Methods for purifying recombinant antibody fragments

Abstract

The present invention relates to a method for purifying a recombinant antibody fragment from inclusion bodies expressed in microbial cells, and more particularly to a method for purifying the recombinant humanized (rHu) antibody fragment ranibizumab from inclusion bodies expressed in microbial cells.

Description

FIELD OF THEINVENTION

[0001] The present invention relates to a method for purifying a recombinant antibody fragment from inclusion bodies expressed in microbial cells, more particularly, the present invention relates to a method for purifying the recombinant humanized (rHu) antibody fragment ranibizumab from inclusion bodies expressed in microbial cells.

[0002] 2. Background of the Invention Recent trends in biopharmaceutical research and development have focused more on the development of antibody fragments. Although a large number of biosimilar therapeutic proteins are under development, significant challenges in the development of biosimilar products include the high cost of downstream processing, non-selective removal of process- and product-related impurities, and proteolytic degradation of the product. Antibody fragments offer certain advantages over full-length monoclonal antibody (mAb) therapeutics, such as improved deep tumor penetration and binding to specific epitopes inaccessible to full-length mAbs. Fabs represent the majority of antibody fragment candidates that have entered clinical development, with three approved by the U.S. Food and Drug Administration. Advances in upstream processing, such as high-titer clones and continuous biomanufacturing, have shifted the focus of the biopharmaceutical industry to improving overall downstream economics. Downstream processing accounts for approximately 60-70% of the total manufacturing costs of monoclonal antibody therapeutics. Downstream capture, intermediate, and final purification steps involve the extensive use of various chromatographic operations.

[0003] Ranibizumab is a VEGF-A antagonist that binds to the active form of human vascular endothelial growth factor-A (VEGF-A) and inhibits its biological function. Excessive upregulation of VEGF-A makes VEGF-A essential for the growth of new blood vessels, leading to angiogenesis and vascular hyperpermeability. Vascular permeability can result in macular edema and choroidal neovascularization, which can cause the wet form of age-related macular degeneration (AMD). Therefore, it is essential to inhibit VEGF-A with VEGF-A antagonists to control and treat the pathological condition of vascular hyperpermeability. Ranibizumab is commercially available as Lucentis® and is administered by intravitreal injection. It is specifically designed and manufactured for intraocular use. Ranibizumab is a recombinant humanized IgG1κ isotype monoclonal antibody fragment designed for intraocular use in the treatment of age-related macular degeneration (ARMD).

[0004] The molecular weight of ranibizumab is about 48.37 kDa, i.e., the light and heavy chains are 23.43 kDa and 24.95 kDa, respectively. The humanized monoclonal antibody fragment is expressed in E. coli cells by recombinant DNA technology and targets human vascular endothelial growth factor A (VEGF-A). In ranibizumab, the 214 residue light chain is linked to the 231 residue N-terminal segment of the heavy chain by a disulfide bond at its C-terminus. Prior art literature provides evidence focusing on the expression of the antibody fragment and its purification. However, recombinant humanized (rHu) ranibizumab produced using E. coli cells forms inclusion bodies in the form of insoluble protein aggregates.

[0005] US Patent Publication No. 2016 / 0289314 discloses the cloning, expression and purification method for preparing ranibizumab.The purification method simply involves the reduction, oxidation and in vitro refolding of solubilized inclusion bodies, followed by the stepwise processing of ultrafiltration and diafiltration-I, anion exchange chromatography, cation exchange chromatography, ultrafiltration and diafiltration-II and filtration.The multiple purification steps of this method lead to low throughput operation, thereby making the method cumbersome.

[0006] Therefore, there is an urgent need to provide a convenient downstream process for obtaining purified antibody fragments. In view of the shortcomings of the downstream purification procedures disclosed in the prior art, the present inventors have attempted to provide a method for purifying antibody fragments. The purified antibody fragments obtained according to the present invention meet the standard Ranimizumab purity and activity standards.

[0007] Object of the invention It is therefore a primary object of the present invention to provide a method for purifying antibody fragments.

[0008] Another object of the present invention is to provide a method for purifying recombinant ranibizumab by integrating a multimodal chromatography purification step with the downstream processing of rHu ranibizumab.

[0009] It is yet another object of the present invention to provide a process that provides approximately a two-fold increase in productivity over existing manufacturing methods.

[0010] Yet another object of the present invention is to provide a method whereby the developed purification platform is applicable to both in vitro refolded and soluble expressed antibody fragments.

[0011] Summary of the Invention The present invention provides a method for purifying recombinant antibody fragments.

[0012] In one aspect, the present invention relates to a method for purifying recombinant human ranibizumab from recombinant host cells, the method comprising obtaining rHu ranibizumab from recombinant host cells in the form of inclusion bodies; solubilizing the inclusion bodies using a solubilization buffer to obtain solubilized rHu ranibizumab; refolding the solubilized rHu ranibizumab using a refolding buffer to obtain refolded rHu ranibizumab; concentrating the refolded rHu ranibizumab by ultrafiltration to obtain concentrated rHu ranibizumab; subjecting the concentrated rHu ranibizumab to sequential multimodal chromatographic purification steps to remove product related impurities, host cell proteins and host cell nucleic acids, followed by diafiltration and ultrafiltration to obtain purified rHu ranibizumab.

[0013] In another aspect, the present invention provides a method for producing a composition comprising: (i) subjecting the concentrated refolded rHu ranibizumab to multimode chromatography, using a buffer having a pH in the range of 4.0 to 10.0 containing 20 mM to 50 mM Tris-HCl and 0 mM to 150 mM NaCl or 20 mM to 50 mM acetate and 0 mM to 150 mM NaCl, the concentrated refolded rHu ranibizumab having a pH in the range of 4.0 to 10.0 is fed to a multimode chromatography resin to capture the rHu ranibizumab and remove impurities; (ii) eluting the rHu ranibizumab into an elution pool in the presence of an elution buffer having a pH in the range of 4.0 to 10.0 comprising 20 mM to 50 mM Tris-HCl and 0 mM to 1000 mM sodium chloride or 20 mM to 50 mM acetate and 0 mM to 1000 mM NaCl; wherein the purified ranibizumab has less than 5% unfolded or misfolded forms of ranibizumab and less than 2% aggregated forms of ranibizumab; (iii) feeding the rHu ranibizumab from step (ii) having a pH in the range of 4.5 to 6.5 using a buffer solution containing 20 mM to 50 mM acetate and 0 mM to 150 mM NaCl and having a pH in the range of 4.0 to 6.5 onto a multi-mode chromatography resin to capture the rHu ranibizumab and further remove impurities; (iv) eluting rHu ranibizumab in the presence of an elution buffer having a pH in the range of 4.0 to 6.5, comprising 20 mM to 50 mM acetate and 0 mM to 1000 mM NaCl; wherein the purified rHu ranibizumab has less than 0.1% unfolded or misfolded forms of rHu ranibizumab and less than 0.3% aggregated forms of rHu ranibizumab; The present invention provides a multi-mode chromatographic purification method comprising:

[0014] In yet another aspect, the present invention provides a multimodal chromatography, wherein the order of phase I and phase II of the multimodal chromatography is interchangeable.

[0015] In yet another aspect, the present invention provides recombinant humanized ranibizumab obtained by downstream steps of ranibizumab purification.

[0016] This Summary is provided to introduce concepts related to methods for purifying a recombinant human antibody fragment (rHu ranibizumab). This Summary is not intended to identify essential features of the claimed invention, nor is it intended for use in determining or limiting the scope of the claimed invention. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 illustrates the process steps of the claimed multi-modal chromatographic purification platform. [Diagram 2]Figure 2 shows the chromatogram of multimode chromatography I. Elution peak I: purified rHu ranibizumab, elution peak II: product related impurities (misfolded and unfolded ranibizumab) and process related impurities (host cell proteins and nucleic acids). [Diagram 3] Figure 3 shows the chromatograms of multimode chromatography II. Flow-through and elution peak I: process related impurities (host cell proteins and nucleic acids). Elution peak II: purified rHu ranibizumab, elution peak III: product related impurities (misfolded and unfolded ranibizumab). [Figure 4] Figure 4 shows the chromatogram of multimode chromatography I in flow-through mode (conductivity of feed material: 6.0 mS / cm, equilibration buffer: 20.0 mM Tris at pH 9.0, refolded rHu ranibizumab was eluted with a 5.0 CV salt gradient). Flow-through: purified rHu ranibizumab (29.69% purity); elution peak I: process-related impurities (host cell proteins and nucleic acids) including rHu ranibizumab, elution peak II: misfolded and unfolded ranibizumab as well as process-related impurities. [Diagram 5] Figure 5 shows the chromatogram of multimode chromatography I in flow-through mode (conductivity of feed material: 12.0 mS / cm, equilibration buffer: 20.0 mM Tris at pH 9.0, refolded rHu ranibizumab was eluted with a 5.0 CV salt gradient). Flow-through: purified rHu ranibizumab (33.12%); elution peak I: process-related impurities (host cell proteins and nucleic acids), elution peak II: misfolded and unfolded ranibizumab and process-related impurities. [Figure 6]Figure 6 shows the chromatogram of multimode chromatography I (equilibration buffer: 20.0 mM Tris pH 9.0, refolded rHu ranibizumab was eluted by a step gradient, first step was 12.5% ​​elution buffer, second step was 100% elution buffer containing 20 mM Tris pH 9.0 and 1.0 M NaCl). Elution peak I: purified rHu ranibizumab (purity 38.25%), elution peak II: product related impurities (misfolded and unfolded ranibizumab) and process related impurities. [Figure 7] Figure 7 shows non-reducing SDS-PAGE for characterization of low and high molecular weight impurities associated with refolded rHu ranibizumab: Lane 1: refolded rHu ranibizumab purified using multi-mode chromatography of the present invention, lane 2: protein molecular weight markers, and lane 3: standard ranibizumab. [Figure 8] Figure 8 shows a reducing SDS-PAGE: lane 1: purified refolded rHu ranibizumab, lane 2: protein molecular weight markers, and lane 3: standard ranibizumab. [Figure 9]Figure 9 shows non-reducing SDS-PAGE of the elution peaks from multimode chromatography I. 1: protein molecular weight markers. 2: standard rHu ranibizumab. 3: elution peak 1 (refolded rHu ranibizumab, 48 kDa, with 38.25% purity with low molecular weight impurities), 4: elution peak 2 (representing all other process related impurities), (conditions for multimode chromatography I: equilibration buffer pH 9.0, refolded rHu ranibizumab was eluted in a 5 CV salt gradient), 5: elution peak 1, 6: elution peak 2, (conditions, equilibration buffer pH 10.0, refolded rHu ranibizumab was eluted in a 5 CV salt gradient); 7: elution peak 1, 8: elution peak 2, (conditions, equilibration buffer pH 10.0, refolded rHu ranibizumab was eluted in a 7.5 CV salt gradient); 9: elution peak 1, 10: elution peak 2, (conditions, equilibration buffer pH 10.0, refolded rHu ranibizumab was eluted in a 10 CV salt gradient). [Figure 10] Figure 10 shows non-reducing SDS-PAGE of purified soluble expressed rHu ranibizumab. 1: standard rHu ranibizumab, 3: purified rHu ranibizumab of the invention. [Figure 11] Figure 11 shows reverse-phase HPLC chromatograms for the purity analysis of purified rHu ranibizumab. A: Standard rHu ranibizumab. B: Purified rHu ranibizumab. [Figure 12] Figure 12 shows size exclusion chromatograms of purified rHu ranibizumab. A: Standard rHu ranibizumab. B: Purified rHu ranibizumab. [Figure 13] Figure 13 shows intact mass analysis by MALDI-TOF: A: standard rHu ranibizumab, B: purified rHu ranibizumab.

[0018] Detailed Description of the Invention The present invention will now be described in detail in connection with certain preferred optional embodiments so that its various aspects may be more fully understood and appreciated.

[0019] Definition: For convenience, before further description of the present invention, some terms employed in the specification, examples and claims are collected here.

[0020] The term "inclusion bodies" refers to insoluble aggregates of a target recombinant protein. Inclusion bodies of a target protein contain a combination of misfolded and partially folded protein. Protein refolding is the process by which a protein acquires its native three-dimensional structure, a biologically functional conformation.

[0021] The term "reducing agent" refers to a substance that causes the reduction of disulfide bonds in proteins, leaving both intramolecular and intermolecular disulfide bonds chemically broken.

[0022] The term "chaotropic agent" refers to a substance that can change the conformation of a protein to make it more water soluble. Examples of such chaotropic agents include ethanol, butanol, urea, guanidinium hydrochloride, thiourea, magnesium chloride, lithium perchlorate, etc.

[0023] The term "host cell protein" refers to all proteins that are expressed by the host cell separately from the target protein during the course of a fermentation or cell culture process.

[0024] As used herein, the term "multimodal chromatography" refers to a chromatographic technique in which a ligand and a protein interact through more than one type of interaction, including ionic interactions, hydrophobic interactions, or hydrogen bonding.

[0025] The present invention provides a method for purifying a recombinant monoclonal antibody fragment from a recombinant host cell, the method comprising: (i) solubilizing inclusion bodies containing the antibody fragment obtained from the recombinant host cells using a solubilization buffer to obtain a solubilized antibody fragment; (ii) refolding the solubilized antibody fragment using a refolding buffer to obtain a refolded antibody fragment; (iii) concentrating the refolded antibody fragment by ultrafiltration to obtain a concentrated antibody fragment; and (iv) subjecting the concentrated antibody fragment to sequential multi-mode chromatographic purification to remove product-related impurities, i.e., host cell proteins and host cell nucleic acids, followed by diafiltration and ultrafiltration to obtain purified rHu antibody fragment. Includes.

[0026] Recombinant ranibizumab is obtained by expressing said protein in recombinant host cells, preferably at equal ratios of heavy and light chain concentration.The recombinant host cell used in the present invention is E. coli.The E. coli host strain used in the present invention is E. coli BL21(DE3).The strain is obtained from Merck Millipore.

[0027] The present invention provides solubilized inclusion bodies containing equal or unequal ratios of the light and heavy chains of recombinant humanized (rHu) ranibizumab in the presence of a solubilization buffer to obtain a solubilized mixture of the light and heavy chains of rHu ranibizumab. The rHu inclusion bodies extracted from recombinant host cells are solubilized using a solubilization buffer containing a chaotropic detergent, a reducing agent and a denaturing agent.

[0028] Chaotropic agents included urea and guanidinium hydrochloride. The pH of the solubilization buffer was maintained within the range of 7 to 10. Reduction of the solubilized inclusion bodies was achieved using a combination of reducing agents including dithiothreitol, β-mercaptoethanol for 30 min to 2 h.

[0029] The chaotropic detergents, reducing agents and denaturing agents are selected from those conventionally used in the art.

[0030] The method for solubilization includes first solubilizing the inclusion bodies obtained from recombinant host cells containing ranibizumab protein in 15 ml of solubilization buffer. The solubilization buffer contains 0.1 M Tris at pH 9.0, 2 mM EDTA and 6 M guanidine hydrochloride as a denaturant, and is treated with these for 30 minutes, followed by reduction in the presence of 5 mM DTT for 1 hour. The soluble and reduced inclusion body solution is oxidized by adding 10 mM oxidized glutathione.

[0031] In another embodiment, in vitro refolding of solubilized rHu ranibizumab is obtained in the presence of a refolding buffer comprising a protein folding agent, a polyol and a surfactant, wherein the pH is maintained between 8.0 and 10.0.

[0032] "In vitro protein refolding" as referred to herein refers to a method for converting misfolded ranibizumab to a correctly folded form. Refolding of ranibizumab was accomplished using a 75-fold dilution in a refolding buffer containing 0.1 M Tris pH 9.0, 0.6 M arginine, 5% sorbitol and 2 mM EDTA at 5°C to 20°C.

[0033] The refolded ranibizumab protein was further concentrated by ultrafiltration using a 5 kDa Ultrasette™ Lab Tangential Flow Filtration device, followed by buffer exchange into 20 mM Tris, pH 9.0. The buffer-exchanged sample was then used as input for multimode chromatography.

[0034] In a most preferred embodiment, the present invention provides a method for producing a method for treating a cancer cell comprising: (a) solubilizing inclusion bodies containing the light and heavy chains of rHu ranibizumab from recombinant host cells to obtain solubilized rHu ranibizumab; (b) refolding the solubilized rHu ranibizumab to obtain refolded rHu ranibizumab; (c) concentrating the refolded rHu ranibizumab by ultrafiltration to obtain concentrated refolded ranibizumab; (d) subjecting the concentrated refolded rHu ranibizumab to multi-mode chromatography, said chromatography step comprising: (i) feeding the concentrated refolded rHu ranibizumab of step (c) having a pH in the range of 4.0 to 10.0 to a multi-mode chromatography resin using a buffer having a pH in the range of 4.0 to 10.0 comprising 20 mM to 50 mM Tris-HCl and 0 mM to 150 mM NaCl or 20 mM to 50 mM acetate and 0 mM to 150 mM NaCl to capture rHu ranibizumab and remove impurities; (ii) eluting rHu ranibizumab into an elution pool in the presence of an elution buffer having a pH in the range of 4.0 to 10.0 comprising 20 mM to 50 mM Tris-HCl and 0 mM to 1000 mM sodium chloride or 20 mM to 50 mM acetate and 0 mM to 1000 mM NaCl; wherein the purified ranibizumab has less than 5% unfolded or misfolded forms of ranibizumab and less than 2% aggregated forms of ranibizumab; (iii) feeding the rHu ranibizumab from step (ii) having a pH in the range of 4.5 to 6.5 using a buffer having a pH in the range of 4.0 to 6.5 comprising 20 mM to 50 mM acetate and 0 mM to 150 mM NaCl to a multi-mode chromatography resin to capture the rHu ranibizumab and further remove impurities; (iv) eluting rHu ranibizumab in the presence of an elution buffer having a pH in the range of 4.0 to 6.5, comprising 20 mM to 50 mM acetate and 0 mM to 1000 mM NaCl; wherein the purified rHu ranibizumab has less than 0.1% unfolded or misfolded forms of rHu ranibizumab and less than 0.3% aggregated forms of rHu ranibizumab; The present invention provides a method for purifying an antibody fragment of recombinant humanized (rHu) ranibizumab from a recombinant host cell, comprising:

[0035] The multimodal chromatography purification steps in steps (i) and (ii) of the above process are referred to as multimodal chromatography Phase I, and the multimodal chromatography purification steps in steps (iii) and (iv) of the above process are referred to as multimodal chromatography Phase II. Phases I and II of multimodal chromatography may be performed sequentially or optionally interchanged in order to remove various process- and product-related impurities associated with rHu ranibizumab.

[0036] Multi-mode chromatography phase I (process steps (i) and (ii)) and phase II (process steps (iii) and (iv)) involve two steps each for the purification of rHu ranibizumab.

[0037] In one aspect, the present invention provides a multimodal chromatography phase I comprising a bind-elute mode of multimodal chromatography and a flow-through mode of multimodal chromatography.

[0038] In another aspect, the present invention provides a multimodal chromatography phase II comprising a bind-elute mode of multimodal chromatography and a flow-through mode of multimodal chromatography.

[0039] Multimodal Chromatography Phase I: The multimodal chromatography mode utilized in phase I is selected from a bind-elute chromatography mode or a flow-through mode. The multimodal chromatography resin I is selected from the group consisting of HEA Hypercel, PPA Hypercel, and BAKERBOND™ XWP 500 Poly PEI-35 or Capto adhere resin.

[0040] The present invention relates to (i) binding the concentrated refolded rHu ranibizumab to multi-mode chromatography resin I using a buffer containing 20 mM to 50 mM Tris-HCl and 0 mM to 150 mM NaCl, having a pH in the range of 8.0 to 10.0, to capture rHu ranibizumab and remove impurities; (ii) eluting rHu ranibizumab into an elution pool in the presence of an elution buffer comprising 20 mM to 50 mM Tris-HCl and 0 mM to 1000 mM sodium chloride, the elution buffer having a pH in the range of 8.0 to 10.0; The present invention provides a multi-mode chromatography phase I with a bind-elute mode of chromatography, comprising:

[0041] The first stage of multimode chromatography phase I with bind-elute chromatography involves binding of refolded rHu ranibizumab and elution of purified refolded rHu ranibizumab. In multimode chromatography resin I, the variation of sample input pH from 8.0 to 10.0 and elution with different salt gradients from 5 CV to 10 CV play an important role in removing impurities and optimizing the purification method.

[0042] In multimode chromatography phase I bind and elute mode, changing the pH of the equilibration buffer from 8.0 to 10.0 alters the charge distribution and three-dimensional conformation of the protein. A combination of ionic interactions, hydrophobic interactions and elution in different salt gradients gave a product with a purity of 38.79% with a recovery of 67.56% in an equilibration buffer of pH 9.0 and a salt gradient of 5 CV.

[0043] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: (i) 20 mM to 50 mM Tris-HCl, and (ii) 0 mM to 150 mM sodium chloride Including, Provide a binding buffer used for binding rHu ranibizumab to multimodal chromatography resin I, having a pH in the range of 8.0 to 10.0.

[0044] In another aspect, the present invention provides a method for producing a composition comprising: (i) 20 mM to 50 mM Tris-HCl, and (ii) 0 mM to 1000 mM sodium chloride Including, Provide an elution buffer used to elute rHu ranibizumab from multimode chromatography resin I into an elution pool, having a pH in the range of 8.0-10.0.

[0045] The present invention relates to (i) feeding the concentrated refolded rHu ranibizumab to a multimode chromatography resin I using a buffer containing 20 mM to 50 mM acetate and 0 mM to 150 mM NaCl, having a pH in the range of 4.0 to 6.5, and an input conductivity in the range of 6.0 mS / cm to 12 mS / cm, to capture rHu ranibizumab and remove impurities; and (ii) eluting the impurities into an elution pool in the presence of an elution buffer comprising 20 mM to 50 mM acetate and 0 mM to 1000 mM sodium chloride and having a pH in the range of 4.0 to 6.5; The present invention provides a multi-mode chromatography phase I with a flow-through mode of chromatography, comprising:

[0046] In the flow-through mode of multimode chromatography phase I, the change in input conductivity of the refolded concentrated fraction of recombinant ranibizumab results in a change in the binding affinity of refolded rHu ranibizumab and impurities. At a sample input conductivity of 12 mS / cm, the impurities bound to the resin and refolded rHu ranibizumab was eluted in the flow-through. Under such conditions, a product with a purity of 33.12% was obtained with a recovery of 76.98%.

[0047] Multimodal Chromatography Phase II: To capture rHu ranibizumab and further remove impurities, the rHu ranibizumab obtained from the eluate of multi-mode chromatography phase I is adjusted to a pH in the range of 4.5-6.5 and fed to multi-mode chromatography resin II using a buffer having a pH in the range of 4.0-6.5 containing 20 mM-50 mM acetate and 0 mM-150 mM NaCl.

[0048] The present invention provides a multi-modal chromatography phase II method carried out using bind-elute chromatography, the method comprising: (i) feeding rHu ranibizumab obtained from the elution pool of multi-mode chromatography phase I having a pH in the range of 4.5-6.5 and an input conductivity in the range of 6.0 mS / cm-12 mS / cm to a multi-mode chromatography resin II using a buffer containing 20 mM-50 mM acetate and 0 mM-150 mM NaCl, having a pH in the range of 4.0-6.5, to capture rHu ranibizumab and further remove impurities; (ii) eluting rHu ranibizumab in the presence of an elution buffer containing 20 mM to 50 mM acetate and 0 mM to 1000 mM NaCl and having a pH in the range of 4.0 to 6.5. Includes.

[0049] The present invention provides a multi-modal chromatography phase II method carried out using a flow-through mode of multi-modal chromatography, the method comprising: (i) feeding rHu ranibizumab obtained from the elution pool of multi-mode chromatography phase I having a pH in the range of 8.0-10.0 to a multi-mode chromatography resin II using a buffer containing 20 mM-50 mM Tris-HCl and 0 mM-150 mM NaCl, having a pH in the range of 8.0-10.0, to capture rHu ranibizumab and further remove impurities; and (ii) eluting rHu ranibizumab in the presence of an elution buffer containing 20 mM to 50 mM Tris-HCl and 0 mM to 1000 mM NaCl and having a pH in the range of 8.0 to 10.0; Includes.

[0050] In a further aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (i) 20 mM to 50 mM acetate, and (ii) 0 mM to 150 mM sodium chloride Including, Provide a binding buffer used for binding rHu ranibizumab to multimode chromatography resin II, having a pH in the range of 4.0 to 6.0.

[0051] In yet another aspect, the present invention provides a method for producing a composition comprising: (i) 20 mM to 50 mM Tris-HCl, and (ii) 0 mM to 1000 mM sodium chloride Including, Provide an elution buffer used to elute rHu ranibizumab from multimode chromatography resin II into an elution pool, having a pH in the range of 4.0-6.0.

[0052] The multimode chromatography resin II is selected from the group consisting of HEA Hypercel, PPA Hypercel, and BAKERBOND™ XWP 500 PolyCSX-35 or Capto MMC resin. In the binding and elution mode of the multimode chromatography resin II, the change in pH of the equilibration buffer from 4.0 to 6.5 results in a change in the charge distribution and three-dimensional conformation of the protein. A combination of ionic interactions, hydrophobic interactions and sample input conductivity resulted in a product of 99.50% purity with a recovery of 46.57% at equilibration buffer pH 5.5 and sample input conductivity of 6.0 mS / cm.

[0053] [Table 1]

[0054] Table 1 above lists the different conditions of equilibration buffer pH and salt concentration that were investigated for optimizing the conditions for rHu ranibizumab recovered in different experiments.

[0055] Increasing the pH increases the negative charge of rHu ranibizumab, leading to stronger electrostatic interactions between rHu ranibizumab and the resin ligands.

[0056] The salt gradient plays an essential role in the selective elution of rHu ranibizumab. It was observed that an elution buffer containing 20 mM Tris-HCl buffer at pH 9.0 and a 5 CV salt elution gradient was useful for producing highly pure rHu ranibizumab of 38.25%. Under such conditions, the maximum purity of refolded rHu ranibizumab was observed, and the input conductivity of the sample and the salt concentration in the elution have an essential influence on the hydrophobic interaction of impurities with the resin to elute the maximum purified refolded rHu ranibizumab.

[0057] [Table 2]

[0058] Table 2 shows the effect of different pH of equilibration buffer and sample loading conditions on the recovery and purity of rHu ranibizumab eluted in multimode chromatography II. The decrease in pH serves to increase the positive charge on rHu ranibizumab, leading to stronger electrostatic interactions between rHu ranibizumab and the resin ligands. The hydrophobicity of protein molecules changes differently at different solution conductivities, which ultimately leads to different binding affinities with the resin. It was observed that an elution buffer containing 20 mM acetate buffer at pH 5.5 and a sample loading conductivity of 6.0 mS / cm was conducive to the production of 99.50% highly pure rHu ranibizumab.

[0059] [Table 3]

[0060] [Table 4]

[0061] Purified ranibizumab was observed in elution peak I in Figure 2 with a purity of 38.79% after elution from multimodal resin chromatography column I. Elution peak II contains misfolded and unfolded ranibizumab as well as process-related impurities such as host cell proteins and nucleic acids.

[0062] FIG. 3 confirms that process related impurities including host cell proteins and nucleic acids were observed in the flow through and elution peak I, purified ranibizumab was observed in elution peak II at 99.50% purity, and product related impurities, e.g., misfolded and unfolded ranibizumab, were observed in elution peak III.

[0063] In another preferred embodiment, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: (i) obtaining rHu ranibizumab from recombinant host cells in the form of inclusion bodies; (ii) solubilizing the inclusion bodies using a solubilization buffer to obtain solubilized rHu ranibizumab; (iii) refolding the solubilized rHu ranibizumab using a refolding buffer to obtain refolded rHu ranibizumab; (iv) concentrating the refolded rHu ranibizumab by ultrafiltration to obtain concentrated rHu ranibizumab; (v) subjecting the concentrated rHu ranibizumab to a series of multimodal chromatographic purification steps to remove product-related impurities, host cell proteins, and host cell nucleic acids and to obtain purified rHu ranibizumab. The present invention provides a pharmaceutical composition comprising ranibizumab obtained by a purification method comprising the steps of:

[0064] In a more preferred embodiment, the present invention provides a recombinant humanized ranibizumab obtained by the method of the present invention.

[0065] In a further aspect, the present invention provides an aqueous composition for intravitreal administration comprising ranibizumab obtained from the method of the present invention, said composition being utilized in the treatment and control of age-related macular degeneration and related diseases.

[0066] [Example] The following examples are offered by way of illustration only, and therefore should not be construed as limiting the scope of the invention in any way.

[0067] Example 1: Multimode chromatography in bind / elute mode (i) Solubilization of inclusion bodies In the present invention, recombinant human ranibizumab expressed in E. coli was used. First, 0.8 g of inclusion bodies were solubilized for 30 minutes in 40 ml of solubilization buffer containing 0.1 M Tris at pH 9.0, 2 mM EDTA, and 6 M guanidine hydrochloride as a denaturant. The inclusion bodies were solubilized for 30 minutes at 25°C using a magnetic stirrer. After solubilization, the IB was centrifuged at 7000 rpm for 10 minutes and filtered using a filter paper with a pore size of 1.0 μm. Dithiothreitol (DTT) was added as a reducing agent so that the concentration of 0.5 M DTT in the solubilization buffer was 5.0 mM. For reduction, the solution was kept stirring for 30 minutes at 25°C. The soluble and reduced inclusion body solution was kept oxidized for 3 hours by adding 10.0 mM oxidized glutathione.

[0068] (ii) Refolding of solubilized IB containing ranibizumab Approximately 40 mL of the solubilized, reduced and oxidized inclusion body solution was added to 960 mL of refolding buffer containing 0.1 M Tris, 0.6 M arginine, 5.0% sorbitol and 2 mM EDTA, 0.60 mM cystine and 0.75 mM cysteine ​​at pH 9.0. The refolding process was carried out at 10±2° C. for 120 hours at 50 rpm.

[0069] (iii) Ultrafiltration and diafiltration of refolded rHu ranibizumab The refolded ranibizumab protein was concentrated by ultrafiltration using a 5kDa Ultrasette™ Lab Tangential Flow Filtration device. The concentrated protein was buffer exchanged into 20mM Tris, pH 9.0 using successive diafiltration procedures. The buffer exchanged sample was then used as input for multimode chromatography.

[0070] (iv) Multimode Chromatography I Multimode chromatography I was performed using polyPEI-35 resin. The pH of concentrated refolded ranibizumab was adjusted to 9.0 and used as the feed material. The experiment to capture refolded rHu ranibizumab was performed with 20 mM Tris pH 9.0 as the equilibration buffer, followed by a 3 CV salt gradient with 20 mM Tris pH 9.0 containing 1 M NaCl. Under such conditions, no protein was observed in the flow-through. RP-HPLC analysis of the elution peak confirmed that purified ranibizumab was observed in elution peak I (38.79% purity) (Figure 2). Elution peak II contains product-related impurities (misfolded and unfolded ranibizumab) as well as process-related impurities (host cell proteins and nucleic acids). In FIG. 2, elution peak 1 of the multi-mode chromatography contained purified rHu ranibizumab, which was characterized at 48 kDa in lane number 3 of the SDS PAGE in FIG.

[0071] (v) Multimode Chromatography II Multimode chromatography II was performed using polyCSX-35 resin. The experiments capturing rHu ranibizumab were performed using 10 mM acetate buffer at pH 5.50 as equilibration buffer. The elution pool of multimode chromatography I was used as feed material for multimode chromatography II. The pH of the feed material was adjusted to pH 5.50 with glacial acetic acid. Elution was performed with a salt-based step gradient with 10 mM acetate buffer at pH 5.50 containing 1 M NaCl. After selective removal of process-related impurities with a 10% gradient, pure ranibizumab was eluted with a 45% elution step gradient. RP-HPLC analysis of these peaks confirmed that process-related impurities (host cell proteins and nucleic acids) were observed in the flow-through and elution peak I, while purified ranibizumab was observed in elution peak II (99.50% purity) and product-related impurities (misfolded and unfolded ranibizumab) were observed in elution peak III (Figure 3). The quality of the product was verified using SDS PAGE analysis and was in good agreement with that of standard ranibizumab (Figure 7, lane number 1, 48 kDa).

[0072] Example 2: Bind-elute multimode chromatography I and II Refolding of rHu ranibizumab and its pretreatment (i) Solubilization of inclusion bodies 0.4 g of IB containing recombinant human ranibizumab expressed in E. coli was dissolved in 20 mL of solubilization buffer containing 0.1 M Tris pH 9.0, 2.0 mM EDTA and 6.0 M guanidine hydrochloride as a denaturant for 30 min. Further processing was performed as per the method of Example 1(i).

[0073] (ii) Refolding of solubilized inclusion bodies Refolding was carried out according to the method disclosed in Example 1(ii). (iii) Ultrafiltration and diafiltration of refolded rHu ranibizumab Ultrafiltration and diafiltration of refolded rHu ranibizumab was performed as described in Example 1(iii).

[0074] (iv) Multimode Chromatography I Multimode chromatography I was performed using polyPEI-35 resin. The concentrated refolded ranibizumab was adjusted to pH 10.0 and used as the feed material. The refolded rHu ranibizumab capture experiment was performed with 20.0 mM Tris pH 10.0 as the equilibration buffer, followed by a 5.0 CV salt gradient with 20.0 mM Tris pH 10.0 containing 1.0 M NaCl. No protein was observed in the flow-through. RP-HPLC analysis of the elution peak confirmed that purified ranibizumab was observed in elution peak I (37.97% purity). Elution peak II contains product-related impurities (misfolded and unfolded ranibizumab) as well as process-related impurities (host cell proteins and nucleic acids).

[0075] (v) Multimode Chromatography II Multimode chromatography II was performed using polyCSX-35 resin. The experiments capturing rHu ranibizumab were performed using 10.0 mM acetate buffer at pH 5.5 as equilibration buffer. The elution pool from multimode chromatography I was used as the feed material for multimode chromatography II. The pH of the feed material was adjusted to pH 5.5 using glacial acetic acid. Elution was performed with a salt-based step gradient with 10 mM acetate buffer at pH 5.5 containing 1.0 M NaCl. After selective removal of process-related impurities with a 10% gradient, pure ranibizumab was eluted with a 45% elution step gradient. RP-HPLC analysis of these peaks confirmed that process-related impurities (host cell proteins and nucleic acids) were observed in the flow-through and elution peak I, while purified ranibizumab was observed in elution peak II (99.50% purity) (Figure 3), and product-related impurities (misfolded and unfolded ranibizumab) were observed in elution peak III.

[0076] Example 3: Multimode chromatography in flow-through mode I and multimode chromatography in bind-and-elute mode II Refolding and pretreatment of rHu Ranibizumab: (i) Solubilization of inclusion bodies Recombinant human ranibizumab expressed in E. coli was used in this study. 0.267 g of inclusion bodies (IB) were dissolved in 13.3 mL of solubilization buffer. Further processing was as described in Example 1(i). (ii) Refolding of solubilized inclusion bodies 13.3 mL of solubilized, reduced and oxidized inclusion bodies were added to 986.7 mL of refolding buffer and the procedure was carried out as in Example 1(ii).

[0077] (iii) Ultrafiltration and diafiltration of refolded rHu ranibizumab The refolded ranibizumab protein was concentrated by ultrafiltration using a 5kDa Ultrasette™ Lab Tangential Flow Filtration device. The concentrated protein was buffer exchanged into 20mM Tris, pH 8.0 using successive diafiltration procedures. The buffer exchanged sample was then used as input for multimode chromatography.

[0078] (iv) Multimode Chromatography I Multimode chromatography I was performed using polyPEI-35 resin. The concentrated refolded ranibizumab was adjusted to pH 8.0 and used as the feed material. The experiment to capture refolded rHu ranibizumab was performed with 20.0 mM Tris pH 8.0 as the equilibration buffer, followed by a 5.0 CV salt gradient with 20.0 mM Tris pH 8.0 containing 1.0 M NaCl. Under such conditions, refolded rHu ranibizumab was observed in the flow-through. RP-HPLC analysis of the flow-through confirmed that purified ranibizumab was observed in the flow-through (31.24% purity). Elution peak I and elution peak II contain product-related impurities (misfolded and unfolded ranibizumab) and process-related impurities (host cell proteins and nucleic acids), respectively.

[0079] (v) Multimode Chromatography II Multimode chromatography II was performed using polyCSX-35 resin. The experiments capturing rHu ranibizumab were carried out using 10.0 mM acetate buffer at pH 5.5 as equilibration buffer. The flow-through of multimode chromatography I was used as feed material for multimode chromatography II. The pH of the feed material was adjusted to pH 5.5 using glacial acetic acid. Elution was performed with a salt-based step gradient with 10.0 mM acetate buffer at pH 5.5 containing 1.0 M NaCl. After selective removal of process-related impurities with a 10% gradient, pure ranibizumab was eluted with a 45% elution step gradient. RP-HPLC analysis of these peaks confirmed that process-related impurities (host cell proteins and nucleic acids) were observed in the flow-through and elution peak I, while purified ranibizumab was observed in elution peak II (99.50% purity) and product-related impurities (misfolded and unfolded ranibizumab) were observed in elution peak III.

[0080] Example 4: Multimode chromatography in flow-through mode I and multimode chromatography in bind-and-elute mode II (i) Solubilization of inclusion bodies Solubilization was carried out as described in Example 2(i). (ii) Refolding of solubilized inclusion bodies Refolding was carried out as described in Example 2(ii). (iii) Ultrafiltration of refolded rHu ranibizumab The refolded ranibizumab protein was concentrated by ultrafiltration using a 5 kDa Ultrasette™ Lab Tangential Flow Filtration device. The concentrated protein sample was then used as input for Multimode Chromatography I.

[0081] (iv) Multimode Chromatography I Multimode chromatography I was performed in flow-through mode using polyPEI-35 resin. The concentrated refolded ranibizumab was adjusted to pH 9.0 and conductivity to 6.0 mS / cm and used as the feed material. The experiment to capture the refolded rHu ranibizumab in the flow-through was performed with 20.0 mM Tris, pH 9.0 as the equilibration buffer, followed by a 5.0 CV salt gradient with 20.0 mM Tris, pH 9.0 containing 1.0 M NaCl to remove impurities. Under this experimental condition, the refolded rHu ranibizumab was observed in the flow-through. RP-HPLC analysis of the flow-through confirmed that purified ranibizumab was observed in the flow-through (purity 29.69%. Elution peak I and elution peak II contain product related impurities (misfolded and unfolded ranibizumab) and process related impurities (host cell proteins and nucleic acids). Figure 4 shows the chromatogram of multimode chromatography I performed for the purification of rHu ranibizumab.

[0082] (v) Multimode Chromatography II Multimode chromatography II was performed using polyCSX-35 resin. The experiments capturing rHu ranibizumab were carried out using 10.0 mM acetate buffer at pH 5.50 as equilibration buffer. The elution pool of multimode chromatography I was used as feed material for multimode chromatography II. The pH of the feed material was adjusted to pH 5.5 using glacial acetic acid. Elution was performed with a salt-based step gradient with 10.0 mM acetate buffer at pH 5.5 containing 1 M NaCl. After selective removal of process-related impurities in a 10.0% gradient, pure ranibizumab was eluted in a 45.0% elution step gradient. RP-HPLC analysis of these peaks confirmed that process-related impurities (host cell proteins and nucleic acids) were observed in the flow-through and elution peak I, while purified ranibizumab was observed in elution peak II (99.50% purity) and product-related impurities (misfolded and unfolded ranibizumab) were observed in elution peak III.

[0083] Example 5: Multimode chromatography in flow-through mode I and multimode chromatography in bind-and-elute mode II (i) Solubilization of inclusion bodies Solubilization of the inclusion bodies was carried out as described in Example 2(i). (ii) Refolding of solubilized inclusion bodies Refolding was carried out as described in Example 2(ii). (iii) Ultrafiltration and diafiltration of refolded rHu ranibizumab Ultrafiltration of refolded rHu ranibizumab was carried out as described in Example 4(iii).

[0084] (iv) Multimode Chromatography I Multimode chromatography I was performed in flow-through mode using polyPEI-35 resin. The concentrated refolded ranibizumab was adjusted to pH 9.0 and conductivity to 12.0 mS / cm and used as the feed material. The experiment to capture the refolded rHu ranibizumab in the flow-through was performed with 20.0 mM Tris, pH 9.0 as the equilibration buffer, followed by a 5.0 CV salt gradient with 20.0 mM Tris, pH 9.0 containing 1.0 M NaCl to remove impurities. Under this experimental condition, the refolded rHu ranibizumab was observed in the flow-through. RP-HPLC analysis of the flow-through confirmed that purified ranibizumab was observed in the flow-through (purity 33.12%) (Figure 5). Elution peaks I and II contain product-related impurities (misfolded and unfolded ranibizumab) and process-related impurities (host cell proteins and nucleic acids). Figure 5 shows the chromatogram of multimode chromatography I performed for the purification of rHu ranibizumab.

[0085] (v) Multimode Chromatography II Multimode chromatography II was performed using polyCSX-35 resin. The experiments capturing rHu ranibizumab were carried out using 10.0 mM acetate buffer at pH 5.5 as equilibration buffer. The elution pool of multimode chromatography I was used as the feed material for multimode chromatography II. The pH of the feed material was adjusted to pH 5.5 using glacial acetic acid. Elution was performed with a salt-based step gradient with 10 mM acetate buffer at pH 5.5 containing 1.0 M NaCl. After selective removal of process-related impurities with a 10.0% gradient, pure ranibizumab was eluted with a 45.0% elution step gradient. RP-HPLC analysis of these peaks confirmed that process-related impurities (host cell proteins and nucleic acids) were observed in the flow-through and elution peak I, while purified ranibizumab was observed in elution peak II (99.50% purity) and product-related impurities (misfolded and unfolded ranibizumab) were observed in elution peak III.

[0086] Example 6: Multimode Chromatography in Bind-and-Elute Mode II followed by Multimode Chromatography in Bind-and-Elute Mode I (i) Solubilization of inclusion bodies The solubilization step was carried out as in the procedure of Example 1(i). (ii) Refolding of solubilized inclusion bodies The refolding step was carried out as per the method of Example 1(ii). (iii) Ultrafiltration and diafiltration of refolded rHu ranibizumab The refolded ranibizumab protein was concentrated by ultrafiltration using a 5kDa Ultrasette™ Lab Tangential Flow Filtration device. The pH of the concentrated protein sample was then adjusted to pH 5.5 using glacial acetic acid. The conductivity of the concentrated refolded protein sample was adjusted to 10.0mS / cm. The host cell protein was precipitated at pH 5.5. The precipitated host cell protein was removed by centrifugation at 8000rpm for 15 minutes, and then the refolded protein supernatant was used as the input for multimode chromatography II.

[0087] (iv) Multimode Chromatography II Multimode chromatography II was performed using polyCSX-35 resin. rHu ranibizumab capture experiments were performed using 10.0 mM acetate buffer at pH 5.50 as equilibration buffer. Elution was performed with a salt-based step gradient with 10.0 mM acetate buffer at pH 5.5 containing 1.0 M NaCl. After selective removal of process-related impurities with a 10% gradient, pure ranibizumab was eluted with a 45% elution step gradient. RP-HPLC analysis of these peaks confirmed that process-related impurities (host cell proteins and nucleic acids) were observed in the flow-through and elution peak I, while purified ranibizumab was observed in elution peak II, and product-related impurities (misfolded and unfolded ranibizumab) were observed in elution peak III.

[0088] (v) Multimode Chromatography I Multimode chromatography I was performed in flow-through mode using polyPEI-35 resin. The pH of the output of multimode chromatography II was adjusted to pH 9.0. The experiment to capture refolded rHu ranibizumab in the flow-through was performed with a 5 CV salt gradient using 20.0 mM Tris pH 9.0 as the equilibration buffer followed by 20.0 mM Tris pH 9.0 containing 1.0 M NaCl to remove impurities. Under this experimental condition, refolded rHu ranibizumab was observed in the flow-through. RP-HPLC analysis of the flow-through confirmed that purified ranibizumab was observed in the flow-through. Elution peak I and elution peak II contain product-related impurities (misfolded and unfolded ranibizumab) and process-related impurities (host cell proteins and nucleic acids).

[0089] Example 7: Multimode chromatography in bind-elute mode I and II Soluble expression and pretreatment of rHu ranibizumab in vivo: (i) Expression of soluble rHu ranibizumab in mutant E. coli E. coli cells containing expressed recombinant human ranibizumab were harvested and resuspended in lysis buffer (20.0 mM Tris, 0.1 mM EDTA, pH 9.0) at a cell pellet:buffer ratio of 1:10 (w / v). Cell lysis was performed using a high-pressure homogenizer at 15000 bar for 20 minutes. To obtain the cell lysate supernatant, the cell lysate was centrifuged at 10000 rpm for 30 minutes at 4°C.

[0090] (ii) Ultrafiltration and diafiltration of refolded rHu ranibizumab Soluble rHu ranibizumab was buffer exchanged into 20.0 mM Tris, pH 9.0 using a continuous diafiltration procedure. The buffer exchanged sample was used as input for multimode chromatography.

[0091] (iii) Multimode Chromatography I Multimode chromatography was performed using polyPEI-35 resin. The concentrated soluble rHu ranibizumab was adjusted to pH 9.0 and used as the feed material. The experiment to capture soluble rHu ranibizumab was performed with 20.0 mM Tris pH 9.0 as the equilibration buffer, followed by a 5.0 CV salt gradient with 20.0 mM Tris pH 9.0 containing 1.0 M NaCl. Under the experimental conditions, no protein was observed in the flow-through. RP-HPLC analysis of the elution peaks confirmed that purified ranibizumab was observed in elution peak I (38.79% purity). Elution peak II contains product-related impurities (misfolded and unfolded ranibizumab) as well as process-related impurities (host cell proteins and nucleic acids).

[0092] (iv) Multimode Chromatography II Multimode chromatography II was performed using polyCSX-35 resin. The experiments capturing rHu ranibizumab were carried out using 10.0 mM acetate buffer at pH 5.50 as equilibration buffer. The elution pool of multimode chromatography I was used as the feed material for multimode chromatography II. The pH of the feed material was adjusted to pH 5.50 with glacial acetic acid. Elution was performed with a salt-based step gradient with 10.0 mM acetate buffer at pH 5.50 containing 1.0 M NaCl. After selective removal of impurities related to the 10.0% salt gradient run, pure rHu ranibizumab was eluted with a 45.0% elution step gradient. RP-HPLC analysis of these peaks confirmed that process-related impurities (host cell proteins and nucleic acids) were observed in the flow-through and elution peak I, while purified ranibizumab was observed in elution peak II (Figure 3), and product-related impurities (misfolded and unfolded ranibizumab) were observed in elution peak III. The product quality was verified using SDS PAGE analysis and was in good agreement with that of standard ranibizumab (lane no. 3, 48 kDa in Figure 10).

[0093] Example 8: Multimode chromatography in bind and elute mode I and II (i) Solubilization of inclusion bodies Solubilization was carried out as in Example 1(i). (ii) Refolding of solubilized inclusion bodies Refolding was carried out as in Example 1(ii). (iii) Ultrafiltration and diafiltration of refolded rHu ranibizumab Filtration was carried out as in Example 1(iii). (iv) Multimode Chromatography I Multimode chromatography I was performed using Capto adhere resin. The concentrated refolded ranibizumab was adjusted to pH 9.0 and used as the feed material. The experiment to capture refolded rHu ranibizumab was performed with a 5.0 CV salt gradient using 20.0 mM Tris pH 9.0 as the equilibration buffer, followed by 20.0 mM Tris pH 9.0 containing 1.0 M NaCl. Under this experimental condition, no protein was observed in the flow-through. RP-HPLC analysis of the elution peak confirmed that purified ranibizumab was observed in elution peak I (38.79% purity). Elution peak II contains product-related impurities (misfolded and unfolded ranibizumab) as well as process-related impurities (host cell proteins and nucleic acids).

[0094] (v) Multimode Chromatography II Multimode chromatography II was performed using Capto MMC resin. The experiments capturing rHu ranibizumab were carried out using 10.0 mM acetate buffer at pH 5.50 as equilibration buffer. The elution pool of multimode chromatography I was used as the feed material for multimode chromatography II. The pH of the feed material was adjusted to pH 5.50 using glacial acetic acid. Elution was performed with a salt-based step gradient with 10 mM acetate buffer at pH 5.50 containing 1.0 M NaCl. After selective removal of process-related impurities in a 10.0% salt gradient, pure rHu ranibizumab was eluted with a 45.0% elution step gradient. RP-HPLC analysis of these peaks confirmed that process-related impurities (host cell proteins and nucleic acids) were observed in the flow-through and elution peak I, while purified ranibizumab was observed in elution peak II and product-related impurities (misfolded and unfolded ranibizumab) were observed in elution peak III.

[0095] Example 9: Analytical characterization of recombinant ranibizumab (i) Absorbance measurement at A280 of ranibizumab samples UV absorbance measurements at 280 nm were used to determine the total protein refolded and present in the chromatographic output. All collected fractions were read at 280 nm using a Nanodrop 2000 and a UV-1800 Shimadzu UV-visible spectrophotometer.

[0096] (ii) Bradford assay for total protein estimation of ranibizumab The orthogonal technique used for total protein estimation was the Bradford assay at 595 nm using a Nanodrop 2000. 5 μl of sample was added to 250 μl of Bradford reagent, followed by mixing on a shaker for 30 seconds. After mixing, the sample was incubated in the presence of dye for 25 minutes and the absorbance at 595 nm was measured.

[0097] (iii) Reversed-Phase HPLC Analysis for Quantitation of Ranibizumab The concentration of rHu ranibizumab in the various chromatographic outputs was determined using a 4.6 mm x 150 mm Aeris™ 3.6 um WIDEPORE XB C18 column on an Agilent 1260 HPLC system (Figure 11). The mobile phase consisted of 0.1% (v / v) TFA in water (solvent A) and 0.1% (v / v) TFA in water, 70% (v / v) acetonitrile, 20% (v / v) isopropanol (solvent B). The flow rate was maintained at 1 ml / min using a linear gradient of A against B at a wavelength of 214 nm. For the purified refolded ranibizumab in Figure 11(b) obtained by the method of the present invention, the peak (retention time: 28.1 min) is similar to the peak obtained in Figure 11(a) for standard ranibizumab.

[0098] (iv) SDS PAGE analysis of ranibizumab samples SDS PAGE analysis for the identification of process and product related impurities for ranibizumab was performed using a 12% (1 mm thick) separation gel under non-reducing (Figure 7) and reducing (Figure 8) conditions with a constant voltage of 120 V for the stacking gel and 100 V for the separation gel. Each sample was boiled for 10 minutes in starting buffer before running the gel. After electrophoretic separation on polyacrylamide gel, 0.05% (w / v) Coomassie Brilliant Blue G-250 in 4:1:5 (water:glacial acetic acid:methanol) was used for protein detection. Analysis of the gel in Figure 7 shows the presence of a 48 kDa protein band in lane 1 for the purified refolded protein, thereby confirming the presence of rHu ranibizumab. The reduced gel result in Figure 8 shows the presence of the light and heavy chains of ranibizumab. Figures 7 and 8 show the biosimilarity with standard ranibizumab.

[0099] (v) ELISA Assay for Quantifying the In Vitro Bioactivity of Ranibizumab Samples Immunoenzymatic assay (Lucentis / Ranibizumab, anti-VEGF ELISA kit for humans from Alpha diagnostic international, USA). Samples containing purified refolded ranibizumab were reacted with VEGF antigen coated on a plate. Bound ranibizumab was detected with horseradish peroxidase (HRP) enzyme-labeled anti-human IgG. The immunological reaction results in the formation of a sandwich complex of solid-phase VEGF-binding antibody-ranibizumab enzyme-labeled antibody. The microtiter strips were washed to remove unbound reactants. The substrate tetramethylbenzidine (TMB) was then reacted. The amount of hydrolyzed substrate was read at 450 nm in a microtiter plate reader, which is directly proportional to the concentration of ranibizumab present.

[0100] (vi) ELISA assay for quantification of ranibizumab host cell proteins (HCPs) Contamination by host cell proteins in the flow-through of anion exchange chromatography was analyzed using a two-site immunoenzymatic assay (Cygnus E. coli HCP Analysis Kit F410). Samples containing E. coli HCP were simultaneously reacted with horseradish peroxidase (HRP) enzyme-labeled anti-E. coli antibodies in microtiter strips coated with affinity-purified capture anti-E. coli antibodies. The immunological reaction results in the formation of a sandwich complex of solid-phase antibody-HCP-enzyme-labeled antibody. The microtiter strips were washed to remove unbound reactants. The substrate tetramethylbenzidine (TMB) was then reacted. The amount of hydrolyzed substrate was read in a microtiter plate reader at 450 nm and is directly proportional to the concentration of E. coli HCP present.

[0101] (vii) SE HPLC analysis of Ranibizumab samples: The aggregate content in the various outputs was determined using SEC-HPLC analysis performed using a Yarra™ 3um SEC-2000 (300×7.8 mm ID) column ( FIG. 12 ). The mobile phase consisted of 20 mM acetate buffer with 50 mM sodium chloride buffer at pH 5.50. The analysis was performed at 25° C. in isocratic mode with a flow rate of 0.5 ml / min. Protein detection was performed at 280 nm using a photodiode array detector.

[0102] (viii) Estimation of double-stranded DNA: The presence of DNA in the output samples was estimated using the Quant-iT™ PicoGreen assay (Invitrogen BioServices India Private Limited, India). A standard curve was generated using double-stranded λ DNA. 0.5 ml of output sample was added to 0.5 ml of diluted Quant-iT™ dsDNA BR reagent and the reaction mixture was incubated for 5 minutes. After 5 minutes, the fluorescence was measured using a fluorescence spectrophotometer (excitation wavelength 480 nm and emission wavelength 520 nm).

[0103] (viii) Intact mass analysis of purified rHu ranibizumab by matrix-assisted laser desorption / ionization (MALDI-TOF) Standard and purified rHu ranibizumab were mixed with sinapinic acid matrix in a 1:1 ratio for MALDI-TOF analysis. The matrix sinapinic acid (10 mg / ml) was prepared in 30% v / v acetonitrile, 0.1% v / v TFA in high purity water. 1 μl of homogenized mixture of sample and matrix was placed on a clean 384-well MALDI plate. The plate was inserted into an AB SCIEX TOF / TOF™ 5800 instrument. The instrument was used in positive ion mode. A 337 nm nitrogen laser was used as the ionization source. A laser intensity of 4000-5000 was used for the analysis of the samples. Analysis of the results was performed using ProteinPilot™ software (Figure 13). Figure 13 shows the comparison of intact mass between purified rHu ranibizumab obtained using the developed purification platform and standard ranibizumab molecules.

[0104] (ix) Peptide fingerprinting by LC-MS Coomassie stained protein bands from SDS-PAGE analysis were cut using a scalpel on a glass plate and chopped into small pieces. Gel pieces were washed with 100 mM NH4HCO3 and 200 μl of 50% acetonitrile and 50% of 50 mM NH4HCO3 were added, vortexed, and incubated for 10 min. The liquid was removed and the gel pieces were washed again with 100% acetonitrile to dehydrate. The acetonitrile was removed and air dried for 10 min in a fume hood. For reduction and alkylation, the gel pieces were covered with 50 μl, 10 mM DTT in 100 mM NH4HCO3 at 56 °C for 45-60 min. The DTT solution was removed and 50 μl of 55 mM iodoacetamide in 100 mM NH4HCO3 was added and incubated for 45 min in the dark at room temperature. The iodoacetamide was removed. Gel pieces were washed with 100 μl of 100 mM NH4HCO3 followed by vortexing for 5 min with 50% acetonitrile + 50% 50 mM NH4HCO3 twice. Gels were dehydrated with 100 μl of acetonitrile as above. Residual liquid was removed and dried using a speedvac. For trypsin digestion, 20 μl of trypsin (12.5 ng / μl) solution was added until it just covered the gel pieces. Gel pieces were rehydrated in a buffer containing 50 mM NH4HCO3 and trypsin for 30 min at 4 °C. Gel pieces were digested overnight at 37 °C. The digested supernatant was transferred to a clean 1.5 mL Eppendorf tube for peptide extraction. 30 μL of 50% acetonitrile and 2% formic acid was added to the gel pieces and incubated for 20 min. The peptide solution was sonicated for 5 min in a water bath without heating. The supernatant was removed and combined with the initial digestion solution. The peptide solution was then dried in a speedvac concentrator. The peptide sample was reconstituted in 3.0% v / v acetonitrile, 0.1% v / v formic acid in high purity water. 4 μL of sample (2.5 μg) was loaded onto an Agilent ZORBAX RRHD 300SB-C18, 0.3×100 mm, 3 μm column and analyzed by a Q Exactive Orbitrap LC-MS / MS system.

[0105] Advantages of the Invention Nearly a two-fold increase in productivity was achieved by combining a multi-mode chromatographic purification step with downstream processing of rHu ranibizumab compared to existing manufacturing methods. Orthogonal analytical studies established analytical equivalence / homogenity between the purified drug obtained using the present invention and standard ranimizumab. The developed purification platform is applicable to both in vitro refolded and soluble expressed antibody fragments. The invention as originally claimed is set forth below. [1] A method for purifying an antibody fragment of recombinant humanized (rHu) ranibizumab expressed in a recombinant host cell as either an inclusion body or a soluble form, the method comprising the steps of: (a) solubilizing inclusion bodies containing the light and heavy chains of rHu ranibizumab from recombinant host cells to obtain solubilized rHu ranibizumab; (b) refolding the solubilized rHu ranibizumab of step (a) to obtain refolded rHu ranibizumab; (c) concentrating the refolded rHu ranibizumab of step (b) by ultrafiltration to obtain concentrated refolded ranibizumab; (d) subjecting the concentrated refolded / soluble form of rHu ranibizumab of step (c) to multi-mode chromatography, said chromatography steps comprising the following steps (i) to (iv); (i) feeding the concentrated refolded rHu ranibizumab of step (c) having a pH in the range of 4.0 to 10.0 to a multi-mode chromatography resin using a buffer having a pH in the range of 4.0 to 10.0 comprising 20 mM to 50 mM Tris-HCl and 0 mM to 150 mM NaCl or 20 mM to 50 mM acetate and 0 mM to 150 mM NaCl, to capture rHu ranibizumab and remove impurities; (ii) eluting rHu ranibizumab into an elution pool in the presence of an elution buffer containing 20 mM to 50 mM acetate and 0 mM to 1000 mM sodium chloride or 20 mM to 50 mM acetate and 0 mM to 1000 mM sodium chloride and having a pH in the range of 4.0 to 10.0; wherein purified ranibizumab has less than 5% unfolded or misfolded forms of ranibizumab and less than 2% aggregated forms of ranibizumab; (iii) feeding the rHu ranibizumab from the elution pool of step (ii) having a pH in the range of 4.5 to 6.5 using a buffer having a pH in the range of 4.0 to 6.5 comprising 20 mM to 50 mM acetate and 0 mM to 150 mM NaCl onto a multi-mode chromatography resin to capture the rHu ranibizumab; (iv) eluting rHu ranibizumab with an elution buffer containing 20 mM to 50 mM acetate and 0 mM to 1000 mM NaCl and having a pH in the range of 4.0 to 6.5; wherein the purified rHu ranibizumab has less than 0.1% unfolded or misfolded forms of rHu ranibizumab and less than 0.3% aggregated forms of rHu ranibizumab. [2] The method according to [1], wherein the mode of multimode chromatography is selected from the group consisting of flow-through multimode chromatography and bind-elute multimode chromatography. [3] (i) The pH condition of the binding-elution mode of multimode chromatography is within the range of 8.0 to 10.0, and (ii) The method according to [2], wherein the pH condition in the flow-through mode of the multi-mode chromatography is within the range of 4.0 to 6.5. [4] Multimode chromatography using flow-through mode (i) applying an input conductivity in the range of 6.0 mS / cm to 12 mS / cm to the concentrated refolded ranibizumab of step (c); and (ii) applying an input conductivity in the range of 6.0 mS / cm to 12 mS / cm to the recombinant ranibizumab obtained in step (ii); The method according to claim 2, comprising: [5] The method according to [1], wherein the multimodal chromatography resin in step (i) is selected from the group consisting of HEA Hypercel, PPA Hypercel, and BAKERBOND™ XWP 500 Poly PEI-35 and / or Capto adhere resin. [6] The method of [1], wherein the multimodal chromatography resin in step (iii) is selected from the group consisting of HEA Hypercel, PPA Hypercel, and BAKERBOND™ XWP 500 PolyCSX-35 and / or Capto MMC resin. [7] The method according to [1], wherein steps (i) and (ii) are interchangeable with steps (iii) and (iv), respectively. [8] The pH of the elution pool containing rHu ranibizumab after a first step of multimodal chromatography is adjusted to correspond to the pH range of a successive multimodal chromatography step, the process comprising: (a) adjusting the pH of the elution pool after the first stage of steps (i) and (ii) of multimodal chromatography to a pH in the range of 4 to 6; or (b) adjusting the pH of the elution pool after the first stage of steps (iii) and (iv) of the multimodal chromatography method to a pH in the range of 8 to 10. The method according to [1] or [7], comprising: [9] Recombinant humanized ranibizumab obtained by the method described in [1].

[10] A pharmaceutical composition comprising the ranibizumab described in [9] together with a pharma- ceutical acceptable carrier.

Claims

1. 1. A method for purifying an antibody fragment of recombinant humanized (rHu) ranibizumab expressed in a recombinant host cell either as an inclusion body or in a soluble form, said method comprising the steps of: (a) solubilizing inclusion bodies containing the light and heavy chains of rHu ranibizumab from recombinant host cells to obtain solubilized rHu ranibizumab; (b) refolding the solubilized rHu ranibizumab of step (a) to obtain refolded rHu ranibizumab; (c) concentrating the refolded rHu ranibizumab of step (b) by ultrafiltration to obtain concentrated refolded ranibizumab; (d) subjecting the concentrated refolded / soluble form of rHu Ranibizumab of step (c) to multimodal chromatography, said chromatography steps comprising the following steps (i) to (iv); (i) feeding the concentrated refolded rHu ranibizumab of step (c) at pH 9 onto a multimodal chromatography resin using a buffer comprising 20 mM to 50 mM Tris-HCl and 0 mM to 150 mM NaCl at pH 9 to capture rHu ranibizumab and remove impurities; (ii) eluting rHu ranibizumab into an elution pool in the presence of an elution buffer comprising 20 mM to 50 mM Tris-HCl and 0 mM to 1000 mM sodium chloride at pH 9; wherein the purified rHu ranibizumab has less than 5% unfolded or misfolded forms of ranibizumab and less than 2% aggregated forms of ranibizumab; (iii) feeding the rHu ranibizumab from the elution pool of step (ii), adjusted to pH 5.5 with glacial acetic acid, onto a multi-modal chromatography resin to capture the rHu ranibizumab; (iv) eluting rHu ranibizumab with an elution buffer containing 10 mM acetate and a step gradient of 0 mM to 1000 mM NaCl, pH 5.5; wherein the purified rHu ranibizumab has less than 0.1% unfolded or misfolded forms of rHu ranibizumab and less than 0.3% aggregated forms of rHu ranibizumab; The salt gradient in step (ii) is 5-10 CV.

2. The method of claim 1, wherein the mode of multimode chromatography is selected from flow-through multimode chromatography and bind-elute multimode chromatography.

3. Multimode chromatography using flow-through mode (i) applying an input conductivity in the range of 6.0 mS / cm to 12 mS / cm to the concentrated refolded ranibizumab of step (c); or (ii) applying an input conductivity in the range of 6.0 mS / cm to 12 mS / cm to the recombinant ranibizumab obtained in step (ii); The method of claim 1 or 2, comprising:

4. 2. The method of claim 1, wherein the multimodal chromatography resin in step (i) is selected from the group consisting of HEA Hypercel, PPA Hypercel, BAKERBOND™ XWP 500 Poly PEI-35 and Capto adhere resin.

5. 2. The method of claim 1, wherein the multimodal chromatography resin in step (iii) is selected from the group consisting of HEA Hypercel, PPA Hypercel, BAKERBOND™ XWP 500 PolyCSX-35 and Capto MMC resin.

6. The method according to claim 1, wherein the purity and yield of purified rHu ranibizumab are 99.50±0.08% and 32.55±2.55%, respectively.

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