WO2021170060A1 - Purification d'anticorps bispécifiques - Google Patents

Purification d'anticorps bispécifiques Download PDF

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WO2021170060A1
WO2021170060A1 PCT/CN2021/078023 CN2021078023W WO2021170060A1 WO 2021170060 A1 WO2021170060 A1 WO 2021170060A1 CN 2021078023 W CN2021078023 W CN 2021078023W WO 2021170060 A1 WO2021170060 A1 WO 2021170060A1
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species
bispecific antibody
hole
target
column
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PCT/CN2021/078023
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English (en)
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Yifeng Li
Ying Wang
Taotao QIN
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Wuxi Biologics (Shanghai) Co., Ltd.
WuXi Biologics Ireland Limited
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Priority to CN202180013617.5A priority Critical patent/CN115066258A/zh
Publication of WO2021170060A1 publication Critical patent/WO2021170060A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/522—CH1 domain

Definitions

  • the present invention is directed to methods of purifying a bispecific antibody (bsAb) species, particularly methods of purifying a bsAb species comprising only one kappa light chain (LC) constant region using KappaSelect affinity chromatography.
  • bsAb bispecific antibody
  • LC light chain
  • bispecific antibodies have long been recognized. Besides, they have also been found useful in detection and diagnostic applications. BsAbs have been constructed in many different formats, which can be broadly divided into two categories: IgG-like bsAbs and Fc-free bsAbs. Each has its own advantages and limits. For instance, while the Fc-free bsAbs exhibit enhanced penetration into solid tumours, IgG-like bsAbs are advantageously capable of providing a longer circulation half-life and supporting secondary immune functions.
  • bsAbs In production of bsAbs, random assembly of four distinct polypeptide chains, i.e. heavy chains (HCs) and LCs from two parental mAbs, can potentially give up to 10 different products while only one of them possesses the desired function.
  • Two technologies i.e., the knobs-into-holes (KiH) technology and the CrossMab technology, have been developed to promote HC heterodimerization and cognate HC-LC pairing, respectively.
  • KiH knobs-into-holes
  • CrossMab technology Two technologies, i.e., the knobs-into-holes (KiH) technology and the CrossMab technology, have been developed to promote HC heterodimerization and cognate HC-LC pairing, respectively.
  • KiH knobs-into-holes
  • CrossMab technology the CrossMab technology
  • homodimerization between the "knob” variants is typically not observed, homodimerization between the "hole” variants can still occur. “Hole-hole” homodimers can account for up to 5%of the total mass. In addition, good heterodimerization yields require approximately equal expression of the two HCs. In the case of KiH, the content of homodimer can be significantly increased if the “hole” chain is expressed in excess.
  • WuXiBody TM (also simply referred to as "WuXiBody” herein below) is a novel bsAb platform developed by WuXi Biologics, in which one Fab arm’s CH1 and CL regions are replaced by T cell receptor (TCR) constant domains [1] . This modification ensures cognate LC-HC pairing.
  • TCR T cell receptor
  • HC heterodimerization is promoted using the KiH technology [2] .
  • the CH1-replaced chain has a "knob" mutation, while the CH1-comprising chain has a "hole” mutation.
  • homodimers represent the most difficult-to-remove species as they exhibit similar physicochemical properties to the target bsAb.
  • Protein A chromatography typically used as a capture step for monoclonal antibodies and Fc fusion proteins, cannot differentiate homodimers from the target bsAb. Meanwhile, it has been reported that Protein A chromatography can partially separate half antibody, which contains one Fc domain, from the intact antibody, which contains the full Fc-region, under linear pH gradient elution [3] .
  • KappaSelect is an affinity medium designed for purification of Fab (kappa) fragments [4] . It specifically binds to the constant region of the kappa LC. It is typically used to separate antibody species containing a kappa LC constant region from those lacking that region.
  • some bsAbs adopt the Fab x single-chain variable fragment (scFv) format, in which one Fab arm is replaced by a scFv [5] .
  • a scFv is constructed by connecting the variable regions of the HC and LC via a linker peptide, and it does not contain the LC constant region.
  • the scFv-Fc chain can be expressed in excess and becomes a major contaminant.
  • KappaSelect provides a straightforward solution to the purification problem, allowing quick removal of scFv-Fc and the corresponding homodimer.
  • the target Fab x scFv bsAb binds to KappaSelect through its Fab arm, while the scFv-Fc chain and the homodimer thereof, which lack the LC constant region, are left in the flow-through (Fig. 2) .
  • KappaSelect can be used to separate antibody species containing a kappa LC constant region from those lacking that region, it is not clear whether this medium can readily separate species containing one kappa LC constant region from those containing two kappa LC constant regions. Although antibody species containing one kappa LC constant region are assumed to bind weaker to KappaSelect than those containing two kappa LC constant regions, it is not clear whether the differentia in affinity is big enough to separate them apart.
  • Contaminating byproducts may exhibit undesired activity should they remain in the final purified product.
  • the monospecific byproducts of bsAb production would likely reduce efficacy of the final bispecific formulation if not separated. Accordingly, there is a need for new and/or improved schemes for purification of a target bsAb from culture harvest, which allow the final purified product to meet the biotechnology industry requirements for the production of diagnostic and therapeutic products.
  • the present invention is based on the finding that antibody species having two kappa LC constant regions bind to the KappaSelect medium much tighter than those having only one kappa LC constant region. Thereby, these two species can be readily separated using KappaSelect resin, an affinity medium specifically binds to the constant region of the kappa LC.
  • the present disclosure provides a method of purifying a target bispecific antibody species comprising one kappa light chain (LC) constant region from a fluid comprising the target bispecific antibody species and at least one byproduct species comprising two kappa LC constant region, wherein the method comprises a step of separating said target bispecific antibody from said at least one byproduct species via the KappaSelect affinity chromatography; and wherein said step of separating comprises:
  • the target bispecific antibody species may be an IgG-like bispecific antibody or a bispecific F (ab) 2 fragment.
  • the bispecific antibody species may be an IgG-like bispecific antibody of the knobs-into-holes (KiH) format.
  • the target bispecific antibody species comprises a kappa LC constant region in one Fab arm and TCR constant domains in place of the CH1 and CL regions in the other Fab arm, and wherein the CH1-replaced chain has a "knob" mutation, while the CH1-comprising chain has a "hole” mutation.
  • the byproduct species may be a homodimer by-product in production of bispecific antibodies.
  • the byproduct species may be a hole-hole homodimer by-product in production of a KiH bispecific antibody.
  • the target bispecific antibody species may be a ⁇ -body and the at least one byproduct species comprises mono ⁇ homodimers and mono ⁇ homodimers.
  • the step (c) of eluting comprises a linear pH gradient elution.
  • the linear pH gradient may be from pH 5.5 to pH 3.0.
  • the step (c) of eluting may be a stepwise pH gradient elution.
  • the stepwise pH gradient elution may comprise a stage at pH 3.0, pH 3.2 or pH 3.5.
  • the stepwise pH gradient elution may comprise a stage at pH 3.5.
  • the step (b) of washing may be conducted by consecutively washing the column with buffers having different pH values.
  • the step (b) of washing may be conducted by consecutively washing the column with buffers of pH7.4 and pH 5.5 respectively.
  • the column was washed three times with buffers of pH7.4, pH 5.5 and pH 5.5, respectively.
  • Figures 1A-1B Schematic representation of an asymmetric IgG-like bsAb based on the WuXiBody TM platform.
  • Fig. 1A Target heterodimer;
  • Fig. 1B Potential hole-hole homodimer.
  • Figure 2 Schematic representation of purification of a Fab x scFv format bsAb using KappaSelect affinity medium.
  • Figure 3 Protein A affinity chromatogram of a harvest of WuXiBody TM bsAb. Inset: non-reducing SDS-PAGE analysis of relevant fractions, including Lane 1: isolated hole-hole homodimer as reference, Lane 2: load, and Lane 3: eluate.
  • Figure 4 KappaSelect chromatogram of a harvest of WuXiBody TM bsAb using a linear pH gradient elution according to an example of the present disclosure. The column was eluted from pH 5.5 to pH 3.0 over 20 CV.
  • Figures 5A-5B KappaSelect chromatograms of a harvest of WuXiBody TM bsAb using a stepwise pH gradient elution according to an example of the present disclosure.
  • Fig. 5A Overlayed three chromatograms at different elution pH values;
  • Fig. 5B Non-reducing SDS-PAGE analysis of relevant fractions from the three runs, including Lane 1: isolated hole-hole homodimer as reference, Lane 2: load, Lane 3: eluate 1 (E1) , Lane 4: eluate 2 (E2) , Lane 5: Protein A eluate (included for comparison) , and Lane 6: strip.
  • Figure 6 Overlayed analytical HIC profiles of a Protein A eluate and a KappaSelect eluate according to an example of the present disclosure.
  • Figures 7A-7B AEX chromatograms of a Protein A eluate (Fig. 7A) and a KappaSelect eluate (Fig. 7B) .
  • Inset non-reducing SDS-PAGE analysis of relevant fractions, including Lane 1: isolated hole-hole homodimer as reference, Lane 2: load and Lanes 3-5: flow-through fractions 1-3 (F1-F3) .
  • Figures 8A-8B Purification of a ⁇ -body.
  • Fig. 8A Strategy of purification as previously described;
  • Fig. 8B Alternative strategy using KappaSelect according to the present invention.
  • KappaSelect is an affinity medium designed for purification of Fab (kappa) fragments. It specifically binds to the constant region of a kappa LC. It is typically used to separate antibody species containing a kappa LC constant region from those lacking that region. However, it is not clear whether this medium can readily separate species containing one kappa LC constant region from those containing two kappa LC constant regions. Although antibody species containing one kappa LC constant region are assumed to bind weaker to KappaSelect than those containing two kappa LC constant regions, it is not clear whether the differentia in affinity is big enough to separate them apart.
  • KappaSelect provides a convenient means to separate antibody species containing one kappa LC constant region from those containing two kappa LC constant regions. This information not only enriches our knowledge of KappaSelect but also has important practical values.
  • KappaSelect can also be used to purify bsAbs based on other formats, such as ⁇ -bodies. Moreover, in the case of ⁇ -body purification, efficiency can be greatly improved using the novel information provided by the current study.
  • the present disclosure provides a method of purifying a target bispecific antibody species comprising one kappa light chain (LC) constant region from a fluid comprising the target bispecific antibody species and at least one byproduct species comprising two kappa LC constant region, wherein the method comprises a step of separating said target bispecific antibody from said at least one byproduct species via the KappaSelect affinity chromatography.
  • LC light chain
  • the bsAb species can be either of the IgG-like bsAb category or the Fc-free category and can be of any format that comprises a kappa LC constant region, including for example Triomabs/quadroma, DVD-Ig (dualvariable domain Ig) , CrossMAb, Two-in-one IgG, Fab x scFv bsAb, especially WuXiBody bsAbs (in which one Fab arm’s CH1 and CL regions are replaced by TCR constant domains) and ⁇ -body and F (ab) 2 fragments thereof.
  • the bsAb species is of the KiH design to facilitate heterodimerization.
  • the byproduct species include those commonly observed in bsAb production, including homodimers, 3/4 antibodies (antibody lacking one LC) , half antibody, HC dimer, free HC, and free LC.
  • the target bsAb species contains one kappa LC constant region, such as a Fab x scFv bsAb, a WuXiBody and a ⁇ -body, while the byproduct species contains two, such as the undesired homodimers.
  • the target bsAb species and the byproduct species are included in a fluid to be loaded onto a chromatography column (i.e., the "load” herein forth) .
  • the fluid can be a cell culture harvest from a recombinant production of bsAb.
  • the cell culture harvest is pre-treated by filtration (e.g., depth filtration) to provide the fluid or "load” .
  • the method of purification according to the present invention is basically based on a separation using KappaSelect medium to separate the target bsAb species into the eluate and optionally, the byproduct (s) into the strip so that the target species can be collected from the appropriate elution fraction (s) .
  • the method further includes one or more upstream or downstream purification processes, such as filtration and an additional chromatography.
  • the step of separating may comprise:
  • the step (c) of eluting is a linear pH gradient elution.
  • the linear pH gradient can be from pH 5.5 to pH 3.0 over a certain folds of CV or a defined time period.
  • the appropriate folds of CV or time period can be determined by a sound judgement of a person with ordinary skills in the art.
  • the appropriate buffer can be determined by a sound judgement of a person with ordinary skills in the art.
  • the step (c) of eluting is a stepwise pH gradient elution.
  • the stepwise pH gradient elution may comprise a stage at pH 3.0, pH 3.2 or pH 3.5, preferably pH3.5.
  • the step (b) of washing is conducted by consecutively washing the column with buffers having different pHs (e.g., pH 7.4 and pH 5.5) .
  • the washing volume is 5 CV each time.
  • the appropriate buffer can be determined by a sound judgement of a person with ordinary skills in the art.
  • the invention is based on the finding in purification of a WuXiBody bsAb, it is clear from the above that the underlying concept of the invention can be extended to other formats. It is demonstrated that the method according to the invention provides a more efficient way to purify a target bsAb.
  • the target bsAb species is a ⁇ -body, and the method using KappaSelect according to the invention poses a more efficient way compared to the conventional protocols.
  • a common HC is included to avoid the HC pairing problem and bispecificity is conferred by two different LCs: one kappa ( ⁇ ) and one lambda ( ⁇ ) [6] .
  • ⁇ -body requires three chains: HC, ⁇ LC and ⁇ LC.
  • mono ⁇ and mono ⁇ homodimers will also be generated when these three chains are coexpressed.
  • ⁇ -bodies can be purified by sequentially running through a KappaSelect affinity chromatography and a LambdaFabSelect affinity chromatography, wherein LambdaFabSelect is an affinity medium that binds to the constant region of a lambda LC.
  • Ammonium sulfate, ethanol, glycine, sodium acetate trihydrate, sodium chloride, sodium hydroxide, sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate dehydrate and Tris (hydroxymethyl) aminomethane were purchased from Merck (Darmstadt, Germany) .
  • Acetic acid and hydrochloric acid were purchased from J. T. Baker (Phillipsburg, NJ, USA) .
  • MabSelect PrismA, KappaSelect and tricorn 5/200 column (inner diameter: 5 mm, length: 200 mm) were purchased from GE Healthcare (Uppsala, Sweden) .
  • POROS 50 HQ and MAbPac HIC-20 (4.6 x 100 mm) column were purchased from Thermo Fisher Scientific (Waltham, MA, USA) .
  • N-Glycosidase (PNGase F) was purchased from New England Biolabs (Ipswich, MA, USA) .
  • 30%acrylamide/bis-acrylamide solution (37.5: 1) and TEMED were purchased from Bio-Rad Laboratories (Hercules, CA, USA) .
  • Ammonium persulfate, Coomassie blue R-250, glycerol, sodium dodecyl sulfate (SDS) , iodoacetamide and Bromophenol blue were purchased from Sigma-Aldrich (St. Louis, MO, USA) .
  • the target bsAb was an asymmetric IgG-like bsAb based on the WuXiBody TM platform. It was expressed in CHO-K1 cells grown in HyClone ActiPro culture medium supplemented with Cell Boost 7a and 7b (the medium and feeding supplements are from GE Healthcare) . The cell culture was allowed to grow for 14 days and then harvested. The culture harvest was clarified by depth filtration. The obtained filtrate contained among others the target bsAb heterodimer, the potential hole-hole homodimer and the hole half antibody.
  • the target bsAb heterodimer was a WuXiBody TM IgG-like bsAb of the KiH design, wherein the CH1 region and the CL region in the knob half antibody was replaced by a C ⁇ domain and a C ⁇ domain of TCR, and the hole-hole homodimer was formed since the hole half antibody without the CH1/CL-replacement was expressed in excess (Figs. 1A-1B) .
  • An AKTA pure 150 system installed with Unicorn software version 7.3 (GE Healthcare, Uppsala, Sweden) was used for column chromatography. pH and conductivity were measured using SevenExcellence S470 pH/Conductivity meter (Mettler-Toledo, Columbus, OH, USA) . Protein concentration was measured using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) . An Agilent 1260 liquid chromatography instrument (Agilent Technologies, Santa Clara, CA, USA) was used for analytical hydrophobic interaction chromatography (HIC) .
  • HIC hydrophobic interaction chromatography
  • MabSelect PrismA (a Protein A affinity medium) was packed in a 0.5 cm diameter column with 16.0 cm bed height.
  • the column volume (CV) is approximately 3 ml.
  • Column load is the culture harvest clarified by depth filtration.
  • the column was loaded at 30 mg protein per ml of medium and run in bind-elute mode.
  • the system was run at a flow rate of 190 cm/hr (residence time: 5 min) .
  • the column was washed consecutively with 50 mM Tris-acetate, 150 mM NaCl, pH 7.4, 50 mM Na-acetate/HAc, 1 M NaCl, pH 5.5 and 50 mM Na-acetate/HAc, pH 5.5, each time with 5 CV.
  • the column was eluted with 50 mM Na-acetate/HAc, pH 3.5.
  • KappaSelect affinity medium was packed in a 0.5 cm diameter column with 16.0 cm bed height.
  • the column volume is approximately 3 ml (CV: ⁇ 3 ml) .
  • Column load is the culture harvest clarified by depth filtration.
  • the column was loaded at 18 mg protein per ml of medium (approximately the medium’s maximum dynamic binding capacity under the selected condition) and run in bind-elute mode.
  • the system was run at a flow rate of 193 cm/hr (residence time: 5 min) .
  • the column was washed consecutively with 50 mM Tris-acetate, 150 mM NaCl, pH 7.4, 50 mM Na-acetate/HAC, 1 M NaCl, pH 5.5 and 50 mM Na-acetate/HAc, pH 5.5, each time with 5 CV.
  • the column was eluted using a pH gradient from pH 5.5 to pH 3.0 (50 mM Na-acetate/HAc, pH 5.5 to 3.0) over 20 CV.
  • stepwise elution the column was eluted with 50 mM Na-acetate/HAc at pH 3.0, pH 3.2 and pH 3.5, respectively.
  • AEX chromatography was performed with POROS’s 50 HQ medium in bind-elute mode using a 0.5 cm diameter column with 15.0 cm bed height (CV: ⁇ 3 ml) .
  • the load was Protein A or KappaSelect eluate pH adjusted to 7.0.
  • the column was loaded at 40 mg protein per ml of medium and run at a flow rate of 177 cm/hr (residence time: 5 min) . After loading, the column was washed consecutively with 50 mM Tris-HAc, pH 7.0 and 50 mM Tris-HAc, pH 8.0, each for 3 CV.
  • the column was eluted with 50 mM Tris-HAc, 130 mM NaCl, pH 8.0.
  • Analytical HIC was conducted using an Agilent system with a MAbPac HIC-20 stainless steel column (4.6 x 100 mm) .
  • Mobile phase A consisted of 100 mM sodium phosphate and 600 mM ammonium sulfate, pH 7.0
  • mobile phase B consisted of 10 mM sodium phosphate, pH 7.0.
  • the column was eluted with a gradient from 0%to 100%B over 30 min (approximately 4.5 CV) at a flow rate of 0.25 ml/min.
  • Samples subjected to HIC analysis were deglycosylated and diluted to 0.5 mg/ml with mobile phase A. 50 ⁇ g of sample was injected per run.
  • Non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE
  • This Example demonstrates attempted separation between antibody species comprising one kappa LC constant region and those comprising two via Protein A chromatography.
  • the culture harvest was clarified by depth filtration as described above.
  • the obtained filtrate was loaded onto a MabSelect PrismA column and the separation chromatography was run as described above.
  • the inset is a photograph of non-reducing SDS-PAGE analysis of the relevant fractions, which includes Lane 1: isolated hole-hole homodimer as reference, Lane 2: load and Lane 3: eluate.
  • the hole-hole homodimer reference was generated by expressing the hole half antibody only and purified by Protein A chromatography followed by size exclusion chromatography. The homodimer produced two bands (Lane 1) .
  • a mass spectrometry analysis indicated presence of a truncated version, which is approximately 1500 Dalton smaller, in addition to the intact version (data not shown) .
  • both the load (Lane 2) and the eluate (Lane 3) contained the hole-hole homodimer, wherein the band of the intact hole-hole homodimer overlaps with that of the target bsAb. Presence of the hole-hole homodimer is indicated by the band slightly below the main band (in both Lane 2 and Lane 3) , which corresponds to the truncated version as in Lane 1. As can be seen then, Protein A chromatography failed to separate the KiH heterodimer from the hole-hole homodimer as they both contain the Fc domain (Fig. 3) .
  • the band seen on the lower part of the gel was identified to be the hole half antibody by mass spectrometry analysis (data not shown) .
  • formation of the hole-hole homodimer was resultant from overexpression of the hole half antibody. Since the homodimer is less stable than the heterodimer, only part of the overexpressed hole half antibodies formed hole-hole homodimers while the rest remained as unpaired half antibodies.
  • Example 1 Separation by KappaSelect Using a Linear pH Gradient elution
  • antibody species comprising different number of kappa LC constant regions were separated using KappaSelect and elution was conducted with a linear pH gradient as described above.
  • the culture harvest was clarified by depth filtration as described above.
  • the obtained filtrate was loaded onto a KappaSelect column and eluted using a linear pH gradient as described above.
  • the inset is a photograph of non-reducing SDS-PAGE analysis, which includes Lane 1: isolated hole-hole homodimer as reference, Lane 2: load, Lanes 3: eluate 1 (E1) , Lane 4: eluate 2 (E2) , Lane 5: Protein A eluate (included for comparison) , and Lane 6: strip.
  • the isolated hole-hole homodimer and the Protein A eluate were obtained as described in the Comparative Example above.
  • the hole-hole homodimer was absent in both eluates E1 and E2 (Lane 3 and Lane 4) , and was mainly found in the strip (Lane 6) .
  • the culture harvest was clarified by depth filtration as described above.
  • the obtained filtrate was loaded onto a KappaSelect column and eluted at pH 3.0, pH 3.2 and pH 3.5, respectively.
  • FIG. 5A exhibits overlayed chromatograms of the three runs at different pH values.
  • Fig. 5B exhibits three photographs of non-reducing SDS-PAGE analysis corresponding to the three runs, each including Lane 1: isolated hole-hole homodimer as reference, Lane 2: load, Lane 3: eluate 1 (E1) , Lane 4: eluate 2 (E2) , Lane 5: Protein A eluate (included for comparison) , Lane 6: strip.
  • SDS-PAGE analysis suggests, in comparison, elution at pH 3.5 allows the most complete removal of the hole-hole homodimer (Fig. 5B) .
  • the Protein A eluate and the KappaSelect eluate were separately subjected to an AEX chromatography as described above.
  • the KappaSelect eluate used for this analysis was eluted at pH 3.5.
  • the hole half antibody and the hole-hole homodimer had a relatively high isoelectric point (pI) (i.e., 7.5) , they would not bind to the AEX column at pH 7.0.
  • the targe KiH bsAb had a much lower pI due to the replacement by TCR constant domains of lower pI, and would bind to the AEX column under the same condition.
  • FIG. 7A-7B The result was shown in Figures 7A-7B.
  • the inset is a photograph of non-reducing SDS-PAGE analysis of the relevant fractions, which includes Lane 1: isolated hole-hole homodimer as reference, Lane 2: load and Lanes 3-5: flow-through fractions 1-3 (F1-F3) .
  • the flow-through from the run with the Protein A eluate contained both the hole half antibody and the hole-hole homodimer (Fig. 7A, Lanes 3-5)
  • the flow-through from the run with the KappaSelect eluate contained only half antibody (Fig. 7B, Lanes 3-5) . This suggests that the hole-hole homodimer byproduct was completely removed by KappaSelect.

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Abstract

L'invention concerne un procédé de purification d'une espèce d'anticorps bispécifique cible comprenant une région constante à chaîne légère (LC) kappa à partir d'un fluide comprenant l'espèce d'anticorps bispécifique cible et au moins une espèce de sous-produit comprenant deux régions constantes LC kappa, ledit procédé comprenant une étape de séparation dudit anticorps bispécifique cible à partir de ladite au moins une espèce de sous-produit par l'intermédiaire de la chromatographie d'affinité KappaSelect.
PCT/CN2021/078023 2020-02-28 2021-02-26 Purification d'anticorps bispécifiques WO2021170060A1 (fr)

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Citations (5)

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WO2013088259A2 (fr) * 2011-10-19 2013-06-20 Novimmune S.A. Procédés de purification d'anticorps
CN103509116A (zh) * 2006-04-05 2014-01-15 艾伯维生物技术有限公司 抗体纯化
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