepharose FF, rProtein A Sepharose FF, rmp Protein A Sepharose FF, MabSelect, MabSelect SuRe, MabSelect SuRe LX, MabSelect Xtra, MabSelect PrismA (GE Healthcare), and Toyopearl rProtein A (Tosoh Bioscience).

[0097] In preferred embodiments, resins with high binding capacity and / or alkaline stability may be used for Protein A affinity chromatography. In some embodiments, the Protein A affinity chromatography uses Protein A, Protein A derivative, or alkali-stabilized Protein A-derived affinity medium. In preferred embodiments, alkali-stabilized Protein A-derived (E. coli) ligands may be used. In some embodiments, the alkali-stabilized Protein A-derived ligand may be coupled to a highly cross-linked agarose matrix, preferably immobilized with a chemically stable thio-ether linkage. In some embodiments, MabSelect SuRe from GE Healthcare Life Sciences is used, which can be rapidly and efficiently cleaned after the run with up to 0.5 M NaOH. The alkali-stabilized ligand of MabSelect SuRe is derived from the B domain of Protein A and essentially lacks the VH3 binding domain giving a higher elution pH. In some embodiments, MabSelect SuRe LX is used, which has a higher binding capacity than MabSelect SuRe.

[0098] In a preferred embodiment, provided is a method for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0099] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate, wherein the affinity chromatography step is a protein A chromatography step;

[0100] (ii) a pH adjustment step to obtain a pH adjusted composition,

[0101] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0102] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixedmode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0103] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), said adjusted pH is maintained throughout steps (ii) to (v) within 2.0 units, preferably 1 .8 units such as 1 .6 units of said adjusted pH.

[0104] In a preferred embodiment, provided is a method for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0105] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate, wherein the affinity chromatography step is a protein A chromatography step comprising an alkali- tolerant Protein A derivative as a ligand;

[0106] (ii) a pH adjustment step to obtain a pH adjusted composition, (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0107] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixedmode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0108] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), said adjusted pH is maintained throughout steps (ii) to (v) within 2.0 units, preferably 1 .8 units such as 1 .6 units of said adjusted pH.

[0109] In a preferred embodiment, provided is a method for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0110] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate, wherein the affinity chromatography step is a protein A chromatography step comprising an alkali- stabilized tetramer variant of domain B of Protein A bound to a cross-linked agarose matrix;

[0111] (ii) a pH adjustment step to obtain a pH adjusted composition,

[0112] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0113] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixedmode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0114] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), said adjusted pH is maintained throughout steps (ii) to (v) within 2.0 units, preferably 1 .8 units such as 1 .6 units of said adjusted pH. In a preferred embodiment, provided is a method for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0115] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate, wherein the affinity chromatography step is a protein A chromatography step;

[0116] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0117] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0118] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixedmode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0119] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v).

[0120] In a preferred embodiment, provided is a method for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0121] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate, wherein the affinity chromatography step is a protein A chromatography step comprising an alkali- tolerant Protein A derivative as a ligand;

[0122] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0123] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through, (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixedmode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0124] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v).

[0125] In a preferred embodiment, provided is a method for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0126] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate, wherein the affinity chromatography step is a protein A chromatography step comprising an alkali- stabilized tetramer variant of domain B of Protein A bound to a cross-linked agarose matrix;

[0127] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0128] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0129] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixedmode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0130] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v).

[0131] In a preferred embodiment, provided is a method for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0132] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate, wherein the affinity chromatography step is a protein A chromatography step; (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0133] (iii) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step

[0134] (ii) followed by an incubation step to obtain an incubated flow-through,

[0135] (iv) subjecting the incubated flow-through obtained in step (iii) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0136] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v).

[0137] In a preferred embodiment, provided is a method for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0138] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate, wherein the affinity chromatography step is a protein A chromatography step comprising an alkali- tolerant Protein A derivative as a ligand;

[0139] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0140] (iii) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0141] (iv) subjecting the incubated flow-through obtained in step (iii) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0142] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v).

[0143] In a preferred embodiment, provided is a method for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0144] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate, wherein the affinity chromatography step is a protein A chromatography step comprising an alkali- stabilized tetramer variant of domain B of Protein A bound to a cross-linked agarose matrix;

[0145] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0146] (iii) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0147] (iv) subjecting the incubated flow-through obtained in step (iii) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0148] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v).

[0149] In the method according to the present invention as provided in the embodiments above, one or several wash steps between the sample load on the Protein A affinity column and the elution of the immunoglobulin from the Protein A column may be included employing special wash buffer(s). In some embodiments, the wash buffer is the buffer used to remove impurities from the Protein A resin without removing significant amounts of the immunoglobulin of interest bound to the Protein A. In some embodiments, the wash buffer may comprise salt and detergent (e.g., polysorbate); salt and solvent (e.g., hexylene glycol); high concentration salt (e.g., high molarity Tris buffer); or salt and polymer (e.g., polyethylene glycol). In some embodiments, the wash buffer may include chaotropic reagents (e.g., urea or arginine) and / or protease inhibitors (e.g., EDTA). In some embodiments, the wash buffer may have a lower pH as the loading buffer and / or a higher pH as the elution buffer.

[0150] In the method according to the present invention as provided in the embodiments above, elution of the immunoglobulin of interest from the Protein A column is carried out using an elution buffer. Preferably, the elution buffer utilized in the method according to the present invention has a low pH and thereby disrupts interactions between Protein A and the immunoglobulin of interest by changing the protein conformation. In preferred embodiments, the elution buffer has a pH in the range from about 2 to about 5, most preferably in the range from about 3 to about 4. Examples of buffers that will control the pH within this range include phosphoric acid, acetic acid, citric acid, glycine, and ammonium buffers, as well as combinations thereof. Preferred buffers are citric acid and acetic acid buffers. In some embodiments, the buffer may have a citric acid concentration of 20 mM to 200 mM, preferably 50 mM to 15 0 mM citrate, most preferably of 100 mM. Other elution buffers are contemplated, including high pH buffers (e.g., pH 9 or more) or buffers comprising a compound or composition such as MgCl2 for eluting the immunoglobulin of interest.

[0151] The Protein A affinity chromatography resin may be regenerated with 0.1 to 0.5 NaOH, preferably within the column (cleaning in place).

[0152] In some embodiments, the term affinity chromatography step or protein A affinity chromatography step includes a viral clearance step on the eluate of the affinity chromatography step or protein A affinity chromatography step.

[0153] In some embodiments, the affinity chromatography step comprises a virus clearance step on the eluate of the affinity chromatography step comprising decreasing the pH of the eluate of the affinity chromatography step. In some embodiments, the protein A affinity chromatography step comprises a virus clearance step on the eluate of the protein A affinity chromatography step comprising decreasing the pH of the eluate of the protein A affinity chromatography step.

[0154] In some embodiments, the affinity chromatography step comprises a virus clearance step on the eluate of the affinity chromatography step comprising decreasing the pH of the eluate of the affinity chromatography step to 3.0 to 4.0. In some embodiments, the protein A affinity chromatography step comprises a virus clearance step on the eluate of the protein A affinity chromatography step comprising decreasing the pH of the eluate of the protein A affinity chromatography step to 3.0 to 4.0. pH adjustment step

[0155] The method of the present invention comprises a pH adjustment step. In a preferred embodiment, the pH adjustment step comprises increasing the pH.

[0156] In a preferred embodiment, the pH adjustment step is carried out immediately after the affinity chromatography step. As described in the previous section, the affinity chromatography step includes a viral clearance step and therefore, the pH adjustment step is carried out immediately after the virus clearance step.

[0157] In preferred embodiments, the pH adjustment comprising increasing the pH occurs only once throughout the method of the invention.

[0158] In some embodiments, a method is provided for obtaining an immunoglobulin with N-terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0159] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0160] (ii) a pH adjustment step to obtain a pH adjusted composition,

[0161] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0162] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixedmode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0163] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), said adjusted pH is maintained throughout steps (ii) to (v) within 2.0 units, preferably 1 .8 units such as 1 .6 units of said adjusted pH.

[0164] In preferred embodiments, after the pH adjustment step (ii) above, said pH is maintained throughout the method of the invention within 2.0 units of the adjusted pH. In preferred embodiments, after the pH adjustment step (ii) above, said pH is maintained throughout the method of the invention within 1.8 units of the adjusted pH. In preferred embodiments, after the pH adjustment step (ii) above, said pH is maintained throughout the method of the invention within 1.6 units of the adjusted pH.

[0165] In some embodiments, a method is provided for obtaining an immunoglobulin with N-terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0166] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0167] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0168] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0169] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixedmode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0170] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v). In preferred embodiments, the pH adjustment step (ii) above, comprises adjusting the pH to 5.0 to 7.0. In preferred embodiments, the pH adjustment step (ii) above, comprises adjusting the pH to 5.0 to 6.8. In preferred embodiments, the pH adjustment step (ii) above, comprises adjusting the pH to 5.0 to 6.6.

[0171] In some embodiments, a method is provided for obtaining an immunoglobulin with N-terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0172] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate followed by a virus clearance step;

[0173] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0174] (iii) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0175] (iv) subjecting the incubated flow-through obtained in step (iii) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0176] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v).

[0177] In some embodiments, a method is provided for obtaining an immunoglobulin with N-terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0178] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate followed by a virus clearance step;

[0179] (ii) a pH adjustment step to obtain a pH adjusted composition,

[0180] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through, (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixedmode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0181] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), said adjusted pH is maintained throughout steps (ii) to (v) within 2.0 units, preferably 1 .8 units such as 1 .6 units of said adjusted pH.

[0182] In preferred embodiments, after the pH adjustment step (ii) above, said pH is maintained throughout the method of the invention within 2.0 units of the adjusted pH. In preferred embodiments, after the pH adjustment step (ii) above, said pH is maintained throughout the method of the invention within 1.8 units of the adjusted pH. In preferred embodiments, after the pH adjustment step (ii) above, said pH is maintained throughout the method of the invention within 1.6 units of the adjusted pH.

[0183] In some embodiments, a method is provided for obtaining an immunoglobulin with N-terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0184] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate followed by a virus clearance step;

[0185] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0186] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0187] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixedmode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0188] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v). In preferred embodiments, the pH adjustment step (ii) above, comprises adjusting the pH to 5.0 to 7.0. In preferred embodiments, the pH adjustment step (ii) above, comprises adjusting the pH to 5.0 to 6.8. In preferred embodiments, the pH adjustment step (ii) above, comprises adjusting the pH to 5.0 to 6.6.

[0189] In some embodiments, a method is provided for obtaining an immunoglobulin with N-terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0190] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate followed by a virus clearance step;

[0191] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0192] (iii) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0193] (iv) subjecting the incubated flow-through obtained in step (iii) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0194] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v).

[0195] In preferred embodiments, the pH adjustment step (ii) above, comprises adjusting the pH to 5.0 to 7.0. In preferred embodiments, the yield of the immunoglobulin is higher as compared to the same method in which the pH is not maintained within 5.0 to 7.0, wherein the yield of the immunoglobulin is given by the main peak in % as measured by ion exchange-high-performance liquid chromatography (HPLC-IEX).

[0196] In preferred embodiments, the pH adjustment step (ii) above, comprises adjusting the pH to 5.0 to 6.8. In preferred embodiments, the yield of the immunoglobulin is higher as compared to the same method in which the pH is not maintained within 5.0 to 6.8, wherein the yield of the immunoglobulin is given by the main peak in % as measured by ion exchange-high-performance liquid chromatography (HPLC-IEX).

[0197] In preferred embodiments, the pH adjustment step (ii) above, comprises adjusting the pH to 5.0 to 6.6. In preferred embodiments, the yield of the immunoglobulin is higher as compared to the same method in which the pH is not maintained within 5.0 to 6.6, wherein the yield of the immunoglobulin is given by the main peak in % as measured by ion exchange-high-performance liquid chromatography (HPLC-IEX).

[0198] Mixed-mode anion exchange chromatography or anion exchange chromatography

[0199] The method of the invention further comprises a mixed-mode anion exchange chromatography step or an anion exchange chromatography step. In a preferred embodiment, the method of the invention uses a mixed-mode anion exchange chromatography step.

[0200] In a preferred embodiment, the anion exchange chromatography step is carried out after the affinity chromatography such as Protein A chromatography step. In a preferred embodiment, it is carried after the affinity chromatography and prior to the polishing chromatography such as cation exchange chromatography step.

[0201] In a preferred embodiment, mixed-mode anion exchange chromatography step is carried out after the affinity chromatography such as Protein A chromatography step. In a preferred embodiment, it is carried after the affinity chromatography and prior to the polishing chromatography such as cation exchange chromatography step.

[0202] The mixed-mode chromatography step is conducted with “mixed-mode media” or “mixed-mode resin”. These terms refer to chromatographic media possessing functional groups consisting of either charged hydrophobic ion exchange ligands or crystalline minerals such as hydroxyapatite or fluorapatite. Instead of “mixed-mode chromatography” the term “multi modal chromatography” or in connection with a specific procedure “hydrophobic charge induction chromatography” is sometimes used.

[0203] Mixed-mode chromatography is an interaction of at least two principles, hydrophobic interaction and ion exchange or metal affinity interaction and ion exchange. Positively charged hydrophobic ligands belong to the group of mixed-mode anion exchanger (e.g., Capto Adhere). Some mixedmode media have zwitterionic character (e.g., Bakerbond ABx). Other mixed-mode media possess hydrophobic ligands which are ionisable and convert from uncharged to positively charged by lowering the pH (e.g., MEP HyperCel). Finally, hydroxyapatite and fluorapatite media have more complex mixed-mode functions by possessing positively charged calcium ions and negatively charged phosphate groups.

[0204] Anion exchange chromatography is a form of ion exchange chromatography (IEX), which is used to separate molecules based on their net surface charge. Anion exchange chromatography, more specifically, uses a positively charged ion exchange resin with an affinity for molecules having net negative surface charges.

[0205] In preferred embodiments, the anion exchange chromatography step reduces the host cell proteins and lipoprotein a (LPA), and host cell DNA, aggregates, fragments, charge variants, endotoxins, and leached Protein A.

[0206] In preferred embodiments, the anion exchange chromatography step is conducted in flow through mode. In preferred embodiments, the pH and ionic strength of the buffer used in the anion exchange chromatography step is adjusted according to the pl of the immunoglobulin to be obtained or purified. The pH and the ionic strength have to be adjusted according to the pl of the immunoglobulin in such a way that the immunoglobulin does not bind to the mixed-mode ligand while residual contaminants to be cleared (DNA, aggregates, leached Protein A, host cell proteins) remain bound. In preferred embodiments, the anion exchange chromatography step is conducted using a phosphate or T ris buffer at a pH range of 6 to 8.5. In some embodiments, the anion exchange chromatography step is conducted using a phosphate or T ris buffer at a pH range of 6 to 7. In some embodiments, the conductivity of the buffer is adjusted by the buffer concentration or by adding salt such as NaCI. In preferred embodiments, the buffer is a sodium phosphate buffer. In preferred embodiments, the buffer is a sodium phosphate buffer in the concentration range of 10 to 50 mM. In a preferred embodiment, the method of the invention uses a mixed-mode chromatography. In a preferred embodiment, the method of the invention uses a mixed-mode anion exchange chromatography comprising hydrophobic and anion exchange functions. More preferred are mixed-mode resins containing positively charged N- benzyl-N-methyl ethanolamine ligands, which are bound to a highly cross-linked agarose matrix. The most preferred mixed-mode resin used is Capto Adhere ImpRes (GE Healthcare Life Science).

[0207] In preferred embodiments, the mixed-mode anion exchange chromatography step reduces the host cell proteins and lipoprotein a (LPA), and host cell DNA, aggregates, fragments, charge variants, endotoxins, and leached Protein A.

[0208] In preferred embodiments, the mixed-mode anion exchange chromatography step is conducted in flow through mode. In preferred embodiments, the pH and ionic strength of the buffer used in the mixed-mode anion exchange chromatography step is adjusted according to the pl of the immunoglobulin to be obtained or purified. The pH and the ionic strength have to be adjusted according to the pl of the immunoglobulin in such a way that the immunoglobulin does not bind to the mixed-mode ligand while residual contaminants to be cleared (DNA, aggregates, leached Protein A, host cell proteins) remain bound. In preferred embodiments, the mixed-mode anion exchange chromatography step is conducted using a phosphate or Tris buffer at a pH range of 6 to 8.5. In some embodiments, the mixed-mode anion exchange chromatography step is conducted using a phosphate or Tris buffer at a pH range of 6 to 7. In some embodiments, the conductivity of the buffer is adjusted by the buffer concentration or by adding salt such as NaCI. In preferred embodiments, the buffer is a sodium phosphate buffer. In preferred embodiments, the buffer is a sodium phosphate buffer in the concentration range of 10 to 50 mM.

[0209] The regeneration (cleaning in place) for the mixed-mode resin may be performed according to the method of the present invention with low pH, high salt, and high pH, e.g., with 10 to 200 mM citric acid, 0.5 to 2 M NaCI, and 10 mM to 1 M NaOH. In a preferred embodiment, provided is a method for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0210] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0211] (ii) a pH adjustment step to obtain a pH adjusted composition,

[0212] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography in flow-through mode to obtain an incubated flow-through, wherein the mixed-mode anion exchange chromatography step comprises N-benzyl-N-methyl ethanolamine as a ligand bound to a cross-linked agarose matrix, or

[0213] (iii-b) a mixed-mode anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through, wherein the mixed-mode anion exchange chromatography step comprises N-benzyl-N-methyl ethanolamine as a ligand bound to a cross-linked agarose matrix,

[0214] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixedmode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0215] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), said adjusted pH is maintained throughout steps (ii) to (v) within 2.0 units, preferably 1 .8 units such as 1 .6 units of said adjusted pH.

[0216] In a preferred embodiment, provided is a method for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0217] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0218] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0219] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography in flow-through mode to obtain an incubated flow-through, wherein the mixed-mode anion exchange chromatography step comprises N-benzyl-N-methyl ethanolamine as a ligand bound to a cross-linked agarose matrix, or (iii-b) a mixed-mode anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through, wherein the mixed-mode anion exchange chromatography step comprises N-benzyl-N-methyl ethanolamine as a ligand bound to a cross-linked agarose matrix,

[0220] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixedmode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0221] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v).

[0222] In a preferred embodiment, provided is a method for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0223] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate, wherein the affinity chromatography step is a protein A chromatography step;

[0224] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0225] (iii) a mixed-mode anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through, wherein the mixed-mode anion exchange chromatography step comprises N-benzyl-N-methyl ethanolamine as a ligand bound to a cross-linked agarose matrix,

[0226] (iv) subjecting the incubated flow-through obtained in step (iii) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0227] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v).

[0228] In a preferred embodiment, provided is a method for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps: (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate, wherein the affinity chromatography step is a protein A chromatography step;

[0229] (ii) a pH adjustment step to obtain a pH adjusted composition,

[0230] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography in flow-through mode to obtain an incubated flow-through, wherein the mixed-mode anion exchange chromatography step comprises N-benzyl-N-methyl ethanolamine as a ligand bound to a cross-linked agarose matrix, or

[0231] (iii-b) a mixed-mode anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through, wherein the mixed-mode anion exchange chromatography step comprises N-benzyl-N-methyl ethanolamine as a ligand bound to a cross-linked agarose matrix,

[0232] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixedmode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0233] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), said adjusted pH is maintained throughout steps (ii) to (v) within 2.0 units, preferably 1 .8 units such as 1 .6 units of said adjusted pH.

[0234] In a preferred embodiment, provided is a method for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0235] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate, wherein the affinity chromatography step is a protein A chromatography step;

[0236] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0237] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography in flow-through mode to obtain an incubated flow-through, wherein the mixed-mode anion exchange chromatography step comprises N-benzyl-N-methyl ethanolamine as a ligand bound to a cross-linked agarose matrix, or

[0238] (iii-b) a mixed-mode anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through, wherein the mixed-mode anion exchange chromatography step comprises N-benzyl-N-methyl ethanolamine as a ligand bound to a cross-linked agarose matrix,

[0239] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixedmode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0240] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v).

[0241] In a preferred embodiment, provided is a method for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0242] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate, wherein the affinity chromatography step is a protein A chromatography step;

[0243] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0244] (iii) a mixed-mode anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through, wherein the mixed-mode anion exchange chromatography step comprises N-benzyl-N-methyl ethanolamine as a ligand bound to a cross-linked agarose matrix,

[0245] (iv) subjecting the incubated flow-through obtained in step (iii) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0246] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v).

[0247] In a preferred embodiment, provided is a method for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0248] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate, wherein the affinity chromatography step is a protein A chromatography step comprising an alkali- tolerant Protein A derivative as a ligand such as an alkali-stabilized tetramer variant of domain B of Protein A bound to a cross-linked agarose matrix;

[0249] (ii) a pH adjustment step to obtain a pH adjusted composition,

[0250] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography in flow-through mode to obtain an incubated flow-through, wherein the mixed-mode anion exchange chromatography step comprises N-benzyl-N-methyl ethanolamine as a ligand bound to a cross-linked agarose matrix, or

[0251] (iii-b) a mixed-mode anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through, wherein the mixed-mode anion exchange chromatography step comprises N-benzyl-N-methyl ethanolamine as a ligand bound to a cross-linked agarose matrix,

[0252] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixedmode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0253] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), said adjusted pH is maintained throughout steps (ii) to (v) within 2.0 units, preferably 1 .8 units such as 1 .6 units of said adjusted pH.

[0254] In a preferred embodiment, provided is a method for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0255] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate, wherein the affinity chromatography step is a protein A chromatography step comprising an alkali- tolerant Protein A derivative as a ligand such as an alkali-stabilized tetramer variant of domain B of Protein A bound to a cross-linked agarose matrix;

[0256] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0257] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography in flow-through mode to obtain an incubated flow-through, wherein the mixed-mode anion exchange chromatography step comprises N-benzyl-N-methyl ethanolamine as a ligand bound to a cross-linked agarose matrix, or (iii-b) a mixed-mode anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through, wherein the mixed-mode anion exchange chromatography step comprises N-benzyl-N-methyl ethanolamine as a ligand bound to a cross-linked agarose matrix,

[0258] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixedmode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0259] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v).

[0260] In a preferred embodiment, provided is a method for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0261] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate, wherein the affinity chromatography step is a protein A chromatography step comprising an alkali- tolerant Protein A derivative as a ligand such as an alkali-stabilized tetramer variant of domain B of Protein A bound to a cross-linked agarose matrix;

[0262] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0263] (iii) a mixed-mode anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through, wherein the mixed-mode anion exchange chromatography step comprises N-benzyl-N-methyl ethanolamine as a ligand bound to a cross-linked agarose matrix,

[0264] (iv) subjecting the incubated flow-through obtained in step (iii) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0265] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v). Incubation step

[0266] The method of the present invention comprises an incubation step to convert the N-terminal glutamine of an immunoglobulin in a composition comprising the immunoglobulin to N-terminal pyro glutamate. Several monoclonal antibodies contain a Gin or Glu residue at the N-terminus of the light chain and / or heavy chain. The formation of PyroGlu from N-terminal Gin or Glu is a post-translational modification observed for monoclonal antibodies during production and storage conditions. PyroGlu formation may occur spontaneously or enzymatically, i.e., glutaminyl cyclase, an enzyme found in plants, animals, and humans, catalyses the reaction.

[0267] In some embodiments, the incubation step is carried out before or after the mixed-mode anion exchange chromatography step. In preferred embodiments, the incubation step is carried out after the mixed-mode anion exchange chromatography step. In preferred embodiments, the incubation step is carried out after the mixed-mode anion exchange chromatography step and before the cation exchange chromatography step.

[0268] In some embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer. In some embodiments, the incubation step is carried out at a temperature from 20 °C to 40 °C. In some embodiments, the incubation step is carried out at a pH from 5.0 to 7.0. In some embodiments, the incubation step is conducted for a duration from 1 hour to 10 days. In some embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0269] In some embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer. In some embodiments, the incubation step is carried out at a temperature from 20 °C to 40 °C. In some embodiments, the incubation step is carried out at a pH from 5.0 to 7.0. In some embodiments, the incubation step is conducted for a duration from 2 hours to 7 days. In some embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0270] In some embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at a temperature from 20 °C to 37 °C. In some embodiments, the incubation step is carried out at a pH from 5.0 to 6.8. In some embodiments, the incubation step is conducted fora duration from 2 hours to 7 days. In some embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0271] In some embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at a temperature from 20 °C to 37 °C. In some embodiments, the incubation step is carried out at a pH from 5.0 to 6.6. In some embodiments, the incubation step is conducted fora duration from 2 hours to 7 days. In some embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0272] In some embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at a temperature from 20 °C to 37 °C. In some embodiments, the incubation step is carried out at a pH from 5.0 to 6.5. In some embodiments, the incubation step is conducted fora duration from 2 hours to 7 days. In some embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0273] In some embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at room temperature. In some embodiments, the incubation step is carried out at a pH from 5.0 to 6.5. In some embodiments, the incubation step is conducted for a duration from 2 hours to 7 days. In some embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0274] In some embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at a temperature from 35 to 40 °C. In some embodiments, the incubation step is carried out at a pH from 5.0 to 6.5. In some embodiments, the incubation step is conducted fora duration from 2 hours to 7 days. In some embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0275] In some embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at a temperature from 35 to 37 °C. In some embodiments, the incubation step is carried out at a pH from 5.0 to 6.5. In some embodiments, the incubation step is conducted fora duration from 2 hours to 7 days. In some embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0276] In preferred embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at a temperature from 35 to 40 °C. In preferred embodiments, the incubation step is carried out at a pH from 5.0 to 6.5. In preferred embodiments, the incubation step is conducted for a duration from 2 hours to 7 days. In preferred embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0277] In preferred embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at a temperature from 35 to 40 °C. In preferred embodiments, the incubation step is carried out at a pH from 5.0 to 6.5. In preferred embodiments, the incubation step is conducted for a duration from 14 hours to 7 days. In preferred embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0278] In preferred embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at a temperature from 35 to 40 °C. In preferred embodiments, the incubation step is carried out at a pH from 5.0 to 6.5. In preferred embodiments, the incubation step is conducted for a duration from 24 hours to 7 days. In preferred embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0279] In some embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer. In some embodiments, the incubation step is carried out at a temperature from 20 °C to 40 °C. In some embodiments, the incubation step is carried out at a pH from 5.0 to 7.0. In some embodiments, the incubation step is conducted for a duration from 1 hour to at least 30 hours such as at least 24 hours. In some embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0280] In some embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer. In some embodiments, the incubation step is carried out at a temperature from 20 °C to 40 °C. In some embodiments, the incubation step is carried out at a pH from 5.0 to 7.0. In some embodiments, the incubation step is conducted for a duration from 2 hours to at least 30 hours such as at least 24 hours. In some embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0281] In some embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at a temperature from 20 °C to 37 °C. In some embodiments, the incubation step is carried out at a pH from 5.0 to 6.8. In some embodiments, the incubation step is conducted for a duration from 2 hours to at least 30 hours such as at least 24 hours. In some embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0282] In some embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at a temperature from 20 °C to 37 °C. In some embodiments, the incubation step is carried out at a pH from 5.0 to 6.6. In some embodiments, the incubation step is conducted fora duration from 2 hours to at least 30 hours such as at least 24 hours. In some embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0283] In some embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at a temperature from 20 °C to 37 °C. In some embodiments, the incubation step is carried out at a pH from 5.0 to 6.5. In some embodiments, the incubation step is conducted fora duration from 2 hours to at least 30 hours such as at least 24 hours. In some embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0284] In some embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at room temperature. In some embodiments, the incubation step is carried out at a pH from 5.0 to 6.5. In some embodiments, the incubation step is conducted for a duration from 2 hours to at least 30 hours such as at least 24 hours. In some embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0285] In some embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at a temperature from 35 to 40 °C. In some embodiments, the incubation step is carried out at a pH from 5.0 to 6.5. In some embodiments, the incubation step is conducted for a duration from 2 hours to at least 30 hours such as at least 24 hours. In some embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0286] In some embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at a temperature from 35 to 37 °C. In some embodiments, the incubation step is carried out at a pH from 5.0 to 6.5. In some embodiments, the incubation step is conducted fora duration from 2 hours to at least 30 hours such as at least 24 hours. In some embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0287] In preferred embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at a temperature from 35 to 40 °C. In preferred embodiments, the incubation step is carried out at a pH from 5.0 to 6.5. In preferred embodiments, the incubation step is conducted fora duration from 2 hours to at least 30 hours such as at least 24 hours. In preferred embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0288] In preferred embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at a temperature from 35 to 40 °C. In preferred embodiments, the incubation step is carried out at a pH from 5.0 to 6.5. In preferred embodiments, the incubation step is conducted fora duration from 14 hours to at least 30 hours such as at least 24 hours. In preferred embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0289] In preferred embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at a temperature from 35 to 40 °C. In preferred embodiments, the incubation step is carried out at a pH from 5.0 to 6.5. In preferred embodiments, the incubation step is conducted for a duration from 24 hours to at least 30 hours such as at least 24 hours. In preferred embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0290] In preferred embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at a temperature from 35 to 40 °C. In preferred embodiments, the incubation step is carried out at a pH from 5.0 to 6.5. In preferred embodiments, the incubation step is conducted for a duration of at least 20 to 24 hours. In preferred embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM.

[0291] In preferred embodiments, the incubation step is carried out with a buffer, preferably a phosphate buffer at a temperature from 35 to 40 °C, preferably 37 °C. In preferred embodiments, the incubation step is carried out at a pH from 5.0 to 6.5. In preferred embodiments, the incubation step is conducted for a duration of at least 20 to 24 hours. In preferred embodiments, the final concentration of the buffer used in the incubation step is from 20 to 200 mM, preferably 50 to 150 mM. In preferred embodiments, the incubation step converts the N-terminal glutamine on an immunoglobulin in a composition comprising the immunoglobulin to N-terminal pyro glutamate. In preferred embodiments, the incubation step reduces the total number of immunoglobulins in a composition comprising an N-terminal glutamine. In preferred embodiments, the incubation step increases the total number of immunoglobulins in a composition comprising an N-terminal pyro glutamate. In preferred embodiments, the incubation step reduces the total number of immunoglobulins comprising an N-terminal glutamine in a composition and increases the total number of immunoglobulins comprising an N-terminal pyro glutamate in the composition. In preferred embodiments, the conversion from N-terminal glutamine to N-terminal pyro glutamate during the incubation step of the present invention is performed non-enzymatically.

[0292] In a preferred embodiment, a method is provided for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0293] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0294] (ii) a pH adjustment step to obtain a pH adjusted composition,

[0295] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0296] (iv subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixedmode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0297] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), said adjusted pH is maintained throughout steps (ii) to (v) within 2.0 units, preferably 1 .8 units such as 1 .6 units of said adjusted pH. In this context, the incubation step itself does not involve a pH adjustment step such that the pH varies more than as defined above.

[0298] In a preferred embodiment, a method is provided for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0299] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate; (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0300] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0301] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixedmode cation exchange chromatography or cation exchange chromatography in bind- elute mode to obtain an incubated eluate,

[0302] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v). In this context, the incubation step itself does not involve a pH adjustment step such that the pH varies more than as defined above.

[0303] In a preferred embodiment, a method is provided for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0304] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0305] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0306] (iii) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0307] (iv) subjecting the incubated flow-through obtained in step (iii) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0308] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v). In this context, the incubation step itself does not involve a pH adjustment step such that the pH varies more than as defined above.

[0309] In preferred embodiments, the incubation step reduces the N-terminal Gin by at least 70%, at least 80%, at least 90%, at least 95% or at least 99%. In preferred embodiments, the incubation step described above comprises the following sequential steps:

[0310] (1) heating the pH adjusted composition of step (ii) up to 35 to 40 °C such as 37 °C;

[0311] (2) maintaining the temperature of (1 ) for at least 20 to 24 hours; and

[0312] (3) cooling down to 18 to 22 °C, to obtain an incubated flow-through.

[0313] In preferred embodiments, the incubation step described above comprises the following sequential steps:

[0314] (1) heating the flow-through of step (iii) or (iii-b) up to 35 to 40 °C such as 37 °C;

[0315] (2) maintaining the temperature of (1 ) for at least 20 to 24 hours; and

[0316] (3) cooling down to 18 to 22 °C, to obtain an incubated flow-through.

[0317] In preferred embodiments, the incubation step described above comprises the following sequential steps:

[0318] (1) heating the pH adjusted composition of step (ii) up to 35 to 40 °C such as 37 °C with a phosphate buffer at pH 5.0 to 7.0 such as 5.0 to 6.5;

[0319] (2) maintaining the temperature of (1 ) for at least 20 to 24 hours; and

[0320] (3) cooling down to 18 to 22 °C, to obtain an incubated flow-through.

[0321] In preferred embodiments, the incubation step described above comprises the following sequential steps:

[0322] (1) heating the flow-through of step (iii) or (iii-b) up to 35 to 40 °C such as 37 °C with a phosphate buffer at pH 5.0 to 7.0 such as 5.0 to 6.5;

[0323] (2) maintaining the temperature of (1 ) for at least 20 to 24 hours; and

[0324] (3) cooling down to 18 to 22 °C, to obtain an incubated flow-through.

[0325] In preferred embodiments, the incubation step described above comprises the following sequential steps:

[0326] (1) heating the pH adjusted composition of step (ii) up to 35 to 40 °C such as 37 °C with 20 to 200 mM phosphate buffer such as 50 to 150 mM at pH 5.0 to 7.0 such as 5.0 to 6.5;

[0327] (2) maintaining the temperature of (1 ) for at least 20 to 24 hours; and

[0328] (3) cooling down to 18 to 22 °C, to obtain an incubated flow-through. In preferred embodiments, the incubation step described above comprises the following sequential steps:

[0329] (1) heating the flow-through of step (iii) or (iii-b) up to 35 to 40 °C such as 37 °C with 20 to 200 mM phosphate buffer such as 50 to 150 mM at pH 5.0 to 7.0 such as 5.0 to 6.5;

[0330] (2) maintaining the temperature of (1 ) for at least 20 to 24 hours; and

[0331] (3) cooling down to 18 to 22 °C, to obtain an incubated flow-through.

[0332] Preferably, (1) heating, (2) maintaining the temperature of (1), and (3) cooling as described above, are performed actively. More preferably, (1) heating and (3) cooling as described above is performed actively under stirring conditions.

[0333] N-terminal glutamine and N-terminal pyro glutamate possess difference in charge and can therefore be separated by ion exchange chromatography. In a preferred embodiment, the incubation step is followed by a cation exchange chromatography. In some embodiments, the charge variants may be detected by ion exchange-high-performance liquid chromatography.

[0334] In a preferred embodiment, a method is provided for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0335] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0336] (ii) a pH adjustment step to obtain a pH adjusted composition,

[0337] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or

[0338] (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0339] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0340] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), said adjusted pH is maintained throughout steps (ii) to (v) within 2.0 units, preferably 1 .8 units such as 1 .6 units of said adjusted pH. In this context, the incubation step itself does not involve a pH adjustment step such that the pH varies more than as defined above. In this context, the incubation step comprises the following sequential steps:

[0341] (1) heating the pH adjusted composition of step (i) or the flow-through of step (iii-b) up to 35 to 40 °C such as 37 °C with 20 to 200 mM phosphate buffer such as 50 to 150 mM at pH 5.0 to 7.0 such as 5.0 to 6.5;

[0342] (2) maintaining the temperature of (1 ) for at least 20 to 24 hours; and

[0343] (3) cooling down to 18 to 22 °C, to obtain an incubated flow-through.

[0344] In a preferred embodiment, a method is provided for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0345] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0346] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0347] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or

[0348] (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0349] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0350] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v). In this context, the incubation step itself does not involve a pH adjustment step such that the pH varies more than as defined above. In this context, the incubation step comprises the following sequential steps: (1) heating the pH adjusted composition of step (i) or the flow-through of step (iii-b) up to 35 to 40 °C such as 37 °C with 20 to 200 mM phosphate buffer such as 50 to 150 mM at pH 5.0 to 7.0 such as 5.0 to 6.5;

[0351] (2) maintaining the temperature of (1 ) for at least 20 to 24 hours; and

[0352] (3) cooling down to 18 to 22 °C, to obtain an incubated flow-through.

[0353] In a preferred embodiment, a method is provided for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0354] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0355] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0356] (iii) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0357] (iv) subjecting the incubated flow-through obtained in step (iii) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0358] (iv) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v). In this context, the incubation step itself does not involve a pH adjustment step. In this context, the incubation step comprises the following sequential steps:

[0359] (1) heating the flow-through of step (iii) up to 35 to 40 °C such as 37 °C with 20 to 200 mM phosphate buffer such as 50 to 150 mM at pH 5.0 to 7.0 such as 5.0 to 6.5;

[0360] (2) maintaining the temperature of (1 ) for at least 20 to 24 hours; and

[0361] (3) cooling down to 18 to 22 °C, to obtain an incubated flow-through.

[0362] In a preferred embodiment, the yield of the immunoglobulin is higher as compared to the same method without an incubation step of (iii-a) or (iii-b), wherein the yield of the immunoglobulin is given by the main peak in % as measured by ion exchange-high-performance liquid chromatography (HPLC-IEX). In another preferred embodiment, the yield of the immunoglobulin is higher as compared to the same method without an incubation step of (iii-b), wherein the yield of the immunoglobulin is given by the main peak in % as measured by ion exchange-high- performance liquid chromatography (HPLC-IEX).

[0363] Cation exchange chromatography

[0364] The method of the present invention further comprises a cation exchange chromatography step or a mixed-mode cation exchange chromatography.

[0365] Mixed-mode chromatography is an interaction of at least two principles, hydrophobic interaction and ion exchange or metal affinity interaction and ion exchange. Negatively charged ligands belong to the cation exchanger mixed-mode (e.g., Capto MMC). Some mixed-mode media have zwitterionic character (e.g., Bakerbond ABx). Other mixed-mode media possess hydrophobic ligands which are ionisable and convert from uncharged to positively charged by lowering the pH (e.g., MEP HyperCel). Finally, hydroxyapatite and fluorapatite media have more complex mixed-mode functions by possessing positively charged calcium ions and negatively charged phosphate groups.

[0366] In some embodiments, the mixed-mode cation exchange chromatography is carried out after the affinity chromatography step. In some embodiments, the mixed-mode cation exchange chromatography step is carried out after the affinity chromatography step and after the mixedmode anion exchange chromatography step. In preferred embodiments, the mixed-mode cation exchange chromatography step is performed immediately after the incubation step. In preferred embodiments, the mixed-mode cation exchange chromatography follows the affinity chromatography step, the anion exchange chromatography step or the mixed-mode anion exchange chromatography step and the incubation step, in that order.

[0367] In a preferred embodiment, a method is provided for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0368] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0369] (ii) a pH adjustment step to obtain a pH adjusted composition,

[0370] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or

[0371] (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through, (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixed-mode cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0372] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), said adjusted pH is maintained throughout steps (ii) to (v) within 2.0 units, preferably 1 .8 units such as 1 .6 units of said adjusted pH.

[0373] In a preferred embodiment, a method is provided for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0374] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0375] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0376] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or

[0377] (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0378] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixed-mode cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0379] (vi) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v).

[0380] In a preferred embodiment, a method is provided for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps: (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0381] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0382] (iii) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0383] (iv) subjecting the incubated flow-through obtained in step (iii) to a mixed-mode cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0384] (iv) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v).

[0385] Cation exchange chromatography relies on charge-charge interactions between the proteins in the sample and the charges immobilized on the resin. In cation exchange chromatography, the molecules to be bound are positively charged and the immobilized functional groups (ligands) are negatively charged. In some embodiments, the cation exchange chromatography step is performed using a resin selected from a group consisting of S-resins (sulphonate), SP resins (sulphopropyl), SiB resins (sulphoisobutyl), SE resins (sulphoethyl), and CM resins (carboxymethyl).

[0386] In some embodiments, the cation exchange chromatography is carried out after the affinity chromatography step. In some embodiments, the cation exchange chromatography step is carried out after the affinity chromatography step and after the mixed-mode anion exchange chromatography step. In preferred embodiments, the cation exchange chromatography step is performed immediately after the incubation step. In preferred embodiments, the cation exchange chromatography follows the affinity chromatography step, the anion exchange chromatography step or mixed-mode anion exchange chromatography step and the incubation step, in that order.

[0387] In general, the cation exchange chromatography step can be performed with all common commercially available cation exchange resins or membranes. Cation exchange resins may be used in the form of pre-packed columns or membranes on which the functional group is fixed. Alternatively, the resins may be purchased as bulk material and the columns are packed by the user. There are no specific limitations as to the capacity and the dimension of the columns other than the usual ones. The person skilled in the art knows the amount of cation exchange resin and the size of the column to be used. This depends on the overall scale of the process. Typical commercially available products include, for example, Macro-Prep High S, Macro-Prep CM, Unosphere Rapid S, Unosphere Rapid S40, Nuvia S, and Nuvia HR-S (Bio-Rad, California, USA), Toyopearl CM, Toyopearl SP, Toyopearl Sulfate 650 F, and Toyopearl GigaCap S (Tosoh Bioscience, Germany), Millipore ProRes S, Fractogel EMD COO-, Fractogel EMD SO3-, Fractogel EMD SE Hicap, Eshmuno CPX (Merck KGaA, Germany), Biosepra CM Ceramic HyperD, Biosepra S Ceramic HyperD, S HyperCel (Pall Corporation, New York, USA), Poros HS, Poros XS (Applied Biosystems, Germany), YMC BioPro SmartSep 30S, YMC BioPro SmartSep 70S (YMC Europe) CM-Sepharose FF, SP-Sepharose FF, S-Sepharose FF, SP- Sepharose HP, SP-Sepharose XL, SP-Sepharose Big Beads, CM-Sephadex, Capto S, Capto SP ImpRes, and Source S (all GE Healthcare, Germany).

[0388] In preferred embodiments, the cation exchange chromatography step is performed with sulfonate, sulfopropyl, or sulfoisobutyl ligands. In some embodiments, the cation exchange chromatography step is performed with sulfonate or sulfopropyl ligands linked to rigid matrices such as highly cross-linked agarose. In some embodiments, the cation exchange chromatography step is performed with Nuvia HR-S, or poly(styrenevinylbenzene), Poros 50 HS, or polymethacrylate, e.g., Fractogel EMD SO3-. The most preferred cation exchange resin is Poros HS 50 with sulfopropyl (-CH2CH2CH2SO3-) ligands bound to a cross-linked poly(styrenedivinylbenzene) matrix.

[0389] In a preferred embodiment, a method is provided for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0390] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0391] (ii) a pH adjustment step to obtain a pH adjusted composition,

[0392] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or

[0393] (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through, (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0394] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), said adjusted pH is maintained throughout steps (ii) to (v) within 2.0 units, preferably 1 .8 units such as 1 .6 units of said adjusted pH.

[0395] In a preferred embodiment, a method is provided for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0396] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0397] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0398] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or

[0399] (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0400] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0401] (vi) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v).

[0402] In a preferred embodiment, a method is provided for obtaining an immunoglobulin with N- terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps: (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0403] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0404] (iii) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0405] (iv) subjecting the incubated flow-through obtained in step (iii) to a cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0406] (iv) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v).

[0407] In some embodiments, the cation exchange chromatography may be equilibrated with a buffer having a pH of about pH within 2.0 units such as 1.8 units or 1.6 units of the adjusted pH of step (i). In some embodiments, the cation exchange chromatography may be equilibrated with a buffer having a pH of about 5.0 to 7.0 such as 5.0 to 6.8 or 5.0 to 6.6 or 5.0 to 6.5.

[0408] In some embodiments, the buffer concentration may be in the range of 10 mM to 100 mM, preferably in the range of 20 mM to 50 mM. In some embodiments, the buffer is citrate, lactate, succinate, formate, acetate, malonate, glycine, MES, PIPES, phosphate, BisTris, or mixtures thereof. In a preferred embodiment, an acetate buffer such as sodium acetate is used.

[0409] The cation exchange chromatography step may separate charge variants of the immunoglobulin and may deplete residual host cell proteins, host cell DNA, aggregates, fragments, viruses, endotoxins, flocculants, and leached Protein A. The immunoglobulin may bind to the resin at a pH value below the isoelectric point (pl) of the immunoglobulin and at low conductivity. The pl of an immunoglobulin refers to the pH at which the protein has a net overall charge equal to zero, i.e. , the pH at which the protein has an equal number of positive and negative charges. Determination of the pl may be accomplished according to techniques established in the prior art, such as isoelectric focusing. Low conductivity according to the the present invention means a conductivity below 5 mS / cm.

[0410] In some embodiments, for elution, an increase in the ionic strength of the elution buffer may be used, provided either by a single step or a gradient. Examples of salts used in the method according to the present invention for the elution of cation exchange chromatography are chloride salts, sulfate salts, phosphate salts, citrate salts, formate salts, or acetate salts. Preferably, NaCI or KCI are used. The ionic strength may be increased to up to 1 M. In a preferred embodiment, the salt gradient elution for the cation exchange chromatography may be performed with sodium acetate with NaCI. The gradient may be generated by mixing two buffers (e.g., starting with 100% Buffer A and mixing with buffer B having a NaCI concentration of 500 mM until 37 % of buffer B is reached). The increasing salt gradient elution may comprise three segments. For example in segment (i) buffer B is increased within 1 to 3 CV, preferably within 1 CV, the percentage of buffer B is raised from 0% to a content in the range of about 5 % to about 50 %, preferably of about 10 % to about 30 %, such as 15 % and in segment (ii) buffer B is increased within 5 to 30 CV, preferably 10 to 20 CV, such as 10 CV, to an content in the range of about 10 % to about 80 %, preferably about 30 % to about 50%, such as about 37%.

[0411] In some embodiments, an increase in the pH of the elution buffer may be used in the method according to the present invention, provided either by a single step or a gradient. Further, an increase in both the pH and ionic strength of the elution buffer may be used for elution, provided either by a single step or a gradient.

[0412] The cation exchange chromatography resin may be regenerated with 1 M NaCI for 2 to 3 column volumes and 1 M NaOH for 2 to 3 column volumes.

[0413] Depth filtration and microfiltration

[0414] The method of the present invention may further comprise one or more depth filtration steps. Depth filters consist of a matrix of fibers or beads, wherein separation takes place throughout the matrix rather than on its surface. Examples of depth filters include without limitation Pall SXL series (e.g., SXLP700416 and SXLPDE2408SP filter capsules, Pall HP series (e.g., PDD1 , PDE2, PDH4, PDK5, PEKM) (Pall Corporation), Millistak series (e.g., XOHC, FOHC, DOHC, AIHC, and BIHC Pod filters), HC-Pro series, Clarisolve series (e.g., 40 MS) (EMD Millipore), or Zeta Plus Series (e.g., 60SP02, 60ZB05, 90ZB08, VR07, and VR05 filter capsules) (3M).

[0415] Preferably, the depth filtration filter utilized in the method according to the present invention is composed of cellulose, inorganic filter aid, and a resin system that imparts a strong positive charge to the filter matrix, as for example Zeta Plus from 3M.

[0416] Preferably, the depth filtration filter utilized in the method of the present invention comprises more than one layer. For example, a double-layer filter may be used for depth-filtration.

[0417] In some embodiments, the process parameters for performing the depth filtration comprise a volumetric load of 10 to 200 L / m2, preferably of 50 to 100 L / m2, a maximum pressure of 0.2 to 2.0 bar, preferably 0.5 to 1 .5 bar and room temperature.

[0418] In contrast to depth filtration, microfiltration is a particle filtration method using membranes whereby separation is achieved by retaining the particles on the surface of a membrane. Preferably, membrane filters with a pore size from about 0.1 to 10 pm are used in the method of the present invention. More preferably, sterilized micro filters with pore sizes of about 0.2 pm are used, as for example Supor EKV (Pall). The use of additional pre-filters with larger pore sizes (0.45 pm, 3 pm) to prevent the decrease in flow by rapid blocking of the small pore sized filters is common.

[0419] In a preferred embodiment, the purification may include one or more filtration steps preceding the first chromatography step. In a more preferred embodiment, the first chromatography step is preceded by a depth filtration and a microfiltration step for cell culture clarification. Additional filtration steps using positively and / or negatively charged membranes may be included in a filtration train clarifying the sample prior to the capture chromatography step. Filter modules, combining different materials, charged and uncharged, are also comprised in a preferred embodiment.

[0420] Virus filtration, Tangential Flow Ultrafiltration / Diafiltration (TF-UF / DF)

[0421] The method of the present invention may comprise one or more ultrafiltration and / or nanofiltration step(s). Ultrafiltration is a form of membrane filtration in which pressure forces a liquid against a semipermeable membrane. Suspended solids and solutes of high molecular weight are retained, while water and low molecular weight solutes pass through the membrane. Ultrafiltration is a commonly used method for separation, purifying and concentrating macromolecular solutions, especially protein solutions. Ultrafiltration may be combined with diafiltration. This mode is suitable for buffer exchange, to remove salts and other microspecies from the solution via repeated or continuous dilution and re-concentration. Ultrafiltration may be performed with stacked membranes in a tangential flow or cross flow filtration system (TFF or TF-UF), especially for processing large sample volumes. Alternatively, hollow fiber systems are commonly used for ultrafiltration. Membrane cut-off sizes range from about 1 to 300 kDa. For immunoglobulins, typical cut offs for the ultrafiltration membranes are 10 to 100 kDa. Within the framework of the present invention, a molecular weight cut off of 30 or 50 kDa for the UF membranes is preferred.

[0422] Nanofiltration is predominantly used for viral filtration and is required for the safety of therapeutic proteins produced in mammalian cell cultures. Nanofiltration steps are usually performed at the end of downstream processes close to filling of the bulk of purified immunoglobulin. The pore sizes of the frequently used nanofilters range between 15 and 35 nm (e.g., Planova (Asahi Kasei), Viresolve (EMD-Millipore)). In the present invention, the terms “nanofiltration”, “virus filtration”, and “virus retentive filtration” are used synonymously.

[0423] Items of the present invention

[0424] 1. A method for obtaining an immunoglobulin with N-terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps: (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0425] (ii) a pH adjustment step to obtain a pH adjusted composition,

[0426] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or

[0427] (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0428] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0429] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), said adjusted pH is maintained throughout steps (ii) to (v) within 2.0 units, preferably 1.8 units such as 1.6 units of said adjusted pH, and wherein the immunoglobulin is IL-6 receptor antibody.

[0430] 2. The method of item 1 , comprising the following sequential steps:

[0431] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0432] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0433] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or

[0434] (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through, (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0435] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v), and wherein the immunoglobulin is IL-6 receptor antibody.

[0436] 3. The method of any one of items 1 or 2, comprising the following sequential steps:

[0437] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0438] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0439] (iii) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0440] (iv) subjecting the incubated flow-through obtained in step (iii) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0441] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v), and wherein the immunoglobulin is IL-6 receptor antibody.

[0442] 4. A method for obtaining an immunoglobulin with N-terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0443] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0444] (ii) a pH adjustment step to obtain a pH adjusted composition,

[0445] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or

[0446] (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0447] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0448] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), said adjusted pH is maintained throughout steps (ii) to (v) within 2.0 units, preferably 1.8 units such as 1.6 units of said adjusted pH, and wherein the immunoglobulin is tocilizumab.

[0449] 5. The method of item 4, comprising the following sequential steps:

[0450] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0451] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0452] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or

[0453] (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0454] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0455] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v), and wherein the immunoglobulin is tocilizumab. 6. The method of any one of items 4 or 5, comprising the following sequential steps:

[0456] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0457] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0458] (iii) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0459] (iv) subjecting the incubated flow-through obtained in step (iii) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0460] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v), and wherein the immunoglobulin is tocilizumab.

[0461] 7. A method for obtaining an immunoglobulin with N-terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0462] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0463] (ii) a pH adjustment step to obtain a pH adjusted composition,

[0464] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or

[0465] (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0466] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate, (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), said adjusted pH is maintained throughout steps (ii) to (v) within 2.0 units, preferably 1.8 units such as 1.6 units of said adjusted pH, and wherein the incubation step is conducted over a period of at least 20 to 24 hours at a temperature from 35 to 40 °C using a phosphate buffer at a final concentration of 50 to 150 mM. and wherein the immunoglobulin is IL-6 receptor antibody.

[0467] 8. The method of item 7, comprising the following sequential steps:

[0468] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0469] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0470] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or

[0471] (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0472] (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0473] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v), and wherein the incubation step is conducted over a period of at least 20 to 24 hours at a temperature from 35 to 40 °C using a phosphate buffer at a final concentration of 50 to 150 mM. and wherein the immunoglobulin is IL-6 receptor antibody. 9. The method of any one of items 7 or 8, comprising the following sequential steps:

[0474] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0475] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0476] (iii) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0477] (iv) subjecting the incubated flow-through obtained in step (iii) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0478] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v), and wherein the incubation step is conducted over a period of at least 20 to 24 hours at a temperature from 35 to 40 °C using a phosphate buffer at a final concentration of 50 to 150 mM. and wherein the immunoglobulin is IL-6 receptor antibody.

[0479] 10. A method for obtaining an immunoglobulin with N-terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:

[0480] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0481] (ii) a pH adjustment step to obtain a pH adjusted composition,

[0482] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or

[0483] (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through, (iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0484] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), said adjusted pH is maintained throughout steps (ii) to (v) within 2.0 units, preferably 1.8 units such as 1.6 units of said adjusted pH, and wherein the incubation step is conducted over a period of at least 20 to 24 hours at a temperature from 35 to 40 °C using a phosphate buffer at a final concentration of 50 to 150 mM. and wherein the immunoglobulin is tocilizumab.

[0485] 11 . The method of item 10, comprising the following sequential steps:

[0486] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0487] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0488] (iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or

[0489] (iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0490] (iv subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0491] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v), and wherein the incubation step is conducted over a period of at least 20 to 24 hours at a temperature from 35 to 40 °C using a phosphate buffer at a final concentration of 50 to 150 mM. and wherein the immunoglobulin is tocilizumab.

[0492] 12. The method of any one of items 10 or 11 , comprising the following sequential steps:

[0493] (i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;

[0494] (ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,

[0495] (iii) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,

[0496] (iv) subjecting the incubated flow-through obtained in step (iii) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,

[0497] (v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v), and wherein the incubation step is conducted over a period of at least 20 to 24 hours at a temperature from 35 to 40 °C using a phosphate buffer at a final concentration of 50 to 150 mM. and wherein the immunoglobulin is tocilizumab.

[0498] Examples

[0499] The methods of the invention are supported and illustrated by reference to the following examples. It is emphasized that these examples should by no means be interpreted as limiting the scope of the invention.

[0500] Example 1 : Incubation parameters promoting N-terminal Gin cyclization

[0501] The following experiments were conducted to identify the effect of the incubation parameters temperature, pH, incubation time, and buffer concentration for the robust conversion from N- terminal Gin to PyroGlu prior to cation exchange chromatography, and thus boosting overall mAb yield.

[0502] Example 1.1 : Effect of incubation temperature and incubation pH on the cyclization of N-terminal Gin

[0503] The clarified cell culture harvest was subjected to Protein A affinity chromatography followed by low pH virus inactivation and mixed-mode anion exchange chromatography.

[0504] To identify the effect of incubation pH on N-terminal Gin conversion to PyroGlu either the load at pH 6.5 or the flow-through at pH 5.0 of the mixed-mode anion exchange chromatography was incubated for 7 days at a temperature of 20 or 37 °C. The effect of incubation temperature and incubation pH on charge variants of Tocilizumab is shown in Table 1 and Table 2.

[0505] Table 1 : Effect of incubation temperature and incubation pH on N-terminal Gin variants and main peak of Tocilizumab (detected by HPLC-IEX)

[0506] Table 2: Effect of incubation temperature and incubation pH on acidic variants and basic variants of Tocilizumab (detected by HPLC-IEX)

[0507] The converted PyroGlu can be detected as an increase of the main peak. Conversion of N- terminal Gin variant was observed at 20 °C for 7 days incubation for both pH 6.5 and pH 5.0, respectively. Incubation at 37 °C resulted in a stronger reduction of N-terminal Gin after 7 days at both pH 6.5 and pH 5.0, respectively. The cyclization of N-terminal glutamine was accelerated by the increasing temperature and independent of the pH within the range of pH 5.0 to pH 6.5. Example 1.2: Effect of phosphate addition on N-terminal Gin variants

[0508] To study the effect of the buffer environment 50 mM Nab^PC was added to the mixed-mode anion exchange flow-through at pH 6.5 and incubated for 50 hours either at room temperature or 37 °C. The addition of 50 mM phosphate reduced N-terminal Gin variants (Table 3) at room temperature and 37 °C, respectively. However, the overall reduction of N-terminal Gin of Tocilizumab was more pronounced at an incubation temperature of 37 °C compared to the incubation at room temperature.

[0509] Table 3: Effect of phosphate addition on the reduction of N-terminal Gin variant (%) of Tocilizumab

[0510] Oxidation of Met256 was monitored because minor oxidation process during the 50 hours long incubation can lead to a quality comparability problem. Therefore, incubation time should be maximized in 50 hours (Table 4).

[0511] Table 4: Met256 oxidation ratio (%) during the incubation

[0512] Example 1.3: Effect of phosphate concentration and incubation time on the cyclization of N- terminal Gin variants

[0513] The phosphate concentration-dependent N-terminal Gin cyclisation was analysed by setting a phosphate concentration of 50 mM, 100 mM, or 150 mM by addition of 2 M NaH2PO4 to mixedmode anion exchange chromatography flow-through at pH 5.5 which was incubated for 24 hours (Table 5). Table 5: Phosphate concentration-dependent effect on the reduction of N-terminal Gin variants of Tocilizumab (detected by HPLC-IEX)

[0514] The percental amount of N-terminal Gin variants of Tocilizumab decreased with increasing phosphate concentration and incubation time regardless of the incubation temperature that was applied. However, the overall reduction of N-terminal Gin variants of Tocilizumab was more pronounced at an incubation temperature of 37 °C compared to the incubation at room temperature. A clear reduction in N-terminal Gin variants could be achieved already after 14 hours of incubating the intermediate mixed-mode anion exchange flow-through at 37 °C and the reduction of N-terminal Gin variants was further increased after 24 hours.

[0515] However, an increased phosphate concentration leads to an increase in conductivity of the buffer solution. Consequently, the loading of the subsequent cation exchange chromatography will require a higher dilution rate resulting in an extended process time.

[0516] Among other parameters, an incubation temperature of 37°C, a phosphate concentration of the intermediate flow-through of 50 mM, and an incubation time of 24 hours was sufficient for the incubation step leading to a significant increase in the conversion from N-terminal Gin to PyroGlu bearing Tocilizumab prior to cation exchange chromatography thereby reducing the amount of N-terminal Gin to be depleted by cation exchange chromatography and thus boost overall mAb yield.

[0517] Moreover, it could be shown that the formation of PyroGlu from N-terminal Gin was independent from the pH of the incubated in-process material / protein solution within the range of pH 5.0 to pH 6.5. Therefore, the incubation step can be performed (directly) prior or following the intermediate chromatography step, i.e. , with the pH adjusted mixed-mode anion exchange load (pH 6.4-6.6) or with the mixed-mode anion exchange flow-through (pH 5.0-5.5) without any further adjustment of the pH. Thus, the incubation step can simply be embedded into the downstream purification train. Example 2: Downstream purification process - Purification process to reduces N- terminal Gin variants of Tocilizumab

[0518] In the following methods are described in which an incubation step was implemented in the downstream purification sequence of Tocilizumab to promote the conversion of N-terminal Gin to PyroGlu, thus increasing process producibility and process yield. The following methods were developed in small scale and applied in pilot scale and commercial scale process for the downstream purification process of Tocilizumab.

[0519] Selection of chromatography media

[0520] Subsequent to an extended screening of commonly used chromatography media for capture, intermediate and polishing, the following chromatography media were selected for downstream purification process of Tocilizumab (Table 6).

[0521] Table 6: Chromatography media.

[0522] MMAEX = mixed-mode anion exchange chromatography; CEX = cation exchange chromatography

[0523] Preparation of cell culture supernatant

[0524] The cell culture harvest was passed through a depth filter (3M) and the clarified harvest was further filtered through a membrane filter with 0.2 pm pore size (LifeAssure 3M). The filtered solution was stored in a 2000L single use mixer (SUM) bag (ThermoFisher) at room temperature until use.

[0525] Protein A affinity chromatography

[0526] The first downstream process step was dedicated to remove the majority of HCP, HCDNA and other impurities. During the two-cycle Protein A capture, the depth-filtered cell culture fluid was applied to a MabSelect SuRe LX (Cytiva) Protein A resin.

[0527] The parameters of the packed column and the used mode and utilities are shown in Table 7. Table 7: Details of protein A affinity chromatography

[0528] Briefly, the column was equilibrated with buffer 1.5 mM Tris-base, 18.5 mM Tris-HCI, 150 mM NaCI, pH 7.2. Intermediate washes were performed with buffer equilibration buffer and 10 mM EDTA, 2.0 M urea, 1.5 M NaCI, 40 mM NaH2PO4x2H2O, pH 7.2. The captured Tocilizumab was eluted from the MabSelect SuRe LX resin with 100 mM citric acid monohydrate buffer (pH 3.5). The eluate was collected in a 500 L SUM bag (ThermoFisher). The MabSelect SuRe LX resin was regenerated by a series of purified water, 0.2 M NaOH, and 3.5 v / v% acetic acid, 100 mM Na2SO4 between the cycles. The column was stored in 20 % ethanol between the batches. The chromatography sequence and parameters of the Protein A affinity chromatography can be seen in Table 8. Table 8: Chromatography sequence of affinity chromatographic step

[0529] The protein A affinity chromatography step comprised a viral clearance step comprising low pH virus inactivation. The low pH virus inactivation was performed in two cycles. Directly after the collection, the pH of the Protein A affinity chromatography eluate was adjusted to pH 3.3±0.1 with 1 M citric acid during constant stirring. After the pH adjustment the Protein A affinity chromatography eluate was transferred into another 500L SUM bag where it was incubated for minimum 60 minutes at room temperature without stirring. pH adjustment step

[0530] The pH of the eluate was adjusted to pH 6.5±0.1 with 2 M Tris (pH 9.0). The in-process materials from the two cycles were stored in 500 L SUM bags (ThermoFisher) at room temperature before the next chromatography step.

[0531] According to the invention, after this pH adjustment step, there is no other pH adjustment step that changes the pH by more than 2.0 units or outside of 5.0 to 7.0.

[0532] The process of pH adjustment is shown in Table 9.

[0533] Table 9: pH adjustment step

[0534] Mixed-mode anion exchange chromatography

[0535] Residual process related impurities (such as HCP, HCDNA, leached protein A and potentially endotoxin) were further decreased in the mixed-mode anion exchange chromatography step. Product related impurities can also be decreased via the mixed-mode anion exchange intermediate purification step. Furthermore, mixed-mode anion exchange chromatography also contributes to viral clearance. The mixed-mode anion exchange chromatography was performed in flow-through mode using the Capto Adhere ImpRes resin (GE Healthcare). Before the load, the conductivity of the neutralized Protein A eluate was adjusted. Therefore, the neutralized Protein A eluate was diluted with purified water by applying an in-line dilution system to a range of 4.5 to 7.5 mS / cm conductivity. The diluted load protein solution was applied to Capto Adhere ImpRes column which was equilibrated with a 50 mM sodium phosphate buffer (17.9 mM Na2HPO4x2H2O, 32.1 mM NaH2PO4x2H2O, pH 6.6). Collection of the flow-through started at 0.1 AU UV (280 nm) absorbance signal into a 500 L SUM bag through a PALL NT7UEDFP1G microfilter. After the protein load the equilibration buffer was applied to the column and the flow-through was collected into the same 500 L SUM bag. Between the two cycles, the Capto Adhere ImpRes resin was regenerated with 1 M NaCI, 100 mM citric acid and 1.0 M NaOH. The column parameters and chromatography sequence are shown in Tables 10 and 11 .

[0536] Table 10: Equipment and process information

[0537] Table 11 : Chromatography sequence of mixed-mode anion exchange chromatography step Incubation step

[0538] The incubation step was implemented into the purification train to facilitate the conversion of N- terminal Gin charge variants to PyroGlu / main peak and acidic variants, thus stabilizing the charge variant profile of the mAb and hence improving biosimilarity of the product and the robustness of the following CEX chromatography by stabilizing the load material charge variants ratio.

[0539] The phosphate ion concentration of the mixed-mode anion exchange flow-through was increased to 50 mM by addition of 2.0 M NaH2PO4x2H2O, and pH was set to 5.5±0.1 using 2 M acetic acid buffer. The pH adjusted mixed-mode anion exchange flow-through from the first cycle was hold till the second mixed-mode anion exchange flow-through collection, pH and phosphate concentration adjustment. When phosphate concentration and pH of both solutions were adjusted, heating up to 37.0 °C and 70 rpm stirring were started. The incubation time was 24 hours minimum, beginning from the time point when the temperature of the in-process material reaches the set point (37°C). Stirring was stopped during the incubation temperature was maintained at 37°C. The solution was cooled down to room temperature under stirring. The utilized equipment and the description of the process can be seen in Table 12.

[0540] Table 12: Equipment and used parameter of the incubation step

[0541] Cation exchange chromatography In the next step of the downstream purification process, a cation exchange chromatography was conducted to further remove low molecular weight (LMW) impurities, acidic charge variants, and HCP from the samples. The cation exchange chromatographic step was performed using Poros 50 HS strong cation exchange resin (ThermoFisher) equilibrated with 50 mM sodium acetate buffer, pH 5.5, and the column was washed with the same buffer. The flow-through of mixedmode anion exchange chromatography was diluted with purified water to adjust the conductivity to 4.0 - 6.0 mS / cm during the load. The chromatograph was operated in bind and elute mode. The bound antibody was eluted with a two-step linear conductivity gradient by mixing buffer CEX-A and buffer CEX-B (50 mM sodium acetate, 500 mM NaCI, pH 5.5) in the following ratio and sequences (see Table 8): 0% CEX-B to 15% CEX-B (Gradient I) and 15% CEX-B to 37% CEX-B (Gradient II). The collection of fractions was started when the eluate UV280nm absorbance value increased above 0.15 AU and stopped when it decreased below 0.15 AU. The Poros HS 50 resin was stripped with 37% 50 mM sodium acetate, 500mM NaCI, pH 5.5, and regenerated with 1 M NaCI and 1 M NaOH and stored in 10 mM NaOH.

[0542] The utilized column parameters and chromatography sequence are shown in Tables 13 to 14.

[0543] Table 13: Equipment and process information

[0544] Table 14: Chromatography sequence of cation exchange chromatography step

[0545] Virus retentive filtration The cation exchange chromatography eluate was filtered with a Millipore Virosolve Pro Magnus Device using Wattson Marlow peristaltic pump. A dual layer prefilter (SARTOPORE 2 - 5447358K2-SS filter) with 0.2 + 0.1 pm pore size was used to protect the filter from blockage. The filters were conditioned with sodium acetate buffer (50 mM sodium acetate, pH 5.5) prior to filtration. The same buffer was used to wash out the residual mAb from the filter train. The pressure was set between 0.9 to 2.0 bar throughout filtration. The filtrate was collected in a TFF feed tank until the filtrate volume reached 450±30 kg, then the procedure was stopped until the TFF concentration was started. The virus retentive filtration was started again when the TFF concentration was started.

[0546] Equipment and conditions of the virus retentive filtration step are shown in Tables 15 and 16.

[0547] Table 15: Equipment and parameters of virus filtration Table 16: Virus filtration sequence

[0548] Tangential flow Ultrafiltration / Diafiltration

[0549] Tangential Flow Ultrafiltration / Diafiltration was carried out to set the concentration of the mAb and replace the solution buffer with the final formulation buffer. The concentration of the virus filtered eluate was performed using PALL ultrafiltration system with a Pellicon 3 cassette with Ultracel 30 kDa membrane (P3C030C10) which was flushed with 4.1 mM Na2HPC>4x2H20, 10.9 mM NaH2 C>4x2H2O, pH 6.5. First, the virus filtrated in-process material was concentrated to 20.0±2.0 g / L (preconcentration). Then, the buffer of the filtrate was exchanged with 10 diavolumes of 4.1 mM Na2HPC>4x2H2O, 10.9 mM NaH2PC>4x2H2O, pH 6.5. The final mAb concentration was set to 35 ±2 g / L. The TMP was set to 0.35 bar during the tangential flow filtration.

[0550] The ultra / diafiltered and concentrated mAb solution was supplemented with 10 times concentrated sucrose stock solution (500 g / L sucrose, 15 mM sodium phosphate pH 6.5) and aseptically filtered (PALL Supor EKV filter, NP5LEKVP1 G) into 20 L and 10 L single-use bags (2D HyClone I Thermo Scientific BioProcess Labtainer). The produced drug substance was stored at -70 °C.

[0551] The equipment and conditions of the tangential flow filtration step is shown in Tables 17 and 18. Table 17: Equipment and parameters of ultrafiltration

[0552] Table 18: Ultrafiltration sequence

[0553] Effect of the incubation step in downstream purification sequence on charge variant profile of Tocilizumab

[0554] The incubation of the intermediate flow-through prior to cation exchange chromatography drastically decreased the N-terminal Gin variants (Table 19). Table 19: Effect of incubation on charge variant profile of Tocilizumab

[0555] The remaining N-terminal Gin variants were eliminated by the following cation exchange chromatography step. Moreover, the removal of the N-terminal Gin variants resulted in a clear increase of the main peak over the downstream purification process. Basic variants were decreased in the cation exchange chromatography step due to the removal of N-terminal Gin which contains a large number of basic variants. Altogether, the purification sequence covering the incubation of the intermediate flow-through prior to polishing chromatography had proven to be most efficient not only with regards to N-terminal Gin conversion to PyroGlu but also in terms of the obtained main peak / mAb yield and thus was chosen for the subsequent purification runs.

[0556] List of References

[0557] 1 . Chelius D, Jing K, Lueras A, Rehder DS, Dillon TM, Vizel A, Rajan RS, Li T, Treuheit MJ, Bondarenko PV (2006) Formation of Pyroglutamic Acid from N-Terminal Glutamic Acid in Immunoglobulin Gamma Antibodies. Anal. Chem. 78, 2370-2376

[0558] 2. Dick LW., Kim C, Qiu D, Cheng KC (2007) Determination of the Origin of the N-Terminal Pyro-Glutamate Variation in Monoclonal Antibodies Using Model Peptides. Biotechnology and Bioengineering 97(3): 544-553

[0559] 3. Liu YD, Goetze AM, Bass RB, Flynn GC (2011) N-terminal Glutamate to Pyroglutamate Conversion in Vivo for Human lgG2 Antibodies. The Journal of Biological Chemistry 286(13): 11211-11217

[0560] 4. Scheinecker C, Smolen J, Yasothan U, Stoll S, Kirkpatrick P (2009) Tocilizumab. Nature Reviews Drug Discovery 8: 273-274

[0561] 5. Schilling S, Wasternack C, Demuth HU (2008) Glutaminyl cyclases from animals and plants: a case of functionally convergent protein evolution. Biol. Chem. 389: 983-991

[0562] 6. Sheppard M, Laskou F, Stapleton PP, Hadavi S, Dasgupta B (2017) Tocilizumab (Actemra). Human Vaccines & Immunotherapeutics 13(9): 1972-1988 7. Tanakaa T, Narazakia M, Kishimoto T (2011) Anti-interleukin-6 receptor antibody, tocilizumab, for the treatment of autoimmune diseases. FEBS Letters 585: 3699-3709

[0563] 8. Xu W, Peng Y, Wang F, Paporello B, Richardson D, Liu H (2013) Method to convert N- terminal glutamine to pyroglutamate for characterization of recombinant monoclonal antibodies. Analytical Biochemistry 436: 10-12

[0564] 9. Yu L, Vizel A, Huff MB, Young M, Remmele RL, He B (2006) Investigation of N-terminal glutamate cyclization of recombinant monoclonal antibody in formulation development. Journal of Pharmaceutical and Biomedical Analysis 42: 455-463

[0565] 10. WO9219759A1

[0566] 11. W02019043096A1

Claims

Claims1. A method for obtaining an immunoglobulin with N-terminal pyro glutamate from a composition comprising the immunoglobulin with N-terminal glutamine, the method comprising the following sequential steps:(i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;(ii) a pH adjustment step to obtain a pH adjusted composition,(iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or(iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,(iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,(v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), said adjusted pH is maintained throughout steps (ii) to (v) within 2.0 units, preferably 1.8 units such as 1.6 units of said adjusted pH.

2. The method of claim 1 , comprising the following sequential steps:(i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;(ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,(iii-a) an incubation step on the pH adjusted composition obtained in step (ii) immediately followed by a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode to obtain an incubated flow-through, or(iii-b) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,(iv) subjecting the incubated flow-through obtained in step (iii-a) or (iii-b) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,(v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (ii) to (v).

3. The method of any one of claims 1 or 2, comprising the following sequential steps:(i) an affinity chromatography step on the composition comprising the immunoglobulin with N-terminal glutamine in bind-elute mode to obtain an eluate;(ii) a pH adjustment step to a pH from 5.0 to 7.0, preferably 5.0 to 6.6 to obtain a pH adjusted composition,(iii) a mixed-mode anion exchange chromatography or anion exchange chromatography in flow-through mode on the pH adjusted composition obtained in step (ii) followed by an incubation step to obtain an incubated flow-through,(iv) subjecting the incubated flow-through obtained in step (iii) to a mixed-mode cation exchange chromatography or cation exchange chromatography in bind-elute mode to obtain an incubated eluate,(v) subjecting the incubated eluate of step (iv) to at least one filtration step to obtain a filtrate comprising the immunoglobulin with N-terminal pyro glutamate, wherein after the pH adjustment is conducted in step (ii), the same pH range is maintained throughout steps (i) to (v).

4. The method of any one of the preceding claims, wherein the incubation step is performed in a buffer.

5. The method of claim 4, wherein the buffer is a phosphate buffer preferably a sodium phosphate buffer.

6. The method of claim 5 or 6, wherein the buffer has a final concentration of 10 to 200 mM, preferably a final concentration of 50 to 150 mM, more preferably a final concentration of 50 mM.

7. The method of any one of the preceding claims, wherein the incubation step is performed for 2 to 35 hours, such as 14 to 35 hours, or at least 24 hours such as 30 hours.

8. The method of any one of the preceding claims, wherein the incubation step is performed at a temperature of 20 to 40 °C such as 35 °C to 39 °C such as 37 °C.

9. The method of any one of the preceding claims, wherein the incubation step is performed non-enzymatically.

10. The method of any one of the preceding claims, wherein the incubation step reduces the N-terminal Gin by at least 70 %, at least 80 %, at least 90%, at least 95% or at least 99%.

11. The method of any one of the preceding claims, wherein the incubation step of (iii-a) comprises the following sequential steps(1) heating the pH adjusted composition of step (i) up to 35 to 40 °C such as 37 °C;(2) maintaining the temperature of (1) for at least 20 to 24 hours; and(3) cooling down to 18 to 22 °C, to obtain an incubated flow-through, or wherein the incubation step of (iii-b) comprises the following sequential steps(1) heating the flow-through of step (iii-b) up to 35 to 40 °C such as 37 °C;(2) maintaining the temperature of (1) for at least 20 to 24 hours; and(3) cooling down to 18 to 22 °C, to obtain an incubated flow-through.

12. The method of claim 11 , wherein heating of step (1), maintaining of step (2), and cooling of step (3) is performed actively.

13. The method of any one of claims 11 and 12, wherein heating of step (1) and cooling of step (3) is performed under stirring conditions.

14. The method of any one of the preceding claims, wherein the affinity chromatography of step (i) is a Protein A affinity chromatography, preferably wherein the Protein A affinity chromatography comprises an alkali-tolerant Protein A derivative as a ligand, preferably analkali-stabilized tetramer variant of domain B of Protein A bound to a cross-linked agarose matrix.

15. The method of any one of the preceding claims, wherein the mixed-mode anion chromatography of step (iii-a) or (iii-b) comprises N-benzyl-N-methyl ethanolamine as a ligand bound to a cross-linked agarose matrix.

16. The method of any one of the preceding claims, wherein the cation exchange chromatography of step (iv) comprises sulfopropyl (-CH2CH2CH2SO3-) as a ligand bound to a cross-linked poly(styrene-divinylbenzene) matrix.

17. The method of any one of the preceding claims, wherein the immunoglobulin is an IgG 1 .

18. The method of claim 17, wherein the lgG1 is an anti-IL-6 receptor antibody.

19. The method of claim 18, wherein the anti-IL-6 receptor antibody is tocilizumab.

20. The method of any one of the preceding claims comprising step (vi) formulating the obtained filtrate from step (v) with a buffer, and at least one further compound selected from a stabilizing agent, a surfactant, and an amino acid to obtain a pharmaceutical composition comprising the immunoglobulin.

21. The method of any one of the preceding claims, wherein the yield of the immunoglobulin is higher as compared to the same method without an incubation step of (iii-a) or (iii-b), wherein the yield of the immunoglobulin is given by the main peak in % as measured by ion exchange- high-performance liquid chromatography (HPLC-IEX).

22. The method of any one of the preceding claims, wherein the yield of the immunoglobulin is higher as compared to the same method without an incubation step of (iii-b), wherein the yield of the immunoglobulin is given by the main peak in % as measured by ion exchange-high- performance liquid chromatography (HPLC-IEX).

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