METHOD TO INCREASE ANTIBODY YIELD DURING ION EXCHANGE CHROMATOGRAPHY
20220411466 · 2022-12-29
Inventors
Cpc classification
B01D15/3809
PERFORMING OPERATIONS; TRANSPORTING
International classification
Abstract
The present invention relates to a method for increasing antibody yield during antibody purification from a sample by ion exchange chromatography in flow-through mode by pre-conditioning the sample with Tris without the use of NaCl to adjust the conductivity.
Claims
1. A method for increasing the yield of an antibody in the flow through of ion exchange chromatography during antibody purification comprising the steps of: a. providing a sample comprising an antibody; b. adjusting the conductivity of the sample; c. processing the adjusted sample by ion exchange chromatography in flow-through mode and d. collecting the flow-through comprising the antibody, wherein the conductivity of the sample in step b) is adjusted with Tris to at least 10 mS/cm and wherein the pH after adjusting the conductivity is in the range of pH 6.5 to 7.5.
2. The method according to claim 1, wherein the sample comprising an antibody is an affinity chromatography eluate.
3. The method according claim 2, wherein the affinity chromatography eluate is a Protein A chromatography eluate with a pH of about 3 to about 4.
4. The method according to claim 3, wherein the pH of about 3 to about 4 of the sample is adjusted to a pH of about 5.2 to about 5.6, preferably to a pH of 5.5.
5. The method according to claim 1, wherein the conductivity of the sample is adjusted to a conductivity between 10 and 50 mS/cm.
6. The method according to claim 5, wherein the conductivity is adjusted to the range of 13 to 30 mS/cm.
7. The method according to claim 5, wherein the conductivity is adjusted to 15 mS/cm.
8. The method according to claim 1, wherein the ion exchange chromatography is multimodal anion exchange chromatography.
9. The method according to claim 1, wherein the monoclonal antibody to be purified is a monoclonal antibody.
10. The method according to claim 9, wherein the monoclonal antibody to be purified is an anti-IL17c antibody.
11. The method according to claim 10, wherein the monoclonal anti-IL17C antibody to be purified comprises a VH of SEQ ID NO: 8 and a VL of SEQ ID NO: 7.
12. The method according to claim 11, wherein the monoclonal anti-IL17C antibody to be purified consists of a heavy chain of SEQ ID NO:10 and a light chain of SEQ ID NO: 9.
13. The method according to claim 1, wherein the yield of the purified antibody in the flow-through is more than 75%.
14. The method according to claim 1, wherein the conductivity of the sample in step b) is adjusted with Tris in the absence of NaCl.
15. A pharmaceutical composition obtained by the method according to claim 1.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0047]
[0048] Antibody purifications may employ 1-, 2-, or 3-step procedures. Numbers refer to the steps. Typical yield and purity expectations are indicated. AC=affinity chromatography; SEC=size exclusion chromatography; IEX=ion exchange chromatography; CIEX=cation exchange chromatography; AIEX=anion exchange chromatography.
[0049]
[0050] Representative flow-through elution chromatograms of samples comprising an antibody with a heavy chain of SEQ ID NO.:10 and a light chain of SEQ ID NO.:9 pre-conditioned with or without Tris and purified by multimodal AIEX. Pre-conditioning: (1) no Tris addition, conductivity: 8.2 [mS/cm], (2) 5% (v/v) Tris addition, conductivity 13.6 mS/cm, (3) 10% (v/v) Tris addition, conductivity 17.2 mS/cm, (4) 20% (v/v) Tris addition, conductivity 25.5 mS/cm. FT: Flow through. CIP: cleaning in place
DETAILED DESCRIPTION OF THE INVENTION
[0051] Protein Purification by Chromatography
[0052] Therapeutic antibody manufacturing is typically divided into i) upstream processing (USP), including production of the antibody protein; ii) downstream processing (DSP), comprising yield of the antibody in a pure form by purification; and iii) final processing to gain product integrity and safety. Typically, the first step of a downstream purification process, following the production phase, involves clarification of the harvested cell culture mixture where one or more of steps of precipitation, flocculation, (depth) filtration and/or centrifugation can be used to separate the desired antibody from cells, cellular debris, and other contaminants. Downstream purification typically includes one or more (orthogonal) chromatographic separation steps based on e.g. affinity, ion-exchange, hydrophobic interaction, hydroxyapatite, chromatofocusing, gel filtration and reverse phase to efficiently remove process and product related impurities. These contaminants include but are not limited to HCPs, leached protein A, product isoforms, high molecular weight (HMW), low molecular weight (LMW), and clipped or degraded product.
[0053] Affinity chromatography refers to the use of a compound that specifically interacts with a desired target protein to be purified. Usually, the compound is immobilized on a resin for the purpose of isolating, purifying, or removing the desired target product. For antibody purification, for example, affinity resins include Protein A obtained from Staphylococcus aureus, Protein G from Streptococcus sp., Protein L from Peptostreptococcus magnus, and recombinant or synthetic versions or peptides of such. These resins include MAbSelect™ (GE Healthcare), Prosep A® (Millipore) and others. For laboratory scale applications, a one-step affinity purification generally achieves satisfactory purity. Protein A chromatography, for example, as the most widely used affinity purification to capture IgG antibodies supports purity of >95% with excellent recovery due to its high specificity to the Fc part of IgGs. Other examples of well-established purification methods include thiophilic adsorption, hydrophobic interaction or aromatic adsorption chromatography, metal chelate affinity chromatography, and size exclusion chromatography. (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75 (1998) 93-102).
[0054] Further removal of aggregates/impurities can be achieved by a combination of one or two further orthogonal chromatographic steps that may include hydroxyapatite, hydrophobic interaction (HIC), and ion exchange chromatography (IEX, e.g. cation exchange (CEX), anion exchange (AEX), or mixed-mode exchange). At manufacturing scale, removal of aggregates and impurities is often achieved through use of IEX after initial antibody affinity chromatography. Commercial multimodal ion exchangers such as Capto MMC and Capto adhere, as well as Capto MMC ImpRes and Capto adhere ImpRes (all from GE Healthcare) can be used for removal of contaminants downstream of the initial affinity capture. IEX separates proteins with differences in surface charge to give a high-resolution separation with high sample loading capacity. This separation is based on reversible electrostatic interactions between a charged protein (i.e. charged amino acid side chains) and an oppositely charged chromatography medium. AEX involves purification of proteins on a resin with positively charged functional groups (e.g. strong anion exchangers with quaternary amine group, or weak anion exchanger with secondary amine group). CEX involves purification of proteins on a resin with negatively charged functional groups (e.g. strong cation exchangers with sulfite groups, or weak cation exchangers with carboxylate anions). Both, AEX and CEX, have been demonstrated to be effective in removing not only aggregates but also other impurities during production scale processes. Each chromatography step, either cation exchange or anion exchange, can be performed in bind and elute or flow-through mode, depending upon the physicochemical properties of the target protein and impurities. Protein molecules vary considerably in their charge properties and exhibit different degrees of interaction with charged chromatography media according to differences in their overall charge, charge density and surface charge distribution. For example, monoclonal antibodies comprise ionizable groups such as carboxyl groups and amino groups. The charge of these groups will depend on the pH. Therefore, depending on an antibody's isoelectric point (pI) the charge of a protein molecule can be manipulated by exposing the bulk product to different pH conditions. Monoclonal IgG1 antibodies typically have basic pIs of around 7-9. In flow-through mode, appropriate pH and conductivity conditions need to be defined to customize the charge of the target antibody such that the antibody will not bind but will flow through the resin, with the majority of impurities bound to the column. AEX chromatography is often run in flow-through mode at neutral to slightly basic pH for removal of impurities such as viruses and DNA, which are expected to bind to the resin while the product is collected in the non-bound fraction. As the mode of separation of an AEX chromatography resin is based on electrostatic interactions factors such as conductivity (controlled by salt concentration) also influence the capability of DNA, host cell protein, aggregates and other impurity clearance in AEX in FT mode.
[0055] The essential core of the present invention is that the conductivity of the sample is adjusted with Tris only. Surprisingly, it was found that the addition of Tris, and not only the adjustment of conductivity (e.g. with NaCl), improves the yield of multimodal AEX chromatography in FT mode. In a head to head comparison of a sample load adjusted with Tris only and a sample load adjusted with NaCl to the same conductivity, it was found that with addition of Tris only, antibody yield after MMC in FT mode is about 5% higher and monomer content slightly lower, but still within the specification.
EMBODIMENTS
[0056] In one embodiment, the disclosure relates to a method for purifying an antibody comprising the steps of: [0057] a. providing a sample with a first pH comprising an antibody; [0058] b. adjusting the first pH of the sample to a second pH; [0059] c. adjusting the conductivity of the sample and the second pH to a third pH; [0060] d. processing the adjusted sample by ion exchange chromatography in flow-through mode and [0061] e. collecting the flow-through comprising the antibody,
[0062] wherein pH and conductivity of the sample in step c) is adjusted with Tris.
[0063] In another embodiment, the disclosure relates to a method for increasing the yield of an antibody comprising the steps of: [0064] a. providing a sample with a first pH comprising an antibody; [0065] b. adjusting the first pH of the sample to a second pH; [0066] c. adjusting the conductivity of the sample and the second pH to a third pH; [0067] d. processing the adjusted sample by ion exchange chromatography in flow-through mode and [0068] e. collecting the flow-through comprising the antibody,
[0069] wherein pH and conductivity of the sample in step c) is adjusted with Tris.
[0070] In a certain embodiment, the disclosure relates to a method for purifying an antibody comprising the steps of: [0071] a. providing a sample with a first pH comprising an antibody; [0072] b. adjusting the first pH of the sample to a second pH; [0073] c. adjusting the conductivity of the sample and the second pH to a third pH; [0074] d. processing the adjusted sample by ion exchange chromatography in flow-through mode and [0075] e. collecting the flow-through comprising the antibody,
[0076] wherein pH and conductivity of the sample in step c) is adjusted with Tris to a conductivity of at least 10 mS/cm.
[0077] In a certain embodiment, the disclosure relates to a method for purifying an antibody comprising the steps of: [0078] a. providing a sample with a first pH comprising an antibody; [0079] b. adjusting the first pH of the sample to a second pH; [0080] c. adjusting the conductivity of the sample and the second pH to a third pH; [0081] d. processing the adjusted sample by ion exchange chromatography in flow-through mode and [0082] e. collecting the flow-through comprising the antibody,
[0083] wherein pH and conductivity of the sample in step c) is adjusted with Tris to a conductivity between 10 and 50 mS/cm. Preferably, the conductivity is adjusted to 15 mS/cm.
[0084] In another embodiment, the disclosure relates to a method for increasing the yield of an antibody comprising the steps of: [0085] a. providing a sample with a first pH comprising an antibody; [0086] b. adjusting the first pH of the sample to a second pH; [0087] c. adjusting the conductivity of the sample and the second pH to a third pH; [0088] d. processing the adjusted sample by ion exchange chromatography in flow-through mode and [0089] e. collecting the flow-through comprising the antibody,
[0090] wherein pH and conductivity of the sample in step c) is adjusted with Tris to a conductivity between 10 and 50 mS/cm. Preferably, the conductivity is adjusted to 15 mS/cm.
[0091] In another embodiment, the disclosure relates to a method for purifying an antibody comprising the steps of: [0092] a. providing a sample with a first pH comprising an antibody; [0093] b. adjusting the first pH of the sample to a second pH; [0094] c. adjusting the conductivity of the sample and the second pH to a third pH; [0095] d. processing the adjusted sample by ion exchange chromatography in flow-through mode and [0096] e. collecting the flow-through comprising the antibody,
[0097] wherein pH and conductivity of the sample in step c) is adjusted with Tris to a conductivity between 10 and 30 mS/cm and a third pH of about 6.5 to 7.5. Preferably, the conductivity is adjusted to 15 mS/cm and the pH to about 7.1.
[0098] In another embodiment, the disclosure relates to a method for increasing the yield of an antibody comprising the steps of: [0099] a. providing a sample with a first pH comprising an antibody; [0100] b. adjusting the first pH of the sample to a second pH; [0101] c. adjusting the conductivity of the sample and the second pH to a third pH; [0102] d. processing the adjusted sample by ion exchange chromatography in flow-through mode and [0103] e. collecting the flow-through comprising the antibody,
[0104] wherein pH and conductivity of the sample in step c) is adjusted with Tris to a conductivity between 10 and 30 mS/cm and a third pH of about 6.5 to 7.5. Preferably, the conductivity is adjusted to 15 mS/cm and the pH to about 7.1.
[0105] In preferred embodiments, the sample in step a) is an affinity chromatography eluate obtained after an affinity chromatography step. Most preferably the sample in step a) is a Protein A chromatography eluate with a first pH of about 3 to about 4, ideally with a first pH of 3.6. Non-limiting examples of affinity chromatography supports include, but are not limited to Protein A,
[0106] Protein G, Protein L and affinity supports comprising the antigen against which the antibody of interest was raised. In certain aspects, the Protein A chromatography resin is selected from ProSep Ultra Plus, MabSelect SuRe™, or Amsphere Protein ATM resins. Prior to sample loading the affinity column is equilibrated with a suitable buffer (e.g. PBS, pH 7.0-7.3). After the sample is loaded onto the column the column is washed one or multiple times using a suitable washing buffer (e.g. PBS, pH 7.0-7.3). The antibody bound to the affinity support can then be eluted using an appropriate elution buffer (e.g. Na-Acetate buffer, pH 3.6).
[0107] In other embodiments, the disclosure relates to a method according to any of the preceding embodiments, wherein the second pH of step b) is adjusted to a pH of about 5.2 to about 5.6. Preferably, the second pH is adjusted to pH 5.5.
[0108] In preferred embodiments, mixed mode or mixed modal or multimodal (“MM”) chromatography may be used as ion exchange chromatography in step d). This mixed mode step can feature either cation or anion exchange or a combination of both. This step can be based on a single type of ion exchanger mixed mode procedure or can include multiple ion exchanger mixed mode steps such as a cation exchange mixed mode step followed by an anion exchange mixed mode step or vice versa. Chromatographic mediums for MM chromatography include, among others, mixtures of the following: anion exchange medium, cation exchange medium, hydrophobic interaction medium, hydrophilic interaction medium, hydrogen bonding, pi-pi bonding, and metal affinity. In some embodiments, an MM chromatographic medium with at least an ion exchange medium, such as an anion exchange medium or a cation exchange medium is used in the MM chromatography. A suitable cation exchange column is a column whose stationary phase comprises anionic groups. An example of such a column is a Capto MMC™, Capto MMC™ ImpRes (GE Healthcare), Nuvia™ cPrime™ (Biorad). In one embodiment, the cation exchange mixed mode chromatography comprises N-benzyl-n-methyl ethanolamine. In another aspect, a suitable anion exchange column is a column whose stationary phase comprises cationic groups. In one embodiment, the mixed mode chromatography is a Capto™ Adhere chromatography or a Capto™ Adhere ImpRes chromatography (GE Healthcare). In one embodiment, the first mixed mode chromatography is carried out in flow-through mode. Prior to loading the sample (e.g. affinity eluate) onto the mixed mode column, the column can be equilibrated using a suitable buffer.
[0109] In other embodiments, the antibody sample (e.g. an affinity chromatography eluate) is prepared for the mixed mode step by adjusting the load, pH, conductivity and ionic strength of the sample.
[0110] In one embodiment, the disclosure relates to a method for purifying an antibody comprising the steps of: [0111] a. providing a sample with a first pH comprising an antibody; [0112] b. adjusting the first pH of the sample to a second pH; [0113] c. adjusting the conductivity of the sample and the second pH to a third pH and the load density; [0114] d. processing the adjusted sample by ion exchange chromatography in flow-through mode and [0115] e. collecting the flow-through comprising the antibody,
[0116] wherein the conductivity of the sample in step c) is adjusted to a conductivity between 10 and 30 mS/cm and a third pH of about 6.5 to 7.5 with a load density of about 10 to about 200 g/L. Preferably, the conductivity is adjusted to 15 mS/cm and the third pH is adjusted to pH 7.1 with a load density from 20 to 40 g/L.
[0117] In another embodiment, the disclosure relates to a method for increasing the yield of an antibody comprising the steps of: [0118] a. providing a sample with a first pH comprising an antibody; [0119] b. adjusting the first pH of the sample to a second pH; [0120] c. adjusting the conductivity of the sample and the second pH to a third pH and the load density; [0121] d. processing the adjusted sample by ion exchange chromatography in flow-through mode and [0122] e. collecting the flow-through comprising the antibody,
[0123] wherein the conductivity of the sample in step c) is adjusted to a conductivity between 10 and 30 mS/cm and a third pH of about 6.5 to 7.5 with a load density of about 10 to about 200 g/L. Preferably, the conductivity is adjusted to 15 mS/cm and the third pH is adjusted to pH 7.1 with a load density from 20 to 40 g/L.
[0124] In one embodiment, the antibody to be purified is applied in a solution with a conductivity of more than 10 mS/cm onto the multimodal anion exchange chromatography resin. In another embodiment, the antibody is applied in a solution with a conductivity in the range of about 10 mS/cm to about 30 mS/cm. In some embodiments, the antibody is applied in a solution with a conductivity of about 15 mS/cm onto the multimodal anion exchange chromatography resin.
[0125] In one aspect, the antibody sample is applied in the range from about 1 to 300 g, about 5 to 200 g, about 10 to 100 g, about 20 to 50 g, 20 to 40 g per liter of resin material in the multimodal anion exchange chromatography step.
[0126] In one embodiment, the present disclosure refers to a method for purifying an antibody specific for IL-17C by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0127] a. providing an affinity chromatography (AC) eluate with a first pH comprising the anti-IL17C antibody; [0128] b. adjusting the first pH of the AC eluate to a second pH; [0129] c. adjusting the conductivity of the eluate and the second pH to a third pH; [0130] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0131] e. collecting the flow-through comprising the anti-IL-17C antibody,
[0132] wherein pH and conductivity of the eluate in step c) is adjusted with 2 M Tris to a Tris concentration of 5% (v/v), 10% (v/v), 15% (v/v), 20% (v/v) or in the range of 5% (v/v) to 20% (v/v) and wherein said antibody comprises a HCDR1 region comprising the amino acid sequence of SEQ ID No.: 1, a HCDR2 region comprising the amino acid sequence of SEQ ID No.: 2, a HCDR3 region comprising the amino acid sequence of SEQ ID No.: 3, a LCDR1 region comprising the amino acid sequence of SEQ ID No.: 4, a LCDR2 region comprising the amino acid sequence of SEQ ID No.: 5 and a LCDR3 region comprising the amino acid sequence of SEQ ID No.: 6 and a variable heavy chain and a variable light chain that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the variable heavy chain of SEQ ID No.: 8 and the variable light chain of SEQ ID No.: 7.
[0133] In another embodiment, the present disclosure refers to a method for purifying an antibody specific for IL-17C by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0134] a. providing an affinity chromatography (AC) eluate with a first pH of about 3 to about 4 comprising an anti-IL-17C antibody; [0135] b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; [0136] c. adjusting the conductivity of the eluate to a conductivity between 10 and 30 mS/cm, preferably 15 mS/cm and the second pH to a third pH of about 7.1; [0137] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0138] e. collecting the flow-through comprising the antibody,
[0139] wherein pH and conductivity of the eluate in step c) is adjusted with 2 M Tris to a Tris concentration of 5% (v/v), 10% (v/v), 15% (v/v), 20% (v/v) or in the range of 5% (v/v) to 20% (v/v) and wherein said antibody comprises a HCDR1 region comprising the amino acid sequence of SEQ ID No.: 1, a HCDR2 region comprising the amino acid sequence of SEQ ID No.: 2, a HCDR3 region comprising the amino acid sequence of SEQ ID No.: 3, a LCDR1 region comprising the amino acid sequence of SEQ ID No.: 4, a LCDR2 region comprising the amino acid sequence of SEQ ID No.: 5 and a LCDR3 region comprising the amino acid sequence of SEQ ID No.: 6 and a variable heavy chain and a variable light chain that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the variable heavy chain of SEQ ID No.: 8 and the variable light chain of SEQ ID No.: 7.
[0140] In another embodiment, the present disclosure refers to a method for purifying an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0141] a. providing an affinity chromatography (AC) eluate with a first pH of about 3 to about 4 comprising an anti-IL17C antibody; [0142] b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; [0143] c. adjusting the conductivity of the eluate to a conductivity between 10 and 30 mS/cm, preferably 15 mS/cm and the second pH to a third pH of about 7.1; [0144] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0145] e. collecting the flow-through comprising the antibody,
[0146] wherein pH and conductivity of the eluate in step c) is adjusted with 2 M Tris to a Tris concentration of 5% (v/v), 10% (v/v), 15% (v/v), 20% (v/v) or in the range of 5% (v/v) to 20% (v/v) and wherein said antibody comprises a HCDR1 region comprising the amino acid sequence of SEQ ID No.: 1, a HCDR2 region comprising the amino acid sequence of SEQ ID No.: 2, a HCDR3 region comprising the amino acid sequence of SEQ ID No.: 3, a LCDR1 region comprising the amino acid sequence of SEQ ID No.: 4, a LCDR2 region comprising the amino acid sequence of SEQ ID No.: 5 and a LCDR3 region comprising the amino acid sequence of SEQ ID No.: 6 and a heavy chain and a light chain that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the heavy chain of SEQ ID No.: 10 and the light chain of SEQ ID No.: 9.
[0147] In another embodiment, the present disclosure refers to a method for increasing the yield of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0148] a. providing an affinity chromatography (AC) eluate with a first pH comprising an snti-IL17C antibody; [0149] b. adjusting the first pH of the AC eluate to a second pH; [0150] c. adjusting the conductivity of the eluate and the second pH to a third pH; [0151] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0152] e. collecting the flow-through comprising the antibody,
[0153] wherein pH and conductivity of the eluate in step c) is adjusted with 2 M Tris to a Tris concentration of 5% (v/v), 10% (v/v), 15% (v/v), 20% (v/v) or in the range of 5% (v/v) to 20% (v/v) and wherein said antibody comprises a HCDR1 region comprising the amino acid sequence of SEQ ID No.: 1, a HCDR2 region comprising the amino acid sequence of SEQ ID No.: 2, a HCDR3 region comprising the amino acid sequence of SEQ ID No.: 3, a LCDR1 region comprising the amino acid sequence of SEQ ID No.: 4, a LCDR2 region comprising the amino acid sequence of SEQ ID No.: 5 and a LCDR3 region comprising the amino acid sequence of SEQ ID No.: 6 and a variable heavy chain and a variable light chain that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the variable heavy chain of SEQ ID No.: 8 and the variable light chain of SEQ ID No.: 7.
[0154] In another embodiment, the present disclosure refers to a method for increasing the yield of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0155] a. providing an affinity chromatography (AC) eluate with a first pH of about 3 to about 4 comprising an anti-IL17C antibody; [0156] b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; [0157] c. adjusting the conductivity of the eluate to a conductivity between 10 and 30 mS/cm, preferably 15 mS/cm and the second pH to a third pH of about 7.1; [0158] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0159] e. collecting the flow-through comprising the antibody,
[0160] wherein pH and conductivity of the eluate in step c) is adjusted with 2 M Tris to a Tris concentration of 5% (v/v), 10% (v/v), 15% (v/v) or 20% (v/v) or in the range of 5% (v/v) to 20% (v/v) and wherein said antibody comprises a HCDR1 region comprising the amino acid sequence of SEQ ID No.: 1, a HCDR2 region comprising the amino acid sequence of SEQ ID No.: 2, a HCDR3 region comprising the amino acid sequence of SEQ ID No.: 3, a LCDR1 region comprising the amino acid sequence of SEQ ID No.: 4, a LCDR2 region comprising the amino acid sequence of SEQ ID No.: 5 and a LCDR3 region comprising the amino acid sequence of SEQ ID No.: 6 and a variable heavy chain and a variable light chain that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the variable heavy chain of SEQ ID No.: 8 and the variable light chain of SEQ ID No.: 7.
[0161] In another embodiment, the present disclosure refers to a method for increasing the yield of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0162] a. providing an affinity chromatography (AC) eluate with a first pH of about 3 to about 4 comprising an anti-IL17C antibody; [0163] b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; [0164] c. adjusting the conductivity of the eluate to a conductivity between 10 and 30 mS/cm, preferably 15 mS/cm and the second pH to a third pH of about 7.1; [0165] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0166] e. collecting the flow-through comprising the antibody,
[0167] wherein pH and conductivity of the eluate in step c) is adjusted with 2 M Tris to a Tris concentration of 5% (v/v), 10% (v/v), 15% (v/v) or 20% (v/v) or in the range of 5% (v/v) to 20% (v/v) and wherein said antibody comprises a HCDR1 region comprising the amino acid sequence of SEQ ID No.: 1, a HCDR2 region comprising the amino acid sequence of SEQ ID No.: 2, a HCDR3 region comprising the amino acid sequence of SEQ ID No.: 3, a LCDR1 region comprising the amino acid sequence of SEQ ID No.: 4, a LCDR2 region comprising the amino acid sequence of SEQ ID No.: 5 and a LCDR3 region comprising the amino acid sequence of SEQ ID No.: 6 and a heavy chain and a light chain that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the heavy chain of SEQ ID No.: 10 and the light chain of SEQ ID No.: 9.
[0168] In preferred embodiments, the adjustment of pH and conductivity of the antibody sample in step c) with Tris leads to an increase in yield of the antibody in the flow-through after multimodal anion exchange chromatography. In an embodiment adjustment with 2 M Tris, pH 7.1 to a concentration of 5% (v/v) leads to an antibody yield of more than or equal to 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% after the multimodal anion exchange chromatography step. In another embodiment adjustment with 2 M Tris, pH 7.1 to a concentration of 10% (v/v) leads to an antibody yield of more than or equal to 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% after the multimodal anion exchange chromatography step. In another embodiment adjustment with of 2 M Tris, pH 7.1 to a concentration of 15% (v/v) leads to an antibody yield of more than or equal to 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% after the multimodal anion exchange chromatography step. In yet another embodiment adjustment with 2 M Tris, pH 7.1 to a concentration of 20% (v/v) leads to an antibody yield of more than or equal to 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% after the multimodal anion exchange chromatography step.
[0169] In one embodiment, the antibody or antibody fragment to be purified is a human, humanized or chimeric antibody or antibody fragment.
[0170] In certain embodiments, the antibody to be purified is an IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM isotype antibody.
[0171] In preferred embodiments, the antibody to be purified is of the IgG isotype or variants thereof. More preferably, the antibody is an IgG1 antibody.
[0172] In one embodiment, the present disclosure refers to the purification of an antibody specific for IL-17C. In other embodiments the mAb to be purified shares more than or equal to 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity in the CDRs compared to the CDRs of SEQ ID NO.: 1, SEQ ID NO.:2, SEQ ID NO.:3 and SEQ ID NO.: 4, SEQ ID NO.: 5, and SEQ ID NO.: 6.
[0173] Antibody preparations to which the invention can be applied can include unpurified or partially purified antibodies from natural, synthetic, or recombinant sources. The antibody sample may be cell culture material, for example, solubilized cells and cell culture supernatant. In certain embodiments, it is a clarified cell culture harvest. The methods of the invention can be used as a polishing step to purify an antibody from any mixture containing the antibody. For example, such a mixture can be a Protein A eluate.
[0174] Further, the present invention is directed toward pharmaceutical compositions comprising one or more antibodies purified by a method described herein.
[0175] The purity of the antibodies of interest in the resultant sample product can be analyzed using methods well known to those skilled in the art, e.g., size-exclusion chromatography, Poros™ A HPLC Assay, HCP ELISA, Protein A ELISA, and western blot analysis.
[0176] In a preferred embodiment, the method provided herein results in a purified antibody having a SEC monomer content of equal to or more than 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%. In another embodiment, the purified protein has a SEC monomer content of 100%.
[0177] In another embodiment, the method provided herein results in a purified antibody with a yield of equal to or more than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.
[0178] In another embodiment, the present disclosure refers to a method for increasing the yield of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0179] a. providing an affinity chromatography (AC) eluate with a first pH comprising an anti-IL17C antibody; [0180] b. adjusting the first pH of the AC eluate to a second pH; [0181] c. adjusting the conductivity of the eluate and the second pH to a third pH; [0182] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0183] e. collecting the flow-through comprising the antibody,
[0184] wherein pH and conductivity of the eluate in step c) is adjusted with Tris and wherein said antibody comprises a HCDR1 region comprising the amino acid sequence of SEQ ID No.: 1, a HCDR2 region comprising the amino acid sequence of SEQ ID No.: 2, a HCDR3 region comprising the amino acid sequence of SEQ ID No.: 3, a LCDR1 region comprising the amino acid sequence of SEQ ID No.: 4, a LCDR2 region comprising the amino acid sequence of SEQ ID No.: 5 and a LCDR3 region comprising the amino acid sequence of SEQ ID No.: 6 and a variable heavy chain and a variable light chain that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the variable heavy chain of SEQ ID No.: 8 and the variable light chain of SEQ ID No.: 7.
[0185] In another embodiment, the present disclosure refers to a method for increasing the yield of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0186] a. providing an affinity chromatography (AC) eluate with a first pH of about 3 to about 4 comprising an anti-IL17C antibody; [0187] b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; [0188] c. adjusting the conductivity of the eluate to a conductivity between 10 and 30 mS/cm, preferably 15 mS/cm and the second pH to a third pH of about 7.1; [0189] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0190] e. collecting the flow-through comprising the antibody,
[0191] wherein pH and conductivity of the eluate in step c) is adjusted with Tris and wherein said antibody comprises a HCDR1 region comprising the amino acid sequence of SEQ ID No.: 1, a HCDR2 region comprising the amino acid sequence of SEQ ID No.: 2, a HCDR3 region comprising the amino acid sequence of SEQ ID No.: 3, a LCDR1 region comprising the amino acid sequence of SEQ ID No.: 4, a LCDR2 region comprising the amino acid sequence of SEQ ID No.: 5 and a LCDR3 region comprising the amino acid sequence of SEQ ID No.: 6 and a variable heavy chain and a variable light chain that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the variable heavy chain of SEQ ID No.: 8 and the variable light chain of SEQ ID No.: 7.
[0192] In another embodiment, the present disclosure refers to a method for increasing the yield of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0193] a. providing an affinity chromatography (AC) eluate with a first pH of about 3 to about 4 comprising an antibody; [0194] b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; [0195] c. adjusting the conductivity of the eluate to a conductivity between 10 and 30 mS/cm, preferably 15 mS/cm and the second pH to a third pH of about 7.1; [0196] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0197] e. collecting the flow-through comprising the antibody,
[0198] wherein pH and conductivity of the eluate in step c) is adjusted with Tris and wherein said antibody comprises a HCDR1 region comprising the amino acid sequence of SEQ ID No.: 1, a HCDR2 region comprising the amino acid sequence of SEQ ID No.: 2, a HCDR3 region comprising the amino acid sequence of SEQ ID No.: 3, a LCDR1 region comprising the amino acid sequence of SEQ ID No.: 4, a LCDR2 region comprising the amino acid sequence of SEQ ID No.: 5 and a LCDR3 region comprising the amino acid sequence of SEQ ID No.: 6 and a heavy chain and a light chain that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the heavy chain of SEQ ID No.: 10 and the light chain of SEQ ID No.: 9.
[0199] In another embodiment, the present disclosure refers to a method for the purification of an antibody specific for IL-17C by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0200] a. providing an affinity chromatography (AC) eluate with a first pH comprising an antibody; [0201] b. adjusting the first pH of the AC eluate to a second pH; [0202] c. adjusting the conductivity of the eluate and the second pH to a third pH; [0203] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0204] e. collecting the flow-through comprising the antibody,
[0205] wherein pH and conductivity of the eluate in step c) is adjusted with Tris and wherein said antibody comprises a HCDR1 region comprising the amino acid sequence of SEQ ID No.: 1, a HCDR2 region comprising the amino acid sequence of SEQ ID No.: 2, a HCDR3 region comprising the amino acid sequence of SEQ ID No.: 3, a LCDR1 region comprising the amino acid sequence of SEQ ID No.: 4, a LCDR2 region comprising the amino acid sequence of SEQ ID No.: 5 and a LCDR3 region comprising the amino acid sequence of SEQ ID No.: 6 and a variable heavy chain and a variable light chain that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the variable heavy chain of SEQ ID No.: 8 and the variable light chain of SEQ ID No.: 7.
[0206] In another embodiment, the present disclosure refers to a method for the purification of an antibody specific for IL-17C by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0207] a. providing an affinity chromatography (AC) eluate with a first pH of about 3 to about 4 comprising an antibody; [0208] b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; [0209] c. adjusting the conductivity of the eluate to a conductivity between 10 and 30 mS/cm, preferably 15 mS/cm and the second pH to a third pH of about 7.1; [0210] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0211] e. collecting the flow-through comprising the antibody,
[0212] wherein pH and conductivity of the eluate in step c) is adjusted with Tris and wherein said antibody comprises a HCDR1 region comprising the amino acid sequence of SEQ ID No.: 1, a HCDR2 region comprising the amino acid sequence of SEQ ID No.: 2, a HCDR3 region comprising the amino acid sequence of SEQ ID No.: 3, a LCDR1 region comprising the amino acid sequence of SEQ ID No.: 4, a LCDR2 region comprising the amino acid sequence of SEQ ID No.: 5 and a LCDR3 region comprising the amino acid sequence of SEQ ID No.: 6 and a variable heavy chain and a variable light chain that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the variable heavy chain of SEQ ID No.: 8 and the variable light chain of SEQ ID No.: 7.
[0213] In another embodiment, the present disclosure refers to a method for the purification of an antibody specific for IL-17C by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0214] a. providing an affinity chromatography (AC) eluate with a first pH of about 3 to about 4 comprising an antibody; [0215] b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; [0216] c. adjusting the conductivity of the eluate to a conductivity between 10 and 30 mS/cm, preferably 15 mS/cm and the second pH to a third pH of about 7.1; [0217] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0218] e. collecting the flow-through comprising the antibody,
[0219] wherein pH and conductivity of the eluate in step c) is adjusted with Tris and wherein said antibody comprises a HCDR1 region comprising the amino acid sequence of SEQ ID No.: 1, a HCDR2 region comprising the amino acid sequence of SEQ ID No.: 2, a HCDR3 region comprising the amino acid sequence of SEQ ID No.: 3, a LCDR1 region comprising the amino acid sequence of SEQ ID No.: 4, a LCDR2 region comprising the amino acid sequence of SEQ ID No.: 5 and a LCDR3 region comprising the amino acid sequence of SEQ ID No.: 6 and a heavy chain and a light chain that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the heavy chain of SEQ ID No.: 10 and the light chain of SEQ ID No.: 9.
[0220] In another embodiment, the present disclosure refers to a method for increasing the yield of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0221] a. providing an affinity chromatography (AC) eluate with a first pH comprising an antibody; [0222] b. adjusting the first pH of the AC eluate to a second pH; [0223] c. adjusting the conductivity of the eluate and the second pH to a third pH; [0224] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0225] e. collecting the flow-through comprising the antibody,
[0226] wherein pH and conductivity of the eluate in step c) is adjusted with Tris in the absence of NaCl and wherein said antibody comprises a HCDR1 region comprising the amino acid sequence of SEQ ID No.: 1, a HCDR2 region comprising the amino acid sequence of SEQ ID No.: 2, a HCDR3 region comprising the amino acid sequence of SEQ ID No.: 3, a LCDR1 region comprising the amino acid sequence of SEQ ID No.: 4, a LCDR2 region comprising the amino acid sequence of SEQ ID No.: 5 and a LCDR3 region comprising the amino acid sequence of SEQ ID No.: 6 and a variable heavy chain and a variable light chain that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the variable heavy chain of SEQ ID No.: 8 and the variable light chain of SEQ ID No.: 7.
[0227] In another embodiment, the present disclosure refers to a method for increasing the yield of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0228] a. providing an affinity chromatography (AC) eluate with a first pH of about 3 to about 4 comprising an antibody; [0229] b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; [0230] c. adjusting the conductivity of the eluate to a conductivity between 10 and 30 mS/cm, preferably 15 mS/cm and the second pH to a third pH of about 7.1; [0231] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0232] e. collecting the flow-through comprising the antibody,
[0233] wherein pH and conductivity of the eluate in step c) is adjusted with Tris in the absence of NaCl and wherein said antibody comprises a HCDR1 region comprising the amino acid sequence of SEQ ID No.: 1, a HCDR2 region comprising the amino acid sequence of SEQ ID No.: 2, a HCDR3 region comprising the amino acid sequence of SEQ ID No.: 3, a LCDR1 region comprising the amino acid sequence of SEQ ID No.: 4, a LCDR2 region comprising the amino acid sequence of SEQ ID No.: 5 and a LCDR3 region comprising the amino acid sequence of SEQ ID No.: 6 and a variable heavy chain and a variable light chain that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the variable heavy chain of SEQ ID No.: 8 and the variable light chain of SEQ ID No.: 7.
[0234] In another embodiment, the present disclosure refers to a method for increasing the yield of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0235] a. providing an affinity chromatography (AC) eluate with a first pH of about 3 to about 4 comprising an antibody; [0236] b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; [0237] c. adjusting the conductivity of the eluate to a conductivity between 10 and 30 mS/cm, preferably 15 mS/cm and the second pH to a third pH of about 7.1; [0238] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0239] e. collecting the flow-through comprising the antibody,
[0240] wherein pH and conductivity of the eluate in step c) is adjusted with Tris in the absence of NaCl and wherein said antibody comprises a HCDR1 region comprising the amino acid sequence of SEQ ID No.: 1, a HCDR2 region comprising the amino acid sequence of SEQ ID No.: 2, a HCDR3 region comprising the amino acid sequence of SEQ ID No.: 3, a LCDR1 region comprising the amino acid sequence of SEQ ID No.: 4, a LCDR2 region comprising the amino acid sequence of SEQ ID No.: 5 and a LCDR3 region comprising the amino acid sequence of SEQ ID No.: 6 and a heavy chain and a light chain that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the heavy chain of SEQ ID No.: 10 and the light chain of SEQ ID No.: 9.
[0241] In another embodiment, the present disclosure refers to a method for the purification of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0242] a. providing an affinity chromatography (AC) eluate with a first pH comprising an antibody; [0243] b. adjusting the first pH of the AC eluate to a second pH; [0244] c. adjusting the conductivity of the eluate and the second pH to a third pH; [0245] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0246] e. collecting the flow-through comprising the antibody,
[0247] wherein pH and conductivity of the eluate in step c) is adjusted with Tris in the absence of NaCl and wherein said antibody comprises a HCDR1 region comprising the amino acid sequence of SEQ ID No.: 1, a HCDR2 region comprising the amino acid sequence of SEQ ID No.: 2, a HCDR3 region comprising the amino acid sequence of SEQ ID No.: 3, a LCDR1 region comprising the amino acid sequence of SEQ ID No.: 4, a LCDR2 region comprising the amino acid sequence of SEQ ID No.: 5 and a LCDR3 region comprising the amino acid sequence of SEQ ID No.: 6 and a variable heavy chain and a variable light chain that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the variable heavy chain of SEQ ID No.: 8 and the variable light chain of SEQ ID No.: 7.
[0248] In another embodiment, the present disclosure refers to a method for the purification of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0249] a. providing an affinity chromatography (AC) eluate with a first pH of about 3 to about 4 comprising an antibody; [0250] b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; [0251] c. adjusting the conductivity of the eluate to a conductivity between 10 and 30 mS/cm, preferably 15 mS/cm and the second pH to a third pH of about 7.1; [0252] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0253] e. collecting the flow-through comprising the antibody,
[0254] wherein pH and conductivity of the eluate in step c) is adjusted with Tris in the absence of NaCl and wherein said antibody comprises a HCDR1 region comprising the amino acid sequence of SEQ ID No.: 1, a HCDR2 region comprising the amino acid sequence of SEQ ID No.: 2, a HCDR3 region comprising the amino acid sequence of SEQ ID No.: 3, a LCDR1 region comprising the amino acid sequence of SEQ ID No.: 4, a LCDR2 region comprising the amino acid sequence of SEQ ID No.: 5 and a LCDR3 region comprising the amino acid sequence of SEQ ID No.: 6 and a variable heavy chain and a variable light chain that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the variable heavy chain of SEQ ID No.: 8 and the variable light chain of SEQ ID No.: 7.
[0255] In another embodiment, the present disclosure refers to a method for the purification of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0256] a. providing an affinity chromatography (AC) eluate with a first pH of about 3 to about 4 comprising an antibody; [0257] b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; [0258] c. adjusting the conductivity of the eluate to a conductivity between 10 and 30 mS/cm, preferably 15 mS/cm and the second pH to a third pH of about 7.1; [0259] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0260] e. collecting the flow-through comprising the antibody,
[0261] wherein pH and conductivity of the eluate in step c) is adjusted with Tris in the absence of NaCl and wherein said antibody comprises a HCDR1 region comprising the amino acid sequence of SEQ ID No.: 1, a HCDR2 region comprising the amino acid sequence of SEQ ID No.: 2, a HCDR3 region comprising the amino acid sequence of SEQ ID No.: 3, a LCDR1 region comprising the amino acid sequence of SEQ ID No.: 4, a LCDR2 region comprising the amino acid sequence of SEQ ID No.: 5 and a LCDR3 region comprising the amino acid sequence of SEQ ID No.: 6 and a heavy chain and a light chain that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the heavy chain of SEQ ID No.: 10 and the light chain of SEQ ID No.: 9.
[0262] In another embodiment, the present disclosure refers to a method for increasing the yield of an antibody during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0263] a. providing an affinity chromatography (AC) eluate with a first pH of about 3 to about 4 comprising an antibody; [0264] b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; [0265] c. adjusting the conductivity of the eluate to a conductivity between 10 and 30 mS/cm, preferably 15 mS/cm and the second pH to a third pH of about 7.1; [0266] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0267] e. collecting the flow-through comprising the antibody,
[0268] wherein pH and conductivity of the eluate in step c) is adjusted with 2 M Tris to a Tris concentration of 5% (v/v), 10% (v/v), 15% (v/v) or 20% (v/v) or in the range of 5% (v/v) to 20% (v/v) wherein said antibody has a heavy chain of SEQ ID No.: 10 and a light chain of SEQ ID No.: 9.
[0269] In another embodiment, the present disclosure refers to a method for purification of an antibody by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0270] a. providing an affinity chromatography (AC) eluate with a first pH of about 3 to about 4 comprising an antibody; [0271] b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; [0272] c. adjusting the conductivity of the eluate to a conductivity between 10 and 30 mS/cm, preferably 15 mS/cm and the second pH to a third pH of about 7.1; [0273] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0274] e. collecting the flow-through comprising the antibody,
[0275] wherein pH and conductivity of the eluate in step c) is adjusted with 2 M Tris to a Tris concentration of 5% (v/v), 10% (v/v), 15% (v/v) or 20% (v/v) or in the range of 5% (v/v) to 20% (v/v) wherein said antibody has a heavy chain of SEQ ID No.: 10 and a light chain of SEQ ID No.: 9.
[0276] In another embodiment, the present disclosure refers to a method for increasing the yield of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0277] a. providing an affinity chromatography (AC) eluate with a first pH of about 3 to about 4 comprising an anti-IL17C antibody; [0278] b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; [0279] c. adjusting the conductivity of the eluate to a conductivity between 10 and 30 mS/cm, preferably 15 mS/cm and the second pH to a third pH of about 7.1; [0280] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0281] e. collecting the flow-through comprising the antibody,
[0282] wherein pH and conductivity of the eluate in step c) is adjusted with Tris in the absence of NaCl and wherein said antibody has a heavy chain of SEQ ID No.: 10 and a light chain of SEQ ID No.: 9.
[0283] In another embodiment, the present disclosure refers to a method for the purification of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0284] a. providing an affinity chromatography (AC) eluate with a first pH of about 3 to about 4 comprising an anti-IL17C antibody; [0285] b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; [0286] c. adjusting the conductivity of the eluate to a conductivity between 10 and 30 mS/cm, preferably 15 mS/cm and the second pH to a third pH of about 7.1; [0287] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0288] e. collecting the flow-through comprising the antibody,
[0289] wherein pH and conductivity of the eluate in step c) is adjusted with Tris in the absence of NaCl and wherein said antibody has a heavy chain of SEQ ID No.: 10 and a light chain of SEQ ID No.: 9.
[0290] In another embodiment, the present disclosure refers to a method for increasing the yield of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0291] a. providing an affinity chromatography (AC) eluate with a first pH of about 3 to about 4 comprising an anti-IL17C antibody; [0292] b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; [0293] c. adjusting the conductivity of the eluate to a conductivity between 10 and 30 mS/cm, preferably 15 mS/cm and the second pH to a third pH of about 7.1; [0294] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0295] e. collecting the flow-through comprising the antibody,
[0296] wherein pH and conductivity of the eluate in step c) is adjusted with 2 M Tris to a Tris concentration of 5% (v/v), 10% (v/v), 15% (v/v) or 20% (v/v) or in the range of 5% (v/v) to 20% (v/v) in the absence of NaCl and wherein said antibody has a heavy chain of SEQ ID No.: 10 and a light chain of SEQ ID No.: 9.
[0297] In another embodiment, the present disclosure refers to a method for the purification of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising the following steps: [0298] a. providing an affinity chromatography (AC) eluate with a first pH of about 3 to about 4 comprising an anti-IL17C antibody; [0299] b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; [0300] c. adjusting the conductivity of the eluate to a conductivity between 10 and 30 mS/cm, preferably 15 mS/cm and the second pH to a third pH of about 7.1; [0301] d. processing the adjusted eluate by ion exchange chromatography in flow-through mode and [0302] e. collecting the flow-through comprising the antibody,
[0303] wherein pH and conductivity of the eluate in step c) is adjusted with 2 M Tris to a Tris concentration of 5% (v/v), 10% (v/v), 15% (v/v) or 20% (v/v) or in the range of 5% (v/v) to 20% (v/v) in the absence of NaCl and wherein said antibody has a heavy chain of SEQ ID No.: 10 and a light chain of SEQ ID No.: 9.
[0304] In other embodiments, conductivity is adjusted to at least 10 mS/cm, to a range of 10 to 50 mS/cm, to a range of 10 to 30 mS/cm, 11 to 30 mS/cm, 12 to 30 mS/cm, 13 to 30 mS/cm, 10 to 29 mS/cm, 10 to 28 mS/cm, 10 to 27 mS/cm, 10 to 26 mS/cm, 11 to 29 mS/cm, 11 to 28 mS/cm, 11 to 27 mS/cm, 11 to 26 mS/cm, 12 to 29 mS/cm, 12 to 28 mS/cm, 12 to 27 mS/cm, 12 to 26 mS/cm, 13 to 29 mS/cm, 13 to 28 mS/cm, 13 to 27 mS/cm, 13 to 26 mS/cm, or 13 to 25 mS/cm.
Definitions
[0305] The term “protein” as used herein refers a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, but one of ordinary skill in the art will understand that the term is not limited to lengthy chains and can refer to a minimal chain comprising two amino acids linked together via a peptide bond. As used herein, a “peptide,” a “peptide fragment,” a polypeptide“,” an “amino acid chain,” an “amino acid sequence,” or any other term used to refer to a chain or chains of two or more amino acids, are generically included in the definition of a “protein,” even though each of these terms can have a more specific meaning. The term “protein” can be used instead of, or interchangeably with any of these terms. The term further includes proteins, which have undergone post-translational or post-synthesis modifications, for example, glycosylation, acetylation, phosphorylation, or amidation.
[0306] A “buffer” is a solution that resists changes in pH by the action of its acid-base conjugate components. Various buffers, which can be employed depending, for example, on the desired pH of the buffer are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975). Non-limiting examples of buffers that will control the pH in this range include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, and ammonium buffers, as well as combinations of these.
[0307] “Tris” or tris(hydroxymethyl)aminomethane is an organic compound with the formula (HOCH2)3CNH2. Synonyms are TRIS, Tris, Tris base, Tris buffer, Trizma, Trisamine, THAM, Tromethamine, Trometamol, Tromethane, Trisaminol. The preferred IUPAC name is 2-Amino-2-(hydroxymethyl)propane-1,3-diol. CAS Registry Number: 77-86-1.
[0308] The term “isoelectric point (pI)” is the pH at which a particular molecule or surface carries no net electrical charge. The pI of a polypeptide is dependent on the amino acids that make up the polypeptide. At a pH below its pI, the polypeptide carries a net positive charge. At a pH above its pI, the polypeptide carries a net negative charge. A polypeptide can therefore be separated on the basis of its ionization status at a given pH. The actual pI of a polypeptide can be affected by factors such as post-translational modification. The actual pI can be determined by experimental methods such as isoelectric focusing.
[0309] The terms “chromatography” refers to any current or future chromatography-based process of purifying one or more target molecules from a sample, e.g. by the removal of impurities and/or other non-target molecules. During chromatography a solute of interest, for example a polypeptide, in a mixture is separated from other solutes in a mixture as a result of differences in rates at which the individual solutes of the mixture migrate through a stationary medium under the influence of a moving phase, or in bind and elute processes. Examples of liquid chromatography purification include, but are not limited to: affinity chromatography, immobilized metal ion affinity chromatography flow-through chromatography, ion exchange chromatography, size-exclusion chromatography, reversed-phase chromatography, simulated moving-bed chromatography, hydrophobic interaction chromatography, gel filtration, chromato-focusing.
[0310] The term “mixed-mode chromatography” or “multimodal chromatography” refers to a purification process using mixed-mode adsorbents, which provide multiple modes of interaction, such as hydrophobic, cation exchange, and hydrogen bonding interaction between the polypeptide of interest and the adsorbent ligands. Commercially available mixed mode chromatography resins include Capto™ MMC, Capto™ MMC ImpRes, Capto Blue, Blue Sepharose™ 6 Fast Flow, Capto™ Adhere, and Capto™ Adhere ImpRes from GE Healthcare Life Sciences or Eshmuno® HCX from EMD Millipore, or Nuvia™ cPrime from Bio-Rad.
[0311] The terms “cation exchange resin,” “cation exchange adsorbent,” or “cation exchange matrix” refer to a solid phase which is negatively charged, and which thus has free cations for exchange with cations in an aqueous solution passed over or through the solid phase. A negatively charged ligand attached to the solid phase to form the cation exchange resin may be, e.g. a carboxylate or sulfonate. Commercially available cation exchange resins include carboxy-methyl-cellulose, sulphopropyl (SP) immobilized on agarose (e.g. SP Sepharose™ XL, SP-Sepharose™ Fast Flow, SP Sepharose™ High Performance, CM Sepharose™ Fast Flow, CM Sepharose™ High Performance, Capto™ S, and Capto™ SP ImpRes from GE Healthcare Life Sciences, or Fractogel® EMD SE HiCap, Fractogel® EMD SO3″, Fractogel® EMD COO″, Eshmuno™ S, and Eshmuno™ CPX from EMD Millipore, or UNOsphere™ S and Nuvia™ S from Bio-Rad).
[0312] The terms “anion exchange resin,” “anion exchange adsorbent,” or “anion exchange matrix” are used herein to refer to a solid phase, which is positively charged, e.g. having one or more positively charged ligands, such as quaternary amino groups, attached thereto. Commercially available anion exchange resins include DEAE Sepharose™ Fast Flow, Q Sepharose™ Fast Flow, Q Sepharose™ High Performance, Q Sepharose™ XL, Capto™ DEAE, Capto™ Q, and Capto™ Q ImpRes from GE Healthcare Life Sciences, or Fractogel® EMD TMAE HiCap, Fractogel® EMD DEAE, and Eshmuno Q from EMD Millipore, or U Osphere™ Q and Nuvia™ Q from Bio-Rad.
[0313] The term “antibody” refers to glycosylated and non-glycosylated immunoglobulins of any of the five major classes (isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) and combinations and variants thereof. As used herein, the term encompasses antibodies form any species (e.g. murine, canine, feline, IgY, etc.) and combinations thereof, e.g. human, humanized, chimeric antibodies. The term refers to monoclonal and polyclonal antibodies as well as to monospecific and multi-specific antibodies (such as bispecific antibodies). As used herein, the term also encompasses fusion proteins comprising an antigen determination portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. As used herein, the term “antibody” includes intact immunoglobulins as well as antibody fragments, that refer to one or more portions of an antibody that retain the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing spatial distribution) an antigen. Examples of binding fragments include, but are not limited to Fab, Fab′, F(ab′)2, Fd, Fv and dAb fragments (Ward et al., (1989) Nature 341:544-546), single chain Fv (scFv) (e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Any naturally occurring, enzymatically obtainable, synthetic, alternative scaffold, or genetically engineered polypeptide that specifically binds an antigen are intended to be encompassed within the term “antibody” as used herein.
[0314] The terms “contaminant” and “impurity” are used interchangeably herein and refer to any objectionable molecule, including a biological macromolecule such as a DNA, an RNA, one or more host cell proteins, endotoxins, lipids and one or more additives which may be present in a sample containing the target protein that is being separated from one or more of the foreign or objectionable molecules using a process of the present invention. Additionally, such a contaminant may include any reagent, which is used in a step that may occur prior to the purification process.
[0315] “High molecular weight (HMW) species” include species having a higher molecular weight than the target protein mass, such as multimers. Multimers include everything other than the monomer of the target protein. For instance, a monomer of an IgG antibody encompasses the traditional tetrameric antibody composition comprising two heavy and light chains. Multimers include species having a higher molecular mass than the target protein mass, such as dimers (two identical proteins associated covalently or non-covalently) and aggregates (covalent or non-covalently associated whole and/or partial proteins).
[0316] “Low molecular weight (LMW) species” include species having a lower molecular weight than the target protein mass, such as clips, and degraded product.
[0317] As used herein, the term “polishing” refers to a downstream processing step which occurs after the initial (affinity) capture step and which is intended to remove residual amounts of impurities that are present in the product stream and which typically have more similarity to the product than the impurities removed during the capture step.
[0318] Methods for the determination of yield or purity of a polypeptide are known to those of skill in the art. Yield or purity of a polypeptide may be determined by any suitable method of analysis (e.g., band intensity on a silver stained gel, polyacrylamide gel electrophoresis, ELISA, HPLC and the like). An exemplary method is size-exclusion chromatography (SEC) high-performance liquid chromatography (HPLC). Purity may be determined using relative “area under the curve” (AUC) values, which can typically be obtained for peaks in a chromatogram, such as an HPLC chromatogram.
[0319] The term “bind and elute mode” refers to a product separation technique in which at least one product contained in a sample (e.g., an Fc region containing protein) binds to a chromatographic resin or media and is subsequently eluted.
[0320] The term “flow-through mode” refers to conditions at which the target protein will flow through while contaminants will bind to the chromatography support.
[0321] The amino acid and encoding nucleic acid sequences in Table 1 are an example of an IL-17C antibody, as well as portions thereof.
TABLE-US-00001 TABLE 1 Exemplary IL-17C antibody sequences Antibody SEQ ID No.: [aa]/[DNA] MAB#1 HCDR1 SEQ ID DYAMH No.: 1 HCDR2 SEQ ID YIGGVGEGTQYAESVKG No.: 2 HCDR3 SEQ ID GFAIRYYGFDY No.: 3 LCDR1 SEQ ID SGDKLGDKYAY No.: 4 LCDR2 SEQ ID QDSKRPS No.: 5 LCDR3 SEQ ID QVFTFPLVTT No.: 6 VL SEQ ID SYELTQPPSVSVSPGQTASITCSGDKLGDKYAYWYQQKP No.: 7 GQSPVLVIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAE DEADYYCQVFTFPLVTTVFGGGTKLTVLGQ VH SEQ ID EVQLLESGGGLVQPGGSLRLSCAASGFTVSDYAMHWVR No.: 8 QAPGKGLEWVSYIGGVGEGTQYAESVKGRFTISRDNSKN TLYLQMNSLRAEDTAVYYCARGFAIRYYGFDYWGQGTLV TVSS Light chain SEQ ID SYELTQPPSVSVSPGQTASITCSGDKLGDKYAYWYQQKP No.: 9 GQSPVLVIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAE DEADYYCQVFTFPLVTTVFGGGTKLTVLGQPKAAPSVTLF PPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAG VETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTH EGSTVEKTVAPTECS Heavy SEQ ID EVQLLESGGGLVQPGGSLRLSCAASGFTVSDYAMHWVR chain No.: 10 QAPGKGLEWVSYIGGVGEGTQYAESVKGRFTISRDNSKN (IgG1) TLYLQMNSLRAEDTAVYYCARGFAIRYYGFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK VL SEQ ID TCCTACGAGCTGACCCAGCCCCCCTCCGTGTCCGTGTC No.: 11 TCCTGGCCAGACCGCCTCCATCACCTGTTCCGGCGACA AGCTGGGCGATAAGTACGCCTACTGGTATCAGCAGAAG CCCGGCCAGTCCCCCGTGCTGGTCATCTACCAGGACT CCAAGCGGCCCTCCGGCATCCCTGAGCGGTTCTCCGG CTCCAACTCCGGCAACACCGCCACCCTGACCATCTCCG GCACCCAGGCCGAGGACGAGGCCGACTACTACTGCCA GGTGTTCACCTTCCCCCTGGTCACCACCGTGTTCGGCG GAGGCACCAAGCTGACCGTGCTGGGCCAG VH SEQ ID GAGGTGCAGCTGCTGGAATCCGGCGGAGGACTGGTGC No.: 12 AGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTC CGGCTTCACCGTGTCCGACTACGCTATGCACTGGGTCC GACAGGCCCCTGGCAAGGGCCTGGAATGGGTGTCCTA TATCGGCGGCGTGGGCGAGGGCACCCAGTACGCTGAG TCTGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTC CAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGG CCGAGGACACCGCCGTGTACTACTGTGCCAGAGGCTT CGCCATCCGGTACTACGGCTTCGACTACTGGGGCCAG GGCACCCTGGTCACCGTGTCTAGC Light chain SEQ ID TCCTACGAGCTGACCCAGCCCCCCTCCGTGTCCGTGTC No.: 13 TCCTGGCCAGACCGCCTCCATCACCTGTTCCGGCGACA AGCTGGGCGATAAGTACGCCTACTGGTATCAGCAGAAG CCCGGCCAGTCCCCCGTGCTGGTCATCTACCAGGACT CCAAGCGGCCCTCCGGCATCCCTGAGCGGTTCTCCGG CTCCAACTCCGGCAACACCGCCACCCTGACCATCTCCG GCACCCAGGCCGAGGACGAGGCCGACTACTACTGCCA GGTGTTCACCTTCCCCCTGGTCACCACCGTGTTCGGCG GAGGCACCAAGCTGACCGTGCTGGGCCAGCCTAAGGC CGCTCCCTCCGTGACCCTGTTCCCCCCATCCTCCGAGG AACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATC TCCGACTTCTACCCTGGCGCCGTGACCGTGGCCTGGA AGGCCGACAGCTCTCCTGTGAAGGCCGGCGTGGAAAC CACCACCCCCTCCAAGCAGTCCAACAACAAATACGCCG CCTCCTCCTACCTGTCCCTGACCCCCGAGCAGTGGAAG TCCCACCGGTCCTACAGCTGCCAGGTCACACACGAGG GCTCCACCGTGGAAAAGACCGTGGCCCCTACCGAGTG CTCC Heavy SEQ ID GAGGTGCAGCTGCTGGAATCCGGCGGAGGACTGGTGC chain No.: 14 AGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTC (lgG1) CGGCTTCACCGTGTCCGACTACGCTATGCACTGGGTCC GACAGGCCCCTGGCAAGGGCCTGGAATGGGTGTCCTA TATCGGCGGCGTGGGCGAGGGCACCCAGTACGCTGAG TCTGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTC CAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGG CCGAGGACACCGCCGTGTACTACTGTGCCAGAGGCTT CGCCATCCGGTACTACGGCTTCGACTACTGGGGCCAG GGCACCCTGGTCACCGTGTCTAGCGCCTCCACCAAGG GCCCCTCCGTGTTCCCTCTGGCCCCCTCCAGCAAGTCC ACCTCTGGCGGCACCGCTGCCCTGGGCTGCCTGGTCA AGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAAC TCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTG CCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCC GTCGTGACCGTGCCCTCCAGCTCTCTGGGCACCCAGA CCTACATCTGCAACGTGAACCACAAGCCCTCCAACACC AAGGTGGACAAGCGGGTGGAACCCAAGTCCTGCGACA AGACCCACACCTGTCCCCCCTGCCCTGCCCCTGAACTG CTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCC CAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGA CCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGA AGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGC ACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACAAC TCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGC ACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAA GGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGA CCATCTCCAAGGCCAAGGGCCAGCCCCGCGAGCCCCA GGTGTACACACTGCCCCCTAGCCGGGAAGAGATGACC AAGAACCAGGTGTCCCTGACCTGTCTGGTCAAGGGCTT CTACCCCTCCGACATTGCCGTGGAATGGGAGTCCAACG GCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGT GCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGC TGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGT GTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACC ACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG
WORKING EXAMPLES
Example 1
[0322] To test the impact of Tris addition (i.e. increasing Tris concentration) to a sample comprising an antibody with heavy chain of SEQ ID NO.:10 and light chain of SEQ ID NO.:9 prior to loading a Capto adhere ImpRes column (GE Healthcare) in flow-through mode on yield and purity, four different purification runs were carried out. One run without Tris addition and three runs with addition of 5, 10 and 20% (v/v) of 2 M Tris pH 7.1 to the sample eluate. Antibody yield and purity (SEC monomer) of the resulting flow-throughs were analyzed. Results are listed in Table 2 and corresponding chromatograms are shown as overlay in
TABLE-US-00002 TABLE 2 Yield and monomer content of the antibody in the flow-through after multimodal AEX chromatography using Capto ® adhere ImpRes (GE Healthcare) 2M Tris pH 7.1 [% (v/v)] none 5 10 20 Conductivity [mS/cm] 8.2 13.6 17.2 25.5 Yield [%] 58.0 78.0 81.9 84.6 SEC Monomer [%] 97.8 97.4 97.0 96.6
Example 2
[0323] To elucidate, that the addition of Tris to the antibody sample as in Example 1, and not only the adjustment of conductivity, improves antibody yield in the flow through after Capto adhere ImpRes chromatography a head-to-head comparison between pre-conditioning of the sample with Tris (run 1) and pre-conditioning of the sample with NaCl (run 2) was performed. For sample preparation, conductivity was adjusted in run 1 with 2 M Tris pH 7.1 and in run 2 with 5 M NaCl to a target conductivity of 15 mS/cm (Table 3).
TABLE-US-00003 TABLE 3 Conductivity PH Start Target Run Start Target [mS/cm] [mS/cm] Comment 1 5.50 7.10 6.80 15.00 Conductivity adjusted with 2M Tris pH 7.1 2 5.49 7.10 7.00 15.00 Conductivity adjusted with 5M NaCl
[0324] Both loads have the same conductivity but differ in their buffer matrix. pH and conductivity measurements were performed at ambient temperature 20° C.±2° C. Purification results and QC data are shown in Table 4.
TABLE-US-00004 TABLE 4 SEC/MALS HMW LMW Yield after Recovery Monomer impurities impurities Purification (rel. to Run Mode [%] [%] [%] [mg] Load) [%] 1 MM-AIEX 96.7 2.1 1.1 675.2 81.5% conductivity adjusted with 2M Tris pH 7.1 2 MM-AIEX 97.1 1.7 1.2 644.6 77.3% conductivity adjusted with 5M NaCl
[0325] Without addition of Tris (run 2), the yield is about 5% less compared to run 1, in which conductivity was adjusted to 15 mS/cm with 2 M Tris, pH 7.1.